p-Aminobenzamidine attenuates cardiovascular dysfunctions in spontaneously hypertensive rats.

AIMS: Diminazene aceturate, a putative ACE2 activator, is susceptible to cleavage resulting in the formation of p-aminobenzamidine (PAB). This study aimed to investigate the effects of PAB in addressing cardiovascular dysfunctions in spontaneously hypertensive rats (SHR). MAIN METHODS: Acute effects of PAB on mean arterial pressure (MAP), heart rate (HR), and aortic (AVC) and mesenteric vascular conductance (MVC) were evaluated in anesthetized SHR. Isolated aortic rings and the Langendorff technique were used to investigate the acute and chronic effects of PAB in the artery and heart. Chronic treatment with PAB (1 mg/kg, gavage) was carried out for 60 days. During this period, systolic blood pressure (SBP) and HR were measured by tail-cuff plethysmography. After the treatment, the left ventricle was collected for histology analyses, western blotting, and ACE2 activity. KEY FINDINGS: Bolus infusion of PAB acutely reduced MAP and increased both AVC and MVC in SHR. Additionally, PAB induced coronary and aorta vasodilation in isolated organs from Wistar and SHR in an endothelial-dependent manner. The chronic PAB treatment in SHR significantly attenuated the increase of SBP and improved the aorta vasorelaxation induced by acetylcholine and bradykinin-induced coronary vasodilation. In addition, chronic treatment with PAB attenuated the cardiomyocyte hypertrophy and extracellular matrix deposition in hearts from SHR. PAB did not alter the protein expression of the AT1, AT2, Mas, ACE, ACE2, or ACE2 activity. SIGNIFICANCE: PAB induced beneficial effects on cardiovascular dysfunctions induced by hypertension, suggesting that this molecule could be used in the development of new drugs for the treatment of cardiovascular diseases.

Apelin-13 Protects Corpus Cavernosum Against Fibrosis Induced by High-Fat Diet in an MMP-Dependent Mechanism.

BACKGROUND: An increased fibrosis of the corpora cavernosa is a prevalent process that underlies most cases of erectile dysfunction. Apelin, an endogenous circulating peptide, has been documented as an important effector on cardiovascular homeostasis, controlling vascular function and reducing fibrosis in multiple pathological conditions. Recently, initial studies have shown that Apelin, acting through the APJ receptor, also modulates penile erection, however, the role of this system on penile structure and intracorporal collagen remodeling has not been investigated yet. AIMS: Here we sought to investigate the effect of chronic Apelin treatment on the corpus cavernosum structure of hyperchOlesterolemic mice. METHODS: Apolipoprotein gene-deleted (ApoE-/-) mice were fed with a Western diet for 11 weeks and received Apelin-13 (2 mg/kg/day) or vehicle during the last 3 weeks. Penile samples were obtained for histological and biochemical analyses to assess the intracorporal collagen content and key proteins expression. Furthermore, the effect of Apelin-13 was evaluated in cultured NIH3T3 mouse fibroblasts stimulated with TGF-ß. OUTCOME: Local expression of Apelin-13 in mouse corpus cavernosum and its protective effect against fibrosis. RESULTS: Apelin and APJ receptor were expressed (gene and protein) within the corpus cavernosum of ApoE-/- mice, indicating a local modulation of the Apelin system. Interestingly, 3 weeks of Apelin-13 treatment strongly reduced intracavernosal collagen content. In addition, Apelin-13 enhanced total matrix metalloproteinase (MMP) activity in the mice penis, which was associated with an increased protein expression of MMP-1, MMP-3, MMP-8, and MMP-9, while tissue inhibitor of metalloproteinase were unaltered. These beneficial actions were not associated with changes in nNOS or eNOS protein expression, intracavernosal reactive oxygen species content, or atherosclerotic plaque deposition. Additionally, in cultured fibroblast, Apelin-13 inhibited TGF-ß-induced fibroblast to myofibroblast differentiation and collagen production, possibly through the activation of ERK1/2 kinase. CLINICAL TRANSLATION: These results point out Apelin/APJ system as a potential target to treat intracavernosal fibrosis-related disorders. STRENGTH & LIMITATIONS: These results provide the first evidence of the Apelin system's positive role on erectile tissue structure/remodeling. Nevertheless, additional functional study addressing erectile response would bring extended validation regarding the relevance of such effect. CONCLUSION: These results suggest a local modulation of the Apelin system within the corpus cavernosum. Remarkably, Apelin-13 reduced intracavernosal fibrosis in hypercholesterolemic mice by: (i) enhancing MMPs expression and activity; and (ii) inhibiting fibroblast differentiation into myofibroblast. Altogether, these results suggest an essential protective role of Apelin, indicating Apelin/APJ system as a promising candidate for the development of fibrosis-associated erectile dysfunction treatments. Sturny M, Anguenot L Costa-Fraga FP, et al. Apelin-13 Protects Corpus Cavernosum Against Fibrosis Induced by High-Fat Diet in an MMP-Dependent Mechanism. J Sex Med 2021;18:875-888.

