ORIGINAL ARTICLE
Journal ofComplement 3 is Involved in
Changing the Phenotype of
Human Glomerular
Mesangial Cells

CellularPhysiology

JIAN-XIN WAN,1 NOBORU FUKUDA,1,2* MORITO ENDO,1,3 YOSHIKO TAHIRA,1
EN-HUI YAO,1 HIROYUKI MATSUDA,1,2 TAKAHIRO UENO,1 AND KOICHI MATSUMOTO1
1

Department of Medicine, Division of Nephrology and Endocrinology, Nihon University School of Medicine, Tokyo, Japan

2

Advanced Research Institute for the Science and Humanities, Nihon University, Tokyo, Japan

3

Faculty of Human Health Science, Hachinohe University, Aomori, Japan

Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement
components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a criticalcomponent of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3
stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of
C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels
were increased in response to IFN-g and TNF-a. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated
DNA synthesis in HMCs in a dose-dependent manner. C3a decreased expression h-caldesmon mRNA, a marker of the contractile
phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 a1 (collagen IV) mRNAs, which are markers of
the synthetic phenotype. C3a decreased expression of a-smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3
reduced the DNA synthesis and proliferation of HMCs, increased expression of h-caldesmon mRNA, and decreased expression of
osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic
phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial
cells in renal diseases.
J. Cell. Physiol. 213: 495–501, 2007. ß 2007 Wiley-Liss, Inc.

Proliferation of glomerular mesangial cells appears to play animportant role in the pathogenesis of progressive glomerularabnormalities leading to glomerulosclerosis. In experimental
models of glomerulopathy, increased mesangial cellproliferation often precedes the development ofglomerulosclerosis with increased extracellular matrix (ECM)
deposition in the mesangium (Pesce et al., 1991; Floege et al.,1992). The search for agents which are capable of favorably
regulating MC proliferation are, therefore, of considerable
clinical importance in the area of progressive glomerular
damage. There are known many similarities between thefunction and structure of the glomerulus and those of thevasculature. Endothelial cells compose both the glomerulus andvasculature, and mesangial cells are modified vascular smoothmuscle cells (VSMCs) derived from the same progenitor cells(Bakris et al., 1996). We found that complement 3 (C3) isexpressed in spontaneously hypertensive rat (SHR)-derived
VSMCs that show the synthetic phenotype and exaggerated
growth. We found that C3 causes VSMCs to convert from thecontractile to the synthetic phenotype to stimulate growth (Linet al., 2004).
Complement components, particularly C3, have been
detected in glomeruli of patients with a variety of renal diseases(Welch, 2001). It is generally accepted that the complement
system is associated with the development of glomerulopathy,
and the beneficial effects of complement inhibition have been
investigated in cases of acute and chronic renal injury (Johnson,1997). Moreover, recent studies have suggested that local
production of complement significantly influences pathologic
conditions in renal disease (Zhou et al., 2001). Complement
activation leads to tissue injury in several ways, including
generation of anaphylactic and chemotactic peptides C3a andß 2 0 0 7 W I L E Y - L I S S , I N C .

C5a by cleavage of C3 and C5, respectively, and formation of
the C5b-9 membrane attack complex. However, little attention
has been given to the direct effects of C3, which is a centralcomponent of the complement system that acts on humanglomerular mesangial cells (HMCs).
In the current study, to examine the contributions of C3 toglomerulopathy, we investigated the role of C3 in phenotype
modulation and proliferation of HMCs.
Materials and Methods
Cell culture

HMCs were purchased from Cambrex BioScience (Walkersville,
MD). HMCs were maintained in RPMI 1640 (Sigma, St. Louis, MO)with 10% fetal bovine serum (FBS) (Gibco Laboratories, Grand
Island, NY) and 0.05 mg/ml gentamicin (Gibco). Experiments were
performed on cells between the fifth and tenth passages.

Contract grant sponsor: Ministry of Education, Science, Sports, andCulture of Japan;
Contract grant number: 15590863.
*Correspondence to: Noboru Fukuda, Department of Medicine,
Nihon University School of Medicine, Ooyaguchi-kami 30-1,
Itabashi-ku, Tokyo 173-8610, Japan.
E-mail: fukudan@med.nihon-u.ac.jp
Received 10 November 2006; Accepted 2 April 2007
DOI: 10.1002/jcp.21129

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Determination of DNA synthesis and cell numbers

TABLE 2. Thermal cycling profiles for PCR

DNA synthesis in HMCs was assessed by 5-bromo-2-deoxyuridine(BrdU) labeling and detection with an ELISA kit (Cell Proliferation
ELISA System, RPN250, Amersham Biosciences, Buckinghamshire,UK). Quiescent HMCs were treated both with and without human
complement 3a (C3a, Calbiochem, Spring Valley, CA) for 24 h andthen incubated with 10 mM BrdU for 2 h. BrdU uptake wasmeasured according to the ELISA protocol. The optical density
was determined in a plate reader at 450 nm (Microplate Reader
MPR-A4i-II, Tosoh, Tokyo, Japan).
Quiescent HMCs were incubated for 24 h in 12-well plates with
RPMI 1640 with 5% FBS containing 0.05 mg/ml gentamicin (Gibco).
HMCs were trypsinized with 0.05% trypsin at 24, 48, and 72 h after
inoculation, and cell numbers were counted with a CoulterCounter (Beckman Coulter, Fullerton, CA).

Target mRNA Denaturation Primer annealing Primer extension Cycles

Total RNA was extracted from cultured HMCs with ISOGEN(Nippon Gene, Toyama, Japan) according to the manufacturer’s
instructions. Aliquots of total RNA were reverse transcribed into
single-stranded cDNA by incubation with avian myeloblastoma
virus reverse transcriptase (Takara Biochemicals, Osaka, Japan).
Diluted cDNA products were then subjected to PCR. Primers used
to amplify complement 3 (C3), h-caldesmon, osteopontin, matrix
Gla, and collagen type1 a1 (collagen IV) transcripts are listed inTable 1. Human 18S ribosomal RNA was amplified as an internal
control. PCR was performed according to the profiles shown in
Table 2. PCR was performed in a DNA thermal cycler (GeneAmp
PCR System 2700, Applied Biosystems, Foster City, CA). The
quality and concentration of amplified PCR products were
determined with the Agilent 2100 Bioanalyzer (Agilent, Palo Alto,CA).
Western blot analysis for C3 and C3a receptor proteins

HMCs or U937 cells (positive control for C3a Receptor) were
disrupted with lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM
NaCl, 0.02% sodium azide, 100 mg/ml phenylmethylsulfonyl
fluoride, 1 mg/ml aprotinin, 1% Triton X-100). Total proteins
were extracted, heated at 958C for 5 min, subjected to 7.5%
SDS–polyacrylamide gel electrophoresis (SDS–PAGE), andelectroblotted onto PVDF membranes (Amersham Biosciences,
Uppsala, Sweden). Blots were incubated with rabbit polyclonal
antibody specific for C3, goat polyclonal antibody specific for C3a
receptor (Santa Cruz Biotechnology, Santa Cruz, CA), or mousemonoclonal antibody specific for a-tubulin (Sigma) as an internal
control and then with goat anti-rabbit IgG, donkey anti-goat IgG orgoat anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA),
respectively, as secondary antibodies. Bound antibodies weredetected by enhanced chemiluminescence (ECL Kit, RPN2106,
Amersham) and exposure to X-ray films. SDS–PAGE molecular
TABLE 1. PCR primers and product sizes

C3
h-caldesmon
Osteopontin
Matrix Gla
Collagen IV
18S

PrimerSequence

5(
3(
5(
3(
5(
3(
5(
3(
5(
3(
5(
3(

5(-GCTGAAGCACCTCATTGTGA-3(5(-CTGGGTGTACCCCTTCTTGA-3(
5(-TCGCCTACCAGAGAATGAC-3(
5(-TTGTTGGGTCGAACTCCTTC-3(
5(-TCAGCTGGATGACCAGAGTG-3(
5(-TGAAATTCATGGCTGTGGAA-3(
5(-CACGAGCTCAATAGGGAAGC-3(
5(-GCTGCTACAGGGGGATACAA-3(
5(-TGGTCCAAGAGGATTTCCAG-3(
5(-TCATTGCCTTGCACGTAGAG-3(
5(-CGACGACCCATTCGAACGTCT-3(
5(-GCTATTGGAGCTGGAATTACCG-3(

PCR, polymerase chain reaction; C3, complement 3.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

94-C, 30 sec
94-C, 30 sec
94-C, 30 sec
94-C, 30 sec
94-C, 30 sec
94-C, 30 sec

58-C, 30 sec
55-C, 30 sec
57-C, 30 sec
56-C, 30 sec
55-C, 30 sec
55-C, 30 sec

72-C, 30 sec
72-C, 30 sec
72-C, 30 sec
72-C, 30 sec
72-C, 30 sec
72-C, 30 sec

35
30
30
30
30
20

weight markers (Prestained SDS–PAGE Standard Broad Range,
Bio-Rad) were run to calibrate the gel. Bands were scanned andquantitated by densitometry with NIH Image Software (NIH Image
1.63f).
Immunofluorescence of a-smooth muscle actin

Reverse transcription-polymerase
chain reaction (RT-PCR) analysis

TargetmRNAC3h-caldesmon
Osteopontin
Matrix Gla
Collagen IV
18 S

Immunocytochemical staining was performed for a-smoothmuscle actin expression. HMCs were immunostained using
monoclonal mouse anti-smooth muscle actin antibody (1A4, 1:200;
Dako, Carpinteria, CA), a marker for the contratile phenotype.
Incubation with the primary antibody dilution was performed at
48C overnight. Incubation with secondary FITC-conjugated goatanti-mouse IgG (1:1,000, Invitrogen, Tokyo, Japan) was performedfor 60 min in the dark. Coverslips were placed over the cells andsealed with manicure. Cells were visualized by fluorescencemicroscopy with standard FITC excitation/emission filter
combinations.
Application of short interfering RNA (siRNA)A duplex siRNA was designed to target human C3 (NCBI:
NM_000064). The target sequence is
CCGAGCCGTTCTCTACAATTA (2,601–2,621 bp). The
siRNA nucleotide sequences for C3 werer(GAGCCGUUCUCUACAAUUA)dTdT (Sense) andr(UAAUUGUAGAGAACGGCUC)dGdG (Antisense).
Meanwhile, a non-silencing siRNA duplex targeting sequence,
AATTCTCCGAACGTGTCACGT, was used as a negative control(control siRNA). Control siRNA nucleotide sequences werer(UUCUCCGAACGUGUCACGU)dTdT (Sense) andr(ACGUGACACGUUCGGAGAA)dTdT (Antisense). The duplex
siRNA to C3 and the control siRNA duplex were synthesized by
Qiagen (Tokyo, Japan). HMCs were seeded in 12-well plates at adensity of 1  105 cells per well to obtain 50% confluence.
Transfection of the siRNA was performed with RNAiFect
Transfection Reagent (Qiagen) in RPMI 1640 (without FBS andantibiotics) per the manufacturer’s instructions to yield a final
siRNA concentration of 3.3 mg/ml in the culture media. Forty-eight
hours after transfection of siRNA, cells were stimulated with
0.1 mM C3a, 10 mg/ml tumor necrosis factor-a (TNF-a, Sigma), or
50 ng/ml interferon-g (IFN-g, Sigma) for 24 h. Control cells weretransfected with the siRNA buffer alone.
Statistical analysis

PCR
product (bp)169

Values are reported as mean  SEM. Statistical analysis was donewith Student’s t-test for unpaired data, two-way analysis of variance(ANOVA), or Duncan’s multiple range test. P < 0.05 wasconsidered statistically significant.

300
153
188
192
312

Results
Expression of C3 and C3a receptor mRNA and protein
in cultured HMCs

HMCs expressed both C3 mRNA and protein. IFN-g and TNFa significantly increased levels of C3 mRNA and protein in
HMCs (Fig. 1A,B). HMCs also expressed C3a receptor mRNAand protein (Fig. 1C,D). C3a receptor mRNA expression wasincreased by exogenous C3a, TNF-a and IFN-g.

C3 CHANGES PHENOTYPE OF MESANGIAL CELLS

Fig. 1. A: Expression of C3 mRNA by RT-PCR analysis of cultured HMCs treated with TNF-a or IFN-g. Quiescent HMCs were incubatedwithout (control) or with 10 mg/ml TNF-a or 50 ng/ml IFN-g for 24 h. The ratio of the abundance of each mRNA to that of 18S rRNA was evaluatedby densitometric analysis. B: Expression of C3 protein by Western blot analysis in HMCs cultured without or with TNF-a and IFN-g.
Quiescent HMCs were incubated without (control) or with 10 mg/ml TNF-a or 50 ng/ml IFN-g for 24 h. The ratio of the abundance of C3
protein to that of a-tubulin was evaluated by densitometric analysis. C: RT-PCR analysis of expression of C3a receptor (C3aR) mRNA bycultured HMCs without or with C3a, TNF-a, and IFN-g. Quiescent HMCs were incubated without (control) or with 0.1 mM C3a, 10 mg/ml TNF-a,or 50 ng/ml IFN-g for 24 h. The ratio of the abundance of each mRNA to that of 18S rRNA was evaluated by densitometric analysis. D: Expressionof C3a receptor protein by Western blot analysis in HMCs or U937 cells. Data are mean W SEM (n U 6). MP < 0.05 versus control cells.

Effects of exogenous C3a on phenotype
and growth of HMCs

C3 convertases, including the spontaneously generated fluidphase convertase, cleave C3 and release the anaphylatoxin C3a.
The effects of exogenous C3a on phenotypic marker mRNAs
and DNA synthesis in HMCs are shown in Figure 2. Exogenous
C3a significantly decreased the abundance of h-caldesmonmRNA in HMCs ( P < 0.05). Exogenous C3a significantly
increased expression of osteopontin, matrix Gla, and collagen
IV mRNAs in HMCs ( P < 0.05). Exogenous C3a (0.001–1.0 mM)
significantly increased DNA synthesis in HMCs in adose-dependent manner ( P < 0.05). These data suggest thatexogenous C3a promotes conversion of HMCs from thecontractile phenotype to the synthetic phenotype andstimulates growth of HMCs.
Effect of C3a on morphology and a-smoothmuscle actin expression in HMCs

C3a treatment did not considerably change morphology of
HMCs. However, C3a treatment for 24 or 48 h markedly
decreased immnofluorescence of a-smooth muscle actin in
HMCs compared to HMCs without C3a treatment (Fig. 3).
Effects of siRNA against C3 on phenotype
and growth of HMCs

To evaluate the contribution of endogenous C3 to thephenotype and growth of HMCs, we designed and synthesized aJOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

duplex siRNA against C3. The effects of the duplex siRNA
specific for C3 on expression of C3 mRNA and protein in
HMCs are shown in Figure 4. siRNA against C3 significantly
decreased levels of both C3 mRNA and protein ( P < 0.05).
The effects of siRNA to C3 on expression of phenotypicmarker mRNAs in HMCs are shown in Figure 5. siRNA against
C3 significantly increased expression of h-caldesmon mRNA in
HMCs ( P < 0.05), whereas siRNA to C3 significantly decreased
levels of osteopontin, matrix Gla, and collagen IV mRNAs in
HMCs ( P < 0.05). These data suggest that suppression ofendogenous C3 causes HMCs to change from the synthetic tothe contractile phenotype in culture.
The effects of siRNA against C3 on growth of HMCs areshown in Figure 6. siRNA to C3 significantly inhibited DNA
synthesis in HMCs in a dose-dependent manner ( P < 0.05).
siRNA to C3 significantly suppressed proliferation of HMCs at
48 and 72 h after transfer of siRNA in the presence of 5% calf
serum ( P < 0.05).
Discussion

The mesangial tissue between the glomerular capillaries
consists of mesangial cells and an intercellular matrix. Mesangial
cells contain contractile processes that attach directly to thecapillaries and regulate capillary diameter and provide
structural support for the glomeruli through contractions.
Mesangial cells contain high levels of myosin and actin filaments
and have various functions, including phagocytosis. Mesangial

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Fig. 2. A–D: Effects of exogenous C3a on expression of phenotype marker mRNAs in cultured HMCs. Quiescent HMCs were incubatedwith or without 0.1 mM C3a, 10 mg/ml TNF-a, or 50 ng/ml IFN-g for 24 h. Total RNA was extracted and h-caldesmon (A), osteopontin (B), matrix
Gla (C), and collagen IV (D) mRNAs were evaluated by RT-PCR analysis. The ratio of the abundance of each mRNA to that of 18S rRNA
was evaluated by densitometric analysis. Data are mean W SEM (n U 6). MP < 0.05 versus control cells. E: Effects of exogenous C3a on DNA
synthesis in cultured HMCs. Quiescent HMCs were incubated without or with 0.001–1.0 mM C3a for 24 h and incorporation of BrdU into DNA
was examined. Data are mean W SEM (n U 8). MP < 0.05 versus control cells.

cells also produce substances, such as extracellular matrix
components, hormones, and cytokines (Stockand and Sansom,
1998). The glomerulus is a highly specialized vascular structurein which mesangial cells serve as specialized pericytes sharing
properties with VSMCs (Schlondorff, 1987). In fact, progressive
glomerulosclerosis shows pathological similarities toarteriosclerosis (Grone et al., 1994; Oda and Keane, 1999),
suggesting that these two entities may share pathogenic
mechanisms.

Fig. 3. Representative immunofluorescence of a-smooth muscle
actin of HMCs treated without or with C3a (0.1 mM) for 24 h (A,B), or
48 h (C,D). Magnification 100T. [Color figure can be viewed in the
online issue, which is available at www.interscience.wiley.com.]

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

VSMCs show either a synthetic or contractile phenotype.
The synthetic phenotype is formed when a cell of thecontractile phenotype loses contractile function and begins toproliferate and produce additional proteins, such as
extracellular matrix components (Ross, 1993; Schwartz, 1997).
Freshly plated VSMCs do not proliferate, and they possess ahigh volume-fraction of myofilaments, show few biosyntheticorganelles, and retain their ability to contract in response to a
vasoconstrictor. These characteristics define the differentiatedcontractile phenotype. After several passages, VSMCs
dedifferentiate and display the synthetic phenotype, which ischaracterized by a reduction in the volume fraction of
myofilaments and an increase in synthetic organelles such as
Golgi, mitochondria, and endoplasmic reticulum. Cells with thesynthetic phenotype produce copious amount of proteases,
growth factors, and cytokines (Mosse et al., 1985; Glukhova
et al., 1990). Similar to VSMCs, mesangial cells with thesynthetic phenotype have increased organelle content andproduction of extracellular matrix components, such as
collagens and fibronectin (Makino et al., 1995).
It has been reported that caldesmon is involved in smoothmuscle contraction as an actin-linked regulatory protein (Sobue
et al., 1985; Marston and Redwood, 1993). Ueki et al. (1987)
reported that h-caldesmon is closely associated withphenotypic modulation of smooth muscle cells. Osteopontin
and matrix Gla are expressed preferentially in passaged cells
that have downregulated production of contractile proteins
and are expressed strongly in dedifferentiated VSMCs(Shanahan et al., 1993). Therefore, we used h-caldesmon as amarker of the contractile phenotype and matrix Gla andosteopontin as markers of the synthetic phenotype in cultured
HMCs treated with exogenous C3a, TNF-a, and IFN-g.
Exogenous C3a decreased expression of h-caldesmon mRNAin HMCs, whereas it increased the expression of matrix Gla andosteopontin mRNAs, indicating that C3a promotes HMCs,

C3 CHANGES PHENOTYPE OF MESANGIAL CELLS

Fig. 4. Effects of siRNA against C3 on expression of C3 mRNA and protein in cultured HMCs. Quiescent HMCs were incubated with
3.3 mg/ml siRNA against C3. Forty-eight hours after transfection of siRNA, cells were incubated with 10 mg/ml TNF-a or 50 ng/ml IFN-g for 24 h.
A: The ratio of the abundance of each mRNA to that of 18S rRNA was evaluated by densitometric analysis. Data are mean W SEM (n U 4).
#
P < 0.05 versus cells without C3 siRNA or control siRNA. MP < 0.05, versus untreated control cells. B: The ratio of C3 protein to a-tubulin was
evaluated by densitometric analysis. Data are mean W SEM (n U 4). #P < 0.05 versus cells without C3 siRNA or with control siRNA. MP < 0.05 versus
control cells.

