Hypoxic Adaptability Of Yak Pulmonary Artery Smooth Muscle Cells Based On Multi-omics Analysis

An increasing number of research studies have demonstrated that acute hypoxia is characterized by vasoconstriction in the pulmonary vasculature,whereas chronic hypoxia describes pulmonary arterial wall remodeling,resulting in irreversible pulmonary hypertension in mammals.Yaks（Bos grunniens）are representative animal species of high altitude areas such as the Qinghai-Tibet Plateau（3,000-5,500 meters above sea level）,and they show no obvious symptoms of vascular remodeling upon long term adaptation.Therefore,the yak is an ideal model for studying hypoxic pulmonary hypertension（HPH）prevention and treatment.Hypoxia inducible factor-1α（HIF-1α）is a key transcription factor for oxygen balance regulation and hypoxia adaptation.It regulates the expression of more than 100 target genes which in turn regulates changes in peripheral oxygen concentration in cells,tissues,and the body.Therefore,in this study,yaks of different ages were selected as research subjects to analyze the distribution and expression of HIF-1αin the lung,and yellow cattle were selected as the normoxic control group.To further study the molecular regulation mechanism of hypoxia on the proliferation of yak pulmonary artery smooth muscle cells,this research was conducted by means of transcriptomics and proteomics.The specific results are shown as follows:1.The distribution and expression of HIF-1αin adult yellow cattle and yak lungs were detected by immunohistochemistry,immunofluorescence,and Western blot techniques.The results showed that HIF-1αwas mainly localized in arterioles and some alveolar cells in adult yellow cattle lung,but mainly expressed in pulmonary artery smooth muscle cells,bronchiolar epithelial cells,and some alveolar cells in adult yak lung.In addition,Western blot results showed that HIF-1αprotein expression in adult yak lung was higher than that of yellow cattle（p<0.01）.Subsequently,the expression and distribution of HIF-1αin yak lungs at different developmental phases were further detected.Co-location of HIF-1αandα-SMA（a marker of smooth muscle cells）was determined by immunofluorescence.The results showed that HIF-1αexpression increased gradually with increasing age of yak,and HIF-1αwas mainly located in pulmonary artery smooth muscle cells（PASMCs）in the adult and senior groups,suggesting that HIF-1αplays an important role in adaptation of pulmonary artery to hypoxia in yak.2.Primary yak pulmonary artery smooth muscle cells（PASMCs）were successfully isolated and cultured.The PASMCs were fusiform and showed a typical"hills and valleys"morphology characteristic of smooth muscle cells after confluence.The primary cells were purified by differential time adherent method,and the purification of primary cells was confirmed by strong positive staining forα-smooth muscle actin and Calponin-1 by immunofluorescence assay.To test the influence of moderate hypoxia on proliferative responses of PASMCs to mitogens,purified cultured PASMCs were exposed to hypoxia（5%O₂）or normoxia（21%O₂）,and cell proliferation was measured by CCK-8 assay or protein expression of Proliferating Cell Nuclear Antigen（PCNA）,a cell proliferation marker.The results showed that PCNA expression in hypoxia group was higher than that in normoxia group and the difference was significant（p<0.01）,indicating that hypoxia could temporarily promote PASMCs proliferation.Moreover,we also tested the expression of pro-apoptosis and anti-apoptosis factors（Bax and Bcl-2）.Under hypoxic conditions,Bax/Bcl-2 ratios decreased as exposure time increased,suggesting that PASMCs apoptosis was inhibited by hypoxia.In addition,we assessed protein expression of HIF-1αin cells exposed to hypoxia for 12 h,24 h,48 h,and 72 h,and found that HIF-1αprotein levels were upregulated when exposed to 5%O₂ in a time-dependent manner,with an upward trend from 12 to 48 h with the highest expression at 48 h,and a downward trend at 48 to 72h,suggesting that hypoxia could induce transient