An optimized and validated 384-well plate assay to test platelet function in a high-throughput screening format.

Despite significant advances in the treatment of cardiovascular diseases, antiplatelet therapies are still associated with a high risk of hemorrhage. In order to develop new drugs, methods to measure platelet function must be adapted for the high-throughput screening (HTS) format. Currently, all assays capable of assessing platelet function are either expensive, complex, or not validated, which makes them unsuitable for drug discovery. Here, we propose a simple, low-cost, and high-throughput-compatible platelet function assay, validated for the 384-well plate. In the proposed assay, agonist-induced platelet activity was assessed by three different methods: (i) measurement of light absorbance, which decreases with platelet aggregation; (ii) luminescence measurement, based on ATP release from activated platelets and luciferin-luciferase reaction; and (iii) automated bright-field microscopy of the wells and further quantification of platelet image area, described here for the first time. Brightfield imaging results were validated by demonstrating the similarity of dose-response curves obtained with absorbance and luminescence measurements after stimulating platelets, pre-incubated with prostaglandin E1 or tirofiban, and demonstrating the similarity of dose-response curves obtained with agonists. Assay quality was confirmed using the Z'-factor, a statistical parameter used to validate the robustness and suitability of an HTS assay. The results showed that, under high rotations per minute (1200 RPM), an acceptable Z'-factor score is reached for absorbance measurements (Z'-factor - 0.58) and automated brightfield imaging (Z'-factor - 0.52), without the need of replicates, while triplicates must be used to achieve an acceptable Z'-factor score (0.54) for luminescence measurements. Using low platelet concentration (4 × 104/µl - 10 µl), the brightfield imaging test was further validated using washed platelets. Furthermore, drug screening was performed with compounds selected by structure-based virtual screening. Taken together, this study presents an optimized and validated assay for HTS to be used as a tool for antiplatelet drug discovery.

Age-related changes in vascular responses to angiotensin-(1-7) in female mice.

The vasodilatory effect of angiotensin-(1-7) seems to vary between sexes, and estradiol (E2) can modulate the magnitude of the Ang-(1-7) vasodilatory response in female rats. However, there are few studies addressing the influence of sex on the age-related vasodilatory effect of Ang-(1-7). Here, we evaluated the vasodilatory response to Ang-(1-7) on vascular ageing. Ang-(1-7) dose-response curves were determined in mice aortic rings from males (old and young) and females (E2 treated/non-treated old and young) mounted in an isolated organ chamber. Abdominal aortic rings were used for protein expression analysis and determination of reactive oxygen species (ROS) and nitric oxide (NO) production. Our results showed that the Ang-(1-7) vasodilatory effect was absent in aorta from old females, contrasting with a full response in vessels from young females. The Ang-(1-7) vasodilatory effect was restored by E2 replacement in old females. A robust increase in Mas receptor, SOD2, NRF-2 and NOX2 expression was observed in aorta from old females, which was normalized by E2. This effect of E2 was also associated with lower production of ROS and normal levels of NO. In conclusion, our data demonstrated that pathways involved in the Ang-(1-7) vasodilatory response in female mice is affected by hormonal changes in ageing and rescued by E2.

Apelin-13 treatment enhances the stability of atherosclerotic plaques.

BACKGROUND: Apelin is an endogenous peptidergic system which modulates cardiovascular function. Recent studies pointed out a fundamental contribution of apelin on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define a beneficial or deleterious role. To better understand apelin function on atherosclerosis, we aimed to investigate apelin-13 treatment effects on atherosclerotic plaques composition. DESIGN: Apolipoprotein E gene-deleted mice were fed on Western-type diet for 11 weeks. Atherosclerotic plaque formation was induced in the carotid artery by a shear stress modifier device, which exposes the same vessel to distinct patterns of shear stress enabling the formation of plaques with different composition. Mice were treated with apelin-13 (2 mg kg-1 day-1 ) or vehicle for the last 3 weeks. RESULTS: Apelin-13 treatment did not alter the lipid content of low shear stress- and oscillatory shear stress-induced plaques in the carotid. However, apelin-13 greatly ameliorated plaque stability by increasing intraplaque collagen content and reducing MMP-9 expression. Furthermore, apelin-13 decreased the infiltration of inflammatory cells (neutrophil and macrophage) and intraplaque reactive oxygen species content. Interestingly, apelin-13 treatment reduced total cholesterol, LDL levels and free fatty acid serum levels, while HDL, triglycerides serum levels were not significantly changed. CONCLUSIONS: Apelin-13 treatment for 3 weeks did not alter the lesion size, but it significantly enhanced the stable phenotype of atherosclerotic plaques and improved serum lipid profile. These results indicate that activation of apelin system decreases plaque vulnerability.

