Expression Of Endothelial Cell Markers Research Articles

Angiogenesis in alkaptonuria.

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of AKU treatment is palliative and little is known about its physiopathology. Neovascularization is involved in the pathogenesis of systemic inflammatory rheumatic diseases, a family of related disorders that includes AKU. Here, we investigated the presence of neoangiogenesis in AKU synovium and healthy controls. Synovium from AKU patients, who had undergone total joint replacement or arthroscopy, or from healthy patients without any history of rheumatic diseases, who underwent surgical operation following sport trauma was subjected to hematoxylin and eosin staining. Histologic grades were assigned for clinical disease activity and synovitis based on cellular content of the synovium. By immunofluorescence microscopy, using different endothelial cell markers, we observed large vascularization in AKU but not in healthy synovium. Moreover, Western blotting and quantification analyses confirmed strong expression of endothelial cell markers in AKU synovial tissues. Importantly, AKU synovium vascular endothelium expressed high levels of β-dystroglycan, a protein previously involved in the regulation of angiogenesis in osteoarthritic synovium. This is the first report providing experimental evidences that new blood vessels are formed in AKU synovial tissues, opening new perspectives for AKU therapy.

Plasma treatment in conjunction with EGM-2 medium increases endothelial and osteogenic marker expressions of bone marrow mesenchymal stem cells

For many tissue engineering applications, an important goal is to create functional tissues in vitro. In order to make such tissues viable, they have to be vascularized. Bone marrow mesenchymal stem cells (BMSCs) are a promising candidate for vascularization. Growth supplements are commonly used to induce BMSCs differentiation, and further improvements in differentiation conditions can be achieved by modifying the cells’ growth environment, such as pretreating the culture dish with gas plasma to modify the surface functional groups that the cells are seeded on. In this work, we compare the effects of different gas plasmas on the growth and differentiation of BMSCs. We treat the growth dish with different plasmas (CO2, N2, and O2) and induce BMSC differentiation on them with endothelial growth medium-2 (EGM-2). We find that EGM-2 by itself does not have strong effect on endothelial differentiation. However, we find that plasma treatment markedly increases the mRNA expression of endothelial cell markers. Interestingly, we also find that the upregulation of osteogenic markers. The data show that plasma treatment in conjunction with EGM-2 can enhance BMSCs differentiation into both endothelial-like cells and osteogenic-like cells. The effects of EGM-2 on BMSCs differentiation are influenced by the plasma-modified surface of the substrate. Our findings provide a method to enhance EGM-2-based cell differentiation, with consequences for tissue engineering and stem cell biology applications.

Involvement of human chorionic gonadotropin in regulating vasculogenic mimicry and hypoxia-inducible factor-1α expression in ovarian cancer cells

BackgroundHuman chorionic gonadotropin (hCG) can play a crucial role in angiogenesis. In the present study, we focused on hCG to gain insight into its potential effects on vasculogenic mimicry (VM) in ovarian cancer cells.MethodsOvarian cancer OVCAR-3 cells were incubated with different concentrations of recombinant hCG in 3-dimensional cultures. VM was identified by morphological observations and vascular endothelial cell marker detection in OVCAR-3 cells. Expression of hCG, hypoxia-inducible factor-1α (HIF-1α), and the endothelial cell markers CD31, VEGF, and factor VIII were detected by reverse transcription polymerase chain reaction and western blotting. The effect of hCG on endothelial cell-marker expression in ovarian cancer cells was further explored using small interfering RNA (siRNA) and plasmid-based approaches.ResultsIncubation of OVCAR-3 cells with recombinant hCG induced vessel-like network formation, which was accompanied by significant elevation of vascular marker expression. Attenuation of hCG expression by siRNA in OVCAR-3 cells suppressed the expression of endothelial cell markers and HIF-1α by tumour cells. Overexpression of hCG in OVCAR-3 cells resulted in increased expression of endothelial cell markers and HIF-1α.ConclusionsHCG was crucial for changing the phenotype of OVCAR-3 cells to endothelial-like cells. The effect of hCG induction on VM in ovarian cancer cells is potentially associated with HIF-1α.

Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function.

The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate.

Neuropsychological, Neurovirological and Neuroimmune Aspects of Abnormal GABAergic Transmission in HIV Infection.

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients with effective suppression of virus replication by combination antiretroviral therapy (cART). Several neurotransmitter systems were reported to be abnormal in HIV-infected patients, including the inhibitory GABAergic system, which mediates fine-tuning of neuronal processing and plays an essential role in cognitive functioning. To elucidate the role of abnormal GABAergic transmission in HAND, the expression of GABAergic markers was measured in 449 human brain specimens from HIV-infected patients with and without HAND. Using real-time polymerase chain reaction, immunoblotting and immunohistochemistry we found that the GABAergic markers were significantly decreased in most sectors of cerebral neocortex, the neostriatum, and the cerebellum of HIV-infected subjects. Low GABAergic expression in frontal neocortex was correlated significantly with high expression of endothelial cell markers, dopamine receptor type 2 (DRD2L), and preproenkephalin (PENK) mRNAs, and with worse performance on tasks of verbal fluency. Significant associations were not found between low GABAergic mRNAs and HIV-1 RNA concentration in the brain, the history of cART, or HIV encephalitis. Pathological evidence of neurodegeneration of the affected GABAergic neurons was not present. We conclude that abnormally low expression of GABAergic markers is prevalent in HIV-1 infected patients. Interrelationships with other neurotransmitter systems including dopaminergic transmission and with endothelial cell markers lend added support to suggestions that synaptic plasticity and cerebrovascular anomalies are involved with HAND in virally suppressed patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s11481-016-9652-2) contains supplementary material, which is available to authorized users.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial‐to‐mesenchymal transition (EndoMT)

PurposeThe fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial‐to‐mesenchymal transition (EndoMT) and/or bone marrow‐derived circulating fibrocytes contribute to the myofibroblast population present in PDR membranes.MethodsEpiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor‐β1 (TGF‐ β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐a (TNF‐a).ResultsAll membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co‐expressed the fibroblast/myofibroblast markers fibroblast‐specific protein‐1 (FSP‐1) and a‐SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP‐1, a‐SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co‐localization of CD45 with FSP‐1 and a‐SMA and co‐localization of CD11b with a‐SMA and matrix metalloproteinase‐9, demonstrating the presence of infiltrating fibrocytes. Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial‐cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP‐1).ConclusionsThese results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial‐to‐mesenchymal transition (EndoMT)

PurposeThe fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial‐to‐mesenchymal transition (EndoMT) and/or bone marrow‐derived circulating fibrocytes contribute to the myofibroblast population present in PDR membranes.MethodsEpiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor‐β (TGF‐β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐a (TNF‐a).ResultsAll membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co‐expressed the fibroblast/myofibroblast markers fibroblast‐specific protein‐1 (FSP‐1) and a‐SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP‐1, a‐SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co‐localization of CD45 with FSP‐1 and a‐SMA and co‐localization of CD11b with a‐SMA and matrix metalloproteinase‐9, demonstrating the presence of infiltrating fibrocytes. Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial‐cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP‐1).ConclusionsThese results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Abstract 4171: Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts

Abstract BACKGROUND: Endothelial-mesenchymal transition (EndMT) is an extreme form of endothelial cell plasticity whereby endothelial cells acquire mesenchymal phenotype. While EndMT is a significant component during embryogenesis, maladaptive EndMT is a key contributor to fibrotic pathologies including cancer. With regard to this, EndMT accounts for up to 40% of cancer-associated fibroblasts (CAFs) that play an important role in cancer progression through potentially oncogenic signals like transforming growth factor- β (TGF-β). Recent investigations have demonstrated that the angiogenic co-receptor Neuropilin-1 (NRP1) is aberrantly expressed in cancer and modulates cancer progression through interaction with various ligands, including TGF-β. However, the relationship and driving mechanisms linking NRP1 and EndMT remain unexplored. METHODS: SiRNA-mediated NRP1 gene knockdown studies using Dharmafect-4 transfection reagent and siNRP1 or scramble control (Ambion) were performed on human umbilical vein endothelial cells (HUVECs), at baseline and after TGF-β stimulation. Total RNA was extracted using Trizol® reagent (Invitrogen), and precipitated with isopropanol. Complementary DNA was synthesized using iScript cDNA synthesis kit (Bio-Rad) and real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green (Bio-Rad) following manufacturer's protocols. Markers of EndMT and mediators of TGF-β1 signalling were evaluated by RT-PCR, western blotting and immunocytochemistry. RESULTS: RT-PCR and western blots measuring NRP1 mRNA and protein levels in HUVEC lysates confirmed successful silencing. Light and fluorescence microscopy revealed marked morphological changes in TGF-β1 stimulated scramble siRNA-transfected HUVECs. TGF-β1 stimulated HUVECs demonstrated a distinct change from “cobblestone-like endothelial cell morphology” to an enlarged spindle shaped appearance consistent with a “fibroblast like morphology”, accompanied by re-arrangement of the cytoskeleton. We further observed that these characteristic EndMT features were revoked upon NRP1 silencing where cells maintained a conventional cobblestone phenotype. Expression of endothelial cell markers, CD-31 and VE-Cadherin, were significantly higher in NRP1-silenced vs. scramble siRNA-treated HUVECs; additionally, NRP1 silencing was associated with significant reduction in expression of the mesenchymal markers α-smooth muscle actin, Slug and N-Cadherin. Loss of NRP1 was accompanied by decreased expression of TGF-β1 and its receptors as well as significant changes in the key downstream effectors of TGF-β1 signalling. CONCLUSION: Our novel findings demonstrating that loss of NRP1 revokes TGF-β-dependent EndMT-like phenotype switching suggesting that NRP1 may represent a novel therapeutic target to limit the source of EndMT derived CAFs and associated cancer aggression. Citation Format: Pratiek N. Matkar, Krishna Kumar Singh, Gerald Prud'homme, Howard Leong-Poi. Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4171. doi:10.1158/1538-7445.AM2015-4171

Abstract 460: Novel compound elicits anti-tumor macrophages associated with tumor regression in breast cancer

Abstract Cancer immuno-therapies targeting components of the immune system have shown great promise for the treatment of various forms of cancer. Most of the immuno-therapies currently under trial intervene in adaptive immune cell pathways; however, the innate immune system has also recently been shown to play a potent immunomodulatory role in the tumor microenvironment. Tumor associated macrophages (TAMs) compose up to 50% of tumor volume. Presently, we demonstrate that administration of a novel compound stimulates rapid tumor regression that is associated with the infiltration of F4/80+ macrophages into tumors in the MMTV-PyMT murine model of breast cancer. A significant increase in tumor-infiltrating macrophages was visible by immunohistochemistry after five days of treatment and macrophages were necessary for tumor regression as demonstrated by cell depletion assays in vivo. Of note, macrophage infiltration correlated with an increase in tumor cell death as shown by elevated expression of the apoptotic protein cleaved-caspase-3 (CC3); conversely, cell proliferation factor Ki67 was decreased. Additionally, compound-treated animals exhibited striking changes in tumor vascularization, with a marked decrease in overall endothelial cell marker expression as displayed by immunohistochemistry. Taken together, these findings reveal a novel role for macrophages in breast tumor regression during treatment with a novel compound that alters the tumor microenvironment. Citation Format: Holly E. Ponichtera, Jennifer L. Guerriero, Alaba O. Sotayo, Anthony Letai. Novel compound elicits anti-tumor macrophages associated with tumor regression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 460. doi:10.1158/1538-7445.AM2015-460

Abstract 614: 1,25-dihydroxyvitamin D Enhances Vegf-stimulated Porcine Adipose-derived Mesenchymal Stem Cells Toward the Endothelial Phenotype Involving Wnt/β-catenin Pathway

