Differentiation Of Vascular Endothelial Cells Research Articles

Pathogenic role of PFKFB3 in endothelial inflammatory diseases.

The differentiation of vascular endothelial cells and the formation of new blood vessels are inseparable from the energy supply and regulation of metabolism. The budding of blood vessels is a starting point of glycolysis pathway in angiogenesis. Phosphofructokinase-2/fructose 2,6-biophosphatase 3 (PFKFB3), a key rate-limiting enzyme in glycolysis, exhibits strong kinase activity. Inhibition of PFKFB3 can reduce the rate of glycolysis, thereby inhibiting the budding of blood vessels, resulting in inhibition of pathological angiogenesis. In this review, the role of PFKFB3 in the angiogenesis of inflammatory diseases was summarized, and the endothelial inflammatory diseases associated with PFKFB3 were reviewed.

Preparation and Biocompatibility of Core-Shell Microspheres for Sequential, Sustained Release of BMP-2 and VEGF

Bone defect repair remains a challenge in orthopedics. This study describes the development and potential effectiveness of vascular endothelial growth factor (VEGF)/bone morphogenetic protein-2 (BMP-2) shell-core microspheres for promoting bone regeneration. Poly(L-lactic acid)/polylactic-co-glycolic acid (PLLA/PLGA) core-shell microspheres loaded with VEGF and BMP-2 were prepared by a coaxial electrospray technique, and their surface morphology, core-shell distribution, and particle size were examined. Different groups of microspheres were prepared with different placement of the growth factors, and the encapsulation efficiency and in vitro release curves were measured. Additionally, the effects of the different groups of microspheres on the proliferation and differentiation of osteoblasts and vascular endothelial cells were investigated. The prepared microspheres had a core-shell structure with good homogeneity and dispersion, a clear boundary, and a smooth surface. On scanning electron microscopy, the mean diameter of the microspheres was similar for all six preparations (P > 0.05). During in vitro release, growth factor was initially released via a brief burst release from the outer shell of the microsphere followed by a slower sustained release. The release of growth factors from the inner core remained relatively slow and sustained. Sequential release of different growth factors was achieved through the inconsistent release rates from the microsphere shell and inner core. All groups of microspheres showed no cytotoxicity, good biocompatibility, and the ability to promote osteoblast proliferation. The microspheres loaded with BMP-2 also promoted osteoblast differentiation, and VEGF-loaded microspheres promoted the proliferation and differentiation of vascular endothelial cells. The BMP-2 (core)/VEGF (shell) microsphere group best promoted osteoblast differentiation. The microspheres prepared in this study exhibited slow sequential release of BMP-2 and VEGF and showed good biocompatibility along with the ability to promote osteoblast differentiation and vascular endothelial cell proliferation.

The Functions and Mechanisms of Basic Fibroblast Growth Factor in Tendon Repair.

Tendon injury is a disorder of the musculoskeletal system caused by overuse or trauma, which is characterized by pain and limitations in joint function. Since tendon healing is slowly and various treatments are generally ineffective, it remains a clinically challenging problem. Recent evidences suggest that basic fibroblast growth factor (bFGF) not only plays an important role in tendon healing, but also shows a positive effect in laboratory experimentations. The purpose of this review is to summarize the effects of bFGF in the tendon healing. Firstly, during the inflammatory phase, bFGF stimulates the proliferation and differentiation of vascular endothelial cells to foster neovascularization. Furthermore, bFGF enhances the production of pro-inflammatory factors during the early phase of tendon healing, thereby accelerating the inflammatory response. Secondly, the cell proliferation phase is accompanied by the synthesis of a large number of extracellular matrix components. bFGF speeds up tendon healing by stimulating fibroblasts to secrete type III collagen. Lastly, the remodeling phase is characterized by the transition from type III collagen to type I collagen, which can be promoted by bFGF. However, excessive injection of bFGF can cause tendon adhesions as well as scar tissue formation. In future studies, we need to explore further applications of bFGF in the tendon healing process.

Network-Based Analysis Reveals Gene Signature in Tip Cells and Stalk Cells.

