AMP-activated protein kinase rescues the angiogenic functions of endothelial progenitor cells via manganese superoxide dismutase induction in type 1 diabetes

Endothelial progenitor cells (EPCs) play an essential role in angiogenesis but are functionally impaired in diabetes. We recently reported that decreased expression of manganese superoxide dismutase (MnSOD) critically contributes to diabetic EPC dysfunction. AMP-activated protein kinase (AMPK) activation has been shown to induce MnSOD and suppress hyperglycemia-induced mitochondrial ROS production in endothelial cells. However, whether AMPK protects EPCs from oxidative stress in diabetes is unknown. We tested the hypothesis that AMPK activation rescues impaired EPC functions through MnSOD induction in type 1 diabetes. Bone marrow-derived EPCs from adult male streptozotocin-induced diabetic mice and normal controls were used. AMPK activity was decreased in diabetic EPCs, indicated by reduced AMPK and acetyl-CoA carboxylase phosphorylation. AMPK activation by treating diabetic EPCs with its selective agonist AICAR rescued their in vitro functions, including Matrigel tube formation, adhesion, and migration. Furthermore, AICAR restored the decreased MnSOD protein and enzymatic activity and suppressed the mitochondrial superoxide level in diabetic EPCs, indicated by MitoSOX flow cytometry. These beneficial effects of AICAR on MnSOD and EPC functions were significantly attenuated by silencing MnSOD or AMPK antagonist compound C pretreatment. Finally, the expression of protein phosphatase 2A, a key enzyme for AMPK dephosphorylation and inactivation, was increased in diabetic EPCs, and its inhibition by siRNA or okadaic acid reversed the deficient AMPK activation and MnSOD level in diabetic EPCs. These findings demonstrate for the first time that AMPK activation rescues impaired EPC functions and suppresses mitochondrial superoxide by inducing MnSOD in type 1 diabetes.     

Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.     

The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local over-expression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration, and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.     

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.     

Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function

Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb Angelica gigas is known to possess potent anti‐inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009 ]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood‐derived AC133+ cells that produce functional EPC progenies. Decursin dose‐dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle‐shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin‐2, angiopoietin receptor Tie‐2, Flk‐1 (vascular endothelial growth factor receptor‐2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose‐dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor‐induced mobilization of circulating EPCs (CD34 + /VEGFR‐2+ cells) from bone marrow and early incorporation of Dil‐Ac‐LDL‐labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild‐type‐ or bone‐marrow‐transplanted mice. Accordingly, decursin attenuated EPC‐derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis. J. Cell. Biochem. 113: 1478–1487, 2012. © 2012 Wiley Periodicals, Inc.     

Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells,Oligodendrocyte Precursor Cells,and Endothelial Progenitor Cells

Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9) has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs). However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs), and endothelial progenitor cells (EPCs). Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM), and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types.     

Association of SIRT1 expression with shear stress induced endothelial progenitor cell differentiation

Shear stress imposed by blood flow is crucial for differentiation of endothelial progenitor cells (EPCs). Histone deacetylase SIRT1 has been shown to play a pivotal role in many physiological processes. However, association of SIRT1 expression with shear stress‐induced EPC differentiation remains to be elucidated. The present study was designed to determine the effect of SIRT1 on EPC differentiation induced by shear stress, and to seek the underlying mechanisms. Human umbilical cord blood‐derived EPCs were exposed to laminar shear stress of 15 dyn/cm² by parallel plate flow chamber system. Shear stress enhanced EPC differentiation toward endothelial cells (ECs) while inhibited to smooth muscle cells (SMCs). The expressions of phospho‐Akt, SIRT1 and histone H3 acetylation (Ac‐H3) in EPCs were detected after exposure to shear stress for 2, 6, 12, and 24 h, respectively. Shear stress significantly activated Akt phosphorylation, augmented SIRT1 expression and downregulated Ac‐H3. SIRT1 siRNA in EPCs diminished the expression of EC markers, but increased the expression of SMC markers, and resulted in upregulation of Ac‐H3. Whereas, resveratrol, an activator of SIRT1, had the opposite effects on both EPC differentiation and histone H3 acetylation. Wortmannin, an inhibitor of PI3‐kinase, suppressed endothelial differentiation of EPCs, decreased SIRT1, and upregulated Ac‐H3 expression. In addition, SIRT1 promoted tube formation of EPCs in matrix gels. These results provided a mechanobiological basis of shear stress‐induced EPC differentiation into ECs and suggest that PI3k/Akt‐SIRT1‐Ac‐H3 pathway is crucial in such a process. J. Cell. Biochem. 113: 3663–3671, 2012. © 2012 Wiley Periodicals, Inc.     

