Final Report Summary - EPCREPAIR (Evaluation of Beneficial Role of Protein Phosphatases and KLF2 in Diabetic Endothelial and Progenitor Cell Repair)
Publishable summary
Background:
Type-2 diabetes is associated with impaired neovascularisation, which involves dysfunction of circulating pro-angiogenic mononuclear cells (CAC) but the specific molecular mechanisms underlying this deleterious consequence of metabolic disorders are incompletely understood. The main objectives of our project was then to develop a model in vitro mimicking the harmful actions of diabetic conditions on CAC in order to test and propose some potent new therapeutic strategies to reverse the dysfunctional phenotype of CAC isolated from diabetic patients or pre-diabetic patients with metabolic syndrome. Previous work from our groups has shown beneficial effects of the shear stress sensitive transcriptional factor KLF2 (Kruppel-like factor 2) on endothelial function especially by promoting an anti-oxidant cellular profile. Our group previously showed major beneficial effects of KLF2 (Kruppel-like factor 2) on endothelial function Interestingly, a recent study pointed out the lifespan regulator p66Shc, a pro-oxidant adaptor protein as a modulator of KLF2 expression. Furthermore, p66Shc has recently been linked to obesity-induced insulin resistance and deleterious consequences of diabetes.
Thus, the study of the putative beneficial role of KLF2 or the deleterious action of its potent regulator p66Shc were particularly investigated aiming to the development of pharmaceutical targets and tools necessary for preventing clinical cardiovascular complications of diabetes. In parallel, considering the specific deleterious role of phosphatases like PTP1B in diabetes and evidenced in endothelial dysfunction by our group, we also focused on studying activation pathways involving deregulated phosphorylation cascades.
Methods:
We successfully isolated and cultured circulating pro-angiogenic cells (CACs) (formerly referred to as EPC) from buffy coats and from fresh peripheral blood obtained from healthy individuals, from type-2 diabetics (DM2), and from patients with the metabolic syndrome (pre-diabetics). We characterized CACs after 7 days of culture as mononuclear cells double positive for UEA lectin and with the capacity to uptake DiLDL, their endothelial and leukocyte markers were also characterized by flow cytometry (FACS) analysis. Different pro-diabetic conditions were tested in vitro followed by Q-PCR analysis for mRNA expression of various phosphatases and factors involved in the vascularization function of CACs.
The number of CACs reaches about 3% of the initial population of PBMCs isolated from buffy coats or fresh blood. Functional analysis of CACs aiming to assess their ability to move and migrate, necessary functions in a context of in vivo revascularization, were performed using live-cell imaging microscopy such as chemotaxis assays. Considering the major functional impact of cellular oxidative status, we followed by live-cell microscopy the cytoplasmic reactive oxygen species (ROS) production in CACs, treated with PA or isolated from patients, using an oxidative sensitive fluorescent probe (CM-DCFHDFA). Lentiviral transductions were also set up and successfully performed on CACs in order to assess the potent beneficial effect of Klf2 over-expression, or silencing of p66Shc or PTP1b. Finally, we transposed our newly learned skills on isolating and culturing CACs from bone-marrow derived mononuclear cells isolated from healthy wild-type mice or obese diabetic mice. We then assessed their revascularization capacity in vivo following the hindlimb reperfusion blood, flow using Laser Doppler Imaging, in mice subjected to a femoral artery ligation.
Results:
Our first clear significant result concerns our data on in vitro treatment of CAC. Despite conflicting reports we did not observe significant action of high glucose on CAC, but a dominant role for free fatty acid (palmitic acid, PA). Analysis of multiple phosphatases expression did not show any significant changes between conditions. Rather, we observed for the first time that this pro-diabetic molecule PA, encountered at high levels in pre-diabetic patients with metabolic syndrome and also in diabetic patients, showed marked actions on CACs. It induces a large increase in the pro-oxidant protein p66Shc mRNA levels while significantly increases oxidative stress. In parallel, PA modulates expression of different vascularization factors. It indeed decreases mRNA levels of PECAM-1 and SDF1alpha but counterintutively increases VEGF-A, without affecting VEGF-B expression. We also observed a diminution of surface expression of different markers known to play a role in vascularizing action of CACs, such as VEGFR2, Tie2 and CXCR4
A major finding of this study is that a pre-treatment with the grape and red wine-derived polyphenol resveratrol prevented the action of PA-induced increase in p66Shc and VEGF-A mRNA expressions and abolished the PA-induced oxidative stress in CACs. In parallel, resveratrol significantly increased the migration capacity of PA-treated CACs towards the chemoattractant factor VEGF-A.
Another interesting and new result consists in the observation that CACs isolated from Type-2 diabetic and metabolic syndrome patients display an increased oxidative stress compared to aged-matched healthy control cells, even after seven days of in vitro culture. Most interestingly, this elevated oxidative status was completely reversed by a pre-treatment with resveratrol which points out these compounds as a putative potent therapeutic tool in the context of metabolic disorders.
Our in vivo experiments highlighted a beneficial action on of bone-marrow (BM)-derived CACs, as injection of cells isolated from wild-type mice improved revascularization, assessed by laser Doppler imaging (LDPI), after hindlimb ischemia. However, BM-derived CACs isolated from obese diabetic mice leprob/ob showed the same functional capacity. It is certainly due to a mild diabetic phenotype encountered in these 4 month-old obese mice, already described in the literature. Hence, genetically over-expression Klf2 or silencing p66Shc in vitro in obese BM-CACs did not further improve the revascularization capacity of these cells.
Conclusion:
Our results shed new light on the protective mechanisms of resveratrol action on diabetic CAC, and in particular its link with the pro-oxidant protein p66Shc. Thus, we show for the first time a new potent target of this polyphenol compound, increasing its therapeutical potential to the restoration of CAC function in diabetic patients.
