The Role And Mechanism Of Piezo1 In Vascular Calcification

Vascular calcification refers to the abnormal deposition of calcium phosphate in the form of hydroxyapatite in the arterial wall.It is an independent risk factor for cardiovascular and cerebrovascular diseases,and is closely related to its morbidity and mortality.Vascular calcification is mainly divided into vascular intima calcification and medial calcification.Intimal calcification is commonly found in intimal plaques in patients with atherosclerosis and is associated with plaque rupture events.Medial calcification is common in the blood vessels of patients with chronic kidney disease,hypertension and diabetes.In the past,vascular calcification was considered to be the passive deposition of calcium phosphate in blood vessels,but a large number of studies in recent years have shown that vascular calcification is an active,cell-mediated pathological process similar to endochondral osteogenesis.The prominent pathological changes of medial calcification are Vascular smooth muscle cells transform from a contractile phenotype to an osteoblast-like phenotype that synthesizes bone matrix proteins.The mechanism driving this osteogenic transformation is not well understood,and possible factors include hyperphosphatemia,hypercalcemia,vascular aging,inflammatory cytokines,oxidative stress,etc.Studies have shown that patients with chronic kidney disease are exposed to high circulating calcium levels Significantly increased the risk of vascular calcification,and long-term calcium supplementation for the prevention of osteoporosis was accompanied by a high incidence of vascular calcification,indicating that hypercalcemia is an important factor in promoting vascular calcification.Growing evidence suggest that vascular endothelial cells play a role in vascular calcification through endothelial-mesenchymal transition,autocrine/paracrine pathways,endothelial cell-derived extracellular vesicles（EVs）,angiogenesis,and mechanotransduction play an important role.In mouse atherosclerotic plaques,increased activity of bone morphogenetic proteins（BMPs）promotes endothelial cells to undergo mesenchymal transition and ultimately calcification.Previous studies have also shown that parathyroid hormone can induce endothelial-mesenchymal transition in endothelial cells and promote the occurrence of vascular calcification in uremic rats.Endothelial cells are under constant mechanical force due to blood pressure and flow in the arterial and venous systems.Blood flowing through vascular endothelial cells generates shear stress.Under normal physiological conditions,a dynamic balance between mechanical shear stress and biological responses maintains endothelial integrity.In contrast,changes in endothelial shear stress induced by hypertension or excessive blood flow can lead to vascular remodeling by disrupting cytoskeletal proteins and vascular function,including loss of endothelial integrity,increased endothelial cell stiffness,altered vasodilation properties,and leukocyte adhesion.The Piezo channel is encoded by two genes called Piezo1 and Piezo2 in humans,which are located on chromosomes 16 and 18,respectively,and the encoded proteins are large multi-channel transmembrane structures.Piezo1channels can be detected in a variety of endothelial cells,which are widespread in the vasculature,lymphatic system,and heart,and cells in these sites can directly sense physiological shear stress in the cardiovascular system.Piezo1 is activated by cell membrane tension induced by high pressure,shear stress,and membrane stretching,allowing the influx of cations,mainly Ca²⁺,and triggering downstream calcium signaling.However,at the inner curvature of the aortic arch,endothelial cells are subject to turbulent flow,and activation of Piezo1 induces endothelial cell inflammation and atherosclerosis.Previous studies have shown that conditional knockout of Piezo1 reduces the accumulation of endothelial inflammatory cells.Furthermore,specific knockout of Piezo1 in endothelial cells attenuated endothelial inflammation and neointimal formation-induced atherosclerosis after partial carotid artery ligation in mice lacking LDL receptors and on a high-fat diet.Studies have shown that chronic exposure to high circulating calcium levels is associated with a significantly increased risk of calcification in patients with chronic kidney disease.However,the mechanism of vascular calcification in the body in the environment of hypercalcemia is not clear,and the Piezo1 channel of vascular endothelial cells can mediate Ca²⁺influx after activation under the action of blood flow shearing force.Therefore,the purpose of this study is to explore the relationship between Piezo1 channel of vascular endothelial cells and vascular calcification to further clarify the mechanism of vascular calcification.Part One The role of Piezo1 in vitamin D-induced vascular calcification in miceObjective:To explore the role of Piezo1 channel of vascular endothelial cells in vascular calcification induced by high calcium