Early Endothelial Cells Research Articles

Fibrate treatment of eEOCs in murine AKI

Early endothelial outgrowth cells (eEOCs) protect mice from acute kidney injury (AKI). Peroxisome proliferator-activated receptor-alpha (PPAR-α) has been shown to mediate renoprotective effects under different experimental conditions. The aim of the study was to investigate consequences of fibrate treatment of murine eEOCs in a cell-based therapeutic approach to AKI. Male C57/Bl6N mice, subjected to unilateral renal ischemia (40min) post-uninephrectomy, were systemically injected with 0.5×10(6) untreated or fenofibrate (FF 1, 5, 10 or 50μm)/clofibrate (CF 1mm) pretreated syngeneic murine eEOCs. Renal function and morphology were analyzed 48h later. Cellular consequences of eEOC treatment with fibrates (FF 1, 5, 10, 50μm, CF 1mm) were evaluated using different in vitro assays (direct cell migration, apoptosis/necrosis, ELISA studies). Administration of untreated eEOCs did not protect mice from AKI. Injection of eEOCs treated with CF (1mm) or FF 50μm did not result in any protection from ischemia-induced renal dysfunction. In vitro analysis showed reduced cellular secretion of vasoprotective vascular endothelial growth factor (VEGF), an effect that was more pronounced with CF; FF increased percentages of apoptotic/necrotic eEOCs, and both substances failed to stimulate migration of cultured cells. With lower FF concentrations (1, 5, 10μm) cell survival was increased and 10μm FF stimulated VEGF secretion. In vivo administration of FF-treated eEOCs (10μm) also did not result in any renoprotective effect. PPAR-α activation using fibrates does not stimulate renoprotective effects of syngeneic murine eEOCs in ischemic AKI, although lower fibrate concentrations significantly activate eEOCs in vitro.

Abstract 18031: Identification of Novel Determinants Towards Endothelial Differentiation Using a Heterokaryon Approach

For directed differentiation of embryonic stem cells (ESCs) to endothelial cells (ECs), we need more information regarding the genes that are activated or suppressed, and their temporal order of activation, during EC specification. Accordingly, we sought to use heterokaryons (the stable fusion product of two different cell types) to identify critical determinants of EC differentiation. The premise is that cytoplasmic factors in the EC that maintain its phenotype will act on the ESC nucleus in the heterokaryon to reprogram it toward EC lineage. Using murine specific probes, or RNAseq, we will identify key genes that are modulated during early EC determination. Accordingly, we employed murine ESC labelled by transduction with retroviruses encoding GFP, and human ECs labelled with CellTracker Red, and induced heterokaryon formation using sendai virus-enveloped protein. Multi-nucleated heterokaryons were generated, which contained punctate murine nuclei and diffusely staining human nuclei. The dual-stained heterokaryons were sorted from the unfused cells and homokaryons by FACS. The extent and timing of differentiation of the murine ESCs within the heterokaryons were examined by probing for EC specific genes (KDR, CD144, vWF and CD31) and ES pluripotency markers (Nanog, Sox2 and Oct4) using murine specific primers. Intriguingly, heterokaryons appear to recapitulate ontogeny, in that early mesodermal genes (eg. KDR) are upregulated in murine ESC at 6h, whereas more EC-specific genes are not upregulated until later (eg. vWF at 24h). Data from RNAseq studies further confirmed the major transcriptional factors and regulators and identified some novel factors for endothelial differentiation. These novel genes will be confirmed using gain- and loss- of function studies of ESCs undergoing differentiation. These studies shall provide new knowledge regarding the hierarchy of genes regulating differentiation to the EC lineage and thus provide insight into vascular development, and ultimately new therapeutic avenues for vascular regeneration.

Comparison of the biological characteristics of early and late endothelial progenitor cells derived from mouse bone marrow

