Rab6 GTPase Contributes To Phenotypic Modulation Of Pulmonary Artery Smooth Muscle Cells Under Hypoxia

Backgroud and Objective:Pulmonary hypertension is a kind of clinical syndrome,which is caused by many factors,such as pulmonary vascular and structural abnormalities,leading to increased vascular resistance and pulmonary artery pressure.Acute or sustained reduction of alveolar oxygen occurs in a variety of lung diseases eventually develop to hypoxic pulmonary hypertension(HPH),results in reconstruction of resistance vessels in the pulmonary vascular bed,and follows by right ventricular hypertrophy and heart failure.HPH is one of the important pathogenesis of many cardiopulmonary diseases,such as chronic obstructive pulmonary disease,chronic pulmonary heart disease and chronic high altitude disease.At present,the treatment of HPH is still based on the primary treatment,and some treatment methods such as vasodilator,oxygen therapy,targeted drugs can not effectively control the progress of hypoxic pulmonary hypertension.Therefore,it is worthy to elucidate the pathogenesis of HPH for clinical significance of prevention and treatment in cardiovascular diseases.Pulmonary arterial smooth muscle cells(PASMCs)are the cellular components of the normal pulmonary arterial wall,located in the middle of blood vessel walls,to maintain the integrity of the vascular structures and mediate the vascular response to hypoxia,and this activity is closely related to the development of HPH.PASMCs are in a reversible state and retain remarkable plasticity,which is different from skeletal and cardiac muscle cells.According to the function and structure of PASMCs,PASMCs are divided into two types:contractive(differentiated)and synthesis(undifferentiated).Recently,several studies have demonstrated that PASMCs can induce a phenotype switch from contractile to proliferative activity and induce excessive proliferation when stimulated by many factors,but phenotypic modulation is a gradual process,so a large number of the middle of the transition type can exist with the same blood vessel walls.In recent years,studies have shown that hypoxia can induce phenotypic modulation of PASMCs,but the specific mechanism involved in the event is poorly understood.Intracellular trafficking between organelles is vital for cell survival and is regulated by members of the Ras-like superfamily.This superfamily includes the Ras,Rho,Arf,Ran and Rab subfamilies.Rab GTPases are by far the largest group,consists of about 200 amino acids.They are monomeric proteins of 20-29 kDa in size,with similar function,structure,and guanine nucleotide binding properties.At present,more than 60 Rab proteins have been found,almost all related to membrane organelles of eukaryotic cells,have different Rab in different membrane proteins,each kind of organelles containing at least more than one Rab protein.As a molecular switch of vesicle transport,Rab protein plays a role in the formation,transport,adhesion,anchoring and fusion of vesicles.Rab6 is a member of the Rab protein family,resides at the medial/trans-Golgi cisternae,as well as negative golgi pipe mesh structure.Some studies have found that Rab6 are involved in a variety of proteins in the endoplasmic reticulum and golgi apparatus between anterograde transport,affect the endoplasmic reticulum folding environment.Previous data suggests that ER stress can also modulate phenotypic transformation in vitro.Therefore,hypoxia induces the phenotypic transformation of the PASMCs and ER stress,Whether Rab6 can influence phenotypic transformation of RPASMCs under hypoxia through the regulation of ER stress needs further study.Taken together,the aim of this study is to investigate whether Rab6 can induce phenotypic modulation of RPASMCs under hypoxia through the regulation of ER stress,as well as to investigate the role of ER stress in the process during hypoxia.Methods:1.Rat pulmonary arterial smooth muscle cells were divided into 4 groups:0,12,24 and 48h after hypoxia,the expression of a-SMA,VIM,Rab6 and GRP78 in PASMCs was determined by WB and the location of a-SMA,VIM and GRP78 was determined by immunofluorescence;2.RPASMCs were transfected with siRNA marked by Cy3 and the efficiency of transfection was observed by immunofluorescence microscopy.Inhibition rate of target protein and mRNA was measured by WB and RT-PCR;3.RPASMCs were transfected with Rab6 siRNA,for inhibiting the expression of Rab6,then were exposed to hypoxia and the location and expression of a-SMA,VIM and GRP78 in RPASMCs was measured by WB and immunofluorescence.Results:1.Compared with hypoxia Oh group,the expression of the phenotype marker gene a-SMA was down-regulated and VIM was up-regulated after 48h hypoxia;Rab6 was up-regulated;the level of GRP78 after 12h of hypoxic stimulation was also increased;2.Rab6 siRNA was transfected into RPASMCs successfully,and they strongly inhibited the expression of Rab6;3.Compared with the hypoxia after pretreatment with NC siRNA,the expression of phenotype marker gene protein a-SMA was up-regulated and VIM expression was down-regulated;the expression of GRP78 in RPASMCs was down-regulated after transfection with Rab6 siRNA under hypoxic conditions.Conclusion:1.Hypoxia can induce RPASMCs to undergo phenotypic modulation and endoplasmic reticulum stress,and ER stress occurs early than phenotypic modulation.2.Rab6 is involved in hypoxia-induced phenotypic modulation and ER stress in RPASMCs.