Perindopril Induces TSP-1 Expression in Hypertensive Patients with Endothelial Dysfunction in Chronic Treatment

Thrombospondin-1 (TSP-1) is a potent endogenous inhibitor of both physiological and pathological angiogenesis, widely studied as a target in drug development for treating cancer. Several studies performed in the cardiovascular field on TSP-1 are contradictory, the role of TSP-1 in the physiopathology of cardiovascular disorders (CVDs) being, for the moment, incompletely understood and may be due to the presence of several domains in its structure which can stimulate many cellular receptors. It has been reported to inhibit NO-mediated signaling and to act on the angiogenesis, tissue perfusion, endothelial cell proliferation, and homeostasis, so we aimed to quantify the effect Perindopril has on TSP-1 plasma levels in hypertensive patients with endothelial dysfunction in comparison with other antihypertensive drugs, such as beta blockers, calcium channel blockers, and diuretics, in a chronic treatment. As a conclusion, patients under treatment with Perindopril had increased plasma levels of TSP-1 compared with other hypertensive patients and with the control group. The results of this study confirms the pleiotropic properties of Perindopril: anti-proliferative, anti-inflammatory, with effects showed by quantifying a single biomarker: TSP-1.


Introduction
Thrombospondin-1 (TSP-1) is a potent endogenous inhibitor of both physiological and pathological angiogenesis, widely studied as a target in the drug development for treating cancer.
TSP-1 is the most studied member of the family of thrombospondins, a family which consists of five multimeric, multidomain calcium-binding glycoproteins that act as regulators of cell-cell and  (2) 69.14 ± 7.18 (2) <0.001 Data are represented as mean ± standard deviation (SD). HTN: hypertension, SBP: systolic blood pressure, DBP: diastolic blood pressure. Statistical significance was considered at a p-value < 0.05. (1) A significant difference when compared with the control group; (2) A significant difference between both hypertensive groups. Table 2 shows the biochemical data for all of the groups. A significant difference between groups can be noticed in what concerns the cholesterol level of Group C (patients under treatment with Perindopril), due to a possible mild dislipidemia. Additionally, the creatinine levels were higher in this group, with mild renal impairment being one of the first choices for angiotensin-converting enzyme (ACE) inhibitors in hypertensive patients, as a result of their action of decreasing the proteinuria and offering renal protection due to the amelioration of ED. Furthermore, it can be noticed that a higher level of systemic inflammation in Group C was statistically significant, reflected through the erythrocyte sedimentation rate (ESR) levels.
Concerning the other parameters, there were no significant statistical differences between the groups having the plasma values being in the normal range. Table 3 shows the principal indicators of the presence/absence of ED in the three groups. The patients from Group A had no endothelial dysfunction (reflected throughout the flow mediated vasodilatation-FMD value), compared with the other two groups of hypertensive patients. In Group B, the degree of ED was higher, compared with Group C and statistically significant. The plasma levels of high-selectivity C reactive protein were higher in Group C, compared with the levels of other groups, and also the number of people who were smokers was higher in this group. The TSP-1 plasma level was also the highest in this group, being very close to the value considered statistical significant (p < 0.05). The intima-media thickness (IMT) values were greater in both groups of hypertensive patients. Additionally, the pentraxin-3 (PTX3) plasma levels were the smallest in the C group who were under treatment with perindopril. The echocardiography showed a septal hypertrophy for all hypertensive patients (IVS > 11 mm) compared with the control group, without any statistical significance between the three groups. ( Table 4). All of the other echocardiographic parameters were within their normal range, in all three groups, without any significant difference. The analyses performed in order to determine the correlations between the plasma levels of TSP-1 and other parameters in both groups of hypertensive patients, and in the control group (Tables 5-7) showed that TSP-1 correlated positively (r > 0.3) only in Group B, with tryglicerides, leucocites, and neutrophils. Table 5. Correlation of TSP-1 plasma levels with all data in Group C.