Objective To observe the biological characteristics and precise role in angiogenesis of endothelial progenitor cells (EPCs).Methods Bone marrow-derived mononuclear cells were isolated and cultured.EPCs were identified by using immunohistochemistry,immunofluorescence and confocal microscopy.Using methyl thiazol tetrazolium(MTT) method,three-dimensional culture and flow cytometry,the characteristics of early and late EPCs were compared.The features of EPCs were observed by transmission electron microscopy.Results EPCs expressed cell surface antigen positive markers.After two weeks the late EPCs had greater proliferation,achieving 1 × 1011/L,and could form vascular-like structure.Leukocyte differentiation antigen 133 (CD133) expression of the early EPCs was up to (88.7 ± 2.4)％,then late EPCs was gradually decreased to (46.4 ± 3.6) ％.But vascular endothelial growth factor receptor-2 (VEGFR-2) expression was increased from (55.3 ±2.5)％ up to (81.1 ± 1.6)％,and there was significant difference between early EPCs and late EPCs (P ＜ 0.05).The late EPCs could be seen in the endothelial cells characteristic cell-Weible-Palade (WP) bodies.Conclusion There are different qualities of early EPCs and late EPCs in the bone marrow.They show some of the same phenotypes but there are also differences.The late EPCs may be an attractive candidate cell for angiogenesis. Key words: Bone marrow; Endothelial progenitor cells ; Endothelial cells ; Angiogenesis

Angiopoietin-1 treated early endothelial outgrowth cells (eEOCs) are activated in vitro and reduce renal damage in murine acute ischemic kidney injury (iAKI)

BackgroundAcute kidney injury (AKI) severely worsens prognosis of hospitalized patients. Early Endothelial Outgrowth Cells act protective in murine acute ischemic renal failure and renoprotective actions of eEOCs have been documented to increase after cell pretreatment with 8-O-cAMP and Melatonin. Angiopoietin-1 is critically involved in maintaining vascular integrity and regeneration. Aim of the study was to analyze the consequences of eEOC treatment with Ang-1 in murine AKI.MethodsAfter 40 minutes of unilateral renal artery clamping with contralateral nephrectomy, male C57/Bl6N mice were injected with either untreated or pretreated (Ang-1) syngeneic murine eEOCs. Two days later serum creatinine levels and morphology were evaluated. Cultured, Ang-1 treated murine eEOCs were analyzed for production/release of proangiogenic and proinflammatory mediators, migratory activity, and cell survival, respectively.ResultsAngiopoietin-1 pretreatment of eEOCs significantly reduced serum creatinine in cell-injected mice. In vitro analysis showed increased migration of Ang-1 treated eEOCs and supernatant from Ang-1 treated eEOCs stimulated migration of cultured mature endothelial cells. In addition, Ang-1 reduced percentages of Annexin V+/PI+ eEOCs. Intrarenal numbers of eEOCs remained unaffected by Ang-1 and eEOCs did not produce more or less proangiogenic/proinflammatory mediators after being stimulated with Ang-1.ConclusionsAngiopoietin-1 pretreatment of eEOCs increases the cells’ renoprotective competence in ischemic AKI. Thus, the armentarium of eEOC agonists in AKI is increasingly being expanded and the treatment of AKI with eEOCs becomes a promising future option.

Laminar shear stress upregulates endothelial Ca2+-activated K+channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade

In endothelial cells (ECs), Ca²⁺-activated K⁺ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca²⁺ levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca²⁺/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca²⁺/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm²) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8-30), whereas oscillatory shear (OS; ± 5 dyn/cm² × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

Bone morphogenetic protein-5 and early endothelial outgrowth cells (eEOCs) in acute ischemic kidney injury (AKI) and 5/6-chronic kidney disease

Early endothelial outgrowth cells (eEOCs) reproducibly have been shown to act protectively in acute ischemic kidney injury (AKI) and chronic kidney injury. Bone morphogenetic protein-5 (BMP-5) acted antifibrotically in human hypertensive nephropathy. The aim of the current study was to analyze effects of BMP-5 treatment in an eEOC-based therapy of murine AKI and 5/6-nephrectomy. Male C57/Bl6N mice were either subjected to unilateral renal artery clamping postuninephrectomy or to 5/6-nephrectomy. Untreated or BMP-5-pretreated murine eEOCs were injected into recipient animals at the time of reperfusion (AKI) or at 2 and 5 days after 5/6-nephrectomy. Analysis of renal function and morphology was performed at 48 h and at 6 wk (AKI) or at 8 wk (5/6 model). Cellular consequences of eEOC treatment were evaluated using different in vitro assays. AKI was mitigated significantly by injecting BMP-5-pretreated eEOCs. Renal function was improved at 48 h [corrected] after cell therapy. In 5/6-nephrectomy, the cells failed to act renoprotectively, [corrected] but proteinuria was reduced after administering untreated eEOCs." Next, the original version read as "BMP-5 acts as a potent eEOC agonist in murine AKI in the short [corrected] term. Cell effects in 5/6-nephrectomy are heterogenous, but untreated cells act antifibrotically [corrected] without any impact on EnMT.

