Screening Of Plasma Biomarkers Of Tissue Ischemia And Hypoxia Based On Multi-omics Technology And Preliminary Study Of The Regulatory Mechanism Of Hypoxia Adaptatio

BackgroundHypoxia is a condition in which the amount of oxygen in a cell falls below normal levels,often leading to changes in metabolites,proteins,and enzymes that can lead to cellular dysfunction.Blood loss is one of the important causes of tissue hypoxia.Due to the decrease of red blood cell and hemoglobin,less oxygen is delivered to tissues,resulting in tissue hypoxia.Therefore,hypoxic tissue injury is a common complication of ischemic diseases,causing damage to a wide variety of human organs such as heart,brain and lung tissues.Early diagnosis and intervention of hypoxic tissue injury have important practical significance to improving the prognosis of patients with ischemic disease.In clinical practice,hemoglobin(Hb),arterial partial pressure of oxygen(PaO2),and oxygen saturation(SaO2)are commonly used to evaluate systemic circulating oxygen content and ischemia,which are used as the basis of whether patients need red blood cell transfusion.However,these indicators can only assess the circulating oxygen content,and are not sensitive and specific to the monitoring of tissue oxygen content.The current monitoring of oxygen content in tissue and cell mainly relies on the implantation of microsensors such as microprobes,polar spectroscopic microcatheter,and Clark-type oxygen electrodes.These techniques are difficult to be widely applied in clinical practice due to their invasiveness.Therefore,finding biomarkers for tissue hypoxia that are easy to obtain and detect can effectively solve the technical problem of monitoring tissue oxygen content,assist clinical diagnosis of tissue hypoxia,and effectively manage the early blood transfusion treatment of patients with ischemic diseases.In recent years,multi-omics techniques including genomics,transcriptomics,metabolomics,and proteomics have been widely used in the exploration of biomarkers and therapeutic targets,becoming indispensable auxiliary tools in molecular and cell biology research.In this study,TMT-based liquid chromatography-tandem mass spectrometry(LC-MS/MS)and untargeted metabolomics were utilized in a hemorrhage rat model.Bioinformatics analysis methods and validation means of parallel reaction monitoring were used to screen tissue hypoxia biomarkers to explore the regulation mechanism of hypoxia and the changes in metabolic map.Objective1.The ischemic hypoxic rat model was established and plasma biomarkers of tissue hypoxia were developed to provide a theoretical basis for its auxiliary diagnosis.2.To explore the effect of hypoxia on endothelial cell functional characteristics and the adaptive regulation mechanism,contributing to the customized management of oxygen supply,oxygen control therapy,and blood transfusion therapy strategies for ischemic diseases.Methods1.Establish the ischemic hypoxia rat model and verify the validityMale F344 Fischer rats aged 10 weeks were randomly selected and divided into normal group,hypoxia group for 7 days and hypoxia group for 15 days.The hypoxia rats were bleeding on day 1,3,5,7,9,11,13,and 15,respectively,through angular vein located at the base of the eye.The volume of the bleeding was calculated as 20%of peripheral blood circulation(sample volume=weight×0.06×20%).The changes of RBC,hemoglobin,and platelets were detected by blood cell counting instrument.The arterial blood samples from abdominal aorta were collected for blood gas analysis.2.Differential expression proteins were screened by combining metabolomics and quantitative proteomicsThe changes of plasma proteins and small molecule metabolites in hypoxia and normoxia rats were detected by LC-MS/MS liquid chromatography-mass spectrometry,HPLC high performance liquid chromatography,and time of flight mass spectrometry(GC-TOF/MS).Bioinformatics analysis was used for GO enrichment and KEGG pathway to obtain candidate biomarkers and signaling pathways related to hypoxia.3.PRM and WB were used to screen the expression of biomarkers in plasma,heart,brain and lung tissuesBy synthesizing specific target peptides of candidate proteins,the expression of these proteins