# AGEs and Glucose Levels Modulate Type I and III Procollagen mRNA Synthesis in Dermal Fibroblasts Cells Culture

Authors: Serban Iren Andreea; Costache Marieta; Dinischiotu Anca
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

In the dermis, fibroblasts play an important role in the turnover of the dermal extracellular matrix. Collagen I and III, the most important dermal proteins of the extracellular matrix, are progressively altered during ageing and diabetes. For mimicking diabetic conditions, the cultured human dermal fibroblasts were incubated with increasing amounts of AGE-modified BSA andD-glucose for 24 hours. The expression of procollagen α2(I) and procollagen α1(III) mRNA was analyzed by quantitative real-time PCR. Our data revealed that the treatment of fibroblasts with AGE-modified BSA upregulated the expression of procollagen α2(I) and procollagen α1(III) mRNA in a dose-dependent manner. High glucose levels mildly induced a profibrogenic pattern, increasing the procollagen α2(I) mRNA expression whereas there was a downregulation tendency of procollagen α1(III) mRNA.


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## Body

## 1. Introduction

Elevated levels of blood glucose give
birth to a vicious cycle of metabolic disturbances within the
intracellular and extracellular environment and lead to a broad array of
diabetes complications.  In the hyperglycemic milieu activation of the aldose
reductase pathway (AR),  protein kinase C (PKC), especially theβ-isoform, and generation of advanced glycation end
products (AGEs) can be noticed [1]. In ageing, the nonenzymatic
glycosylation of proteins or Maillard reaction occurs, which is a consequence of  the elevated levels of glucose and is accelerated in
diabetes [2].  The glycation reaction
starts when the amino groups of a protein react nonenzymatically
with glucose to forma Schiff base, stabilized through Amadori
rearrangement, and it represents an early temporary step in the glycation process.
In the advanced stage, complex reactions that lead to the formation
of AGEs occur [3]. In ageing and diabetes, the
AGEs levels of long life proteins of the extracellular matrix are increased [4]. These products cause
cellular dysfunctions by multiple mechanisms, including receptor-independent
and receptor-dependent processes, and can directly influence the structural
integrity of the vessel wall and underlying basement membranes
through excessive cross-linking of matrix molecules such as collagen and  through disruption of matrix-matrix and matrix-cell interactions [5]. The formation of
AGEs in skin collagen favors cross-linking reactions, resulting in decreased degradability and impaired dermal regeneration [6]. AGEs can bind to fibroblast
cell membranes and may contribute to the progression of skin ageing [7].The receptor for AGE (RAGE) is a multiligand receptor belonging to the immunoglobulin superfamily found in a widerange of cell types, including endothelial cells (EC), mononuclear phagocytes (MP), lymphocytes, vascular smooth muscle (VSMC), and neurons [8]. Recently, it was demonstrated that RAGE is highly expressed in skin and upregulated by
AGEs and tumor necrosis factor-alpha (TNF-α) [9].Collagen type I and III are the
major structural components of the dermal extracellular matrix (ECM),
representing over 70% and, respectively, 15% of skin dry weight and providing the dermis with tensile strength and stability [10]. Collagen metabolism is a
complex process requiring a balance between synthesis and degradation through the action of cytokines and matrix metalloproteinase (MMPs).
The most important profibrogenic cytokine is the transforming growth
factor-beta (TGF-β1) [11].The aim of this study was to investigate the influence of high glucose concentration and AGEs mimicking diabetic conditions onprocollagen 1α2 (PCol 1 α2) and procollagen 3α1 (PCol 3 α1) genes expression in cultured
human skin fibroblasts.


## 2. Materials and Methods

### 2.1. Preparation and Identification of AGE-Modified BSA

Solutions of 1.6 MD-glucose (Sigma-Aldrich company) and 100 mg/mL bovine serum albumin (BSA Sigma-Aldrich, RIA grade, fraction V) have been coincubated insterile PBS 10 mM,  pH 7.4 (Gibco) for 6 weeks under aerobic conditions at 37˚C, in the presence of 3 mM NaN3 (Merck) to prevent bacterial growth [12]. Controls with BSA solution only were simultaneously kept
under the same conditions.  After 6
weeks, the free sugar was removed by dialysis against 10 mM PBS, pH 7.4 for 48 hours. The protein concentration (70 mg BSA/mL)
was determined by Bradford method [13]. The formation of AGE-modified BSA (AGE-BSA) was analyzed by
fast protein liquid chromatography (FPLC), SDS-PAGE,
and by fluorescence spectroscopy.Fast protein liquid chromatography
An FPLC automated system (ÄKTA FPLC-Amersham Pharmacia Biotech)
with a size exclusion column (Superdex 200 HR 10/30) was used for separation of glycated and unglycated BSA. The samples of 1.55 μg /μL prepared in 10 mM PBS pH 7.4 were eluted in 5% acetonitrile (Merck) and 10 mM PBS pH
7.4, at a constant flow rate of 0.8 mL/min, and their absorption at 280 nm was automatically recorded.Gel electrophoresis
AGE-BSA cross-linking and aggregation was investigated by 7.5% SDS-PAGE) with Mini Protean Bio-Rad equipment [14] and protein bands were stained by Coomassie Brilliant Blue R-250 (Sigma-Aldrich).AGE-BSA linked fluorescence assay
The fluorescence detection of Maillard compounds was done using the parameter λex370 nm/λem440 nm and for pentosidine-like products, λex335 nm/λem385 nm was used. The fluorescence emission spectra between 380 and 600 nm (370 nm excitation) and between 350 and 500 nm (335 nm excitation) were scanned using a JASCO FP 750 spectrofluorometer [15].