Fig. 5. Effects of siRNA to C3 on expression of phenotypic marker mRNAs in HMCs. Quiescent HMCs were incubated with 3.3 mg/ml siRNA
to C3. Forty-eight hours after transfection of siRNA, cells were incubated with 10 mg/ml TNF-a, 50 ng/ml IFN-g, or 0.1 mM C3a for 24 h. Total
RNA was extracted and h-caldesmon (A), osteopontin (B), and matrix Gla (C) mRNAs were evaluated by RT-PCR analysis. The ratio of theabundance of each mRNA to that of 18S rRNA was evaluated by densitometric analysis. Data are mean W SEM (n U 4). #P < 0.05 versus cells
without C3 siRNA or with control siRNA. MP < 0.05 versus untreated control cells.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

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Fig. 6. Effects of siRNA to C3 on growth of HMCs. A: Effects of siRNA to C3 on basal DNA synthesis of cultured HMCs. Quiescent HMCs
were incubated with 100–1,000 nM siRNA to C3 or control siRNA and incorporation of BrdU into DNA was examined. MP < 0.05 versus control
siRNA. B: Effects of C3 specific siRNA on proliferation of HMCs. HMCs were inoculated at 1 T 105 cells/ml in RPMI 1640 containing 5% calf
serum and 3.3 mg/ml siRNA to C3, 3.3 mg/ml control siRNA, or no siRNA (control cells). HMCs were replenished with RPMI 1640 containing
5% calf serum and 3.3 mg/ml siRNA to C3 and control siRNA every 24 h. Cells were trypsinized at 24, 48, and 72 h after inoculation, and cell number
was determined with a Coulter Counter. Data are mean W SEM (n U 4). MP < 0.05 versus control siRNA or control cells.

transition from the contractile to the synthetic phenotype.
Exogenous C3a decreased expression of a-smooth muscle
actin in HMCs. In addition, exogenous C3a stimulated DNA
synthesis in HMCs. These findings suggest that C3a causes
HMCs to convert to the synthetic phenotype and that it
stimulates the growth of HMCs.
Exogenous C3a also significantly increased the expression ofcollagen IV mRNA in HMCs. Collagen IV is the predominantmolecule in the matrix surrounding mesangial cells (Setty et al.,1998). Glomerulosclerosis is characterized by excessive
deposition and accumulation of ECM components. Thesefindings suggest that the increased production of collagen IV is
associated with the increased number of intracellular organelles
that occurs with the C3-induced switch to the synthetic
phenotype and that is increased production of collagen isassociated with deposition of more ECM.
Although the major site of production of complement
proteins is the liver, complement biosynthesis has been
detected in many other organs and cell types, including
indigenous cells in the kidney (Morgan and Gasque, 1997; Dahaand van Kooten, 2000). Local production of complement has
been reported to be associated with tissue damages against avariety of conditions (Laufer et al., 2001). The complement
system plays an important role in host defenses against various
pathogens, whereas inappropriate activation of thecomplement system can damage host tissues. A pivotal step inthe complement cascade is generation of activated C3 (Li et al.,1999), which occurs through the classical, alternative, or lectin
pathway. The alternative pathway is initiated continuously inplasma by the spontaneous activation of C3, termed ‘‘tick-overof C3.’’ Once C3 has been activated, C3 cleavage is amplified
and C5 convertases, such as C4b2a3b and C3bBb3b, areformed. Cleavage of C5 by the C5 convertase results in therelease of the potent anaphylatoxin C5a and the formation of
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

the membrane attack complex (MAC), which triggers various
cellular-metabolic pathways. However, significant cleavage of
C5 should not have occurred in the present study because theinitial pathway of the complement cascade was not activated.
C3 can be synthesized by embryonic cells (Hong et al., 1991)
and enhances the growth of cancer cells (Di Renzo et al., 1999).
Taken together, these findings suggest that C3 may participate
in cell differentiation and proliferation.
In the cardiovascular field, C3 is known to be increased inatherosclerotic lesions and to contribute to the development of
atherosclerosis in vivo (Seifert and Kazatchkine, 1988; Buono
et al., 2002). Recently, we demonstrated by microarray analysis
that C3 mRNA is produced in smooth muscle from SHR in vivo
but is not expressed in smooth muscle from Wistar Kyoto rats.
In contrast, there were no differences in levels of mRNAs for
C4, C8, and C9 in aortic smooth muscle cells from SHR and
Wistar Kyoto rats (Lin et al., 2004). These data suggest that
expression of C3 in VSMCs is independent of that of othercomplement system components and that C3 has unique effects
on VSMCs.
Synthesis of C3 has been reported in HMCs. A significant
increase in C3 expression was found in kidney biopsy specimensfrom patients with immune-mediated glomerulonephritis andwith interstitial nephritis (Sacks et al., 1993a; Welch et al.,1993). Expression of C3 is tissue specific and is regulated byimmune complexes and several cytokines. IL-1 and other proinflammatory cytokines, such as TNF-a and IL-6, stimulate C3synthesis in several cell types (Volanakis, 1995). In the present
study, synthesis of C3 was detected in cultured HMCs as inprevious studies (Sacks et al., 1993b; van den Dobbelsteen et al.,1994) and was increased in the presence of IFN-g and TNF-a.
Thus, endogenous C3 is increased by cytokines such as IFN-g
and TNF-a, which may contribute to the pathogenesis ofprogressive glomerulosclerosis.

C3 CHANGES PHENOTYPE OF MESANGIAL CELLS

To observe the effects of inhibiting endogenous C3 on
expression of the synthetic phenotype and proliferation ofHMCs, we designed a duplex siRNA against C3 to inhibit
endogenous C3. This siRNA was effective in inhibiting C3
production in HMCs and caused an increase in the expressionof h-caldesmon mRNA and a decrease in the expression of
osteopontin, matrix Gla, and collagen IV mRNAs. These data
indicate that C3 contributes directly to the synthetic phenotypeof HMCs. Moreover, siRNA to C3 reduced the higher basal
DNA synthesis and proliferation observed in HMCs with thesynthetic phenotype.
In conclusion, C3 is produced by HMCs and contributes tothe exaggerated growth and expression of the synthetic
phenotype of HMCs. C3 may underly the synthetic phenotype
and exaggerated growth of HMC. A therapeutic approach
utilizing siRNA to target C3 may be effective in the treatment ofprogressive renal disease.
Acknowledgments

This work was supported in part by a Grant-in Aid to Nihon
University for the High-Tech Research Center from theJapanese Ministry of Education, Science, Sports, and Culture
and by the Ministry of Education, Science, Sports, and Culture ofJapan (15590863).
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Journal of Chromatography B, 859 (2007) 272–275

Short communication

Optimization and validation of a high-performance liquid chromatographic
method with UV detection for the determination of pyrrole–imidazolepolyamides in rat plasma
Akiko Fukasawa a , Takashi Nagashima a , Takahiko Aoyama a , Noboru Fukuda b ,Hiroyuki Matsuda b , Takahiro Ueno c , Hiroshi Sugiyama d ,Hiroki Nagase b , Yoshiaki Matsumoto a,∗a Department of Clinical Pharmacokinetics, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japanb Advanced Research Institute for the Science and Humanities, Nihon University, Tokyo 102-0073, Japanc Department of Medicine, Division of Nephrology and Endocrinology, Nihon University School of Medicine, Tokyo 173-8610, Japand Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan

Received 18 July 2007; accepted 26 September 2007
Available online 5 October 2007

Abstract
A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination ofplasma pyrrole (Py)–imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma
with methanol, Py–Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mm × 15.0 cm TOSOH Co., Japan) column
maintained at 40 ◦ C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0–10 min, A: 100–20%,
B: 0–80% linear gradient; 10–15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method
was used to determine the plasma concentration time profiles of Py–Im polyamides after intravenous injection.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Pyrrole–imidazole polyamide; HPLC; Rat plasma; Pharmacokinetics

1. Introduction
Pyrrole (Py)–imidazole (Im) polyamides are small synthetic
molecules composed of the aromatic rings of N-methylpyrrole
and N-methylimidazole amino acid [1]. Py–Im polyamides
were purified by HPLC using Chemcobond 5-ODS-H column, 0.1% acetic acid and acetonitrile 0–50% linear gradient,
40 min. The detection wavelength was set at 254 nm [2,3]. Synthetic polyamides can bind to specific nucleotide sequencesin the minor groove of double-helical DNA with high affinity
and specificity. Sequence-specific DNA recognition by Py–Im
polyamide depends on the side-by-side paring of Py and Im; thePy/Im pair targets the CG base pair, Im/Py recognizes the GC
base pair, and Py/Py binds to both AT and TA base pairs [4]. Various types of sequence-specific DNA-binding Py–Im polyamides

have been developed to regulate gene expression by targeting thepromoter regions of enhancer and transcription factor binding
elements in vitro [5]. Recently, Matsuda et al. [6] reported that
the Py–Im polyamide targeting the rat transforming growth factor (TGF)-␤1 is a novel gene-silencing agent for the treatmentof progressive renal diseases in Dahl-S rats. These observations
suggested that the Py–Im polyamide could be a useful bioprobe
for molecular biology and a potential medicine.
The aims of the present study are to develop a sensitive andspecific HPLC assay for the quantitation of Py–Im polyamides
in rat plasma and to propose its application to pharmacokinetic
studies.
2. Experiment2.1. Chemicals and reagents∗ Corresponding author. Tel.: +81 47 465 7182; fax: +81 47 465 7182.

E-mail address: matsuy@pha.nihon-u.ac.jp (Y. Matsumoto).
1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2007.09.032

Py–Im polyamides (A) and (B) were provided by Gentier
Biosystems Co., Ltd. (Japan). Their chemical structures are

A. Fukasawa et al. / J. Chromatogr. B 859 (2007) 272–275

273

Fig. 1. Chemical structures of Py–Im polyamides (A) and (B). The molecular weight of Py–Im polyamide (A) is 1035.12 and that of Py–Im polyamide (B) is 1665.78.

shown in Fig. 1. Py–Im polyamide (A) was composed of AcImPyPy-ImPyPy-␤-Dp (Dp: N,N-dimethylaminopropylamide).
Py–Im polyamide (B) was composed of Ac-PyPy-␤-PyImPy-PyPyPy␤-ImPy-␤-Dp. The molecular weights of Py–Im
polyamides (A) and (B) were calculated from the sum of the standard atomic weights of all the atoms [7]. Py–Im polyamide (A)was 1035.12 and Py–Im polyamide (B) was 1665.78. Acetonitrile and methanol were HPLC-grade from Wako Pure Chemical
Industries, Ltd. Acetic acid was of the highest quality from Kanto
Chemical Co., Inc. The water was purified by distillation.

rat while under anesthesia with pentobarbital sodium (Dainippon Sumitomo Pharma Co.). The cannulation treatment was
performed at least 1 day prior to the experiment. A Py–Im
polyamide aqueous solution was injected into the jugular vein
at a single dose of 2.0 mg/kg. Blood samples (0.5 ml) were collected at 0, 10, 30, 60, 90 and 120 min after the injection. Aftereach sampling, the blood drawn was replaced with an equalvolume of saline.

2.2. Chromatographic system

Blood samples were centrifuged in a 1.5 ml microcentrifuge
at 10,000 × g at 4 ◦ C. Each sample was immediately transferred to a heparinized microcentrifuge tube and centrifuged
for 10 min. Collected plasma samples (50 ␮l) were then vortexmixed with 100 ␮l of methanol for 10 s. The mixture wascentrifuged for 5 min to precipitate protein. Supernatants were
centrifuged for 5 min again and 30 ␮l of each supernatant was
directly injected into the chromatograph.

The analyses of Py–Im polyamides were carried out usinga Shimadzu LC-20A HPLC system (Tokyo, Japan) witha reversed-phase TSK-GEL ODS-80TM (4.6 mm × 15.0 cm,
TOSOH Co., Japan) column maintained at 40 ◦ C. The mobile
phase solvent A was 0.1% acetic acid and the solvent B was
acetonitrile (0–10 min, A: 100–20%, B: 0–80% linear gradient;
10–15 min, A: 40%, B: 60%). The flow rate was set at 1.0 ml/min.
The detection wavelength was set at 310 nm. Injection volume
was 30 ␮l.
2.3. Animals and blood sampling
Male Wistar rats (14 weeks old) weighing 280–300 g wereobtained from Sankyo Lab Service Corporation. The animals
were maintained in a temperature-controlled room on a 12-h
light:12-h dark cycle and allowed free access to food and tap
water. A polyethylene tube (0.58 mm, I.D. 0.96 mm, O.D.) was
inserted into the left femoral artery and right jugular vein of the

2.4. Preparation of plasma samples

2.5. Stock solution and standards
Primary stock solutions of Py–Im polyamide (A) or (B)(2.0 mg/ml) were prepared in water and stored at 4 ◦ C until analysis. Working solutions with concentrations of 20 and 200 ␮g/ml
were prepared by adding water. Py–Im polyamide (A) or (B) calibration standards were prepared fresh daily at concentrations of
0, 1, 2, 5, 10, 20, 50, 100 and 200 ␮g/ml by spiking 45 ␮l ofblank rat plasma with 5 ␮l of water (for “zero” standard sample) or Py–Im polyamides (A) and (B) working solution. In thesame manner, quality control (QC) samples at concentrations of274

A. Fukasawa et al. / J. Chromatogr. B 859 (2007) 272–275

1, 5, 20, and 100 ␮g/ml were freshly prepared to evaluate theaccuracy and precision of the HPLC method.

Table 1
Recovery of Py–Im polyamides (A) and (B) from plasma (n = 3)

2.6. Linearity of calibration curve

Theoreticalconcentration(␮g/ml)Calibration curves were constructed by plotting the peak areaof Py–Im polyamide. The linearity of the calibration curves wasevaluated by linear regression analysis.

1
5
20
100

Recovery (%)
Py–Im polyamide (A)Py–Im polyamide (B)Mean ± S.D.

CV (%)

Mean ± S.D.

CV (%)

83.7 ± 7.5
103.5 ± 4.5
97.4 ± 1.7
97.7 ± 2.7

8.9
4.4
1.7
2.8

109.0 ± 1.2
87.3 ± 4.4
93.4 ± 11.1
92.7 ± 2.8

1.1
5.1
11.9
3.0

2.7. Recovery
To assess the extraction recovery, two series of Py–Im
polyamide QC samples were prepared as described above one
with rat plasma and another without plasma. The set without
plasma was prepared by adding water instead of plasma. The
extraction recovery was determined from the ratio of peak areaof water standard to that of the corresponding plasma standard.
2.8. Sensitivity
The lower limit of quantification (LLOQ) was determined
during the evaluation of the linear range of the calibration curve.
LLOQ was defined as the lowest concentration yielding a precision with coefficients of variation (CV) of less than 20% andaccuracy within 15% of the theoretical value (i.e., accuracy
between 85 and 115%) for both intra- and inter-day analysis.
2.9. Accuracy and precision
For the intra- and inter-day precision and accuracy of theassay, Py–Im polyamide (A) and (B) QC samples were prepared
as described above. The intra-day precision was calculated fromthe CV for QC samples in three replicate analyzed on the sameday, and the inter-day precision was determined by the analysis
of QC samples in three consecutive days. The accuracy of each
set of measurements was calculated by comparing the means
with nominal values, and was expressed in percent. The criterion
for the acceptability of precision was that the relative standard
deviation for each concentration level did not exceed ±15%except for the LLOQ, which did not exceed ±20%. Similarly, for
accuracy, the mean value did not exceed ±15% of the nominal
concentration except for the LLOQ, where the limit was the
LLOQ.

2.10. Assay application
The present method was used to determine the concentrationsof Py–Im polyamides (A) and (B) in rat plasma after intravenous injection of Py–Im polyamide (A) and (B) aqueous solutions.
3. Results and discussion
3.1. Separation
The UV absorbances of Py–Im polyamides (A) and (B)were determined from wavelengths of 200–400 nm. Py–Im
polyamides (A) and (B) had a maximum UV absorption at
310 nm (data not shown). Therefore, the UV detection wavelength at 310 nm was measured. Chromatograms of blankplasma and plasma spiked with Py–Im polyamides (A) and(B) (5 ␮g/ml) are shown in Fig. 2. Py–Im polyamides (A) and(B) were well separated from co-extracted material under thedescribed chromatographic conditions at approximate retention times of 9.7 and 10.5 min, respectively. The peak shapes
were satisfactory and completely resolved from one another.
No interference with constituents from the plasma matrix was
observed.
3.2. Linearity of calibration curves
Calibration curves for Py–Im polyamides (A) and (B) werelinear over the concentration range of 1–200 ␮g/ml in rat plasma.
The mean (±S.D.) regression equation from three replicate calibration curves on different days for Py–Im polyamide (A) was
Y = 24,206 (±1413)X − 7715(±7112) and for Py–Im polyamide(B) was Y = 32,876(±924)X − 8753(±12,146), where Y isFig. 2. Representative chromatograms of (A) blank rat plasma, (B) blank rat plasma spiked with Py–Im polyamide (A) (5 ␮g/ml), and (C) blank rat plasma spiked
with Py–Im polyamide (B) (5 ␮g/ml). Approximate retention times: Py–Im polyamide (A) (5 ␮g/ml) = 9.7 min; Py–Im polyamide (B) (5 ␮g/ml) = 10.5 min.

A. Fukasawa et al. / J. Chromatogr. B 859 (2007) 272–275

275

Table 2
Accuracy and precision of the HPLC method for determining
Theoretical concentration (␮g/ml)

Intra-dayObserved concentration(mean ± S.D.; ␮g/ml)

Inter-dayAccuracy (%)

CV (%)

Observed concentration(mean ± S.D.; ␮g/ml)

Accuracy (%)

CV (%)

Py–Im polyamide (A) concentration in plasma samples (n = 3)1
1.0 ± 0.155.6 ± 0.1
20
20.3 ± 0.3
100107.7 ± 4.0

102.2
111.8
101.4
107.7

5.3
1.6
1.4
3.7

1.1 ± 0.2
5.3 ± 0.3
18.3 ± 1.8
103.6 ± 3.7

106.7
106.3
91.3
103.6

14.4
6.4
9.7
3.5

Py–Im polyamide (B) concentration in plasma samples (n = 3)1
1.0 ± 0.155.0 ± 0.2
20
19.5 ± 0.1
100103.7 ± 2.7

102.8
100.8
97.5
103.7

10.0
3.5
0.6
2.6

1.0 ± 0.2
5.2 ± 0.2
18.2 ± 1.4
103.2 ± 3.3

102.2
104.0
90.8
103.2

15.0
3.1
7.9
3.2

peak area and X is the concentration of the analyte. R2 ofPy–Im polyamide (A) was 0.9997(±0.0003) and R2 of Py–Im
polyamide (B) was 0.9983(±0.0018).
3.3. Recovery
The efficiencies of the extraction procedure for the two analytes at QC levels (1, 5, 20, and 100 ␮g/ml) are presented
in Table 1. The recovery ranged between 83.7 and 103.5%
for Py–Im polyamide (A) and 87.3 and 109.0% for Py–Im
polyamide (B).
3.4. Precision, accuracy and sensitivity
Table 2 shows the intra- and inter-day precision and accuracy of Py–Im polyamides (A) and (B). The intra- and inter-day
accuracies (% deviation) were within ±20% for the LLOQ and±15% for the other QC samples. The intra- and inter-day assay
precisions (CV) were also within the acceptable range of 20%
for LLOQ and 15% for the other QC samples. The LLOQ wasdetermined as 1 ␮g/ml for both Py–Im polyamides (A) and(B).