In vivo trafficking of endothelial progenitor cells their possible involvement in the tumor neovascularization

Circulating endothelial progenitor cell (EPCs) have been reported to contribute to vasculogenesis in adult organisms. To investigate the possible recruitment of EPCs and organization to form tumor vasculature, we investigated the in vivo real-time trafficking of EPCs non-invasively by using positron emission tomography (PET). A conditionally immortalized endothelial cell line derived from rat bone marrow (TR-BME1) was labeled with [2-(18)F] 2-fluoro-2-deoxy-D-glucose (FDG) and chased the accumulation in the rat tumor with PET. TR-BME1 cells were accumulated in the tumor tissues time-dependently. To investigate that the accumulation of the cells is specific or not, rats were previously irradiated with gamma-ray to suppress the influence of non-labeled EPCs derived from its bone marrow and used for PET analysis. The accumulation of TR-BME1 cells in the tumor was enhanced in gamma-ray-irradiated rats compared with that of non-irradiated ones, suggesting that TR-BME1 cells accumulated in the tumor specifically like as EPCs. Then the involvement of matrix metalloproteinases (MMPs) in EPC recruitment was examined. An inhibitor of MMP, MMI270, which suppressed invasion and tube formation abilities of TR-BME1 cells, only slightly suppressed the accumulation of TR-BME1 cells in the tumor of rats. These results suggest that EPCs are recruited in the tumor tissues for formation of tumor vasculature, and demonstrate the usefulness of TR-BME1 cells for studies on EPC related phenomena.     

Sitagliptin‐mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.     

Diabetes alters subsets of endothelial progenitor cells that reside in blood, bone marrow, and spleen

Circulating endothelial progenitor cells (EPCs) derived from the bone marrow (BM) participate in maintaining endothelial integrity and vascular homeostasis. Reduced EPC number and function result in vascular complications in diabetes. EPCs are a population of cells existing in various differentiation stages, and their cell surface marker profiles change during the process of mobilization and maturation. Hence, a generally accepted marker combination and a standardized protocol for the quantification of EPCs remain to be established. To determine the EPC subsets that are affected by diabetes, we comprehensively analyzed 32 surface marker combinations of mouse peripheral blood (PB), BM, and spleen cells by multicolor flow cytometry. Ten subsets equivalent to previously reported mouse EPCs significantly declined in number in the PB of streptozotocin-induced diabetic mice, and this reduction was reversed by insulin treatment. The PI(-)Lin(-)c-Kit(-)Sca-1(+)Flk-1(-)CD34(-)CD31(+) EPC cluster, which can differentiate into mature endothelial cells in vitro, was the highest population in the PB, BM, and spleen and occurred 61 times more in the spleen than in the PB. The cell number significantly decreased in the BM as well as in the PB but paradoxically increased in the spleen under diabetic conditions. Insulin treatment reversed the decrease of EPC subsets in the BM and PB and reversed their increase in spleen. A similar tendency was observed in some of the major cell populations in db/db mice. To the best of our knowledge, we are the first to report spatial population changes in mouse EPCs by diabetes in the blood and in the BM across the spleen. Diminished circulating EPC supply by diabetes may be ascribed to impaired EPC production in the BM and to decreased EPC mobilization from the spleen, which may contribute to vascular dysfunction in diabetic conditions.     

Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress.

Endothelial progenitor cells (EPCs), circulating in peripheral blood, migrate toward target tissue, differentiate, and contribute to the formation of new vessels. In this study, we report that shear stress generated by blood flow or tissue fluid flow can accelerate the proliferation, differentiation, and capillary-like tube formation of EPCs. When EPCs cultured from human peripheral blood were subjected to laminar shear stress, the cells elongated and oriented their long axes in the direction of flow. The cell density of the EPCs exposed to shear stress was higher, and a larger percentage of these cells were in the G2-M phase of the cell cycle, compared with EPCs cultured under static conditions. Shear stress markedly increased the EPC expression of two vascular endothelial growth factor receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase-1, and an intercellular adhesion molecule, vascular endothelial-cadherin, at both the protein and mRNA levels. Assays for tube formation in the collagen gels showed that the shear-stressed EPCs formed tubelike structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that EPCs are sensitive to shear stress and that their vasculogenic activities may be modulated by shear stress.     

Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin α(v)/β(3), and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.     

