Aging is associated with chronic sub-clinical inflammation that underlies the increased risk of atherosclerosis and cardiovascular disease. It is manifested by chronic low-amplitude increases in the plasma levels of the major acute phase proteins, such as C reactive protein (CRP), serum amyloid A (SAA) and alpha1 acid glycoprotein (AGP) in the elderly. The long term objective of the proposed research is to deepen our understanding for the relationship between aging, chronic inflammation and regulation of the acute phase proteins. We recently found that ceramide, a second messenger molecule that mediates the acute stimulation of CRP, AGP and SAA during inflammation is chronically increased in liver from old rats and mice. We hypothesize that increased level of ceramide in aging cause chronic activation of C/EBP transcription factor and increased expression of CRP, AGP and SAA. Our objectives are to decipher the role of ceramide increases in aging - associated increase(s) in the expression of APP and to determine to what extent this is similar to the mechanisms that regulate the inflammation-induced changes in APP secretion. We propose the following specific aims: (I) To identify which pathway for ceramide generation leads to induction of acute phase proteins (APP) mRNA increases. Using adenovirus-mediated gene transfer we will overexpress acid and neutral SMases in primary mouse hepatocytes and will test which of the enzymes is sufficient to up-regulate the level of mRNA for three of the major APP in mouse, SAP, AGP and SAA. To identify the subcellular pool of ceramide involved in signaling, subcellular localization of overexpressed ASMase and NSMase will be studied and compared to the localization of the excess ceramide generated during aging and inflammation (2) To test in vitro whether active ASMase or NSMase is required for the induction of APP mRNA by aging and inflammation. In this specific aim we will identify the mechanism for generation of ceramide and induction of APP expression during inflammation and aging. Using hepatocytes from ASMase(-/-) mice and specific inhibitors of NSMase, we will identify which of SMases is indispensable for APP up-regulation. (3) To decipher the role of acid and neutral SMase in APP regulation in vivo. Using acid sphingomyelinase knockout mice, we will validate the conclusions drawn from Sp.
Aim 1 and 2 in a physiological context. The results of these studies will elucidate the factors that regulate ceramide level in inflammation and during aging. An understanding of the cellular and molecular mechanisms responsible for this regulation will help to define how ceramide exerts its effects and its role in cardiovascular disease, atherosclerosis and, perhaps other diseases of the elderly, such as stroke and Alzheimer disease.
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