### 2.2. Cell Culture

Human dermal fibroblasts were obtained from
skin biopsy sampled from the inferior pubian region of young normal female patients (average age 30 ± 2.3 years) by employing explants technique [16]. All patients gave informed written consent to tissue collection, which was conducted under a protocol approved by the Ethical Commission of National Institute of Endocrinology C.I.
Parhon. The cells (2 × 104/mL) were grown in DMEM (Sigma-Aldrich) medium supplemented with 10% fetal
bovine serum (Sigma-Aldrich),
sterile antimycotic solution 1X: penicillin 100 IU/mL,
streptomycin 0.1 mg/mL and amphotericin 0.25 μg/mL (Sigma-Aldrich), D-glucose 5.5 mM, 2% glutamine (Sigma-Aldrich), 0.22% NaHCO3 (Sigma-Aldrich), and 0.47% HEPES (Sigma-Aldrich) in a 5% CO2 humidified atmosphere, at 37˚C.


### 2.3. Treatment of Cells

The 70% confluent fibroblasts
cultures (passage 3–5) were
maintained 24 hours in the growth medium containing 0.5% fetal bovine serum for synchronization of cells cycle. Then they were treated with varying amounts ofD-glucose:
5.5 mM (normoglycemic), 11 mM, 22 mM, and 33 mM (hyperglycemic) for 24 hours. The osmolarity of the
medium was adjusted with D-mannitol after the addition of glucose, in order to have the same osmolarity in all samples. Other flasks of cells were treated with different amounts of sterile AGE-modified BSA, and BSA was added in order to adjust the total protein concentration to 5 mg/mL. The effect of
D-glucose,
D-mannitol, or AGE-BSA on cell viability was assessed by estimating the percent
of cells excluding Trypan blue. There was no significant effect of D-glucose,
D-mannitol, or AGE-BSA on cells viability. Over 95% of the cells excluded the
dye.


### 2.4. Real-Time PCR for Collagen Type I and III Genes Expression

RNA of treated cells was extracted using TRI Reagent kit (Sigma-Aldrich)
according to the manufacturer’s recommendations [17] and by Chomezynski method [18]. RNA integrity and purity were electrophoretically verified by ethidium bromide staining and by OD260/OD280 nm absorption ratio. The specific sense and antisense oligonucleotide primers for target genes and for reference gene (Table 1) were
designed with Beacon Designer programs (Premier Biosoft), based on the
published genes sequences. 1 μg aliquots of total RNA of each sample were
reverse-transcribed into cDNA using Bio-Rad iScript cDNA Synthesis Kit,
following the recommendations of the supplier. Real-time PCR was performed in
the Bio-Rad iCycler iQTM in a final reaction mixture of 25 μL
consisting of 4 μL diluted template cDNA, 12.5 Bio-Rad 2x iQ SYBERTM Green Supermix, and 10 pmol of each forward and reverse primers (Applied Biosystems). The following real-time PCR
experimental run protocol was used: denaturation program (95˚C for 8 minutes),
amplification, and quantification program repeated 45 times (95˚C for 30 seconds,
54˚C for 30 seconds, and 72˚C for 30 seconds with a single fluorescence
measurement), melting curve program repeated 80 times for 10 seconds (55˚C–95˚C with a
heating rate of 0.5˚C per second and a continuous florescence measurement).
Melting curve analysis showed a single product for each transcript with melting
temperatures as follows: for PCol 1α2, 89˚C; for PCol 3α1, 91˚C; and for 18 S RNA, 89˚C.  In order to calculate the relative expression
ratio (R) [19] of target genes (PCol 1α2 and PCol 3α1) versus a reference gene, (18 S RNA) it was necessary to determine the crossing points (CPs)
or cycle threshold (CT), the real-time PCR amplification efficiencies (Es), and
the linearity for each transcript (CT is defined as the number of cycle at which
the fluorescence signal is greater than a defined threshold in the logarithmic
phase of amplification). Real-time PCR efficiencies were calculated from the
given slope of a calibration curve CT=f(dilution series of cDNA for each gene) in iCycler iQTM software, according to the equation:
(1)E=10(−1/slope)⁢,
see  [20]. The investigated transcripts  showed good real-time PCR efficiency rates for 1.93 PCol 1α2, 2.12 for PCol 3α1, and 1.89 for 18 S RNA with high
linearity (Pearson correlation coefficient r = 0.998). Generally, the relative
expression ratio (R) of  the target gene
is calculated based on E and CT deviation of a sample versus a control and expressed in comparison with a reference gene according to Pfaffl equation [19]: (2)R=(Etarget)ΔCTtarget(control-sample)(Eref)ΔCTref(control-sample), where Etarget is the real-time PCR efficiency of target gene
transcript, Eref is the real-time PCR efficiency of a reference
gene transcript, ΔCTtarget is the CT deviation of control-sample of target gene transcript, and ΔCTref is the CT deviation of control-sample of reference gene transcript.Table 1
Sequences of human primers used for real-time PCR.
Gene (mRNA)Oligonucleotide primer sequence (5′-3′)Amplification fragmentAnnealing temperature (˚C)CalculateUseProcolagen 1α2 (PCol 1α2) senseGTGGTTACTACTGGATTGACC33153.454Procolagen 1α2 (PCol1α2) antisenseTTGCCAGTCTCCTCATCCATProcolagen 3α1 (Pcol3α1) senseGGAGTAGCAGTAGGAGGAC915454Procolagen 1α1 (PCol3α1) antisenseAACCAGGATGACCAGATGTA18S RNA senseCTCAACACGGGAAACCTCAC13353.55418S RNA antisenseTTATCGGAATTAACCAGACAAATCGAll experiments were done twice
and samples were run in triplicate each time, the data having been expressed as the means ± standard deviation. The statistical
significance of differences between the experiments was evaluated using
Student’s t-test.P values < .05 were considered to be
statistically significant.