The Biphasic Effects of Oxidized-Low Density Lipoprotein on the Vasculogenic Function of Endothelial Progenitor Cells

Late-outgrowth endothelial progenitor cells (EPCs) are stress-resistant and responsible for reparative functions in the cardiovascular system. Oxidized-LDL (oxLDL) plays a critical role in cardiovascular disease pathogenesis. However, it is largely unknown what the impacts of oxLDL are on late-outgrowth EPCs. This study aimed to investigate the concentration-related effects of oxLDL on EPC functions and related angiogenesis, in vitro and in vivo. In this study, early and late-outgrowth EPCs were generated from circulating human mononuclear cells. oxLDL may regulate EPC vasculogenic function via the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Lower concentrations (5 μg/mL) of oxLDL can potentiate EPC tube formation in vitro and in vivo by activating eNOS mechanisms, which are mediated by p38 MAPK- and SAPK/JNK-related pathways. Higher concentrations of oxLDL (10-50 μg/mL) impaired EPC function via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways and consequent inhibition of eNOS activity, which could be reversed by anti-oxidants (diphenylene iodonium and apocynin) and gp91^phox siRNA. In conclusion, oxLDL has concentration-dependent biphasic effects on human late-outgrowth EPC tube formation in vitro and in vivo.     

Effects of rapamycin on number activity and eNOS of endothelial progenitor cells from peripheral blood

The aim of this investigation is to determine whether rapamycin treatment has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days in culture, attached cells were stimulated with rapamycin (in a series of final concentrations: 0.1, 1.0, 2.0 and 5.0 g/ml) for 6, 12, 24 and 48 h. EPCs were characterized as adherent cells, double positive for DiLDL uptake and lectin binding by direct fluorescence staining. EPC proliferation and migration were determined using the MTT assay and a modified version of the Boyden chamber assay, respectively. An EPC adhesion assay was performed by replating the cells on fibronectin-coated dishes; adherent cells were then counted. Tube formation activity was assayed by using a tube formation assay kit and endothelial nitric oxide synthase (eNOS) was assayed by Western blot analysis. Incubation of isolated human MNCs with rapamycin decreased the number of EPCs present; rapamycin also decreased EPCs proliferative, migratory, adhesive, tube formation capacity and eNOS production in a concentration- and time-dependent manner. Rapamycin was found to decrease the number, proliferative, migratory, adhesive and tube formation capacities of the EPCs, and also was found to decreases eNOS in the EPCs.     

Improved angiogenic activity of endothelial progenitor cell in diabetic patients treated with insulin plus metformin

Type 2 diabetes (T2DM) is associated with an increased vascular disease. Moreover, endothelial progenitor cell (EPC) function is impaired in diabetic patients. Decreased EPC number plays a critical role in reduced endothelial repair and development of the vascular disorder. To determine the effect of metformin and insulin plus metformin on functional activity of EPCs, 130 participants were divided into three groups (group 1: healthy control; group 2: metformin; group 3: insulin plus metformin). The concentration of EPCs in the circulation was first quantified. Thereafter, circulating EPCs (cEPCs) were harvested and the biological features of these cells including proliferative, clonogenicity, tubulogenic, and migratory properties were analyzed after expansion. The serum protein levels of some proangiogenic factors were also measured. Our results showed greater numbers of cEPCs in control and in diabetic patients treated with insulin plus metformin than in metformin-treated patients. Insulin plus metformin therapy was associated with augmented proliferative, clonogenicity, migratory, and tubulogenic activity of cEPCs in patients with T2DM. Increased serum concentrations of angiogenic factors were also observed in patients treated with insulin plus metformin. Western blot analysis showed increased protein levels of pTie-2/Tie2 and Pakt/AKT in cEPCs harvested from T2DM, treated with insulin metformin plus. This study showed that treatment with insulin plus metformin in diabetic patients is associated with increased mobilization of EPCs into the circulation, with potential beneficial effect in vascular protection in diabetic patients.     

Endothelial progenitor cells in diabetes mellitus

Diabetes mellitus is associated with an increased risk of cardiovascular disease due to its negative impact on the vascular endothelium. The damaged endothelium is repaired by resident cells also through the contribution of a population of circulating cells derived from bone marrow. These cells, termed endothelial progenitor cells (EPCs) are involved in maintaining endothelial homeostasis and contributes to the formation of new blood vessels with a process called postnatal vasculogenesis. The mechanisms whereby these cells allow for protection of the cardiovascular system are still unclear; nevertheless, consistent evidences have shown that impairment and reduction of EPCs are hallmark features of type 1 and type 2 diabetes. Therefore, EPC alterations might have a pathogenic role in diabetic complications, thus becoming a potential therapeutic target. In this review, EPC alterations will be examined in the context of macrovascular and microvascular complications of diabetes, highlighting their roles and functions in the progression of the disease.