Hypertension is a major risk factor for .cardiovascular and renal diseases. Inflammation and components of the extracellular matrix (EM) have a negative impact on the physiology and function of end target organs such as the arteries, heart and kidneys in hypertension. Blocking angiotensin-converting enzyme (ACE) decreases angiotensin II (Ang II) and increases kinins, leading to decreased cardiovascular inflammation, hypertrophy and collagen. ACE inhibitors (ACEi) increase plasma Ac-SDKP, a negative regulator of cell proliferation present in plasma and tissue. In hypertension and heart failure, Ac-SDKP prevents monocyte/macrophage infiltration and fibrosis in the aorta, kidneys and left ventricle (LV). By virtue of its anti- fibrotic and anti-inflammatory effects, Ac-SDKP was able to improve renal function in hypertension, diabetes and other experimental models of renal diseases. However, the mechanism(s) or receptor(s) involved in Ac- SDKP's cardiovascular and renal effects are not fully understood. We hypothesize that Ac-SDKP exerts its anti-inflammatory and anti-fibrotic effects on the cardiovascular and renal systems in hypertension via specific receptor(s) located on the plasma membrane, contributing to end organ protection.
In aim I we will identify and characterize Ac-SDKP receptors using pharmacological tools [ l]Hpp-Aca-SDKP, 5(6)FAM-SDKP and new analogues of Ac-SDKP), proteomic technology, and cloning techniques.
In aim II we will 1) perform a more extensive examination of the structural activity of Ac-SDKP in order to a) develop potent antagonists that lack partial agonistic activity and b) improve the affinity of the radio-iodinated peptide;and 2) characterize the Ac-SDKP receptor in fibroblasts and macrophages (rat and human), using [ l]-Hpp- Aca-SDKP and newly developed antagonists;and 3) compare rat cardiac fibroblasts and human cardiac fibroblasts for the inhibitory effect of Ac-SDKP or analogues on collagen synthesis and proliferation.
In aim we will study whether Ac-SDKP receptor activity depends on mechanisms closely linked to the regulation of receptor internalization.
In aim I V We will determine 1) the effect of Ac-SDKP on the non-receptor tyrosine kinase Src and HB-EGF on Ang II and ET-1-stimulated transactivation of the EGFR;2) whether Ac-SDKP inhibits the effects of calcium ionophores or EGF on p42/44 MAPK and collagen synthesis;3) whether PLC, EGFR, cSrc, calmodulin kinase or IP3 inhibitors attenuate MAPK activity and collagen synthesis to the same extent as Ac-SDKP in response to Ang II or ET-1;and 4) whether inhibition of MAP kinase activation by Ac- SDKP is mediated by MAP kinase phosphatase-1, using selective inhibitors of phosphatases and specific SiRNAs. This project will provide important new information on the d the mechanism of action of Ac-SDKP. Consequently, it will identify another component (Ac-SDKP) as part of the multiple mediators participating in the cardioprotective effects of ACEi in hypertension.
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