Embryonic Endothelial Progenitor Cells Research Articles

Clonal and Scalable Endothelial Progenitor Cell Lines from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can be used as a renewable source of endothelial cells for treating cardiovascular disease and other ischemic conditions. Here, we present the derivation and characterization of a panel of distinct clonal embryonic endothelial progenitor cells (eEPCs) lines that were differentiated from human embryonic stem cells (hESCs). The hESC line, ESI-017, was first partially differentiated to produce candidate cultures from which eEPCs were cloned. Endothelial cell identity was assessed by transcriptomic analysis, cell surface marker expression, immunocytochemical marker analysis, and functional analysis of cells and exosomes using vascular network forming assays. The transcriptome of the eEPC lines was compared to various adult endothelial lines as well as various non-endothelial cells including both adult and embryonic origins. This resulted in a variety of distinct cell lines with functional properties of endothelial cells and strong transcriptomic similarity to adult endothelial primary cell lines. The eEPC lines, however, were distinguished from adult endothelium by their novel pattern of embryonic gene expression. We demonstrated eEPC line scalability of up to 80 population doublings (pd) and stable long-term expansion of over 50 pd with stable angiogenic properties at late passage. Taken together, these data support the finding that hESC-derived clonal eEPC lines are a potential source of scalable therapeutic cells and cell products for treating cardiovascular disease. These eEPC lines offer a highly promising resource for the development of further preclinical studies aimed at therapeutic interventions.

Predicting in vivo therapeutic efficacy of bioorthogonally labeled endothelial progenitor cells in hind limb ischemia models via non-invasive fluorescence molecular tomography

Human embryonic stem cells-derived endothelial progenitor cells (hEPCs) were utilized as cell therapeutics for the treatment of ischemic diseases. However, in vivo tracking of hEPCs for predicting their therapeutic efficacy is very difficult. Herein, we developed bioorthogonal labeling strategy of hEPCs that could non-invasively track them after transplantation in hind limb ischemia models. First, hEPCs were treated with tetraacylated N-azidomannosamine (Ac4ManNAz) for generating unnatural azide groups on the hEPCs surface. Second, near-infrared fluorescence (NIRF) dye, Cy5, conjugated dibenzocylooctyne (DBCO-Cy5) was chemically conjugated to the azide groups on the hEPC surface via copper-free click chemistry, resulting Cy5-hEPCs. The bioorthogonally labeled Cy5-hEPCs showed strong NIRF signal without cytotoxicity and functional perturbation in tubular formation, oxygen consumption and paracrine effect of hEPCs in vitro. In hind limb ischemia models, the distribution and migration of transplanted Cy5-hEPCs were successfully monitored via fluorescence molecular tomography (FMT) for 28 days. Notably, blood reperfusion and therapeutic neovascularization effects were significantly correlated with the initial transplantation forms of Cy5-hEPCs such as ‘condensed round shape’ and ‘spread shape’ in the ischemic lesion. The condensed transplanted Cy5-hEPCs substantially increased the therapeutic efficacy of hind limb ischemia, compared to that of spread Cy5-hEPCs. Therefore, our new stem cell labeling strategy can be used to predict therapeutic efficacy in hind limb ischemia and it can be applied a potential application in developing cell therapeutics for regenerative medicine.

Pancreatic acinar differentiation is guided by differential laminin deposition

Endothelial cells play multiple roles during pancreas organogenesis. First, they are required to instruct endoderm-derived pancreatic progenitor cells to initiate branching morphogenesis. Later, blood vessels promote β-cell differentiation but also limit acinar development. In this work, we show how endothelial cells might signal to pancreatic progenitors and spatially regulate acinar differentiation. Using an ex vivo culture system of undifferentiated E12.5 pancreata, we demonstrate that embryonic endothelial progenitor cells and their conditioned medium prevent the expression of two members of the pro-acinar transcriptional PTF1L-complex. This effect is not mediated by SPARC, a protein abundantly released in the medium conditioned by endothelial progenitors. On the contrary, heterotrimeric laminin-α1β1γ1, also produced by endothelial progenitor cells, can repress acinar differentiation when used on its own on pancreatic explants. Lastly, we found that laminin-α1 is predominantly found in vivo around the pancreatic trunk cells, as compared to the tip cells, at E14.5. In conclusion, we propose that expression or deposition of laminin-α1β1γ1 around the trunk cells, where blood vessels are predominantly localized, prevent acinar differentiation of these cells. On the contrary, transient decreased expression or deposition of laminin-α1β1γ1 around the tip cells would allow PTF1L-complex formation and acinar differentiation.