Fig. 3. Plasma concentration time profile after intravenous administration(2.0 mg/kg) of Py–Im polyamides (A) () and (B) (䊉). The data was shownas the mean ± S.D. (n = 3).

assay was validated to apply the requirements of pharmacokinetic studies.
Acknowledgments

3.5. Application
The plasma concentrations of Py–Im polyamides (A) and (B)were determined at 0, 10, 30, 60, 90 and 120 min after injection.
The plasma concentration time profile after intravenous administration (2.0 mg/kg) of Py–Im polyamides (A) and (B) in the
rat are shown in Fig. 3. The retention time of Py–Im polyamide(A) extracted from plasma (4.5 ␮g/ml) and water (5.5 ␮g/ml)
were the same time at 9.8 min. The retention time of Py–Im
polyamide (B) extracted from plasma (2.5 ␮g/ml) and water(5.6 ␮g/ml) were the same at 10.5 min. The respective retention time of Py–Im polyamides extracted from plasma and water
were consistent.
In conclusion, the newly developed HPLC method for thedetermination of plasma Py–Im polyamides in rats is simple,
sensitive and specific, and the method can be used for the analysis of large numbers of plasma samples in other species. The

This work was supported in part by a grant from “Academic
Frontier” Project for Private Universities from the Ministry of
Education, Culture, Sports, Science and Technology of Japan(2006–2010).
References[1] J.W. Trauger, E.E. Baird, P.B. Dervan, Nature 382 (1996) 559.
[2] T. Bando, A. Narita, I. Saito, H. Sugiyama, Chem. Eur. J. 20 (2002) 4781.
[3] Y.M. Lai, N. Fukuda, T. Ueno, H. Matsuda, S. Saito, K. Matsumoto, H.
Ayame, T. Bando, H. Sugiyama, H. Mugishima, K. Serie, J. Pharmacol.
Exp. Ther. 315 (2) (2005) 571.
[4] S. White, E.E. Baird, P.B. Dervan, Chem. Biol. 4 (8) (1997) 569.
[5] M.S. Murty, H. Sugiyama, Biol. Pharm. Bull. 27 (4) (2004) 468.
[6] H. Matsuda, N. Fukuda, T. Ueno, Y. Tahira, H. Ayame, W. Zhang, T. Bando,
H. Sugiyama, S. Saito, K. Matsumoto, H. Mugishima, K. Serie, J. Am. Soc.
Nephrol. 17 (2) (2006) 422.
[7] M.E. Wieser, Pure Appl. Chem. 78 (11) (2006) 2051.

Original article 671

Chimeric DNA–RNA hammerhead ribozyme targetingtransforming growth factor-b1 mRNA ameliorates
renal injury in hypertensive rats
Yoshiko Tahiraa, Noboru Fukudaa,b,d, Morito Endoc, Takahiro Uenoa,
Hiroyuki Matsudaa, Satoshi Saitoa, Koichi Matsumotoa and Hideo Mugishimad
Objective Transforming growth factor (TGF)-b is a criticalfactor in the progression of renal injury, regardless of the
primary etiology. Such injury is characterized by glomerularsclerosis and tubulointerstitial fibrosis. To develop aribozyme-based therapy for progressive renal diseases, we
examined the effects of chimeric DNA–RNA hammerhead
ribozyme targeting TGF-b1 mRNA on glomerulosclerosis insalt-loaded, stroke-prone spontaneously hypertensive rats
(SHR-SP) and salt-sensitive Dahl (Dahl-S) rats.
Methods The chimeric DNA–RNA ribozyme to TGF-b1 wasdelivered by polyethylenimine to cultured mesangial cells
from SHR-SP in vitro and to glomeruli in SHR-SP in vivo. The
chimeric ribozyme reduced expression of TGF-b1 mRNA andprotein, which was accompanied by inhibition of expressionof extracellular matrix molecules such as fibronectin andcollagen type I in mesangial cells from SHR-SP in vitro.
Results One intraperitoneal injection of 200 mg of chimeric
DNA–RNA ribozyme to TGF-b1 in vivo markedly ameliorated
thickening of capillary artery walls and glomerulosclerosis insalt-loaded SHR-SP and Dahl-S rats without a reduction inblood pressure. The chimeric ribozyme reduced expressionof TGF-b1 and connective tissue growth factor (CTGF)
mRNAs in renal cortex in salt-loaded Dahl-S rats. Chimericribozyme to TGF-b1 significantly reduced levels of protein in
urine in the Dahl-S rats.

Introduction
A number of studies have identified transforming growth
factor (TGF)-b as a critical factor in kidney diseases suchas glomerulosclerosis [1] and mesangioproliferative glomerulonephritis [2,3]. TGF-b mRNA and protein are
expressed strongly in various animal models of progressive renal diseases such as hypertensive renal sclerosis,
diabetic nephropathy and focal renal sclerosis [4–6].
Because there are still no effective treatments for progressive renal diseases, gene therapy is now being considered as a strategy. Ribozymes are RNA molecules thatcatalytically cleave a phosphodiester bond in the appropriate target RNAs in a sequence-specific manner,
thereby inhibiting the expression of specific gene products. Ribozymes have progressed from being objects ofscientific study to potential therapeutic agents for treatment of both acquired and inherited diseases [7]. We

Conclusion These results suggest that chimeric DNA–RNA
ribozyme to TGF-b1 may be useful as a gene therapy for
progressive tissue injury in a wide variety of renal diseases,
including hypertensive nephrosclerosis. J Hypertens
25:671–678 Q 2007 Lippincott Williams & Wilkins.

Journal of Hypertension 2007, 25:671–678
Keywords: gene therapy, glomerulosclerosis, hypertension, ribozyme,transforming growth factor-b1
aDepartment of Medicine, Nihon University School of Medicine, Division of
Nephrology and Endocrinology, Tokyo, bAdvanced Research Institute for the
Science and Humanities, Nihon University, Tokyo, cHachinohe University, Facultyof Human Health Science and dDepartment of Advanced Medicine, Division of
Cell Regeneration and Transplantation, Nihon University School of Medicine,Tokyo, Japan

Correspondence and requests for reprints to Noboru Fukuda, MD, PhD,
Department of Medicine, Nihon University School of Medicine, Ooyaguchi-kami
30-1, Itabashi-ku, Tokyo 173-8610, Japan
Tel: +81 3 3972 8111; fax: +81 3 3972 8666;e-mail: fukudan@med.nihon-u.ac.jp
Sponsorship: This work was supported in part by a Grant-in-Aid for the HighTech Research Center from the Japanese Ministry of Education, Science, Sports,
and Culture to Nihon University and from the Ministry of Education, Science,Sports, and Culture of Japan (15590863).
Conflict of interest: none.
Received 13 April 2006 Revised 4 October 2006
Accepted 18 October 2006

have demonstrated that ribozyme to TGF-b1 inhibits
growth of vascular smooth muscle cells in vitro [8,9] andneointima formation of arteries after angioplasty in vivo
[10]. However, ribozyme therapy for progressive renal
diseases has not yet been reported.
Experimental evidence supports the notion that thepathogenesis of glomerulosclerosis with nephrosclerosis
involves glomerular hypertension, mesangial dysfunction, such as mesangiolysis, and increased mesangial
matrix [11]. Glomerular hypertension results fromsystemic hypertension and leads to increased glomerular
capillary pressure with subsequent glomerular endothelial damage and sclerosis [12,13]. Stroke-prone spontaneously hypertensive rats (SHR-SP) show severe
cardiovascular organ damage such as stroke, ventricular
hypertrophy and glomerulosclerosis associated with
TGF-b [5]. Salt-sensitive Dahl (Dahl-S) rats also show

0263-6352 ß 2007 Lippincott Williams & Wilkins

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

672 Journal of Hypertension

2007, Vol 25 No 3

glomerulosclerosis, which is known to be associated with
TGF-b [4].

in a dark environment and embedded in paraffin for lightmicroscopy with eriochrome black T stain.

In the current study, to develop ribozyme therapy for
progressive renal diseases, we examined the in-vivo
effects of chimeric DNA–RNA hammerhead ribozyme
targeting TGF-b1 mRNA on glomerulosclerosis in SHRSP and Dahl-S rats.

Reverse transcription–polymerase chain reaction

Methods
Our investigation conformed to standards of the Guide for
the Care and Use of Laboratory Animals [14].
Synthetic chimeric DNA–RNA hammerhead ribozyme

The 38-base chimeric DNA–RNA hammerhead ribozyme, which contained deoxyribonucleotides instead ofribonucleotides at noncatalytic residues and two phosphorothioate linkages at the 30 terminus, was designed tocleave the GUC sequence in the rat TGF-b1 mRNA [7].
The TGF-b1 specific ribozyme and a mismatch ribozyme with a single base change in the catalytic loop
region were synthesized with the use of a DNA/RNA
synthesizer (Model 394; Applied Biosystems Inc., Foster
City, California, USA).
Isolation of glomeruli and culture of mesangial cells

Glomeruli were isolated from the kidneys of 4-week-old
male SHR-SP/Izumo rats (SHR Corporation, Funabashi,
Chiba, Japan). Glomeruli were isolated with a gradedsieve technique as described previously [15]. The renal
cortex was excised and minced into small pieces understerile conditions. The minced cortex was pressed
through a 200-mm sieve and suspended in RPMI 1640
medium (Gibco Laboratories, Grand Island, New York,USA). The suspension was passed through a 120-mm
sieve, and glomeruli were collected on the surface ofthe sieve and resuspended in RPMI 1640. Mesangial cells
were isolated from explants of whole glomeruli according
to the differential growth capacities of glomerular epithelial and mesangial cells [15].
Delivery of chimeric DNA–RNA ribozyme to TGF-b1
in vitro and in vivo

For in-vitro delivery, mesangial cells were transfectedwith 1.0 mmol/l fluorescein isothiocyanate (FITC)labeled chimeric DNA–RNA ribozyme to TGF-b1 with
polyethylenimine for 120 min. After 24 h, cells were fixedwith acetone at 48C for 10 min and examined via fluorescence microscopy. Total intracellular FITC signal, as
well as distribution of signal to the cytoplasmic or nuclear
compartment, were noted.
For in-vivo delivery, 500 mg/kg of FITC-labeled chimeric
DNA–RNA ribozyme to TGF-b1 in polyethylenimine
was injected into SHR-SP subcutaneously, intraperitoneally or intravenously. After 24 h, kidneys were removed
from SHR-SP and fixed in 10% neutral buffered formalin

mRNA was extracted from cultured mesangial cells of
SHR-SP and renal cortex from Dahl-S rats loaded with
1% salt water. Aliquots of mRNA were reverse transcribed into single-stranded cDNA by incubation withavian myeloblastoma virus reverse transcriptase (Takara
Biochemicals, Otsu, Shiga, Japan). Diluted cDNA products were then subjected to polymerase chain reaction(PCR). The primers used for amplification were as follows:(1) TGF-b1: forward 50 -GCCCTGGATACCAACTAC
TGCT-30 , reverse 50 -AGGCTCCAAATGTAGGGGCAGG-30 , product 161 base-pair (bp);(2) fibronectin: forward 50 -TGCCACTGTTCTCCTA
CGTG-30 , reverse 50 -ATGCTTTGACCCTTACA
CGG-30 , product 312 bp;(3) collagen type I: forward 50 -GGTGCTAGATCAG
GAGCAGG-30 , reverse 50 -ATGCCCACTCCCTAA
CAGTG-30 , product 182 bp; and(4) connective tissue growth factor (CTGF): forward 50 ATCCCTGCGACCCACACAAG-30 , reverse 50 -TT
ACGTCTGGCGTCGAAGGC-30 , product 145 bp;(5) human 18S ribosomal RNA was used as an internal
control, the primers were as follows: forward 50 TCAAGAACGAAAGTCGGAGG-30 , reverse 50 -GG
ACATCTAAGGGCATCACA-30 , product 312 bp.
PCR was performed in a DNA thermal cycler (PerkinElmer Cetus, Norwalk, Connecticut, USA), and products
were separated by electrophoresis on 1.5% agarose gels,stained with ethidium bromide and visualized byultraviolet illumination.
Western blot analysis

Mesangial cells of SHR-SP (105 cells/cm2) and renal
cortex from Dahl-S rats loaded with 1% salt were disrupted with lysis buffer [50 mmol/lTris–HCl (pH8.0),
150 mmol/l NaCl, 0.02% sodium azide, 100 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1% Triton
X-100]. Total proteins were extracted and purified with
100 ml of chloroform and 400 ml of methanol. Protein
samples were boiled for 3 min and subjected to electrophoresis on 8% polyacrylamide gels and then transblotted to nitrocellulose membranes (BioRad Laboratories,
Hercules, California, USA). Blots were incubated withrabbit pan-specific polyclonal antibody for TGF-b (R &D Systems, Minneapolis, Minnesota, USA) or mousemonoclonal antibody specific for a-tubulin as a control(Sigma BioScience, St. Louis, Missouri, USA), diluted
1 : 500 in 5% nonfat milk in TBST solution (10 mmol/l
Tris–HCl, pH 8.0, 150 mmol/l NaCl, and 0.05% Tween20) for 3 h at room temperature. The membrane wasincubated with goat anti-mouse IgG for 1 h at room
temperature, then washed with TBST once for 15 min,

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Ribozyme to TGF-b1 prevents renal injury Tahira et al.

and four times for 5 min. Immune complexes on themembrane were detected by the enhanced chemiluminescence method (Amersham Pharmacia Biotech,
Buckinghamshire, UK).
Experimental design

Twelve-week-old male SHR-SP or 7-week-old male
Dahl-S rats of the Iwai substrain (Seac Yoshitomi Ltd,
Fukuoka, Japan) were loaded with 1% salt water for 6 or 8
weeks, respectively. After 4 weeks of loading with salt
water, the control group received an intraperitoneal
injection of 1.5 ml of saline. The mismatch or ribozyme
group received an intraperitoneal injection of mismatchribozyme or chimeric DNA–RNA ribozyme to TGFb1 (200 mg/body weight) with polyethylenimine (Fig. 1).
Systolic blood pressure (SBP) was measured by the tailcuff method.
Histological examination

Kidneys removed from SHR-SP or Dahl-S rats were fixedin 10% neutral buffered formalin and embedded inparaffin for light microscopic study. Sections (2 mm thick)were stained with Masson trichrome (MT) stain or hematoxylin–eosin (HE). Histological examination was done
by a pathologist without any prior knowledge of theexperimental groups. To semiquantify the glomerularmatrix, 50 glomeruli were selected randomly. The percentage of each glomerulus occupied by mesangial matrix
was estimated and given a score of 0, normal; 1, involvement of up to 25% of the glomerulus; 2, involvement of
25–50% of the glomerulus; 3, involvement of 50–75% of
the glomerulus; or 4, involvement of 75–100% of theglomerulus. The glomerular injury score (GIS) was
obtained by the following formula: [(0  n0) þ (1  n1) þ(2  n2) þ (3  n3) þ (4  n4)]/50. To semiquantify thetubulointerstitial area, 20 areas of renal cortex wereselected randomly. The percentage of each area that
Fig. 1SHR-SP (n = 6)

Ribozyme (i.p.)

SacrificeSalt loading
12-week-old

16-week-old

Ribozyme (i.p.)

18-week-old

SacrificeDahl-S rat (n = 6)

673

showed sclerofibrotic change was estimated and assigned a score of 0, normal; 1, involvement of less than
10% of the area; 2, involvement of 10–30% of the area; 3,
involvement of 30–50% of the area; or 4, involvement of
more than 50% of the area. The tubulointerstitial injury
score (TIS) was calculated as [(0  n0) þ (1  n1) þ(2  n2) þ (3  n3) þ (4  n4)]/20.
Statistical analysis

Results are given as the mean  SEM. The significanceof differences between mean values was evaluated byStudent’s t-test for unpaired data and by two-way analysis
of variance (ANOVA) followed by Duncan’s multiple
range test.

Results
Delivery of chimeric DNA–RNA ribozyme to TGF-b1

FITC-labeled chimeric ribozyme was observed in thecytosol of cultured mesangial cells in vitro, and thetransfection efficiency was approximately 20% (Fig. 2b).
FITC-labeled chimeric ribozyme was clearly visible inglomeruli from SHR-SP 24 h after subcutaneous, intraperitoneal or intravenous administration (Fig. 2c, e, f),but not visible in glomeruli 72 h after subcutaneousadministration (Fig. 2d). FITC-labeled chimeric ribozyme was also visible in capillary arteries 24 h after
subcutaneous administration (Fig. 2c). Intravenous andintraperitoneal administration were more effective
than subcutaneous administration for delivery of theribozyme.
Effects of chimeric DNA–RNA ribozyme to TGF-b1 on
expression of TGF-b1, fibronectin, and collagen type I
mRNAs and TGF-b1 protein in mesangial cells from
SHR-SP

Levels of TGF-b1 mRNA were lower with chimeric
DNA–RNA ribozyme to TGF-b1 (0.1 and 1.0 mmol/l)than with mismatch ribozyme in mesangial cells from
SHR-SP. This reduction was dose dependent (Fig. 3a).
Chimeric DNA–RNA ribozyme to TGF-b1 (1.0 mmol/l)also reduced levels of TGF-b1 protein (Fig. 3b). Ribozyme to TGF-b1 decreased levels of fibronectin mRNA
at a concentration of 1.0 mmol/l and collagen type I
mRNA at concentrations of 0.1 and 1.0 mmol/l in comparison to expression levels with mismatch ribozyme(Fig. 3a).

Salt loading
7-week-old

11-week-old

15-week-old
Urine samplingExperimental protocols for treatment of salt-loaded stroke-prone
spontaneously hypertensive rats (SHR-SP) and salt-sensitive Dahl
(Dahl-S) rats with chimeric DNA–RNA ribozyme targeting transforming
growth factor (TGF)-b1 mRNA.

Effects of chimeric DNA–RNA ribozyme to TGF-b1 on
expression of TGF-b1 and CTGF mRNAs and TGF-b1
protein in renal cortex from Dahl-S rats

Intraperitoneal administration of 200 mg of chimeric
DNA–RNA ribozyme to TGF-b1 markedly reduced
expression of TGF-b1 and CTGF mRNAs and TGFb1 protein in renal cortex from salt-loaded Dahl-S rats
(Fig. 4).

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674 Journal of Hypertension

2007, Vol 25 No 3

Fig. 2

Delivery of chimeric DNA–RNA ribozyme to transforming growth factor (TGF)-b1 in vitro and in vivo. For in-vitro delivery, mesangial cells weretransfected with 1.0 mmol/l fluorescein isothiocyanate (FITC)-labeled chimeric ribozyme with polyethylenimine for 120 min. Cells were fixed withacetone at 48C for 10 min and examined via fluorescent microscopy without (a) or with (b) ultraviolet light. For in-vivo delivery, 200 mg of FITClabeled chimeric DNA–RNA ribozyme to TGF-b1 polyethylenimine was injected into stroke-prone spontaneously hypertensive rats (SHR-SP)
subcutaneously (c), intraperitoneally (d,e), or intravenously (f). After 24 h (c, e, f) or 72 h (d), kidneys were removed, fixed in 10% neutral-bufferedformalin in the dark, and embedded in paraffin for light microscopy with eriochrome black T stain. Large arrows indicate the localization of labeledribozyme in glomeruli and small arrows indicate capillary arteries.

Effects of chimeric DNA–RNA ribozyme to TGF-b1 on
blood pressure and nephrosclerosis in SHR-SP and
Dahl-S rats

The renal cortex of salt-loaded SHR-SP and of saltloaded Dahl-S rats showed marked thickening ofcapillary artery walls and severe glomerulosclerosis withglomerular ischemia due to obstruction of the afferent
artery. Intraperitoneal administration of 200 mg of chimeric DNA–RNA ribozyme to TGF-b1 or mismatchribozyme had no effect on blood pressure in salt-loaded
SHR-SP and Dahl-S rats with salt-loading (Table 1).

