Treatment with CB2 Agonist JWH-133 Reduces Histological Features Associated with Erectile Dysfunction in Hypercholesterolemic Mice

Hypercholesterolemia is one of the most important risk factors for erectile dysfunction, mostly due to the impairment of oxidative stress and endothelial function in the penis. The cannabinoid system might regulate peripheral mechanisms of sexual function; however, its role is still poorly understood. We investigated the effects of CB2 activation on oxidative stress and fibrosis within the corpus cavernosum of hypercholesterolemic mice. Apolipoprotein- E-knockout mice were fed with a western-type diet for 11 weeks and treated with JWH-133 ( selective CB2 agonist) or vehicle during the last 3 weeks. CB2 receptor expression, total collagen content, and reactive oxygen species ( ROS) production within the penis were assessed. In vitro corpus cavernosum strips preparation was performed to evaluate the nitric oxide ( NO) bioavailability. CB2 protein expression was shown incavernosal endothelial and smooth muscle cells of wild type and hypercholesterolemic mice. Treatment with JWH-133 reduced ROS production and NADPH-oxidase expression in hypercholesterolemic mice penis. Furthermore, JWH-133 increased endothelial NO synthase expression in the corpus cavernosum and augmented NO bioavailability. The decrease in oxidative stress levels was accompanied with a reduction in corpus cavernosum collagen content. In summary, CB2 activation decreased histological features, which were associated with erectile dysfunction in hypercholesterolemic mice.

An Oral Formulation of Angiotensin-(1-7) Reverses Corpus Cavernosum Damages Induced by Hypercholesterolemia

Danielle Gomes Passos Silva, Fabiana Pereira Da Costa Fraga, Nikolaos Stergiopulos

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 MeasuresThe 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. Fraga-Silva RA, Costa-Fraga FP, Savergnini SQ, De Sousa FB, Montecucco F, da Silva D, Sinisterra RD, Mach F, Stergiopulos N, da Silva RF, and Santos RAS. An oral formulation of angiotensin-(1-7) reverses corpus cavernosum damages induced by hypercholesterolemia. J Sex Med 2013;10:2430-2442.

Wiley-Blackwell2013

Diminazene Protects Corpus Cavernosum Against Hypercholesterolemia-Induced Injury

Younouss Faye, Rafaela Fernandes Da Silva, Fabiana Pereira Da Costa Fraga, Nikolaos Stergiopulos, Mikaël Nils Sturny

IntroductionAngiotensin-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. AimHere, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia-induced corpus cavernosum (CC) injury. MethodsHypercholesterolemic 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 MeasuresACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures. ResultsACE2 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. ConclusionACE2 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. Fraga-Silva RA, Costa-Fraga FP, Montecucco F, Sturny M, Faye Y, Mach F, Pelli G, Shenoy V, da Silva RF, Raizada MK, Santos RAS, and Stergiopulos N. Diminazene protects corpus cavernosum against hypercholesterolemia-induced injury. J Sex Med 2015;12:289-302.

Wiley-Blackwell2015

Differential effects of reduced cyclic stretch and perturbed shear stress within the arterial wall