Embryonic stem cell-derived endothelial progenitor cells reduce recurrent miscarriage in a mouse miscarriage model

Embryonic stem cell-derived endothelial progenitor cells reduce recurrent miscarriage in a mouse miscarriage model

Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, β1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane.

Neutrophil Elastase-Generated Fragment of Vascular Endothelial Growth Factor-A Stimulates Macrophage and Endothelial Progenitor Cell Migration.

Elastase released from neutrophils as part of the innate immune system has been implicated in chronic diseases such as emphysema and cardiovascular disease. We have previously shown that neutrophil elastase targets vascular endothelial growth factor-A (VEGF) for partial degradation to generate a fragment of VEGF (VEGFf) that has distinct activities. Namely, VEGFf binds to VEGF receptor 1 but not to VEGF receptor 2 and shows altered signaling compared to intact VEGF. In the present study we investigated the chemotactic function of VEGF and VEGFf released from cells by neutrophil elastase. We found that endothelial cells migrated in response to intact VEGF but not VEGFf whereas RAW 264.7 macrophages/monocytes and embryonic endothelial progenitor cells were stimulated to migrate by either VEGF or VEGFf. To investigate the role of elastase-mediated release of VEGF from cells/extracellular matrices, a co-culture system was established. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with neutrophil elastase. Elastase treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, elastase treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment.

Morphogenesis of the lymphatic vasculature: A focus on new progenitors and cellular mechanisms important for constructing lymphatic vessels.

Lymphatic vessels serve crucial roles in the regulation of tissue fluid homeostasis, dietary lipid absorption and immune cell trafficking. Defects in lymphatic vessel morphogenesis and function have been associated with lymphedema, obesity, hypertension and tumour metastasis. Morphogenetic events important for construction of the lymphatic vasculature during development include the specification and emergence of lymphatic endothelial progenitor cells, their differentiation and assembly into interconnected vessels and vascular remodeling, ultimately giving rise to a functional vascular network. Despite the embryonic origins of lymphatic endothelial progenitor cells being long debated, work performed over the last decade had overwhelmingly supported at least a great majority of progenitor cells arising from the venous vasculature. Here, we review the most recent advances in the field of lymphatic vessel morphogenesis, with a focus on studies that have identified novel sources of embryonic lymphatic endothelial progenitor cells, together with the cellular mechanisms by which lymphatic vessels are initially assembled.

The Developing Role of Neuregulin1 In Cardiac Regenerative Stem Cell Therapy

Myocardial infarction, heart failure, and chronic ischemic heart disease account for the majority of the cardiovascular burden. The current treatment strategies focus on limiting the progression of disease and preserving cardiac myocardium. The goal of stem cell therapy, on the other hand, is to reverse or replace damaged cardiac tissue. Over the past two decades many studies have been conducted to understand stem cell performance, survival, and the potential for cardiac repair. Neuregulin1, an epidermal growth factor family member, promotes embryonic stem cell differentiation into the cardiac lineage and improves survival in bone marrow-derived mesenchymal stem cell and embryonic endothelial progenitor cells. Current clinical trials are actively pursuing Neuregulin1's therapeutic potential in the areas of heart failure and cardiac ischemia. It is the intent of this paper to review the current knowledge of Neuregulin1 in stem cell biology and discuss the potential of using Neuregulin1 to improve stem cell therapy for cardiac repair.

Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.

