Functional And Mechanistic Characterization Of The Histone Deacetylase Sirt7 In Vascular Calcification

BackgroundVascular calcification is a significant risk factor for cardiovascular complications in patients with chronic kidney disease（CKD）.Previous studies including ours have shown that vascular calcification resembles physiological bone formation and mineralization.Hyperphosphatemia is frequently observed in CKD patients,and it is a significant risk factor for vascular calcification.Previous Studies have reported that high phosphate mimicking that observed in CKD patients directly induces vascular calcification in vitro through up-regulation of the osteogenic genes such as Alpl,Runx2and Bmp2.Currently,there are no drugs available for the treatment of vascular calcification.Therefore,understanding of the underlying mechanisms through which vascular calcification occurs may help to develop novel therapeutics for vascular calcification in CKD patients.The histone deacetylase Sirt7 belongs to the sirtuin family,which are NAD⁺-dependent protein deacetylases.The substrates of Sirt7 include histone and non-histone proteins.Sirt7 regulates its downstream gene expression or protein functions through altering their acetylation levels.Previous studies have reported the crucial role of Sirt7in cardiovascular diseases,however,its role in vascular calcification under CKD conditions remains to be fully elucidated.ObjectiveIn the present study,we have investigated the role of Sirt7 in vascular calcification under CKD conditions using cell and animal models.Our study may provide a potential therapeutic target for the treatment of vascular calcification in patients with CKD.Methods（1）Validation of VSMCs in vitro calcification modelPrimary murine VSMCs were isolated as we previously described.In order to ensure the biological stability of VSMCs,2-4 generation of cells were used for subsequent experiments.VSMCs were treated with normal phosphate（1 m M Pi）and high phosphate（3 m M Pi）for 0 day,7 days or 14 days in vitro.Calcium content was determined by alizarin red staining and calcium quantitative assay.RT-q PCR was used to detect the m RNA expression level of osteogenic marker such as Runx2,Bmp2,Opn and Alpl.（2）Validation of vascular calcification mouse modelSeven-week-old C57BL/6 male mice were injected with a dose of Vitamin D₃(5X 10⁵ IU kg^-1,6μl/g)subcutaneously for 3 days,and kept for another 4 days.Vascular calcification in these mice was determined by alizarin red staining and calcium quantitative assay.RT-q PCR was used to detect the m RNA expression level of the osteogenic markers such as Runx2,Bmp2,Opn and Alpl.Sirt7 expression was measured by RT-q PCR and western blot.（3）Sirt7 inhibits high phosphate-induced vascular calcificationAortic rings were cultured ex vivo as explants,and treated with normal phosphate（1 m M Pi）or high phosphate（5 m M Pi）in the absence or presence of 10m M NAM（nicotinamide,a Sirtuins inhibitor）for 6 days.Calcium content was determined by alizarin red staining.VSMCs were treated with normal phosphate（1 m M Pi）or high phosphate（3 m M Pi）in the absence or presence of NAM for 9days.Calcium content was determined by alizarin red staining and calcium quantitative assay.VSMCs were treated with normal phosphate（1 m M Pi）or high phosphate（3 m M Pi）,and transfected with scramble si RNA or Sirt7 si RNA for up to 9 days.Calcium content was determined by alizarin red staining and calcium quantitative assay.RT-q PCR was used to detect the m RNA expression level of Runx2,Bmp2,Opn,Alpl and Osterix.Western blot was used to detect the protein expression level of Runx2,Bmp2,and Osterix.（4）High-throughput RNA sequencingVSMCs were transfected with scramble si RNA or Sirt7 si RNA.After 48 hours,RT-q PCR was used to confirm the knockdown efficiency.The remaining samples were sent to Aksomics for high-throughput RNA sequencing and subsequent bioinformatics analysis.（5）Measurement of reactive oxygen species（ROS）levelVSMCs were treated with normal phosphate（1 m M Pi）or high phosphate（3 m M Pi）,and transfected with scramble si RNA or Sirt7 si RNA.After 48 hours,cells were incubated with H2DCFDA at 37℃for 30 min in the dark,and then the ROS level was measured by FACs.（6）Cell cycle analysisVSMCs were treated with normal phosphate（1 m M Pi）or high phosphate（3 m M Pi）,and transfected with scramble si RNA or Sirt7 si RNA for 48 hours,the cells were treated with RNase and stained with propidium iodide.Cell cycle was analyzed using flow cytometry.（7）Measurement of cellular senescenceVSMCs were treated with normal phosphate（1 m M Pi）or high phosphate（3 m M Pi）,and transfected with scramble si RNA or Sirt7 si RNA for 9 days,the cells were fixed and stained with SA-β-gal following manufacturer’s instructions.Images were recorded by Light microscope and the blue cells represent senescent cells.ResultsConsistent with previous reports,we showed that high phosphate induced VSMC in vitro calcification and osteogenic differentiation.Interestingly,Sirt7 expression was dramatically down-regulated in calcified arteries from mice administered with high dose vitamin D₃.Our further studies showed that knockdown of Sirt7 expression in VSMCs enhanced high phosphate induced calcium deposition.Interestingly,knockdown of Sirt7 did not alter m RNA expression of Osterix but significantly increased its protein expression.Our RNA-sequencing and bioinformatic analysis revealed that Sirt7 downstream genes were associated with cell cycle and cellular ROS.We further demonstrated that knockdown of Sirt7 expression increased the cell cycle arrest in G₀/G₁ phase,and attenuated the expression of key cell cycle regulators cyclin D1,CDK-4 and p-RB.In accordance with these observations,silencing of Sirt7 using si RNA promoted the cellular senescence of VSMCs.However,knockdown of Sirt7 in VSMCs did not alter cellular ROS levels.ConclusionIn summary,our study demonstrates the key inhibitory role of Sirt7 in vascular calcification.Mechanistically,Sirt7 attenuates vascular calcification through the regulation of cell cycle,cellular senescence and Osterix protein expression.Our study provides a potential therapeutic target for vascular calcification in patients with CKD patients.