Thrombospondin-1 Correlation
Age (years) --r = 0.025, p < 0.001 HTN duration (months) --r = 0.022, p < 0.001 SBP (mmHg) --r = 0.005, p < 0.001 DBP (mmHg) --r = 0.045, p < 0.001 Cholesterol (mg/dL) --r = 0.0004, p < 0.001 Triglicerides (mg/dL) --r = 0.01, p < 0.001 Creatinine (mg/dL) --r = 0.001, p < 0.001 Glucose (mg/dL) --r = 0.015, p < 0.001 Potassium (mmol/L) --r = 0.005, p < 0.001 Heart rate (beats/min) -- --, no correlation. Table 6. Correlation of TSP-1 plasma levels with all data in Group B. Table 7. Correlation of TSP-1 plasma levels with all data in Group A. Figure 1 shows that patients treated with the lowest concentration of Perindopril (5 mg/day) had the lowest TSP-1 plasma levels, compared with those who were under treatment with the highest concentration of Perindopril available on the market (10 mg/day). Figure 1 shows that patients treated with the lowest concentration of Perindopril (5 mg/day) had the lowest TSP-1 plasma levels, compared with those who were under treatment with the highest concentration of Perindopril available on the market (10 mg/day).    Figure 1 shows that patients treated with the lowest concentration of Perindopril (5 mg/day) had the lowest TSP-1 plasma levels, compared with those who were under treatment with the highest concentration of Perindopril available on the market (10 mg/day).

Discussion
Perindopril belongs to the third generation of ACE inhibitors, intensively prescribed and studied. ACE inhibitors are a group of drugs used and listed as first-line agents in the treatment of hypertension, congestive heart failure, myocardial infarction, and left ventricular systolic dysfunction, and are either used alone or in combination with other types of drugs with different action mechanisms [30][31][32][33][34][35][36]. All of the positive effects on the pathologies mentioned previously are the consequences of their mechanism of action: firstly, they block the angiotensin (Ang)-converting enzyme the enzyme that transforms Ang I (found in the vascular endothelium of lungs and other organs and in lower concentrations in the vascular plasma) in Ang II, decreasing the circulating levels of Ang II. Due to the decreased synthesis of Ang II, the plasmatic levels of Ang I are increased, and through a feed-back phenomenon, Ang II induces the stimulation of the rennin synthesis and lowers the levels of angiotensinogen, due to its high consumption. The decreased levels of Ang II induce decreased effects of the peptide on the vascular tonus, the synthesis and liberation of aldosteron, the sodium-potassium balance, etc.
Secondly, due to the fact that ACE is identical with the enzyme that degrades bradykinin (BK), kinase II, Perindopril and other ACE inhibitors increase the bradykinin concentration and stimulate the action of endogenous kinins to produce nitric oxide (NO), prostaciclins, and EDHF (endotheliumderived hyperpolarizing factor), through the stimulation of bradykinin B2 receptors, having, as a consequence, the vascular protective effects and the reversed ED installed [34,36,37]. The inhibition of the BK degradation is the main mechanism (known at the moment) by which ACE inhibitors induce cardiovascular protection. This effect may differ between ACE inhibitors as it depends on the degree of the tissue affinity and on their binding to BK. Additionally, ACE inhibitors have a higher

