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.     

Human mature endothelial cells modulate peripheral blood mononuclear cell differentiation toward an endothelial phenotype

Circulating endothelial progenitor cells (EPCs) can contribute to neovascularization, even if the mechanisms by which they interact with mature endothelial cells remain unclear. The interactions between human coronary artery endothelial cells (HCAECs) and peripheral blood mononuclear cells (PBMCs) during their early differentiation towards an EPC phenotype were investigated. A co-culture model, in which the two cell types share the same culture medium in the absence of any exogenous angiogenic stimulus, was used. The role of hypoxia was assessed by pretreating HCAECs with 3% O₂ before co-culture setting. Since we have previously shown that both adherent and suspended PBMCs display a significant increase in endothelial marker expression within the 2nd day of culture in an angiogenic environment, the role of HCAECs on early PBMC differentiation was evaluated in both adherent and suspended cell fractions.A 3-day co-culture period increased the expression of VEGF-R2, VE-cadherin, α_vβ₃- and α₅-integrin in both the adherent and suspended PBMCs, assessed by cytofluorimetric analysis, and up-regulated VEGF-R1 mRNA assessed by real-time RT-PCR. HCAECs influenced PBMC adhesion, transendothelial migration and cell organization on Matrigel. Hypoxia modulated either PBMC differentiation or their functional properties. These data strongly suggest that endothelium may support the differentiation of PBMCs into EPCs.     

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.     

Distinct Tie2 tyrosine phosphorylation sites dictate phenotypic switching in endothelial progenitor cells

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial and their progenitor cells, nominated as endothelial progenitor cells (EPCs). Tie2/Tek is a tyrosine kinase receptor expressed by endothelial cells that induces signal transduction pathways involved in endothelial biology. To address the potential importance of the various tyrosine residues of Tie2 in EPC development, we generated a series of Tie2 tyrosine mutated (Y1106F, Y1100F, and Y1111F) EPCs and then assess the biological features of these cells. Clonogenic, tubulogenic, proliferative, migratory, and functional properties of these cells were analyzed. Next, GFP-positive EPCs containing Tie2 tyrosine mutations were systemically transplanted into sublethaly irradiated mice to analyze the potency of these cells for marrow reconstitution. We found that mutation in the Tie2 tyrosine 1106 residue directed EPCs toward a mature endothelial phenotype, which was associated with augmented tubulogenic and migratory properties, and increased phosphorylation of the active site (tyrosine 992) as well as increased vascular perfusion in the in vivo Matrigel plug assay. Moreover, transplantation of 1106 Tie2 mutant EPCs failed to reconstitute the bone marrow after myeloablation, whereas transplantation of EPCs with the 1100 or 1111 Tie2 tyrosine mutation resulted in bone marrow engraftment, leading to improved survival of recipient mice. Our findings demonstrate that the tyrosine 1106 residue in Tie2 plays a key role to maintain the stemness features of EPCs.     

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.     

Angiotensin II promotes NO production, inhibits apoptosis and enhances adhesion potential of bone marrow-derived endothelial progenitor cells

Endothelial progenitor cells (EPCs) participate in the processes of postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. The level of EPCs present has been found to be directly associated with the outcome of cardiovascular diseases, and could be regulated by stimulatory or inhibitory factors. Given the close relationship between angiotensin II (AngII) and the cardiovascular system, we investigated the effect of AngII on the activities of bone marrow (BM)-derived EPCs. Cells were isolated from BM of rats by density gradient centrifugation. Administration of AngII significantly promoted nitric oxide (NO) release, inhibited EPC apoptosis and enhanced EPC adhesion potential. All of these AngII-mediated effects on EPCs were attenuated by pretreatment with valsartan or L-NAME. Moreover, both LY294002 and wortmannin abolished the anti-apoptotic effect of AngII. Western blot analyses indicated that endothelial NO synthase (eNOS) protein and phosphorylated Akt increased with the treatment of AngII in EPCs. Thus, AngII improved several activities of EPCs through AngII type 1 receptor (AT1R), which may represent a possible mechanism linking AngII and AT1R with angiogenesis. Additionally, AngII-induced NO synthesis through eNOS in EPCs regulates apoptosis and adhesion, and the PI3-kinase/Akt pathway has an essential role in AngII-induced antiapoptosis signaling.     