High levels of oncomiR‐21 contribute to the senescence‐induced growth arrest in normal human cells and its knock‐down increases the replicative lifespan

Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21CIP1 and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21CIP1, and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.

Cell-Cell Interactions Influence Vascular Reprogramming by Prox1 during Embryonic Development

Lymphangiogenesis is a highly regulated process that involves the reprogramming of venous endothelial cells into early lymphatic endothelial cells. This reprogramming not only displays a polarized expression pattern from the cardinal vein, but also demonstrates vascular specificity; early lymphatics only develop from the cardinal vein and not the related dorsal aorta. In our transgenic model of lymphangiogenesis, we demonstrate that Prox1 overexpression has the ability to reprogram venous endothelium but not early arterial endothelial cells in vivo, in spite of the fact that Prox1 expression is forced onto both vascular beds. Our observations suggest that this specificity during embryogenesis may be due to cell-cell interactions between the developing arterial endothelial cells and smooth muscle cells. These conclusions have far reaching implications on how we understand the vascular specificity of lymphangiogenesis.

Multifunctional P(PEGMA)-REDV conjugated titanium surfaces for improved endothelial cell selectivity and hemocompatibility.

Pre-vascularization of scaffolds using endothelial cells (ECs) and preservation of hemocompatibility are effective strategies to improve the long-term viability of tissue engineered constructs. The current work reports a multifunctional titanium (Ti) surface for simultaneous enhancement of EC selectivity while preserving hemocompatibility. This is achieved by REDV conjugation on surface-grafted PEGMA polymer brushes via surface-initiated atom transfer radical polymerization (ATRP) on a dopamine (DOPA)-modified Ti surface. Our results showed that the proliferation and attachment of human umbilical vein endothelial cells (HUVECs) were substantially improved by P(PEGMA)-REDV conjugation compared to pristine Ti surfaces, whilst no significant effects were observed for the mesenchymal stem cells (MSCs), thus confirming the selectivity of REDV for ECs. Platelets adhesion assay further revealed that the immobilization of PEGMA polymer brushes led to the amelioration of surface hemocompatibility, and this enhancement was not negated by the conjugation of REDV. The current multifunctional Ti-surface can potentially be useful in tissue engineered constructs for bone and dental applications as it allows for early and selective EC attachment and improved hemocompatibility whilst at the same time supporting MSC proliferation and growth.

Angiopoietin-2 modulates eEOC-mediated renoprotection in AKI in a dose-dependent manner

Early endothelial outgrowth cells (eEOCs) significantly protect mice from acute kidney injury (AKI). Angiopoietin-2 (Ang-2) has been shown to be critically involved in vascular repair and homeostasis. The aim of this study was to investigate consequences of Ang-2 treatment of syngeneic murine eEOCs in a cell-based therapeutic approach for AKI. Male 8- to 12-week-old C57/Bl6N mice, subjected to unilateral renal ischemia (40 minutes) postuninephrectomy were systemically injected with 0.5 × 10(6) untreated or Ang-2-pretreated syngeneic murine eEOCs. Renal function and morphology were analyzed 48 hours later. Cellular consequences of eEOC treatment with Ang-2 were evaluated using different in vitro assays (direct and indirect migration, apoptosis/necrosis, ELISA studies). Administration of untreated eEOCs did not protect mice from AKI. Ang-2 dose-dependently modulated cell effects in AKI. While incubating the cells at a concentration of 200 ng/mL (1 hour) did not have any effect on renal function, doubling the concentration (400 ng/mL) resulted in significant renoprotection of cell-injected mice. With 800 ng/mL, cell injection dramatically worsened renal function of treated animals. In vitro analysis showed significantly accelerated migration of cultured mature endothelial cells after incubation with supernatant from Ang-2-treated eEOCs (200 and 400 ng/mL). These effects were most pronounced with 400 mg/mL. In addition, Ang-2 promoted survival of eEOCs. Cellular releases of VEGF and IL-6 were decreased by Ang-2, while TGF-β levels in the medium of Ang-2-stimulated eEOCs were not different from those in untreated cells. Ang-2 acts as modulator of eEOCs in AKI. The migration analysis indicates that the Ang-2 significantly alters indirect (paracrine) activity of eEOCs, thus promoting renoprotection in a dose-dependent manner.