Intraperitoneal administration of chimeric DNA–RNA
ribozyme to TGF-b1 markedly ameliorated thickening of
the capillary artery wall and glomerulosclerosis in kidneysof salt-loaded SHR-SP and Dahl-S rats. Administrationof mismatch ribozyme had no effect on thickening or
glomerulosclerosis (Fig. 5).
Intraperitoneal administration of chimeric DNA–RNA
ribozyme to TGF-b1 significantly (P < 0.05) decreased
the GIS and TIS in kidney from salt-loaded SHR-SP and
Dahl-S rats. Mismatch ribozyme had no effect on GIS or

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Ribozyme to TGF-b1 prevents renal injury Tahira et al.

675

Fig. 3

Effects of chimeric DNA–RNA ribozyme to transforming growth factor (TGF)-b1 on expression of TGF-b1, fibronectin and collagen type I mRNAs (a)and TGF-b1 protein (b) in cultured mesangial cells from stroke-prone spontaneously hypertensive rats (SHR-SP). Levels of TGF-b1, fibronectin andcollagen type I mRNAs were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The ratio of the abundance of TGF-b1,
fibronectin and collagen type I mRNAs to that of 18S rRNA was evaluated by densitometric analysis. Data are the means of experiments carried out
in duplicate (a). Levels of TGF-b1 protein in renal cortex were evaluated by Western blot analysis. The abundance of TGF-b1 protein was evaluatedby densitometric analysis. Data are the means of experiments carried out in duplicate (b).

Fig. 4

Effects of chimeric DNA–RNA ribozyme to transforming growth factor (TGF)-b1 on expression of TGF-b1 and connective tissue growth factor(CTGF) mRNAs and TGF-b1 protein in renal cortex of salt-loaded salt-sensitive Dahl (Dahl-S) rats. Seven-week-old male Dahl-S rats were given 1%
salt water for 6 or 8 weeks. After 4 weeks of salt loading, the mismatch or ribozyme group received an intraperitoneal injection of mismatch ribozyme
or chimeric DNA–RNA ribozyme to TGF-b1 (200 mg/body weight). Levels of TGF-b1 and CTGF mRNAs were evaluated by reverse transcriptionpolymerase chain reaction (RT-PCR). The ratio of the abundance of TGF-b1 and CTGF mRNAs to that of 18S rRNA was evaluated by densitometricanalysis. Data are the mean  SEM (n ¼ 4). P < 0.05 versus mismatch ribozyme (a). Levels of TGF-b1 protein in renal cortex were evaluated byWestern blot analysis. The abundance of TGF-b1 protein was evaluated by densitometric analysis. Data are the means of experiments carried out in
duplicate (b).

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676 Journal of Hypertension

2007, Vol 25 No 3

Systolic blood pressure in salt-loaded, stroke-prone
spontaneously hypertensive rats (SHR-SP) and salt-sensitive Dahl
(Dahl-S) rats treated without (control) or with mismatch ribozyme(mismatch) or chimeric DNA–RNA hammerhead ribozyme
targeting transforming growth factor (TGF)-b1 mRNA (ribozyme)

Table 1

Glomerular injury score (GIS) or tubulointerstitial injury
score (TIS) in salt-loaded, stroke-prone spontaneously
hypertensive rats (SHR-SP) and salt-sensitive Dahl (Dahl-S) rats
treated without (control) or with mismatch ribozyme (mismatch) or
chimeric DNA–RNA hammerhead ribozyme targeting transforming
growth factor (TGF)-b1 mRNA (ribozyme)

Table 2

Systolic blood pressure (mmHg)

Control
Mismatch
Ribozyme

SHR-SP

Dahl-S272  16275  22277  10214  28224  36205  20

Strain

Group

GIS

TIS

SHR-SP

Control
Mismatch
Ribozyme
Control
Mismatch
Ribozyme

0.87  0.01
0.84  0.12
0.68  0.06
1.01  0.07
1.02  0.14
0.64  0.09

0.67  0.10
0.65  0.18
0.50  0.07
0.72  0.18
0.78  0.12
0.54  0.26

Dahl-Sn ¼ 6 in each group.

TIS in kidney from salt-loaded SHR-SP and Dahl-S rats
(Table 2).

 P < 0.05 versus mismatch. n ¼ 4 in each group.

excretion of protein by 50% (P < 0.05). Administration of
mismatch ribozyme had no effect on proteinuria (Fig. 6).

Effects of chimeric DNA–RNA ribozyme to TGF-b1 on
proteinuria in Dahl-S rats

Discussion

Intraperitoneal administration of chimeric DNA–RNA
ribozyme to TGF-b1 to salt-loaded Dahl-S rats decreased

In the present study, salt-loaded SHR-SP and Dahl-S rats
experience significant thickening of the capillary arteries,

Fig. 5

Histological findings in renal cortex of salt-loaded, stroke-prone spontaneously hypertensive rats (SHR-SP) and salt-sensitive Dahl (Dahl-S) rats
treated with chimeric DNA–RNA ribozyme to transforming growth factor (TGF)-b1. Twelve-week-old male or 7-week-old male SHR-SP and Dahl-S
rats were loaded with 1% salt water for 6 or 8 weeks, respectively. After 4 weeks of salt loading, the mismatch or ribozyme group received an
intraperitoneal injection of mismatch ribozyme or chimeric DNA–RNA ribozyme to TGF-b1 (200 mg/body weight). Renal cortex was fixed and stainedwith Masson trichrome for SHR-SP or hematoxylin–eosin for Dahl-S rats. Bar ¼ 20 mm.

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Ribozyme to TGF-b1 prevents renal injury Tahira et al.

Fig. 6

Effects of chimeric DNA–RNA ribozyme to transforming growth factor(TGF)-b1 on proteinuria in salt-loaded, salt-sensitive Dahl (Dahl-S) rats.
Data are the mean  SEM (n ¼ 4). P < 0.05 versus mismatch ribozyme
and control.

glomerulosclerosis, atrophic tubules and interstitial
fibrosis in kidney in response to the development ofhypertension. We have demonstrated previously that
expression of TGF-b1 mRNA is higher in glomeruli
and capillaries of renal cortex from SHR-SP than in thoseof normotensive Wistar–Kyoto rats [5]. In addition,
Dahl-S rats also developed severe hypertension, glomerulosclerosis, thickening of capillary artery walls and interstitial fibrosis in response to salt loading [4].
TGF-b1 stimulates extracellular matrix formation[16,17]. TGF-b-induced production of extracellularmatrix proteins in glomeruli causes renal damage in rats
with thymidylate synthase complementing protein(Thy-1)-induced nephritis [18], diabetic nephropathy[19] and interstitial nephritis induced by obstructive
nephropathy [20]. Moreover, TGF-b has recently beenreported to induce epithelial–mesenchymal transformation in renal tissue, which plays a critical role in thepathogenesis of nephritis [21]. Thus, TGF-b may associate the nephrosclerosis and interstitial nephropathy by its
effects on extracellular matrix formation and epithelial–mesenchymal transformation in kidney.
Ribozymes hybridize to and cleave a target RNA. Oncethe target RNA is cleaved, the ribozyme can dissociatefrom the cleaved products and repeat this process withanother RNA molecule [22]. Thus, ribozyme does not
require any cellular components and has high specificity
for inhibiting expression of a target gene. One problem
with ribozymes is rapid degradation in tissue, which
diminishes the availability of the ribozyme and reduces
their efficiency as a gene therapy. There are a number ofmodifications that can improve stability, specificity andefficacy of ribozymes. Our chimeric DNA–RNA hammerhead ribozyme to rat TGF-b1 mRNA contained

677

deoxyribonucleotides instead of ribonucleotides at noncatalytic residues to enhance catalytic turnover andimprove stability, as reported previously [23]. In addition,
two deoxyribonucleotides at the 30 -terminus of the chimeric DNA–RNA ribozyme were modified with phosphorothioate linkages to improve resistance to nucleases[24]. Moreover, to increase uptake of the ribozyme, wedelivered our ribozyme with polyethylenimine because
liposome-complexed molecules are preferentially transported to the cytoplasm [25].
In the present study, the chimeric DNA–RNA ribozyme
to TGF-b1 with polyethylenimine was transferred to
cultured mesangial cells in vitro and to glomeruli in
SHR-SP by subcutaneous, intraperitoneal or intravenous
injection. The chimeric ribozyme reduced expression of
TGF-b1 and inhibited expression of extracellular matrix
proteins, such as fibronectin and collagen type I, inmesangial cells from SHR-SP in vitro, suggesting thatribozyme to TGF-b1 may inhibit extracellular matrix
formation in glomeruli.
In the present experiments, a single injection of chimeric
DNA–RNA ribozyme to TGF-b1 markedly ameliorated
capillary artery hypertrophy and glomerulosclerosis insalt-loaded SHR-SP without any reduction in blood pressure. Because the effect of the ribozyme on glomerulosclerosis in SHR-SP was unexpectedly strong, to verify
the efficiency we examined the effects of this ribozyme
on glomerulosclerosis and proteinuria in salt-loaded
Dahl-S rats. One injection of chimeric DNA–RNA ribozyme to TGF-b1 also markedly improved the capillaryartery hypertrophy and glomerulosclerosis and significantly reduced proteinuria in salt-loaded Dahl-S rats
without any reduction in blood pressure. These findings
indicate that TGF-b contributes to both capillary artery
hypertrophy and glomerulosclerosis in SHR-SP and
Dahl-S rats, which were efficiently ameliorated by chimeric ribozyme independent of blood pressure. The
chimeric ribozyme also reduced expression of TGF-b1
and CTGF mRNAs in renal cortex in salt-loaded Dahl-S
rats. CTGF is a potent growth factor that stimulates
growth of mesenchymal cells, including mesangial cells,
and formation of extracellular matrix downstream of
TGF-b1 [26]. These findings suggest that TGF-b1
causes glomerulosclerosis through induction of CTGF
in Dahl-S rats. Ribozyme to TGF-b1 significantly
decreased GIS and TIS of renal cortex in salt-loaded
SHR-SP and Dahl-S rats, suggesting that TGF-b1 may
also be involved in renal interstitial fibrosis due to extracellular matrix formation in these hypertensive rats.
Dahly et al. [27] injected anti-TGF-b antibody every
day for 2 weeks into salt-loaded Dahl-S rats and foundthat the antibody significantly reduced blood pressure,
proteinuria and the degree of glomerulosclerosis andrenal interstitial fibrosis. In the present study, the chimeric ribozyme to TGF-b1 ameliorated the renal injury

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678 Journal of Hypertension

2007, Vol 25 No 3

and proteinuria with only a single injection and without
reduction of blood pressure in salt-loaded Dahl-S rats,suggesting that this ribozyme is an efficient strategy toameliorate progressive renal injury.

15

16

17

Theoretically, nuclease-resistant ribozymes can be
designed to cleave any target RNA. The recently completed sequencing of the human genome and of the
genomes of several pathogenic species provide a large
number of potential ribozyme targets that may benefit
clinical medicine. These ribozymes can be chemicallymodified to prevent biological degradation, and thus,
nuclease-resistant synthetic ribozymes are emerging as
a new and broadly useful class of therapeutic agents.
Gene therapy by nucleic acid-based therapeutics is currently in its infancy, but preliminary studies suggest that
this type of therapy may be effective against diseases
caused by genes encoding nontractable drug targets. We
plan to develop chimeric DNA–RNA ribozyme targeting
TGF-b1 mRNA as a clinical therapy for progressive renal
diseases such as glomerulonephritis and hypertensive
nephrosclerosis.

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Biochemical and Biophysical Research Communications 353 (2007) 182–188
www.elsevier.com/locate/ybbrc

A novel approach for myocardial regeneration with educated cord
blood cells cocultured with cells from brown adipose tissue
Yoshihiro Yamada a,*, Shin-ichiro Yokoyama b, Noboru Fukuda b, Hiroyasu Kidoya a,
Xiao-Yong Huang a, Hisamichi Naitoh a, Naoyuki Satoh a, Nobuyuki Takakura a,*a

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, 565-0871, Japan
Second Department of Internal Medicine, Nihon University School of Medicine, Ooyaguchi-kami 30-1, Itabashi-ku, Tokyo 173-8610, Japanb

Received 22 November 2006
Available online 11 December 2006

Abstract
Umbilical cord blood (CB) is a promising source for regeneration therapy in humans. Recently, it was shown that CB was a source of
mesenchymal stem cells as well as hematopoietic stem cells, and further that the mesenchymal stem cells could diﬀerentiate into a numberof cells types of mesenchymal lineage, such as cardiomyocytes (CMs), osteocytes, chondrocytes, and fat cells. Previously, we reported
that brown adipose tissue derived cells (BATDCs) diﬀerentiated into CMs and these CMs could adapt functionally to repair regionsof myocardial infarction. In this study, we examined whether CB mononuclear cells (CBMNCs) could eﬀectively diﬀerentiate into
CMs by coculturing them with BATDCs and determined which population among CBMNCs diﬀerentiated into CMs. The results showthat BATDCs eﬀectively induced CBMNCs that were non-hematopoietic stem cells (HSCs) (educated CB cells: e-CBCs) into CMs in vitro. E-CBCs reconstituted infarcted myocardium more eﬀectively than non-educated CBMNCs or CD34-positive HSCs. Moreover, wefound that e-CBCs after 3 days coculturing with BATDCs induced the most eﬀective regeneration for impaired CMs. This suggests thate-CBCs have a high potential to diﬀerentiate into CMs and that adequate timing of transplantation supports a high eﬃciency for CM
regeneration. This strategy might be a promising therapy for human cardiac disease.
 2006 Elsevier Inc. All rights reserved.
Keywords: Stem cell; Cord blood; Adipose tissue; Myocardial regeneration; Cell fusion

Myocardial regeneration is currently a popular topic in
cardiac medicine, and research in regenerative medicine hasadvanced in an explosive manner. Many cell types such asbone marrow mesenchymal stem cells (BM-MSCs) [1–3],
embryonic stem (ES) cells [4,5], and cardiac tissue stem
cells [6,7] have been found to undergo myocardial diﬀerentiation and can be used as a source for cardiomyocytes(CMs). Additional cell types may also prove to have cardiac diﬀerentiation ability. With regard to human therapy,
umbilical cord blood (CB) is a promising source because
transplantation of CB has already been established forpatients with blood diseases. Moreover, usage of CB over*Corresponding authors. Fax: +81 6 6879 8314.
E-mail addresses: yamaday@biken.osaka-u.ac.jp (Y. Yamada), ntakaku@biken.osaka-u.ac.jp (N. Takakura).
0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.12.017

comes considerable problems encountered with othersources of CMs, such as allergenic, ethical, and tumorigenic issues. Furthermore, CB contains both hematopoietic
stem cells (HSCs) [8] and MSCs [9]. Also, stem cells are
more abundant in CB than in adult human peripheral
blood or bone marrow (BM) and stem cells in CB have ahigher proliferative potential associated with an extended
life span and longer telomeres [10–12].
Indeed, CD34+ cells derived from human CB homed toinfarcted hearts and reduced the size of the infarcted area;
this was not through direct diﬀerentiation into CMs, but
through enhancing neovascularization [13,14]. These studies showed no evidence of myocytes of human origin in theinfracted myocardium; however, it was reported that unrestricted somatic stem cells (USSCs) from human CB could
diﬀerentiate into CMs in vitro and in vivo [15]. SuchY. Yamada et al. / Biochemical and Biophysical Research Communications 353 (2007) 182–188

USSCs are ﬁbroblastic in appearance and negative forhematopoietic cell markers, such as c-kit, CD34, andCD45. USSCs injected into immunosuppressed pig modelof myocardial infarction (MI) improved perfusion and wallmotion, reduced infarct scar size, and enhanced cardiac
function. USSCs seem to be a useful source for myocardial
regeneration; however, they are a rare population, therefore, expansion of USSCs is required for application toclinical therapy. In spite of these challenges for the repairof CM, to date, no clinical studies of CB have been reported. Previously, we reported that brown adipose tissue
derived cells (BATDCs) included cardiac progenitor cells
and they eﬀectively diﬀerentiated into CMs in vitro andin vivo [16]. This indicated that our culture system of BATDCs contained diﬀerentiation molecular cues for CMs.
When BATDCs were injected into MI rats, they diﬀerentiated into CMs as well as endothelial cells (ECs) andsmooth muscle cells (SMCs), supported the growth of resident cells and vascular cells, and restored cardiac function.
This suggested a potential therapeutic use for BATDCs inhuman ischemic heart disease. However, in humans, BAT
exists only in the embryonic stage and infants, therefore,
it is diﬃcult to obtain BATDCs to treat adult cardiac disease. In this study, we examined whether mononuclear cells
from CB can diﬀerentiate into CMs upon coculturing withBATDCs. Moreover, usefulness of CB cells that were
exposed to BATDCs for MI repair was evaluated.
Materials and methods
Cell preparation and ﬂow cytometry. Brown adipose tissue (BAT) was
dissected from postnatal day (P1) to P7 neonates of C57BL/6 mice. BAT
was dissociated by DispaseII (Roche, Mannheim, Germany), drawn
through a 23G needle and prepared as single cell suspension as previously
reported [16]. Human CB mononuclear cells (CBMNCs) were purchasedfrom Cambrex (Baltimore, MD). The cell-staining procedure for the ﬂow
cytometry was also as previously described [17]. The monoclonal antibodies (mAbs) used in immunoﬂuorescence staining were anti-humanCD45, -34, and -HLA–ABC mAbs (Pharmingen, SanDiego, CA). All
mAbs were puriﬁed and conjugated with ﬂuorescein isothiocyanate(FITC), PE (phycoerythrin), biotin or allophycocyanin (APC). Biotinylated antibodies were visualized with PE-conjugated streptavidin (Pharmingen) or APC-conjugated streptavidin (Pharmingen). Cells wereincubated for 5 min on ice with CD16/32 (FccIII/II Receptor) (1:100)(Fcblock, Pharmingen) prior to staining with primary antibody. Cells
were incubated in 5% fetal calf serum/phosphate-buﬀered saline (FCS/
PBS; washing buﬀer) with primary antibody for 30 min on ice, and washedtwice with washing buﬀer. Secondary antibody was added and the cells
were incubated for 30 min on ice. After incubation, cells were washedtwice with, and suspended in, the washing buﬀer for ﬂuorescence-activatedcell sorter (FACS) analysis. The stained cells were analyzed and sorted by
EPICS Flowcytometer (BECKMAN COULTER, San Jose, CA). The
sorted cells were added to 24-well dishes (Nunc, Roskilde, Denmark), precoated with 0.1% gelatin (Sigma, St. Louis, USA), and cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM; Sigma), supplemented with
10% FCS and 10 5M 2-mercaptoethanol (2-ME), at 37 C in a 5% CO2
incubator.
Cell coculture. BATDCs were prepared as described above. WhenBATDCs and human (h) CBMNCs were cocultured in contact conditions,1  105 BATDCs were plated per well of a 24-well plate and cultured for 7days, and then, 1  105 CBMNCs, or 1  104 CD34+ HSCs were cultured
with BATDCs for 10 days. Staining was performed with anti-cardiac

183

troponinT (Santa Cruz), -MEF-2C (Cell signaling) and -HLA ABC
antibodies (Pharmingen).
Supplemental information reveals the Materials and methods for RTPCR analysis, Immunohistochemistry, FISH staining, and procedure formouse myocardial infarction (MI) model and echocardiography.

