# Different Effects of Angiotensin Converting Enzyme Inhibitors on Endothelin-1 and Nitric Oxide Balance in Human Vascular Endothelial Cells: Evidence of an Oxidant-Sensitive Pathway

Authors: Giovambattista Desideri; Davide Grassi; Giuseppe Croce; Raffaella Bocale; Sergio Tiberi; Stefano Evangelista; Stefano Necozione; Ferdinando Di Orio; Claudio Ferri
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Angiotensin converting enzyme inhibitors (ACE-I) are known to affect the endothelial function decreasing the formation of the potent vasoconstrictor angiotensin II and increasing the bioavailability of the vasodilators bradykinin and nitric oxide. In order to test the potential differences among the class of ACE-I, we have evaluated their effects on endothelin-1 and nitric oxide availability, and on reactive oxygen species generation in human vascular endothelial cells (HUVECs) obtained from umbilical cord veins. HUVECs were cultured in the presence of either sulfhydryl-containing ACE-I (zofenoprilat or captopril) or nonsulfhydryl-containing ACE-I (enalaprilat or lisinopril). After 24 hours, all ACE-I reduced endothelin-1 secretion and increased nitric oxide production. However, zofenoprilat
(−42% after 8 hours of incubation) was more effective (P<.05) than enalaprilat
(−25%), lisinopril (−21%), and captopril (−30%) in reducing endothelin-1 secretion. Similarly, zofenoprilat (+110% after 8 hours of incubation) was more effective (P<.05) than enalaprilat (+64%), lisinopril (+63%), and captopril (+65%) in increasing nitrite plus nitrate production. The effect of ACE-I on the nitric oxide availability and on endothelin-1 secretion is mediated by the activation of bradykinin
B2 receptor being reverted, at least in part, by a specific antagonist. Zofenoprilat and, to a lesser extent, captopril also blunted tumour necrosis factor (TNF)α-stimulated production of reactive oxygen species and decreased TNFα-stimulated glutathione consumption in endothelial cells. In conclusion, structural differences among the class of ACE-I play a role in their interaction with mediators that influence the homeostasis of the endothelium. The marked antioxidant effects exerted by zofenoprilat due to its sulphydryl group and lipophilia seem to be responsible for its greater endothelium protective activity.


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## Body

## 1. Introduction

Angiotensin  converting enzyme (ACE), also known as
kininase II, is a bivalent dipeptidyl  carboxyl metallopeptidase present both as
a membrane-bound form in epithelial, neuroepithelial, and endothelial cells,
including the vascular ones, and as a soluble form in different body fluid,
including blood [1]. Due to its ability to cleave the C-terminal dipeptide from
a number of peptides, ACE can either convert the inactive decapeptide
angiotensin I to the active octapeptide angiotensin II or inactivate kinins
[1]. Thus, ACE strategically modulates the balance between the vasoconstrictive
and salt-retentive renin-angiotensin system and the vasodilatory and
natriuretic kallikrein-kinin one [1]. As a consequence, after the initial use
as antihypertensive drugs [2], ACE-inhibitors (ACE-Is) rapidly became a
fundamental tool also in treating congestive heart failure, left ventricular
dysfunction after myocardial infarction, diabetic and nondiabetic nephropathies
[2–4].Despite of the successful use in all of the above
conditions, the mechanisms responsible for the vascular benefits exerted by
ACE-I are not fully understood. ACE-I are able to improve both endothelium-dependent [5] and
endothelium-independent [6] vascular relaxation. However, the endothelial
effects of ACE-I are not only dependent on decrease of angiotensin II formation
and increase of bradykinin bioavailability [2, 5, 6]. In this regard, it has
been suggested that the vascular action of ACE-I could be also related to their
ability to reduce production of endothelin-1 (ET-1) [7], one of the most potent vasoconstrictor [8], through an increased nitric oxide (NO) production
[7, 9] leading to a down-regulation of ET-1 gene expression [9].In this regard, sulfhydryl containing ACE-I can act as
antioxidants by scavenging superoxide anion [10] as well as nonsuperoxide
radicals [11]. Since unscavenged superoxide anion quenches NO to give the
pro-oxidant compound peroxynitrite [12], which is unable to down-regulate (or
even up-regulates) ET-1 gene expression, sulfhydryl containing ACE-I could be
particularly effective in decreasing ET-1 secretion by cultured HUVECs in increasing NO production [13].To address this topic, we compared the effects of
zofenoprilat and captopril, that are, two sulfhydryls containing ACE-I, with those of
enalaprilat and lisinopril, two nonsulfhydryls containing ACE-I, on ET-1 secretion and NO
production by human vascular endothelial cells (HUVECs). In addition, to assess
the ACE-I antioxidant properties, their effects on intracellular content of the
endogenous free radical scavenger reduced glutathione (GSH) [14, 15] and the
generation of reactive oxygen species were also evaluated.