Therapeutic potential of human-induced pluripotent stem cell-derived endothelial cells in a bleomycin-induced scleroderma mouse model

Vascular injury and destruction of endothelial cells (ECs) are the early events in scleroderma (SSc) patients. This study aims to investigate the therapeutic potential of human-induced pluripotent stem cell-derived ECs (hiPSC-ECs) to treat SSc. We have assessed the functional differentiation of hiPSC-ECs and compared them with human embryonic stem cell-derived ECs (hESC-ECs) by a variety of in vitro experimental approaches. Additionally, we evaluated the therapeutic potential of hiPSC-ECs in a bleomycin-induced SSc mouse model. Our results demonstrated that hiPSC-ECs and hESC-ECs showed similar maximum expressions of FLK1 (early EC marker) at day five during differentiation. After sorting and culturing, the FLK1-positive cells exhibited spindle and subsequent endothelial cobblestone morphology in EGM2 medium. The hESC-ECs and hiPSC-ECs also expressed late EC markers CD31 (68% and 75%), CD144 (50% and 61%), CD146 (46% and 61%), and DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL) uptake (55% and 63%), respectively. They additionally formed capillary-like structures on Matrigel. Analyses of the transplantation of sorted CD31-positive hiPSC-ECs into the bleomycin-induced SSc mouse model demonstrated that these cells participate in recovery of the damaged vessels. There was a reduction in collagen content; the number of total and degranulated mast cells returned to their normal state, and bleomycin-induced wounds as well as skin fibrosis improved four weeks after transplantation of hiPSC-ECs. Our findings have shown that the differentiation process from hESCs and hiPSCs to vascular cell components is similar. Additionally, this is the first study to determine the therapeutic potential of vascular cells from hiPSCs in the treatment of an SSc model. In the future, with further validation, these may be used as an appropriate source for the treatment of SSc patients.

Differential Regulation of Bone Marrow-Derived Endothelial Progenitor Cells and Endothelial Outgrowth Cells by the Notch Signaling Pathway

Endothelial progenitor cells (EPCs) are heterogeneous populations of cells that participate in vasculogenesis and promote tissue regeneration. However the different roles of EPC populations in vasculogenesis and tissue regeneration, as well as their regulation and mechanisms remain elusive. In the present study, we cultured bone marrow (BM)-derived early EPCs (EEPCs) and endothelial outgrowth cells (EOCs), and investigated their roles in liver regeneration and their regulation by the Notch signaling pathway. We found that Notch signaling exhibited different effects on the proliferation and migration of EEPCs and EOCs. Our results also showed that while EEPCs failed to form vessel-like structures in a three dimensional sprouting model in vitro, EOCs could sprout and form endothelial cords, and this was regulated by the Notch signaling. We further showed that, by using a conditional knockout model of RBP-J (the critical transcription factor mediating Notch signaling), Notch signaling differentially regulates EEPCs and EOCs. In a partial hepatectomy (PHx) model, EEPCs Notch-dependently benefitted liver regeneration with respect to liver function and hepatocyte proliferation and apoptosis. In contrast, EOCs appeared not directly involved in the recovery of liver function and the increase of hepatocytes. These data suggested that the RBP-J-mediated Notch signaling differentially regulated the two types of EPCs, which showed different roles in liver regeneration.

Impairment and Differential Expression of PR3 and MPO on Peripheral Myelomonocytic Cells with Endothelial Properties in Granulomatosis with Polyangiitis

Background. Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune-mediated diseases characterized by vasculitic inflammation of respiratory tract and kidneys. Clinical observations indicated a strong association between disease activity and serum levels of certain types of autoantibodies (antineutrophil cytoplasm antibodies with cytoplasmic [cANCA in GPA] or perinuclear [pAN CA in MPA] immunofluorescence). Pathologically, both diseases are characterized by severe microvascular endothelial cell damage. Early endothelial outgrowth cells (eEOCs) have been shown to be critically involved in neovascularization under both physiological and pathological condition. Objectives. The principal aims of our study were (i) to analyze the regenerative activity of the eEOC system and (ii) to determine mPR3 and MPO expression in myelo monocytic cells with endothelial characteristics in GPA and MPA patients. Methods. In 27 GPA and 10 MPA patients, regenerative activity blood-derived eEOCs were analyzed using a culture-forming assay. Flk-1+, CD133+/Flk-1+, mPR3+, and Flk-1+/mPR3+ myelomonocytic cells were quantified by FACS analysis. Serum levels of Angiopoietin-1 and TNF-α were measured by ELISA. Results. We found reduced eEOC regeneration, accompanied by lower serum levels of Angiopoietin-1 in GPA patients as compared to healthy controls. In addition, the total numbers of Flk-1+ myelomonocytic cells in the peripheral circulation were decreased. Membrane PR3 expression was significantly higher in total as well as in Flk-1+ myelomonocytic cells. Expression of MPO was not different between the groups. Conclusions. These data suggest impairment of the eEOC system and a possible role for PR3 in this process in patients suffering from GPA.