Results
Diﬀerentiaion of CBMNCs into CMs by coculturing withBATDCs in vitro
Previously, we reported that BATDCs diﬀerentiate into
CMs spontaneously; this suggests that BATDCs produce
molecules that induce self diﬀerentiation into CMs by anautocrine loop. Moreover, CBMNCs contained cells witha potential to diﬀerentiate into various cell types, such as
osteoblasts, chondrocytes, and CMs. Therefore, to determine whether molecules produced from BATDCs induce
CBMNCs to diﬀerentiate into CMs eﬀectively, we cultured
CBMNCs with BATDCs and observed the diﬀerentiationof CBMNCs into CMs. At ﬁrst, dissociated BATDCs from
P1 to P7 neonatal mice were cultured on 0.1% gelatin-coated dishes. After 1 week, human CBMNCs were added andcocultured with BATDCs. After coculturing CBMNCs
with BATDCs for 14 days, among HLA-positive
CBMNCs, nuclear located MEF2C positive and cardiac
troponinT-positive cells (Fig. 1A and B), or cardiac troponinI-positive cells (Fig. 1C) were eﬀectively produced. In
contrast, sorted CD34+38 HSC population from
CBMNCs, which was previously reported to diﬀerentiate
into CMs [18], was diﬀerentiated into cardiac troponinTpositive CMs (Fig. 1E); however, the frequency of CM differentiation from HSC population was lower than thatfrom total CBMNCs (Fig. 1I). CBMNCs alone did not differentiate into cardiac troponinT-positive or MEF2C-positive cells spontaneously under the same culture medium
without coculturing with BATDCs (Fig. 1G, H, and I).
The expression of CM-speciﬁc genes in CBMNCs educatedby culturing with BATDCs
Next, we evaluated the length of time required for
CBMNCs to become committed CM lineage cells whencocultured with BATDCs. For this purpose, we attempted
to coculture CBMNCs with BATDCs for 1 to 7 days and
HLA+CD45 CD34 non-hematopoietic cells [we termedthem educated CB cells (e-CBCs)] were then sorted andmRNA was extracted from the cells as indicated inFig. 2A. Because mature hematopoietic cells from cocultured CBMNCs did not diﬀerentiate into CMs (data notshown) and HSC population barely diﬀerentiated into
CMs (Fig. 1), we deduced that cardiac stem/progenitor
cells were more abundant in non-hematopoietic cells andwere therefore CD45 CD34 . We analyzed the expressionof CM-speciﬁc genes on days 0, 3, and 7 as indicated inFig. 2B and conﬁrmed that cardiac actin, myosin lightchain 2v, and speciﬁc transcriptional factor, such as184

Y. Yamada et al. / Biochemical and Biophysical Research Communications 353 (2007) 182–188

Fig. 1. CBMNCs can diﬀerentiate into CMs upon coculturing with BATDCs. Immunocytochemical analysis of human CBMNCs (A–D), andCD34+CD38 HSCs (E, F) cocultured with BATDCs from wild type mice, or CBMNCs (G, H) cultured without BATDCs for 14 days. (A) Expression ofcardiac troponinT (green) and MEF2C (red). (B) Human HLA expression (blue) in the same ﬁeld as (A). (C, E, and G) Expression of cardiac troponinI(green) and nuclear staining with PI (red). (D, F, and H) Human HLA expression (blue) in the same ﬁelds as (C, E, and G), respectively. Scale bar in (H)indicates 5 lm. (I) Quantitative evaluation of diﬀerentiated cardiac troponinT and MEF2C-positive CMs among adhering HLA-positive CB-derived cells.
Data for CBMNC, and CD34+CD38 HSCs cocultured with BATDCs (b-adipocyte) and CBMNCs cultured without BATDCs are displayed. Results
represent means ± SD of ﬁve independent experiments.

GATA-4 and Nkx2.5 were expressed on day 3 of coculture(Fig. 2B). MHC alpha and beta mRNAs were notexpressed on day 3; however, they started to be expressedaround day 7.
Educated CBMNCs in non-hematopoietic lineage
contributed to myocardial regeneration
As indicated in Fig. 2, 3 days of coculturing with BATDCs was enough for commitment of CBMNCs into CM
lineage. Next, in order to determine whether e-CBCs could
eﬀectively contribute to the regeneration of the heart, we
injected the e-CBCs into the hearts of nude rats after the
induction of an acute MI as indicated in Fig. 3A. At ﬁrst,
we cocultured CBMNCs with BATDCs for 3 days, puriﬁed
HLA+CD45 CD34 cells (e-CBCs) by FACS and injected
the cells into the hearts of experimental MI nude rats ateach of ﬁve sites at the border of the infarcted tissue. As
a control, infarcted hearts were injected with either equalvolumes and numbers of CBMNCs that were not exposed
to BATDCs (non-e-CBCs), or CD34+38 HSCs directly
sorted from freshly isolated CBMNCs. Upon injection of
e-CBCs, donor-derived human HLA- and SA-positive cells

were detected abundantly in the infarct border zone
(Fig. 3B, a, b, and c; 23.4 ± 3.1% of total cardiomyocytes
in one ﬁeld), but the contribution to CMs by the injection
with non-e-CBCs and CD34+38 HSCs in the MI was 20(Fig. 3B, d, e, and f; 1.1 ± 0.3%) and 15-fold (Fig. 3B, g,h, and i; 1.5 ± 0.3%), respectively, less than that of e-CBCs.
e-CBC-derived SA-positive CMs also expressed connexin
43 (Fig. 3Bc), indicating that transplanted e-CBC-derived
CMs formed gap junctions with host CMs. Moreover,
the assessment of cardiac function by echocardiography
revealed that the hearts injected with e-CBCs showed
improved contractions of movement of the infarcted anterior walls and reduced left ventricular remodeling compared with the hearts injected with non-e-CBCs or
CD34+38 HSCs (Fig. 4).
To clarify the origin of CMs in recipient tissue,
donor-derived human chromosomes and host-derived rat
chromosomes were simultaneously detected by using
species-speciﬁc chromosome probes using ﬂuorescentin situ hybridization (FISH) analysis. In this analysis,
we used centromere probes, because 5 lm thick slices
may not always include sex chromosomes in the
nuclei as previously described [18]. Result showed that

Y. Yamada et al. / Biochemical and Biophysical Research Communications 353 (2007) 182–188

185

Fig. 2. Expression of CM-speciﬁc genes in e-CBCs. (A) Design of experiment for the isolation of human e-CBCs. CBMNCs were cocultured withBATDCs for 1 to 10 days, and then CD45 CD34 human HLA+ cells were sorted and total RNA was extracted. (B) PCR analysis was performed with
CM-speciﬁc primers in e-CBCs after coculturing with BATDCs for 0 day (D0; freshly isolated CBCs without exposure to BATDCs), 3 days (D3) and 7
days (D7). Distilled water (DDW) and CMs from embryos (CM) were used as a negative and a positive control, respectively. GAPDH was used as aninternal control.

implanted e-CBCs of human origin transferred into
female nude rats formed CMs that stained only withprobes speciﬁc for human chromosomes (Fig. 3C, a andb). We also checked the serial confocal imaging to exclude
the possibility that they arose from cell overlay as
previously reported [18], but could not observe any
superimposed cells (data not shown). This indicated thatin vivo cardiac diﬀerentiation of e-CBCs was not inducedby the fusion mechanism. In contrast, when
CD34+CD38 HSCs were implanted into MI induced
nude rats, human HLA-positive CMs were stained withboth human and rat chromosome probes (data notshown). This indicated that generation of CM-derived
HSCs was due to the fusion mechanism between
donor-derived cells and host CMs as previously reported[18,19].
Discussion
So far, various kinds of sources for CMs, such as adult
BM HSCs [19,20], MSCs [1–3], and ES cells [4,5] have beenreported; however, there is some controversy regarding theeﬃciency of cardiomyoplasty. In terms of the myocardial
regeneration therapy for human, human CB cells seem tobe a safe and useful source compared to other sources,
because these cells have already been utilized in CB transplantation for managing patients with blood disease.

However, no clinical trials using CB cells to treat heart disease have been reported.
In this study, we raised two important points. The ﬁrst isthat e-CBCs of non-hematopoietic origin were more eﬀectively diﬀerentiated into CMs compared with
CD34+CD38 HSCs in vitro. Moreover, we showed that eCBCs diﬀerentiation into CMs was not through the cellfusion mechanism. On the other hand, CMs derived from
CD34+CD38 HSCs were generated through cell fusion withhost CMs in vivo. Previously, it was reported that MSCs but
not HSCs from BM could migrate into the heart and diﬀerentiate into CMs in mouse MI model [21]. This suggested
that MSCs were the predominant source for myocardial
regeneration. Our report is the ﬁrst to show that non-hematopoietic cells can be used as CM source and how these compare with HSCs in human cord blood cells.
The second is the new strategy for myocardial regeneration using CB cells and BATDCs. Using the coculturingmethod described here, CB cells were eﬀectively induced
to diﬀerentiate into CMs in vitro. Moreover, e-CBCs wereeﬀectively diﬀerentiated into CMs in immunodeﬁcient rat
MI model and improved the cardiac function. Furthermore, we found that an adequate duration of coculturingof CB cells with BATDCs was critical for CM regeneration. In the present method, three days of coculturing
was the most eﬀective to produce CMs from e-CBCs andimprove the CM function and this timing was consistent186

Y. Yamada et al. / Biochemical and Biophysical Research Communications 353 (2007) 182–188

Fig. 3. CBMNCs cocultured with BATDCs contributed to cardiac regeneration. (A) Strategy for transplantation. After human CBMNCs were cocultured
for 3 days with BATDCs from mice, human HLA+CD45 CD34 cells (e-CBCs) were sorted by FACS and injected into the border zone of ischemia
induced nude rats. As a control, CBMNCs that were not cocltured (non-e-CBCs) and freshly isolated CD34+CD38 HSCs were used. (B) CM
development from injected e-CBCs (a–c), non-e-CBCs (d–f), and CD34+CD38 HSCs in MI induced heart. (a, d, and g) Expression of SA (green). (b, e,
and h) human HLA (red). (c, f, and i) are merged image of (a and b), (d and e), and (g and h), respectively, and stained with anti-connexin 43 antibody(blue). Arrows in (c, f, and i) indicate the regions in which human CB-derived connexin positive CMs make tight junction with resident host CMs. Scalebar in (i) indicates 20 lm. (C) (a) Expression of human HLA (red) and SA (green) in the site of implantation of e-CBCs in MI induced heart. Nuclei werecounter stained with TOPRO3 (blue). (b) FISH staining in a serial section of (a). Green colors indicate rat chromosome, and red color indicates humanchromosome. Nuclear staining was performed with TOPRO3 (blue). Cells expressing human chromosome (red) in the nuclei indicate that these cells were
derived from e-CBCs and did not fuse with host CM expressing only rat chromosome (green). Arrowheads indicate nuclei from host CMs and arrow
indicates human nuclei in e-CBCs (a, b). Scale bar indicates 5 lm.

Fig. 4. e-CBCs transplantation improved cardiac function. LV diameter (LV end-diastolic diameter) and function (Ejection fraction) were assessed byechocardiography at 0, 3, 7, 14, and 28 days following myocardial infarction and injection of saline, e-CBCs, non-e-CBCs, and HSCs (CD34+). Note thatin case of injection of e-CBCs, enlargement of LV diameter was reduced and LV function was signiﬁcantly improved compared with injection of saline,
non-e-CBCs, and HSCs. Each data point is the mean of ﬁve determinations; bars denote ±SD.

with the expression of Nkx2.5 and GATA-4 on CBMNCs(Fig. 2). In order to determine how e-CBCs diﬀerentiatedinto CMs, we tested a number of growth and survival factors and found that Akt activation seemed to play a role inthe diﬀerentiation of CBMNCs into CMs (Supplemental
data 1). Akt, a serine threonine kinase, transduces powerfulsurvival signals in many systems [22,23]. Recently, it wasreported that overexpression of Akt1 in MSCs increased
the post-transplantation viability of these cells andenhanced their therapeutic eﬃciency [24]. In fact, intramyocardial injection of MSCs that had been transfected with aretroviral vector containing the Akt gene resulted in the

Y. Yamada et al. / Biochemical and Biophysical Research Communications 353 (2007) 182–188

diﬀerentiation of MSCs into CMs and led to the preventionof ventricular remodeling and to the restoration of cardiac
function after MI. In order to examine the survival signals
in e-CBCs in each culture day as indicated, we checked thephosphorylation level of Akt (p-Akt) in e-CBCs on days 0,
3, and 7. Before extraction of cell lysate, e-CBCs were
exposed to hypoxic condition for 24 h. The p-Akt level of
e-CBCs on day 3 was 10-fold higher than that on day 7
and it was higher (1.7·) than that on day 0 before coculturing with BATDCs (Supplement data 1). The resistance ofcell to apoptosis induced by hypoxic stimuli was proportional to the level of p-Akt in e-CBCs, i.e., it was higher
on day 3 compared to day 0 and day 7. This anti-apoptotic
eﬀect might contribute to the high incidence of CMs
derived from e-CBCs and to prevention of cardiac remodeling caused by conditions such as hypoxia, inﬂammation,and mechanical stress, and many endogenous factors suchas angiotensin II, endothelin-1, and norepinephrine [25] inMI model.
We have as yet not clariﬁed the precise mechanism
whereby CB cells expressed high levels of p-Akt in hypoxic
conditions after short-term coculturing with BATDCs.
BATDCs were derived from adipose tissue, which possessed many beneﬁcial factors, such as vascular endothelial
growth factor (VEGF), hepatocyte growth factor (HGF),
angiopoietin-1, and so on [16]. Therefore, these factors
might support the high level of p-Akt in e-CBCs. Identiﬁcation of such factors may enable eﬀective myocardial
regeneration. Use of CBCs together with such factors forprotection against cell apoptosis may enable the application of the strategy described here to the clinic for managing patients of ischemic disease.
Acknowledgments
We thank Miss M. Sato for technical support. This
study was supported in part by a grant from the Ministryof Education, Culture, Sports, Science, and Technologyof Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at doi:10.1016/
j.bbrc.2006.12.017.
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European Journal of Pharmacology 549 (2006) 98 – 106
www.elsevier.com/locate/ejphar

Effects of G-CSF on cardiac remodeling and arterial hyperplasia in rats
Yuxin Li a , Noboru Fukuda a,b,⁎, Shin-Ichiro Yokoyama a , Yoshiaki Kusumi c , Kazuhiro Hagikura a ,Taro Kawano a , Tadateru Takayama a , Taro Matsumoto d , Aya Satomi d , Junko Honye a ,Hideo Mugishima d , Masako Mitsumata c , Satoshi Saito a,dbaDepartment of Internal Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan
Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo 102-8275, Japanc
Department of Pathology, Nihon University School of Medicine, Tokyo 173-8610, Japand
Department of Advanced Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan

Received 16 March 2006; received in revised form 26 July 2006; accepted 2 August 2006
Available online 15 August 2006

Abstract
Although granulocyte colony-stimulating factor (G-CSF) has been shown to prevent cardiac remodeling after acute myocardial infarction, themechanism and safety of G-CSF treatment acute myocardial infarction remain controversial. The purpose of the present study was to investigate ina rat model the mechanisms underlying the beneficial effect of G-CSF in acute myocardial infarction and to determine whether G-CSF treatment
aggravates vascular remodeling of injured artery after acute myocardial infarction. Sprague–Dawley rats received transplanted bone marrow cells
from green fluorescent protein (GFP) transgenic rats. Acute myocardial infarction was induced by ligation of the left coronary artery. After 24 h,
the right carotid artery was injured with a balloon catheter. G-CSF (100 μg/kg/day) or saline was injected subcutaneously for 5 consecutive days
after induction of acute myocardial infarction. G-CSF treatment significantly improved left ventricle function and reduced infarct size in rats withacute myocardial infarction. Expression of mRNA for the angiogenic cytokines was significantly higher in the infarction border area in the G-CSF
group than in the control group. The surviving cardiomyocytes in infarction area were more in the G-CSF group. GFP-positive cells were gatheredin the infarction border area in both groups; G-CSF did not increase cardiac homing of GFP-positive bone marrow cells in contrast to control
group. Most GFP-positive cells were CD68-positive (macrophages). It was difficult to find bone marrow-derived cardiomyocytes in the infarcted
area. G-CSF treatment inhibited neointima formation and increased reendothelialization of the injured artery. GFP-positive cells were identified
most in the adventitia of the injured artery. A few cells in the neointima and reendothelialization were GFP positive. In conclusion, administrationof G-CSF appears to be effective for treatment of left ventricular remodeling after acute myocardial infarction and does not aggravate vascularremodeling. The effect of G-CSF on cardiac and vascular remodeling may occur mainly through a direct action on the heart and arteries.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Cytokine; Coronary disease; Infarction; Stem cell; Restenosis

1. Introduction
Myocardial infarction is a common cause of cardiac morbidity and mortality, and left ventricular remodeling after
myocardial infarction is critical for progression to heart failure.
Several cytokines, including granulocyte colony-stimulating
factor (G-CSF), erythropoietin, and leukemia inhibitory factor,
have been reported to reduce cardiac remodeling after myo⁎ Corresponding author. Department of Internal Medicine, Nihon University
School of Medicine, 30-1 Oyaguchi-Kamimachi, Itabashi-ku, Tokyo 173-8610,
Japan. Tel.: +81 3 3972 8111; fax: +81 3 3972 8666.
E-mail address: fukudan@med.nihon-u.ac.jp (N. Fukuda).
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.08.006

cardial infarction (Moon et al., 2003; Orlic et al., 2001; Zouet al., 2003). G-CSF improves cardiac function and reduces
mortality after myocardial infarction in animal model (Adachi
et al., 2004; Kawada et al., 2004; Minatoguchi et al., 2004;
Ohtsuka et al., 2004; Orlic et al., 2001). G-CSF has various
effects, for example, on proliferation, survival, and differentiation of hematopoietic cells and on mobilization of bonemarrow cells (Avalos, 1996; Berliner et al., 1995). Mobilization of bone marrow cells, including stem and progenitor
cells, to the myocardium and transdifferentiation of these cells
into heart cells has been considered a main mechanism to
improve cardiac remodeling after myocardial infarction (Adachi
et al., 2004; Kawada et al., 2004; Minatoguchi et al., 2004; OrlicY. Li et al. / European Journal of Pharmacology 549 (2006) 98–106

99

Table 1
RT-PCR primers used in the study
Primer nameNucleotide sequence

PCR product length (bp)

VEGF

Sense: 5′-ACTGGACCCTGGCTTTACTG-3′
Antisense: 5′-ACGCACTCCAGGGCTTCATC-3′
Sense: 5′-AGGAGAGGACCTGAAACTGTCTT-3′
Antisense: 5′-ATTCCTGGGCTCTGCAGGCATAG-3′
Sense: 5′-GTGGCTGGAAAAACTTGAGA-3′
Antisense: 5′-TGGATTTCAAGACGGGATGT-3′
Sense: 5′-GACCAGTGGGCATCGCTACG-3′
Antisense: 5′-CTGGTTGGCTGATGCTACTG-3′
Sense: 5′-TGCGAGCCCAGTCCAAGAGA-3′
Antisense: 5′-ACAGGGTAACTCAAAGGCTC-3′
Sense: 5′-TGCCACCATCACTCAATACC-3′
Antisense: 5′-AAACGCCAATAGCACGGTGA-3′
Sense: 5′-AAGCGGCTCTACTGCAAG-3′
Antisense: 5′-AGCCAGACATTGGAAGAAACA-3′
Sense: 5′-ACCCTTCCAGGTGAGGTCT-3′
Antisense: 5′-CCACCTCTACATCGAACCT-3′

256

Flt-1
Angiopoietin-1
Angiopoietin-2
Tie-1
Tie-2
Basic-FGF
TGFβ

214
201
170
270
217
372
161

VEGF, vascular endothelial growth factor; basic-FGF, basic fibroblast growth factor; TGFβ, transforming growth factor β.

et al., 2001). However, recent studies (Harada et al., 2005;
Ohtsuka et al., 2004) have shown that G-CSF acts directly on
cardiomyocytes to reduce apoptotic cell death and protect theinfarcted heart via activation of the Stat3 pathway. Thus, G-CSF
may inhibit cardiac remodeling by several mechanisms.
However, administration of G-CSF may have negative effects
in patients with acute myocardial infarction. G-CSF induced
mobilization of progenitors from bone marrow may lead totransdifferentiation into vascular smooth muscle cells, which may
aggravate restenosis (Shoji et al., 2004; Simper et al., 2002). Thus,
G-CSF may worsen plaque formation and restenosis in coronary
arteries. The administration of G-CSF to patients with acute
myocardial infarction undergoing percutaneous coronary intervention remains controversial (Hill et al., 2005; Kang et al., 2004;
Petzsch et al., 2004; Ripa et al., 2004; Valgimigli et al., 2005).
The purpose of the present study was to investigate in a rat
model the mechanisms underlying the beneficial effect of G-CSF
in acute myocardial infarction and to determine whether G-CSF
treatment aggravates vascular remodeling of injured artery after
myocardial infarction.

subcutaneously immediately after induction of myocardial
infarction and was continued daily for additional consecutive
4 days.