## 2. Materials and Methods

### 2.1. Cells

HUVECs were harvested from fresh human
umbilical cord veins cultured until
the third passageas previously described [7, 16, 17]. The purity of the endothelial cell monolayer
was confirmed by their cobblestone morphological pattern and by cell staining
with a monoclonal antibody specific for von Willebrand factor [17]. Newly confluent cells in culture medium were lifted with
trypsinization; the trypsin was inhibited with 20% foetal calf serum, and cells
were washed in culture medium. After 10 minutes of centrifugation (1100 rpm,
20°C), the supernatant was removed and HUVECs were resuspended in culture
medium (3 mL) and then used for the experiments.HUVECs were incubated either with zofenoprilat (the active
form of zofenopril), or enalaprilat (the active form of enalapril), or
lisinopril or captopril for various times up
to 24 hours. The above experiments were repeated in the presence of
either bradykinin, orDes-Arg9-[Leu8]-BK, that is, a
bradykinin B1
receptor
antagonist, or D-Arg-[Hyp3,Thi5,8,D-phe7]-BK, that
is, a bradykinin B2 receptor antagonist. Finally, experiments were also repeated in
the presence of the NO synthase competitive inhibitor Nω-nitro-L-arginine methyl ester (L-NA).Zofenoprilat was obtained from Menarini Ricerche SpA, Firenze, Italy.
Angiotensin II was purchased by Clinalfa (Laufelfingen,
Switzerland).
The other reagents were purchased by Sigma (St Louis, Mo, US). If it is not otherwise
specified, all the tested substances have been added to culture medium to a
final concentration of10−8M, a concentration that fully inhibited
the human recombinant ACE for all the antagonists under study [18].


### 2.2. Endothelin-1

The peptide was assayed as previously described [16].
In brief, the culture medium derived from each well was centrifuged at 3.000 rpm for 10 minutes. The supernatant was subsequently freeze-dried,
reconstituted  in starting high performance liquid
chromatography buffer, injected onto C18
columns
(Pharmacia, Uppsala, Sweden), and eluted over 70 minutes using a linear
gradient of 15–75%
acetonitrile/0.1% trifluoroacetic acid in water. Fractions were collected each
minute and evaporated before reconstitution in assay buffer (50 mmol/L
phosphate buffer, pH 7.4, containing 0.9% NaCl, 0.05% NaN3, and 0.5%
bovine serum albumin). Endothelin-1 immunoreactivity was then assayed on
reconstituted samples by a sensitive radioimmunoassay (Peninsula Laboratories, Belmont, Calif,
USA).
Interassay and intra-assay variations were <10%. Cross-reactivity of the
ET-1 antibody with endothelin-2 and endothelin-3 was <7%, according to the
supplier.


### 2.3. Nitric Oxide

NO production by HUVECs was assessed by
evaluating the concentration of NO metabolite (NOx), that is, nitrite plus
nitrate, in culture medium. Briefly, NOx concentrations were evaluated by
colorimetric detection of nitrite after conversion of all sample nitrate to
nitrite (Assay Design Inc., Ann Arbor,
Mich, USA)
as previously described [7].