A comparison of human cord blood- and embryonic stem cell-derived endothelial progenitor cells in the treatment of chronic wounds

Endothelial progenitor cells (EPCs) promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. Therefore, EPCs have been highlighted as an alternative cell source for wound healing. EPCs can be isolated from various sources, including the bone marrow, cord blood, and adipose tissue. However, several recent studies have reported that isolating EPCs from these sources has limitations, such as the isolation of insufficient cell numbers and the difficulty of expanding these cells in culture. Thus, human embryonic stem cells (hESCs) have generated great interest as an alternative source of EPCs. Previously, we established an efficient preparation method to obtain EPCs from hESCs (hESC-EPCs). These hESC-EPCs secreted growth factors and cytokines, which are known to be important in angiogenesis and wound healing. In this study, we directly compared the capacity of hESC-EPCs and human cord blood-derived EPCs (hCB-EPCs) to benefit wound healing. The number of hESC-EPCs increased during culture and was always higher than the number of hCB-EPCs during the culture period. In addition, the levels of VEGF and Ang-1 secreted by hESC-EPCs were significantly higher than those produced by hCB-EPCs. After transplantation in a mouse dermal excisional wound model, all EPC-transplanted wounds exhibited better regeneration than in the control group. More importantly, we found that the wounds transplanted with hESC-EPCs showed significantly accelerated re-epithelialization. Thus, hESC-EPCs may be a promising cell source for the treatment of chronic wounds.

Ex Vivo Reconstitution of Arterial Endothelium by Embryonic Stem Cell-Derived Endothelial Progenitor Cells in Baboons

There is an increasing need for an animal model that can be used to translate basic research into clinical therapy. We documented the differentiation and functional competence of embryonic stem cell (ESC)-derived endothelial cells in baboons. Baboon angioblasts were sequentially differentiated from embryoid body cultures for 9 days in an angioblast differentiation medium with varying concentrations of BMP-4, FLT-3 ligand, stem cell factor, thrombopoietin, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and knockout serum replacement. Real-time polymerase chain reaction results showed that ESC-derived angioblasts downregulated NANOG and OCT3/4, upregulated T-brachyury and GATA2, and moderately expressed CD34; they did not express CD144, TEK, or VWF, and varied in levels of CD31 expression. Several populations of putative angioblasts appeared 3 days and 9 days after differentiation, as identified by flow cytometry. Angioblasts at this stage exhibited dual paths of differentiation toward hematopoietic and vascular fates. To examine whether derived angioblasts could reconstitute the endothelium, we built an ex vivo culture system and seeded fluorescently labeled angioblast cultures onto a denuded segment of the femoral artery. We found that the seeded cells were able to grow into the endothelium on the interior surface of denuded artery segments within 5 days after seeding. After 14 days of ex vivo culture, the transplanted cells expressed CD31, an endothelial marker. The control arteries, seeded with vehicle only, did not harbor cells with endothelial markers. We conclude that ESC-derived angioblasts are promising therapeutic agents for repairing damaged vasculature, and that the baboon model will be vital for optimizing therapies for human clinical studies.

Endothelial progenitors encapsulated in bioartificial niches are insulated from systemic cytotoxicity and are angiogenesis competent

Intrinsic stem cells (SC) participate in tissue remodeling and regeneration in various diseases and following toxic insults. Failure of tissue regeneration is in part attributed to lack of SC protection from toxic stress of noxious stimuli, thus prompting intense research efforts to develop strategies for SC protection and functional preservation for in vivo delivery. One strategy is creation of artificial SC niches in an attempt to mimic the requirements of endogenous SC niches by generating scaffolds with properties of extracellular matrix. Here, we investigated the use of hyaluronic acid (HA) hydrogels as an artificial SC niche and examined regenerative capabilities of encapsulated embryonic endothelial progenitor cells (eEPC) in three different in vivo models. Hydrogel-encapsulated eEPC demonstrated improved resistance to toxic insult (adriamycin) in vitro, thus prompting in vivo studies. Implantation of HA hydrogels containing eEPC to mice with adriamycin nephropathy or renal ischemia resulted in eEPC mobilization to injured kidneys (and to a lesser extent to the spleen) and improvement of renal function, which was equal or superior to adoptively transferred EPC by intravenous infusion. In mice with hindlimb ischemia, EPC encapsulated in HA hydrogels dramatically accelerated the recovery of collateral circulation with the efficacy superior to intravenous infusion of EPC. In conclusion, HA hydrogels protect eEPC against adriamycin cytotoxicity and implantation of eEPC encapsulated in HA hydrogels supports renal regeneration in ischemic and cytotoxic (adriamycin) nephropathy and neovascularization of ischemic hindlimb, thus establishing their functional competence and superior capabilities to deliver stem cells stored in and released from this bioartificial niche.

