O65 SARCOPLASMIC CALCIUM ATPASE (SERCA) PROTEOLYSIS BY MATRIX METALLOPROTEINASE (MMP)-2 CONTRIBUTES TO VASCULAR DYSFUNCTION IN EARLY HYPERTENSION

Abstract

Background and objective: Hypertension-induced increased matrix metalloproteinase (MMP)-2 activity proteolyzes extra- and intracellular proteins in aortas and contributes to vascular dysfunction. MMP-2 proteolyzes sarcoplasmic reticulum calcium ATPase (SERCA) in the heart during ischemia and reperfusion injury, thus impairing cardiac function. SERCA activity is also impaired in the arteries of hypertensive animals. We investigated whether hypertension-induced increased MMP-2 activity in aorta contributes to SERCA proteolysis, vascular remodeling and dysfunction. Methods: Aortas from male Sprague-Dawley (SD) rats, incubated or not with angiotensin II (AngII) and MMP inhibitors, ONO-4817 or doxycycline (Doxy), were used for zymography and co-immunoprecipitation of SERCA with MMP-2. Male SD rats were also submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with Doxy from third to seventh day post-surgery. Systolic blood pressure (SBP) was assessed basally and daily by tail-cuff plethysmography. After 7 days, aortas were collected for gel and in situ zymography, western blot to SERCA, ATPase activity assay, vascular reactivity to phenylephrine and acetylcholine, Ki-67 immunofluorescence and hematoxylin/eosin stain. A7r5 cells were incubated or not with AngII, in the presence or absence of SERCA inhibitor, tapsigargin, to assess intracellular calcium concentrations. Results: AngII increased MMP-2 activity in aortas and MMP-2 co-immunoprecipitated with SERCA. SBP was increased in 2K-1C rats and Doxy did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Intracellular calcium concentrations were increased in A7r5 cells incubated with AngII and/or SERCA inhibitor, tapsigargin. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and Doxy decreased Ki-67 cell proliferation. In 2K-1C rats, arterial contraction to phenylephrine was accentuated but Doxy ameliorated only the impaired relaxation to acetylcholine. Conclusion: Doxy reduced MMP-2 activity in the aortas of 2K-1C rats and prevented the ATPase activity impairment and MMP-2-induced SERCA proteolysis and vascular dysfunction.