application) Heparan sulfates (HSs) and heparin, proposed as endogenous defending molecules in the """"""""glomerular self-defense"""""""", are inhibitors of mesangial cell growth in human proliferative nephritides, in experimental renal disease models, and in cell culture, but the side activities of heparin, such as anti-coagulant activity, have limited its potential therapeutical applications. My previous studies have shown that HSs and heparin are inhibitors of rat mesangial cell (RMC) proliferation in culture, but that endogenous HS oligosaccharides synthesized by RMCs are much more potent than heparin on an equivalent mass basis. We hypothesize that a defined fine structure(s) in the HS oligosaccharides is required to bind to a specific receptor(s) on the mesangial cell surface, thereby inhibiting RMC proliferation. The overall purpose of this proposal is to adapt Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) for the analysis of the fine structure of the RMC HS oligosaccharides and to deduce the essential structures required for their anti-mitogenic activity. We propose: 1) to adapt the FACE methods to separate, identify and quantitate fluorotagged derivatives prepared from digests of heparin/HS with the eliminase heparitinase enzymes; 2a) to use RMCs cultured with [3H]glucosamine and [35S]sulfate to label the intact HS proteoglycans and the HS oligosaccharides generated from them via intracellular endoglycosidases; and 2b) to prepare chemical amounts (>200 pg quantities) of the HS oligosaccharides and of the intact HS chains following proteinase K digestion of the parent proteoglycans; 3a) to determine the concentration dependence of the anti-mitogenic activity of the HS chains and of the HS oligosaccharides, and their binding constants to the RMCs; and 3b) to modify selectively the HS chains, the HS oligosaccharides and heparin by specific enzymes (heparitinase lyases) and by fluorotagging, and to test the products for anti-mitogenic and binding activities; 4a) to separate sub-fractions of bioactive and inactive HS oligosaccharides by affinity binding to RMCs at 4 degrees C; and 4b) to determine the fine structures of the active (bound) and inactive (unbound) HS oligosaccharides using FACE analyses and thereby identify key structures required for the antimitogenic activity. If successful, this project can provide a head start for defining the molecular basis of HSs in regulating mesangial cell growth and mechanism(s) underlying this function, and designing suitable compounds for clinical treatment of proliferative nephritides using HSs as model molecules.
| Lauer, Mark E; Hascall, Vincent C; Wang, Aimin (2007) Heparan sulfate analysis from diabetic rat glomeruli. J Biol Chem 282:843-52 |
| Wang, Aimin; Hascall, Vincent C (2004) Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion. J Biol Chem 279:10279-85 |