NF κB Activation in Embryonic Endothelial Progenitor Cells Enhances Neovascularization via PSGL-1 Mediated Recruitment: Novel Role for LL37

Embryonal endothelial progenitor cells (eEPCs) are capable of inducing therapeutic angiogenesis in a chronic hind limb model. However, the proportion of eEPCs recruited to the ischemic tissue appears to be a limiting step for the induction of cell-based therapeutic neovascularization. In the present study, we primed eEPCs with the human cathelicidin LL37 (hCAP-18) ex vivo to selectively enhance the eEPC-dependent gain of perfusion in vivo and elucidated the mechanism of action of LL37 on eEPCs. Seven days after femoral artery excision, 5 x 10(6) eEPCs (wt, wild type; p65t, transiently p65 transient; p65s, stable p65-transfected; LL37-eEPCs, LL37 peptide preincubated) were retroinfused into the anterior tibial vein. Recruitment of diI-labeled eEPCs in the ischemic gastrocnemic muscle was investigated 2 days later, whereas collateral growth and perfusion score (obtained by fluorescent microspheres) were assessed at day 7 and day 35 and are given as percentage of day 7 level. Capillary/muscle fiber ratio in the ischemic lower limb was obtained at day 35. Embryonic EPC recruitment in vitro and in vivo was found elevated after LL37 and p65t pretreatment, but not in p65s-eEPCs displaying increased IkappaBalpha or after LL37 in IkappaB-DN overexpressing eEPCs. Using LL37- and p65t-eEPCs, collateral growth (181 +/- 10% and 165 +/- 8%, respectively) surpassed that of wt-eEPCs (135 +/- 7%), increasing perfusion ratio (208 +/- 20% and 210 +/- 17% vs. 142 +/- 12% in wt-eEPCs, respectively), whereas p65s-eEPCs exerted no additive effect (collateral growth 130 +/- 8%; perfusion ratio 155 +/- 15%). Moreover, p65t-eEPC-induced neovascularization was abrogated by blocking antibodies against E-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). We conclude that NF kappaB activation by LL37 or transient p65-transfection increases functionally relevant eEPC recruitment to ischemic muscle tissue via induction of PSGL-1 and E-selectin.

Bone marrow-derived angiogenic cells restore lung alveolar and vascular structure after neonatal hyperoxia in infant mice

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury, we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)), embryonic endothelial progenitor cells, mouse embryonic fibroblasts (control), or saline were then injected into the pulmonary circulation. At 21 days of age, saline-treated mice had enlarged alveoli, reduced septation, and a reduction in vascular density. In contrast, mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia.

Activated Local Immunity by CC Chemokine Ligand 19-Transduced Embryonic Endothelial Progenitor Cells Suppresses Metastasis of Murine Ovarian Cancer