## 2.1. Preparation and Identification of AGE-Modified BSA

Solutions of 1.6 MD-glucose (Sigma-Aldrich company) and 100 mg/mL bovine serum albumin (BSA Sigma-Aldrich, RIA grade, fraction V) have been coincubated insterile PBS 10 mM,  pH 7.4 (Gibco) for 6 weeks under aerobic conditions at 37˚C, in the presence of 3 mM NaN3 (Merck) to prevent bacterial growth [12]. Controls with BSA solution only were simultaneously kept
under the same conditions.  After 6
weeks, the free sugar was removed by dialysis against 10 mM PBS, pH 7.4 for 48 hours. The protein concentration (70 mg BSA/mL)
was determined by Bradford method [13]. The formation of AGE-modified BSA (AGE-BSA) was analyzed by
fast protein liquid chromatography (FPLC), SDS-PAGE,
and by fluorescence spectroscopy.Fast protein liquid chromatography
An FPLC automated system (ÄKTA FPLC-Amersham Pharmacia Biotech)
with a size exclusion column (Superdex 200 HR 10/30) was used for separation of glycated and unglycated BSA. The samples of 1.55 μg /μL prepared in 10 mM PBS pH 7.4 were eluted in 5% acetonitrile (Merck) and 10 mM PBS pH
7.4, at a constant flow rate of 0.8 mL/min, and their absorption at 280 nm was automatically recorded.Gel electrophoresis
AGE-BSA cross-linking and aggregation was investigated by 7.5% SDS-PAGE) with Mini Protean Bio-Rad equipment [14] and protein bands were stained by Coomassie Brilliant Blue R-250 (Sigma-Aldrich).AGE-BSA linked fluorescence assay
The fluorescence detection of Maillard compounds was done using the parameter λex370 nm/λem440 nm and for pentosidine-like products, λex335 nm/λem385 nm was used. The fluorescence emission spectra between 380 and 600 nm (370 nm excitation) and between 350 and 500 nm (335 nm excitation) were scanned using a JASCO FP 750 spectrofluorometer [15].


## 2.2. Cell Culture

Human dermal fibroblasts were obtained from
skin biopsy sampled from the inferior pubian region of young normal female patients (average age 30 ± 2.3 years) by employing explants technique [16]. All patients gave informed written consent to tissue collection, which was conducted under a protocol approved by the Ethical Commission of National Institute of Endocrinology C.I.
Parhon. The cells (2 × 104/mL) were grown in DMEM (Sigma-Aldrich) medium supplemented with 10% fetal
bovine serum (Sigma-Aldrich),
sterile antimycotic solution 1X: penicillin 100 IU/mL,
streptomycin 0.1 mg/mL and amphotericin 0.25 μg/mL (Sigma-Aldrich), D-glucose 5.5 mM, 2% glutamine (Sigma-Aldrich), 0.22% NaHCO3 (Sigma-Aldrich), and 0.47% HEPES (Sigma-Aldrich) in a 5% CO2 humidified atmosphere, at 37˚C.


