Search

Delayed diabetic wound healing is, in part, the result of inadequate endothelial progenitor cell (EPC) proliferation, mobilization, and trafficking. Recently, we developed a serum-free functional culture system called the quality and quantity culture (QQc) system that enhances the number and vasculogenic potential of EPCs. We hypothesize that QQc restoration of diabetic EPC function will improve wound closure. To test this hypothesis, we measured diabetic c-kit(+)Sca-1(+)lin(-) (KSL) cell activity in vitro as well as the effect of KSL cell-adoptive transfer on the rate of euglycemic wound closure before and after QQc. KSL cells were magnetically sorted from control and streptozotocin-induced type I diabetic C57BL6J bone marrow. Freshly isolated control and diabetic KSL cells were cultured in QQc for 7 days and pre-QQc and post-QQc KSL function testing. The number of KSL cells significantly increased after QQc for both diabetic subjects and controls, and diabetic KSL increased vasculogenic potential above the fresh control KSL level. Similarly, fresh diabetic cells form fewer tubules, but QQc increases diabetic tubule formation to levels greater than that of fresh control cells (P < 0.05). Adoptive transfer of post-QQc diabetic KSL cells significantly enhances wound closure compared with fresh diabetic KSL cells and equaled wound closure of post-QQc control KSL cells. Post-QQc diabetic KSL enhancement of wound closure is mediated, in part, via a vasculogenic mechanism. This study demonstrates that QQc can reverse diabetic EPC dysfunction and achieve control levels of EPC function. Finally, post-QQc diabetic EPC therapy effectively improved euglycemic wound closure and may improve diabetic wound healing.

In stem cell biology, CD34+ or CD133+ hematopoietic stem cells (HSCs) give rise to two types of endothelial progenitor cell (EPC) colonies: primitive and definitive EPC-colony forming units (primitive EPC-CFU and definitive EPC-CFU), which can be morphologically defined. Based on their morphology, an evaluation of the number or the ratio of each EPC colony constitutes the Endothelial Progenitor Cell Clonogenic Forming Assay (EPC-CFA), a novel assay to quantify the differentiation of colony forming EPCs. This assay system allows us to practically evaluate the vasculogenic potential of primary or cultured stem cell populations, i.e., mononuclear cells or fractionated stem cells (CD34+ or CD133+ cells) in peripheral blood, bone marrow, or umbilical cord blood. EPC-CFA can be used not only for basic research in vascular biology but also for evaluating the vascular reparative activity of patients with cardiovascular diseases. This review summarizes the underlying concepts and significance of the EPC-CFA in vascular biology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Endothelial progenitor cells (EPCs) play a critical role in restoration of ischemic diseases. However, the actual status of EPC development and the mechanisms of EPC dysfunctions in patients with various ischemic diseases remain unknown., Methods: To investigate the detailed function of EPCs in experimental murine models, we have established an EPC colony forming assay (EPC-CFA) in murine EPCs. The abilities of murine EPCs in differentiation, adhesive capacity, proliferative potency, and transplantation in vitro and in vivo were then examined., Results: Peripheral blood mononuclear cells (PB-MNCs), bone marrow mononuclear cells (BM-MNCs) or bone marrow c-Kit+/Sca-1+ lineage negative (BM-KSL) cells differentiated into two types of EPC colony forming units (EPC-CFUs), large sized EPC (large-EPC)-CFUs and small sized EPC (small-EPC)-CFUs. Gene expression analysis demonstrated that both EPC-CFU-derived cells expressed eNOS, Flk-1 and VE-cadherin, markers of endothelial cells (ECs), although the small-EPCs derived from small-EPC-CFU were higher in number and showed more immature features (higher population of KSL cells). Functionally, the large-EPCs derived from large-EPC-CFU had higher adhesive capacity but lower proliferative potency than small-EPCs, showing improved tubular forming capacity and incorporation potency into primary EC-derived tube formation. Importantly, hindlimb ischemia increased the frequencies of large-EPC-CFUs differentiated from PB-MNCs and bone marrow. Actually, transplantation of large-EPCs into ischemic hindlimb enhanced neovascularization in hindlimb ischemia model, although small-EPCs or murine ECs did not, suggesting that large-EPC-CFUs might play an important role in restoration of ischemic diseases., Conclusions: We demonstrated, using a murine ischemia model, that the EPC-CFA could be a useful way to investigate the differentiation levels of murine EPCs, further providing a crucial clue that large-EPC-CFU status may be more functional or effective EPCs to promote neovascularization.