Angiogenesis occurs during various physiological or pathological processes such as wound healing and tumor growth. Differentiation of vascular endothelial cells into tip cells and stalk cells initiates the formation of new blood vessels. Tip cells and stalk cells are endothelial cells with different biological characteristics and functions. The aim of this study was to determine the mechanisms of angiogenesis by exploring differences in gene expression of tip cells and stalk cells. Raw data were retrieved from NCBI Gene Expression Omnibus (GSE19284). Data were reanalyzed using bioinformatics methods that employ robust statistical methods, including identification of differentially expressed genes (DEGs) between the stalk and tip cells, Weighted Gene Correlation Network Analysis (WGCNA), gene ontology and pathway enrichment analysis using DAVID tools, integration of Protein-Protein Interaction (PPI) networks and screening of hub genes. DEGs of stalk and tip cells were grouped as dataset A. Gene modules associated with differentiation of stalk and tip cells screened by WGCNA were named dataset B. Further, we retrieved existing markers of angiogenesis from previous experimental studies on tip and stalk cells which we called dataset C. Intersection of datasets A, B and C was used as a candidate gene. Subsequently, we verified the results applying Quantitative Polymerase Chain Reaction (Q-PCR) to our clinical specimen. In general, the Q-PCR results coincide with the majority of the expression profile. We identified five candidate genes, including ESM1, CXCR4, JAG1, FLT1 and PTK2, and two pathways, including Rap1 signaling pathway and PI3K-Akt signaling pathway in vascular endothelial cells that differentiate into tip cells and stalk cells using bioinformatics analysis. Bioinformatics approaches provide new avenues for basic research in different fields such as angiogenesis. The findings of this study provide new perspectives and a basis for the study of molecular mechanisms of vascular endothelial cell differentiation into stalk and tip cells. Genes and pathways identified in this study are potential biomarkers and therapeutic targets for angiogenesis in the tumor.

Abstract 12338: Promotion of Arteriolar Differentiation of Vascular Endothelial Cells by PKD-1/CD36 Signaling-Mediated Chromatin Remodeling

Vessel narrowing and occlusions reduce blood supply to end tissues, leading to myocardial infarction in the heart or tissue loss in the peripheral vasculature. Effective approaches are needed to establish de novo arteriogenesis to treat these conditions. An essential first step is to understand mechanisms of arteriolar differentiation, in which transcriptional reprogramming of vascular endothelial cells (ECs) may be critical. Our previous studies indicate that protein kinase D (PKD-1) is required for arterial differentiation and plays an essential role in proarteriogenic reprogramming via transcriptional inhibition of antiangiogenic receptor CD36 via histone deacetylase HDAC7. We hypothesize that PKD-1/CD36 signaling initiates an epigenetic reprogramming of ECs for arteriolar differentiation via chromatin remodeling. To test this hypothesis, we isolated primary heart and lung ECs from wild-type control, EC-specific cd36 KO or pkd-1 KO mice. Total RNA was extracted for expression of arteriogenic genes by RT-qPCR. The proteins were collected for Western blotting and ChIP assays. Our results showed that when CD36 was downregulated in primary ECs from hearts and lungs, the expression levels of PKD-1 and arteriogenic genes EphB2 and DLL4 were significantly elevated compared to controls (p < 0.05). Intriguingly, we observed a significant increase in protein expression of neuropilin1. In contrast, in heart ECs from the EC-specific pkd-1 KO mice, arteriogenic gene expressions were downregulated whereas CD36 expression was significantly upregulated. Using the ChIP assay, we measured the interactions between H3K9 on the CD36, DLL4, or EphB2 promoters using ECs from EC-specific cd36 and pkd-1 KO mice. In conclusion, our results indicate that an epigenetic reprogramming regulates arteriolar differentiation at a transcriptional level by chromatin remodeling. PKD-1 signaling may dictate CD36 transcriptional levels, thereby reprogramming ECs for arteriolar differentiation by inducing an arteriogenic gene signature, which may contribute to de novo arteriogenesis under ischemic conditions.

Blood vessel formation in cerebral organoids formed from human embryonic stem cells

Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.