Background:
Type-2 diabetes is associated with impaired neovascularisation, which involves dysfunction of circulating pro-angiogenic mononuclear cells (CAC) but the specific molecular mechanisms underlying this deleterious consequence of metabolic disorders are incompletely understood. The main objectives of our project was then to develop a model in vitro mimicking the harmful actions of diabetic conditions on CAC in order to test and propose some potent new therapeutic strategies to reverse the dysfunctional phenotype of CAC isolated from diabetic patients or pre-diabetic patients with metabolic syndrome. Previous work from our groups has shown beneficial effects of the shear stress sensitive transcriptional factor KLF2 (Kruppel-like factor 2) on endothelial function especially by promoting an anti-oxidant cellular profile. Our group previously showed major beneficial effects of KLF2 (Kruppel-like factor 2) on endothelial function Interestingly, a recent study pointed out the lifespan regulator p66Shc, a pro-oxidant adaptor protein as a modulator of KLF2 expression. Furthermore, p66Shc has recently been linked to obesity-induced insulin resistance and deleterious consequences of diabetes.
Thus, the study of the putative beneficial role of KLF2 or the deleterious action of its potent regulator p66Shc were particularly investigated aiming to the development of pharmaceutical targets and tools necessary for preventing clinical cardiovascular complications of diabetes. In parallel, considering the specific deleterious role of phosphatases like PTP1B in diabetes and evidenced in endothelial dysfunction by our group, we also focused on studying activation pathways involving deregulated phosphorylation cascades.
Methods:
We successfully isolated and cultured circulating pro-angiogenic cells (CACs) (formerly referred to as EPC) from buffy coats and from fresh peripheral blood obtained from healthy individuals, from type-2 diabetics (DM2), and from patients with the metabolic syndrome (pre-diabetics). We characterized CACs after 7 days of culture as mononuclear cells double positive for UEA lectin and with the capacity to uptake DiLDL, their endothelial and leukocyte markers were also characterized by flow cytometry (FACS) analysis. Different pro-diabetic conditions were tested in vitro followed by Q-PCR analysis for mRNA expression of various phosphatases and factors involved in the vascularization function of CACs.
The number of CACs reaches about 3% of the initial population of PBMCs isolated from buffy coats or fresh blood. Functional analysis of CACs aiming to assess their ability to move and migrate, necessary functions in a context of in vivo revascularization, were performed using live-cell imaging microscopy such as chemotaxis assays. Considering the major functional impact of cellular oxidative status, we followed by live-cell microscopy the cytoplasmic reactive oxygen species (ROS) production in CACs, treated with PA or isolated from patients, using an oxidative sensitive fluorescent probe (CM-DCFHDFA). Lentiviral transductions were also set up and successfully performed on CACs in order to assess the potent beneficial effect of Klf2 over-expression, or silencing of p66Shc or PTP1b. Finally, we transposed our newly learned skills on isolating and culturing CACs from bone-marrow derived mononuclear cells isolated from healthy wild-type mice or obese diabetic mice. We then assessed their revascularization capacity in vivo following the hindlimb reperfusion blood, flow using Laser Doppler Imaging, in mice subjected to a femoral artery ligation.
Results:
Our first clear significant result concerns our data on in vitro treatment of CAC. Despite conflicting reports we did not observe significant action of high glucose on CAC, but a dominant role for free fatty acid (palmitic acid, PA). Analysis of multiple phosphatases expression did not show any significant changes between conditions. Rather, we observed for the first time that this pro-diabetic molecule PA, encountered at high levels in pre-diabetic patients with metabolic syndrome and also in diabetic patients, showed marked actions on CACs. It induces a large increase in the pro-oxidant protein p66Shc mRNA levels while significantly increases oxidative stress. In parallel, PA modulates expression of different vascularization factors. It indeed decreases mRNA levels of PECAM-1 and SDF1alpha but counterintutively increases VEGF-A, without affecting VEGF-B expression. We also observed a diminution of surface expression of different markers known to play a role in vascularizing action of CACs, such as VEGFR2, Tie2 and CXCR4
A major finding of this study is that a pre-treatment with the grape and red wine-derived polyphenol resveratrol prevented the action of PA-induced increase in p66Shc and VEGF-A mRNA expressions and abolished the PA-induced oxidative stress in CACs. In parallel, resveratrol significantly increased the migration capacity of PA-treated CACs towards the chemoattractant factor VEGF-A.
Another interesting and new result consists in the observation that CACs isolated from Type-2 diabetic and metabolic syndrome patients display an increased oxidative stress compared to aged-matched healthy control cells, even after seven days of in vitro culture. Most interestingly, this elevated oxidative status was completely reversed by a pre-treatment with resveratrol which points out these compounds as a putative potent therapeutic tool in the context of metabolic disorders.
Our in vivo experiments highlighted a beneficial action on of bone-marrow (BM)-derived CACs, as injection of cells isolated from wild-type mice improved revascularization, assessed by laser Doppler imaging (LDPI), after hindlimb ischemia. However, BM-derived CACs isolated from obese diabetic mice leprob/ob showed the same functional capacity. It is certainly due to a mild diabetic phenotype encountered in these 4 month-old obese mice, already described in the literature. Hence, genetically over-expression Klf2 or silencing p66Shc in vitro in obese BM-CACs did not further improve the revascularization capacity of these cells.
Conclusion:
Our results shed new light on the protective mechanisms of resveratrol action on diabetic CAC, and in particular its link with the pro-oxidant protein p66Shc. Thus, we show for the first time a new potent target of this polyphenol compound, increasing its therapeutical potential to the restoration of CAC function in diabetic patients.