expression of HIF-1α.3.Transcriptome analysis of yak PASMCs in normoxic group and hypoxic group showed that 13330 genes expressed in the two groups,and the number of differentially expressed genes in only normoxic or hypoxic group were 277 and 328 respectively.A total of 239 differentially expressed genes（DEGs）between groups were selected by the condition p-value<0.05 and|log2FC|≥1,including 192 up-regulated genes and 47down-regulated genes.GO enrichment analysis showed that DEGs were significantly enriched in functions relating to extracellular regions,response to hypoxia and response to decreased oxygen levels.KEGG enrichment analysis showed that DEGs mainly participated in HIF-1 signaling pathway,TGF-beta signaling pathway,and glycolysis/gluconeogenesis pathway.Twelve differentially expressed genes were selected for q RT-PCR detection,and seven DEGs（ACVRL1,FZD2,AQP1,ID1,EGLN3,BNIP3,DDIT4）relating to angiogenesis and hypoxia were selected from GO enrichment analysis.Five DEGs（EGLN1,VEGFA,HK2,BMP6,CXCL8）associated with HIF-1 signaling,TGF-beta signaling,and cytokine-cytokine receptor interactions were selected from KEGG enrichment analysis.The quantitative validation results were consistent with transcriptome sequencing results,indicating that the sequencing results were reliable.4.Proteomic TMT analysis of yak PASMCs in normoxic and hypoxic groups showed that the number of peptides and proteins were 19635 and 7823,respectively.A total of 248 differentially expressed proteins（DEPs）between groups were selected by the condition p-value<0.05,up-regulation differential ratio≥1.2,down-regulation differential ratio≤0.8,including 187 up-regulation proteins and 61 down-regulation proteins.GO enrichment analysis showed that DEPs were significantly enriched in functions related to extracellular regions,DNA replication initiation,and MCM complex.KEGG enrichment analysis showed that DEPs were mainly involved in DNA replication,cell cycle,TGF-beta signaling pathway,ECM-receptor interaction,HIF-1signaling pathway,MAPK signaling pathway,and NF-kappa B signaling pathway.We selected several differentially expressed proteins from the above mentioned proteins for Western blot analysis.The results showed that after hypoxia treatment,the expression levels of HIF-1α,HIF-2α,and HIF-3αin PASMCs of yaks were higher than that in normal oxygen treatment,and the differences were significant（p<0.05）;the protein expression of HO-1,VEGF（HIF-1αdownstream genes）,and TGF-β1（in TGF-βpathway）were also significantly up-regulated（p<0.05）;In MAPK pathway,the phosphorylation levels of p38 and JNK proteins in hypoxia group were also higher than those in the normoxic group（p<0.05）.5.To explore whether HIF-1αaffects proliferation and apoptosis of yak PASMCs,we induced HIF-1αexpression by DMOG,a competitive inhibitor of prolyl hydroxylase（PHD）.DMOG treatment significantly increased HIF-1αprotein level,and with increased concentration,HIF-1αand PCNA protein levels also increased,while the ratio of Bax/Bcl-2 protein content decreased.Subsequently,expression of HIF-1αwas inhibited by LW6 under hypoxia,and protein levels of HIF-1α,PCNA,Bax,and Bcl-2 were also detected.The results were opposite to those of DMOG treatment.In conclusion,we successfully isolated and cultured yak PASMCs for the first time and showed that HIF-1αwas mainly concentrated in the medial layer of small pulmonary arteries,and also found that 5%O₂ oxygen concentration could temporarily promote PASMCs proliferation and HIF-1αexpression.Transcriptomics and proteomics studies showed that hypoxia can activate key factors in vascular development,hypoxia response,DNA replication,cell cycle,and TGF-β/HIF-1/MAPK pathway.Finally,we proved that HIF-1αcan promote the proliferation and inhibit the apoptosis of PASMCs in yak through drug treatment.Therefore,this study provides a theoretical basis for further studies on the adaptation mechanism of yak lung in hypoxic environment,and also provides a new idea for the development of treatments of hypoxic pulmonary hypertension.