## 2.3. Treatment of Cells

The 70% confluent fibroblasts
cultures (passage 3–5) were
maintained 24 hours in the growth medium containing 0.5% fetal bovine serum for synchronization of cells cycle. Then they were treated with varying amounts ofD-glucose:
5.5 mM (normoglycemic), 11 mM, 22 mM, and 33 mM (hyperglycemic) for 24 hours. The osmolarity of the
medium was adjusted with D-mannitol after the addition of glucose, in order to have the same osmolarity in all samples. Other flasks of cells were treated with different amounts of sterile AGE-modified BSA, and BSA was added in order to adjust the total protein concentration to 5 mg/mL. The effect of
D-glucose,
D-mannitol, or AGE-BSA on cell viability was assessed by estimating the percent
of cells excluding Trypan blue. There was no significant effect of D-glucose,
D-mannitol, or AGE-BSA on cells viability. Over 95% of the cells excluded the
dye.


## 2.4. Real-Time PCR for Collagen Type I and III Genes Expression

RNA of treated cells was extracted using TRI Reagent kit (Sigma-Aldrich)
according to the manufacturer’s recommendations [17] and by Chomezynski method [18]. RNA integrity and purity were electrophoretically verified by ethidium bromide staining and by OD260/OD280 nm absorption ratio. The specific sense and antisense oligonucleotide primers for target genes and for reference gene (Table 1) were
designed with Beacon Designer programs (Premier Biosoft), based on the
published genes sequences. 1 μg aliquots of total RNA of each sample were
reverse-transcribed into cDNA using Bio-Rad iScript cDNA Synthesis Kit,
following the recommendations of the supplier. Real-time PCR was performed in
the Bio-Rad iCycler iQTM in a final reaction mixture of 25 μL
consisting of 4 μL diluted template cDNA, 12.5 Bio-Rad 2x iQ SYBERTM Green Supermix, and 10 pmol of each forward and reverse primers (Applied Biosystems). The following real-time PCR
experimental run protocol was used: denaturation program (95˚C for 8 minutes),
amplification, and quantification program repeated 45 times (95˚C for 30 seconds,
54˚C for 30 seconds, and 72˚C for 30 seconds with a single fluorescence
measurement), melting curve program repeated 80 times for 10 seconds (55˚C–95˚C with a
heating rate of 0.5˚C per second and a continuous florescence measurement).
Melting curve analysis showed a single product for each transcript with melting
temperatures as follows: for PCol 1α2, 89˚C; for PCol 3α1, 91˚C; and for 18 S RNA, 89˚C.  In order to calculate the relative expression
ratio (R) [19] of target genes (PCol 1α2 and PCol 3α1) versus a reference gene, (18 S RNA) it was necessary to determine the crossing points (CPs)
or cycle threshold (CT), the real-time PCR amplification efficiencies (Es), and
the linearity for each transcript (CT is defined as the number of cycle at which
the fluorescence signal is greater than a defined threshold in the logarithmic
phase of amplification). Real-time PCR efficiencies were calculated from the
given slope of a calibration curve CT=f(dilution series of cDNA for each gene) in iCycler iQTM software, according to the equation:
(1)E=10(−1/slope)⁢,
see  [20]. The investigated transcripts  showed good real-time PCR efficiency rates for 1.93 PCol 1α2, 2.12 for PCol 3α1, and 1.89 for 18 S RNA with high
linearity (Pearson correlation coefficient r = 0.998). Generally, the relative
expression ratio (R) of  the target gene
is calculated based on E and CT deviation of a sample versus a control and expressed in comparison with a reference gene according to Pfaffl equation [19]: (2)R=(Etarget)ΔCTtarget(control-sample)(Eref)ΔCTref(control-sample), where Etarget is the real-time PCR efficiency of target gene
transcript, Eref is the real-time PCR efficiency of a reference
gene transcript, ΔCTtarget is the CT deviation of control-sample of target gene transcript, and ΔCTref is the CT deviation of control-sample of reference gene transcript.Table 1
Sequences of human primers used for real-time PCR.
Gene (mRNA)Oligonucleotide primer sequence (5′-3′)Amplification fragmentAnnealing temperature (˚C)CalculateUseProcolagen 1α2 (PCol 1α2) senseGTGGTTACTACTGGATTGACC33153.454Procolagen 1α2 (PCol1α2) antisenseTTGCCAGTCTCCTCATCCATProcolagen 3α1 (Pcol3α1) senseGGAGTAGCAGTAGGAGGAC915454Procolagen 1α1 (PCol3α1) antisenseAACCAGGATGACCAGATGTA18S RNA senseCTCAACACGGGAAACCTCAC13353.55418S RNA antisenseTTATCGGAATTAACCAGACAAATCGAll experiments were done twice
and samples were run in triplicate each time, the data having been expressed as the means ± standard deviation. The statistical
significance of differences between the experiments was evaluated using
Student’s t-test.P values < .05 were considered to be
statistically significant.