Synergies of accelerating differentiation of bone marrow mesenchymal stem cells induced by low intensity pulsed ultrasound, osteogenic and endothelial inductive agent

In terms to investigate the effect of low-intensity pulsed ultrasound (LIPUS) for differentiation of bone marrow mesenchymal stem cells (BMSCs) and the feasibility of simultaneously inducing into osteoblasts and vascular endothelial cells within the cell culture medium in which two inductive agents are added at the same time with or without LIPUS. Cells were divided into a non-induced group, an osteoblast-induced group, a vascular endothelial-induced group, and a bidirectional differentiation-induced group. Each group was further subdivided into LIPUS and non-LIPUS groups. The cell proliferation in each group was measured by MTT assay. Cell morphological and ultrastructural changes were observed by inverted phase contrast microscopy and transmission electron microscopy. The differentiation of BMSCs was detected by confocal microscopy, flow cytometry and quantitative RT-PCR. Results demonstrated that both osteoblast and vascular endothelial cell differentiation markers were expressed in the bidirectional differentiation induction group and early osteogenesis and angiogenesis appeared. The cell proliferation, differentiation rate and expression of osteocalcin and vWF in the LIPUS groups were all significantly higher than those in the corresponding non-LIPUS group (p < .05), suggesting LIPUS treatment can promote the differentiation efficiency and rate of BMSCs, especially in the bidirectional differentiation induction group. This study suggests the combination of LIPUS and dual-inducing agents could induce and accelerate simultaneous differentiation of BMSCs to osteoblasts and vascular endothelial cells. These findings indicate the method could be applied to research on generating vascularized bone tissue with a shape and function that mimics natural bone to accelerate early osteogenesis and angiogenesis.

A sulfonated reversible thermal gel for the spatiotemporal control of VEGF delivery to promote therapeutic angiogenesis.

Despite medical and surgical advancements for the treatment of cardiovascular disease, mortality and morbidity remain high. Therapeutic angiogenesis has been one approach to address the major clinical need for a more effective treatment to restoring blood flow in ischemic organs and tissues, but current progress in angiogenic drug delivery is inadequate at providing sufficient bioavailability without causing safety concerns. An injectable sulfonated reversible thermal gel composed of a polyurea conjugated with poly(N-isopropylacrylamide) and sulfonate groups has been developed for the delivery of angiogenic factors. The thermal gel allowed for the spatiotemporal control of vascular endothelial growth factor release with a decreased initial burst release and reduced release rate in vitro. A subcutaneous injection mouse model was used to evaluate efficacious vascularization and assess the inflammatory response due to a foreign body. Thermal gel injections showed substantial vascularization properties by inducing vessel formation, recruitment and differentiation of vascular endothelial cells, and vessel stabilization by perivascular cells, while infiltrating macrophages due to the thermal gel injections decreased over time. These results demonstrated effective localization and delivery of angiogenic factors for therapeutic angiogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3053-3064, 2018.

Efficient differentiation of vascular endothelial cells from dermal-derived mesenchymal stem cells induced by endothelial cell lines conditioned medium

ObjectiveTo directionally-differentiate dermis-derived mesenchymal stem cells (DMSCs) into vascular endothelial cells (VECs) in vitro, providing an experimental basis for studies on the pathogenesis and treatment of vascular diseases. MethodsAfter separation by adherent culture, VEC line supernatant, vascular endothelial growth factor (VEGF), bone morphogenetic protein-4 and hypoxia were used for the differentiation of VECs from DMSCs. The cell type was authenticated by flow cytometry, matrigel angiogenesis assay in vitro, and immunofluorescent staining during differentiation. The VEGF concentration was investigated by enzyme-linked immunosorbent assay. ResultsAfter 28 days of differentiation, the cell surface marker CD31 was significantly positive (80%–90%) by flow cytometry in the VEC line-conditioned culture, which was significantly higher than in the other groups. Differentiated DMSCs had the ability to ingest Dil-ac-LDL and vascularize in the conditioned culture, but not in the other groups. In the VEC line supernatant, the concentration of VEGF was very low. The VEGF concentration changed along with the differentiation into VECs in the medium of the conditioned culture group. ConclusionVEC line supernatant can induce the differentiation of DMSCs into VECs, possibly through the pathway except VEGF.

Correlation between Contrast-Enhanced Ultrasound and Microvessel Density via CD31 and CD34 in a rabbit VX2 lung peripheral tumor model.