### 2.4. Measurements of Intracellular Glutathione Redox Status and Oxidative Stress

Intracellular glutathione (GSH) concentration was
measured according to the method previously described by our group [15]. In
brief, 2×106 HUVECs were firstly diluted in 1 mL isotonic saline +
HCl (10 mmol/L) and then lysed in acetone, thawed four times, and centrifuged
for 15 minutes at 4°C. Supernatants were deproteinized with 10%
5-sulfosalicylic acid and used for total GSH determination, that is,
glutathione (GSH) + GSH disulphide (GSSG), by the enzymatic method described by
Anderson [19]. For GSSG determination, 0.1 mL deproteinized supernatants were
treated with 2 μL 2-vinylpyridine,
neutralized with triethanolamine at a final pH of 6.5 and assayed after 1 hour
incubation. Then, endothelial cell GSH content was calculated by subtracting
GSSG from total intracellular GSH concentrations.Intracellular oxidative stress was measured at
baseline and after incubation with tumour necrosis factor (TNF)α according to Wu and Juurlink’s method [14].
In brief, cultured HUVECs were loaded with the permeable agent
5-(6)-carboxy-2′-7′-dichlorodihydrofluorescein (DCHF) ester for 60 minutes. In
the presence of intracellular esterases permeable DCHF ester is converted to
its impermeable counterpart. This latter is oxidized to the fluorescent DCF by
strong oxidants such as hydroxyl radicals [15, 20]. Then, intracellular
oxidative stress was quantified by monitoring DCF content in HUVECs with
fluorimeter with excitation at 495 nm and emission at 525 and expressed as
percent of control.


### 2.5. Statistical Analysis

Changes in migration were analyzed by ANOVA followed
by Bonferroni’s test. Multiple comparisons were analyzed by ANOVA
followed by post hoc analysis to adjust the significance level.
Statistical significance was considered asP<.05. Data are given as the mean ±SD of four
experiments.


## 2.1. Cells

HUVECs were harvested from fresh human
umbilical cord veins cultured until
the third passageas previously described [7, 16, 17]. The purity of the endothelial cell monolayer
was confirmed by their cobblestone morphological pattern and by cell staining
with a monoclonal antibody specific for von Willebrand factor [17]. Newly confluent cells in culture medium were lifted with
trypsinization; the trypsin was inhibited with 20% foetal calf serum, and cells
were washed in culture medium. After 10 minutes of centrifugation (1100 rpm,
20°C), the supernatant was removed and HUVECs were resuspended in culture
medium (3 mL) and then used for the experiments.HUVECs were incubated either with zofenoprilat (the active
form of zofenopril), or enalaprilat (the active form of enalapril), or
lisinopril or captopril for various times up
to 24 hours. The above experiments were repeated in the presence of
either bradykinin, orDes-Arg9-[Leu8]-BK, that is, a
bradykinin B1
receptor
antagonist, or D-Arg-[Hyp3,Thi5,8,D-phe7]-BK, that
is, a bradykinin B2 receptor antagonist. Finally, experiments were also repeated in
the presence of the NO synthase competitive inhibitor Nω-nitro-L-arginine methyl ester (L-NA).Zofenoprilat was obtained from Menarini Ricerche SpA, Firenze, Italy.
Angiotensin II was purchased by Clinalfa (Laufelfingen,
Switzerland).
The other reagents were purchased by Sigma (St Louis, Mo, US). If it is not otherwise
specified, all the tested substances have been added to culture medium to a
final concentration of10−8M, a concentration that fully inhibited
the human recombinant ACE for all the antagonists under study [18].


## 2.2. Endothelin-1

The peptide was assayed as previously described [16].
In brief, the culture medium derived from each well was centrifuged at 3.000 rpm for 10 minutes. The supernatant was subsequently freeze-dried,
reconstituted  in starting high performance liquid
chromatography buffer, injected onto C18
columns
(Pharmacia, Uppsala, Sweden), and eluted over 70 minutes using a linear
gradient of 15–75%
acetonitrile/0.1% trifluoroacetic acid in water. Fractions were collected each
minute and evaporated before reconstitution in assay buffer (50 mmol/L
phosphate buffer, pH 7.4, containing 0.9% NaCl, 0.05% NaN3, and 0.5%
bovine serum albumin). Endothelin-1 immunoreactivity was then assayed on
reconstituted samples by a sensitive radioimmunoassay (Peninsula Laboratories, Belmont, Calif,
USA).
Interassay and intra-assay variations were <10%. Cross-reactivity of the
ET-1 antibody with endothelin-2 and endothelin-3 was <7%, according to the
supplier.


