The Mechanism Of Heparin's Inhibitory Effect On Hypoxia-induced Pulmonary Hypertension

Heparin, a glycosaminoglycan, has been used as an anticoagulant for more than 50 years. Besides anticoagulation, heparin has a variety of other biological activities, such as regulation of lipid metabolism, control of cell attachment to various proteins in the extracellular matrix, binding with acid and basic fibroblast growth factors, and inhibition of vascular smooth muscle cell (SMC) proliferation.The balance between cell proliferation and cell quiescence is regulated by a variety of cell cycle modulators. Cyclin dependent kinase (CDK) is a major regulator of the transition between the phases of the cell cycle. Cyclin/CDK complexes are composed of a regulatory subunit, cyclin, and an active kinase subunit, CDK. P27 is a primary negative regulator of CDK in SMC and play an important role in the inhibition of CDK activity.P27 is a important regulator of G1/S restriction point.An important pathological feature of pulmonary hypertension is increased medial thickening of the pulmonary artery attributable to hypertrophy and hyperplasia of pulmonary artery SMC (PASMC). Thompson and Khoury also have reported that heparin inhibits PASMC proliferation in vitro and in vivo. To date, however, the mechanism by which heparin inhibits PASMC proliferation has not been elucidated.Materials and methods1. Animals Twenty-four male SD rats were randomly divided into three groups .Group A: normoxia and injected normal saline , group B: hypoxia and injected normal saline, group C: hypoxia and injected heparin. The mice were placed in a hypoxic chamber 8hrs/d or exposed to normoxia for 2 weeks. Oxygen concentration was maintained at (10±0.5)% by controlling the flow rates of N₂. Cage concentration of O₂ was checked every 30 minutes. The heparin-treated mice were given 300 U/kg of heparin peritoneal twice daily for 14 days. In control groups, mice were given 0.4 mL of saline peritoneal twice daily.2. Measurement of right ventricular systolic pressure (RVSP)After two weeks of hypoxia ,RVSP was measured with the use of a single lumen catheter passed through the right external jugular vein to instead of the pulmonary artery pressure.3. Preparation of the sample of lung tissue.After measurement of the RVSP, recipe right lung for immunohistochemical test and left lung for RT-PCR test. The results of the immunohistochemical test were displayed by gray scale. The results of RT-PCR were displayed by the ratio of the strap of p27's absorbance on that of GAPDH.4. Statistical analysisThe results were described by (X±S) ,and analyzed by SPSS software. It isstatistical significant when p<0.05.Result1. RVSPRVSP was tested after two weeks of hypoxia. Analyse the results by Newman-Keul test. The seriatim from higher to lower is group B, group C and group A. It was statistical significant when compared either two groups of the three(p<0.01)2. Results of innumohistochemicalThe group C's gray scale were less than group A's, and group A's were less than group B's. It was statistical significant when compared either two groups of the three(p<0.01) 3. Results of RT-PCRThe results of the RT-PCR test showed that the ratio of group C was higher than that of group A ,and the ratio of group A was higher than that of group B. It was statistical significant when compared either two groups of the three(p<0.01).ConclusionP27 plays a critical role in the inhibitory effect of heparin on hypoxia-induced pulmonary hypertension. Hypoxia can inhibited the formation of p27.