Ribeiro, Maurício Serra

Introduction: Renin-Angiotensin System (RAS) regulates the cardiovascular system and has been implicated in the development of abdominal aortic aneurysm (AAA). RAS modulates function via balancing the ACE/Chymase/AngII-AT1 receptor and ACE-2/Ang(1-7)-Mas receptor axis. Preferential activation of the ACE/Chymase/AngII-AT1 receptor pathway of the RAS contributes to dysregulation of cardiovascular system and disease while the Ang(1-7)-MasR axis contributes to anti-inflammatory pathways. Therefore, identifying the RAS axis mechanism involved in growth, progression, or the risk of rupture in AAA humans could bring a novel treatment perspective. Hypothesis: We hypothesize that in the AAA, the local RAS is altered such as it favours the ACE/AngII/AT1R axis while decreasing the ACE-2/Ang(1-7)/MasR pathway, with consequent AAA development. Objective: We sought to investigate the expression of RAS components in the AAA human aorta. Methods: Aorta tissue from AAA patients (n=21) was collected during the AAA conventional repair surgery, and aorta control aortas were obtained from organ donors (Control, n=11). Protein expression of renin, chymase, ACE, ACE-2 AT1, AT2, and MAS receptors was analyzed by Western Blotting. Results: Our AAA cohort consisted mostly males (76.6% AAA vs. 63.6% Control, p=0.68), smokers or ex-smokers (90.4% AAA vs. 36.3% Control, p=0.002) and most of them had hypertension(71.4% AAA vs. 36.3% Control, p=0.07). Analysis of protein expression in the AAA group showed that ACE and AT1 receptors were upregulated compared with Control (p=0.05; p=0.02, respectively,), while chymase expression was decreased in AAA compared to the control (p=0.03). Additionally, the AAA group showed downregulation in ACE-2 and AT2 compared with Control (respectively, p=0.01, p=0.03). There was no significant difference in the expression of renin and MAS receptors between groups (p=0.52, p=0.41, respectively,). Conclusions: Our data show that the ACE/AT1R axis was upregulated while ACE-2 and AT2 receptors were downregulated in the AAA aorta. Our results implicate local RAS in AAA development and warrant further studies.

Elastase-2 (ELA-2) enzyme generates angiotensin-II (AngII) in arteries and is implicated in abnormal vascular remodeling in animal models.Our group demonstrated previously that ELA-2 knockout (ELA-2KO) mice were less susceptible to develop an aorta dilation in a response to AngII infusion. In this study, we hypothesize that susceptibility to an aorta dilation and abdominal aortic aneurysm (AAA) is mediated by AngII receptors (AT1 and AT2). Methods: Male wild type (C57bl/6, WT) and ELA-2KO (CELA-2aTm1Bdr) mice were treated with either saline or AngII (2.28g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. Mice were divided into four groups: WT treated with saline (WT+SAL, n=5), ELA-2KO treated with saline (ELA-2KO+SAL, n=5), WT treated with AngII (WT+AngII,n=5), and ELA-2KO treated with AngII (ELA-2KO+AngII, n=5). Protein expression of AT1 and AT2 receptor, CD68, and α-actin was measured by immunofluorescence in formalin-fixed, paraffin-embedded aorta tissue. ANOVA and Kruskal Wallis tests were used to compare the average fluorescent intensity between groups. Results: AngII treatment was associated with increased AT1 receptor expression in a WT group, but not with AT2 receptor expression (vs. WT+SAL,p=0.06 and p=0.31, respectively). The ELA-2KO group showed no difference in AT1 receptor expression in a response to AngII infusion compared to a ELA-2KO+SAL group (p=0.81). While ELA-2KO+Ang II mice had lower AT2 expression compared to ELA-2KO+SAL (p=0.05). The α-actin expression was similar in all groups. WT+AngII group expressed more CD68 expression in a response to an AngII treatment that was increased in a WT group (p=0.03), but not in a ELA-2KO group. Conclusion: Our data demonstrate that Ang II treatment did not increase an expression of AT1 receptor in ELA-2KO, but was associated with a downregulation of AT2 receptors. Taken together, this suggests That ELA-2KO mice resistance to developing an aorta dilation/AAA is related to downregulation of Ang II receptors. Our results also highlight the importance of the local/tissue response in the pathophysiology of AAA.