## 3. Results

### 3.1. Evidence of AGE-Modified BSA Formation

The AGEs content in the preparations was assessed by fluorescence
measurements, SDS-PAGE analysis, and gel filtration studies.Fluorescence Assays
The fluorescence level measured at 385 nm  emission wavelength after a 335 nm excitation wavelength was 3.77 fluorescence units (RFU) for control BSA and 74.9 RFU for AGE-BSA (Figure1(a)). At 440 nm emission
after a 370 nm excitation was 3.7 RFU for control BSA and 65.7 RFU for AGE-BSA. All fluorescence recorded was done at 1 mg/mL protein (Figure 1(b)).Fluorescence emission spectra in RFU (relative fluorescence
units)/1 mg BSA; 6 weeks incubation of BSA (100 mg/mL) at
37˚C in PBS 10 mM pH 7.4; curve (1) unglycated BSA (control); curve  (2) BSA + 1.6 M D-glucose (AGE-modified BSA). (a) Fluorescence emission spectra of samples at 335 nm excitation. (b) Fluorescence
emission spectra of samples at 370 nm excitation.
(a)(b)Gel electrophoresis
SDS-PAGE analysis has shown the formation of an AGE-BSA monomer of 77.049 kDa and a 155.84 kDa dimer, whereas the monomer of control BSA was of 68.66 kDa (Figure2).(a) and (b): SDS-PAGE electrophoretic profiles of 6 weeks
glycation of BSA (100 mg/mL) at 37˚C in PBS 10 mM pH 7.4; lanes 1 and 3: BSA + 1.6 D-glucose (AGE-modified BSA) (loaded with 10 μg and 5 μg protein, resp.); lanes 2 and 4: unglycated BSA (control) (loaded with 10 μg and 5 μg protein, resp.); lane 5: molecular weight marker (MWM 105 Bio-Rad). This was carried out using a 4% stacking and 7.5% resolving gel and Coomassie blue staining. (c) Corelation MW-RF.
(a)(b)(c)Chromatographic studies
The FPLC elution pattern of BSA control sample showed only one peak corresponding to a 67.96 kDa molecular weight (retention volume 13.20 mL), while the glycated BSA (AGE-BSA) presented two peaks of 83.29 kDa (retention volume 12.78 mL) and 161.46 kDa
(retention volume 10.81 mL). The peak with
the retention volume at 12.78 mL showed a slightly increase in molecular weight
and a major increase in 280 nm absorbance in comparison with the
unglycated BSA peak. The peak with the retention volume 10.81 mL corresponds to
a dimer with high molecular weight of 161.46 kDa of the glycated BSA monomer of 83.29 kDa (Figure3). The increase in molecular
weight of glycated BSA monomer and the formation of glycated BSA dimer with a
higher molecular mass is probably due to the ability of AGEs compounds to
generate intra- and intermolecular cross-linkings. Chromatographic
data are in accordance with the SDS-PAGE results.Figure 3
FPLC separation of glycated BSA on Superdex 200 HR 10/30 column, 155 μg protein/100 μL injection volumes: 6
weeks incubation of BSA (100 mg/mL) at 37˚C in 10 mM PBS pH 7.4; curve (1) BSA + 1.6 M D-glucose (AGE-modified BSA); curve (2) unglycated BSA (control).


### 3.2. Influence of High Glucose Concentration on the Expression of Procollagen Type I and III in Cultured Human Dermal Fibroblasts

The influence of high glucose
concentration (mimicking diabetic conditions) on
steady-state levels of theprocollagen 1α2 and procollagen 3α1 mRNA was determined. Confluent monolayer
fibroblasts were treated with 11 mM, 22 mM, and 33 mM D-glucose
for 24 hours. The same type of cells treated with 5.5 mM D-glucose (normoglycemic conditions) was used as control. The mRNA expression was analyzed by quantitative real-time PCR relative
to 18 S RNA. The high level in glucose compared to the control (5.5 mM glucose)
resulted in a moderate increase of the relative expression ration (R) of procollagen α2(I) as follows: at 11 and at 22 mM glucose, the relative
expression ratio (R) increased to 1.33 ± 0.051-fold (P < .05) and to 1.28 ± 0.048-fold (P < .05), respectively,
and at 33 mM glucose the increase was 1.64 ± 0.063-fold
P < .02. In the case of
procollagen α1(III) mRNA at all
concentration of glucose used in the cells treatment,  there was a trend for downregulation compared to the control (Figure 4).Figure 4
The relative expression ratios (R) for procollagen 1α2 and for procollagen 3α1 genes after 24 hours
glucose treatment of culture human dermal fibroblasts. R was expressed in arbitrary units. The data are shown as the mean ± SD for two independent experiments run in triplicate each time with significant differences compared
to control (5.5 mM glucose) at
*P < .05,
**P < .02,
and ***P < .01.


