The Roles And Mechanisms Of Rho A/ROCK1 Signaling In Human Calcific Aortic Valve Disease Via Regulation Of Warburg Effect

BackgroundCalcific aortic valve disease(CAVD)is an increasingly-frequent,also chronic and progressive disorder that has been identified as the independent risk factor for myocardial infarction,cardiovascular death and all-cause death.The most serious matter in terms of CAVD is that there is no effective pharmacology approach to prevent or reduce the progression of valvular calcification,which therefore contributes to the stubbornly high morbidity and mortality.Potentially destructive hemodynamic forces the valve leaflets confronted might trigger and aggravate the calcific progress.The aberrant differentiation of valvular interstitial cells to osteogenic lineages is a vibrantly regulated osteogenic process,which promotes valvular calcification and is identified as the theoretical basis of the ectopic ossification of aortic valves.However,how the osteogenic differentiation of valvular interstitial cells is initiated by abnormal hemodynamics remains unclear.Thus,it is very necessary and urgent to intensively elucidate the mechanisms underlying the initiation effect of possible mechanosensitive signaling on the osteogenic differentiation of valvular interstitial cells,so as to delay the CAVD progression effectively.Ras homolog gene family,member A(Rho A)/Rho-associated protein kinase 1(ROCK1),a mechano-sensing signaling,widely distributed in cardiovascular system that could transduce mechanical signals into cellular responses.Recently,several animal studies indicated that Rho A/ROCK signaling involves in the regulation of CAVD progression.Nonetheless,the regulatory role of Rho A/ROCK1 in human CAVD models has not been reported,additionally the mechanisms underlying any positive effect of Rho A/ROCK on CAVD require further study.Metabolic reprogramming is a mechanism by which cells meet energy demands themselves to sustain proliferation,growth and differentiation.The Warburg effect,a type of metabolic reprogramming which has been widely investigated,manifesting as a preference for glycolysis to generate energy even under adequate oxygen conditions,is extensively documented in atherosclerosis and skeletal development,which share similar pathophysiological calcification features to those of CAVD.Studies have reported that Rho A/ROCK1 could regulate tumor invasion and metastasis through mediating mechano-reactive metabolism,suggesting Rho A/ROCK1 signaling might play crucial roles in CAVD via regulating VICs’metabolic reprogramming.Therefore,a further understanding of the role and mechanism of Rho A/ROCK1 in human CAVD through regulation of metabolic reprogramming of VICs might contribute to exploring the therapeutic target for CAVD treatment.Objectives1.To identify the differential expression levels of Rho A/ROCK1 signaling and Warburg effect-associated proteins in human CAVD processes.2.To elucidate the role of Rho A/ROCK1 signaling in human VICs’ Warburg effect and to further clarify their osteogenic mechanism.MethodsⅠ.Human aortic valve tissues collection and analysis1.Procurement and pretreatment of clinical specimens:A total of 34 human aortic valve tissues were obtained from CAVD patients(n=22)and aortic dissection patients(n=12)as non-CAVD controls between October 2021 and October 2022.All samples were resected aseptically and intraoperatively,rinsed with cold sterile phosphate buffered saline to prepare valve stromal cells,or preserved in liquid nitrogen or 4%paraformaldehyde immediately.2.Detection of calcium and osteogenic differentiation:Alizarin red staining was used confirm calcium deposition on tissues.Western blot and immunohistochemistry were used to analyze osteogenic marker proteins in both groups.3.Detection of Rho A/ROCK1 signaling and Warburg effect associated key proteins:Western blot and immunohistochemistry were used to detect Rho A/ROCK1 signaling activation levels,also Warburg effect-related key transporters and enzymes expressions in both groups.Ⅱ.Human VICs isolation,identification,and osteogenic induction in vitro/vivo.1.Human VICs isolation and culture:Human VICs were isolated using the type II collagenase twice-digestion method.VICs in all in vitro experiments were utilized from passages 2 to 7.2.Human VICs identification:Immunofluorescence was used to test the expressions of interstitial and endothelial cell markers,Vimentin and CD31,to determine the purity of isolated VICs.3.Human VICs osteogenic induction and identification:To stimulate calcification in vitro,human VICs were cultured with inorganic phosphate-osteogenic medium(complete media supplemented with 2mM sodium dihydrogen phosphate,75 mM ascorbic acid,100nM dexamethasone and 10-7 mM insulin)for 7 days.Western blot,Alizarin red staining and ALP staining were used to detect osteogenic associated proteins,calcium deposition and ALP content of VICs by time gradient.4.Detection of Rho A/ROCK1 signaling and Warburg effect associated key proteins,also metabolic pattern in osteogenic induction model in vitro and vivo:Western blot was used to confirm the expression levels of Rho A/ROCK1 signaling and Warburg effect associated key proteins at different time point.Seahorse was used to examine the OCR and ECAR of human VICs.