Increases in local angiotensin II (ANGII) signaling have been implicated in a number of diseases, including atherosclerosis, hypertension, and aneurysm. Endothelial dysfunction is strongly associated with increased ANGII signaling and the pathogenesis of these diseases. While increases in Runx2 have been implicated in cardiovascular calcification and vascular stiffening in diabetes, little is known regarding the role of Runx2 in the regulation of endothelial function. In the present study, we hypothesized that Runx2 is a key mediator of impaired vascular function and increased vascular wall stiffness provoked by ANGII. Wild-type mice (WT) and littermates that were deficient in one copy of Runx2 (HET) were implanted with osmotic minipumps containing saline or a pressor dose of ANGII (1000 ng/kg/min). Blood pressure was measured non-invasively using tail-cuff methods (CODA) and mice were sacrificed following 14 days of treatment. Changes in endothelium-dependent relaxation (relaxation to acetylcholine, R ACH ) and passive pressure-diameter relationships were measured in vitro (Living Systems arteriograph). In WT mice, treatment with ANGII for 14 days resulted in profound impairment in R ACH (85 ± 4% vs 47 ± 14% p<0.05) and slight reductions in vascular diameter at high distending pressures (~10% reduction in maximal diameter). While Runx2 deficiency did not alter R ACH or vascular distensibility/compliance in saline-treated mice, loss of one copy of Runx2 in ANGII-treated mice dramatically improved R ACH compared to their wild-type, ANGII-treated littermates. Interestingly, impairments in the pressure-diameter relationship with ANGII infusion were not affected by loss of one copy of Runx2. Critically, reductions in Runx2 did not significantly alter the pressor response to ANGII. In conclusion, our data suggest that Runx2 may play an integral role in the pathogenesis of ANGII-induced endothelial dysfunction, with these changes occurring independently of alterations in extracellular matrix remodeling and vascular stiffness. These data suggest that Runx2 may play a key, context-dependent role mediating vascular responses to ANGII.

Endothelial dysfunction and arterial stiffening major risk factors associated with progression of atherosclerosis and increased risk of acute coronary events. Recent data suggest sirtuins may play a role in protecting against a variety age-related diseases, and we recently reported that sirtuins are reduced in the aging vasculature. The roles of SIRT3 (a deacetylase that is largely restricted to the mitochondria) in the regulation of vasomotor function and arterial stiffness, however, remains poorly understood. Therefore, we hypothesized that genetic inactivation of SIRT3 in hypercholesterolemic mice will impair endothelial function and reduce vascular compliance compared to hypercholesterolemic mice with normal levels of SIRT3. We used Ldlr -/- /ApoB 100/100 (LA) mice that were either wild-type for SIRT3 (LA-SIRT3 +/+ ) or null for SIRT3 (LA-SIRT3 -/- ) and fed a western diet for 12 months. At time of sacrifice, aorta was removed for isolated organ chamber baths to measure changes in endothelium-dependent relaxation to acetylcholine (ACH), and carotid arteries were harvested to assess changes in vascular compliance using a pressurized cannulation system. Surprisingly, losses of SIRT3 did not impair relaxation to ACH in aorta compared to LA-SIRT3 +/+ mice (38.2±4.4%, 37.0±4.4%, respectively). Furthermore, reductions in SIRT3 did not impact vascular compliance or distensibility in carotid arteries from the same animals. In conclusion, in contrast to our hypothesis, inactivation of SIRT3 does not impact vasomotor function or conduit vessel compliance in advanced atherosclerosis. Importantly, this and may be explained by a “floor-effect”, and future work examining the effect of SIRT3 overexpression on vascular function and pathology will be instrumental in determining its biological role and ultimate therapeutic utility.

