Growing evidence supported a tight relationship between Ang II and inflammation, because Ang II up-regulates the inflammatory response and inflammatory T-cells are fully equipped with RAS components and deliver new synthesized Ang II to sites of inflammation, independently of the activation of systemic and tissuebased RAS [1-3]. Human circulating T-cells but not B-cells are endowed with autonomous cell-based RAS [4].

Ang II participates in the recruitment of T-cells via the direct activation of inflammatory cells or via the up-regulation of the synthesis of adhesion molecules and/or chemokines by resident cells [2,3].

The ACE gene expression and enzymatic activity have a key-role in the increase and decrease in Ang II synthesis associated to systemic, local or cell-based RAS activation or inhibition [5]. An increased expression of ACE gene and activity was reported in circulating T-cells in hypertensive patients with low grade inflammation [6]; augmented levels of tissue ACE strictly related to the amount of infiltrated inflammatory T-cells were shown in coronary plaques and microvessels of left ventricular tissues of unstable angina patients [7,8], but the direct involvement of T-cells based RAS in unstable angina is still a matter of debate. In acute coronary syndromes (ACS) an imbalance of T-cell homeostasis does occur and a higher systemic frequency of activated T-cells compared with those with stable angina was reported; T-cell repertoire perturbation plays a pivotal role in coronary instability [9-12]; however, at the best of our knowledge whether and how an up-regulation of circulating T-cell based ACE gene expression and activity could participate to ACS inflammation was not studied.

The present study was aimed at investigating the pattern of T-cell based ACE gene expression and enzymatic activity (assayed in pellet and in supernatant) in cultured circulating T-cells obtained by ACS patients with high (>3 mg/dl) sensible protein C reactive (hsCRP) values. The same data were also obtained in T-cell from control subjects with normal hsCRP values and from hypertensives with low grade inflammation (hsCRP>2 mg/dl).

Subjects investigated

T-lymphocytes were obtained from 11 (6 males, 5 females, 57-69 years) patients with unstable angina (UA) in class IIIB of Braunwald’s classification admitted to our Coronary Care Unit and from 15 (9 males, 6 females, 45-67 years) subjects with World Health Organization stage II essential hypertension who attended to our outpatients’ clinic. Ten (6 males, 4 females, 39-56 years) normotensive subjects were enrolled in the study as control group comparable for age and sex to anginal patients and hypertensive group.

Diagnosis of UA patients was performed according to the 2015 European Society of Cardiology Guidelines for the management of ACS in patients presenting without persistent ST elevation [13].

Only patients with recent onset (7 days before admission) of angina who had at least 2 episodes of angina at rest or 1 episode lasting more than 20 minutes during the preceding 24 hours associated with transient ischemic ST-segment changes and high sensitivity troponin T levels <0.1 ng/mL (troponin T–negative) were included in the study. UA patients received nitrates.

Exclusion criteria were secondary forms of arterial hypertension, ischemic heart disease/acute coronary syndrome (only for hypertensive and controls) or stroke in the previous 6 months, impaired glucose tolerance or diabetes mellitus, renal failure (creatinine clearance <60 ml/min), presence of a major illness such as cancer, liver disease, chronic and acute inflammatory disease or infectious disease in the previous 3 months. No subject was on treatment with ACE-inhibitors, AT1 receptor antagonists, diuretics, statin or anti-inflammatory drugs. Main demographic and clinical characteristics of enrolled patients are shown in table 1.

Table 1: Clinical characteristics of controls, unstable angina and hypertensive patients.

According to hsCRP values at enrollment, only hypertensive patients with low grade inflammation (hsCRP>2 mg/l) had been included; assay of hsCRP levels and other general chemical tests were done as a part of a routine medical check-up.

The protocol of this study complies with the principles of the Helsinki declaration and was approved by the Ethical Committee of our Institution. All patients gave their written informed consent to participate and to have their data analyzed for the study.

