Study On Regulative Effect And Mechanism Of Vascular Smooth Muscle Cells Using Adipose-derived Stem Cells With Platelet-rich Plasma In The Rat

Background:Endovascular invasive treatment solves the problem of limb ischemia in elderly patients with lower extremity arteriosclerosis occlusion,but the maintenance time is short and can not avoid the occurrence of postoperative restenosis,resulting in high recurrence rate.Lower extremity atherosclerotic occlusion is one of the most common diseases in vascular surgery,and especially in developed and developing countries,and is the third leading cause of cardiovascular disease incidence after coronary atherosclerotic heart disease and cerebral infarction[1].Patients may experience decreased skin temperature,intermittent claudication of the lower extremities,resting pain,and even gangrenous tissue infections due to lower extremity arterial occlusion.In the United States,more than 60,000 amputations related to peripheral arterial disease are performed each year[2,3],which seriously affects the quality of life and physical health of people.Open revascularization surgery requires patients having better systemic conditions,which most elderly patients can’t afford.With the development of medical technology,significant advancements have been made in treatment modalities,including revascularization surgery,vascular interventional therapy,and drug therapy.The long-term effect of vascular interventional therapy as the main treatment modality is still unsatisfactory,and even the most advanced techniques,such as drug-coated balloon angioplasty and drug-coated stenting are unable to prevent the occurrence of post-operative restenosis[4-6].Atherosclerosis leading to stenosis and vascular occlusion,which is closely related to phenotypic conversion of vascular smooth muscle cells(VSMC)[7,8].Hippo-Yap pathway is an important pathway for the phenotypic transformation of VSMC.Inhibiting this pathway may inhibit the phenotypic transformation of VSMC.VSMC play an important role in maintaining the structural and functional stability of blood vessels.By regulating blood flow to target tissues through contraction and relaxation,and thus exhibit stronger phenotypic plasticity during restenosis[9].VSMC switch from the contractile phenotype to synthetic phenotype,also known as the dedifferentiated phenotype,after receiving stimulation or mechanical injury,mainly manifesting as hypocontractile function.Synthetic VSMC are more secretory and can proliferate and migrate in large numbers,producing extracellular matrix proteins that lead to functional disorders,vascular wall thickening,luminal narrowing,reduced vascular compliance,and vascular remodeling[10-12].Phenotypic switching to a synthetic phenotype is a prerequisite for the proliferation of VSMC,and the ability to effectively control this phenotypic switch is one of the keys to the treatment of cardiovascular disease.It has also been shown that the Hippo-Yap pathway plays an important role in restenosis in rodents,as a sensing mechanism for immune homeostasis,which is activated by receiving external stimuli,and thus involved in regulating cellular responses to alterations in stimuli[13-14].Adipose-derived stem cells(ADSC)are of great research value in the field of regenerative medicine,due to their ease of harvesting,multidirectional differentiation potential,proliferative capacity,and modulation of immune cell activation[15-18].Platelet rich plasma(PRP)has been used in several clinical areas,especially in tissue regeneration,because of its multiple cytokine content.The immunomodulatory properties of mesenchymal stem plasma may be enhanced when combined with PRP[19].Co-culture of PRP with ADSC increased the proliferative capacity of ADSC and promoted angiogenesis[20-21].Most studies related to stem cell therapy for lower extremity atherosclerosis occlusion have focused on revascularization and angiogenesis,but studies on whether adipose stem cells can promote VSMC phenotypic transformation have not been reported.Purpose:Therefore,the study focused on whether ADSC combined with PRP can inhibit VSMC phenotypic transformation and regulate Hippo-Yap pathway,and further elucidated the possibility of ADSC combined with PRP as seed cells to enhance the treatment of lower limb atherosclerotic occlusive disease in vivo.Its mechanism on VSMC phenotypic transformation to provide new directions and strategies for non-surgical treatment modalities for restenosis of lower extremity atherosclerotic occlusive disease.Methods:In this study,we constructed an in vitro cell model by culturing platelet-derived growth factor(PDGF)with VSMC,constructed an in vivo animal model of lower limb atherosclerotic occlusion in rats by guidewire injury combined with high-fat diet feeding.and divided into the ADSC group,PRP group,ADSC+PRP group,ADSC+inhibitor group,and ADSC+PRP+inhibitor group,according to the different treatment methods.The migration of VSMC was detected by cell scratch test and the proliferation of VSMC was detected by cell proliferation test.The expression levels of SMA-22α,YAP,and TNF-αwere detected at the cellular level and in arterial vascular tissues by PCR and Western Blot.Immunohistochemical staining was used to detect the animal model specimens,and the hemodynamic conditions associated with lower limb arteries were detected by color Doppler ultrasound.Results:1.In vitro results(1)The results of cell scratch test showed that ADSC+PRP group promoted the migration of smooth muscle cells in the disease model compared with ADSC group and PPP group.The cell migration ability of ADSC+PRP group and ADSC group have decreased after adding Hippo inhibitor(XMU-MP-1).(2)The results of cell proliferation test showed that ADSC+PRP group did not increase the proliferation of smooth muscle cells compared with ADSC group and PRP group.But the cell proliferation ability of ADSC+PRP group and ADSC group increased after adding Hippo inhibitor(XMU-MP-1).(3)The results of cell immunohistochemistry(Ki67)showed that ADSC+ PPP group did not promote the proliferation of smooth muscle cells compared with ADSC group and PRP group.But the cell proliferation ability of ADSC+ PRP group and ADSC group increased after adding Hippo inhibitor((XMU-MP-1).(4)PCR and Western Blot experiments showed that ADSC combined with PRP significantly increased the expression of SM-22α,decreased the expression level of OPN,TNF-α,YAP in comparison with the model group.In contrast,the addition of Hippo inhibitor decreased SM-22αexpression level and increased OPN,TNF-α and YAP expression.2.In vivo results(1)The expression of TNF-α in vascular tissue was increased in the in vivo animal model of lower extremity arteriosclerosis occlusion constructed by guidewire injury combined with high-fat diet feeding.The expression of Yap in smooth muscle cells is increase,the expression of SM-22α is decrease.(2)The results of in vivo experiments by color Doppler ultrasonography showed that ADSC combined with PRP significantly improved the degree of luminal stenosis in a model of lower extremity atherosclerotic occlusion.(3)PCR and Western Blot experiments showed that ADSC combined with PRP significantly increased the expression of SM-22α,decreased the expression level of OPN,TNF-α,YAP in comparison with the model group.In contrast,the addition of Hippo inhibitor decreased SM-22α expression level and increased OPN,TNF-α and YAP expression.(4)The staining of pathological specimens of femoral artery in different treatment groups was compared and analyzed.It was found that the number of contractile smooth muscle cells and intimal thickening decreased in ADSC+PRP group,and the number of proliferative smooth muscle cells decreased significantly compared with the model group.After adding the inhibitor,the number of contractile smooth muscle cells in ADSC+PRP+XMU-MP-1 group was lower than that in the treatment group.ConclusionIn summary,this study found that during the development of lower extremity atherosclerotic occlusive disease,smooth muscle cells are converted from the contractile phenotype to the synthetic phenotype;ADSC combined with PRP significantly reduced the level of stenosis in atherosclerotic occlusion and promoted the conversion of VSMC to the contractile phenotype;and ADSC may promoted the conversion of smooth muscle cells to the contractile phenotype through the Hippo-Yap pathway.