Vascular remodeling with hypertrophy, hyperplasia and distal extension of pulmonary artery smooth muscle cells (PASMC) is a major component of the pulmonary vascular resistance in pulmonary hypertension. Several agents have become available to treat remodeling but none have been very satisfactory. Heparin is a normal constituent of the lung and is antiproliferative for PASMC. This grant continues our studies to understand how the cluster of varying glycosaminoglycans known as heparin work as an antiproliferative agent and to develop them, or their derivatives, into effective therapies for pulmonary hypertension. Unfractionated heparin contains a central protein core with many carbohydrate side chains which vary in content from batch to batch. We have found some of these batches to be much more antiproliferative for PASMC than others and to be better at inhibiting the development of hypoxia induced hypertension and remodeling in mice, rats, guinea pigs and now pigs. Low molecular weight (LMW) heparins are the carbohydrate side chains of heparin cleaved off the protein core and are not very antiproliferative. We have created more potent antiproliferative LMW heparins by adding bulky hexanoylated (H) or butanoylated (B) groups. We have also shown that heparin inhibits PASM proliferation by stimulating the cyclin kinase p27. We have preliminary evidence that heparin may do this by suppressing ERK and Rho/ROCK activity. With this background we plan the following specific aims: 1. Determine the structures in heparin's carbohydrate side chains which mediate the antiproliferative action on PASMC by: a. identifying the importance of the 2-O sulfonate groups on the hexuronic acid residues as well as the proportions of basic sugar residues, i.e. N- acetylation and N-sulfonation of glucosamine in the repeating disaccharide units necessary to give maximum antiproliferative activity, b. altering the chemical structure of our H and B LMW heparins to match features identified as enhancing the antiproliferative strength in step 1a, to make these LMW heparin derivatives even more potent, and c. increasing the length of the H and B LMW heparins to octa and decanoylated chains to try to achieve even greater antiproliferative potency. 2. Determine in a preclinical model if potent antiproliferative heparins or heparin derivatives can inhibit or reverse established pulmonary hypertension in hypoxic pigs. 3. Determine the mechanism by which heparin enhances p27 activity (ERK, Rho/ROCK;etc), build on our pilot data that strongly antiproliferative heparins inhibit RhoA/ROCK while weakly antiproliferative heparins do not, and verify our findings in heparin treated hypoxic mice.
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