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

Léa Liliane Colette Anguenot, Maiia Bragina, Rafaela Fernandes Da Silva, Augusto Martins Lima, Fabiana Pereira Da Costa Fraga, Nikolaos Stergiopulos, Mikaël Nils Sturny
ELSEVIER SCI LTD, 2021
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Résumé

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-beta. 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-beta-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. Copyright (C) 2021 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.

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Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis

Maiia Bragina, Babak Ebadi Shirmard, Fabiana Pereira Da Costa Fraga, Nikolaos Stergiopulos, Mikaël Nils Sturny

Background: The renin-angiotensin system (RAS) plays an important role in erectile function. The RAS contains 2 major axes: one deleterious, composed of ACE-Ang II-AT1 receptor, and another protective, composed of ACE2-Ang-(1-7)-Mas receptor. While aging is a well-known cause for development of male sexual disorders, little is known about local regulation of the RAS in age-related erectile dysfunction (ED). Aim: The present study aimed to assess regulation of the RAS in aging-associated ED rat model and evaluate possible options for disease management through pharmacological modulation of the RAS. Methods: Penile tissues were harvested from 3-, 12-, and 24-month-old Wistar rats. Local expression of major RAS components and ED markers was measured by RT-PCR. Protein expression of RAS components was assessed by western blot. Collagen deposition was measured by Sirius Red and immunohistochemical staining. Evaluation of collagen content was also performed in penile sections of Mas-knockout mice by Sirius Red and Masson's trichrome stainings. Finally, the effect of Ang-(1-7) pretreatment on TGF-beta-induced myofibroblast activation was studied in primary cavernosal and immortalized fibroblasts. Outcomes: Experimental results highlighted the essential role of the RAS in modulation of cavernosal fibrosis. Results: The present study demonstrates local expression of angiotensinogen mRNA alongside with major RAS components, which suggests local autonomous functioning of the RAS within penile tissue. Gene expression analysis revealed strong positive correlation between ACE-Ang II-AT1 axis with markers for inflammation and fibrosis. While corpus cavernosum from 24-month-old rats was characterized by increased collagen deposition, protein expression of ACE, AT1, and Mas was shown to be upregulated in the penile tissue of this group. At the same time, penile sections from Mas-knockout mice (FVB/N background) were also shown to have increased collagen deposition. Finally, it was demonstrated that Ang-(1-7) treatment of primary cavernosal and immor-talized fibroblasts was able to alleviate TGF-beta-induced fibroblast-to-myofibroblast transition. Clinical Translation: The present study suggests Ang-(1-7) treatment as a possible strategy for pharmacological management of fibrosis-associated ED in aging. Strengths & Limitations: The link between the RAS and penile fibrosis, indicated by a holistic screening of different ED markers, was confirmed by in vivo and in vitro data. However, results, presented in the manuscript, need to be further reinforced by human data. Important to note, the main goal of the study was to characterize RAS regulation in aging condition rather than state any causal relationships. Conclusion: Present study characterizes RAS regulation in aging-associated ED and indicates its important role in cavernosal fibrosis. Copyright (C) 2020, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.

ELSEVIER SCI LTD2020

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Capillary Morphogenesis Gene 2 (CMG2) is a 55kDa single-pass transmembrane protein best described as the main Anthrax Toxin Receptor. However, CMG2 physiological role remains elusive and understanding better its in vivo function was the main objective of my thesis. My first aim was to investigate the prevalence of intrinsically disordered domains in the human transmembrane proteome. Using prediction tools, I found that 50% of transmembrane proteins had at least one domain of minimum 30 amino-acids predicted as intrinsically disordered (IDD), these proteins being mainly involved in cell signaling and adhesion. The majority of the IDDs were localized in the cytoplasm, and the intrinsic disorder containing proteins were significantly more phosphorylated and interacted on average with more proteins than fully ordered proteins, corroborating their role in signaling. CMG2 loss-of-function mutations is the known cause of a rare inherited genetic disorder called Hyaline Fibromatosis Syndrome (HFS). HFS patients develop subcutaneous nodules, gingival hypertrophy, contracture of the large joints, and in the most severe cases diarrhea and premature death. In a second part of my thesis, I was involved in understanding the molecular defects underlying missense mutations localized in the ectodomain and a highly conserved cytoplasmic domain of CMG2. We found that CMG2 was able to bind the actin cytoskeleton through Talin and Vinculin when free of its extracellular ligand. Interestingly, the HFS mutation p.C218R, altered the cytoskeleton release and led to a CMG2 protein able to bind both the extracellular ligand and the actin cytoskeleton. Strikingly, we observed that all but one cytoplasmic missense mutation hindered actin binding, a defect potentially relevant to HFS pathogenesis. Furthermore, we showed that the Src-like kinase family are involved in the release of Talin upon ligand binding and that CMG2 binding to its extracellular ligand is critical for the correct orientation of the mitotic spindle of zebrafish epiblast cells, which allows cell division along the Animal/Vegetal axis. Through the analysis of cmg2 knock-out mice, along with the study of HFS patient samples, we aimed at better defining CMG2 function. We showed that type VI Collagen was strongly accumulating in the cmg2 KO mice uterus tissue leading to failure of parturition, an accumulation that was also observed in HFS patient nodules. In addition, we observed that CMG2 was able to interact and potentiate MT1-MMP, and that type VI Collagen is a bona fide ligand for CMG2, its binding leading to the phosphorylation of the receptor. With the scope to understand the HFS patient subcutaneous nodules pathogenesis, we investigated the gene expression profile of fibroblasts derived from non-affected and affected tissues by RNA sequencing. Fibroblasts derived from affected tissues appeared to differentially express genes involved in the TGFBeta pathway and cytoskeleton, such as alpha smooth muscle actin, a marker of myofibroblast activation. We observed the presence of myofibroblasts in the patient nodules, and showed that CMG2 was limiting their activation process, loss-of-function mutations in CMG2 leading to an exacerbated differentiation. As we observed CMG2 interaction with the TGFBeta receptor 2, we postulated that CMG2 influence the TGFBeta pathway, a process that is altered in HFS patient and potentially lead to the formation of nodules.

