Study On The Mechanisms Of Calcium And HIF In Hypoxia Induced PASMC Proliferation

Background and Objective: Oxygen is essential for the organism. Hypoxia is a common pathologic process in many diseases; it is also the direct cause of high altitude diseases. Both acute and chronic hypoxic exposure can cause hypoxic pulmonary hypertension (HPH), which is characterized by pulmonary vasoconstriction (HPV) and vascular remodeling. The relationship between Ca²⁺ and HPV has been investigated thoroughly, and it is believed that the reduced electron transport of mitochondria respiratory chain induces alterations of reactive oxygen species around the voltage operated K+ channels, which inhibit the Kv channel, reduced cell membrane potential, and triggers Ca²⁺ influx, the increase of [Ca²⁺]_I cause pulmonary arterial smooth muscle cells (PASMCs) constrict, and lead to pulmonary hypertension. But the relation of disturbed Ca²⁺ homeostasis and pulmonary vascular remodeling is not clear; especially which are the key points in calcium signal transduction need to be answered. In fact, the remodeling of pulmonary vascular wall involves reprogramming of a series of genes under hypoxic exposure. More and more evidences proved that hypoxia inducible factor (HIF) is the key to regulate hypoxic responses. HIF-1 was first discovered in 1992, later the other subtypes of HIF-2/3/4 were also found, which are all heterodimers, including the same HIF-βwith different HIF-α. Some experiments showed HIFs take part in the development of HPH, involved vaso-active substances and promote vascular remodeling, but disagreements remain over the interpretation of the action of HIFs and Ca²⁺. Since disturbed Ca²⁺ homeostasis and HIF are the two keys in regulating signaling and transcription respectively in cellular response to hypoxia, it is reasonable to suggest there might be close relationship between the two keys. In light of this hypothesis, the temporal characteristics of HIF expression, the involvement of Ca²⁺ signal transduction in hypoxic induced proliferation, and the relationship between Ca²⁺ and HIF-2α, were investigated in the model of cultured PASMCs under hypoxic condition.Methods:Rat PASMCs in primary culture were obtained from extension of small slices of pulmonary arterial media cultured for several days, and were confirmed by light microscope, immunocytochemistry. Studies were conducted on the 3-7 passage of PASMCs cultured in vitro either in normoxic or hypoxic condition (2% O2). The viability of PASMCs cultured under normoxic and hypoxic condition was examined in time course at 6 h, 12 h, 24 h, and 48 h respectively. Then the time point with the most significant change of cell viability was chosen for the following experiments.In order to study the involvement of Ca²⁺ signal transduction in hypoxic proliferation, PASMCs in medium supplemented with 10% serum in the proliferative states were then deprivated of serum for about 24 h culture to be arrested in the quiescence stage, and then divided into different groups: the normoxic, the hypoxic, the hypoxic + L-type calcium blocker nifedipine (NFD, 10μM), the hypoxic + calmodulin antagonist trifluoperazine (TFP, 10μM), the hypoxic + intracellular calcium chelator BAPTA/AM (20μM) and the hypoxic + inhibitor of nuclear factor-kappaB PDTC (20μM) group. The nomoxic group was cultured under 5% CO2 + 20% O2 and the hypoxic groups under 5% CO2 + 2% O2. The viability of PASMCs was measured by MTT method, the cell cycle using flow cytometry, the proliferating cell nuclear antigen (PCNA) using immunofluorescence staining.To clarify the temporal characteristics of HIF expression, PASMCs were arrested with deprivation of serum for 24 h and then cultured in vitro either in normoxic or hypoxic condition (2% O2), the changes of mRNA expression of HIF-1α, HIF-2αand HIF-βwere determined by RT-PCR, and the changes of protein were examined by Western blotting, at 12 h, 24 h, 48 h, 72 h incubation.To investigate the relationship between Ca²⁺ and HIF-2α, the arrested PASMCs were cultured in vitro either in normoxic or hypoxic condition (2% O2), some groups were added with L-type calcium blocker nifedipine (NFD, 10μM), T-type calcium blocker amiloride (100μM), the calmodulin antagonist trifluoperazine (TFP, 7.5μM), intracellular calcium chelator BAPTA/AM (15μM) respectively, after 24 h further incubation, the alterations of HIF-2αmRNA expression were determined by RT-PCR, and the changes of protein were examined by Western blotting. Results:1. The viability of PASMCs increased significantly at 12 h exposure to hypoxia, compared with normoxic group, the percentage of G0/G1 decreased, S and G2/M increased, the proliferation index (PI) increased about 1.6 times and the staining of PCNA was strong positive.2. The L-type calcium blocker nifedipine (NFD, 10μM), intracellular calcium chelator BAPTA/AM (20μM), the calmodulin antagonist trifluoperazine (TFP, 10μM) and the inhibitor of nuclear factor-κB PDTC (20μM) inhibited the proliferation of PASMCs induced by hypoxia significantly. The viability and PI decreased and the expression of PCNA was reduced.3. The expression of HIF-βmRNA and protein did not change significantly under hypoxic condition.4. The expressions of HIF-1αand HIF-2αmRNA did not change under hypoxia. While the protein levels of HIF-2αand HIF-1αwere upregulated significantly after onset of hypoxia with different patterns. The protein level of HIF-2αreached and stayed at high level to form a plateau from 12 h, but the protein level of HIF-1αincreased to a peak at 48 h then declined after longer exposure to hypoxia.5. There were no obvious effects of NFP on HIF-2αmRNA and protein levels.6. Amiloride reduced the expression of HIF-2αmRNA, but without obvious effects on the protein level. Either BAPTA/AM or TFP suppressed HIF-2αmRNA and protein significantly.Conclusions:1. Calcium signal transduction take part in hypoxic-induced proliferation of PASMCs, CaM and NF-kappaB are the two key points.2. The expression of HIF-βmRNA and protein in PASMCs were constitutive and not affected by hypoxic exposure.3. The HIF-1αand HIF-2αwere regulated at post transcription level, their protein levels in response to to low oxygen tension show different characteristic .4. The response of HIF-2αexpression in PASMCs to hypoxia is regulated by calcium signal transduction, especially via CaM.