## 2.3. Nitric Oxide

NO production by HUVECs was assessed by
evaluating the concentration of NO metabolite (NOx), that is, nitrite plus
nitrate, in culture medium. Briefly, NOx concentrations were evaluated by
colorimetric detection of nitrite after conversion of all sample nitrate to
nitrite (Assay Design Inc., Ann Arbor,
Mich, USA)
as previously described [7].


## 2.4. Measurements of Intracellular Glutathione Redox Status and Oxidative Stress

Intracellular glutathione (GSH) concentration was
measured according to the method previously described by our group [15]. In
brief, 2×106 HUVECs were firstly diluted in 1 mL isotonic saline +
HCl (10 mmol/L) and then lysed in acetone, thawed four times, and centrifuged
for 15 minutes at 4°C. Supernatants were deproteinized with 10%
5-sulfosalicylic acid and used for total GSH determination, that is,
glutathione (GSH) + GSH disulphide (GSSG), by the enzymatic method described by
Anderson [19]. For GSSG determination, 0.1 mL deproteinized supernatants were
treated with 2 μL 2-vinylpyridine,
neutralized with triethanolamine at a final pH of 6.5 and assayed after 1 hour
incubation. Then, endothelial cell GSH content was calculated by subtracting
GSSG from total intracellular GSH concentrations.Intracellular oxidative stress was measured at
baseline and after incubation with tumour necrosis factor (TNF)α according to Wu and Juurlink’s method [14].
In brief, cultured HUVECs were loaded with the permeable agent
5-(6)-carboxy-2′-7′-dichlorodihydrofluorescein (DCHF) ester for 60 minutes. In
the presence of intracellular esterases permeable DCHF ester is converted to
its impermeable counterpart. This latter is oxidized to the fluorescent DCF by
strong oxidants such as hydroxyl radicals [15, 20]. Then, intracellular
oxidative stress was quantified by monitoring DCF content in HUVECs with
fluorimeter with excitation at 495 nm and emission at 525 and expressed as
percent of control.


## 2.5. Statistical Analysis

Changes in migration were analyzed by ANOVA followed
by Bonferroni’s test. Multiple comparisons were analyzed by ANOVA
followed by post hoc analysis to adjust the significance level.
Statistical significance was considered asP<.05. Data are given as the mean ±SD of four
experiments.


## 3. Results

### 3.1. ACE-I Counteracts Endothelin-1 Secretion by HUVEC: Evidence of Different Pathway

All tested ACE-I at10−8M reduced
spontaneous ET-1 secretion by HUVECs to a level which was similar to that
induced by bradykinin (see Figure 1(a)). Preincubation with the bradykinin B2 receptor antagonist D-Arg-[Hyp3,Thi5,8,D-phe7]-BK
but not the bradykinin B1 receptor antagonist Des-Arg9-[Leu8]-BK
both at 10−6M abolished
the inhibitory effect of ACE-I on ET-1 secretion by HUVECs (see Figures 1(b)–1(f)). These data
suggested that bradykinin B2 receptor stimulation was involved in
the inhibitory effect of the four tested ACE-Is on ET-1 secretion. Although all
the four tested drugs counteracted spontaneous ET-1 secretion by HUVECs,
zofenoprilat was more effective than the other ACE-I in this setting. In
addition, the inhibitory effect of zofenoprilat on ET-1 secretion was only
partially counteracted by bradykinin B2 receptor inhibition (see Figure
1(c)). This finding indicated that bradykinin  B2 receptor
stimulation does
not represent the only pathway involved in the inhibitory effect of
zofenoprilat on ET-1 production by HUVECs.Effects of10−8M captopril (CPT,
(a) and (b)), zofenoprilat (ZOF,
(a) and (c)), lisinopril (LIS,
(a) and (d)), enalaprilat (ENA,
(a) and (e)) and bradykinin (BK,
(a) and (f)) on endothelin-1 secretion (expressed as % of control) by vascular
endothelial cells derived from umbilical cord vein after 8 hours of incubation
both alone and in the presence of either Des-Arg9-[Leu8]-BK,
that is, a bradykininB1 receptor antagonist (B1ra, 10−6M), or D-Arg-[Hyp3,Thi5,8,D-phe7]-BK,
that is, a bradykinin B2 receptor antagonist (B2ra, 10−6M), or the NO synthase competitive inhibitor
Nω-nitro-L-arginine methyl ester (L-NA, 3×10−3M).  (A) P<.0008
or less versus CPT,
ZOF, LIS, ENA, and BK; (B) P<.04  or less versus ZOF; (C) P<.0005
or less versus CPT+B1ra; (D) P<.0003 or less versus ZOF+B1ra,
ZOF+B2ra, and ZOF+L-NA; (E) P<.002  versus ZOF+B1ra; (F) P<.0001
versus LIS+B1ra; (G) P<.0002 or less versus ENA+B1ra; (H) P<.0001
versus BK+B1ra.
(a)(b)(c)(d)(e)(f)