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.

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., (Copyright © 2012 Wiley Periodicals, Inc.)

Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony-forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood-CD133(+) cells for 7 days produced a 52.9-fold increase in total cell number and 3.28-fold frequency in definitive EPC colony development, resulting in a 203.9-fold increase in estimated total definitive EPC colony number in vitro. Pre- or post-QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer-tipped conductance catheter examinations 28 days post-MI revealed significant preservation of left ventricular (LV) function in rats receiving pre- or post-QQc cells compared with those receiving phosphate-buffered saline. Assessments of global LV contractility indicated a dose-dependent effect of pre- or post-QQc cells and the superior potency of post-QQc cells over pre-QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post-QQc cells compared with pre-QQc cells. Our optimal serum-free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration.

Endothelial progenitor cells (EPCs) have been isolated and shown to be effective in animal models of ischemia, and many groups involved in clinical trials have demonstrated that EPC therapy is safe and feasible for the treatment of critical limb ischemia and cardiovascular diseases. However, many issues in the field of EPC biology, especially in regards to the proper and unambiguous molecular characterization of these cells still remain unresolved, hampering not only basic research but also the effective therapeutic use and widespread application of these cells. In this review, we introduce the recent concept of EPC identification in terms of hematopoietic and nonhematopoietic EPCs along with the development of EPC biology research. Furthermore, we define the role of circulating EPCs in postnatal neovascularization to illustrate the future direction of EPC therapeutic applications. Next, we review on-going medical applications of EPC for cardiovascular and peripheral vascular diseases, introduce the practical example of therapeutic application of EPCs to patients with ischemic disease, and discuss about the feedback of clinical researches., (Copyright © 2011 AlphaMed Press.)

Although endothelial progenitor cells (EPCs) differentiate from minor populations of stem cells in bone marrow (BM), the differential role of hematopoietic stem cell (HSC) subpopulations in EPC development is largely unclear. Morphological characterization of EPC colonies has revealed that c-kit+/Sca-1+/lineage (Lin)-(KSL) cells mainly develop small EPC-colony forming units (CFUs) not large EPC-CFUs. In contrast, c-kit+/Sca-1-/Lin- (KL) cells develop large EPC-CFUs not small EPC-CFUs. Neither c-kit-/Sca-1+/Lin- (SL) cells nor c-kit-/Sca-1-/Lin- (L) cells develop EPC-CFUs to an appreciable extent. Hindlimb ischemia enhances formation of large EPC-CFUs from all HSC subpopulations, suggesting an important role for ischemia in functional EPC development. Real time RT-PCR analysis shows that KSL, KL and SL cells but not L cells express various factors at high levels, maintaining a BM-EPC pool. In hindlimb ischemia, transplanted KSL, KL and SL cells efficiently differentiate into endothelial lineage cells in situ and augment capillary density. The percentage of Ki-67+ cycling cells among transplanted cells in ischemic tissue was also greater for KSL, KL and SL cells than L cells. Moreover, the frequency of VEGF- or SDF-1-expressing cells was higher transplanted KSL, KL or SL cells than L cells. Thus, KSL, KL and SL cells are not different in their angiogenic competence under ischemic conditions. In conclusion, although KSL cells are clearly the most potent contributors to EPC development, KL and SL cells may also contribute to neovascularization via both autocrine and paracrine mechanisms in response to ischemic signals., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133(+) cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133(+) cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.