### 3.3. Effect of AGE-Modified BSA on the Expression of Procollagen Type I and III in Cultured Human Dermal Fibroblasts

Confluent monolayers human skin fibroblasts were exposed to increasing concentrations of AGE-modified BSA or BSA as control in culture medium containing 0.5% fetal bovine serum for 24 hours and mRNA expression forprocollagen1α2 and procollagen3α1 was determined by real-time PCR. The treatment with increasing AGE-BSA levels adjusted to
concentrations of 5 mg/mL protein with BSA upregulated the mRNA expression of
procollagen α2(I) and procollagen α1(III)
compared to the control (5 mg/mL BSA) (Figure 5). In response to 1 mg/mL of AGE-BSA, the relative expression ratio (R) for procollagen α2 (I) was 2.03 ± 0.15-fold (P < .05) and for procollagen
α1(III) was 3.4 ± 0.3-fold (P < .05) on average. In the
case of 2 mg/mL AGE-BSA treatment, the real upregulation ratio was, on average,
3.57 ± 0.26-fold (P < .05) and 6.22 ± 0.27 (P < 0.01) for procollagen α2 (I) and, respectively, for procollagen
α1(III). The treatment of cultured dermal fibroblasts with 4 mg/mL AGE-BSA increased
relative expression ratio (R) to 4.14 ± 0.14-fold (P < .01) and to 7.4 ± 0.32-fold (P < .01)  for procollagen α2(I) and, respectively, for procollagen
α1(III). These results showed that AGE-BSA upregulated
the procollagen α1(III) mRNA expression to a great extent in comparison to
procollagen α2(I) mRNA expression and
this stimulation appeared to have a dose-dependent effect.Figure 5
The relative expression ratios (R) for procollagen 1α2 and for procollagen 3α1 genes after 24 hours
incubation with AGE-BSA of culture human dermal fibroblasts. R was expressed in arbitrary units. The data are shown as the mean ± SD for two independent experiments run in triplicate each time with significant differences compared to control (5 mg/mL BSA) at *P < .05 and ***P < .01.


## 3.1. Evidence of AGE-Modified BSA Formation

The AGEs content in the preparations was assessed by fluorescence
measurements, SDS-PAGE analysis, and gel filtration studies.Fluorescence Assays
The fluorescence level measured at 385 nm  emission wavelength after a 335 nm excitation wavelength was 3.77 fluorescence units (RFU) for control BSA and 74.9 RFU for AGE-BSA (Figure1(a)). At 440 nm emission
after a 370 nm excitation was 3.7 RFU for control BSA and 65.7 RFU for AGE-BSA. All fluorescence recorded was done at 1 mg/mL protein (Figure 1(b)).Fluorescence emission spectra in RFU (relative fluorescence
units)/1 mg BSA; 6 weeks incubation of BSA (100 mg/mL) at
37˚C in PBS 10 mM pH 7.4; curve (1) unglycated BSA (control); curve  (2) BSA + 1.6 M D-glucose (AGE-modified BSA). (a) Fluorescence emission spectra of samples at 335 nm excitation. (b) Fluorescence
emission spectra of samples at 370 nm excitation.
(a)(b)Gel electrophoresis
SDS-PAGE analysis has shown the formation of an AGE-BSA monomer of 77.049 kDa and a 155.84 kDa dimer, whereas the monomer of control BSA was of 68.66 kDa (Figure2).(a) and (b): SDS-PAGE electrophoretic profiles of 6 weeks
glycation of BSA (100 mg/mL) at 37˚C in PBS 10 mM pH 7.4; lanes 1 and 3: BSA + 1.6 D-glucose (AGE-modified BSA) (loaded with 10 μg and 5 μg protein, resp.); lanes 2 and 4: unglycated BSA (control) (loaded with 10 μg and 5 μg protein, resp.); lane 5: molecular weight marker (MWM 105 Bio-Rad). This was carried out using a 4% stacking and 7.5% resolving gel and Coomassie blue staining. (c) Corelation MW-RF.
(a)(b)(c)Chromatographic studies
The FPLC elution pattern of BSA control sample showed only one peak corresponding to a 67.96 kDa molecular weight (retention volume 13.20 mL), while the glycated BSA (AGE-BSA) presented two peaks of 83.29 kDa (retention volume 12.78 mL) and 161.46 kDa
(retention volume 10.81 mL). The peak with
the retention volume at 12.78 mL showed a slightly increase in molecular weight
and a major increase in 280 nm absorbance in comparison with the
unglycated BSA peak. The peak with the retention volume 10.81 mL corresponds to
a dimer with high molecular weight of 161.46 kDa of the glycated BSA monomer of 83.29 kDa (Figure3). The increase in molecular
weight of glycated BSA monomer and the formation of glycated BSA dimer with a
higher molecular mass is probably due to the ability of AGEs compounds to
generate intra- and intermolecular cross-linkings. Chromatographic
data are in accordance with the SDS-PAGE results.Figure 3
FPLC separation of glycated BSA on Superdex 200 HR 10/30 column, 155 μg protein/100 μL injection volumes: 6
weeks incubation of BSA (100 mg/mL) at 37˚C in 10 mM PBS pH 7.4; curve (1) BSA + 1.6 M D-glucose (AGE-modified BSA); curve (2) unglycated BSA (control).


