The Role Of KLF5in Hypoxic Pulmonary Arterial Hypertension

Part OneRole of KLF5in hypoxia-induced pulmonary artery hypertensionBackground: Hypoxic pulmonary artery hypertension (PAH) is a complex systemic disease, and small pulmonary arterial remodeling underlies pathological alteration of hypoxic PAH. KLF5, a zinc-finger type transcription factor, is involved in proliferation, apoptosis, differentiation and migration of carcinoma cells. Its expression is found to be significantly increased in human pulmonary artery smooth muscle cells (HPASMC) from patients with pulmonary hypertension, and KLF5regulates cardiovascular remodeling. However, the role of KLF5in the pathogenesis of hypoxic PAH is largely undefined.Methods:Male Sprague-Dawley rats weighing200-250g were exposed to hypoxic (10%O2) or normoxic conditions. Hemodynamic analyses, pulmonary artery media thickness and right ventricular hypertrophy were measured in vivo. Paraffin sections from rats were stained with hematoxylin and eosin for evaluation of degree of vascular remodeling. HPASMC were maintained under normoxic condition or hypoxic conditions (5%O2) in a modular incubator. Cell viability and cell apoptosis were assessed using CCK-8test and Annexin V/PI assay. Cells were labeled with bromodeoxyuridine (BrdU) or prodium iodide (PI) to detect proliferating cells or identify different stages of cell cycle. The transwell migration assays were conducted to measure the capacity of cell motility. Immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed to examine the protein levels and the mRNA expression was analyzed by real-time quantitative PCR. The direct interaction of KLF5and HIF-1α were analyzed by co-immunoprecipitation (co-IP) assay in cells exposed to normoxia or hypoxia. Small interfering RNAs (siRNAs) targeting KLF5or HIF-1α, as well as lentiviral vectors expressing KLF5were designed and transfected into HPASMC. Lentiviral vectors encoding short hairpin RNA (shRNA) targeting rat KLF5were conducted and injected into rats via tail vein.Results:Mean pulmonary arterial pressure (mPAP) was elevated in rats exposed to hypoxia, with markedly distal artery medial thickening and right ventricular hypertrophy. Ki-67/PCNA positive cells in pulmonary artery media were increased after hypoxia exposure, while the number of TUNEL positive cells in hypoxic rats was dramatically decreased. Hypoxia promoted the proliferation and migration of HPASMC and inhibited cell apoptosis, which can be reversed by the knockdown of KLF5or HIF-la. Overexpression of KLF5upregulated the protein level of HIF-la and was immunoprecipitated with HIF-la. Cell cycle regulators cyclin B1, cyclin D1and apoptosis-related proteins including bax, bcl-2, survivin, caspase-3and caspase-9were involved in the regulation of KLF5-HIF-1α induced HPASMC survival. Lentiviral vector-mediated knockdown of KLF5significantly downregulated HIF-1α expression and attenuated hypoxic PAH endpoints in rats.Conclusions:Hypoxia promoted the proliferation and migration of PASMC and inhibited cell apoptosis, resulting in vascular remodeling. The study implied that KLF5-HIF-1α axis plays a key part in the pathogenesis of hypoxia-induced vascular remodeling and may represent a potential novel therapeutic target for hypoxic PAH. Part TwoKLF5promotes hypoxia-induced survival and inhibits apoptosis in non-small cell lung cancer cells via HIF-1αObjectives: Transcription factor Kriippel-like factors5(KLF5) is overexpressed in a wide range of tumor tissues and acts as a prognostic factor in cancer. However, the role of KLF5in non-small cell lung cancer is not clear. Hypoxia plays a vital part in the development of cancer via Hypoxia-inducible factor1(HIF-1). The study was performed to explore the role of KLF5in hypoxic lung cancer cells and the potential association between KLF5and HIF-la.Methods: We performed CCK-8test to measure cell viability, colony forming assay for cell clonality, CFSE labeling assay for cell proliferation and annexin-v/PI assay for cell apoptosis in A549cells exposed to20%or1%O2. Small interfering RNA (siRNA) was synthesized to suppress the expression of KLF5or HIF-1a. Co-immunoprecipitate assay was performed to assess the direct relationship between KLF5and HIF-la. PCR and immunoblot were conducted to detect the expression of mRNA and proteins.Results: The study showed that hypoxia increased cell viability, clonality and proliferation and inhibited cell apoptosis in A549cells. The expression of HIF-1a and KLF5were increased time-dependently in hypoxia. KLF5or HIF-1a knockdown inhibited hypoxia-induced cell survival and promoted cell apoptosis by actively down-regulating cyclin B1, survivin and up-regulating caspase-3. KLF5was revealed to co-immunoprecipitate with HIF-1a and hypoxia increased the amount of KLF5and HIF-1a complex. Moreover, silencing of KLF5decreased HIF-1a expression while KLF5was not affected by HIF-1a inhibition in hypoxia, confirming the effect of KLF5on up-regulation of HIF-1a.Conclusions:The study identified hypoxia as a tumor promoter by triggering KLF5â†'HIF-1αâ†'cyclin B1/survivin/caspase-3in lung cancer cells.