Endothelin-1 role in NLRP3 activation in smooth muscle tissue of corpora cavernosa

Author(s)

Rafael Sobrano Fais

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-03-03

Examining board members

Fernando Silva Carneiro; Edson Antunes; Riccardo Lacchini; Luciana Venturini Rossoni

• Advisor: Fernando Silva Carneiro

Abstract

Introduction: Erectile dysfunction (ED) is defined as the inability to achieve or maintain an erection of the penis for satisfactory sexual performance. ED is often associated with risk factors for the development of cardiovascular diseases such as arterial hypertension, and/or metabolic diseases such as dyslipidemia and diabetes. Arterial hypertension has become a growing public health problem, as it is an important risk factor for the onset of cardiovascular, cerebrovascular and renal diseases. In addition, ED occurs twice as often in patients with arterial hypertension as compared to normotensive patients. Vascular damage in hypertension occurs, in part, due to the increase in inflammatory mediators. Similarly, several studies show that increased expression of inflammatory mediators are closely linked to the development of ED. ED is strongly influenced by increased expression of endothelin-1 (ET-1) in a DOCA/salt model of hypertension, due to its contractile and pro-inflammatory effects. The NLRP3 receptor is the most studied member of the family of the inflammasome, a multiprotein complex of the innate immune system that triggers caspase-1 activation and maturation of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β). The increased activity of the NLRP3 receptor promotes vascular alterations, cardiac and renal hypertrophy, in addition to increased blood pressure. However, it is still unknown whether NLRP3 contributes to the pro-inflammatory action of ET-1. Hypothesis: NLRP3 mediates chronic proinflammatory effects of ET-1 on CC of DOCA/salt animals, constituting an important mechanism for the genesis of ED. Objective: To investigate the role of NLRP3 in ET-1-induced inflammatory process modulation and ED associated with the DOCA/salt hypertension model. Methods: C57BL/6 (WT) and NLRP3 deficient (NLRP3-/-) mice were uninefrectomized and received a silicone pellet containing deoxycorticosterone acetate (DOCA, 2.4 mg/Kg/day) or empty for 21 days. These WT animals were treated with bosentan (gavage 30 mg/Kg/day) in the last 10 days. The mice systolic blood pressure was checked by tail cuff before and on days 3, 7, 10, 14 and 21 after surgery. The levels of ET-1 were assessed in the serum, whereas IL-1β, TNF-α and IL-6 were measured in the corpora cavernosa (CC). We also evaluated caspase-1 activity using the FLICA fluorescent probe. The expression of caspase-1, pro-caspase-1, IL-1β and pro-IL-1β was evaluated by Western blotting. Erectile function was determined by electrically stimulating the cavernous nerve at frequencies from 0.2 to 20 Hz. The ratio between the mean plateau of intracavernous pressure and mean arterial pressure (ICP/MAP) was obtained and analyzed. ICP/MAP was also performed in response to ET-1 injection (0.2492 µg/Kg) in the CC of WT mice with previous administration of MCC950 (0.42646 µg/Kg) or vehicle in the CC. CC reactivity was assessed by relaxation mediated by electrical stimulus, for acetylcholine and sodium nitroprusside, while contraction was induced by phenylephrine. We evaluated, by flow cytometry, the infiltration of immune system cells in the CC12 (macrophages, neutrophils, Th1 and Th17 lymphocytes). The expression of ICAM-1, VCAM-1, of CCL2 smooth muscle alpha-actin and collagen deposition was determined. Finally, WT or RAG-1 deficient mice (RAG-1-/-) were uninefrectomized and received a silicone pellet containing DOCA (2.4 mg/kg/day) or empty for 21 days. In order to verify the effect of neutrophil depletion on erectile function, WT mice were treated with antibody for neutrophil depletion (BE0075-1, 200 µg intraperitoneal injection) or vehicle on days 12 and 17 after surgery. Mice systolic blood pressure was measured by tail cuff before, and on days 3, 7, 10, 14 and 21 after surgery. ICP/MAP was also recorded in this group of animals. Results: DOCA/salt hypertensive mice had increased levels of TNF-α, of caspase-1 activity, expression of caspase-1 and IL-1β in CC. Bosentan treatment or the absence of NLRP3 reversed or prevented these changes. In addition, bosentan treatment, pharmacological inhibition of NLRP3 or the absence of NLRP3 restored erectile function (ICP/MAP), relaxation for acetylcholine and sodium nitroprussitate in the DOCA / salt mice. There was an increase in the recruitment of neutrophils and Th1 lymphocytes to the DOCA/salt mice CC. However, treatment with bosentan or the absence of NLRP3 prevented this increase. In addition, the increment in VCAM-1, ICAM-1 and CCL2 promoted by DOCA/salt hypertension was prevented by treatment with bosentan or the NLRP3 gene deletion. Additionally, we observed that treatment with bosentan or the absence of NLRP3 prevents the in collagen or smooth muscle deposition increment in the DOCA/salt mice. Finally, treatment with BE0075-1 completely restored the increase in blood pressure, IL-1β expression, ICP/MAP, the relaxation for acetylcholine and for sodium nitroprusside in DOCA/salt mice. On the other hand, the RAG-1 gene deletion completely prevented the increase in blood pressure, caspase-1 expression, attenuated the impairment in erectile function and restored relaxation to acetylcholine, but not to sodium nitroprusside. Conclusion: NLRP3 is an important mechanism for the genesis of ED, through increased cytokine release, recruitment of neutrophils and Th1 lymphocytes, promoted by ET-1 in CC. (AU)

• FAPESP's process: 16/07641-0 - The NLRP3 role in erectile dysfunction in hypertensive model DOCA / salt and its possible activation by ET-1
• Grantee: Rafael Sobrano Fais
• Support Opportunities: Scholarships in Brazil - Doctorate