Experimental procedures

T-lymphocyte isolation: Fifty milliliters of peripheral venous blood were drawn from the antecubital vein, collected in a sterile flask containing 3.8% sodium citrate (9/1, vol/vol) and T-lymphocytes isolation was carried out according to our previous report [4,6] (Figure 1). Magnetic beads conjugated with antihuman CD 3 (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) were used for positive selection of T-lymphocytes. In each incubation experiment, mRNA expression of ACE, INF-γ and ACE activity and Ang II concentrations were studied in cell pellet. ACE activity was also investigated in the supernatants, since ACE can be shed by T-cells [6]. To study the effect of stimulation by Ang II on ACE expression and activity and Ang II concentrations, T-lymphocytes were cultured in humidified atmosphere with 5% for 6 or 18 or 24 h with and without 0.1 pmol/l Ang II addition to the culture medium.

Figure 1: The flow chart illustrates total population studied divided in 3 cohorts with parameters investigated in each of them.

Real-Time PCR for the assay of ACE gene expression and RTPCR analysis for the assay of INF-gamma gene expression in T-cells from controls, hypertensive and anginal patients

Isolation of total RNA and real time PCR for ACE gene expression was performed as detailed in our previous paper [6]. Relative ACE gene expression was calculated as 2^-ΔCt (ΔC_t = C_t of the target gene minus C_t of beta-actin). mRNAs for INF-gamma were semi-quantified versus the housekeeping gene GAPDH by RT-PCR (Applied Biosystems) using specific primers.

Assay of ACE activity in the T-cell pellet and in the supernatant of cultured T-cells from controls, hypertensive and anginal patients

T-cell ACE activity was measured by the quantification of hyppuric acid (HA), produced during the enzymatic reaction of ACE on a synthetic substrate hyppuril-hystidyl-leucine (HHL) and assayed by HPLC analysis using a spectrophotometric detector at 228 nm absorbance [6].

Measurement of plasma renin activity (PRA), Ang II and hsCRP

PRA measurement was performed with a commercial kit (Sorin Biomedica). Ang II levels were determined by radioimmunoassay after C18 Sep-Pak cartridge extraction and HPLC separation [6]. hsCRP was measured by a commercial kit (Beckman, Brea, CA) with the lowest detection point of 0.1 mg/dl [6].

Statistical analysis

Data have been stored in a dedicated data-base and analyzed by IBM-SPSS 20 for Windows^® statistical software (IBM-SPSS Inc., USA). Variables were reported as mean ± SD. Within-groups comparisons (data at baseline vs. 24 h; baseline vs. 24 h plus Ang II and 24 h vs. 24 h plus angiotensin II) were made by paired Student t-test analysis. In order to analyze differences in ACE gene expression and enzymatic activity throughout the time-course among the three groups, an analysis of variance (ANOVA) for repeated measures was used. Statistical significance was taken as p<0.05 for all calculations.

ACE gene expression and ACE activity without Ang II stimulation in T-cells from control subjects, ACS and hypertensive patients

Figure 2 shows the pattern of ACE gene expression in T-cells cultured without Ang II stimulation in controls and in patients: in control subjects T-cell ACE gene expression was detectable at baseline and did not significantly change at 18 and 24 hours of cell-incubation. T-cells from ACS patients showed significantly higher (p<0.001) ACE mRNA levels than those expressed by T-cells from controls at baseline and throughout cell-incubation. Hypertensive patients had higher (p<0.01) T-cells ACE mRNA levels than those observed in T-cells from controls, but lower (p<0.01) than those by T-cells from ACS patients. No changes in ACE gene expression were observed during the incubation time.

Figure 2: T-lymphocytes ACE gene expression without and with Ang II–stimulation in controls, anginal and hypertensive patients.

Table 2 shows the different pattern of ACE activity assayed in the pellet and in the supernatant of T-cells cultured without Ang II stimulation: in cultured T-cell from control subjects, ACE activity was detectable in the pellet at baseline and at 24 hours. In the supernatant T-cell ACE activity from controls was no detectable at baseline and was very low at 24 hours of incubation. In anginal patients T-cell ACE activity was significantly higher (p<0.001) than that assayed in controls both in the pellet and in the supernatant; specifically, at 24 hours T-cell ACE activity did not change in the pellet, but significantly increased in the supernatant (p<0.001 vs controls). In hypertensive patients T-cell ACE activity in the pellet and in the supernatant was significantly (p<0.001) higher than those assayed in controls at baseline and at 24 hours and not significantly different when compared with ACS levels; in the supernatant ACE activity was significantly (p<0.001) lower than those observed in ACS patients at 24 hours (Table 2).