in plasma,heart,brain and lung tissues was quantitatively detected.Western blotting was used to verify the comparability and reliability,and the correlation and tissue specificity of these candidate biomarkers with hypoxia were further analyzed.4.Investigate the effect of hypoxia on endothelial cell function and its adaptive regulation mechanismThe hypoxia model of TeloHAEC cells was established in vitro on the premise of excluding ischemia.Protein expression,angiogenesis,and cell function were measured at 0,6,12,24,48 and 72h after culture at 3%oxygen concentration for 72 hours.The expression of candidate biomarkers of cardiac hypoxia was reconfirmed at the cellular level by western blot.Cell activity was detected by MTT assay,and cell cycle,proliferation,and apoptosis were detected by flow cytometry.Results1.The ischemic hypoxia rat model was establishedIn this study,blood samples of rats in three groups were collected 7 times on days 1,3,5,7,11,13 and 15,respectively.The red blood cells(RBC),hemoglobin(Hb)and arterial partial pressure of oxygen(PaO2)decreased significantly(P<0.001)at day 15 compared to day 1;The platelet count(PLT)showed a slight increase trend and the difference was statistically significant(Day 1 vs Day 15:408.4±172.8 vs 922.0±253.6109/L,P<0.05).Besides,the arterial oxygen pressure was also decreased from 117.0±5.3 mmHg to 82.8±7.2 mmHg,indicating that pulmonary dysfunction and anoxia had occurred.Together,these results showed that we have successfully established a rat ischemic-hypoxic model.2.Twenty-two tissue hypoxia candidate proteins and six significantly enriched signaling pathways were identified by mass spectrometryThe 22 candidate proteins were screened as follows:Vascular cell adhesion protein 1(Vcaml),Vitronectin(Vtn),Vitamin K-dependent plasma glycoprotein(Proz),Arachidonate 15-lipoxygenase(Alox15),Thrombospondin-4(Thbs4),Cytosol aminopeptidase(Lap3),secreted phosphoprotein(OPN),Vitamin K-dependent plasma glycoprotein(Pros1),endosialin,Serum paraoxonasel(Pon1),Galectin-3-binding protein(Gal-3),Transferrin receptor protein 1(Tfrc),Matrix metalloproteinase(Mmp2),14-3-3 protein zeta(Ywhaz),14-3-3 protein epsilon(Ywhae),Tenascin C(Tnc),Collagen type Ⅰalpha 2(Colla2),Endoplasmin(Hsp90b1),Heat shock 70 kDa protein 4(Hspa4),Protein kinase C substrate 80 K-H(Prkcsh),Ctype lectin domain family 3 member B(Clec3b),Filamin A-interacting protein 1-like(Filip 11).Differential expressed proteins are mainly enriched in the following six signaling pathways:rno04610 complement and coagulation cascades was the most significantly enriched pathway,followed by rno04912 GnRH signaling,rno04926 relaxin signaling,rno04141 protein processing in endoplasmic reticulum,rno04933 AGE-RAGE signaling in diabetic complications,and rno04610 PI3K-Akt signaling pathway.Nine differential expression proteins are involved in the PI3K-Akt signaling pathway.Complement and coagulation cascades and PI3K-Akt signaling pathway may be the main response pathways of ischemic hypoxia,which are associated with the high expression of downstream target genes such as proliferation,angiogenesis,and migration.3.Tissue correlation validation of candidate biomarkers for tissue hypoxiaCombined with PRM targeted proteomics and western blot,the expression of candidate biomarkers was repeatedly screened.It was found that the differential expressed proteins in plasma showed different trends in heart,lung,and brain tissues.Hemoglobin-stabilizing protein(Ahsp),Serum paraoxonasel(Ponl)and Galectin-3-binding protein(Gal-3)were significantly elevated in plasma and heart,which may be indicative of cardiac hypoxia.Osteopontin(OPN)increased significantly in plasma and brain tissue,which may be a reminder of brain hypoxia.The elevated expression of endosialin and Gal-3 in plasma and lung tissue may be suggestive of pulmonary hypoxia.The expression levels of arachidonate 15-lipoxygenase(Alox15),transferrin receptor(Tfrc)and protein kinase C(Prkcsh)in plasma,heart,brain and lung were significantly increased,while the expression levels of filamin A-interacting protein 1-like(Filip11)and collagen type Ⅰα2(Colla2)were significantly decreased.These proteins may serve as potential biomarkers of circulating