environment.Methods:1.Establishment and grouping of animal models:32 6-week-old clean and healthy male C57BL/6J mice were randomly divided into four groups after 1 week of adaptive feeding:control group（intraperitoneal injection of an appropriate amount of normal saline）,vitamin D group（20 mg/kg,subcutaneous injection）,vitamin+Yoda1（2.6 mg/kg,intraperitoneal injection）,vitamin D+Gs MTx4（0.8 mg/kg,intraperitoneal injection）,8 animals in each group,treated for ten consecutive days,during the experiment,the general condition of the animals was observed and the daily food intake and body weight were recorded.2.Analysis of serum biochemical indicators:After ten days of treatment,the mice were anesthetized with 2%sodium pentobarbital solution 45 mg/100g intraperitoneally,and the eyeball was removed.The blood collection tube was centrifuged at 3000 rpm/min for 15 min and then taken out to observe whether the serum was separated.The upper serum was aspirated with a pipette and packed in centrifuge tubes.Serum levels of calcium,phosphorus,alkaline phosphatase（Alkaline phosphatase,ALP）,serum creatinine（Creatinine,Crea）and blood urea nitrogen（BUN）were detected with an automatic biochemical analyzer.3.Ultrasound detection of vascular function:After ten days of treatment,mice were anesthetized with 2%isoflurane inhalation with a small animal anesthetic agent,and ultrasound data were collected 2 or 3 min after the start of anesthesia.The collected records include:thickness（mm）of aortic arch and common carotid artery,vascular tone and common carotid pulse wave velocity（PWV,m/s）associated with vascular stiffness were calculated.4.Determination of vascular calcium content:After blood collection,the mice were routinely fixed and sterilized,incised along the midline of their abdomen,and the ascending aorta was quickly taken out for determination by the o-cresol peptide complex ketone method.Calcium content was normalized to protein content.Results:1.Changes in body weight and food intake of mice in each group:The weight of the control group of mice was relatively stable during the treatment period,while the weight of the mice in the vitamin D group,the vitamin D+Yoda1 group and the vitamin D+Gs MTx4 group started to decrease significantly on the second day of treatment（P<0.01）and continued.During the whole observation period,the body weight of mice in each group also decreased significantly with the increase of treatment days（P<0.01）.2.Biochemical index test results:Compared with the control group,the levels of serum calcium,alkaline phosphatase,serum creatinine and blood urea nitrogen in the vitamin D group were significantly increased（P<0.05）,but the serum phosphorus level had no significant change.3.Vascular ultrasound test results:After ten days of treatment,compared with the control group,the thickness of the aortic arch and common carotid artery of the mice in the vitamin D group,the vitamin D+Yoda1 group and the vitamin D+Gs MTx4group did not change significantly.However,the aorta,common carotid artery tone and PWV of the common carotid artery in the vitamin D group were all higher than those in the control group（P<0.05）,and Yoda1 could significantly enhance the changes of the above indexes caused by vitamin D.On the contrary,Gs MTx4 could alleviate the changes（P<0.05）.4.The effect of vitamin D,Yoda1 and Gs MTx4 on calcium content in blood vessels of mice:Compared with the control group,the calcium content in the blood vessels of the mice in the vitamin D group was significantly increased（P<0.05）.In addition,Yoda1 aggravated vitamin D-induced increases in vascular calcium,while Gs MTx4 decreased the blood vessel calcium content（P<0.05）.Conclusion:The vascular calcification model can be successfully established by applying supertherapeutic doses of vitamin D.Openers of Piezo1 channels aggravated vascular calcification while their inhibitors attenuated vascular calcification,suggesting that activation of Piezo1 is involved in vascular calcification.Part Two The role of Piezo1 in hypercalcemia-induced umbilical vein endothelial cell injuryObjective:To observe the damage effect of Piezo1 channel opening on human umbilical vein endothelial cells cultured in vitro under high calcium environment.Methods:1.Detection of cell viability:Human umbilical vein endothelial cells（HUVECs）were cultured in vitro,and the cells were incubated with different concentrations of Ca Cl₂ for different durations of time to detect the viability of HUVECs with CCK-8 kit.The effect of co-incubation with Yoda1 on the cell viability was observed.2.Detection of cell damage indicators:Incubate cells with Ca Cl₂ 5m M for 5 days as a high calcium cell damage model,and observe the effect of activating Piezo1 channel on it.Detection indicators include:using NO kit to detect the concentration of NO in cell supernatant;RT-PCR detection of HUVECs-induced NO synthase（e NOS）,matrix metalloproteinase-2（MMP-2）,proliferating cell nuclear antigen（PCNA）,vascular endothelium Growth factor-A（VEGFA）m RNA expression;Western blot detection of HUVECs e NOS,MMP2 protein