2. Materials and methods
All animal care and handling were performed in accordancewith the guidelines specified by the National Institutes of Health
Guide for the Care and Use of Laboratory Animals and wereapproved by Institutional Animal Care and Use Committee.
2.1. Rat myocardial infarction model and G-CSF
administration
Eight-week-old male Sprague–Dawley (SD) rats were usedin this study. Myocardial infarction was induced by permanent
ligation of the left coronary artery with 6–0 polypropylene
suture as described previously (Adachi et al., 2004; Orlic et al.,2001). Rats were randomly assigned to one of two groups:
100 μg/kg/day G-CSF (Kirin-Amgen Inc., G-CSF group) or thesame volume of normal saline (control group) was injected

Fig. 1. Representative M-mode echocardiograms in normal rats (Normal, left
ventricular ejection fraction: 93%, left ventricular internal diameter at enddiastole: 5.52 mm) and in rats with myocardial infarction (MI) with G-CSF (leftventricular ejection fraction: 60%, left ventricular internal diameter at enddiastole: 8.80 mm) or without G-CSF (Control, left ventricular ejection fraction:
33%, left ventricular internal diameter at end-diastole: 11.02 mm) treatment.
Two-dimensional images and M-mode tracing were recorded from theparasternal short axis view 28 days after induction of MI.

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Table 2
Echocardiographic analyses before and after 28 days of MI
Control
Pre-MI

G-CSF
Post-MIN

10

10Left ventricular internal diameter at end-diastole (mm)Left ventricular internal diameter at end-systole (mm)Left ventricular ejection fraction (%)
Fractional shortening (%)

5.83 ± 0.08
1.90 ± 0.13
95.6 ± 0.9
66.8 ± 2.4

10.50 ± 0.66
8.93 ± 0.53
35.9 ± 1.5
15.2 ± 0.7

PChangePre-MI

Post-MI

10

10

4.67 ± 0.64
7.03 ± 0.41
59.7 ± 1.5
51.7 ± 2.4

5.53 ± 0.24
1.93 ± 0.22
94.6 ± 1.0
64.4 ± 2.99.43 ± 0.42
7.15 ± 0.66
53.8 ± 5.9
24.6 ± 3.5

Change3.90 ± 0.54
5.23 ± 0.76
40.8 ± 6.3
39.9 ± 5.2

0.140
0.039
0.004
0.032

Data are expressed as mean ± S.E.M.

2.2. Rat carotid artery balloon injury model
Balloon injury was performed 24 h after induction of acute
myocardial infarction. The right common carotid artery was
exposed at the level of bifurcation through a midline neck incision, as described previously (Griese et al., 2003; Qian et al.,2002). A 2F Fogarty arterial embolectomy catheter (Baxter
Healthcare Corp.) was inserted through the external carotid artery,
inflated with 0.2 ml air, and pulled five times along the common
carotid artery (area injured by the catheter ∼3 cm in length).
2.3. Transplantation of bone marrow cells from green
fluorescent protein transgenic rats to SD rats
To evaluate the distribution of bone marrow cells in the heart
after myocardial infarction and in the injured carotid artery,bone marrow cells from green fluorescent protein (GFP)
transgenic rats (SD TgN [act-EGFP] OsbCZ-004, Japan SLC,
Inc.) were transplanted into four-week-old male SD rats.
Recipient rats were irradiated with a lethal dose (1300 rad) of
X-rays and were injected with 1.3 × 108 GFP bone marrow cells
from tail vein. One month after transplantation, 300 μl bloods
was collected from the tail vein and subjected to fluorescenceactivated cell sorting (FACS) analysis to determine the bonemarrow replacement rate. A greater than 90% bone marrow
chimerism rate was considered successful replacement. Aftertransplantation success, acute myocardial infarction and carotid
artery balloon injury model were performed.

administration, 200 μl peripheral blood was harvested from thetail vein for blood cell counting.
2.6. Angiogenic cytokine levels in peripheral blood
One, 3, 7, 14, and 28 days after induction of myocardial
infarction, rats were killed and peripheral blood (10 ml) wascollected to measure plasma levels of vascular endothelial
growth factor (VEGF), basic fibroblast growth factor (basicFGF), hepatocyte growth factor (HGF), and interleukin-8 by
enzyme-linked immunosorbent assay (ELISA).
2.7. Expression of angiogenic cytokines in the myocardial
infarction border area
One, 3, 7, 14, and 28 days after induction of myocardial
infarction, rats were killed, coronary blood was washed out withsaline, the infarction border area of the heart (100 mg) wasremoved and minced, and total RNA was extracted by the
guanidinium thiocyanate-phenol chloroform method. Reverse
transcription-polymerase chain reaction (RT-PCR) was performed to determine expression of the myocardial cytokines
VEGF and receptor Flt-1, Angiopoietin-1 and -2 and receptors
Tie-1 and -2, basic-FGF, and transforming growth factor β1(TGFβ1). Aliquots of total RNA (1 μg/20 μl) were reversetranscribed into single-stranded cDNA with 0.25 U/μl avian
myeloblastoma virus reverse transcriptase (Life Sciences, Inc.) in

2.4. Echocardiography
Transthoracic echocardiography (ProSound SSD-5500SV,
Aloka Co., Ltd) was performed with a 10 MHz imaging
transducer. Before and 28 days after induction of myocardial
infarction, two-dimensional images and M-mode tracing wererecorded from the parasternal short axis view. Left ventricularinternal diameter at end-diastole, left ventricular internal
diameter at end-systole, left ventricular ejection fraction, andfractional shortening were evaluated by two investigators
blinded to the groups.
2.5. Peripheral blood cell countIn separate experiments, three rats were injected subcutaneously with 100 μg/kg/day G-CSF for 5 consecutive days.
Before and 1, 2, 3, 4, 6, 7, 8, 10, and 15 days after G-CSF

Fig. 2. Peripheral blood cell counts after G-CSF administration in rats. Three
Sprague–Dawley rats were injected subcutaneously with 100 μg/kg/day G-CSF
for 5 consecutive days. Before and 1, 2, 3, 4, 6, 7, 8, 10, and 15 days after G-CSF
administration, 200 μl peripheral blood was harvested from the tail vein forblood cell counting. On day 4 after treatment, total white blood cells increased
3.7-fold, neutrophils increased 16-fold, monocytes increased 9.5-fold, andlymphocytes increased 0.9-fold. WBC, total leukocytes; NEUT, neutrocytes;
LYMPH, lymphocytes; MONO, monocytes.

Y. Li et al. / European Journal of Pharmacology 549 (2006) 98–106

10 mM Tris–HCl (pH 8.3), 5 mM MgCl2, 50 mM KCl, 1 mM
deoxy-NTPs, and 2.5 μM random hexamers. Five microliters ofthe diluted cDNA product was mixed with 10 mM Tris–HCl(pH 8.3), 50 mM KCl, 4 mM MgCl2, 0.025 U/ml Taq DNA
polymerase (Takara BioInc.), and 0.2 μM of each primer in a total
volume of 25 μl. The primers used in this study are listed inTable 1. To confirm noncontamination of genomic DNA,
identical RT-PCR amplification reactions were performed in theabsence of reverse transcriptase. PCR was performed with a DNA
thermal cycler (PerkinElmer).
2.8. Morphometric analysis of infarct size and artery neointima
formation
Twenty-eight days after induction of myocardial infarction,
rats were killed by lethal injection of sodium pentobarbital

101(100 mg/kg body weight, i.p.) and then perfused with saline
followed by 10% formalin at physiologic pressure. Six transverse
segments of equivalent thickness from the base to the apex of theheart and five segments of equivalent thickness of the carotid
artery were embedded in paraffin. From each segment, 4-μmthick sections were cut and stained with hematoxylin–eosin and
Masson trichrome stain. Scarred areas of the heart were stained
blue with Masson trichrome stain. Infarct size measurements were
performed as described previously (Grimm et al., 1998; Pfeffer
et al., 1979). In brief; the lengths of the infarcted surfaces were
measured and expressed as a percentage of the total circumference × 100. Final infarct size was calculated as the average of all
segments from each heart.
For morphometric analysis of neointima formation, thecarotid artery luminal area and internal elastic membrane weretraced. The cross-sectional area between the lumen and theFig. 3. RT-PCR analysis of angiogenic cytokine expression in the infarction border area. Sprague–Dawley rats with myocardial infarction (MI) were treated with G-CSF (G-CSF
group) or saline (control group) for 5 consecutive days. Hearts were removed 1, 3, 7, 14, and 28 days after induction of MI. Rat 18S ribosomal RNA (rRNA) served as aninternal control, the ratio of the abundance of cytokine mRNA to that of 18S rRNA was evaluated by densitometric analysis. Data are the means of experiments carried out in
duplicate. G-CSF increased angiogenic cytokines expressions immediately after administration and the effect continued 7 to 28 days. *P b 0.05 vs. control group. VEGF, vascularendothelial growth factor; Ang-1, angiopoietin-1; Ang-2, angiopoietin-2; basic-FGF, basic fibroblast growth factor; TGFâ, transforming growth factor β.

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internal elastic membrane was hyperplastic neointimal area
(Ohlstein et al., 2000). Restenosis rate was calculated as
hyperplastic neointimal area/internal elastic membrane area.

groups at different time points. Between-group differenceswere analyzed by two-tailed Student's t-test. Probability
values (P) less than 0.05 were considered statisticallysignificant.

2.9. Immunohistochemistry
3. Results
Paraffin blocks of the third segment of heart and all segmentsof carotid artery were used for immunohistochemistry. Immunohistochemical evaluation of the heart focused on the infarction
area and infarction border area. Endothelial cells were identified
with antibody against von Willebrand factor (vWF, 1:100;
DakoCytomation). Smooth muscle cells were identified with
anti-smooth muscle actin antibody (1A4, 1:100; DakoCytomation). Inflammatory cells were identified with anti-CD68 antibody(1:50, Serotec). Cardiomyocytes were identified with anti-alphasarcomeric muscle actin antibody (αSr-1, 1:40; DakoCytomation). GFP cells were identified with anti-GFP antibody (1:200,
MBL). For cell counting, digital photographs were taken at
magnification ×400, and 10 random high power fields from eachsample were chosen and quantified in a blinded manner.
2.10. Statistical analysis
Data are shown as mean ± S.E.M. One-way analysis ofvariance (ANOVA) was performed for comparisons between

3.1. Effect of G-CSF on hemodynamic changes
There were no significant differences in heart rate between
the control and G-CSF groups before and after myocardial
infarction. Representative M-mode echocardiograms after
myocardial infarction are shown in Fig. 1. Changes in left
ventricular internal diameter at end-diastole, left ventricularinternal diameter at end-systole, left ventricular ejection
fraction, and fractional shortening after myocardial infarction
are shown in Table 2. After myocardial infarction, left
ventricular internal diameter at end-diastole and left ventricularinternal diameter at end-systole increased, left ventricularejection fraction and fractional shortening decreased inboth control and G-CSF-treated rats, whereas the increases inleft ventricular internal diameter at end-systole and thedecreases in left ventricular ejection fraction and fractional
shortening were significantly less in the G-CSF group than in
the control group.

Fig. 4. Histologic analysis of heart and artery neointima formation. (A, B) Masson trichrome staining of heart 28 days after myocardial infarction. (A) Control group(n = 10). (B) G-CSF group (n = 10). Left ventricle free wall was thinner and the infarct area was replaced by fibrotic tissue in control group, while infarction wall
thickness was maintained and surviving myocardium was observed in G-CSF group. (C, D) Effect of G-CSF on neointima formation in rat carotid artery 4 weeks after
balloon injury. (C) Control group (n = 10). (D) G-CSF group (n = 10). G-CSF treatment reduced neointima formation in injured artery.

Y. Li et al. / European Journal of Pharmacology 549 (2006) 98–106

103

3.2. Effects of G-CSF on cell counts and angiogenic cytokines
in peripheral blood

ately from 1 day after myocardial infarction and continued until
7 to 28 days after myocardial infarction.

G-CSF treatment induced a significant transient increase in
the number of white blood cells, which decreased immediately
after termination of treatment. On day 4 after treatment, total
white blood cells increased 3.7-fold, neutrophils increased 16fold, monocytes increased 9.5-fold, and lymphocytes increased
0.9-fold (Fig. 2).
There were no differences in plasma levels of VEGF, basicFGF, HGF, and interleukin-8 between control and G-CSF groups.

3.4. Histologic analysis of heart and artery neointima
formation

3.3. G-CSF increased expression of angiogenic cytokines in theinfarction border area
G-CSF treatment increased the expression of most of theangiogenic cytokines analyzed in the infarction border area after
myocardial infarction (Fig. 3). Expression increased immedi-

On day 28 after myocardial infarction, the left ventricle freewall was very thin in the control group (0.32 ± 0.02 mm, n = 10),showing complete transmural infarction with fibrosis (Fig. 4A),
whereas G-CSF treatment preserved left ventricle wall thickness(0.85 ± 0.06 mm, n = 10, P b 0.05 vs. control group) in theinfarcted area (Fig. 4B). The size of the infarcted area wassignificantly larger in the control group (51.7 ± 6.2%) than in the
G-CSF group (34.1 ± 2.7%, P b 0.05 vs. control group).
The effect of G-CSF treatment on neointima formation in the
carotid artery 4 weeks after myocardial infarction and balloon
injury is shown in Fig. 4C–D. Morphometric analysis of fiveserial segments with equivalent thickness showed a 35% decrease

Fig. 5. Immunohistochemistry of infarction area and infarction border area. (A, B) Anti-alpha-sarcomeric muscle actin (αSr-1) staining of infarction area. (A) Control
group. (B) G-CSF group. Surviving cardiomyocytes occupied the infarction area from the endocardium to the epicardium, preserving the wall thickness in the G-CSF
group. (C, D) GFP-positive cells in infarction border area. (C) Control group. (D) G-CSF group. (E) The ratio of GFP-positive cell number to total cell number (43 ± 10%
vs. 41 ± 3%, respectively, P N 0.05). (F) Proportion of GFP-positive cells co-expressing CD68 (67 ± 13% vs. 70 ± 15%, respectively, P N 0.05). (G) Proportion of GFPpositive cells co-expressing α1A4 (6 ± 3% vs. 7 ± 4%, respectively, P N 0.05).

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Y. Li et al. / European Journal of Pharmacology 549 (2006) 98–106

in neointimal area in the G-CSF group compared with that in thecontrol group (43.9 ± 6.2% vs. 28.6 ± 4.7%, P b 0.05).
3.5. Immunohistochemical evaluation of the heart after
myocardial infarction
Immunohistochemical staining for αSr-1 was performed todetect surviving cardiomyocytes in the infarction area. The

number of surviving cardiomyocytes was greatly reduced in thecontrol group compared to those in the G-CSF group. Only afilmy layer of αSr-1-positive cells was identified on theendocardial side, and most of the infarction area was replacedby fibrotic tissue in the control group (Fig. 5A), whereas
surviving cardiomyocytes occupied the infarction area from theendocardium to the epicardium, preserving the wall thickness in
the G-CSF group (Fig. 5B).
G-CSF did not increase cardiac homing of GFP-positivebone marrow cells in the infarction border area. GFP-positive
cells were gathered in the infarction border area in hearts fromboth the control and G-CSF groups (Fig. 5C, D, and E). Most
GFP-positive cells were also CD68 positive (Fig. 5F), and some
GFP-positive cells were also α1A4 positive (Fig. 5G). Few
GFP-positive cells were αSr-1 positive (proportion of GFPpositive cells co-expressing αSr-1 was less than 1% in bothgroups).
3.6. Immunohistochemical evaluation of the carotid artery
after balloon injury
The effect of G-CSF treatment on reendothelialization of the
carotid artery was analyzed by vWF immunostaining 2 weeks
after balloon injury. G-CSF treatment induced reendothelialization of the injured artery. A nearly complete and continuous
lining of vWF-positive cells was observed along the lumen of
the injured artery in the G-CSF group (Fig. 6B) compared withpatchy and interrupted vWF-positive staining of the injured
artery in the control group (Fig. 6A).
To confirm the source of new endothelial cells and neointimalcells, we analyzed GFP staining of injured carotid artery fromrats transplanted with GFP bone marrow cells at different time
points after balloon injury. One week after injury, the elastic
membrane was extended by balloon inflation; some GFPpositive cells were gathered in the adventitia, clinging to theexternal elastic membrane (Fig. 6D). No GFP-positive cells wereidentified in normal artery without balloon injury (Fig. 6C). Two
weeks after injury, GFP-positive cells were distributed in apatchy pattern near the adventitia in the media and neointima ininjured artery in both the control and G-CSF groups (Fig. 6E–F).
Four weeks after injury, neointima formation and GFP-positiveFig. 6. Immunohistochemistry of carotid artery. (A, B) von Willebrand factor(vWF) staining 2 weeks after balloon injury. Interrupted vWF-positive cells
were observed in the control group (A). Continuous lining of vWF-positive cells
was observed along the lumen of the injured artery in the G-CSF group (B). (C,D) GFP staining 1 week after balloon injury with G-CSF treatment. No GFPpositive cells were identified in left normal carotid artery without balloon injury(C). Some GFP-positive cells were gathered in the adventitia (arrows) after
balloon injury (D). (E, F) GFP staining 2 weeks after balloon injury. GFPpositive cells were distributed in a patchy pattern near the adventitia in the media
and neointima in injured artery in both the control (E) and G-CSF groups (F). (G,H) GFP staining 4 weeks after balloon injury. (G) control group. (H) G-CSF
group. Neointima formation and GFP-positive cell distribution were similar to
those 2 weeks after injury. (I, J) GFP staining 4 weeks after balloon injury at
higher magnification. (I) Control group. (J) G-CSF group. Arrows show theinternal elastic lamina. Most GFP-positive cells were located in the adventitia of
the artery, and a few neointimal cells and new endothelial cells were GFP
positive.

