title: Internal Pudental Artery Dysfunction in Diabetes Mellitus Is Mediated by NOX1-Derived ROS-, Nrf2-, and Rho Kinase-Dependent Mechanisms.
abstract: Oxidative stress plays an important role in diabetes mellitus (DM)-associated vascular injury. DM is an important risk factor for erectile dysfunction. Functional and structural changes in internal pudendal arteries (IPA) can lead to erectile dysfunction. We hypothesized that downregulation of nuclear factor E2-related factor 2 (Nrf2), consequent to increased nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1)-derived reactive oxygen species (ROS), impairs IPA function in DM. IPA and vascular smooth muscle cells from C57BL/6 (control) and NOX1 knockout mice were used. DM was induced by streptozotocin in C57BL/6 mice. Functional properties of IPA were assessed using a myograph, protein expression and peroxiredoxin oxidation by Western blot, RNA expression by polymerase chain reaction, carbonylation by oxyblot assay, ROS generation by lucigenin, nitrotyrosine, and amplex red, and Rho kinase activity and nuclear accumulation of Nrf2 by ELISA. IPA from diabetic mice displayed increased contractions to phenylephrine (control 138.5±9.5 versus DM 191.8±15.5). ROS scavenger, Nrf2 activator, NOX1 and Rho kinase inhibitors normalized vascular function. High glucose increased ROS generation in IPA vascular smooth muscle cell. This effect was abrogated by Nrf2 activation and not observed in NOX1 knockout vascular smooth muscle cell. High glucose also increased levels of nitrotyrosine, protein oxidation/carbonylation, and Rho kinase activity, but reduced Nrf2 activity and expression of Nrf2-regulated genes (catalase [25.6±0.05%], heme oxygenase-1 [21±0.1%], and
  
  
  
  quinone oxidoreductase 1 [22±0.1%]) and hydrogen peroxide levels. These effects were not observed in vascular smooth muscle cell from NOX1 knockout mice. In these cells, high glucose increased hydrogen peroxide levels. In conclusion, Rho kinase activation, via NOX1-derived ROS and downregulation of Nrf2 system, impairs IPA function in DM. These data suggest that Nrf2 is vasoprotective in DM-associated erectile dysfunction.
authors.affiliation: From the Ribeirao Preto Medical School (R.A.-L., K.B.N., F.S.C., R.C.T.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Brazil; and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (A.C.M., A.H., R.M.T.). rheurelopes@gmail.com.
authors.forename: Rhéure
authors.initials: R
authors.lastname: Alves-Lopes
authors.affiliation: From the Ribeirao Preto Medical School (R.A.-L., K.B.N., F.S.C., R.C.T.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Brazil; and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (A.C.M., A.H., R.M.T.).
authors.forename: Karla B
authors.initials: KB
authors.lastname: Neves
authors.affiliation: From the Ribeirao Preto Medical School (R.A.-L., K.B.N., F.S.C., R.C.T.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Brazil; and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (A.C.M., A.H., R.M.T.).
authors.forename: Augusto C
authors.initials: AC
authors.lastname: Montezano
authors.affiliation: From the Ribeirao Preto Medical School (R.A.-L., K.B.N., F.S.C., R.C.T.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Brazil; and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (A.C.M., A.H., R.M.T.).
authors.forename: Adam
authors.initials: A
authors.lastname: Harvey
authors.affiliation: From the Ribeirao Preto Medical School (R.A.-L., K.B.N., F.S.C., R.C.T.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Brazil; and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (A.C.M., A.H., R.M.T.).
authors.forename: Fernando S
authors.initials: FS
authors.lastname: Carneiro
authors.affiliation: From the Ribeirao Preto Medical School (R.A.-L., K.B.N., F.S.C., R.C.T.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Brazil; and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (A.C.M., A.H., R.M.T.).
authors.forename: Rhian M
authors.initials: RM
authors.lastname: Touyz
authors.affiliation: From the Ribeirao Preto Medical School (R.A.-L., K.B.N., F.S.C., R.C.T.) and Faculty of Pharmaceutical Sciences of Ribeirao Preto (K.B.N.), University of Sao Paulo, Brazil; and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (A.C.M., A.H., R.M.T.).
authors.forename: Rita C
authors.initials: RC
authors.lastname: Tostes
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000150:complications*; D004195:Disease Models, Animal; D007172:Erectile Dysfunction / Q000209:etiology / Q000503:physiopathology; D005260:Female; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D000074624:NADPH Oxidase 1; D051267:NF-E2-Related Factor 2 / Q000235:genetics*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D010413:Penis / Q000098:blood supply*; D011897:Random Allocation; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction
chemical_list: D000975:Antioxidants; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D017382:Reactive Oxygen Species; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614711:NOX1 protein, mouse
keywords: NOX1; Nrf2; Rho-associated kinases; diabetes mellitus; erectile dysfunction