SIRT1Enhances Therapeutic Efficacy Of Aged Mesenchymal Stem Cells In Myocardial Infarction Via Improving MSCs Senescence And Function

Mesenchymal stem cells (MSCs) are thought to play important roles in tissue regeneration and repair. However, the senescence of MSCs increases with age, which will not only impair the efficacy of tissue regeneration and repair but also compromise aged MSCs-based therapy for tissue engineering and cell transplantation. Allogenic MSCs might be good candidates for aged recipients, however, allogenic MSCs have the risk of transition from an immunoprivileged state to an immunogenic state after their differentiation, and furthermore, no one knows the long-term safety and efficacy of allogenic MSCs nowadays.In this regard, MSCs from young donors might not be the best candidate for aged recipients and we should use young MSCs for aged recipients with caution. Thus, strategies to ameliorate the senescent phenotype and function of aged MSCs are needed. However, how to improve age-related biological properties of MSCs remains an open question. Of note is that SIRT1, a NAD+dependent deacetylase, could not only improve aging and aging-related diseases but also exert beneficial effects on cellular metabolism, cellular survival, cellular angiogenesis and cell differentiation via deacetylating various histones and nonhistones. No report about the role of SIRT1on age-related MSCs senescence and function is available nowadays. Here, this study will investigate the effect and mechanisms of SIRT1on age-related senescence and function of MSCs and furthermore to evaluate whether SIRT1could enhance aged MSCs-based therapy for myocardial infarction (MI), which might shed new light on the treatment of age-related MSCs senescence and function. Part1The effect and mechanisms of SIRT1on age-related senescence of MSCsObjective:We performed this study to investigate the role of SIRT1in age-related MSCs senescence and to explore the related mechanisms.Methods:(1) We first compared the alterations (cellular senescence, cell proliferation and the expression and activity of SIRT1) of MSCs derived from young (4-week-old) and aged (18-month-old) rats. Senescence-associated β-galactosidase (SA-β-gal) assay and Cell Counting Kit-8(CCK-8) test were conducted to evaluate the senescence and proliferation respectively. Quantitative real-time PCR and western blot were undertaken to assess SIRT1expression while the histone deacetylase assay kit was used to detect SIRT1deacetylase activity.(2) We next constructed young MSCs SIRT1knockdown model and aged MSCs SIRT1overexpression model via infecting lentiviruses. The relationships between SIRT1and senescence or proliferation of MSCs was analyzed in these models.(3) Western blot was performed to assess the expression of P16and P21, two aging-related proteins, in young MSCs SIRT1knockdown model and aged MSCs SIRT1overexpression model.(4) Immunofluorescence staining of gamma H2A.X and TRF2was undertaken to evaluate telomere DNA damage in young MSCs SIRT1knockdown model and aged MSCs SIRT1overexpression model.(5) The Telomeric Repeat Ampliation Protocol (TRAP) assay was performed to detect telomerase activity while quantitative real-time PCR was conducted to analyze the expression of telomerase reverse transcriptase (TERT) and telomere length in young MSCs SIRT1knockdown model and aged MSCs SIRT1overexpression model.(6) Quantitative real-time PCR and western blot were conducted to evaluate the expression of shelterin in young MSCs SIRT1knockdown model and aged MSCs SIRT1overexpression model.Results:(1) Cellular senescence increased and cell proliferation decreased significantly in aged MSCs compared to young MSCs. Meanwhile, the expression and activity of SIRT1was down-regulated significantly in aged MSCs compared with young MSCs.(2) Knockdown of SIRT1in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1in aged MSCs reversed the senescent phenotype and stimulated cell proliferation.(3) Knockdown of SIRT1in young MSCs promoted the expression of P16and P21whereas overexpression of SIRT1in aged MSCs inhibited the expression of P16and P21.(4) Knockdown of SIRT1in young MSCs induced telomere DNA damage whereas overexpression of SIRTl in aged MSCs inhibited telomere DNA damage.