Although tumor microenvironments play a key role in successful tumor immunotherapy, effective manipulation of local immunity is difficult because of the lack of an appropriate target system. It is well known that bone marrow-derived endothelial progenitor cells (EPCs) are actively recruited during tumor angiogenesis. Using this feature, we attempted to establish a novel therapeutic modality that targets tumor vessels of multiple metastases using embryonic endothelial progenitor cells (eEPCs) transduced with an immune-activating gene. The eEPCs were retrovirally transduced with the mouse CC chemokine ligand 19 (CCL19) gene, a lymphocyte-migrating chemokine. The mouse ovarian cancer cell line OV2944-HM-1 (HM-1) was inoculated subcutaneously into B6C3F1 mice, along with CCL19-tranduced eEPCs (eEPC-CCL19), resulting in immunologic activity and tumor-inhibitory effects. In this model, eEPC-CCL19 showed tumor repression accompanied by increased tumor-infiltrating CD8+ lymphocytes compared with the control group. In contrast, no tumor repression was observed when the same experiment was done in immunodeficient (SCID) mice, suggesting a crucial role of T-cell function in this system. Next, we established a lung metastasis model by injecting HM-1 cells or B16 melanoma cells via the tail vein. Subsequent intravenous injection of eEPC-CCL19 leads to a decrease in the number of lung metastasis and prolonged survival. Antitumor effects were also observed in a peritoneal dissemination model using HM-1. These results suggest that systemic delivery of an immune-activating signal using EPCs can alter the tumor immune microenvironment and lead to a therapeutic effect, which may provide a novel strategy for targeting multiple metastases of various malignancies.

Abstract 3955: Chemokines Fused to a Fractalkine Backbone and a GPI-Anchor Attract Embryonic Endothelial Progenitor Cells to the Site of Ischemia

Recruitment of endothelial progenitor cells to the sites of ischemia is a prerequisite for efficient therapeutic neovascularization via vasculogenesis. Chemokines play a major role in the homing of EPCs at the ischemic vasculature, a mechanism fading in chronic ischemia. To overcome this limitation, we constructed an artificial adhesion molecule consisting of a GPI-anchor, a fractalkine-backbone and an SDF-1 head (SDF-1-fra-GPI), which was applied for enhanced recruitment of embryonic EPCs (eEPCs: CXCR4++, Tie2++, Thrombomodulin++, CD34-, MHCI-, vWF inducible, eNOS inducible) in vitro and in vivo . Methods: In a flow chamber adhesion assay, Control plasmids (pcDNA or GPI-SDF-1 cDNA) were compared to the SDF-1-fra-GPI construct for eEPC recruitment 24h after liposomal transfection of rat endothelial cells. In vivo, in rabbits (n=5 per group) at day 7 (d7) after femoral artery excision, 1 mg of the SDF-1-fra-GPI or eGFP cDNA was transfected into the ischemic limb. At d9, ischemic hindlimbs were retroinfused with 5x10 6 eEPCs. Angiography was performed for collateral quantification and frame count score at d9 and d37 (% of d9), capillary density was assessed via PECAM-1-staining (capillaries/muscle fiber = c/mf). Results: In vitro, eEPC adhesion (16±12 cells/field) was increased to a higher extent by SDF-1-fra-GPI (79±13) than SDF1-GPI (54±8) or control vector (37±8). In vivo , eEPC adhesion in the ischemic hindlimb after SDF-1-fra-GPI transfection compared to mock transfection (30±3 vs. 9±1 cells/field). Whereas capillary density was unaffected (1.66±0.30 SDF-1-Fra-GPI vs. 1.56±0.29 eEPCs), collateral growth (152±10% SDF-1-fra-GPI vs. 124±13%) as well as perfusion score (198±17% SDF-1-fra-GPI vs.160±6% eEPCs) further increased after SDF-1-fra-GPI transfection (controls: 1.24±0.12 c/mf, collaterals 105±8%, perfusion score 112±11%). We conclude that recruitment of EPCs expressing CXCR4 (the SDF-1 receptor) may benefit from pre-treatment of the recipient vasculature with SDF-1-Fra-GPI, an artificial adhesion molecule. This approach might be valuable for enhancing EPC recruitment in the scenario of therapeutic neovascular-ization of chronic ischemic syndromes.