Liver fibrosis (LF) caused by chronic liver damage has been considered as an irreversible disease. As alternative therapy for liver transplantation, there are high expectations for regenerative medicine of the liver. Bone marrow (BM)- or peripheral blood-derived stem cells, including endothelial progenitor cells (EPCs), have recently been used to treat liver cirrhosis. We investigated the biology of BM-derived EPC in a mouse model of LF. C57BL/6J mice were subcutaneously injected with carbon tetrachloride (CCl(4)) every 3 days for 90 days. Sacrificed 2 days after final injection, whole blood (WB) was collected for isolation of mononuclear cells (MNCs) and biochemical examination. Assessments of EPC in the peripheral blood and BM were performed by flow cytometry and EPC colony-forming assay, respectively, using purified MNCs and BM c-KIT(+), Sca-1(+), and Lin(-) (KSL) cells. Liver tissues underwent histological analysis with hematoxylin/eosin/Azan staining, and spleens were excised and weighed. CCl(4)-treated mice exhibited histologically bridging fibrosis, pseudolobular formation, and splenomegaly, indicating successful induction of LF. The frequency of definitive EPC-colony-forming-units (CFU) as well as total EPC-CFU at the equivalent cell number of 500 BM-KSL cells decreased significantly (p < 0.0001) in LF mice compared with control mice; no significant changes in primitive EPC-CFU occurred in LF mice. The frequency of WB-MNCs of definitive EPC-CFU decreased significantly (p < 0.01) in LF mice compared with control mice. Together, these findings indicated the existence of impaired EPC function and differentiation in BM-derived EPCs in LF mice and might be related to clinical LF.

Background: Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow., Methodology/principal Findings: CD34(+) cells, c-Kit(+)/Sca-1(+)/Lin(-) (KSL) cells, c-Kit(+)/Lin(-) (KL) cells and Sca-1(+)/Lin(-) (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34(+) cells showed the lowest EPC colony forming activity, CD34(+) cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+) cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+) cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+) cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others., Conclusion: These findings suggest that mouse CD34(+) cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

Development of a bioartificial glomerulus, a hemofilter in which the inner surface of hollow fibers is endothelialized, requires expandable, nonimmunogenic, antithrombogenic, and highly permeable endothelial cells. We used human umbilical cord blood CD133(+) endothelial progenitor cells (EPCs) to evaluate the feasibility of application of EPCs for bioartificial glomerulus. Numbers of adhered CD133(+) EPCs (adhered EPCs) was approximately 25 to 30 times as great in the expansion culture group as in the non-expansion group. Adhered EPCs had endothelial cell features, including the expression of CD31, Kinase domain region, von Willebrand factor, vascular endothelial-cadherin, positive for Ulex europeus agglutinin I staining, and up-take of acetylated low-density lipoprotein. Adhered EPCs secreted 6-keto-prostaglandin F(1alpha) identically to that secreted by human umbilical vein endothelial cells (HUVECs). The cells also expressed messenger RNA for phospholipase A(2), cyclooxygenase (COX)-1, COX-2, prostaglandin I(2) synthase, tissue plasminogen activator, and thrombomodulin (TM). TM protein in adhered EPCs properly activated protein C. Scanning electron microscopy revealed the suppression of platelet adhesion and aggregation on the surface of cell monolayer. Adhered EPCs treated with 50 microg/mL of cytochalasin B induced a larger diameter and a greater number of fenestrae, subsequently producing significantly more ultrafiltration than the non-treated cell. These results suggest that CD133(+) EPCs would potentially be applicable in bioartificial glomerulus.

Despite the fact that endothelial progenitor cells (EPCs) are important for postnatal neovascularization, their origins, differentiation, and modulators are not clear. Here, we demonstrate that Lnk, a negative regulator of hematopoietic stem cell proliferation, controls endothelial commitment of c-kit(+)/Sca-1(+)/Lineage(-) (KSL) subpopulations of bone marrow cells. The results of EPC colony-forming assays reveal that small (primitive) EPC colony formation by CD34(-) KSLs and large (definitive) EPC colony formation by CD34((dim)) KSLs are more robust in lnk(-/-) mice. In hindlimb ischemia, perfusion recovery is augmented in lnk(-/-) mice through enhanced proliferation and mobilization of EPCs via c-Kit/stem cell factor. We found that Lnk-deficient EPCs are more potent actors than resident cells in hindlimb perfusion recovery and ischemic neovascularization, mainly via the activity of bone marrow-EPCs. Similarly, lnk(-/-) mice show augmented retinal neovascularization and astrocyte network maturation without an increase in indicators of pathogenic angiogenesis in an in vivo model of retinopathy. Taken together, our results provide strong evidence that Lnk regulates bone marrow-EPC kinetics in vascular regeneration. Selective targeting of Lnk may be a safe and effective strategy to augment therapeutic neovascularization by EPC transplantation.