To evaluate the tumor angiogenesis in lung peripheral VX2 tumor model by contrast-enhanced ultrasound (CEUS) and to determine the correlation between CEUS parameters and microvessel density (MVD) calculated via CD31 and CD34 expression. VX2 pulmonary tumors were created in eight Japanese white rabbits by implanting a VX2 sarcoma into the lower portion of the right lung through ultrasound guidance. Tumors were allowed to grow for 14-21 days to achieve a diameter of 7-15 mm, and were examined by CEUS using a SonoVue contrast agent. The results were recorded as digital video images, and the time-intensity curves and hemodynamic parameters were analyzed. Pathological tumor specimens were immediately obtained after the ultrasound examinations. Tumor specimens were stained with hematoxylin and eosin (H&E) and expressed as CD31 and CD34. The different endothelial cell markers were determined by immunohistochemical staining. MVD was calculated via CD 31 and CD34, and the relationship between CEUS parameters and MVD was analyzed. Two distinct types of microvessels were identified in lung peripheral VX2 tumors: differentiated (CD34+) and undifferentiated (CD31+) vessels. A significant correlation was found between CEUS parameters and undifferentiated MVD (CD31+ vessels) in lung peripheral VX2 tumors (p<0.05). A reverse correlation was observed between different MVDs. Two different degrees of differentiation of vascular endothelial cells (CD31 and CD34) exist in the rabbit lung peripheral VX2 tumor model. CD31 MVD can more effectively evaluate tumor angiogenesis compared with CD34 MVD. CEUS, as a non-invasive imaging method, can effectively evaluate tumor angiogenesis in rabbit peripheral lung cancer.

Preparation of the micro/nano structures of the biomimetic coating stent for loading MiRNA126 by four-beam laser interference

We demonstrated the preparation of drug loading micro structures on the surface of metal coronary stent by using laser interference and provide a good substrate for the next step by using the method of miRNA-126 for the first time to our knowledge. Compared with the drug used in the existing drug eluting stent, miRNA-126 has a better effect on promoting the differentiation of vascular endothelial cells, inhibiting the inflammatory reaction and the proliferation of smooth muscle cells. The laser wavelength is 1064nm, the single pulse energy is 2J, the pulse width is 8ns and the exposure time is 80s. When the incident angle is 2.8°, between beam angle is 90°, we got the bionic micro nano structure witch cycle is 10.21μm. Different cycles can be obtained by changing the angle of incidence and the angle between the laser beam, After a preliminary comparison test, the cycle 10.21μm bracket micro nano structure is better than cycle 5.077μm and 20.9μm in the assembly adhesion force and loading capacity.

Sry-type HMG box 18 contributes to the differentiation of bone marrow-derived mesenchymal stem cells to endothelial cells

ObjectiveMesenchymal stem cells (MSC) have shown therapeutic potential to engraft and either differentiate into or support differentiation of vascular endothelial cells (EC), smooth muscle cells and cardiomyocytes in animal models of ischemic heart disease. Following intracoronary or transendocardial delivery of MSCs, however, only a small fraction of cells engraft and the majority of those persist as an immature cell phenotype. The goal of the current study was to decipher the molecular pathways and mechanisms that control MSC differentiation into ECs. Vascular endothelial growth factor (VEGF-165) treatment is known to enhance in vitro differentiation of MSCs into ECs. We tested the possible involvement of the Sry-type HMG box (Sox) family of transcription factors in this process. Method and ResultsMSCs were isolated from the bone marrow of Yucatan microswine and underwent a 10 day differentiation protocol. VEGF-165 (50ng/ml) treatment of MSCs in vitro induced a significant increase in the protein expression of VEGFR-2, Sox9 and Sox18, in addition to the EC markers PECAM-1, VE-cadherin and vWF, as determined by Western blot or flow cytometry. siRNA-mediated knockdown of Sox18, as opposed to Sox9, in MSCs prevented VEGF-165-mediated induction of EC markers and capillary tube formation. Inhibition of VEGFR-2 signaling (SC-202850) reduced Sox18 and reduced VEGF-165-induced differentiation of MSCs to ECs. ConclusionHere we demonstrate that VEGF-165 mediates MSC differentiation into ECs via VEGFR-2-dependent induction of Sox18, which ultimately coordinates the transcriptional upregulation of specific markers of the EC phenotype.