## 3.2. Influence of High Glucose Concentration on the Expression of Procollagen Type I and III in Cultured Human Dermal Fibroblasts

The influence of high glucose
concentration (mimicking diabetic conditions) on
steady-state levels of theprocollagen 1α2 and procollagen 3α1 mRNA was determined. Confluent monolayer
fibroblasts were treated with 11 mM, 22 mM, and 33 mM D-glucose
for 24 hours. The same type of cells treated with 5.5 mM D-glucose (normoglycemic conditions) was used as control. The mRNA expression was analyzed by quantitative real-time PCR relative
to 18 S RNA. The high level in glucose compared to the control (5.5 mM glucose)
resulted in a moderate increase of the relative expression ration (R) of procollagen α2(I) as follows: at 11 and at 22 mM glucose, the relative
expression ratio (R) increased to 1.33 ± 0.051-fold (P < .05) and to 1.28 ± 0.048-fold (P < .05), respectively,
and at 33 mM glucose the increase was 1.64 ± 0.063-fold
P < .02. In the case of
procollagen α1(III) mRNA at all
concentration of glucose used in the cells treatment,  there was a trend for downregulation compared to the control (Figure 4).Figure 4
The relative expression ratios (R) for procollagen 1α2 and for procollagen 3α1 genes after 24 hours
glucose treatment of culture human dermal fibroblasts. R was expressed in arbitrary units. The data are shown as the mean ± SD for two independent experiments run in triplicate each time with significant differences compared
to control (5.5 mM glucose) at
*P < .05,
**P < .02,
and ***P < .01.


## 3.3. Effect of AGE-Modified BSA on the Expression of Procollagen Type I and III in Cultured Human Dermal Fibroblasts

Confluent monolayers human skin fibroblasts were exposed to increasing concentrations of AGE-modified BSA or BSA as control in culture medium containing 0.5% fetal bovine serum for 24 hours and mRNA expression forprocollagen1α2 and procollagen3α1 was determined by real-time PCR. The treatment with increasing AGE-BSA levels adjusted to
concentrations of 5 mg/mL protein with BSA upregulated the mRNA expression of
procollagen α2(I) and procollagen α1(III)
compared to the control (5 mg/mL BSA) (Figure 5). In response to 1 mg/mL of AGE-BSA, the relative expression ratio (R) for procollagen α2 (I) was 2.03 ± 0.15-fold (P < .05) and for procollagen
α1(III) was 3.4 ± 0.3-fold (P < .05) on average. In the
case of 2 mg/mL AGE-BSA treatment, the real upregulation ratio was, on average,
3.57 ± 0.26-fold (P < .05) and 6.22 ± 0.27 (P < 0.01) for procollagen α2 (I) and, respectively, for procollagen
α1(III). The treatment of cultured dermal fibroblasts with 4 mg/mL AGE-BSA increased
relative expression ratio (R) to 4.14 ± 0.14-fold (P < .01) and to 7.4 ± 0.32-fold (P < .01)  for procollagen α2(I) and, respectively, for procollagen
α1(III). These results showed that AGE-BSA upregulated
the procollagen α1(III) mRNA expression to a great extent in comparison to
procollagen α2(I) mRNA expression and
this stimulation appeared to have a dose-dependent effect.Figure 5
The relative expression ratios (R) for procollagen 1α2 and for procollagen 3α1 genes after 24 hours
incubation with AGE-BSA of culture human dermal fibroblasts. R was expressed in arbitrary units. The data are shown as the mean ± SD for two independent experiments run in triplicate each time with significant differences compared to control (5 mg/mL BSA) at *P < .05 and ***P < .01.