Thrombopoietin (TPO), a physiological regulator of megakaryocyte and platelet development, is a multifunctional positive regulator in early hematopoiesis by hematopoietic stem cells. In this study, we investigated the effect of TPO on endothelial progenitor cells (EPCs) for therapeutic vasculogenesis in vitro and in vivo, and the intracellular signaling mechanism exerting the activity of EPCs. 7-day culture-expanded EPCs derived from human peripheral blood mononuclear cells were applied to each assay. Flow cytometry demonstrated the expression of c-Mpl, the receptor of TPO, in cultured EPCs. In vitro experiments revealed enhanced migration and survival of cultured EPCs by TPO. In vivo, TPO was intramuscularly administered into the foci of ischemic hindlimbs in athymic nude mice, immediately followed by intravenous injection of cultured EPCs, to assess the booster effect of TPO on vascular regeneration. At day 4 post-transplantation, transplanted EPCs were 1.7-fold higher in TPO-treated animals compared to control. At day 28, blood perfusion was recovered in the TPO-treated group, accompanied by an increase in microvascular density. The signaling transduction pathway underlying TPO-mediated activities of cultured EPCs was assessed by Western blotting. TPO induced sequential phosphorylations of Akt to p70S6kinase through mTOR. Inhibition of the PI3-kinase/Akt/mTOR/p70S6kinase signaling pathway negated the biological functions of cultured EPCs, either migration (by LY294002 for PI3-kinase and Rapamycin for mTOR) or survival and tubulogenesis (by Rapamycin). These findings provide evidence that TPO possesses booster potential for therapeutic vasculogenesis, by activating the PI3-kinase/Akt/mTOR/p70S6kinase pathway crucial to the biological activities of EPCs.

Background: Despite accumulating evidence that proves the pivotal role of endothelial progenitor cells (EPCs) in ischemic neovascularization, the key signaling cascade that regulates functional EPC kinetics remains unclear., Methods and Results: In this report, we show that inactivation of specific Jagged-1 (Jag-1)-mediated Notch signals leads to inhibition of postnatal vasculogenesis in hindlimb ischemia via impairment of proliferation, survival, differentiation, and mobilization of bone marrow-derived EPCs. Bone marrow-derived EPCs obtained from Jag-1-/- mice, but not Delta-like (Dll)-1-/- mice, demonstrated less therapeutic potential for ischemic neovascularization than EPCs from the wild type. In contrast, a gain-of-function study using 3T3 stromal cells overexpressing Notch ligand revealed that Jag-1-mediated Notch signals promoted EPC commitment, which resulted in enhanced neovascularization. The impaired neovascularization in Jag-1-/- mice was profoundly rescued by transplantation of Jag-1-stimulated EPCs., Conclusions: These data indicate that specific Jag-1-derived Notch signals from the bone marrow microenvironment are critical for EPC-mediated vasculogenesis, thus providing an important clue for modulation of strategies for therapeutic neovascularization.

We have already confirmed that cell sheet transplantation can improve damaged heart function via continuous cytokine secretion. In this study, we hypothesized that cytokine-secreting cell sheets co-cultured with an endothelial cell source may be more effective for repairing ischemic myocardium. Confluent rat fibroblasts cultured on temperature-responsive culture dishes were harvested as contiguous cell sheets by temperature reduction. Green fluorescent protein (GFP)-positive endothelial progenitor cells (EPCs) were seeded on fibroblast sheets to create co-cultured cell sheets, and sandwich-like constructs were engineered by stacking of the co-cultured cell sheets. These constructs were transplanted into rat myocardial infarction models. Cardiac function and histology were assessed in four groups: the sham operation (C) group, the isolated EPC injection (E) group, the transplantation of triple-layer fibroblast sheets (F) group, and the transplantation of triple-layer sandwich-like constructs (E + F) group. Echocardiography showed significant improvement of the fractional shortening in the E + F group in comparison with the C group (0.25 +/- 0.05 vs. 0.16 +/- 0.02). On histological examination, significantly less connective tissue formation was observed in the E, F, and E + F groups when compared to the C group (C, E, F, and E + F groups: 53 +/- 2%, 41 +/- 4%, 40 +/- 4%, and 32 +/- 7%, respectively). Additionally, increased blood vessel formation was detected in the E, F, and E + F groups compared with the C group (C, E, F, and E + F groups: 1.9% +/- 0.6%, 6.7% +/- 0.6%, 7.8% +/- 0.9%, and 10.2% +/- 2.4%, respectively). Furthermore, GFP-staining demonstrated that the newly formed blood vessels were composed of the co-cultured EPCs. Transplantation of cell sheets co-cultured with an endothelial cell source may be a new therapeutic strategy for myocardial tissue regeneration.