Y. Li et al. / European Journal of Pharmacology 549 (2006) 98–106

cell distribution were similar to those 2 weeks after injury(Fig. 6G–H). Most GFP-positive cells were located in theadventitia of the artery, and a few neointimal cells and new
endothelial cells were GFP positive (Fig. 6I–J).
4. Discussion
In the present study, G-CSF treatment in rats prevented
cardiac remodeling and improved cardiac function after acute
myocardial infarction by preserving the number of cardiomyocytes in the infarction area. G-CSF treatment also accelerated
reendothelialization and inhibited neointima formation in rat
carotid artery after balloon injury.
Since Orlic et al. first reported the beneficial effects of G-CSF
on infarcted heart (Orlic et al., 2001), some studies haveconfirmed that G-CSF improves cardiac function after myocardial infarction (Adachi et al., 2004; Harada et al., 2005; Kawada
et al., 2004; Minatoguchi et al., 2004; Ohtsuka et al., 2004).
Most of these studies have shown that G-CSF improves cardiac
function by mobilizing bone marrow cells to the myocardium
and transdifferentiating these cells into heart cells. However, a
recent study (Harada et al., 2005) and the results of the present
study suggest that G-CSF acts directly on cardiomyocytes.
Harada et al. (2005) showed that cardiomyocytes and cardiac
fibroblasts express G-CSF receptor and that G-CSF exerts direct
protective effects on cardiomyocytes via activation of the G-CSF
receptor. In the present study, although G-CSF increased
neutrophils in peripheral blood approximately 16-fold andincreased total leukocytes approximately 4-fold, it did notincrease blood cytokine levels and did not increase cardiac
homing of bone marrow cells, suggesting that the increased
cardiac expression of various angiogenic factors in the infarction
border area may be the result of a direct effect of G-CSF on heart.
In our experiments, most bone marrow-derived cells in theinfarcted area were CD68-positive (macrophages) or α1A4
positive (vascular smooth muscle cells, myofibroblasts, or bonemarrow stromal cells). However, it was difficult to find bonemarrow-derived cardiomyocytes in the infarcted area. Thus thetransdifferentiation of bone marrow cells into cardiomyocytes
remains to be confirmed; if it did occur, the number of transdifferentiated cardiomyocytes may be too low to explain G-CSFinduced amelioration of cardiac remodeling and improvement offunction (Murry et al., 2004; Nadal-Ginard et al., 2003; Wagers
et al., 2002). Most surviving cardiomyocytes in the infarcted area
were not derived from bone marrow. Thus, these cardiomyocytes
may be associated with the direct protective effect of G-CSF
against cardiomyocytes death. Surviving cardiomyocytes in theinfarction area may play an important role in preventing cardiac
remodeling and in maintaining heart function.
Given previous reports that G-CSF mobilizes bone marrowcells to migrate to the infarcted heart (Kawada et al., 2004;
Minatoguchi et al., 2004; Ohtsuka et al., 2004), it is possiblethat mobilized bone marrow progenitors transdifferentiate into
vascular smooth muscle cells, potentially aggravating plaqueformation and restenosis in percutaneous coronary interventiontreated vessels (Simper et al., 2002). There have been reports ofclinical administration of G-CSF in patients with acute105

myocardial infarction who underwent percutaneous coronary
intervention (Hill et al., 2005; Kang et al., 2004; Petzsch et al.,
2004; Ripa et al., 2004; Valgimigli et al., 2005). The MAGIC
trial reported an unexpectedly high rate of in-stent restenosis ininfarct-related vessels after G-CSF treatment (Kang et al.,2004). Hill et al. (2005) reported potential adverse outcomes insome coronary artery disease patients after G-CSF administration. whereas other groups reported that G-CSF was safe anddid not induce coronary artery restenosis in patients with acute
myocardial infarction (Petzsch et al., 2004; Ripa et al., 2004;
Valgimigli et al., 2005). To investigate the effect of post-acute
myocardial infarction G-CSF administration on treated vessels
remodeling, rat carotid arteries were injured by balloon catheter
after acute myocardial infarction. G-CSF showed effect of
inducing reendothelialization and inhibiting neointima formation in injured artery after acute myocardial infarction.
Vascular injury leads to pathologic repair and remodelinginvolving vascular smooth muscle cell migration and proliferation(Pauletto et al., 1994; Schwartz, 1998). Endothelial cell loss is amajor contributing factor to the pathologic repair of injured
vessels. The origin of neointimal vascular smooth muscle cells
and new endothelial cells is controversial (Campbell et al., 2001;
Han et al., 2001; Hu et al., 2002a,b; Kong et al., 2004; Shoji et al.,
2004; Tanaka et al., 2003). Hu et al. (2002a) showed that smoothmuscle cells in the neointima and atherosclerotic lesions inallografts are not derived from bone marrow, whereas, somestudies (Campbell et al., 2001; Han et al., 2001) reported that bonemarrow-derived cells are recruited as a complementary source ofsmooth muscle-like cells, and few resident smooth muscle cells
are available to effect repair. In the present study, GFP-positive
cells were localized most in the adventitia of the injured artery inboth the control and G-CSF groups, in contrast to normal artery, inwhich no GFP-positive cells were identified. These results suggest that injury itself stimulates the migration of bone marrowcells to the injured region. Few neointimal smooth muscle cells or
new endothelial cells were GFP positive. It is possible that bonemarrow cells do not provide a main source of neointimal smoothmuscle cells and new endothelial cells. Thus, most bone marrowcells may not differentiate into vascular smooth muscle cells andmay not aggravate neointimal hyperplasia.
In conclusion, results of the present study suggest that GCSF treatment in rat model is safe and effective for treatment ofleft ventricle remodeling after acute myocardial infarction and it
does not aggravate vascular remodeling. Moreover, effects may
be mediated by direct action of cardiomyocytes rather thanregenerating myocardium.
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Development of Gene Silencing Pyrrole-Imidazole
Polyamide Targeting the TGF-␤1 Promoter for Treatment of
Progressive Renal Diseases
Hiroyuki Matsuda,* Noboru Fukuda,*†‡ Takahiro Ueno,* Yoshiko Tahira,*
Hirohito Ayame,§ Wen Zhang,§ Toshikazu Bando,§ Hiroshi Sugiyama,§ Satoshi Saito,†
Koichi Matsumoto,* Hideo Mugishima,† and Kazuo Serie储
Departments of *Internal Medicine and †Advanced Medicine and ‡Advanced Research Institute of the Sciences andHumanities, Nihon University, Tokyo Japan; §Department of Chemistry, Graduate School of Science, Kyoto University,
Kyoto, Japan; and 储Gentier Biosystems Incorporation, Tokyo, Japan
Pyrrole-imidazole (Py-Im) polyamides are nuclease-resistant novel compounds that inhibit gene expression by binding to theminor groove of DNA. A Py-Im polyamide that targets rat TGF-␤1 was designed as a gene-silencing agent for progressive renal
diseases, and the distribution and the effects of this polyamide on renal injury were examined in Dahl-salt sensitive (Dahl-S)
rats. For identification of transcription factor binding elements for activation of the rat TGF-␤1 gene, recombinant TGF-␤1
reporter plasmids were transfected into HEK-293 cells, and promoter activity was measured. Py-Im polyamide was designed
to the activator protein-1 binding site of the rat TGF-␤1 promoter. This Py-Im polyamide showed strong, fast, and specificbinding to the target DNA in gel mobility shift and Biacore assays. Py-Im polyamide significantly inhibited TGF-␤1 promoter
activity and expression of TGF-␤1 mRNA and protein in rat mesangial cells. Intravenously administered fluorescein-labeledpolyamide distributed to the kidney of rats. Py-Im polyamide significantly inhibited expression of TGF-␤1 mRNA and proteinin the renal cortex of Dahl-S rats and reduced the increase in urinary protein and albumin in Dahl-S rats independent ofchanges in blood pressure. These results indicate that Py-Im polyamide that targets TGF-␤1 will be a novel gene-silencing
agent for the TGF-␤1–associated diseases, including progressive renal diseases.
J Am Soc Nephrol 17: 422– 432, 2006. doi: 10.1681/ASN.2005060650

T

he use of reverse genetics to inactive genes can aid in
the elucidation of gene function and may also be helpful in the treatment of viral infections, cancer, and otherdiseases that involve aberrant gene expression. Gene function
can be inactivated at the DNA level by homologous recombination and at the RNA level by antisense DNA, ribozymes, andsiRNA. However, these compounds are degraded easily by
nucleases. Therefore, suitable chemical modifications or drugdelivery systems are necessary for their therapeutic applications.
Pyrrole-imidazole (Py-Im) polyamides first were identified
from duocarmycin A and distamycin A, which recognize andbind DNA with sequence specificities and are small synthetic
molecules that are composed of the aromatic rings of N-methylpyrrole and N-methylimidazole amino acids (1–3). Synthetic
Py-Im polyamides bind to specific nucleotide sequences in theminor groove of double-helical DNA with high affinity andblock binding of specific proteins. Therefore, Py-Im polyamides

Received June 22, 2005. Accepted November 2, 2005.
Published online ahead of print. Publication date available at www.jasn.org.
Address correspondence to: Dr. Noboru Fukuda, Department of Internal Medicine, Nihon University School of Medicine, Ooyaguchi-kami 30-1, Itabashi-ku,
Tokyo 173-8610, Japan. Phone: ⫹81-3-3972-8111; Fax: ⫹81-3-3972-8666; E-mail:fukudan@med.nihon-u.ac.jp
Copyright © 2006 by the American Society of Nephrology

may be useful tools in molecular biology medicine. Py-Im
polyamides are resistant to nucleases and do not require particular delivery systems (4). Various types of sequence-specificDNA-binding Py-Im polyamides have been developed to regulate gene expression by targeting the promoter regions ofenhancer and transcription factor binding elements (5). Bindingof enhancers to the major and minor grooves of DNA can beinhibited by the minor groove-binding Py-Im polyamides (6).
DNA recognition by Py-Im polyamides depends on a code ofside-by-side pairing of Py and Im in the minor groove; pairingof Im opposite Py (Im/Py) targets a G-C base pair, and Py/Im
targets a C-G base pair. Py/Py targets either a T-A or an A-T
base pair (3). The binding constants and specificity of Py-Im
polyamides are comparable to those of transcription factors.
Dickinson et al. (7) reported the genes of the HIV were silenced
by Py-Im polyamides to their regulatory sequences.
A number of studies have identified TGF-␤1 as a criticalfactor in kidney diseases such as glomerulosclerosis (8) andmesangioproliferative glomerulonephritis (9,10). TGF-␤1 stimulates the proliferation of mesangial cells and the production of
extracellular matrix (11,12). TGF-␤1 has been reported to induce Thy-1–associated nephritis (13), diabetic nephropathy(14), and interstitial nephritis associated with obstructive nephropathy (15) in rats. TGF-␤1 was reported recently to induce
epithelial-mesenchymal transformation in renal tissue, which
ISSN: 1046-6673/1702-0422

J Am Soc Nephrol 17: 422– 432, 2006

Pyrrole-Imidazole Polyamide to TGF-␤1 Prevents Renal Injury

plays a critical role in the pathogenesis of nephritis (16). Thus,
TGF-␤1 induces nephrosclerosis and interstitial nephropathy
by its effects on mesangial cell growth, extracellular matrix
formation, and epithelial-mesenchymal transformation. TGF-␤1 isexpressed highly in various animal models of progressive renal
disease such as hypertensive renal sclerosis, diabetic nephropathy, and focal renal sclerosis (17–20). Dahl salt-sensitive(Dahl-S) rats show glomerulosclerosis, which is known to be
associated with TGF-␤1 (17). To develop a novel gene-silencing
agent for the treatment of progressive renal diseases, we designed and synthesized a Py-Im polyamide targeting TGF-␤1
and examined its distribution and effects on TGF-␤1 expression
and renal injury in Dahl-S rats.

Materials and Methods
This study conformed to the standards of the Guide for the Care andUse of Laboratory Animals published by the National Institutes of Health(NIH Publication No. 85-23, revised 1996).

Synthesis of Polyamide Targeting Rat TGF-␤1

Polyamide targeting rat TGF-␤1 (polyamide) was designed to span
the boundary of the activator protein-1 (AP-1) binding site (⫺2303 to⫺2297) of the TGF-␤1 promoter (Figure 1A). Numbering refers to thestart of the open reading frame as ⫹1 (21). Mismatch polyamide (mismatch) was designed not to bind transcription binding sites of thepromoter. Polyamides were synthesized according to previously described methods (22) (Figure 1B).

DNA Binding Assays
Fluorescein-labeled DNA corresponding to ⫺2289 to ⫺2310 including the AP-1 binding site and 2-bp mutated DNA were synthesized forgel mobility shift assays. One micromole of the DNA was incubatedwith 50 ␮M polyamide or mismatch for 1 h at 37°C. Moreover, fluo-

423

rescein-labeled appropriate DNA was incubated with polyamide or
mismatch, and then it was incubated with AP-1 extract (Promega,
Madison, WI) again. Those resulting complexes were separated byelectrophoresis and visualized by luminescent image analyzer LAS3000 (Fujifilm, Tokyo, Japan).
Kinetics of binding of polyamide and mismatch to target sequence
were assessed by surface plasmon resonance technique with a molecular interaction model. Biotin double-strand DNA was immobilized to
streptavidin-functionalized sensorchip SA (Biacore, Uppsala, Sweden).
Measurements of binding curves of polyamide or mismatch to the
DNA and data processing were performed on a Biacore X (Biacore)according to recommended protocols.

Construction and Activity of Rat TGF-␤1 Promoter Plasmidand Effect of Polyamide on Promoter Activity
Rat TGF-␤1 promoter cloned into the KpnI site of the pGL3-basic
vector was a gift from Dr. Kyoung Lim (Ajou University School of
Medicine, Suwon, Korea). The promoter deletion mutant (⫺2676 to 72)
was cleaved with KpnI and BpuI102I and self-ligated to the promoterpGL3-basic vector fragment with DNA blunting kit (TaKaRa; Otsu,
Shiga, Japan). Other deletion mutants were generated by cleaving with
KpnI and SacI, XhoI, PvuII, Van91I, NsiI, BstPI, SacII, or Bsu36I to obtainpromoter terminating at ⫺2424, ⫺2153, ⫺1715, ⫺1474, ⫺1327, ⫺1104,⫺742, and ⫺513 to 72.
HEK-293 cells were seeded onto 24-well plates and grown in DMEM
(Sigma-Aldrich, St. Louis, MO) with 20% calf serum. At 70 to 90%
confluence, a mixture of reporter plasmid (1 ␮g/well) and phRG-TK
vector (0.01 ␮g/well; Promega) as an internal control was used totransfect cells with Lipofectamine2000 (Invitrogen, Carlsbad, CA) as
described previously (23) and then changed to flesh medium in thepresence or absence 1.0 mM phorbol 12-myristate 13-acetate (PMA;
Sigma-Aldrich). Cells were incubated for an additional 24 h andscraped into 100 ␮l of cold lysis buffer (PBS [pH 7.4] and 1 mM PMSF).
Luciferase activity was measured with a Dual-luciferase reporter assay

Figure 1. Target sequence and structure of the synthetic pyrrole-imidazole (Py-Im) polyamide that targets the rat TGF-␤1 promoter.
(A) The polyamide was designed to bind to the rat TGF-␤1 promoter adjacent to the activator protein-1 (AP-1) binding site (⫺2303
to ⫺2297) in the ⫺2424 to ⫺2153 region of the deletion mutant. (B) Structure of the rat TGF-␤1–specific polyamide and mismatchpolyamide (mismatch). Mismatch was designed not to bind transcription binding sites of the promoter. Polyamides weresynthesized by solid-phase methods and were purified by HPLC (0.1% AcOH/CH3CN 0 to 50% linear gradient, 0 to 40 min, 254
nm through a Chemcobond 5-ODS-H column).

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Journal of the American Society of Nephrology

system (Promega) and a TD-20/20 luminometer (Turner Designs,
Sunnyvale, CA) (24).
For evaluation of the effect of polyamide on promoter activity, HEK293 cells were transfected with rat TGF-␤1 promoter plasmid andincubated with 0.1 or 1.0 ␮M polyamide or 1.0 ␮M mismatch in thepresence of 1.0 ␮M PMA for 24 h. Luciferase activity then was measured.

J Am Soc Nephrol 17: 422– 432, 2006

Oriental Yeast, Tokyo, Japan) ad libitum for 2 wk. One milligram ofpolyamide or mismatch was dissolved in 100 ␮l of DMSO plus 100 ␮lof H2O and injected via the tail veins of HS rats every 2 d for 2 wk (total
7 mg of polyamide per rat). Control rats received injections of 100 ␮l of
DMSO plus 100 ␮l of H2O (Figure 2). Systolic BP (SBP) was measured
by the tail-cuff method. Urine was collected in metabolic cages for 24 hevery week. Urinary protein and albumin excretion were determinedwith a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

Distribution of Fluorescein-Labeled Polyamide In Vitro and
In Vivo
Glomeruli were isolated from the kidneys of 4-wk-old Wistar-Kyoto
rats (SHR Corp., Funabashi, Chiba, Japan) by a graded-sieve technique
as described previously (25). Mesangial cells were isolated from explants of whole glomeruli according to the differential growth capacities of glomerular epithelial and mesangial cells.
Mesangial cells were seeded onto plates and grown in RPMI-1640
medium (Sigma-Aldrich) with 10% FCS. After 24 h, cells were incubated with 2 ␮M fluorescein-labeled polyamide in RPMI-1640 medium
for 2 h. Cells were washed, and fresh RPMI-1640 was added. After 22 h,cells were viewed at ⫻200 under live cell conditions and then fixed in
4% paraformaldehyde for 10 min. Nuclei were stained with Hoechst
33342 (Invitrogen) and viewed again.
Five milligrams of fluorescein-labeled polyamide was injected into
Wistar rats (250 g body wt) via the tail vein. After 24 h, the kidneys,aorta, heart, and brain were removed, and frozen specimens were madeand viewed at ⫻200. Subsequently, 50 to 100 mg of each tissue washomogenized in DMSO (Sigma-Aldrich). Each sample was centrifuged
and freeze-dried. The precipitate was reconstituted with 50 ␮l of N,Ndimethylformamide (Sigma-Aldrich). Urine was also collected for 24 h
in metabolic cages. Fluorescein-labeled polyamide in 20 ␮l of N,Ndimethylformamide and 20 ␮l of urinary samples were subjected to
HPLC (0.01% CHO2NH4/CH3CN 0 to 100% linear gradient from 0 to 30
min through a Chemcobond 5-ODS-H column [Chemco Scientific,
Osaka, Japan]). Fluorescence was measured with a fluorescence detector (FP-2020 Plus; Nihon Bunko, Tokyo, Japan).

In Vivo Experimental Design
Seven-week-old male Dahl-S rats (CLEA Japan, Tokyo, Japan) wereused in this study. Rats were divided into two groups and were fed
0.3% NaCl (low-salt [LS]) or 8% NaCl (high-salt [HS]) diet (both from

Determination of mRNA Expression
For in vitro experiments, mesangial cells were incubated with 0.1 or
1.0 ␮M polyamide or 1.0 ␮M mismatch in the presence of 1.0 ␮M PMA
in RPMI-1640 medium with 0.5% FCS for 8 h. Total RNA was isolated
and reverse-transcribed as described previously (18). For in vivo experiments, total RNA was isolated from 20 mg of renal cortical tissue from
Dahl-S rats and reverse-transcribed.
Real-time quantitative PCR was performed with cDNA diluted fourtimes and TaqMan Universal Master Mix and an ABI 7500 sequence
detector (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Assay-on-Demand primers and probes (TGF␤1, Rn00572010-m1; connective tissue growth factor [CTGF],
Rn00573960-g1; collagen type 1 ␣1, Rn00801649-g1; fibronectin,
Rn00569575-m1 and TaqMan Rodent GAPDH control reagents) werepurchased from Applied Biosystems. Real-time PCR data were analyzed with a standard curve. Correlation coefficients for the standard
curves all were ⬎0.90.

Measurement of TGF-␤1 Protein
TGF-␤1 protein levels in urine and conditioned medium were determined by enzyme immunoassay (TGF-␤1 Emax ImmunoAssay System;
Promega) as described previously (26). Mesangial cells were treatedwith 0.1 or 1.0 ␮M polyamide or 1.0 ␮M mismatch in the presence of 1.0
␮M PMA for 24 h. Conditioned medium was collected and diluted with
TGF-␤1 sample buffer. Because this assay detects only active TGF-␤1
protein, each sample was acidified to convert latent TGF-␤1 to theactive form. Urine samples from Dahl-S rats that were fed an HS dietfor 2 wk were collected for 24 h and diluted 1:1000 in sample buffer.

Figure 2. Experimental protocols for administration of Py-Im polyamide that targets TGF-␤1 to Dahl-salt sensitive (Dahl-S) rats.

J Am Soc Nephrol 17: 422– 432, 2006

Pyrrole-Imidazole Polyamide to TGF-␤1 Prevents Renal Injury

TGF-␤1 Immunofluorescence

Unstained 3-␮m-thick paraffin sections of renal cortex were deparaffinized, hydrated, and heated for antigen unmasking. Sections were
blocked with Serum Blocking Reagent G (R&D Systems, Minneapolis,
MN) for 15 min. The slides then were incubated with diluted primary
antibody (mAb to TGF-␤1; R&D Systems) for 3 h at room temperature,
washed in PBS, and incubated with fluorescein-conjugated polyclonalgoat anti-mouse antibody (Dako, Carpinteria, CA) for 30 min at room
temperature. After being washed in PBS, sections were incubated with
Hoechst 33342 and viewed at ⫻200.