Background: Cell-based therapy using adipose-derived mesenchymal stem cells (AMSCs) is an attractive option for re-endothelialization post-angioplasty procedures. Vitamin D levels in circulating blood may regulate AMSC-based re-endothelialization of injured arteries. To our knowledge, the role of vitamin D and vascular endothelial growth factor (VEGF) in the differentiation of adipose-derived AMSCs into endothelial cells (ECs) has not been examined. In this study, we investigated the effect of vitamin D on VEGF-induced differentiation of MSCs into ECs. Methods and Results: MSCs isolated from porcine adipose tissue were CD11b-CD34-CD44+CD73+CD90+ and showed characteristics of MSCs. The MSCs were stimulated and differentiated into ECs with endothelial growth media (EGM+50ng/ml of VEGF) and EGM media containing 10nM of calcitriol (EGM+50ng/ml of VEGF +10nM calcitriol) for 10 days. The EC differentiation was assessed by mRNA expression by qPCR and protein expression by flow cytometry for endothelial cell markers. Calcitriol enhanced EGM+VEGF-induced differentiation of MSCs into ECs, as revealed by 2-fold increase in mRNA and 4-fold increase in protein expression of EC markers. Angiogenesis assay and acetylated low density lipoprotein (LDL) uptake assay were used to assess the endothelial functionality that showed significant increase in capillary tube sprouting, and an increase in LDL uptake by the differentiated cells in response to EGM +VEGF+ calcitriol. Examination of the findings from Wnt Pathway array revealed a 3-fold decrease in β-catenin and 3-fold increase in KREMEN1 protein in the cells treated with EGM + VEGF+calcitriol. Conclusion: Vitamin D significantly enhanced VEGF-induced differentiation of MSCs into endothelial cells. Thus, vitamin D status of the patients undergoing coronary intervention might regulate the development of thrombosis and intimal hyperplasia.

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model.

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.

MMP‐2 and MT1‐MMP Silencing Induces the Differentiation of Porcine Adipose‐Derived Mesenchymal Stem Cells to Endothelial Cells

Rationale: The molecular mechanisms that control the ability of adipose-derived mesenchymal stem cells (ADMSCs) to remodel 3-dimensional extracellular matrix barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of AMSCs to endothelial cells (ECs). Methods: MSCs were isolated from porcine abdominal adipose tissue, and characterized by CD44+, CD90+, CD105+, CD14-, and CD45-. The plasticity of AMSCs was detected by multi-lineage differentiation. The mRNA transcripts, protein expression of EC marker, MMPs and TIMPs, and enzyme activity of MMP-2 were analyzed. Results: The differentiation of ADMSCs to ECs was confirmed by protein and mRNA expression of EC markers. The mRNA transcripts for MMP-2 and MT1-MMP were significantly increased during the differentiation. Western blot, zymography and ELISA showed an elevated MMP-2 and MT1-MMP expression and activity. MMP2 and MT1-MMP silencing showed significant increase in the expression of EC markers, formation of capillary tubes, and acetylated-LDL uptake. Conclusion We, for the first time, report that the up-regulation of MMP2 and MT1-MMP has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs can induce the differentiation. These findings could have significant clinical impact in developing better therapeutic approaches to re-endothelialize coronary arteries following interventional procedures or inducing angiogenesis to regenerate myocardium.

Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial-to-mesenchymal transition (EndoMT)

Myofibroblasts expressing α-smooth muscle actin (α-SMA) are the key cellular mediator of fibrosis. Fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) are characterized by the accumulation of a large number of myofibroblasts. We explored the hypothesis that proliferating endothelial cells via endothelial-to-mesenchymal transition (EndoMT) and/or bone marrow-derived circulating fibrocytes contribute to the myofibroblast population present in PDR epiretinal membranes. Epiretinal membranes from 14 patients with PDR were studied by immunohistochemistry. All membranes contained neovessels expressing the endothelial cell marker CD31. CD31+ endothelial cells co-expressed the fibroblast/myofibroblast markers fibroblast-specific protein-1 (FSP-1) and α-SMA, indicative for the occurrence of endoMT. In the stroma, cells expressing FSP-1, α-SMA, the leukocyte common antigen CD45, and the myelomonocytic marker CD11b were detected. Double labeling showed co-localization of CD45 with FSP-1 and α-SMA and co-localization of CD11b with α-SMA and matrix metalloproteinase-9, demonstrating the presence of infiltrating fibrocytes. In addition, we investigated the phenotypic changes that take place in human retinal microvascular endothelial cells following exposure to transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Retinal microvascular endothelial cells changed morphology upon cytokine exposure, lost the expression of endothelial cell markers (endothelial nitric oxide synthase and vascular endothelial-cadherin) and started to express mesenchymal markers (calponin, snail, transgelin and FSP-1). These results suggest that endothelial cells as well as circulating fibrocytes may differentiate into myofibroblasts in the diabetic eye and contribute to pathologic fibrosis in PDR.

