Cyclin-Dependent Kinase Inhibitor p27 ^Kip1 , But Not p21 ^WAF1/Cip1 , Is Required for Inhibition of Hypoxia-Induced Pulmonary Hypertension and Remodeling by Heparin in Mice

Abstract

Abstract-Heparin has growth inhibitory effects on pulmonary artery smooth muscle cell (PASMC) in vitro and in vivo.However, the mechanism has not been fully defined. In this study, we investigated the role of cyclin-dependent kinase inhibitors, p21 WAF1/cip1 (p21) and p27 Kip1 (p27), in the inhibitory effect of heparin on PASMC proliferation in vitro and on hypoxia-induced pulmonary hypertension in vivo using p21 and p27-null mice. In vitro, loss of the p27 gene negated the inhibitory effect of heparin on PASMC proliferation, but p21 was not critical for this inhibition. In vivo, heparin significantly inhibited the development of hypoxia-induced pulmonary hypertension and remodeling, as evidenced by decreased right ventricular systolic pressure, ratio of right ventricular weight to left ventricle plus septum weight, and percent wall thickness of pulmonary artery, in p21 ϩ/ϩ , p21 Ϫ/Ϫ , p27 ϩ/ϩ , and p27 ϩ/Ϫ , but not in p27 Ϫ/Ϫ mice. We also observed that hypoxia decreased p27 expression significantly in mouse lung, which was restored by heparin. Heparin inhibited Ki67 proliferative index in terminal bronchial vessel walls in p27 ϩ/ϩ and p27 ϩ/Ϫ , but not in p27 Ϫ/Ϫ mice exposed to hypoxia. Therefore, we conclude that the cyclin-dependent kinase inhibitor p27, but not p21, is required for the inhibition of hypoxic pulmonary vascular remodeling by heparin. Kip1 Ⅲ p21 WAF1/cip1 Ⅲ heparin Ⅲ pulmonary hypertension Ⅲ hypoxia Ⅲ mouse H eparin, a glycosaminoglycan, has been used as an anticoagulant for more than 50 years. 1 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. 2 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). 3,4 Our previous studies have shown that antiproliferative heparins significantly inhibit pulmonary vascular remodeling induced by hypoxia in rodents 5-7 and PASMC proliferation in culture. 8 -10 Other investigators also have reported that heparin inhibits PASMC proliferation in vitro and in vivo. 8,11 To date, however, the mechanism by which heparin inhibits PASMC proliferation has not been elucidated.The balance between cell proliferation and cell quiescence is regulated by a variety of cell cycle modulators. Cyclindependent kinase (CDK) is a major regulator of the transition between the phases of the cell cycle. 12 Cyclin/CDK complexes are composed of a regulatory subunit, cyclin, and an active kinase subunit, CDK. The cyclin/CDK complexes are controlled by both positive and negative regulators. 13 p21 WAF1/cip1

Cyclic Stretch Affects Pulmonary Endothelial Cell Control of Pulmonary Smooth Muscle Cell Growth

“…BPAECs and BPASMCs were isolated from bovine pulmonary arteries that were purchased as a bovine heart with pulmonary arteries attached as previously described (19). Briefly, under sterile conditions, a clamp was placed on the artery just above the cardiac muscle and below the main branches.…”

“…After 72 hours of growth, the cells were harvested with trypsin/EDTA (Cambrex Corp.) and counted using a Coulter Counter ZM (Coulter Electronics, Hialeah, FL). The percent growth was calculated as (net cell growth in treated medium/net cell growth in standard medium) 3100, where the net cell growth 5 cell growth in standard or treated medium -cell growth in growth arrest medium, as previously described (19). The percent static growth was calculated (net cell growth in media containing lyophilized conditioned media from stretched BPAECs/net cell growth in media containing lyophilized conditioned media from static BPAEC) 3 100.…”

Total flavonoids from Ampelopsis megalophylla suppress proliferation of vascular smooth muscle cells in vivo and in vitro

“…Kip1 is an essential suppressor of hypoxic pulmonary vascular remodeling (Yu et al, 2005). In the present study, the amount of p27…”

Hypoxic Pulmonary Vascular Smooth Muscle Cell Proliferation

“…The suppressive effect of hypoxia on p27 expression has been demonstrated in mice with pulmonary hypertension induced by hypoxia [8]. However, the expression of p27, which blocks the cell cycle at the G0/1 phase, is regulated by several mechanisms including transcription, protein degradation and translation [43][44][45].…”

“…These results suggest that hypoxic enhancement of PASMC proliferation contributes to the progression of hypoxia-associated small pulmonary arterial remodeling and secondary pulmonary hypertension. In animals, hypoxia has been shown to cause pulmonary vascular wall thickening by inducing PASMC proliferation [7][8][9]. Most commonly, pulmonary vascular remodeling is studied in rats or mice exposed to 10% oxygen hypoxia for 2 to 8 weeks [9,10].…”