Statistical Analyses
Values are reported as mean ⫾ SEM. A t test was used for unpaired
data. Two-way ANOVA and Duncan multiple range tests were also
used. P ⬍ 0.05 was considered statistically significant.

425

with deletion of ⫺2424 in the presence of PMA. There was no
significant difference in promoter activity between the deletionof ⫺2153 and ⫺2424 in the absence of PMA, and deletion of⫺2153 was not affected by PMA. These results suggest that the⫺1104 to ⫺742 region contains the basal transcriptional regulatory element and the 2.5-kb transcription start site and thatthe ⫺2424 to ⫺2153 region contains a highly sensitive PMA
response element.
We searched for transcription factor binding sites between⫺2424 and ⫺2153 with TFSEACH (http://mbs.cbrc.jp/research/db/TFSEARCH.html) (30). This region contained an
AP-1 binding site (⫺2303 to ⫺2297). Polyamide was designed
to bind to this region (Figure 1A).

Results

Binding and Specificity of Polyamide to Double-Stranded
DNA and Inhibition of AP-1 Complex Binding

For identification of transcription factor elements that are
responsible for activation of the rat TGF-␤1 gene, a rat TGF-␤1
promoter-pGL3-basic luciferase reporter chimeric plasmid wascreated (Figure 3). The start site of the rat 2.5-kb TGF-␤1 transcript (892 nucleotides upstream of ATG) (24) is at a positionhomologous to that of human TGF-␤1 (858 and 852 nucleotides
upstream of ATG) (27) and mouse (856 nucleotides upstream of
ATG) (28). A comparison of this conserved region with that ofother species revealed 91.4% overall identity with the mousesequence and 81.7% identity with the human sequence (29).
Deletion of ⫺742 markedly reduced promoter activity compared with deletion of ⫺1104 in the presence or absence ofPMA. Deletion of ⫺2153 reduced promoter activity compared

Gel mobility shift and Biacore assays allow for the determination of the binding affinity and specificity of polyamides fortarget DNA. Polyamide bound the appropriate 21-bp doublestranded DNA but did not bind the 2-bp mutated DNA,
whereas mismatch did not show binding to appropriate DNA(Figure 4A). As shown in Figure 4B, a single mobility band wasobserved when the DNA were incubated with AP-1 or treatment with mismatch. Polyamide inhibited AP-1 binding totarget DNA.
Figure 4, C and D, shows kinetics of polyamide and mismatch bindings with target double-strand DNA obtained fromfitting resulting sensorgrams (Biacore assay). Fast binding ofpolyamide to the target sequence occurred relative to that of
mismatch to allow match binding to reach equilibrium at highRat TGF-␤1 Promoter Analysis

Figure 3. Deletion analysis of TGF-␤1 promoter activity. Bars indicate the upstream region of the TGF-␤1 gene. Bold arrow
indicates the start site for the 2.5-kb transcript as identified previously in mice. Open arrows indicate the start sites for the 1.9-kb
transcript and 1.4-kb transcript in rats. HEK-293 cells were transfected with recombinant plasmids and stimulated with 1.0 ␮Mphorbol 12-myristate 13-acetate (PMA). The transcription factor binding sites between ⫺2424 and ⫺2153 were searched withTFSEACH. Narrow arrow indicates the consensus AP-1 binding site at ⫺2303 to ⫺2297 upstream of ATG. *NS versus control with
PMA.

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Journal of the American Society of Nephrology

J Am Soc Nephrol 17: 422– 432, 2006

Figure 4. (A) Gel mobility shift assay. Fluorescein-labeled DNA corresponding to the AP-1 binding site (5⬘-GGAAACTTGAGTCAGGTGGGC) and 2-bp mutated DNA (5⬘-GGAAAGGTGAGTCAGGTGGGC) were synthesized and incubated with Py-Im
polyamides for 1 h at 37°C and loaded onto a 20% polyacrylamide gel. Lane 1, single-stranded DNA; lane 2, double-stranded
DNA; lane 3, DNA with polyamide targeting TGF-␤1 (polyamide); lane 4, 2-bp mutated DNA with polyamide; lane 5, DNA withmismatch polyamide (mismatch). (B) After the fluorescein-labeled appropriate DNA was incubated with polyamides, theresulting complexes were incubated with AP-1 extract for 30 min at 37°C and loaded onto a 4% polyacrylamide gel. Lane 1,
double-stranded DNA; lane 2, DNA with AP-1; lane 3, DNA with polyamide; lane 4, DNA with polyamide and AP-1; lane 5, DNA
with mismatch; lane 6, DNA with mismatch and AP-1. Arrows indicate shifted complex. (C and D) Typical surface plasmon
resonance sensorgrams for the interaction of polyamide and mismatch with the DNA immobilized on the surface of a sensor chip
SA (Biacore assays). The binding response in resonance units is changed with time. The data are fitted to Langmuir double
molecular interaction model with mass transport. All experiments were done using the same DNA immobilization level sensor
chips in HBS-EP buffer (0.01 M HEPES [pH 7.4], 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20) with 0.1% DMSO at 25°C.
Concentrations for polyamide are 0, 1, 5, 10, 20, 30, 50, 75, 100, and 150 nM from lowest to top curves. Concentrations for mismatchare 0, 0.1, 0.2, 0.6, 0.8, 1, 5, 7.5, and 10 ␮M from lowest to top curves.
concentrations. Dissociation equilibrium constant (KD) was
1.43 ⫻ 10⫺9 and 9.76 ⫻ 10⫺7 M for the interaction of polyamide
and mismatch with the DNA, respectively (Table 1).

manner. Mismatch did not affect luciferase activity or expression of TGF-␤1 mRNA and protein in response to PMA.

Effect of Polyamide on TGF-␤1 Promoter Activity andExpression of TGF-␤1

Distribution of Polyamide In Vitro and In Vivo

Treatment with PMA significantly increased luciferase activity in HEK-293 cells that were transfected with rat TGF-␤1
promoter plasmid. Polyamide was significantly and in a concentration-dependent manner decreased luciferase activity
stimulated by PMA (Figure 5A). In mesangial cells, polyamide
decreased expression of TGF-␤1 mRNA (Figure 5B) and protein(Figure 5C) stimulated by PMA in a concentration-dependent

The distribution of fluorescein-labeled polyamide in mesangial cells after a 24-h incubation is shown in Figure 6A. Fluorescein-labeled polyamide was present in all nuclei by 96 h(data not shown).
The in vivo distribution of fluorescein-labeled polyamide in
the kidney, aorta, heart, and brain 24 h after intravenous injection is shown in Figure 6B. Fluorescein-labeled polyamide
strongly localized in nuclei of nephrotubuli and glomeruli.

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J Am Soc Nephrol 17: 422– 432, 2006

427

Table 1. Kinetic constants for polyamide binding and mismatch bindinga

Polyamide binding
Mismatch binding

KD (M)ka (1/Ms)kd (1/s)Specificity

1.43 ⫻ 10⫺99.76 ⫻ 10⫺7

5.97 ⫻ 105
7.68 ⫻ 102

8.54 ⫻ 10⫺4
7.49 ⫻ 10⫺4685aKinetic constants were calculated from the surface plasmon resonance sensorgrams for the interaction of polyamide andmismatch with the DNA immobilized on the surface of a sensor chip SA (Biacore assays, Figure 4C). Polyamide, pyrroleimidazole polyamide targeting rat TGF-␤1 promoter; mismatch, mismatch polyamide; KD, dissociation equilibrium constant;ka, association rate constant; kd, dissociation rate constant. Specificity is defined as KA (polyamide binding)/KA (mismatchbinding).

Figure 5. (A) Effect of polyamide targeting TGF-␤1 on rat TGF-␤1 promoter activity. Rat TGF-␤1 promoter plasmid with luciferase
was transfected into HEK-293 cells, and cells were incubated with polyamide in the absence or presence of 1.0 ␮M PMA for 24 h.
Promoter activity was measured with a dual-luciferase reporter assay system. (B) Effect of polyamide on expression of TGF-␤1
mRNA and protein in mesangial cells. Mesangial cells from Wistar-Kyoto rats were incubated with polyamide in the presence orabsence of 1.0 ␮M PMA for 8 h. The abundance of TGF-␤1 mRNA was determined by real-time PCR analysis. (C) Effect ofpolyamide on expression of TGF-␤1 protein in mesangial cells. Mesangial cells were incubated with polyamide in the presence orabsence of 1.0 ␮M PMA for 24 h. Conditioned medium was collected, and total (latent and active) TGF-␤1 protein was measured
by ELISA. Values are mean ⫾ SEM (n ⫽ 8). *P ⬍ 0.05 versus treatment with PMA alone.
Fluorescein-labeled polyamide also localized in nuclei of aortic
midlayer smooth muscle. Fluorescein-labeled polyamide did
not distribute considerably in heart or brain.
HPLC analysis of fluorescein-labeled polyamide in urine,
kidney, aorta, heart, and brain is shown in Figure 6C. Fluorescein-labeled polyamide was clearly detected in urine, kidney,
and aorta but not in heart or brain.

Effect of Polyamide on Urinary Protein and TGF-␤1
Expression in Dahl-S Rats
Intravenous administration of polyamide or mismatch did
not affect the body weights of Dahl-S rats (Figure 7A). SBP was
remarkably increased in HS rats compared with LS rats. Polyamide or mismatch did not affect SBP (Figure 7B). Both urinary
protein and albumin were significantly increased in HS rats
compared with levels in LS rats. Treatment with polyamide
significantly reduced the increased urinary protein and albumin in HS rats. Mismatch did not affect urinary protein oralbumin levels (Figure 7, C and D).

The effects of polyamide on the expression of TGF-␤1, CTGF,
collagen type 1 ␣1, and fibronectin mRNA in the renal cortex of
Dahl-S rats are shown in Figure 8, A through D. Expression ofthese mRNA was significantly higher in HS rats than in LS rats.
Treatment with polyamide significantly decreased expressionof these mRNA in HS rats. Mismatch did not affect expressionof these mRNA.
The effects of polyamide on urinary excretion of TGF-␤1
protein in Dahl-S rats is shown in Figure 8E. Urinary excretionof TGF-␤1 protein was significantly higher in HS rats than in LS
rats. Treatment with polyamide significantly decreased urinary
excretion of TGF-␤1 protein in HS rats. Mismatch did not affecturinary excretion of TGF-␤1 protein in HS rats.

Immunofluorescence of TGF-␤1 in Renal Cortex

Immunofluorescence of TGF-␤1 in the renal cortex of Dahl-S
rats is shown in Figure 9. TGF-␤1 staining in glomeruli andnephrotubuli in HS rats was stronger than that in LS rats.

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Figure 6. Distribution of fluorescein-labeled Py-Im polyamide in vitro and in vivo. (A) Mesangial cells from Wistar-Kyoto rats wereincubated with fluorescein-labeled polyamide and examined by fluorescence microscopy under live cells condition and then fixedand viewed again. Nuclei were stained with Hoechst 33342 (blue). (B) Five milligrams of fluorescein-labeled polyamide wasinjected into Wistar rats intravenously. Twenty-four hours after injection, the kidneys, aorta, heart, and brain were removed, andfrozen specimens were made. (C) HPLC analysis for the fluorescein-labeled polyamide in the urine, renal cortex, aorta, heart, andbrain. Fluorescein-labeled polyamide was subjected into HPLC. Arrow indicates fluorescein-labeled polyamide positive control.
Magnification, ⫻200 in A and B.

Treatment with polyamide considerably reduced TGF-␤1 staining in glomeruli and nephrotubuli in HS rats. Mismatch did notaffect TGF-␤1 staining.

Discussion
Synthetic polyamides have been reported to bind target sites
within nucleosomes and may influence chromatin structure(1,2). Because polyamides can be readily designed and synthesized to target any sequence of interest, they may be useful instudies of gene function and perhaps in gene therapy. Polyamides are removed from duplex DNA during transcription;
therefore, inhibition of transcription of coding regions is difficult. Polyamides also can inhibit the formation of mRNA in
mammalian cells by alkylation of coding regions (31,32). Extensive inhibition of gene expression by alkylating polyamides toa coding region may provide a powerful tool for the genesuppression therapy of viral infections and cancers. Inhibitionof gene expression by nonalkylating polyamides that target
regulatory sequences is more physiologically relevant than thatby alkylating polyamides because this strategy suppresses theenhancing effect of transcription factors and preserves the baseline expression of the target gene. Thus, suppression of gene
expression by nonalkylating polyamides may be more efficientin the treatment of chronic nonmalignant diseases. Studies ofpolyamides have recently focused on the structural characterization of transcription factor–DNA complexes within promoter sequences.
To design a polyamide that targets rat TGF-␤1, we analyzed
the rat TGF-␤1 promoter structure and PMA-stimulated activity in promoter deletion mutants. Positive-regulatory elementsthat were stimulated by PMA were found at bases ⫺2424 to⫺2153, which contain an AP-1 binding site. AP-1 elements
respond to AP-1 transcription factors such as Jun homodimers
or Fos/Jun heterodimers, PMA, angiotensin II, and v-Src. Stim-

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Pyrrole-Imidazole Polyamide to TGF-␤1 Prevents Renal Injury

429

Figure 7. Changes in body weight (A), systolic BP (B), urinary protein (C), and urinary albumin (D) in Dahl-S rats before and after
treatments with Py-Im polyamide targeting the TGF-␤1 promoter (polyamide). Dahl-S rats were fed a 0.3% NaCl (low salt; E) or
8% NaCl (high salt; 䡺) diet. Dahl-S rats that were fed a high-salt diet also received an intravenous injection of 1 mg of polyamide(F) or mismatch polyamide (mismatch; ‚) every 2 d (total 7 mg) for 2 wk. *P ⬍ 0.05 versus Dahl-S rats that were fed a high-salt
diet without polyamide.
ulation of TGF-␤1 promoter activity occurs by binding to theAP-1 element (33,34). Polyamide targeting TGF-␤1 was designed not to cover AP-1 consensus sequences but spans theboundary of AP-1 binding site with the intention of obtaining
specificity to the promoter. This polyamide showed strong, fast,and specific binding to the target DNA in gel mobility shift andBiacore assays. Fluorescein-labeled polyamide distributed sufficiently in the kidney without any delivery systems and localized in the nuclei of cultured cells for long periods of time. Bestet al. (35,36) reported that fluorescein-polyamide conjugates
exhibit good nuclear uptake in a wide variety of cell lines. In
our in vivo experiments, polyamide administered intravenously
was delivered sufficiently to the kidney and aorta and was
localized to nuclei. Thus, this polyamide bound to target DNA
in vitro and in vivo. HPLC analysis also confirmed that thepresence of polyamide administered intravenously was in theurine, kidney, aorta, liver, and lung. Polyamide was not delivered in heart and brain. It is possible that the differentiatedcardiac tissue does not express TGF-␤1 in normal heart, andthis polyamide does not pass through the blood-brain barrier.
Nucleic acid medicines such as antisense DNA, ribozymes,
and decoy have been developed as gene-silencing agents. De-

coys, in particular, inhibit the binding of target transcription
factors in a manner similar to polyamides. However, because
these agents are degraded easily by nucleases, they requiredrug-delivery systems for sufficient distribution into organs.
Because polyamides are completely resistant to nucleases andcan be delivered into organs without delivery systems, polyamides will be more feasible gene-silencing medicines.
Dahl-S rats are useful models of human progressive renal
disease. Dahl-S rats develop severe hypertension, glomerulosclerosis with thickening of capillary artery walls, and interstitial fibrosis in response to salt loading. Tamaki et al. (17) reported increased levels of TGF-␤1, plasminogen activator
inhibitor-1, fibronectin, and collagen type 1 in the renal cortex
of Dahl-S rats. In these experiments, expression of TGF-␤1
mRNA and protein was enhanced in the renal cortex of HS rats.
Treatment with polyamide significantly inhibited this increased
expression of TGF-␤1 mRNA and protein. Expression of mRNAfor CTGF, a potent growth factor that stimulates the proliferation of mesenchymal cells, including mesangial cells, and induces the production of extracellular matrix downstream of
TGF-␤1 signaling (37–39), was inhibited by polyamide. However, when cell cultures were treated with exogenous TGF-␤1,

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Figure 8. (A through D) Effect of Py-Im polyamide targeting the TGF-␤1 promoter (polyamide) on the expression of TGF-␤1,connective tissue growth factor (CTGF), collagen type 1 ␣1, and fibronectin mRNA in the renal cortex from Dahl-S rats. Dahl-S
rats were fed a 0.3% NaCl (low salt) or 8% NaCl (high salt) diet. Dahl-S rats that were fed a high-salt diet also received an
intravenous injection of 1 mg polyamide or mismatch every 2 d (total 7 mg) for 2 wk. Total RNA then was extracted from the renal
cortex. The abundance of all mRNA was determined by real-time PCR. (B) Effect of polyamide on the urinary excretion TGF-␤1
protein in Dahl-S rats. Two weeks after treatment with polyamide, urine was collected for 24 h. TGF-␤1 protein levels were
determined by ELISA. *P ⬍ 0.05 versus Dahl-S rats that were fed a high-salt diet without polyamide.

Figure 9. Immunofluorescence analysis of TGF-␤1 in the renal cortex of Dahl-S rats that were treated with Py-Im polyamide
targeting the TGF-␤1 promoter (polyamide). Dahl-S rats were fed a 0.3% NaCl (low salt) or 8% NaCl (high salt) diet. Dahl-S ratsthat were fed a high-salt diet also received an intravenous injection of 1 mg polyamide or mismatch every 2 d (total 7 mg) for 2
wk. Sections were generated and incubated with an mAb to TGF-␤1 and a fluorescein-conjugated polyclonal goat anti-mouse
antibody. Nuclei were stained with Hoechst 33342 (blue). Magnification, ⫻200.

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Pyrrole-Imidazole Polyamide to TGF-␤1 Prevents Renal Injury

polyamide did not block CTGF production. Because the CTGF
promoter also has an AP-1 site, this finding further supports the
specificity of polyamide for the TGF-␤1 promoter. Expressionof downstream extracellular matrix components such as collagen type 1 and fibronectin were also increased in the renal
cortex of HS rats. This, too, was suppressed significantly bytreatment with polyamide. In addition, the increased urinary
protein and albumin in HS rats was significantly decreased bytreatment with polyamide without a reduction in BP. Thesefindings suggest that the polyamide ameliorated the renal damages by inhibition of TGF-␤1 in organs and extracellular matrix
components in the renal cortex of Dahl-S rats.
In our study, although an HS diet did not induce considerable renal damage in Dahl-S rats during a 2-wk period, treatment with polyamide significantly suppressed TGF-␤1 immunofluorescence in the glomeruli and nephrotubuli of HS rats.
Dahly et al. (40) injected anti–TGF-␤ antibody into salt-loaded
Dahl-S rats every day for 2 wk and found a significant decrease
in BP, proteinuria, and degree of glomerulosclerosis and renal
interstitial fibrosis. Polyamide completely inhibited the increases in proteinuria and albuminuria in salt-loaded Dahl-S
rats, along with suppression of TGF-␤1 staining in glomeruli
and nephrotubuli. These findings suggest that polyamide may
be feasible gene-silencing agent for the treatment of progressive
renal diseases.
We believe that polyamides will be important gene-silencing
agents in the postgenome era. Polyamides can be readily designed and synthesized to target any gene. In the future, controlof gene expression by polyamides may ameliorate diseases thatare not treatable with current medicines.

Acknowledgments

8.

9.

10.

11.

12.

13.

14.

15.

This work was supported in part by Grants-in-Aid from the Japanese
Ministry of Education, Science, Sports, and Culture of Japan (15590863).

16.

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