Diminazene enhances stability of atherosclerotic plaques in ApoE-deficient mice.

Angiotensin (Ang) II contributes to the development of atherosclerosis, while Ang-(1-7) has atheroprotective actions. Accordingly, angiotensin-converting enzyme 2 (ACE2), which breaks-down Ang II and forms Ang-(1-7), has been suggested as a target against atherosclerosis. Here we investigated the actions of diminazene, a recently developed ACE2 activator compound, in a model of vulnerable atherosclerotic plaque. Atherosclerotic plaque formation was induced in the carotid artery of ApoE-deficient mice by a shear stress (SS) modifier device. The animals were treated with diminazene (15mg/kg/day) or vehicle. ACE2 was strongly expressed in the aortic root and low SS-induced carotid plaques, but poorly expressed in the oscillatory SS-induced carotid plaques. Diminazene treatment did not change the lesion size, but ameliorated the composition of aortic root and low SS-induced carotid plaques by increasing collagen content and decreasing both MMP-9 expression and macrophage infiltration. Interestingly, these beneficial effects were not observed in the oscillatory SS-induced plaque. Additionally, diminazene treatment decreased intraplaque ICAM-1 and VCAM-1 expression, circulating cytokine and chemokine levels and serum triglycerides. In summary, ACE2 was distinctively expressed in atherosclerotic plaques, which depends on the local pattern of shear stress. Moreover, diminazene treatment enhances the stability of atherosclerotic plaques.

Diminazene protects corpus cavernosum against hypercholesterolemia-induced injury.

INTRODUCTION: Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin angiotensin system, which breaks down angiotensin II and forms angiotensin-(1-7). In erectile tissues, it has been documented that angiotensin II contributes to the development of erectile dysfunction (ED), while treatment with angiotensin-(1-7) improves penile erection. However, the expression and function of ACE2 in erectile tissues have never been investigated. AIM: Here, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia-induced corpus cavernosum (CC) injury. METHODS: Hypercholesterolemic apolipoprotein E knockout (ApoE(-/-) ) mice, a well-known model of ED, were treated with diminazene aceturate (DIZE), an ACE2 activator compound, or vehicle for 3 weeks. Reactive oxygen species (ROS), collagen content, and protein expression of ACE2, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) subunits were evaluated in the penis of DIZE-treated and untreated ApoE(-/-) mice. Functional studies were performed in CC strips. MAIN OUTCOME MEASURES: ACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures. RESULTS: ACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, ACE2 was downregulated in penis of hypercholesterolemic mice with ED, suggesting a protective role of ACE2 on the CC homeostasis. In accordance with that, pharmacological ACE2 activation by DIZE treatment reduced ROS production and NADPH oxidase expression, and elevated nNOS and eNOS expression and NO bioavailability in the penis of ApoE(-/-) mice. Additionally, DIZE decreased collagen content within the CC. These beneficial actions of DIZE on the CC were not accompanied by improvements in atherosclerotic plaque size or serum lipid profile. CONCLUSION: ACE2 is expressed in erectile tissue and its reduction is associated with hypercholesterolemia-induced ED. Additionally, treatment with DIZE improved hypercholesterolemia-induced CC injury, suggesting ACE2 as a potential target for treating ED. .

An increased arginase activity is associated with corpus cavernosum impairment induced by hypercholesterolemia.

