Lunyin Yu

Cyclin-Dependent Kinase Inhibitor p27Kip1, But Not p21WAF1/Cip1, Is Required for Inhibition of Hypoxia-Induced Pulmonary Hypertension and Remodeling by Heparin in Mice

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, p21WAF1/cip1 (p21) and p27Kip1 (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+/+, p21−/−, p27+/+, and p27+/−, but not in p27−/− 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+/+ and p27+/−, but not in p27−/− 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.

Deficiency of the NHE1 gene prevents hypoxia-induced pulmonary hypertension and vascular remodeling.

RATIONALE Our previous studies found that Na(+)/H(+) exchanger (NHE) activity played an essential role in pulmonary artery smooth muscle cell (PASMC) proliferation and in the development of hypoxia-induced pulmonary hypertension and vascular remodeling. Other investigators recently observed increased expression of the NHE isoform 1 (NHE1) gene in rodents with pulmonary hypertension induced by hypoxia. However, a causal role for the NHE1 gene in pulmonary hypertension has not been determined. OBJECTIVES To determine the causal role of the NHE1 gene in pulmonary hypertension and vascular remodeling. METHODS We used NHE1-null mice to define the role of the NHE1 gene in the development of pulmonary hypertension and remodeling induced by hypoxia and to delineate the NHE1 regulatory pathway. MEASUREMENTS AND MAIN RESULTS After 2 weeks of exposure to hypoxia, in contrast to wild-type hypoxic littermates, there was no significant increase in right ventricular systolic pressure, in the ratio of right ventricular to left ventricular plus septal weight [RV/(LV + S)], or in medial wall thickness of the pulmonary arterioles in homozygous mice (NHE1(-/-)). There was a significant decrease in Rho kinase (ROCK1 and ROCK2) expression, accompanied by an increase in p27 expression in NHE1(-/-) mice. CONCLUSIONS Our study demonstrated that deficiency of the NHE1 gene prevented the development of hypoxia-induced pulmonary hypertension and vascular remodeling in mice and revealed a novel regulatory pathway associated with NHE1 signaling.

Silencing of Sodium-Hydrogen Exchanger 1 Attenuates the Proliferation, Hypertrophy, and Migration of Pulmonary Artery Smooth Muscle Cells via E2F1

We previously found that deficiency of the sodium-hydrogen exchanger 1 (NHE1) gene prevented hypoxia-induced pulmonary hypertension and vascular remodeling in mice, which were accompanied by a significantly reduced proliferation of pulmonary artery smooth muscle cells (PASMCs), and which decreased the medial-wall thickness of pulmonary arteries. That finding indicated the involvement of NHE1 in the proliferation and hypertrophy of PASMCs, but the underlying mechanism was not fully understood. To define the mechanism by which the inhibition of NHE1 decreases hypoxic pulmonary hypertension and vascular remodeling, we investigated the role of E2F1, a nuclear transcription factor, in silencing the NHE1 gene-induced inhibition of the proliferation, hypertrophy, and migration of human PASMCs. We found that: (1) silencing of NHE1 by short, interfering RNA (siRNA) significantly inhibited PASMC proliferation and cell cycle progression, decreased hypoxia-induced hypertrophy (in terms of cell size and protein/DNA ratio) and migration (in terms of the wound-healing and migration chamber assays); (2) hypoxia induced the expression of E2F1, which was reversed by NHE1 siRNA; and (3) the overexpression of E2F1 blocked the inhibitory effect of NHE1 siRNA on the proliferation, hypertrophy, and migration of PASMCs. The present study determined that silencing the NHE1 gene significantly inhibited the hypoxia-induced proliferation, hypertrophy, and migration of human PASMCs via repression of the nuclear transcription factor E2F1. This study revealed a novel mechanism underlying the regulation of hypoxic pulmonary hypertension and vascular remodeling via NHE1.

Thrombospondin-1 null mice are resistant to hypoxia-induced pulmonary hypertension.

Background and objectiveChronic hypoxia induces pulmonary hypertension in mice. Smooth muscle cell hyperplasia and medial thickening characterize the vasculature of these animals. Thrombospondin-1 null (TSP-1-/-) mice spontaneously develop pulmonary smooth muscle cell hyperplasia and medial thickening. In addition, TSP-1 produced by the pulmonary endothelium inhibits pulmonary artery smooth muscle cell growth. Based on these observations we sought to describe the pulmonary vascular changes in TSP-1-/- mice exposed to chronic hypoxia.MethodsWe exposed TSP-1-/- and wild type (WT) mice to a fraction of inspired oxygen (FiO2) of 0.1 for up to six weeks. Pulmonary vascular remodeling was evaluated using tissue morphometrics. Additionally, right ventricle systolic pressures (RVSP) and right ventricular hypertrophy by right ventricle/left ventricle + septum ratios (RV/LV+S) were measured to evaluate pulmonary hypertensive changes. Finally, acute pulmonary vasoconstriction response in both TSP-1-/- and WT mice was evaluated by acute hypoxia and U-46619 (a prostaglandin F2 analog) response.ResultsIn hypoxia, TSP-1-/- mice had significantly lower RVSP, RV/LV+S ratios and less pulmonary vascular remodeling when compared to WT mice. TSP-1-/- mice also had significantly lower RVSP in response to acute pulmonary vasoconstriction challenges than their WT counterparts.ConclusionTSP-1-/- mice had diminished pulmonary vasoconstriction response and were less responsive to hypoxia-induced pulmonary hypertension than their wild type counterparts. This observation suggests that TSP-1 could play an active role in the pathogenesis of pulmonary hypertension associated with hypoxia.

