Évora, Paulo Roberto Barbosa

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.

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.

To study the effect of hypoxia on vascular tone in human internal mammary artery (IMA), segments of IMA were suspended in organ chambers and contracted with norepinephrine (at a dose producing 30% of maximal contraction). Exposure of IMA segments with endothelium to hypoxia (partial pressure of oxygen, 38 +/- 4 mmHg) resulted in a transient relaxation (47 +/- 6%) followed by constriction (177 +/- 8%) (n = 14). IMA segments without endothelium exhibited a gradual decrease in tension that almost completely counteracted vasoconstriction. The initial transient endothelium-dependent relaxation could be blocked by indomethacin (10(-6)M) and was associated with a 51% increase in 6-ketoprostaglandin F1 alpha production (n = 22, P < 0.05). The endothelium-dependent contraction to hypoxia could be attenuated by indomethacin (n = 6, P < 0.05) or NG-monomethyl-L-arginine (10(-5)M; n = 9, P < 0.05) and was completely blocked by combination of these agonists (n = 6, P < 0.05). These experiments indicate that on exposure to hypoxia, the human IMA exhibits an initial prostacyclin-mediated relaxation followed by contraction due to the production of a constrictor prostanoid in addition to the inhibition of basal production of endothelium-derived relaxing factor.

BACKGROUND When used to reverse the anticoagulant effect of heparin, protamine sulfate often causes vasodilation that can lead to systemic hypotension. Protamine is rich in the basic amino acid arginine, which is the precursor of endothelial cell synthesis of nitric oxide, and nitric oxide is the active component of endothelium-derived relaxing factor (EDRF). METHODS AND RESULTS To determine whether the hypotensive effect of protamine could be due to stimulated release of EDRF, we studied rings (4-5 mm) of canine coronary, femoral, and renal artery suspended in organ chambers containing physiological salt solution (37 degrees C and 95% O2-5% CO2). Arterial rings with and without endothelium were contracted with prostaglandin F2 alpha (2 x 10(-6) M) and exposed to increasing concentrations of protamine (final organ bath concentration, 40-400 micrograms/ml). In arterial segments without endothelium, protamine caused only a modest decrease in tension. However, protamine induced concentration-dependent relaxation in all arterial segments with endothelium, which was significantly greater than in segments without endothelium (p less than 0.05). The endothelium-dependent relaxation induced by protamine was inhibited by NG-monomethyl-L-arginine (L-NMMA) (10(-5) M), but L-NMMA had no effect on rings without endothelium. The action of L-NMMA could be reversed by L-arginine (10(-4) M) but not D-arginine (10(-4) M). CONCLUSIONS This study demonstrates that protamine stimulates the release of EDRF from arterial endothelium, and that endothelium-dependent vasodilation may be an important cause of systemic hypotension during protamine infusion.