The molecular regulation of arteriovenous specification and maintenance.

The formation of a hierarchical vascular network, composed of arteries, veins, and capillaries, is essential for embryogenesis and is required for the production of new functional vasculature in the adult. Elucidating the molecular mechanisms that orchestrate the differentiation of vascular endothelial cells into arterial and venous cell fates is requisite for regenerative medicine, as the directed formation of perfused vessels is desirable in a myriad of pathological settings, such as in diabetes and following myocardial infarction. Additionally, this knowledge will enhance our understanding and treatment of vascular anomalies, such as arteriovenous malformations (AVMs). From studies in vertebrate model organisms, such as mouse, zebrafish, and chick, a number of key signaling pathways have been elucidated that are required for the establishment and maintenance of arterial and venous fates. These include the Hedgehog, Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-β (TGF-β), Wnt, and Notch signaling pathways. In addition, a variety of transcription factor families acting downstream of, or in concert with, these signaling networks play vital roles in arteriovenous (AV) specification. These include Notch and Notch-regulated transcription factors (e.g., HEY and HES), SOX factors, Forkhead factors, β-Catenin, ETS factors, and COUP-TFII. It is becoming apparent that AV specification is a highly coordinated process that involves the intersection and carefully orchestrated activity of multiple signaling cascades and transcriptional networks. This review will summarize the molecular mechanisms that are involved in the acquisition and maintenance of AV fate, and will highlight some of the limitations in our current knowledge of the molecular machinery that directs AV morphogenesis.

Microenvironment influences vascular differentiation of murine cardiovascular progenitor cells

We examined the effects of the microenvironment on vascular differentiation of murine cardiovascular progenitor cells (CPCs). We isolated CPCs and seeded them in culture exposed to the various extracellular matrix (ECM) proteins in both two-dimensional (2D) and 3D culture systems. To better understand the contribution of the microenvironment to vascular differentiation, we analyzed endothelial and smooth muscle cell differentiation at both day 7 and day 14. We found that laminin and vitronectin enhanced vascular endothelial cell differentiation while fibronectin enhanced vascular smooth muscle cell differentiation. We also observed that the effects of the 3D electrospun scaffolds were delayed and not noticeable until the later time point (day 14), which may be due to the amount of time necessary for the cells to migrate to the interior of the scaffold. The study characterized the contributions of both ECM proteins and the addition of a 3D culture system to continued vascular differentiation. Additionally, we demonstrated the capability bioengineer a CPC-derived vascular graft.

Effects of Hydrocortisone, Glycerophosphate and Retinol on the Differentiation of Mesenchymal Stem Cells and Vascular Endothelial Cells to Osteoblasts

Vascular calcification, which causes occlusion and rupture of the vascular, is often observed in patients in the advanced stages of arteriosclerosis. One of the best procedures for inhibiting the accumulation of vascular calcification is to obstruct the differentiation of mesenchymal stem cells (MSCs) and/or vascular endothelial cells (VECs) in the vascular to osteoblasts. In this study, we evaluated the biochemical and genetic characteristics of the process of differentiation of MSCs and VECs to osteoblasts. C3H10T1/2 MSCs, TKD2 VECs and MC3T3-E1 preosteoblasts (POBs) were cultured in medium containing both hydrocortisone and glycerophosphate. These compounds showed strong effects promoting the differentiation of VECs as well as POBs, although the effect was weak in the MSCs. Moreover, C3H10T1/2 MSCs and TKD2 VECs were cultured in medium containing 10 mM retinol, after which the alkali phosphatase (ALP) activity of the MSCs and production of calcified nodules of TKD2 were significantly increased, whereas the marker genes for the osteoblasts were not. These results suggest that retinol does not have an effect in inducing the differentiation of VECs to osteoblasts, but rather exhibits a strong promoting effect on differentiation.