Discussion
Perindopril belongs to the third generation of ACE inhibitors, intensively prescribed and studied. ACE inhibitors are a group of drugs used and listed as first-line agents in the treatment of hypertension, congestive heart failure, myocardial infarction, and left ventricular systolic dysfunction, and are either used alone or in combination with other types of drugs with different action mechanisms [30][31][32][33][34][35][36]. All of the positive effects on the pathologies mentioned previously are the consequences of their mechanism of action: firstly, they block the angiotensin (Ang)-converting enzyme the enzyme that transforms Ang I (found in the vascular endothelium of lungs and other organs and in lower concentrations in the vascular plasma) in Ang II, decreasing the circulating levels of Ang II. Due to the decreased synthesis of Ang II, the plasmatic levels of Ang I are increased, and through a feed-back phenomenon, Ang II induces the stimulation of the rennin synthesis and lowers the levels of angiotensinogen, due to its high consumption. The decreased levels of Ang II induce decreased effects of the peptide on the vascular tonus, the synthesis and liberation of aldosteron, the sodium-potassium balance, etc.
Secondly, due to the fact that ACE is identical with the enzyme that degrades bradykinin (BK), kinase II, Perindopril and other ACE inhibitors increase the bradykinin concentration and stimulate the action of endogenous kinins to produce nitric oxide (NO), prostaciclins, and EDHF (endothelium-derived hyperpolarizing factor), through the stimulation of bradykinin B 2 receptors, having, as a consequence, the vascular protective effects and the reversed ED installed [34,36,37]. The inhibition of the BK degradation is the main mechanism (known at the moment) by which ACE inhibitors induce cardiovascular protection. This effect may differ between ACE inhibitors as it depends on the degree of the tissue affinity and on their binding to BK. Additionally, ACE inhibitors have a higher affinity for bradykinin sites compared with the affinity for Ang I, so they primarily inhibit BK degradation and then the production of Ang II [38].
We choose Perindopril for this study because of its pharmacokinetics and pharmacodynamic properties: the highest bioavailability (75%), the highest terminal elimination half-life of the major active ingredient (30-120 h), a higher time to reach maximum plasma concentration, the highest affinity for BK binding sites when compared with other substances from its class: Enalapril, Lisinopril, Quinapril, Ramipril [39], high lipophilicity and tissue penetration among other ACE inhibitors [38], prolonged duration of action [39], prolonged inhibition of ACE (>48 h) [40], and 24 h efficacy, which implies a low number of administrations per day and a higher compliance for patients to the treatment [39]. Moreover, it has been shown to exert potent anti-apoptotic actions on the endothelium and on the cardiac myocytes [38]. Regarding the study that we performed, the results presented in Tables 2 and 3 clearly suggest the fact that, although the patients under chronic treatment with Perindopril have more risk factors compared with those under treatment with other antihypertensive drugs (beta blockers, calcium channel blockers, diuretics)-this conclusion being based on higher cholesterol levels, BMI, number of smokers, level of systemic inflammation (ESR and hs-CRP plasma levels)-the degree of ED is lower in this group (reflected by the FMD level) and the degree of local endothelial inflammation is also lower in the same group (reflected through the PTX3 plasma levels, an acute inflammatory marker, secreted by the endothelial cells). We can conclude that, based on these parameters, the endothelial function is better preserved by an ACE inhibitor than any other antihypertensive drugs (beta blockers, calcium channel blockers, diuretics) in a chronic treatment.
As for the TSP-1 plasma levels, although the difference between the groups have not reached the level of statistical significance (p = 0.08, p < 0.05), the plasma values of this biomarker are higher in the group of patients treated with Perindopril (Group C). Based on the evidence reported in the literature and discussed in the introduction about the effects of TSP-1 on the vascular endothelium (although they are contradictory and opposing), in our study, and based on the studies reported by others, the low levels of TSP-1 might be an inducer of VSMC proliferation and that higher levels of TSP-1 could induce an anti-proliferative effect on VSMCs by reducing its density (throughout the binding of TSP-1 C-terminal domain to CD47-binding peptide) [40][41][42][43]. Thus, in normal low levels, TSP-1 could act as an inducer of VSMC proliferation and, in pathological conditions (e.g., atherosclerosis), whereas TSP-1 is upregulated, high levels of TSP-1 could act as an anti-proliferative agent, although the effects of TSP-1 could be different in function of the pathology concerned [6].
TSP-1 has been also reported to play an important role in decreasing inflammation and, thus, having an anti-inflammatory action, due to its interaction with CD47 receptor expressed on T-cells and polymorphonuclear cells [44]. So, based on our study, we may say that, in Group C, the patients under chronic treatment with Perindopril, have a more decreased level of endothelial inflammation, reflected through the TSP-1 plasma levels and not only through PTX3.
No correlation had been found between TSP-1 serum levels and the disease severity and/or duration, as Tables 5 and 6 show.
The data found in the literature about the relationship between TSP-1 and leucocytes [45][46][47][48] confirms the same idea found in our study, that TSP-1 plays an important role in the recruitment of monocytes and macrophages to the site of tissue inflammation or injury. In the present study, we found positive correlation between TSP-1 and triglycerides, leucocytes, and neutrophils, correlations that show the local, vascular, inflammatory site in patients under chronic treatment with other antihypertensive drugs, except Perindopril. This is one more argument that Perindopril controls better the local endothelial inflammation and the vascular function.
In a study performed by Xia et al. in 2011 [49], authors suggested that TSP-1 expression might be involved in the regulation of fibroblast phenotypes and that it could prevent the left ventricular hypertrophy in pressure-overloaded hearts, so we may add another positive effect proved by this study for Perindopril: its anti-fibrotic effect.
We also found that the TSP-1 plasma levels are lower for men than for women. This study has several limitations, including the relatively low number of patients examined and the short observation time. Additionally, the study results cannot explain the molecular mechanisms by which Perindopril increases plasma TSP-1 levels in hypertensive patients with endothelial dysfunction; therefore, additional studies are needed to fully assess the effects of TSP-1 on vasculature.