expression;immunofluorescence detection of HUVECs PCNA,VEGFA protein expression.Results:1.Activation of Piezo1 reduced the survival rate of HUVECs:With the prolongation of Ca²⁺（5 mmol/L）treatment time,the survival rate of HUVECs decreased.When the treatment time was 5d,the inhibition rate reached more than 50%（P<0.01）.Considering the influence of cell growth and metabolism,we determined the dosing incubation time as 5d.Yoda1 5μM reduced the survival rate of HUVECs to 30%（P<0.05）,and Gs MTx4 1.25μM could abolish the effect of Yoda1.2.Activation of Piezo1 aggravates the injury of HUVECs induced by Ca Cl₂:（1）Compared with the control group,the content of NO in the Ca Cl₂group was significantly decreased（P<0.05）,and the Ca Cl₂+Yoda1 group was significantly decreased（P<0.01）,while Gs MTx4 could resist the NO reduction caused by Yoda1（P<0.05）.（2）Compared with the control group,the m RNA levels of e NOS,MMP2,PCNA,and VEGFA in the Ca Cl₂ group were significantly decreased（P<0.05）,and the addition of Yoda1 further decreased the above indexes（P<0.05）,but the si RNA knockdown Piezo1 group decreased less than the Yoda1 group（P<0.05）.（3）Compared with the control group,the protein expression levels of e NOS,MMP2,PCNA and VEGFA in the Ca Cl₂ group were significantly decreased（P<0.05）,and the addition of Yoda1 further reduced the above indexes（P<0.05）.The Piezo1 knockdown group with si RNA decreased to a lesser extent（P<0.05）.Conclusion:A high calcium environment can impair vascular endothelial function by reducing the expression of marker genes for vascular endothelial cell migration,proliferation and growth.The opening of Piezo1 channel can further lead to high calcium-mediated endothelial cell damage.Part Three The role of Piezo1 in the osteogenic transformation of vascular smooth muscleObjective:To observe the effect of activating Piezo1 channel of HUVECs on osteotransformation of co-cultured vascular smooth muscle cells.Methods:1.A cell co-culture system was established in the Transwell plate:mouse vascular smooth muscle cells were cultured in the lower chamber of the Transwell plate,and HUVECs were cultured in the upper chamber.HUVECs were incubated with physiological calcium（control）,high calcium and high calcium+Yoda1 for 5d,and then the chamber was transferred to the lower chamber of the well plate and co-cultured with mouse vascular smooth muscle cells for 5d.The total RNA and protein of vascular smooth muscle cells were extracted and detected.The m RNA and protein expression levels of SOX9 and Runx2 in each group of cells were used to verify the effect of endothelial cell secretion on the osteogenic transformation of smooth muscle cells.2.Detection of ALP activity in cells:The ALP activity of vascular smooth muscle cells in each group was detected and calculated with a microplate reader.3.Determination of cellular calcium content:The o-cresol peptide complex ketone method was used to determine and calculate the calcium content of vascular smooth muscle cells in each group.4.Apoptotic number of smooth muscle cells:The Apoptosis detection kit of Annexin V-FITC was used to detect and calculate the number of apoptosis of vascular smooth muscle cells in each group.Results:1.SOX9 and Runx2 m RNA levels in smooth muscle cells:Compared with the control group,the m RNA levels of SOX9 and Runx2in the Ca Cl₂ group were significantly increased（P<0.05）,and the addition of Yoda1 further increased the above indexes（P<0.05）.2.Expression levels of SOX9 and Runx2 proteins in smooth muscle cells:Compared with the control group,the levels of SOX9 and Runx2 in the Ca Cl₂ group were significantly increased（P<0.05）.Yoda1 further increased the levels of SOX9 and Runx2 in cells（P<0.05）.3.Smooth muscle cell ALP activity:Compared with the control group,the ALP activity of the cells in the Ca Cl₂ group was significantly increased（P<0.05）,and the ALP activity of the cells was further increased by Yoda1（P<0.05）.4.Smooth muscle cell calcium content:Compared with the control group,the calcium content of the cells in the Ca Cl₂ group was significantly increased（P<0.05）,and the calcium content of the cells was further increased by Yoda1（P<0.05）.5.Apoptotic number of smooth muscle cells:Compared with the control group,the number of apoptotic cells in the Ca Cl₂ group was significantly increased（P<0.05）,and the number of apoptotic cells was further increased by Yoda1（P<0.05）.Conclusion:A high calcium environment can promote the transformation of vascular smooth muscle to an osteogenic phenotype by injuring vascular endothelial cells.Opening of Piezo1 channel can further lead to hypercalcemia-mediated conversion of vascular smooth muscle to an osteogenic phenotype.Summary:This study demonstrated at the animal and cellular level that Piezo1 channel-mediated Ca²⁺influx can aggravate vascular endothelial injury induced by high calcium environment,and can further lead to the transformation of vascular smooth muscle to an osteogenic phenotype.The findings provide a new experimental basis for preventive and therapeutic strategies for vascular calcification.