Effect of chemokine receptor CXCR4 on hypoxia-induced pulmonary hypertension and vascular remodeling in rats

BackgroundCXCR4 is the receptor for chemokine CXCL12 and reportedly plays an important role in systemic vascular repair and remodeling, but the role of CXCR4 in development of pulmonary hypertension and vascular remodeling has not been fully understood.MethodsIn this study we investigated the role of CXCR4 in the development of pulmonary hypertension and vascular remodeling by using a CXCR4 inhibitor AMD3100 and by electroporation of CXCR4 shRNA into bone marrow cells and then transplantation of the bone marrow cells into rats.ResultsWe found that the CXCR4 inhibitor significantly decreased chronic hypoxia-induced pulmonary hypertension and vascular remodeling in rats and, most importantly, we found that the rats that were transplanted with the bone marrow cells electroporated with CXCR4 shRNA had significantly lower mean pulmonary pressure (mPAP), ratio of right ventricular weight to left ventricular plus septal weight (RV/(LV+S)) and wall thickness of pulmonary artery induced by chronic hypoxia as compared with control rats.ConclusionsThe hypothesis that CXCR4 is critical in hypoxic pulmonary hypertension in rats has been demonstrated. The present study not only has shown an inhibitory effect caused by systemic inhibition of CXCR4 activity on pulmonary hypertension, but more importantly also has revealed that specific inhibition of the CXCR4 in bone marrow cells can reduce pulmonary hypertension and vascular remodeling via decreasing bone marrow derived cell recruitment to the lung in hypoxia. This study suggests a novel therapeutic approach for pulmonary hypertension by inhibiting bone marrow derived cell recruitment.

Long-term exposure to hypoxia inhibits tumor progression of lung cancer in rats and mice

BackgroundHypoxia has been identified as a major negative factor for tumor progression in clinical observations and in animal studies. However, the precise role of hypoxia in tumor progression has not been fully explained. In this study, we extensively investigated the effect of long-term exposure to hypoxia on tumor progression in vivo.MethodsRats bearing transplanted tumors consisting of A549 human lung cancer cells (lung cancer tumor) were exposed to hypoxia for different durations and different levels of oxygen. The tumor growth and metastasis were evaluated. We also treated A549 lung cancer cells (A549 cells) with chronic hypoxia and then implanted the hypoxia-pretreated cancer cells into mice. The effect of exposure to hypoxia on metastasis of Lewis lung carcinoma in mice was also investigated.ResultsWe found that long-term exposure to hypoxia a) significantly inhibited lung cancer tumor growth in xenograft and orthotopic models in rats, b) significantly reduced lymphatic metastasis of the lung cancer in rats and decreased lung metastasis of Lewis lung carcinoma in mice, c) reduced lung cancer cell proliferation and cell cycle progression in vitro, d) decreased growth of the tumors from hypoxia-pretreated A549 cells, e) decreased Na+-K+ ATPase α1 expression in hypoxic lung cancer tumors, and f) increased expression of hypoxia inducible factors (HIF1α and HIF2α) but decreased microvessel density in the lung cancer tumors. In contrast to lung cancer, the growth of tumor from HCT116 human colon cancer cells (colon cancer tumor) was a) significantly enhanced in the same hypoxia conditions, accompanied by b) no significant change in expression of Na+-K+ ATPase α1, c) increased HIF1α expression (no HIF2α was detected) and d) increased microvessel density in the tumor tissues.ConclusionsThis study demonstrated that long-term exposure to hypoxia repressed tumor progression of the lung cancer from A549 cells and that decreased expression of Na+-K+ ATPase was involved in hypoxic inhibition of tumor progression. The results from this study provide new insights into the role of hypoxia in tumor progression and therapeutic strategies for cancer treatment.