Shear Stress Regulates Late EPC Differentiation via Mechanosensitive Molecule-Mediated Cytoskeletal Rearrangement

Background

Previous studies have demonstrated that endothelial progenitor cells (EPCs), in particular late EPCs, play important roles in endothelial maintenance and repair. Recent evidence has revealed shear stress as a key regulator for EPC differentiation. However, the underlying mechanisms regulating the shear stress–induced EPC differentiation have not been understood completely. The present study was undertaken to further investigate the effects of shear stress on the late EPC differentiation, and to elucidate the signal mechanism involved.

Methodology/Principal Finding

In vitro and in vivo assays revealed that cytoskeletal remodeling was involved in the shear stress-upregulated expression of endothelial markers vWF and CD31 in late EPCs, with subsequently increased in vivo reendothelialization after arterial injury. Moreover, shear stress activated several mechanosensitive molecules including integrin β₁, Ras, ERK1/2, paxillin and FAK, which were all involved in both cytoskeletal rearrangement and cell differentiation in response to shear stress in late EPCs.

Conclusions/Significance

Shear stress is a key regulator for late EPC differentiation into endothelial cells, which is important for vascular repair, and the cytoskeletal rearrangement mediated by the activation of the cascade of integrin β₁, Ras, ERK1/2, paxillin and FAK is crucial in this process.     

Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model

Background

Cilostazol(CLZ) has been used as a vasodilating anti-platelet drug clinically and demonstrated to inhibit proliferation of smooth muscle cells and effect on endothelial cells. However, the effect of CLZ on re-endothelialization including bone marrow (BM)-derived endothelial progenitor cell (EPC) contribution is unclear. We have investigated the hypothesis that CLZ might accelerate re-endothelialization with EPCs.

Methodology/Principal Findings

Balloon carotid denudation was performed in male Sprague-Dawley rats. CLZ group was given CLZ mixed feed from 2weeks before carotid injury. Control group was fed normal diet. CLZ accelerated re-endothelialization at 2 weeks after surgery and resulted in a significant reduction of neointima formation 4 weeks after surgery compared with that in control group. CLZ also increased the number of circulating EPCs throughout the time course. We examined the contribution of BM-derived EPCs to re-endothelialization by BM transplantation from Tie2/lacZ mice to nude rats. The number of Tie2-regulated X-gal positive cells on injured arterial luminal surface was increased at 2 weeks after surgery in CLZ group compared with that in control group. In vitro, CLZ enhanced proliferation, adhesion and migration activity, and differentiation with mRNA upregulation of adhesion molecule integrin αvβ3, chemokine receptor CXCR4 and growth factor VEGF assessed by real-time RT-PCR in rat BM-derived cultured EPCs. In addition, CLZ markedly increased the expression of SDF-1α that is a ligand of CXCR4 receptor in EPCs, in the media following vascular injury.

Conclusions/Significance

CLZ promotes EPC mobilization from BM and EPC recruitment to sites of arterial injury, and thereby inhibited neointima formation with acceleration of re-endothelialization with EPCs as well as pre-existing endothelial cells in a rat carotid balloon injury model. CLZ could be not only an anti-platelet agent but also a promising tool for endothelial regeneration, which is a key event for preventing atherosclerosis or restenosis after vascular intervention.     

Shear stress augments the endothelial cell differentiation marker expression in late EPCs by upregulating integrins

Vascular endothelial cell injury has been implicated in the onset of atherosclerosis. A number of previous studies have demonstrated that endothelial progenitor cells (EPCs), in particular late EPCs, play important roles in endothelial maintenance and repair. Recent evidence has revealed shear stress as a key regulator for EPC differentiation. However, the detailed events that contribute to the shear stress-induced EPC differentiation, in particular the mechanisms of mechanotransduction, remain to be identified. The present study was undertaken to further confirm the effects of shear stress on the late EPC differentiation, and to investigate the role of integrins in this procedure. Shear stress was observed to increase the expression of endothelial cell differentiation markers, such as vWF and CD31, in late EPCs isolated from rat bone marrow. Shear stress moreover enhanced the mRNA expression of integrin subunits β(1) and β(3) in a time-dependent manner, and also upregulated specific integrins in late EPCs plated on substrates containing various extracellular matrix (ECM) proteins. In addition, the shear stress-induced vWF and CD31 expression were found to be related to the levels of integrin β(1) and β(3), and were inhibited in late EPCs treated with RGD peptide (Gly-Arg-Gly-Asp-Asn-Pro, GRGDNP) that blocks the binding of integrins to the extracellular matrix. Additionally, this increase was also attenuated by both anti-β(1) integrin and anti-β(3) integrin antibodies. The integrin subunits β(1) and β(3) thus play important roles in regulating the shear stress-induced endothelial cell differentiation marker expression in late EPCs. This may provide novel insights into the mechanisms of mechanotransduction in shear stress-mediated late EPC differentiation.     