Synergistic effect of angiotensin II on vascular endothelial growth factor-A-mediated differentiation of bone marrow-derived mesenchymal stem cells into endothelial cells.

IntroductionIncreased levels of angiotensin II (Ang II) and activity of Ang II receptor type 1 (AT1R) elicit detrimental effects in cardiovascular disease. However, the role of Ang II receptor type 2 (AT2R) remains poorly defined. Mesenchymal stem cells (MSCs) replenish and repair endothelial cells in the cardiovascular system. Herein, we investigated a novel role of angiotensin signaling in enhancing vascular endothelial growth factor (VEGF)-A-mediated differentiation of MSCs into endothelial cells (ECs).MethodsBone marrow was aspirated from the femurs of Yucatan microswine. MSCs were extracted via ficoll density centrifugation technique and were strongly immunopositive for MSC markers, CD44, CD90, and CD105, but negative for hematopoietic markers, CD14 and CD45. Subsequently, naïve MSCs were differentiated for 10 days in varying concentrations and combinations of VEGF-A, Ang II, and AT1R or AT2R antagonists. Markers specific to ECs were determined by FACS analysis.ResultsAT1R and AT2R expression and cellular localization was demonstrated in MSCs stimulated with VEGF-A and Ang II via quantitative RT-PCR and immunofluorescence, respectively. Differentiation of naïve MSCs in media containing Ang II (2 ng/ml) plus low-dose VEGF-A (2 ng/ml) produced a significantly higher percentage of cells that were positive for expression of EC markers (for example, platelet endothelial cell adhesion molecule, vascular endothelial Cadherin and von Willebrand factor) compared to VEGF-A alone. Ang II alone failed to induce EC marker expression. MSCs differentiated with the combination of Ang II and VEGF-A were capable of forming capillary tubes using an in vitro angiogenesis assay. Induction of EC marker expression was greatly attenuated by co-treatment of Ang II/VEGF-A with the AT2R antagonist PD123319, but not the AT1R antagonist telmisartan.ConclusionsWe report the presence of functional AT2R receptor on porcine bone marrow-derived MSCs, where it positively regulates EC differentiation. These findings have significant implications toward therapeutic approaches based on activation of AT2R, which could be a means to stimulate regeneration of damaged endothelium and prevent vascular thrombosis.

Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells.

Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs) differentiate into endothelial cells (ECs) in the presence of VEGF-A in vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, we examined the effect of atherogenic cytokines (IL-6, TNFα, and Ang II) on EC differentiation and function. MSCs (CD44+, CD73+, CD90+, CD14−, and CD45−) were isolated from the bone marrow of Yucatan microswine. Naïve MSCs cultured in differentiation media containing VEGF-A (50 ng/mL) demonstrated increased expression of EC-specific markers (vWF, PECAM-1, and VE-cadherin), VEGFR-2 and Sox18, and enhanced endothelial tube formation. IL-6 or TNFα caused a dose-dependent attenuation of EC marker expression in VEGF-A-stimulated MSCs. In contrast, Ang II enhanced EC marker expression in VEGF-A-stimulated MSCs. Addition of Ang II to VEGF-A and IL-6 or TNFα was sufficient to rescue the EC phenotype. Thus, Ang II promotes but IL-6 and TNFα inhibit VEGF-A-induced differentiation of MSCs into ECs. These findings have important clinical implications for therapies intended to increase cardiac vascularity and reendothelialize coronary arteries following intervention.

