title: Hsp70 regulation on Nox4/p22phox and cytoskeletal integrity as an effect of losartan in vascular smooth muscle cells.
abstract: A series of signaling cascades are activated after angiotensin II binds to angiotensin II type I receptor (AT1R), a peptide that is an important mediator of oxidative stress. Hsp70 regulates a diverse set of signaling pathways through interactions with proteins. Here, we tested the hypothesis of angiotensin II AT1R inhibition effect on Hsp70 interaction with Nox4/p22phox complex and Hsp70 leading to actin cytoskeleton modulation in spontaneously hypertensive rats (SHR) vascular smooth muscle cells (VSMCs). SHR and Wistar-Kyotto rats (VSMCs from 8 to 10 weeks) were stimulated with angiotensin II (100 nmol/L) for 15 min (AII), treated with losartan (100 nmol/L) for 90 min (L), and with losartan for 90 min plus angiotensin in the last 15 min (L + AII). Whereas SHR VSMCs exposure to angiotensin II overexpressed AT1R and Nox4 nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and slightly downregulated caveolin-1 expression, losartan decreased AT1R protein levels and increased caveolin-1 and Hsp70 expression in SHR VSMC membranes. Immunoprecipitation and immunofluorescence confocal microscopy proved interaction and colocalization of membrane translocated Hsp70 and Nox4/p22phox. Increased levels of Hsp70 contrast with the decreased immunoprecipitation of Nox4/p22phox and RhoA in membranes from SHR VSMCs (L) vs SHR VSMCs (AII). Hsp72 depletion resulted in higher Nox4 expression and increased NADPH oxidase activity in VSMCs (L + AII) from SHR when contrasted with nontransfected VSMCs (L + AII). After Hsp72 knockdown in SHR VSMCs, losartan could not impair angiotensin II-enhanced stress fiber formation and focal adhesion assembly. In conclusion, our data showing a negative regulation of Hsp70 on Nox4/p22phox demonstrates a possible mechanism in explaining the antioxidative function joined to cytoskeletal integrity modulation within the effects of losartan in VSMCs from SHR.
authors.affiliation: IMBECU-CONICET (National Council of Scientific and Technical Research of Argentina), Mendoza, Argentina.
authors.forename: Andrea Fernanda
authors.initials: AF
authors.lastname: Gil Lorenzo
authors.forename: Victoria
authors.initials: V
authors.lastname: Bocanegra
authors.forename: María Eugenia
authors.initials: ME
authors.lastname: Benardon
authors.forename: Valeria
authors.initials: V
authors.lastname: Cacciamani
authors.forename: Patricia G
authors.initials: PG
authors.lastname: Vallés
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology*; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D050884:HSP72 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019808:Losartan / Q000494:pharmacology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D017208:Rats, Wistar; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000959:Antihypertensive Agents; D051242:Caveolin 1; D018840:HSP70 Heat-Shock Proteins; D050884:HSP72 Heat-Shock Proteins; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; D020742:rhoA GTP-Binding Protein; D019808:Losartan