Table 2: ACE activity in pellet and supernatant and Ang II pellet concentration of T-lymphocytes from controls, unstable angina and hypertensive patients without or with the addition of 10^-13 mol/l Ang II in cell culture medium.

ACE gene expression and ACE activity in cultured T-cell from control subjects, anginal and hypertensive patients after Ang II stimulation

In control subjects the addition of 0.1 pmol/l Ang II to the T-cell culture medium caused a significant (p<0.01 vs no Ang II stimulus) increase in T-cell ACE mRNA level at the 24 hours of incubation (Figure 2). In T-cells from controls Ang II stimulus caused a significant increase (p<0.05) in T-cell pellet ACE activity at 24 hour and was associated with the spontaneous release of about the 20% of total T-cell ACE activity into the supernatant (Table 2).

Also in T-cells from anginal patients the addition of Ang II to the cell medium significantly (p<0.001) increased T-cell ACE mRNA levels; the rate of increase was higher than that observed in T-cell from control subjects and the highest peak value for ACE gene expression was reached at 18 hours. In T-cell from hypertensive patients the Ang II stimulus caused a significant (p<0.01 vs no Ang II addition) early (peak at 6 hours) increase in T-cell ACE gene expression that was respectively significantly higher (p<0.001) and lower (p<0.001) than that shown by T-cells from controls and from anginal patients respectively (Figure 2).

In T-cells from anginal and hypertensive patients the addition of Ang II to the cell medium was not associated with an increase in the ACE activity of the T-cell pellet, but with a significant increase in the ACE activity in the supernatant that reached about the 64% of the pellet ACE activity in cultured T-cells from hypertensives and about the 260% in cultured T-cells from ACS patients.

Effect of Ang II–stimulation on T-cell INF-gamma gene expression

In controls and in patients, T-cell INF-gamma gene expression was detectable under baseline condition and did not significantly change at 18 and 24 hours of cell- Conversely, the addition of Ang II to the T-cell culture was associated with a significant increase in T-cell INF-gamma mRNA levels, that peaked at the 24^th hour without differences between values observed in T-cells from controls, anginal and hypertensive patients (data not shown).

Ang II concentration in the pellet of cultured T-cells from controls, unstable angina and hypertensive patients

In the T-cell pellet from control subjects Ang II concentration was 176.5+17.9 fmol/mg proteins at baseline and did not significantly change after 24 hours of incubation; Ang II levels significantly increased after the addition of Ang II to the culture medium (Table 3). In T-cell pellet from anginal patients Ang II concentrations was significantly higher (p<0.01) than that assayed in controls both without and with Ang II stimulation (Table 3).

Table 3: Ang II concentration in pellet of T-lymphocytes from controls, unstable angina and hypertensive patients without or with the addition of 10^-13 mol/l Ang II in cell culture medium.

Ang II concentration in T-cell pellet from hypertensives was always significantly (p<0.01) higher than those assayed in controls (Table 3) and differed from ACS Ang II concentrations at baseline and at 24 hours with Ang II stimulation.

Plasma renin activity in controls, angina and hypertensive patients

In the control subjects, PRA in venous blood was 10.5+1.5 pmol. L^-1.min^-1 (range, 4.5 to 15.2 pmol. L^-1.min^-1). PRA levels in anginal and hypertensive patients were 10.1+1.2 pmol. L^-1.min^-1 (range, 4.8 to 14.7 pmol. L^-1.min^-1) and 11.2+0.9 pmol. L^-1.min^-1(range, 4.0 to 16.4 pmol. L^-1.min^-1) respectively, without significant differences between the three groups studied.