hypoxia.4.To explore the effect of hypoxia on endothelial cell function and its adaptive regulation mechanism4.1 Hypoxia activates the transcriptional activity of HIF,promotes the expression of Endosialin,VEGF,and Gal-3,and stimulates angiogenesisThe hypoxia model of TeloHAEC cells with 3%oxygen concentration was established in vitro.After 6 hours of hypoxia,the DNA binding activity of HIF-la was significantly enhanced.The downstream proteins Endosialin,VEGF,and Gal-3 showed an increasing trend with the prolongation of hypoxia,indicating endothelial cell proliferation and neovascularization.4.2 Hypoxia regulates iron metabolismIn 15 days of hypoxia,the plasma transferrin increased continuously(Day 1 vs Day 11:303.6±98.6 vs 442.6±71.2 mg/dL,P<0.05),while the serum iron decreased within one week(Day 1 vs Day 7:29.0±5.3 vs 18.6±5.1 μmol/L,P<0.05),and then increased gradually(Day 7 vs Day 15:18.6±5.1 vs 40.4±7.4 μmol/L,P<0.001).The results showed that ferritin levels were rapidly depleted in the early stage of ischemic hypoxia.Prolonged hypoxia can regulate iron metabolism to meet physiological needs.Monitoring of transferrin and serum iron levels may have implications for the diagnosis of ischemic hypoxia.4.3 Hypoxia affects cell activity and proliferationThe cell activity of TeloHAEC cells decreased within 24h of hypoxia(normoxic group vs 24h hypoxia group:0.33±0.06%vs 0.28±0.04%,P<0.05),and the proliferation rate decreased.After 48h culture,the activity of hypoxia cell increased(normoxic group vs 48h hypoxia group:0.48±0.08%vs 0.58±0.11%,P<0.05),the proliferation rate was also significantly increased.These results indicate that short-term hypoxia can reduce cell activity and proliferation rate,while long-term chronic hypoxia can promote cell proliferation.4.4 Short-term hypoxia leads to cell Gl/S cycle arrestAfter 12 hours of hypoxia,the number of TeloHAEC cells in S-phase decreased significantly,and the percentage of G0/G1 phase cells increased,indicating G1 phase arrest.In 12h and 72h hypoxia groups,the distribution rates of TeloHAEC cells were 9.1±3.4%and 14.3±4.2%in S phase,and 75.0±3.7%and 66.7±9.6%in G0/G1 phase,respectively,indicating that G1/S cycle arrest was improved.These data suggest that cell adaptation to hypoxia is related to the adjustment of cycle dynamics.Short-term hypoxia can induce G1/S cycle arrest,and this phenomenon is transient and reversible.4.5 Hypoxia induced cell apoptosisTeloHAEC cells were cultured under hypoxia for 6,12,24,48 and 72 h,and the number of early apoptotic cells increased at each time point.The number of late apoptotic and necrotic cells increased significantly after 72 hours of hypoxia.Conclusion1.In this study,22 tissue hypoxia candidate proteins and 6 signaling pathways were identified.Complement and coagulation cascades and PI3K-Akt signaling pathway may be the main response pathways of ischemic hypoxia,which are associated with the high expression of downstream target genes such as proliferation,angiogenesis and migration.2.The expression of hypoxia markers in heart,lung,and brain is tissue specific.Ahsp Pon1 and Gal-3 may play a suggestive role in cardiac hypoxia.OPN may be a reminder of brain hypoxia.Endosialin and Gal-3 may be indicative of lung hypoxia.Alox l5,Tfrc,Filip 11,Col1a2,and Prkcsh may serve as potential biomarkers for circulating hypoxia.3.Hypoxia affects endothelial cell function and promotes hypoxia adaptation.Hypoxia activates HIF-la transcription and promotes angiogenesis.Hypoxia regulates the expression of transferrin and ferritin,and adaptively regulates iron metabolism.The effect of hypoxia on cell function is time-dependent.The adaptation of TeloHAEC cells to hypoxia was related to the adjustment of cycle dynamics.Short-term hypoxia reduced the proliferation activity of TeloHAEC cells and induced G1/S cell cycle arrest.Long-term hypoxia can promote cell proliferation and reverse G1/S cycle stagnation to a certain extent.This adaptive regulatory mechanism may depend on HIF-la accumulation or degradation.4.The multi-omics strategies may be a powerful way to explore the biomarkers and mechanisms,and maybe a new tool to help apply biomedical research findings to clinical practice and treatment management.