INTRODUCTION: Hypercholesterolemia is a prevalent risk factor for the development of erectile dysfunction (ED), mostly due to an increase in oxidative stress and impaired nitric oxide (NO) bioavailability within the penis. Arginase is an enzyme that shares the common substrate L-arginine with NO synthase. Augmented arginase activity reduces NO production and is associated with ED development. However, the contribution of arginase hyperactivity in hypercholesterolemia-induced ED is unknown. AIM: In the present study, we investigated the activity and role of arginase in the corpus cavernosum of hypercholesterolemic mice. METHODS: Apolipoprotein E (ApoE) gene-deleted mice fed with a Western-type diet for 11 weeks were treated with the selective arginase inhibitor, N-ω-Hydroxy-L-norarginine (NOHA), or vehicle (saline 0.9%) during the last 9 weeks. Arginase activity and expression were measured in penis protein extraction. Reactive oxygen species (ROS) content within the corpus cavernosum was measured by dihydroethidium staining. Functional in vitro studies were performed using cavernosal strips mounted in an isometric organ bath to evaluate NO production. MAIN OUTCOME MEASURE: Arginase activity and its role in modulating NO and ROS production within the corpus cavernosum of hypercholesterolemic mice is the main outcome measure. RESULTS: Total arginase activity and arginase type II protein expression were increased in hypercholesterolemic mice compared with wild-type mice. The long-term treatment with NOHA normalized this alteration. Moreover, pharmacological arginase inhibition by NOHA attenuated the augmented ROS production within the corpus cavernosum of ApoE(-/-) mice, which increased the NO-dependent response in cavernosal strips. CONCLUSION: These evidences indicate that arginase hyperactivity is associated with ED induced by hypercholesterolemia, suggesting that this enzyme is a potential target for treating ED.

Treatment with Angiotensin-(1-7) reduces inflammation in carotid atherosclerotic plaques.

Angiotensin (Ang)-(1-7), acting through the receptor Mas, has atheroprotective effects; however, its role on plaque vulnerability has been poorly studied. Here, we investigated the expression of the renin-angiotensin system (RAS) components in stable and unstable human carotid plaques. In addition, we evaluated the effects of the chronic treatment with an oral formulation of Ang-(1-7) in a mouse model of shear stress-determined carotid atherosclerotic plaque. Upstream and downstream regions of internal carotid plaques were obtained from a recently published cohort of patients asymptomatic or symptomatic for ischaemic stroke. Angiotensinogen and renin genes were strongly expressed in the entire cohort, indicating an intense intraplaque modulation of the RAS. Intraplaque expression of the Mas receptor mRNA was increased in the downstream portion of asymptomatic patients as compared to corresponding region in symptomatic patients. Conversely, AT1 receptor gene expression was not modified between asymptomatic and symptomatic patients. Treatment with Ang-(1-7) in ApoE-/- mice was associated with increased intraplaque collagen content in the aortic root and low shear stress-induced carotid plaques, and a decreased MMP-9 content and neutrophil and macrophage infiltration. These beneficial effects were not observed in the oscillatory shear stress-induced plaque. In vitro incubation with Ang-(1-7) did not affect ICAM-1 expression and apoptosis on cultured endothelial cells. In conclusion, Mas receptor is up regulated in the downstream portions of human stable carotid plaques as compared to unstable lesions. Treatment with the oral formulation of Ang-(1-7) enhances a more stable phenotype in atherosclerotic plaques, depending on the local pattern of shear stress forces.

An oral formulation of angiotensin-(1-7) reverses corpus cavernosum damages induced by hypercholesterolemia.

INTRODUCTION: The renin angiotensin system plays a crucial role in erectile function. It has been shown that elevated angiotensin-II levels contribute to the development of erectile dysfunction (ED). Oppositely, angiotensin-(1-7) (Ang-[1-7]) mediates penile erection by activation of receptor Mas. Recently, we have developed a formulation based on Ang-(1-7) inclusion in cyclodextrin (CyD) [Ang-(1-7)-CyD], which allows for the oral administration of Ang-(1-7). AIM: In the present study, we evaluated the effects of chronic treatment with Ang-(1-7)-CyD on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemic mice. METHODS: Apolipoprotein(Apo)E-/- mice fed a Western-type diet for 11 weeks received Ang-(1-7)-CyD or vehicle during the final 3 weeks. Collagen content and reactive oxygen species (ROS) production within the corpus cavernosum were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) subunits (p67-phox and p22-phox), and AT1 and Mas receptors in the penis was assessed by Western blotting. Nitric oxide (NO) production was measured by Griess assay in the mice serum. Cavernosal strips were mounted in an isometric organ bath to evaluate the endothelial function. MAIN OUTCOME MEASURES: The effect of Ang-(1-7)-CyD treatment on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemia-induced ED. RESULTS: Ang-(1-7)-CyD treatment reduced collagen content in the corpus cavernosum of ApoE-/- mice. This effect was associated with an attenuation of ROS production and a diminished expression of NADPH. Furthermore, Ang-(1-7)-CyD treatment augmented the expression of nNOS and eNOS in the penis and elevated vascular NO production. Importantly, these effects were accompanied by an improvement in cavernosal endothelial function. CONCLUSION: Long-term treatment with Ang-(1-7)-CyD reduces penile fibrosis associated with attenuation of oxidative stress. Additionally, cavernosal endothelial function in hypercholesterolemic mice was markedly improved. These results suggest that Ang-(1-7)-CyD might have significant therapeutic benefits for the treatment of erectile dysfunction.