Materials and Methods
This prospective, comparative study was performed in the Cardiology Clinic of the City Hospital of Timisoara (a city in the western part of Romania), from February 2015 to July 2016. It involved a number of 351 patients, 105 normotensive patients (Group A), and 246 hypertensive patients (Group B and C), matched by age and sex.

Patient Selection
The normotensive patients (Group A), the control group, were initially examined for suspicion of HTN, but the diagnosis had not been confirmed by the 24 h ambulatory monitoring device.
Patients in Group C (n = 129) were under chronic treatment with Perindopril (5 or 10 mg/day), an angiotensin-converting enzyme (ACE) inhibitor.
Hypertensive patients were included under the following criteria: age >18 years old, who were diagnosed with essential arterial hypertension (blood pressure levels >140/90 mmHg) for at least one year and had received monotherapy with one of the drugs mentioned above.
Patients with other pathologies including atherosclerotic disease, diabetes, coronary artery disease, heart failure, kidney disease, asthma, hepatic disease, and acute or chronic inflammatory conditions were excluded. Additionally, patients treated with other ACE inhibitors or ARBs (antagonist receptor blockers) were excluded from this study due to the similar action in reversing the endothelial dysfunction.
All of the participants from the present study agreed to participate voluntarily and provided written informed consent.
Hypertensive patients (Group B and C) underwent screening including a physical examination and a medical history (duration of hypertension, current treatment, familial history, and other associated medical pathologies).

Laboratory Analysis
Patients were fasted for >10 h and venous blood samples were withdrawn early in the morning, in a temperature-controlled room. Standard biochemical analysis (e.g., serum glucose, creatinine, triglycerides, total cholesterol) was performed in the hospital laboratory by routine methods. Plasma concentrations of hs-CRP (high-sensitivity C-reactive protein), PTX3 (pentraxin-3), and TSP-1 were determined at Bioclinica SA Laboratory in Timisoara. PTX3 and TSP-1 plasma levels were determined by quantitative sandwich enzyme immunoassays (R and D Systems, Minneapolis, MN, USA) and plasma hs-CRP levels were measured using CRP Ultra Kits (Abbott Diagnostics, Wiesbaden, Hesse, Germany), using a highly sensitive immunoturbidimetric method. All of these methods are standardized methods, so we consider that there is no need to repeat the methods here.

Arterial Pressure
Blood pressure was measured three times in the right brachial artery of each patient, in the same temperature-controlled room, after 30 min of rest, with the patient in a supine position. The reported value of pressure was expressed as the mean of three measurements. We calculated the mean arterial blood pressure as follows: (2 × diastolic pressure + systolic pressure)/3.