Hypoxia, Leptin, and Vascular Endothelial Growth Factor Stimulate Vascular Endothelial Cell Differentiation of Human Adipose Tissue-Derived Stem Cells

The plasticity of human adipose tissue-derived stem cells (hASCs) is promising, but differentiation in vitro toward endothelial cells is poorly understood. Flow cytometry demonstrated that hASCs isolated from excised fat tissue were positive for CD29, CD44, CD70, CD90, CD105, and CD166 and negative for the endothelial marker CD31, and the hematopoietic cell markers CD34 and CD133. hASCs differentiated into adipocytes after cultivation in adipogenic medium. Exposure of hASCs for 10 days under hypoxia (3% oxygen) in combination with leptin increased the percentage of CD31(+) endothelial cells as well as CD31, VE-Cadherin, Flk-1, Tie2, von Willebrand factor, and endothelial cell nitric oxide synthase mRNA expression. This was enhanced on co-incubation of vascular endothelial growth factor (VEGF) and leptin, whereas VEGF alone was not sufficient. Moreover, hASCs cultured on a matrigel surface under hypoxia/VEGF/leptin, showed a stable branching network. Hypoxic conditions significantly decreased apoptosis as evaluated by cleaved caspase-3, and increased prolyl hydroxylase domain 3 mRNA expression. Hypoxia increased expression of VEGF as well as leptin transcripts, which were significantly inhibited on co-incubation with either VEGF or leptin or a combination of both. Furthermore, leptin treatment of hypoxic cells increased the expression of the long/signaling form of the leptin receptor (ObRL), which was augmented on co-incubation with VEGF. The observed endothelial differentiation was dependent on the Akt pathway, as co-administration with Akt inhibitor abolished the observed effects. In conclusion, our data demonstrate that hASCs can be efficiently differentiated to endothelial cells by mimicking the hypoxic and pro-angiogenic microenvironment of adipose tissue.

VEGF promotes cardiac stem cells differentiation into vascular endothelial cells via the PI3K/Akt signaling pathway

Recent research suggested that cardiac stem cells (CSCs) may have the clinical application for cardiac repair. However, their characteristics and the regulatory mechanisms of their growth and differentiation have not been fully investigated. Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. But the homing role of VEGF for CSCs is unclear. In this report, CSCs were isolated, purified, and expanded in vitro from rat heart. VEGF, SU5416 (VEGF receptor blocker), and Wortmannin (PI3K/Akt signaling pathway inhibitor) were used for differentiation into vascular endothelial cells (VECs). Real-time qPCR was selected to confirm the role of PI3K/Akt signaling pathway in VECs differentiation from rat CSCs. The result of real-time qPCR demonstrated that PI3K/Akt signaling pathway plays an important role in rat CSCs differentiated into VECs. So, our research provides a theoretical basis and experimental evidence for therapeutic application of rat CSCs to treat cardiac repair.

Transcriptional Regulation of Endothelial Cell and Vascular Development

The establishment and maintenance of the vascular system is critical for embryonic development and postnatal life. Defects in endothelial cell development and vessel formation and function lead to embryonic lethality and are important in the pathogenesis of vascular diseases. Here, we review the underlying molecular mechanisms of endothelial cell differentiation, plasticity, and the development of the vasculature. This review focuses on the interplay among transcription factors and signaling molecules that specify the differentiation of vascular endothelial cells. We also discuss recent progress on reprogramming of somatic cells toward distinct endothelial cell lineages and its promise in regenerative vascular medicine.

Profiling of transcriptional and epigenetic changes during directed endothelial differentiation of human embryonic stem cells identifies FOXA2 as a marker of early mesoderm commitment

IntroductionDifferentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage.MethodsWe performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes.ResultsWe identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC–EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation.ConclusionThis enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease.

Exploitation of in silico potential in prediction, validation and elucidation of mechanism of anti-angiogenesis by novel compounds: comparative correlation between wet lab and in silico data

This study describes in silico validation of the wet lab data from aeroplysinin-1, curcumin and halofuginone using Autodock Tools 4.0. The inhibition patterns of the vascular endothelial cell differentiation and capillary tube formation mediated by anti-angiogenic growth factors from these test compounds were found quite comparable with the in silico results using angiogenic targets (EFGR, bFGF and VEGFR-1). Successful validation of the wet lab results of the selected angiogenic targets by in silico method has led to exploit the hidden potential of in silico tools in preliminary screening of the unknown compounds for anti-angiogenic potential in a cost-effective manner.