## 4. Discussion

Tissue remodeling of extracellular matrix (ECM) is an essential and
dynamic process associated with physiological responses and it involves the production and deposit of newly synthesized ECM components, as well as the degradation of ECM. The balance of these processes results in either preservation or alteration of the structure and functions of the support tissue [21]. Resorption of the ECM is mediated by MMPs, whereas generation of ECM is predominantly achieved through
the production of collagen. Degradation of ECM generally characterizes
pathological states such as arthritis or tumor invasion, whereas the increased
generation of ECM underlies fibrotic diseases. Both processes are strictly
regulated by complex networks of cellular and molecular interaction [22]. Mediators such as the
profibrotic cytokines (TGF-β1,
interleukins, and connective tissue growth factor-CTGF) released by
resident cells, for examples, skin fibroblasts, or infiltrating leukocytes,
monocytes, or macrophages may play a central role in the turnover of the dermal
ECM. Expansion of ECM in fibrosis occurs in many tissues, including skin, as
part of the end-organ complications in diabetes and chronic hyperglycemia. The
formation of AGEs is considered as causative factor in diabetic tissue
fibrosis. Elevated levels of blood glucose ignite a vicious cycle of
metabolic disturbances with activation of multiple pathways. For
example, it was demonstrated that AR is implicatedin the adverse
cellular response to high levels of glucose [23]. Multiple studies
indicate that expression and activity of AR are increased in experimental models and human tissuesin diabetes, including the diabetic kidney [24]. In addition to AR, isoforms
of protein kinase C family, especially PKCβ isoform, has been
associated with enhanced activity in hyperglycemia [25].  In vivo, pharmacologic
blockade of PKCβ has been associated with improved
vascular function in diabeticrats, as well as amelioration of
accelerated mesangial expansion and expression of genes such as
TGF-β1 and extracellular matrix components [11].  Our results
suggest that high levels of glucose may influence the expansion of ECM and the process of skin aging through mild stimulation of procollagen1α2 gene expression and
possibly of other ECM proteins. A potential mediator for this effect is
probably TGF-β1, which is regulated by PKCβ.
Increased glucose flux through glycolysis and AR pathway leads to increased intracellular NADH/NAD+ ratio, which causes an inhibition of NAD+-dependent
enzyme glyceraldehydes-3-phosphate dehydrogenase (GAPDH), which in turn will result in an increase in the dihydroxyacetone phosphate and glyceraldehyde-3-phosphate
levels. These triosephosphates can be converted into PKC activator
diacylglycerol, transformation accelerated by the increase in NADH/NAD+ ratio.Another consequence of
elevated levels of glucose is the Maillard reaction. In
the advanced step, complex reactions that lead to the formation of
AGEs occur [26]. Numerous studies have
suggested a link among glucose-modified proteins, Amadori products,
AGE, and activation of PKCβ isoforms. In cultured
mesangial cells, inhibitors of PKCβ isoforms prevented the
glycated albumin-induced increased expression of collagen IV [27]. Some in vivo studies showed
that infusion of AGE-modified murine serumal bumin into nondiabetic
mice for 4 weeks caused upregulation of glomerular α1(IV) collagen,
laminin β1, and TGF-β1 transcriptionin the kidney [28].In our experimental conditions,  the AGE-modified BSA concentrations used appeared to be high, but the doses
used in the treatment of cells (1, 2, and 4 mg/mL) represent the concentration
of glycated protein not of AGEs products. By fluorescence, gel filtration
chromatography, and SDS-PAGE assays, we have highlighted the formation of AGEs and the cross-linking of glycated BSA but we did not measure the level of AGEs compounds. On the other hand, fibroblasts
can suffer in vivo directly from the effects of AGEs
formed during the degradation of matrix proteins that have a long life and
important amounts of these compounds can accumulate in time.Finally,
by means of real-time PCR, we revealed that AGE-modified BSA interacts with
cultured human dermal fibroblasts and influence their function by significant
upregulation in a dose-dependent manner of bothprocollagen 1α2 and procollagen 3α1 genes expression. In addition, at all AGE-BSA doses used for the
cultured fibroblasts treatments, the ratio of procollagen α1(III)/procollagen α2(I) mRNA remained constant to
approximately 1.7 on average.This could probably alter the turnover
of collagenous ECM in the skin and contribute to a decreased tensile strength
and mechanical stability of connective tissues and a difficult healing in
diabetes. Twigg et al. [29]  have reported that in confluent monolayers of
cultured human dermal fibroblasts, the connective tissue growth
factor (CTGF) is upregulated at the mRNA
and protein levels by AGEs. Later, they have shown that CTGF
contributes significantly, in human dermal fibroblasts, to AGEs
upregulation of fibronectin, another ECM component like collagen, through a PKC-dependent mechanism [30]. In the same year Okano et al. [7] demonstrated that AGEs which are accumulating in elastin, fibronectin, and collagens bind to fibroblast membranes at concentrations of 2.5–40 mg/mL. Our data are in accord with other
studies which described AGEs increased collagen production in normal rat kidney
fibroblasts [31]. Recently, Lohwasser et al. demonstrated for the first time that RAGE protein is highly expressed in human
skin and in cultured human skin fibroblasts and AGE adduct upregulated RAGE
expression and induced significantly upregulated expression of CTGF, TGF-β1,
and procollagen α1(I) [9]. Also RAGE induction through
AGE-BSA and TNF-α was shown before in human umbilical vein endothelial cells [32] and through AGE-BSA in normal
rat kidney fibroblasts [31].It seems that the most important ECM-related protein genesPcol 1α2 and Pcol 3α1 are upregulated in the
presence of AGEs and to a less extent by high levels of glucose in cultured human dermal fibroblast
possibly by receptor-independent/dependent pathways.



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*Source: 473603-2008-04-03.xml*