Vascular Tube Formation Research Articles

Abstract A37: MicroRNA-26b potentiates cytotoxicity of malignant glioma cells by radiation

Abstract MicroRNA-26b is known to be upregulated in high grade glioma and to play a crucial role in epithelial to mesenchymal transition (EMT). In this study, we evaluated the potential of targeting miRNA-26b to overcome the radioresistance of human malignant glioma cells. We also aimed to elucidate the mechanisms of radiosensitization by miR-26b. U251 human glioma cells having activation of PI3K signaling due to deletion of PTEN were transfected with pre-miR-26b or control pre-miRNA. Ectopic overexpression of miR-26b attenuated the expression of p-AKT and enhanced the cytotoxicity of U251 cells by radiation. Overexpression of miR-26b prolonged radiation-induced gamma H2AX foci formation and attenuated the expression of p-DNA-PKcs. Increased expression of mature miR-26b also suppressed invasion, migration, vascular tube formation, and led to down-regulation of HIF-1alpha, VEGF and MMP-2 expression. In contrast, overexpression of miR-26b did not increased radiaton toxicity of normal human astrocytes. Taken together, these findings suggest that miR-26b could be an attractive target for overcoming radioresistnace of human malignant glioma cells via unique modulation of the cancer pro-survival signaling and EMT. Citation Format: David J. Lee, Yeo-Hyun Hwang, In Ah Kim. MicroRNA-26b potentiates cytotoxicity of malignant glioma cells by radiation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A37.

The Role of EGFL7 in Trophoblast Cells and Endothelial Cells during Placental Development

The ability to form and maintain a functional system of contiguous hollow tubes is a critical feature of vascular endothelial cells (ECs). Lumen formation, or tubulogenesis, occurs in blood vessels during both vasculogenesis and angiogenesis in the embryo. Formation of vascular lumens takes place prior to the establishment of blood flow and to vascular remodeling which results in a characteristic hierarchical vessel organization. While epithelial lumen formation has received intense attention in past decades, more recent work has only just begun to elucidate the mechanisms controlling the initiation and morphogenesis of endothelial lumens. Studies using in vitro and in vivo models, including zebrafish and mammals, are beginning to paint an emerging picture of how blood vessels establish their characteristic morphology and become patent. In this article, we review and discuss the molecular and cellular mechanisms driving the formation of vascular tubes, primarily in vivo, and we compare and contrast proposed models for blood vessel lumen formation.

Protein kinase C regulates FLT1 abundance and stimulates its cleavage in vascular endothelial cells with the release of a soluble PlGF/VEGF antagonist

FLT1 and its soluble form (sFLT1) arise as alternate transcripts from the same gene and sFLT1 can antagonize the effect of vascular endothelial growth factor (VEGF) on its cognate receptors. We investigated the effect of VEGF and protein kinase C (PKC) activation on sFLT1 abundance. We demonstrated that VEGF stimulates sFLT1 and FLT1 mRNA and protein levels in vascular endothelial cells via VEGFR2 and PKC. Using an FLT1 expression vector with N and C-terminal epitope tags, we show that PKC activation increases the cleavage of FLT1 into an N-terminal extracellular fragment and a C-terminal intracellular fragment with the cleavage occurring adjacent to the transmembrane domain. The trafficking and glycosylation inhibitors brefeldin, monensin and tunicamycin substantially reduced cleavage and release of the N-terminal ectodomain of FLT1 and inhibited secretion of the isoforms of sFLT1. The shed FLT1 ectodomain can bind VEGF and PlGF and inhibit VEGF-induced vascular tube formation thus confirming that it is functionally equivalent to the alternately spliced and secreted sFLT1 isoforms.

Potential anti-angiogenic sulfates of andrographolide

A new regioisomer of andrographolide, 17-hydro-9-dehydro-andrographolide (1), and five new sulfates of andrographolide (2–6) were isolated from Xiyanping, a China licensed anti-inflammatory drug derived from andrographolide through sulfation reaction. Their chemical structures were elucidated by spectroscopic and chemical methods. The inhibition effects of these compounds on angiogenesis were evaluated by rat aortic ring assay. Compounds 1 and 3 exhibited strong inhibitory activities on vascular endothelial cell tube formation in rat aorta ring at the concentration of 1 μM. Compounds 4 and 5 showed moderate suppression on angiogenesis at 10 μM.