Assessment of Flow-Mediated Dilation (FMD)
The assessment of flow-mediated dilation is, nowadays, the most common, non-invasive technique and is used to evaluate the vascular endothelial function in humans. It was developed in 1992 and, since its inception, ongoing efforts have been made to perform the original methodology in a more accurate, exact, and precise method. It consists of the capacity of the blood vessel to adapt to the increasing of blood pressure, a process that is dependent of the endothelium production of NO [50][51][52][53].
This procedure was performed after the patient fasted, stopped taking the vasoactive medication, and did not smoke for at least 10 h, based on the literature data. After resting for 10 min in a quiet room, the patient was placed in a supine position. His arm was placed in a comfortable position and the brachial artery was imaged above the antecubital fosa in the longitudinal plane, using a linear transducer in 9 MHz mode. The diameter of the brachial artery was measured manually with electronic calipers. In order to produce the local ischemia (to evaluate the ability of the endothelium to produce NO), the cuff of a manometer was inflated to a pressure of 50 mmHg higher than the systolic blood pressure of the patient. After 5 min of hyperemia, the cuff was released, increasing the diameter of the brachial artery. The maximum diameter was measured 1 min after the cuff release and the FMD was defined as the percent change in diameter from the rest to 1 min after the ischemia, considering a 20% increase as normal (healthy endothelium) and under 20% as endothelial dysfunction.

Carotid Intima-Media Thickness (IMT)
Carotid intima-media thickness was determined, as agreed in the Mannheim Consensus [54], at the common carotid artery at baseline in both carotid arteries, with the same equipment, a General Electric medical system VIVID S5 (General Electric Co., Horten, Norway) equipped with a 9 MHz linear array transducer (General Electric Co., Horten, Norway). Patients were examined in a supine position and the IMT was given by the built-in software (5432774-167, General Electric Co., Horten, Norway) of the ultrasound system. The reported value was the mean values of three measurements in each patient for the left and for the right carotid, 1 cm before the bifurcation. The following were considered as normal values: 0.9-1.1 mm, and values >1.2 mm as the presence of atherosclerotic plaque.

Echocardiography
The echocardiography was performed using a high-resolution ultrasonography medical system, VIVID S5, to assess the effects of hypertension on the structures and functions of the heart. We excluded patients with low ejection fractions (<40%), to avoid the interference in our results regarding the ED. The parameters evaluated included the diameter of the left atrium (DLA), the dimensions of the interventricular septum (IVS), posterior wall of the left ventricle (PWLV), the end diastolic diameter of the left ventricle (EDLV), the ejection fraction (EF), and the shortening fraction (SF).
All of the measurements were performed by the same certified physician.

Statistical Analysis
The numerical data was presented as mean ± standard deviation (SD) and the categorical data was presented as the frequency (%). Differences among groups were analyzed using ANOVA and Kruskal-Wallis tests. Correlations between groups or variables were found using Spearman's correlation coefficient. All statistical analyses were performed using SPSS v.17 statistical software (v.17, SPSS Inc., Chicago, IL, USA). A p-value < 0.05 was considered statistically significant.

Compliance with the Ethical Standards
The present study has been approved by the Ethical Committee (29 June 2016) of the "Victor Babes" University of Medicine and Pharmacy, Timisoara, Romania, no. 7/2016. All procedures performed in this study with human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Conclusions
The present study is innovative because, to our knowledge, no other study assessed the efficacy of Perindopril in hypertensive patients with endothelial dysfunction by quantifying TSP-1 plasma levels, compared with other antihypertensive drugs such as beta blockers, calcium channel blockers, and diuretics. Compared with other antihypertensive drugs that were studied here, Perindopril has better properties in reversing and controlling the processes of endothelial dysfunction. In the future, a study that quantifies the effects of an ARB (angiotensin receptor blocker) on TSP-1 plasma levels in hypertensive patients with ED will be necessary.