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)-derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER alpha as well as downregulated gene expressions of ER beta. Under the physiological concentrations of estrogen (17beta-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing beta-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER alpha, rather than ER beta in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER alpha.

Nicotine has been demonstrated to stimulate postnatal angiogenesis, having an antiapoptotic effect on endothelial cells. Given the extent of this angiogenesis-promoting effect, we hypothesized that nicotine may also stimulate postnatal vasculogenesis on endothelial progenitor cells (EPCs). Our analyses reveal some intriguing results using an in vitro assay with 2 x 10(-6) M of nicotine (smoker's average nicotine concentration and the dose of nicotine replacement therapy). The proliferation and migration activities of human EPCs cultured from peripheral blood mononuclear cells of non-smoking healthy volunteers were not affected by nicotine. The effect of nicotine on EPC survival was significantly enhanced under serum starvation on the ratio of Hoechest 33342-stained pyknotic nuclear cells as well as Annexin-V-stained cells to total cells. Furthermore, the antiapoptotic effect of nicotine was blocked completely by nicotinic acetylcholine receptor (nAChR) antagonist hexamethonium. Next, we verified how nicotine acts in vivo. Nicotine (100 ng/ml) was administered orally for 7 days before and 4 weeks after injection of cultured EPCs (1 x 10(5) /mouse) into the tail veins of 8-week-old athymic nude mice with ischemic hindlimbs. Laser doppler imaging analysis indicated that blood perfusion in the ischemic hindlimb was significantly enhanced in EPCs plus nicotine, as compared with EPCs alone. These findings suggest nicotine improves blood flow following EPC transplantation in patients with ischemic diseases.

Background: Although potential participation of bone marrow-derived circulating endothelial progenitor cells (EPCs) to neoangiogenesis has been proposed, the precise molecular mechanisms of EPC recruitment to vascular endothelium has not been fully elucidated., Methods and Results: Peripheral blood mononuclear cells were isolated from healthy volunteers and cultured for 7 days to obtain EPCs. Tumor necrosis factor-alpha-activated human umbilical vein endothelial cells (HUVECs) supported significantly more rolling and adhesion of EPCs compared with inactivated HUVEC monolayer. Pretreatment of activated HUVEC with an adhesion-blocking mAb to E-selectin significantly reduced EPCs adhesion to HUVECs. When HUVECs were transduced with a recombinant adenovirus of E-selectin (AdRSVE-sel) or that of beta-galactosidase (AdRSVLacZ), E-selectin-transduced but not LacZ-transduced HUVECs exhibited significantly more EPC rolling as well as adhesion. Further, effect of AdRSVE-sel or AdRSVLacZ was examined in mouse hind limb ischemic model. AdRSVE-sel-transduced mice showed significantly less limb necrosis and higher laser Doppler ratio when compared with AdRSVLacZ-transduced mice. Interestingly, blood flow recovery of ischemic limb observed in AdRSVE-sel-transduced mice was more prominent when combined with EPC administration compared with that of AdRSVLacZ-transduced mice., Conclusions: Endothelial E-selectin plays a crucial role in EPC-endothelial interaction in vitro. The importance of E-selectin was also confirmed in vivo even in the absence of exogenous EPC. These data provide molecular background for novel cell-based therapy for ischemic atherosclerosis.

Background: Recent data have indicated that estradiol can modulate the kinetics of endothelial progenitor cells (EPCs) via endothelial nitric oxide synthase (eNOS)-dependent mechanisms. We hypothesized that estradiol could augment the incorporation of bone marrow (BM)-derived EPCs into sites of ischemia-induced neovascularization, resulting in protection from ischemic injury., Methods and Results: Myocardial infarction (MI) was induced by ligation of the left coronary artery in ovariectomized mice receiving either 17beta-estradiol or placebo. Estradiol induced significant increases in circulating EPCs 2 and 3 weeks after MI in estradiol-treated animals, and capillary density was significantly greater in estradiol-treated animals. Greater numbers of BM-derived EPCs were observed at ischemic sites in estradiol-treated animals than in placebo-treated animals 1 and 4 weeks after MI. In eNOS-null mice, the effect of estradiol on mobilization of EPCs was lost, as was the functional improvement in recovery from acute myocardial ischemia. A decrease was found in matrix metalloproteinase-9 (MMP-9) expression in eNOS-null mice under basal and estradiol-stimulated conditions after MI, the mobilization of EPCs by estradiol was lost in MMP-9-null mice, and the functional benefit conferred by estradiol treatment after MI in wild-type mice was significantly attenuated., Conclusions: Estradiol preserves the integrity of ischemic tissue by augmenting the mobilization and incorporation of BM-derived EPCs into sites of neovascularization by eNOS-mediated augmentation of MMP-9 expression in the BM. Moreover, these data have broader implications with regard to our understanding of the role of EPCs in post-MI recovery and on the sex discrepancy in cardiac events.