### 3.2. Increased NO Availability Is Responsible for the Inhibitory Effect of ACE-I on Endothelin-1 Secretion by HUVECs

All tested ACE-I at10−8M and bradykinin
at 10−8M significantly increased spontaneous NOx concentrations in
culture medium (see Figure 2(a)). ACE-I and bradykinin related changes in NOx
concentrations were counteracted by previous incubation of HUVECs with the
bradykinin B2 receptor antagonist D-Arg-[Hyp3,Thi5,8,D-phe7]-BK
at 10−6M (see Figures 2(b)–2(f)) while the
bradykinin B1 receptor antagonist Des-Arg9-[Leu8]-BK
at 10−6M was completely ineffective in this setting (see Figures 2(b)–2(f)). The
preincubation of HUVECs with the NO synthase inhibitor L-NA at 3×10−3M completely abolished the effects of ACE-I and bradykinin on NOx production (see
Figures 2(b)–2(f)). In
addition, L-NA also counteracted the inhibitory effect of ACE-I on ET-1
production (see Figures 1(b)–1(f)). These data
suggest that increased NO production plays a pivotal role in the inhibitory
effect of ACE-I on endothelin-1 secretion by HUVECs.Although all four tested ACE-I were effective in
increasing NOx concentrations in culture medium, this effect was more evident
in the presence of zofenoprilat (see Figure 2(a)). In addition, bradykinin B2 receptor inhibition was only partially effective in counteracting
zofenoprilat-induced increment of NOx concentrations in culture medium (see Figure
2(c)).Effects of10−8M captopril (CPT,
(a) and (b)), zofenoprilat (ZOF,
(a) and (c)), lisinopril (LIS,
(a) and (d)), enalaprilat (ENA,
(a) and (e)), and bradykinin (BK,
(a) and (f)) on nitric oxide production, as evaluated by nitrite plus nitrate
concentrations in culture medium (expressed as % of control) by vascular
endothelial cells derived from umbilical cord vein after 8 hours of incubation
both alone and in the presence of either Des-Arg9-[Leu8]-BK,
that is, a bradykinin B1 receptor antagonist (B1ra, 10−6M), or D-Arg-[Hyp3,Thi5,8,D-phe7]-BK,
that is, a bradykinin B1 receptor antagonist (B2ra, 10−6M), or the NO synthase competitive inhibitor Nω-nitro-L-arginine methyl ester (L-NA,
3×10−3M). (A) P<.0001 versus control; (B) P<.02
or less versus CPT, LIS, ENA, and BK and P<.0001 versus control; (C) P<.0003
or less versus control, CPT+B2ra and CPT+L-NA; (D) P<.0001 versus
control and ZOF+L-NA and P<.003 versus ZOF+B2ra; (E) P<.003
versus control and ZOF+L-NA; (F) P<.0004 or less versus control,
LIS+B2ra and LIS+L-NA; (G) P<.0006 or less versus control, ENA+B2ra
and ENA+L-NA; (H) P<.0001 versus control, BK+B2ra and BK+L-NA.
(a)(b)(c)(d)(e)(f)