Ⅲ.The function analysis of Rho A/ROCK1 signaling on human VICs osteogenic differentiation and metabolic reprogramming.Y27632,BAY876 and BX795 were used to block-up Rho A/ROCK1 signaling and inhibit Warburg effect of human VICs,respectively.Western blot,Seahorse,Alizarin red staining and ALP staining were used to assess the function of Rho A/ROCK1 on metabolic reprogramming and osteogenic differentiation of human VICs,as well as the role of Warburg effect in the calcification of human VICs.IV.The mechanism analysis of Rho A/ROCK1-mediated metabolic switching on human VICs osteogenic differentiation.During the osteogenic differentiation of human VICs,real-time quantitative PCR was used to test RUNX2 mRNA expressions;immunoprecipitation was used to test ubiquitination levels of RUNX2;western blot was used to test AMPK phosphorylation levels and RUNX2 protein expression.Then,treat human VICs with Y27632,BAY876,BX795 and Compound C,respectively,and observe whether the above detection indicators changed.ResultsⅠ.Human aortic valve tissues collection and analysisAlizarin red staining result demonstrated prominent calcified lesion formation on valve tissues from CAVD patients.The protein expressions of Rho A,ROCK1,GLUT1,HK2,LDHA,PDK1,PFK1,RUNX2 and OPN,but not ROCK2,were significantly upregulated in CAVD valve samples contrast to that of controls by western blot and immunohistochemistry.Also,increased MYPT1 phosphorylation levels in CAVD group were showed by western blot.Ⅱ.Human VICs isolation,identification,and osteogenic induction in vitro/vivo.Immunofluorescence results confirmed positive staining of Vimentin while negative staining of CD31,indicating a suitable VICs purity for follow-up experiments.Western blot,Alizarin red staining and ALP staining verified upregulated protein expressions of Rho A/ROCK1 signaling,Warburg effect and osteogenic associated markers,as well as increased calcium deposition and ALP contents,during the osteogenic differentiation of human VICs.Ⅲ.The function analysis of Rho A/ROCK1 signaling on human VICs osteogenic differentiation and metabolic reprogramming.Osteogenic human VICs behaved metabolic preference to Warburg effect as their characteristic features,which was detected by western blot,Seahorse,Alizarin red staining and ALP staining.The metabolic reprogramming to Warburg effect and the osteogenic differentiation of VICs were significantly inhibited by ROCK1 activity blocking-up.By contrast,inhibiting metabolic switching to Warburg effect alleviated calcification of VICs,while not affected Rho A/ROCK1 activation levels.Ⅳ.The mechanism analysis of Rho A/ROCK1-mediated metabolic switching on human VICs osteogenic differentiation.The expression of RUNX2 mRNA in osteogenic VICs was markedly increased as demonstrated by real-time quantitative PCR.As shown in western blot analysis,the half-life of RUNX2 was also increased,with AMPK phosphorylation levels downregulated.Immunoprecipitation showed that RUNX2 ubiquitination was decreased in VICs after treatment with osteogenic medium.Further studies showed that Y27632,BAY876 or BX795 treatment all could decrease the endogenous RUNX2 protein abundance in calcified VICs,but did not affect their mRNA levels,additionally,RUNX2 ubiquitination levels were markedly increased and the half-life of RUNX2 was obviously shortened;accompanied by upregulated AMPK phosphorylation levels.Furthermore,downregulation of AMPK activity by Compound C alleviated the RUNX2 protein reduction and RUNX2 half-life shortening promoted by Y27632,BAY876 or BX795 in VICs without affecting RUNX2 mRNA expressions,as shown in rescue experiment.Conclusion1.During human CAVD progress,Rho A/ROCK1 signaling and Warburg effect play crucial roles in osteogenic differentiation of human VICs and calcification of human aortic valves.2.Rho A/ROCK1 aggravated human VICs calcification via promoting VICs’ metabolic reprogramming to Warburg effect,which subsequently reduced AMPK activity.AMPK inhibition reduced RUNX2 ubiquitin-proteasome degradation,thus promoted RUNX2 accumulation,and contributed to a final calcification of VICs and aortic valves.These indicated Rho A/ROCK1 might become a therapeutic target for CAVD treatment.