Metabolic syndrome (MS), overlapping type 2 diabetes, hyperlipidemia, and/or hypertension, owing to high-fat diet, poses risk for cardiovascular disease. A critical feature associated with such risk is the functional impairment of endothelial progenitor cells (EPCs). Dipeptidyl dipeptidase-4 inhibitors (DPP-4 i) not only inhibit degradation of incretins to control blood glucose levels, but also improve EPC bioactivity and induce anti-inflammatory effects in tissues. In the present study, we investigated the effects of such an inhibitor, MK-06266, in an ischemia model of MS using diet-induced obese (DIO) mice. EPC bioactivity was examined in MK-0626-administered DIO mice and a non-treated control group, using an EPC colony-forming assay and bone marrow cKit+ Sca-1+ lineage-cells, and peripheral blood-mononuclear cells. Our results showed that, in vitro, the effect of MK-0626 treatment on EPC bioactivities and differentiation was superior compared to the control. Furthermore, microvascular density and pericyte-recruited arteriole number increased in MK-0626-administered mice, but not in the control group. Lineage profiling of isolated cells from ischemic tissues revealed that MK-0626 administration has an inhibitory effect on unproductive inflammation. This occurred via a decrease in the influx of total blood cells and pro-inflammatory cells such as neutrophils, total macrophages, M1, total T-cells, cytotoxic T-cells, and B-cells, with a concomitant increase in number of regeneration-associated cells, such as M2/M ratio and Treg/T-helper. Laser Doppler analysis revealed that at day 14 after ischemic injury, blood perfusion in hindlimb was greater in MK-0626-treated DIO mice, but not in control. In conclusion, the DPP-4 i had a positive effect on EPC differentiation in MS model of DIO mice. Following ischemic injury, DPP-4 i sharply reduced recruitment of pro-inflammatory cells into ischemic tissue and triggered regeneration and reparation, making it a promising therapeutic agent for MS treatment. [ABSTRACT FROM AUTHOR]

Notch signaling is involved in cell fate decisions during murine vascular development and hematopoiesis in the microenvironment of bone marrow. To investigate the close relationship between hematopoietic stem cells and human endothelial progenitor cells (EPCs) in the bone marrow niche, we examined the effects of Notch signals [Jagged-1 and Delta-like ligand (Dll)-1] on the proliferation and differentiation of human CD133+ cell-derived EPCs. We established stromal systems using HESS-5 murine bone marrow cells transfected with human Jagged-1 (hJagged-1) or human Dll-1 (hDll-1). CD133+ cord blood cells were co-cultured with the stromal cells for 7 days, and then their proliferation, differentiation, and EPC colony formation was evaluated. We found that hJagged-1 induced the proliferation and differentiation of CD133+ cord blood EPCs. In contrast, hDll-1 had little effect. CD133+ cells stimulated by hJagged-1 differentiated into CD31+/KDR+ cells, expressed vascular endothelial growth factor-A, and showed enhanced EPC colony formation compared with CD133+ cells stimulated by hDll-1. To evaluate the angiogenic properties of hJagged-1- and hDll-1-stimulated EPCs in vivo, we transplanted these cells into the ischemic hindlimbs of nude mice. Transplantation of EPCs stimulated by hJagged-1, but not hDll-1, increased regional blood flow and capillary density in ischemic hindlimb muscles. This is the first study to show that human Notch signaling influences EPC proliferation and differentiation in the bone marrow microenvironment. Human Jagged-1 induced the proliferation and differentiation of CD133+ cord blood progenitors compared with hDll-1. Thus, hJagged-1 signaling in the bone marrow niche may be used to expand EPCs for therapeutic angiogenesis. [ABSTRACT FROM AUTHOR]