### 3.3. ACE-I Reduces Intracellular Oxidative Stress and
Increases GSH Content

HUVECs preincubation with10−8M of
zofenoprilat and captopril, but not with enalaprilat and lisinopril, was resulted
in a significant decrease of TNFα-stimulated
generation of reactive oxygen species (see Figure 3(a)). Although both
sulfhydryls containing ACE-I reduced TNFα-stimulated
reactive oxygen species generation in cultured HUVECs, zofenoprilat was more
effective than captopril in this setting (see Figure 3(a)). In keeping with this,
zofenoprilat but not captopril, lisinopril, and enalaprilat significantly
protected HUVECs against the GSH decrease observed after incubation with TNFα (see Figure
3(b)).Effects of10−8M captopril
(CPT), zofenoprilat (ZOF), lisinopril (LIS), and enalaprilat on TNFα-induced
intracellular oxidative stress as evaluated by dichlorofluorescein (DCF,
(a)) and glutathione (GSH, (b)) content. (A) P<.01 or less versus control and TNF+ZOF
and P<.002 versus TNF+CPT; (B) P<.0001 versus control and P<.01 versus
TNF+ZOF. (C) P<.0001 versus TNF, TNF+CPT, TNF+LIS, and TNF+ENA and P<.02
versus TNF+ZOF; (D) P<.0004 or less versus TNF, TNF+CPT, TNF+LIS, and
TNF+ENA.
(a)(b)


## 3.1. ACE-I Counteracts Endothelin-1 Secretion by HUVEC: Evidence of Different Pathway

All tested ACE-I at10−8M reduced
spontaneous ET-1 secretion by HUVECs to a level which was similar to that
induced by bradykinin (see Figure 1(a)). Preincubation with the bradykinin B2 receptor antagonist D-Arg-[Hyp3,Thi5,8,D-phe7]-BK
but not the bradykinin B1 receptor antagonist Des-Arg9-[Leu8]-BK
both at 10−6M abolished
the inhibitory effect of ACE-I on ET-1 secretion by HUVECs (see Figures 1(b)–1(f)). These data
suggested that bradykinin B2 receptor stimulation was involved in
the inhibitory effect of the four tested ACE-Is on ET-1 secretion. Although all
the four tested drugs counteracted spontaneous ET-1 secretion by HUVECs,
zofenoprilat was more effective than the other ACE-I in this setting. In
addition, the inhibitory effect of zofenoprilat on ET-1 secretion was only
partially counteracted by bradykinin B2 receptor inhibition (see Figure
1(c)). This finding indicated that bradykinin  B2 receptor
stimulation does
not represent the only pathway involved in the inhibitory effect of
zofenoprilat on ET-1 production by HUVECs.Effects of10−8M captopril (CPT,
(a) and (b)), zofenoprilat (ZOF,
(a) and (c)), lisinopril (LIS,
(a) and (d)), enalaprilat (ENA,
(a) and (e)) and bradykinin (BK,
(a) and (f)) on endothelin-1 secretion (expressed as % of control) by vascular
endothelial cells derived from umbilical cord vein after 8 hours of incubation
both alone and in the presence of either Des-Arg9-[Leu8]-BK,
that is, a bradykininB1 receptor antagonist (B1ra, 10−6M), or D-Arg-[Hyp3,Thi5,8,D-phe7]-BK,
that is, a bradykinin B2 receptor antagonist (B2ra, 10−6M), or the NO synthase competitive inhibitor
Nω-nitro-L-arginine methyl ester (L-NA, 3×10−3M).  (A) P<.0008
or less versus CPT,
ZOF, LIS, ENA, and BK; (B) P<.04  or less versus ZOF; (C) P<.0005
or less versus CPT+B1ra; (D) P<.0003 or less versus ZOF+B1ra,
ZOF+B2ra, and ZOF+L-NA; (E) P<.002  versus ZOF+B1ra; (F) P<.0001
versus LIS+B1ra; (G) P<.0002 or less versus ENA+B1ra; (H) P<.0001
versus BK+B1ra.
(a)(b)(c)(d)(e)(f)