Tyler Nerton Thacher

Due to the pulsatile nature of blood flow, arteries are constantly exposed to dynamic mechanical forces; the pulsatility continuously stretches the vessel wall and the flow creates a frictional force on the interior surface. These stresses, referred to as cyclic circumferential stretch and shear stress, are known to determine arterial structure and morphology; modulation of which leads to the progression of vascular diseases such as hypertension and atherosclerosis. Yet the individual contributions of cyclic stretch and shear stress, with regards to vascular disease, have yet to be revealed. In this thesis I wish to identify the role of reduced cyclic stretch in the development of endothelial dysfunction and vascular remodeling, develop an experimental model for studying the autonomous effects of shear stress and cyclic stretch and how these two stimuli individually modulate markers of vascular disease in different regions of the vascular wall. I will begin by introducing the different structural and cellular components of the vascular wall and their individual functions. From here I will introduce how hemodynamic forces transmitted to the vascular wall due to the pulsatile nature of blood flow play an essential role maintaining arterial health and function. And as such, how deviations from a physiologic hemodynamic range can have catastrophic implications for the vasculature. Next I will introduce how certain hemodynamic conditions can stimulate cellular dysfunction and how this relates to initiation and progression of vascular disease. In the first paper, we set out to determine if reduction of cyclic stretch could be a factor which induces remodeling of the arterial wall. We found that reducing compliance caused a decrease in vascular smooth muscle function, as well as inducing switch in smooth muscle cell phenotype. Arteries exposed to a reduced cyclic stretch also exhibited increased matrix degradation and cellular proliferation than those allowed to stretch physiologically. These findings accent the importance of cyclic stretch in the maintenance of a differentiated and fully functional phenotype of vascular smooth muscle cells, as well as in the regulation of migratory properties, proliferation and matrix turnover in the vascular wall. In the second paper we investigated how reduction of cyclic stretch influences endothelial dysfunction and modulation of nitric oxide bioavailability. We observed that reduced compliance significantly decreases the activity of the enzyme responsible for producing nitric oxide (eNOS). Overall production of reactive oxygen species were also increased by reducing compliance, which we were able to attribute to stimulation of the superoxide generating NAD(P)H oxidase. We found that experimentally reduced compliance also caused a significant decrease in endothelial function, as assessed with bradykinin dependent vascular relaxation. The results from this study point out how reduced arterial compliance interrupts the eNOS activation pathway and increases vascular levels of oxidative stress, which together could explain the measured decreases on endothelial functionality. In the third article we used our experimental model to investigate how shear stress and cyclic stretch independently stimulate the vascular wall. We found that both oscillatory flow and reduced stretch are detrimental to endothelial function, whereas oscillatory flow alone, dominated total endogenous vascular wall superoxide anion production. Yet when superoxide anion production was analyzed in just the endothelial region we observed that it was modulated more significantly by reduced cyclic stretch than by oscillatory shear, emphasizing an important distinction between shear and stretch mediated effects to the vascular wall. Analysis of eNOS and nitro-tyrosine, the by-product of superoxide anion and nitric oxide, proved that they too are more significantly negatively modulated by oscillatory flow, than by reduced stretch. The findings from this study point out how shear and stretch stimulate regions of the vascular wall differently, affecting NO bioavailability and contributing to vascular disease. The goal of the fourth article is to further investigate how shear stress and cyclic stretch modulate markers of vascular remodelling in different regions of the arterial wall. We demonstrated that while total superoxide production, fibronectin expression and gelatinase activation are predominantly mediated by shear stress, their expression in the endothelial region is mediated by reduced cyclic stretch, which correlates well with results from total MMP-2 expression. By plotting intensity versus radius for these markers of vascular remodeling we are able to see that superoxide production and gelatinase activity follows trends indicating their expression is in part mediated by stress distributions through out the vascular wall, while fibronectin and p22-phox were much less or not at all. Most importantly these findings, when coupled with our results from tissue reactive studies, suggest that the arterial remodeling process triggered in the endothelial region due to reduced stretch causes the most significant changes in arterial smooth muscle function. Perturbed shear stress and reduced arterial compliance have both been implicated in the initiation and progression of vascular disease: this work provides a new perspective into how these stimuli are perceived through out the vascular wall. To conclude, this body of work has shown that cyclic stretching due to the pulsatile nature of blood flow is an essential stimulus regulating arterial remodeling and endothelial viability. We have performed experiments permitting the autonomous effects of cyclic stretch and shear stress to be studied. Yet more importantly, we have shown how cyclic stretch and shear stress stimulate the vascular wall in different regions and compared this data to arterial functionality studies. These results have indicated that although reduced cyclic stretch may not stimulate the total expression of certain markers of vascular disease as much as an OSC shear stress, it does so in specific regions of the vascular wall which can in fact have a more detrimental effect on arterial function. As reduced arterial cyclic stretching is associated with the aging process, this work gives insight into the progression of vascular disease over the course of a person's life time.

EPFL2009