Thickening of the media with intrusion on the vascular lumen contributes substantially to the pulmonary hypertension seen in primary or secondary pulmonary hypertension. Heparin has been shown to be antiproliferative and antihypertrophic for systemic and pulmonary artery smooth muscle cells (PASMC) in vitro and has been variably effective at inhibiting in vivo remodeling in several systemic and pulmonary models. We have found that commercial heparin lots vary widely in their antiproliferative activity. However, an effective antiproliferative heparin in vitro for PASMC proliferation can prevent hypoxic pulmonary hypertension in mice, rats and guinea pigs and can reverse it in guinea pigs even in the presence of continued hypoxia. We have also shown that heparin's ability to inhibit PASMC proliferation and in vivo pulmonary hypertension correlates with its ability to prevent mitogen stimulation of the Na+/H+ antiporter. We have found that highly specific antagonists of the Na+/H+ antiporter such as dimethyl amiloride (DMA) can inhibit PASMC proliferation in response to growth factors in vitro and can substantially prevent hypoxic pulmonary hypertension and remodeling in rats. As we dissect the chemistry of antiproliferative heparins we have shown, among other things, that the protein core is unimportant and that 3-0-sulfate is not an important feature in full-length heparin. We have made new heparin derivatives by O-acetylating heparin with butanoyl and hexanoyl which are more potent antiproliferative agents on PASMC than native heparins and are non-anticoagulant. We have found that in PASMC heparin stimulates the production of the cell cyclin kinase inhibitors p21 and p27 which are inhibitors of cell proliferation in other cells. With this progress we hope to continue our pursuit of an effective treatment for pulmonary hypertension with the following specific aims: 1) Continue examining strongly versus weakly antiproliferative heparins in order to discover the reasons for the differences with a goal of amplifying the antiproliferative potency and perhaps divorcing it from the anticoagulative and osteopenic properties. 2) Determine in the pig, as a preclinical trial in a large mammal, if heparin or heparin fragments or the Na+/H+ inhibitor DMA are effective at preventing hypoxic pulmonary hypertension and 3) Determine if heparin's mechanism of action in preventing SMC proliferation is via stimulation of the cyclin kinase inhibitors p21 and p27. Thus, this proposal may lead to new therapeutic agents for humans with pulmonary hypertension and may elucidate a new understanding of how heparin prevents PASMC growth.
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