## 3.2. Increased NO Availability Is Responsible for the Inhibitory Effect of ACE-I on Endothelin-1 Secretion by HUVECs

All tested ACE-I at10−8M and bradykinin
at 10−8M significantly increased spontaneous NOx concentrations in
culture medium (see Figure 2(a)). ACE-I and bradykinin related changes in NOx
concentrations were counteracted by previous incubation of HUVECs with the
bradykinin B2 receptor antagonist D-Arg-[Hyp3,Thi5,8,D-phe7]-BK
at 10−6M (see Figures 2(b)–2(f)) while the
bradykinin B1 receptor antagonist Des-Arg9-[Leu8]-BK
at 10−6M was completely ineffective in this setting (see Figures 2(b)–2(f)). The
preincubation of HUVECs with the NO synthase inhibitor L-NA at 3×10−3M completely abolished the effects of ACE-I and bradykinin on NOx production (see
Figures 2(b)–2(f)). In
addition, L-NA also counteracted the inhibitory effect of ACE-I on ET-1
production (see Figures 1(b)–1(f)). These data
suggest that increased NO production plays a pivotal role in the inhibitory
effect of ACE-I on endothelin-1 secretion by HUVECs.Although all four tested ACE-I were effective in
increasing NOx concentrations in culture medium, this effect was more evident
in the presence of zofenoprilat (see Figure 2(a)). In addition, bradykinin B2 receptor inhibition was only partially effective in counteracting
zofenoprilat-induced increment of NOx concentrations in culture medium (see Figure
2(c)).Effects of10−8M captopril (CPT,
(a) and (b)), zofenoprilat (ZOF,
(a) and (c)), lisinopril (LIS,
(a) and (d)), enalaprilat (ENA,
(a) and (e)), and bradykinin (BK,
(a) and (f)) on nitric oxide production, as evaluated by nitrite plus nitrate
concentrations in culture medium (expressed as % of control) by vascular
endothelial cells derived from umbilical cord vein after 8 hours of incubation
both alone and in the presence of either Des-Arg9-[Leu8]-BK,
that is, a bradykinin B1 receptor antagonist (B1ra, 10−6M), or D-Arg-[Hyp3,Thi5,8,D-phe7]-BK,
that is, a bradykinin B1 receptor antagonist (B2ra, 10−6M), or the NO synthase competitive inhibitor Nω-nitro-L-arginine methyl ester (L-NA,
3×10−3M). (A) P<.0001 versus control; (B) P<.02
or less versus CPT, LIS, ENA, and BK and P<.0001 versus control; (C) P<.0003
or less versus control, CPT+B2ra and CPT+L-NA; (D) P<.0001 versus
control and ZOF+L-NA and P<.003 versus ZOF+B2ra; (E) P<.003
versus control and ZOF+L-NA; (F) P<.0004 or less versus control,
LIS+B2ra and LIS+L-NA; (G) P<.0006 or less versus control, ENA+B2ra
and ENA+L-NA; (H) P<.0001 versus control, BK+B2ra and BK+L-NA.
(a)(b)(c)(d)(e)(f)


## 3.3. ACE-I Reduces Intracellular Oxidative Stress and
Increases GSH Content

HUVECs preincubation with10−8M of
zofenoprilat and captopril, but not with enalaprilat and lisinopril, was resulted
in a significant decrease of TNFα-stimulated
generation of reactive oxygen species (see Figure 3(a)). Although both
sulfhydryls containing ACE-I reduced TNFα-stimulated
reactive oxygen species generation in cultured HUVECs, zofenoprilat was more
effective than captopril in this setting (see Figure 3(a)). In keeping with this,
zofenoprilat but not captopril, lisinopril, and enalaprilat significantly
protected HUVECs against the GSH decrease observed after incubation with TNFα (see Figure
3(b)).Effects of10−8M captopril
(CPT), zofenoprilat (ZOF), lisinopril (LIS), and enalaprilat on TNFα-induced
intracellular oxidative stress as evaluated by dichlorofluorescein (DCF,
(a)) and glutathione (GSH, (b)) content. (A) P<.01 or less versus control and TNF+ZOF
and P<.002 versus TNF+CPT; (B) P<.0001 versus control and P<.01 versus
TNF+ZOF. (C) P<.0001 versus TNF, TNF+CPT, TNF+LIS, and TNF+ENA and P<.02
versus TNF+ZOF; (D) P<.0004 or less versus TNF, TNF+CPT, TNF+LIS, and
TNF+ENA.
(a)(b)