Angiotensin-converting enzyme 2 activation improves endothelial function.

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

An orally active formulation of angiotensin-(1-7) produces an antithrombotic effect.

INTRODUCTION AND OBJECTIVE: The heptapeptide angiotensin-(1-7) is a component of the renin-angiotensin system, which promotes many beneficial cardiovascular effects, including antithrombotic activity. We have recently shown that the antithrombotic effect of angiotensin-(1-7) involves receptor Mas-mediated NO-release from platelets. Here, we describe an orally active formulation based on angiotensin-(1-7) inclusion in cyclodextrin [Ang-(1-7)- CyD] as an antithrombotic agent. Cyclodextrins are pharmaceutical tools that are used to enhance drug stability, absorption across biological barriers and gastric protection. METHOD: To test the antithrombotic effect of Ang-(1-7)-CyD, thrombus formation was induced in the abdominal vena cava of spontaneously hypertensive rats that were pretreated either acutely or chronically with Ang-(1-7)-CyD. Male Mas-knockout and wild-type mice were used to verify the role of the Mas receptor on the effect of Ang-(1-7)-CyD. RESULTS: Acute or chronic oral treatment with Ang-(1-7)-CyD promoted an antithrombotic effect (measured by thrombus weight; all values are, respectively, untreated vs. treated animals) in spontaneously hypertensive rats (acute: 2.86 ± 0.43 mg vs. 1.14 ± 0.40 mg; chronic: 4.27 ± 1.03 mg vs. 1.39 ± 0.68 mg). This effect was abolished in Mas-knockout mice (thrombus weight in Mas wild-type: 0.76 ± 0.10 mg vs. 0.37 ± 0.02 mg; thrombus weight in Mas-knockout: 0.96 ± 0.11 mg vs. 0.87 ± 0.14 mg). Furthermore, the antithrombotic effect of Ang-(1-7)-CyD was associated with an increase in the plasma level of Angiotensin-(1-7). CONCLUSION: These results show for the first time that the oral formulation Ang-(1-7)-CyD has biological activity and produces a Mas-dependent antithrombotic effect.

An orally active formulation of angiotensin-(1-7) produces an antithrombotic effect

INTRODUCTION AND OBJECTIVE: The heptapeptide angiotensin-(1-7) is a component of the renin-angiotensin system, which promotes many beneficial cardiovascular effects, including antithrombotic activity. We have recently shown that the antithrombotic effect of angiotensin-(1-7) involves receptor Mas-mediated NO-release from platelets. Here, we describe an orally active formulation based on angiotensin-(1-7) inclusion in cyclodextrin [Ang-(1-7)- CyD] as an antithrombotic agent. Cyclodextrins are pharmaceutical tools that are used to enhance drug stability, absorption across biological barriers and gastric protection. METHOD: To test the antithrombotic effect of Ang-(1-7)-CyD, thrombus formation was induced in the abdominal vena cava of spontaneously hypertensive rats that were pretreated either acutely or chronically with Ang-(1-7)-CyD. Male Mas-knockout and wild-type mice were used to verify the role of the Mas receptor on the effect of Ang-(1-7)-CyD. RESULTS: Acute or chronic oral treatment with Ang-(1-7)-CyD promoted an antithrombotic effect (measured by thrombus weight; all values are, respectively, untreated vs. treated animals) in spontaneously hypertensive rats (acute: 2.86 + 0.43 mg vs. 1.14 + 0.40 mg; chronic: 4.27 + 1.03 mg vs. 1.39 + 0.68 mg). This effect was abolished in Mas-knockout mice (thrombus weight in Mas wild-type: 0.76 + 0.10 mg vs. 0.37 + 0.02 mg; thrombus weight in Mas-knockout: 0.96 + 0.11 mg vs. 0.87 + 0.14 mg). Furthermore, the antithrombotic effect of Ang-(1-7)-CyD was associated with an increase in the plasma level of Angiotensin-(1-7). CONCLUSION: These results show for the first time that the oral formulation Ang-(1-7)-CyD has biological activity and produces a Mas-dependent antithrombotic effect.