Biological Surgery

See related article, pages 751–760 Despite the many therapeutic breakthroughs in cardiovascular science, ischemic coronary and peripheral vascular diseases are still a major health problem throughout the world, and many patients do not respond or are not candidates for available medical and/or surgical treatments. Recently, identification of adult progenitor cells capable of contributing to tissue regeneration has raised the possibility that cell-based therapy could be used for the repair of ischemic heart or limb. There is compelling evidence that circulating endothelial progenitor cells (EPCs) play a role in tissue repair after a myocardial infarction, and higher levels of circulating EPCs are predictive of better short and long-term outcomes.1 EPCs were initially described as a subset of CD34+ hematopoietic stem cells that coexpress vascular endothelial growth factor receptor-2 (VEGFR-2), also known as Flk-1 or KDR. VEGFR-2–positive cells derived from embryonic stem cells can differentiate into both endothelial cells and smooth muscle cells, reproducing the vascular organization process.2 Smooth muscle progenitor cells (SMPCs) were identified more recently and reported to be present in bone marrow, circulating blood, vascular adventitia, and other tissues.3,4 Both EPCs and SMPCs can be mobilized by stromal cell–derived factor-1, a chemokine whose expression increases in response to tissue hypoxia based on regulation by the transcription factor, hypoxia-inducible factor-1,5 suggesting that EPCs and SMPCs may play pivotal roles in repairing injury to the vascular network. In addition, several cell types, including bone marrow cells, embryonic EPCs, and mesenchymal stem cells, have been tested in vivo for their effect on restoring blood supply and/or muscle function in ischemic heart and limbs. The …

Thymosin β4 Is an Essential Paracrine Factor of Embryonic Endothelial Progenitor Cell–Mediated Cardioprotection

Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin beta4 (Tbeta4) as a mediator of eEPC-mediated cardioprotection. In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tbeta4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5x10(6) cells) or cells transfected with Tbeta4 shRNA when indicated or 15 mg Tbeta4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32+/-4% to 90+/-2% after eEPC application, an effect sensitive to shRNA transfection compared with Tbeta4 (45+/-7%). In vivo, infarct size decreased with eEPC application (38+/-4% versus 54+/-4% of area at risk; P<0.01), an effect abolished by Tbeta4 shRNA (62+/-3%). Segmental subendocardial shortening improved after eEPC treatment (22+/-3% versus -3+/-4% of control area) unless Tbeta4 shRNA was transfected (-6+/-4%). Retroinfusion of Tbeta4 mimicked eEPC application (infarct size, 37+/-3%; segmental endocardial shortening, 34+/-7%). Myeloperoxidase activity (3323+/-388 U/mg in controls) was decreased by eEPCs (1996+/-546 U/mg) or Tbeta4 alone (1455+/-197 U/mg) but not Tbeta4 shRNA-treated eEPCs (5449+/-829 U/mg). Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to Tbeta4.

Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation

Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.

1050. Mouse Embryonic Endothelial Progenitor Cells Home to Atherosclerotic Lesions after Intravenous Delivery

The integrity of the endothelial monolayer plays a critical role in preventing the pathogenesis of atherosclerosis. This study was designed to investigate whether mouse embryonic endothelial progenitor cells (eEPCs) participate in endothelial repair and limit atherosclerotic lesion formation. We isolated mesodermal cells from C57BL/6 embryos at embryonic day 7.5, the onset of vasculogenesis. These isolated eEPCs displayed unlimited stem-cell-like growth and a stable phenotype in culture. RNA analysis showed that these cells express high levels of the stem cell markers cKit, Sca-1, and Flk-1, as well as the endothelial cell-specific genes Tie2, vWF, and ICAM-2. These cells also expressed high levels of the angiogenic factors VEGF- alpha and TGF-beta. eEPCs formed tube like structures when cultured in Matrigel with endothelial cell growth supplement. Furthermore, in vitro differentiation of eEPCs by retinoic acid and c-AMP activated a strong expression (>10-fold) of PSGL-1 (P- selectin glycoprotein ligand 1), which might play a significant role in homing of eEPCs in situ. We transduced eEPCs with AAV2/8-LacZ and injected 1 × 106 cells I.V. into genetically defined atherosclerosis susceptible mice (LDb; Ldlr-/-Apobec1-/-, n=3). eEPCs were detected in the aortic arch of the aorta where severe atherosclerotic lesions were present. This result indicates that eEPCs can home to the atherosclerotic lesion in vivo and may provide potential ways to interfere with lesion development.