Use of human serum for human corneal endothelial cell culture

Background/aimsTo study human corneal endothelial cells (HCECs) cultured in vitro with human serum (HS) supplemented media (HS-SM) compared with HCEC cultured in fetal bovine serum (FBS) supplemented media (FBS-SM).MethodsOne cornea...

AngioMatrix, a signature of the tumor angiogenic switch-specific matrisome, correlates with poor prognosis for glioma and colorectal cancer patients.

Angiogenesis represents a rate-limiting step during tumor progression. Targeting angiogenesis is already applied in cancer treatment, yet limits of anti-angiogenic therapies have emerged, notably because tumors adapt and recur after treatment. Therefore, there is a strong need to better understand the molecular and cellular mechanisms underlying tumor angiogenesis. Using the RIP1-Tag2 transgenic murine model, we identified 298 genes that are deregulated during the angiogenic switch, revealing an ingression/expansion of specific stromal cell types including endothelial cells and pericytes, but also macrophages and perivascular mesenchymal cells. Canonical TGF-β signaling is up-regulated during the angiogenic switch, especially in tumor-associated macrophages and fibroblasts. The matrisome, comprising extracellular matrix (ECM) and ECM-associated molecules, is significantly enriched, which allowed us to define the AngioMatrix signature as the 110 matrisomal genes induced during the RIP1-Tag2 angiogenic switch. Several AngioMatrix molecules were validated at expression level. Ablation of tenascin-C, one of the most highly induced ECM molecules during the switch, resulted in reduced angiogenesis confirming its important role. In human glioma and colorectal samples, the AngioMatrix signature correlates with the expression of endothelial cell markers, is increased with tumor progression and finally correlates with poor prognosis demonstrating its diagnostic and therapeutic potential.

Impairing Eukaryotic Elongation Factor 2 Kinase Activity Decreases Atherosclerotic Plaque Formation

We tested whether loss of eukaryotic elongation factor 2 kinase (eEF2K) activity in macrophages suppresses development of atherosclerosis by transplanting bone marrow from mice with mutant eEF2K into ldlr−/− mice. Sixteen weeks after high-fat diet feeding, mutant eEF2K hematopoietic chimeras had a dramatically reduced level of atherosclerotic plaque formation. M1-skewed macrophages from eEF2K knock-in mice have less tumour necrosis factor-α release and a lesser ability to induce expression of endothelial cell markers, providing a potential explanation for the role of eEF2K. Because eEF2K activity in cells of the hematopoietic compartment contributes to atherosclerosis development, drugs inhibiting eEF2K might have a beneficial effect in treatment of atherosclerosis.

Endothelial Lineage Differentiation from Induced Pluripotent Stem Cells Is Regulated by MicroRNA-21 and Transforming Growth Factor β2 (TGF-β2) Pathways

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.

Activin ligands are required for the re-activation of Smad2 signalling after neurulation and vascular development in Xenopus tropicalis.

The importance of Transforming Growth Factor β (TGFβ) signalling during early development has been well established. In particular, Nodal ligands have been shown to play essential roles for the specification and the patterning of the mesendoderm, axes formation and organogenesis. Activin ligands, like Nodal, signal by inducing the phosphorylation of the intracellular signal transducers Smad2 and Smad3. However, the roles of Activins during embryonic development are much less understood. Here, we report that during Xenopus tropicalis development two waves of Smad2 phoshorylation can be observed, first during gastrulation and then a second one after neurulation. Using a knock-down approach, we show that the second wave of Smad2 phosphorylation depends on activinβa (actβa) and activinβb (actβb) expression. Knocking down the expression of actβa, or treating the embryos with a chemical inhibitor inhibiting TGFβ receptor I (TGFβRI) activity after neurulation result in a decrease of the expression of endothelial cell markers and a lack of blood flow in Xenopus tadpoles. Taken together these data suggest that Activin ligands play an important role during vascular development in Xenopus tropicalis embryos.