## 4. Discussion

The ability of ACE-I to counteract ET-1 production by
endothelial cells [7, 9] has been proposed as a relevant contributor to the
well-known vascular protective effects exerted by ACE-inhibition [7, 21].
Indeed, although tonic ET-1 production by endothelial cells physiologically
contributes to vascular tone [21, 22], this peptide has per se all the biological potential to contribute to the onset
and progression of atherosclerotic vascular damage [8, 21]. The current report
provides evidence that ACE-I,
tested at concentrations that fully inhibited the ACE, do not share in common
similar efficacy in counteracting
ET-1 release from vascular endothelial cells. Indeed, we found that
zofenoprilat was more effective than captopril, lisinopril, and enalaprilat in
reducing ET-1 secretion from cultured HUVECs. In addition, our data demonstrate
that different intracellular pathways
are involved in the inhibitory effects of the four tested ACE-Is on ET-1
secretion. In this context, it has been
previously demonstrated that the inhibitory effect of ACE-I on ET-1 production
by HUVECs is due to a bradykinin B2 receptor-mediated increase in NO
production by HUVECs [9].As known, oxygen derived free radical can inactivate
NO [12]. In turn, NO represents a barrier against oxidants such as unscavenged
superoxide anion [23]. Thus, it is reasonable to speculate that the greater
effects observed with zofenoprilat in reducing ET-1 secretion and increasing NO
production by cultured HUVECs might have been due to its antioxidant properties
[11, 24]. In keeping with this, zofenoprilat and at lesser extent captopril,
but not lisinopril and enalaprilat, were able to decrease generation of
reactive oxygen species induced by TNFα in
HUVECs. Further, zofenoprilat but not the two nonsulfhydryl containing ACE-I
lisinopril and enalaprilat blunted the GSH  decrease in HUVECs induced by TNFα.
Since sulfhydryls containing
ACE-I are supposed to act as antioxidants as the endogenous free radical
scavenger GSH [2, 10, 11], both these findings suggest that the sulfhydryl
group can be the responsible for the effect of zofenoprilat in reducing ET-1
production, that is, because of sulfhydryl-related scavenging capability and
the consequent decrease in NO inactivation by endogenous oxidants. In agreement
with this hypothesis, Cominacini et al. [24] demonstrated
that zofenoprilat,
but not enalapril, protected the intracellular proinflammatory pleiotropic
mediator nuclear factor κB against
oxidant-induced activation and was able to spare GSH from consumption induced
by oxLDL. Likewise, sulfhydrylic ACE-Is have been reported to protect cultured
endothelial cells against the damage induced by both superoxide and
nonsuperoxide radicals [11] and to decrease LDL susceptibility to oxidation in
hypertensive patients [25, 26].However, the presence of a sulphydryl group in
zofenoprilat molecule does not completely explain our findings. Indeed, the
other sulfhydryl containing ACE-I tested in our study, captopril, was mildly
but not significantly more effective than lisinopril and enalaprilat in our
tests. These data agree with previous findings obtained in leucocytes and
endothelial cells, demonstrating that captopril poorly scavenged newly generated
superoxide anion [27, 28]. In this regard, zofenoprilat displays higher
lipophilicity than captopril, suggesting it could exert more pronounced
intracellular effects [18]. Worth mentioning in this regard, the recent
evidence by Soardo et al. [29] demonstrating a stronger inhibitory effect of
zofenoprilat on alcohol-induced ET-1 production by endothelial cells in
comparison to carvedilol, a beta adrenoceptor blocker with known antioxidant
activity [30].In conclusion, the sulfhydryl containing ACE-I
zofenoprilat, that is, the active drug of the prodrug zofenopril [18], was more
effective than the nonsulfhydryl containing ACE-I lisinopril and enalapril and
the sulfhydryl containing one captopril in reducing ET-1 secretion by cultured
HUVECs and improving NO bioavailability. These findings likely reflect
different antioxidant power between the four tested ACE-Is. Since both
increased ET-1 production and decreased NO bioavailability are deeply involved
in the pathophysiology of atherosclerosis [22], reciprocal changes in ET-1 and
NO production by the vascular endothelium could contribute to the benefits
deriving from clinical use of ACE inhibitors [2]. The presence of a sulfhydryl
group confers to ACE inhibitors some ancillary properties, such as greater
protection against LDL oxidation [26] and nuclear factor κB
activations [24], scavenging of superoxide anion [10] and nonsuperoxide radical
[11], and, as demonstrated in this study, more pronounced favourable effects on
ET-1/NO balance in vascular endothelial cells. Whether or not these endothelial
effects of zofenoprilat could contribute to the observed cardiovascular
benefits deriving from zofenopril treatment [31] remains to be elucidated.



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*Source: 305087-2008-12-01.xml*