Hypoxia Does neither Stimulate Pulmonary Artery Endothelial Cell Proliferation in Mice and Rats with Pulmonary Hypertension and Vascular Remodeling nor in Human Pulmonary Artery Endothelial Cells

Background: Hypoxia results in pulmonary hypertension and vascular remodeling due to induction of pulmonary artery cell proliferation. Besides pulmonary artery smooth muscle cells, pulmonary artery endothelial cells (PAECs) are also involved in the development of pulmonary hypertension, but the effect of hypoxia on PAEC proliferation has not been completely understood. Methods: We investigated PAEC proliferation in mice and rats with hypoxia-induced pulmonary hypertension and vascular remodeling as well as in human PAECs under hypoxia. Results and Conclusion: We did not find significant PAEC proliferation in chronically hypoxic rats or mice. There was a slight decrease in proliferation in mice and rats with pulmonary hypertension and vascular remodeling. We also did not find significant human PAEC proliferation and cell cycle progression under different levels of oxygen (1, 2, 3, 5 and 10%) for one day, although the same conditions of hypoxia induced significant proliferation and cell cycle progression in pulmonary artery smooth muscle cells and pulmonary artery fibroblasts. Exposure to hypoxia for 7 days also did not increase PAEC proliferation. These results demonstrated that hypoxia alone is not a stimulus to PAEC proliferation in vivo and in vitro. The present study provides a novel role for PAECs in hypoxia-induced pulmonary hypertension and vascular remodeling.

Heparin Inhibits Pulmonary Artery Smooth Muscle Cell Proliferation through Guanine Nucleotide Exchange Factor–H1/RhoA/Rho Kinase/p27

Ras homolog gene family member A (RhoA) through Rho kinase kinase (ROCK), one of its downstream effectors, regulates a wide range of cell physiological functions, including vascular smooth muscle cell (SMC) proliferation, by degrading cyclin-dependent kinase inhibitor, p27. Our previous studies found that heparin inhibition of pulmonary artery SMC (PASMC) proliferation and pulmonary hypertension was dependent on p27 up-regulation. To investigate whether ROCK, a regulator of p27, is involved in regulation of heparin inhibition of PASMC proliferation, we analyzed ROCK expression in the lungs from mice and from human PASMCs exposed to hypoxia, and investigated the effect of ROCK expression in vitro by RhoA cDNA transfection. We also investigated the effect of guanine nucleotide exchange factor (GEF)-H1, an upstream regulator of RhoA, on heparin inhibition of PASMC proliferation by GEF-H1 cDNA transfection. We found that: (1) hypoxia increased ROCK expression in mice and PASMCs; (2) overexpression of RhoA diminished the inhibitory effect of heparin on PASMC proliferation and down-regulated p27 expression; and (3) overexpression of GEF-H1 negated heparin inhibition of PASMC proliferation, which was accompanied by increased GTP-RhoA and decreased p27. This study demonstrates that the RhoA/ROCK pathway plays an important role in heparin inhibition on PASMC proliferation, and reveals that heparin inhibits PASMC proliferation through GEF-H1/RhoA/ROCK/p27 signaling pathway, by down-regulating GEF-H1, RhoA, and ROCK, and then up-regulating p27.

Antitumor effect of butanoylated heparin with low anticoagulant activity on lung cancer growth in mice and rats.

Whole unfractionated heparin can modestly decrease tumor growth, but the dose of heparin is limited by its anticoagulant properties. To overcome this limitation, we modified the chemical structure of heparin and have prepared a heparin derivative by O-acylating low molecular weight heparin with butyric anhydride, producing a more potent antiproliferative compound, which is only weakly anticoagulant so that the dose may be escalated without threat of hemorrhage. In this study, we investigated the effect of this chemically modified heparin, butanoylated heparin, on the growth of lung cancer in vitro and in vivo. We found that butanoylated heparin a) significantly inhibited lung cancer cell proliferation in vitro and lung cancer growth in mice and rats; b) had very low anticoagulant effect; c) had no significant toxicity on heart, liver, kidney and lung; d) significantly although modestly induced apoptosis and decreased expression of the cell proliferation pathway consisting of mutant p53, phospho-Rb and E2F1 expression in the tumor tissues. We also found that butanoylated heparin significantly inhibited CXCL12 and CXCR4 expression, suggesting that CXCL12/CXCR4 axis may be involved in regulation of tumor growth inhibition by heparin. We concluded that chemically modified butanoylated heparin has potent antiproliferative activity against lung cancer and may represent a new chemical therapeutic agent for cancer patients.

Significance of the 2-O-sulfo group of L-iduronic acid residues in heparin on the growth inhibition of bovine pulmonary artery smooth muscle cells

Heparin inhibits the growth of several cell types in vitro, including bovine pulmonary artery smooth muscle cells (BPASMCs). To understand more about the heparin structure required for endogenous activity, chemically modified derivatives of native heparin and glycol-split heparin, namely, 2-O-desulfonated iduronic/glucuronic acid residues in heparin, and 2-O-desulfonated iduronic residues in glycol-split heparin were prepared. These were assayed for their antiproliferative potency on cultured BPASMCs. All of the 2-O-desulfonated heparin derivatives had significantly decreased less antiproliferative activity on BPASMCs. These results suggest that the 2-O-sulfo group of iduronic acid residues in heparins major sequence is essential for the antiproliferative properties of heparin. The size of heparin does not affect the growth-inhibitory properties of heparin on BPASMCs at the three dose levels examined.