EPFL2015

Chargement

In Vivo Studies on the Control of Gene Expression and Protein Degradation

Karolina Bojkowska

Cellular homeostasis is maintained by tightly regulated gene networks, controlled notably at the levels of transcription, mRNA processing, and protein post-translational modification and turnover. This thesis focuses on two of these regulatory steps, namely gene transcription and protein stability. I first explored the impact of the KRAB-ZFP/KAP1 gene regulation system on liver function. KRAB-ZFPs constitute the largest family of transcription factors encoded by mouse and humans, yet their physiological roles and gene targets remain mostly elusive. Conversely, their molecular mechanism of action has been rather well characterized, as they can recognize DNA in a sequence-specific manner and recruit the universal co-factor KAP1 (also known as TRIM28), which in turn serves as a scaffold for various heterochromatin-inducing effectors. Several studies have shed some light on the roles played by this system in embryonic tissues, but its functions in the adult remain almost completely unknown. As chromatin regulation has been shown to be a major factor in the control of metabolism, we decided to investigate the role of KRAB-ZFP/KAP1 in the liver, an organ central to this process. For this, we generated conditional liver-specific Kap1 KO mice, the study of which revealed a strikingly sex-specific phenotype, with early-onset steatosis and age-related tumorigenesis restricted to males, contrasting with the mild metabolic disturbances recorded in Kap1-deleted females. Underlying this phenotype, our combined transcriptome and chromatin analyses revealed that KAP1 controls sexually dimorphic genes notably involved in hormone, drug and xenobiotic metabolism. Finally, by using a recently developed technique for direct RNA quantification, we established the range of KRAB-ZFPs expressed in the liver and likely responsible for at least part of the observed effects. This study thus demonstrates the central role of the KRAB/KAP1 system in control of sexual dimorphism, responses to xenobiotic stress and metabolic control in the liver, and further links disturbances in these processes with hepatic carcinogenesis. In parallel, in an effort to pursue a multidisciplinary training combining skills in biology and chemistry, I worked towards the development of a method to study the half-life of proteins in vivo. Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Capitalizing on an in vitro technology previously developed in Kai Johnsson's laboratory, I demonstrated how chemical labeling can be used to measure the half-life of resident intracellular and extracellular proteins in living mice. Our approach is based on labeling of SNAP-tag-fusion proteins with fluorescent probes. First, we verified that SNAP-tag substrates have wide bio-availability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then applied near-infrared probes to perform non-invasive imaging of in vivo labeled tumors. Finally, we used SNAP-mediated chemical pulse-chase labeling to measuring in vivo the half-life of different extra- and intracellular proteins, including KAP1. Importantly, this tagging technology can be applied to any protein amenable to expression as a fusion partner, whether cell-associated or secreted. Furthermore, because the SNAP-tag chemical labeling allows for a large array of probes also suited for experiments aimed at manipulating and monitoring protein function in vivo, such as cross-linking, pull-down or FRET, this methodology opens broad perspectives for studying protein function in living animals.

EPFL2011

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In Vivo Studies on the Control of Gene Expression and Protein Degradation

Karolina Bojkowska

EPFL2011

Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis

Maiia Bragina, Babak Ebadi Shirmard, Fabiana Pereira Da Costa Fraga, Nikolaos Stergiopulos, Mikaël Nils Sturny

ELSEVIER SCI LTD2020

EPFL2015