Akap200 suppresses the effects of Dv-cbl expression in the Drosophila eye

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury.     

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.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Shear stress activates Tie2 receptor tyrosine kinase in human endothelial cells

The receptor tyrosine kinase (RTK) Tie2 is expressed predominantly on endothelial cells. Tie2 is critical for vasculogenesis during development and could be important for maintaining endothelial cell survival and integrity in adult blood vessels. Although most RTKs are activated by shear stress in the absence of ligand activation, the effect of shear stress on Tie2 is unknown. Therefore, we examined the effect of shear stress on Tie2 phosphorylation in primary cultured endothelial cells. Interestingly, shear stress (20 dyne/cm(2)) produced a rapid, marked, and sustained Tie2 phosphorylation, while it produced a rapid but slight and transient phosphorylation of insulin receptor and VEGF receptor 2 (Flk1). In addition, Tie2 phosphorylation in response to shear stress was velocity-dependent, while phosphorylation of insulin receptor and Flk1 was not. Shear stress also produced Akt phosphorylation in a time-, velocity-, and PI 3-kinase-dependent manner. Accordingly, shear stress suppressed serum deprivation-induced endothelial cell apoptosis. Taken together, our results indicated that activation of Tie2/PI 3-kinase/Akt in response to shear stress could be an important signaling cascade for maintaining endothelial survival and integrity in blood vessels.     

Slight up-regulation of Kir2.1 channel promotes endothelial progenitor cells to transdifferentiate into a pericyte phenotype by Akt/mTOR/Snail pathway

It was shown that endothelial progenitor cells (EPCs) have bidirectional differentiation potential and thus perform different biological functions. The purpose of this study was to investigate the effects of slight up-regulation of the Kir2.1 channel on EPC transdifferentiation and the potential mechanism on cell function and transformed cell type. First, we found that the slight up-regulation of Kir2.1 expression promoted the expression of the stem cell stemness factors ZFX and NS and inhibited the expression of senescence-associated β-galactosidase. Further studies showed the slightly increased expression of Kir2.1 could also improve the expression of pericyte molecular markers NG2, PDGFRβ and Desmin. Moreover, adenovirus-mediated Kir2.1 overexpression had an enhanced contractile response to norepinephrine of EPCs. These results suggest that the up-regulated expression of the Kir2.1 channel promotes EPC transdifferentiation into a pericyte phenotype. Furthermore, the mechanism of EPC transdifferentiation to mesenchymal cells (pericytes) was found to be closely related to the channel functional activity of Kir2.1 and revealed that this channel could promote EPC EndoMT by activating the Akt/mTOR/Snail signalling pathway. Overall, this study suggested that in the early stage of inflammatory response, regulating the Kir2.1 channel expression affects the biological function of EPCs, thereby determining the maturation and stability of neovascularization.     

Electronegative LDL circulating in smokers impairs endothelial progenitor cell differentiation by inhibiting Akt phosphorylation via LOX-1

Endothelial progenitor cells (EPCs), important for endothelial regeneration and vasculogenesis, are reduced by cigarette smoking. To elucidate the mechanisms, we examined the effects of electronegative LDL, circulating in chronic smokers, on EPC differentiation. Using ion-exchange chromatography, we purified smoker LDL into five subfractions, L1-L5. In matched, nonsmoking healthy subjects, L5, the most electronegative subfraction, was either absent or scanty. Sustained L5 treatment inhibited CD31 and KDR expression and EPC differentiation, whereas L1-L4 had no effect. L5 also inhibited telomerase activity to accelerate EPC senescence in correlation with reduced Akt phosphorylation. Transfection of day 3 EPCs with dominant negative Akt constructs inhibited CD31 and KDR expression, stalled EPC differentiation, and promoted early senescence. In contrast, transfection with constitutively active Akt rendered the EPCs resistant to L5, allowing normal maturation. L5 upregulated the lectin-like oxidized low density lipoprotein receptor 1 (LOX-1), and pretreatment of EPCs with TS20, a LOX-1-neutralizing antibody, blocked internalization of L5 by EPCs and prevented L5-mediated inhibition of EPC differentiation. Mixing L5 with L1 to physiological L5/L1 ratios did not attenuate L5's effects. These findings suggest that cigarette smoking is associated with the formation of L5, which inhibits EPC differentiation by impairing Akt phosphorylation via the LOX-1 receptor.     

Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.