Our data show that in patients with unstable angina (ACS) circulating T-cell based ACE mRNA expression and enzymatic activity were markedly increased when compared to levels found in T-cell from controls and hypertensive patients and resulted in a de novo enhanced cell-based Ang II production. Only in cultured T-cell from anginal patients ACE activity was detectable not only in the pellet but also in the supernatant under baseline conditions; remarkably, the addition of Ang II stimulus to these cultured T-cells caused the almost complete release of ACE activity in the medium. This pattern of T-cell ACE genetic expression and enzymatic activity was peculiar of enrolled ACS patients with hsCRP >3 mg/dl and was markedly different not only from that observed in T-cell from controls but also from hypertensives with low grade inflammation (hsCRP >2 mg/dl). The normal PRA values found in ACS patients showed that the activation of T-cell RAS was selective and occurred in the absence of systemic RAS involvement. According to our data in ACS patients activated T-cell not only contribute to the acute systemic inflammatory reaction of unstable angina as cells of adaptive immunity [9,10], but also through the autonomous up-regulation of T-cell based RAS [5,6]. The increase in the synthesis of Ang II sustained by activated T-cells could have clinical relevance when it occurs into coronary plaques or microvessels where circulating T-cells are known to migrate [14,15]. No systemic effect can be hypothesized because of the low quote of Ang II synthesize by T-cells when compared to those produced by tissuebased or systemic RAS and because of the very short half-life of Ang II [16,17].

The enhanced de novo Ang II production by T-cells could start a dangerous positive loop in inflamed tissues and further amplify the activation of the immune response [18]. Remarkably, our in vitro findings of the marked release of ACE enzymatic activity in the supernatant by cultured T-cell from anginal patients, in vivo could translate in the local activation by de novo T-cell synthesized Ang II of other cells endowed with AT1-R such as endothelial and monocyte cells inside coronary plaques or microvessels [19,20]. Data available in literature reported that under Ang II stimulus AT-1 receptors of T-cells are upregulated 10 times and that T-cells exposed to Ang II have an increased ability to secrete TNF-alfa and INF-gamma as also confirmed in our in vitro experiments. In agreement with our in vitro findings, in myocardium of unstable angina patients an increased immunostaining for INF-gamma and TNF-alfa was shown next to inflammatory cells and was proved to be dumped in patients under pharmacological ACE inhibition [7,16]. Moreover, T-cells are involved in the process of endothelial dysfunction that contributes to the plaque destabilization in unstable angina and several studies have shown that T-cells infiltrate into atherosclerotic plaques and co-localize with ACE positivity at immunostaining [21-25]. According to our findings the instability of a plaque so to be a culprit lesion could be mediated by an increased de novo local Ang II production by activated circulating Tcells migrated inside plaques [17,18,26].

Finally, the local increased Ang II production by circulating cultured T-cells shown in our ACS patients could in vivo induce an oxidative burst in the endothelium, smooth cells , macrophages and Tcells of ischemic myocardium. Indeed, experimental studies reported that Ang II stimulus was shown to induce a marked increase in the ability of T-cells to produce reactive oxygen species, which in turn amplify the immune response [27,28].

Limits

Since different T-cell populations are known to operate with opposite roles on the regulation of the immune system, we are not able to specify whether T-cell ACE and Ang II upregulation was linked to a specific T-cell subpopulation. In experimental models, 8 weeks of exogenous Ang II treatment accelerated the development of atherosclerosis and prompted a switch from a stable-to-unstable plaque that was associated with a change in CD4⁺ T lymphocyte activity; valsartan inhibited the effect of Ang II and significantly increased the frequency of Th2 and Treg regulatory cells [29]. In different atherosclerotic-prone models the proatherogenic properties of Th1 were shown and evidence from clinical investigations was collected that the upregulation of Th1 immune response was overwhelming in patients with ACS [30] and that an Ang II-due imbalance between Th1 and Th2 response might play a role in plaque rupture [29,30]. As a whole these findings strongly suggest that the activation of T-cell RAS found in angina patients could play a role in the dysregulation in T-cell populations underlying ACS. Dedicated studies are needed to investigate this issue.

In ACS patients with a systemic inflammatory reaction marked by high hs-CRP levels, a selective activation of circulating cultured T-cell based RAS did occur with enhanced ACE gene expression and activity that resulted in an increased de novo Ang II synthesis. Our in vitro findings strongly support that in vivo , once migrated into inflamed tissues, activated T-cells from ACS patients can autonomously increase the local concentration of ACE and Ang II and amplify the inflammatory reaction underlying coronary plaque activation and microvessel damage.