(5) Knockdown of SIRT1in young MSCs down-regulated TERT expression and telomerase activity whereas overexpression of SIRT1in aged MSCs up-regulated TERT expression and telomerase activity. Telomere length was not influenced by SIRT1until the manipulated young or aged MSCs were cultured more than8passages.(6) Knockdown of SIRT1in young MSCs enhanced the expression of tripeptidyl peptidase1(TPP1), a key component of the shelterin, whereas overexpression of SIRT1in aged MSCs inhibited TPP1expression.Conclusion:SIRT1quenches age-related MSCs senescence by mechanisms that include enhancing TPP1expression, increasing telomerase activity and reducing DNA damage. Part2SIRT1enhances therapeutic efficacy of aged mesenchymal stem cells in myocardial infarction via lightening MSCs senescence and heightening pro-angiogenesis and stress resistance.Objective:We performed this study to demonstrate whether SIRT1could ameliorate the senescent phenotype and function of aged MSCs and then enhance aged MSCs-based therapy for myocardial infarction (MI).Methods and results:Sixty Sprague-Dawley (SD) female rats experienced left anterior descending coronary artery ligation were randomly assigned to receiving intramyocardial injection of cell culture medium (DMEM group), SIRT1overexpression vector treated-aged MSCs (SIRT1-aged MSC group) obtained from aged male SD rats or empty vector treated-aged MSCs (vector-aged MSC group). Another twenty sham operated rats underwent open-chest surgery without coronary ligation and any other intervention served as controls (sham group). The survival and engraftment of the male donor cells in the female recipient ischemic hearts was evaluated by quantification of rat Y-chromosome-specific sry gene expression by real-time PCR at1and3day after cell therapy. We found that SIRT1-aged MSC therapy resulted in significantly higher sry gene expressions compared with vector-aged MSCs group, reaching five-and two-fold as high as empty vector MSCs at1and3day after cell transplantation respectively. At day28after cell therapy, cardiac function was evaluated by transthoracic echocardiography and hemodynamic studies; Masson’s trichrome staining was performed to analyze fibrous tissue; immunofluorescence staining of CD31and a-SMA in the border zone of the infarcted heart was conducted to assess angiogenesis. Our data indicated that echocardiographic parameters ((left ventricular end-diastolic diameters (LVEDD), left ventricular end-systolic diameters (LVESD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)) and hemodynamic parameters (left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximum dP/dt (dP/dt max) and minimum dP/dt (dP/dt min)) were improved significantly in SIRT1-aged MSCs group compared with DMEM group while these parameters were not changed significantly in vector-aged MSCs compared with DMEM group. In line with this, the collagen volume fraction was reduced in the vector-aged MSCs and in the SIRT1-aged MSCs group, however, only SIRT1-aged MSCs group reach statistical significance compared with the DMEM group. There were more blood vessel density in the border zone of ischemic hearts in SIRT1-aged MSCs group compared to DMEM or vector-aged MSCs group, however, there were not significant difference between DMEM group and vector-aged MSCs group.In addition, we also analyzed the effect of SIRT1overexpression on senescence (see "Part1"), pro-angiogenesis and stress response of aged MSCs. Matrigel tube formation assay was performed to study the capillary-like tube formation of human umbilical vein endothelial cells (HUVEC) induced by conditioned medium from aged MSCs modified with SIRT1or not. The stress response capability of aged MSCs modified with SIRT1or not was evaluated in H2O2-induced MSCs damage model. SIRT1overexpression ameliorated aged MSCs senescent phenotype (see "Part1") and recapitulated the pro-angiogenesis property of MSCs and conferred the anti-stress response capabilities, as indicated by increases in pro-angiogenic factors, angiopoietin1(Angl) and basic fibroblast growth factor (bFGF), expressions and a decrease in anti-angiogenic factor thrombospondin-1(TBS1) at mRNA levels, and increases in Bcl-2/Bax ratio at protein level.Conclusion:Up-regulating SIRT1expression could enhance the efficacy of aged MSCs-based therapy for MI which is related to the amelioration of senescent phenotype and hence improved biological function of aged MSCs.