Objective: Elastase-2 (ELA-2), a chymotrypsyn-serine protease elastase family member 2A, is expressed in several tissues such as the lungs, heart, liver, kidneys, brain, and vessels. ELA-2 was found to generate angiotensin II (AngII) in arteries and contribute to resistance arteries and vascular remodeling in animal models. It has also been shown that continuous infusion of AngII induces abdominal aortic aneurysm (AAA) in mice. Therefore, we sought to investigate the role of ELA-2 in an aortic dilation, which is a risk factor to AAA development, in a mouse model. Design and method: Male wild type (C57bl/6, WT) and ELA-2 knockout (CELA-2aTm1Bdr; ELA-2 KO) mice were treated with saline or angiotensin II (AngII, 2.28 g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. To evaluate the role of elastase-2 in a AAA development, mice were divided into four groups: WT treated with saline (WT+SAL), ELA-2KO treated with saline (ELA-2KO+SAL), WT treated with AngII (WT+AngII), and ELA-2KO treated with Ang II (ELA-2KO+AngII). The vascular ultrasound (US) was done before treatment and once a week during 28 days of treatment. The abdominal aorta measurements such as maximum aortic diameter, aortic para-renal diameter, aortic infra-renal diameter, and aortic supra-renal diameter were collected. A dilated aorta is a risk factor aortic rupture/aneurysm. Statistical analyses were performed by the Shapiro Wilk Normality test, two-way ANOVA with Sidak's multiple comparisons test. This study is approved by the Ethics Committee (CEUA-131/2019). Results: The most dilated aorta as measured by the maximum aorta diameter was detected in WT+AngII group and was 60% higher than in the WT+Saline (p = 0.01). There was no significant difference in the maximum aorta diameter between ELA-KO+SAL vs. ELA-2KO+AngII (p = 0.8). The measurement of dilation in aortic para-renal and aortic infra-renal showed that AngII induced a dilation in WT+AngII (p < 0.05), while not modified these parameters in ELA-2KO+AngII group. The aortic supra-renal diameter parameters and weight showed no differences between groups. Conclusions: These results suggest that ELA-2KO mice might be less susceptible to develop an aorta dilation. Therefore, Elastase-2 is a candidate factor that contributes to the formation and/or development of abdominal aortic aneurysm.

Objective: The aim of this study was to analyze the outcomes of a standardized protocol using routine CSFD, neuromonitoring, LL reperfusion, and selective TASP to prevent SCI during F-BEVAR. Background: SCI is to be the most devastating complication for the patient, family, and surgeon, with impact on patient’s quality of life and long-term prognosis. An optimal standardized protocol may be used to improve outcomes. Methods: Patients enrolled in a prospective, nonrandomized single-center study between 2013 and 2018. A SCI prevention protocol was used for TAAAS or complex abdominal aneurysms with ≥5-cm supraceliac coverage including CSFD, neuromonitoring, LL reperfusion, and selective TASP. Endpoints included mortality and rates of SCI. Results: SCI prevention protocol was used in 170 of 232 patients (73%) treated by F-BEVAR. Ninety-one patients (55%) had changes in neuromonitoring, which improved with maneuvers in all except for 9 patients (10%) who had TASP. There was one 30-day or in-hospital mortality (0.4%). Ten patients (4%) developed SCIs including in 1% (1/79) of patients with normal neuromonitoring and 10% (9/91) of those who had decline in neuromonitoring (P = 0.02). Permanent paraplegia occurred in 2 patients (1%). Factors associated with SCI included total operating time (odds ratio 1.5, 95% confidence interval 1.1–2.2, P = 0.02) and persistent changes in neuromonitoring requiring TASP (odds ratio 15.7, 95% confidence interval 2.9–86.2, P = 0.001). Conclusion: This prospective nonrandomized study using a standardized strategy to prevent SCI was associated with low incidence of the SCI during F-BEVAR. Permanent paraplegia occurred in 1%.

Background and PurposeAngiotensin II (Ang II), whose generation largely depends on angiotensin‐converting enzyme (ACE) activity, mediates most of the renin‐angiotensin‐system (RAS) effects. Elastase‐2 (ELA‐2), a chymotrypsin‐serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II‐generation in resistance arteries are unknown. We hypothesized that ELA‐2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI.Experimental ApproachConcentration‐effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA‐2 knockout (ELA‐2KO) mice subjected to left anterior descending coronary artery ligation (MI).Key ResultsMI size was similar in WT and ELA‐2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA‐2KO mice. Ang I‐induced contractions increased in WT mice subjected to MI (MI‐WT) compared with sham‐WT mice. No differences were observed in Ang I reactivity between arteries from ELA‐2KO and ELA‐2KO subjected to MI (MI‐ELA‐2KO). Ang I contractions increased in arteries from MI‐WT versus MI‐ELA‐2KO mice. Chymostatin attenuated Ang I‐induced vascular contractions in WT mice, but did not affect Ang I responses in ELA‐2KO arteries.Conclusions and ImplicationsThese results provide the first evidence that ELA‐2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA‐2 as a key player in ACE‐independent dysregulation of the RAS.