Inflammation Associated With Obesity: Relationship With Blood and Bone Marrow Endothelial Cells

The purpose of this study was to assess the inflammatory nature of obesity and its effect on blood and bone marrow endothelial cell populations. Obese patients (BMI ≥30) had significantly higher concentrations of the inflammatory marker C-reactive protein (CRP) (P = 0.03) and lower concentrations of the anti-inflammatory cytokine interleukin-10 (IL-10) (P = 0.05). This cytokine profile is consistent with obesity being an inflammatory condition and is further supported by the significant correlation between total white blood cell count and BMI (r = 0.15; P = 0.035). High BMI was associated with significantly lower numbers of early endothelial cells (CD45(-)/CD34(+)) in the bone marrow (r = -0.20; P = 0.0068). There was also a significant inverse correlation between BMI and a more mature endothelial cell phenotype (CD45(-)/31(+)) in the blood (r = -0.17; P = 0.02). In addition, there was a significant correlation between BMI- and endothelial-related cells of hematopoietic origin (CD133(+)/VEGFR-2(+)) in the bone marrow (r = -0.26; P = 0.0007). Patients with higher plasma IL-10 and insulin-like growth factor (IGF) concentrations had higher numbers of endothelial phenotypes in the bone marrow suggesting a protective effect of these anti-inflammatory cytokines. In conclusion, this work confirms the inflammatory nature of obesity and is the first to report that obesity is associated with reduced endothelial cell numbers in the bone marrow of humans. These effects of obesity may be a potential mechanism for impaired tissue repair in obese patients.

Peritoneal macrophages are distinct from monocytes and adherent macrophages

ObjectivePeritoneal macrophages are used in many studies related to atherosclerosis. In situ, they are non-adherent and upon culturing, they adhere and function as scavengers of modified lipoproteins and dead apoptotic cells. They also produce growth factors, suggesting that they may provide life-supporting function as well. In this study, we propose that macrophage adherence plays a major role in their function and propose a novel concept that non-adherent macrophages are poor scavengers and may delay the process of apoptosis by secretion of growth factors. Methods and resultsWe analyzed non-adherent and adherent macrophages for changes in receptor expression, growth factor production and function by microarrays, real-time PCR, and western blot analyses. Our results indicate that adherent macrophages have increased expression of scavenger receptors as compared to fresh peritoneal cells. While genes for many growth factors were expressed in both non-adherent and adherent macrophages, the milk fat globule-epidermal growth factor 8 protein (MFG-E8) that recognizes and takes up apoptotic cells was specifically enhanced in non-adherent cells. Furthermore, early apoptotic endothelial cells demonstrated signs of delayed apoptosis when incubated in the presence of peritoneal lavage fluid that was shown to contain MFG-E8. Functional arrays indicated that peritoneal non-adherent macrophages represent a class of macrophages, distinct from either blood monocytes or adherent cultured macrophages. ConclusionsThese results suggest that the adherence status of macrophages may play a major role in their functions.

Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature

BackgroundIn vivo studies demonstrate that the Prox1 transcription factor plays a critical role in the development of the early lymphatic system. Upon Prox1 expression, early lymphatic endothelial cells differentiate from the cardinal vein and begin to express lymphatic markers such as VEGFR-3, LYVE-1 and Podoplanin. Subsequent in vitro studies have found that differentiated vascular endothelial cells can be reprogrammed by Prox1 to express a lymphatic gene profile, suggesting that Prox1 can initiate the expression of a unique gene signature during lymphangiogenesis. While the in vitro data suggest that gene reprogramming occurs upon Prox1 expression, it is not clear if this is a direct result of Prox1 in vascular endothelial cells in vivo.ResultsOverexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature. Consequent to this overexpression, embryos suffer from gross edema that results in embryonic lethality at E13.5. Furthermore, hemorrhaging and anemia is apparent along with clear defects in lymph sac development. Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development.ConclusionWe present a novel mouse model that addresses the importance of Prox1 in early embryonic lymphangiogenesis. It is clear that there needs to be a measured pattern of expression of Prox1 during embryonic development. Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.

Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities

BackgroundThe term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs).MethodsHuman blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types.ResultsOur data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs.ConclusionsThis study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

Human ALDH-Bright Bone Marrow Cells Produce Paracrine Factors That Protect Endothelial Cells From Hypoxic and Nutritional Stress.

Abstract 3056Poster Board II-1032Human bone marrow cell populations isolated by sorting cells on the basis of high aldehyde dehydrogenase expression (ALDHbr cells) include endothelial, mesenchymal, and other progenitor cells (Gentry et al, 2007, Cytotherapy 9, 259). These cell populations home to and are retained at ischemic endothelium, induce angiogenesis, and effect restoration of tissue perfusion in a mouse hind limb ischemia model (Cappoccia et al, 2009 Blood 113, 5340.) Critical limb ischemia patients treated with ALDHbr cells in a Phase I/II clinical trial showed increased limb perfusion. We are studying paracrine and contact dependent mechanisms by which ALDHbr cells may repair damaged endothelium by measuring the protective effects of ALDHbr cells on early passage human umbilical vein endothelial cells (HUVEC) exposed to hypoxic and nutritional stress. ALDHbr cells migrated through membrane filters in response to supernatants conditioned by hypoxic HUVECs more rapidly than to normoxic HUVEC supernatants. ALDHbr cells attached to HUVECs that had been induced to form tubular structures on Matrigel®, and more ALDHbr cells decorated HUVEC tubules under hypoxic than normoxic conditions. ALDHbr cells and HUVECs expressed several adhesion molecule-ligand pairs, including VLA-1/VCAM, that are regulated by hypoxia and could mediate these interactions. While HUVEC tubules fell apart under hypoxic conditions, adding ALDHbr cells in a transwell culture system for 24 hours preserved the branching structure of hypoxic HUVEC tubule networks. Gene array studies demonstrated that ALDHbr cells highly express several angiogenic growth factors, cytokines and matrix remodeling molecules. Expression of proteins corresponding to many of these gene products, including members of the ephrin-Eph receptor family that can direct endothelial growth, has been confirmed by flow cytometry. Additionally, 29 angiogenic factors including angiopoietin 2, VEGF-A,C, and D, and MMP2 were upregulated under hypoxic conditions. Gene array and flow cytometry showed that hypoxic HUVECs expressed surface receptors for many of the angiogenic factors expressed by ALDHbr cells. In hypoxic transwell cultures, ALDHbr cells specifically induced HUVECs to express six angiogenic factors that were not induced by hypoxia alone. ALDHbr cells protected HUVECs from apoptosis and necrosis induced by serum starvation when added to cultures at the time of or 24 hours following medium shift. Thus, in addition to providing progenitor cells that can potentially participate in angiogenesis, ALDHbr cell populations can mediate repair of ischemic injury by releasing a variety of angiogenic and protective factors at sites of endothelial damage. DisclosuresSmith:Aldagen, Inc.: Employment. White:Aldagen, Inc.: Employment. Gentry:Aldagen, Inc.: Employment. Balber:Aldagen, Inc: Employment, Equity Ownership.

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

Aims: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. Methods: Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (β-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas β-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions’ integrity. Results: All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with β-gal ranged from 0.1 to 54% of the transfected ECs (mean ± SD: 7 ± 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation. Conclusions: Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.

Transcriptional profiling of CD31+ cells isolated from murine embryonic stem cells

Identification of genes involved in endothelial differentiation is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8-days old embryoid bodies by immuno-magnetic separation using platelet endothelial cell adhesion molecule-1 (also known as CD31) expressed on both early and mature endothelial cells. CD31(+) cells exhibit endothelial-like behavior by being able to incorporate DiI-labeled acetylated low-density lipoprotein as well as form tubular structures on matrigel. Quantitative and semi-quantitative PCR analysis further demonstrated the increased expression of endothelial transcripts. To ascertain the specific transcriptomic identity of the CD31(+) cells, large-scale microarray analysis was carried out. Comparative bioinformatic analysis reveals an enrichment of the gene ontology categories angiogenesis, blood vessel morphogenesis, vasculogenesis and blood coagulation in the CD31(+) cell population. Based on the transcriptomic signatures of the CD31(+) cells, we conclude that this ES cell-derived population contains endothelial-like cells expressing a mesodermal marker BMP2 and possess an angiogenic potential. The transcriptomic characterization of CD31(+) cells enables an in vitro functional genomic model to identify genes required for angiogenesis.