ANGPTL4 is a secreted tumor suppressor that inhibits angiogenesis

Tumor suppressors with extracellular function are likely to have advantages as targets for cancer therapy, but few are known. Here, we focused on angiopoietin-like 4 (ANGPTL4), which is a secreted glycoprotein involved in lipoprotein metabolism and angiogenesis, is methylation-silenced in human cancers, but has unclear roles in cancer development and progression. We found a deletion mutation in its coiled-coil domain at its N-terminal in human gastric cancers, in addition to hypermethylation of the ANGPTL4 promoter CpG islands. Forced expression of wild-type ANGPTL4, but not ANGPTL4 with the deletion, at physiological levels markedly suppressed in vivo tumorigenicity and tumor angiogenesis, indicating that the latter caused the former. Tumor-derived ANGPTL4 suppressed in vitro vascular tube formation and proliferation of human umbilical vascular endothelial cells, partly due to suppression of ERK signaling. These showed that ANGPTL4 is a genetically and epigenetically inactivated secreted tumor suppressor that inhibits tumor angiogenesis.

Organosulfur Garlic Compounds Induce Neovasculogenesis in Human Endothelial Progenitor Cells through a Modulation of MicroRNA 221 and the PI3-K/Akt Signaling Pathways

Human endothelial progenitor cells (EPCs) play crucial roles in the prevention of ischemic injury via neovasculogenesis. Frequent garlic consumption is reportedly associated with a low incidence of cardiovascular diseases (CVD). However, the molecular mechanisms by which garlic extracts, including diallyl disulfide (DADS) and diallyl trisulfide (DATS), exert an effect on neovasculogenesis have not been elucidated yet. The current study investigated the effects of these organosulfur compounds on neovasculogenesis by using vascular tube formation assay, Western blotting assay, real-time polymerase chain reaction (RT-PCR), and immunohistochemical (IHC) staining assays in both in vitro and in vivo models. The current study demonstrates that DADS and DATS dose-dependently enhance the neovasculogenesis of human EPCs in vitro. The mechanism of actions included the up-regulation of the c-kit protein, as well as the phosphorylation (i.e., activation) of the Akt and ERK 1/2 signaling molecules in human EPCs. Furthermore, DATS suppressed the expression of microRNA (miR) 221 in vitro. In a mouse xenograft model of neovasculogenesis, DATS consumption induced the formation of new blood vessels at a dosage of 10 mg/kg of body weight/day. It is suggested that garlic consumption enhances neovasculogenesis in human EPCs and thereby probably exerts a preventive effect against ischemic injuries.

MicroRNA-1 enhances the angiogenic differentiation of human cardiomyocyte progenitor cells

Instigated by the discovery of adult cardiac progenitor cells, cell replacement therapy has become a promising option for myocardial repair in the past decade. We have previously shown that human-derived cardiomyocyte progenitor cells (hCMPCs) can differentiate into cardiomyocyte-, endothelial-, and smooth muscle-like cells in vitro, and in vivo after transplantation in a mouse model of myocardial infarction, resulting in preservation of cardiac function. However, to allow successful repopulation of the injured myocardium, it is of key importance to restore myocardial perfusion by the formation of new vasculature. Several studies have shown that microRNAs regulate vascular differentiation of different stem/progenitor cells. Here, we show that miR-1 is upregulated in hCMPCs during angiogenic differentiation. Upregulation of miR-1 enhanced the formation of vascular tubes on Matrigel and within a collagen matrix, and also increased hCMPC motility, as shown by planar and transwell migration assays. By western blot, qRT-PCR and luciferase reporter assays, miR-1 was found to directly target and inhibit the expression of sprouty-related EVH1 domain-containing protein 1 (Spred1). Knocking down Spred1 phenocopies the functional effect seen for miR-1 upregulation. Using a systems biology approach, we found that in hCMPCs, miR-1 is proposed to control a network of genes predominantly involved in angiogenesis-related processes, including the Spred1 pathway. Our data shows that by upregulation of miR-1, the angiogenic differentiation of hCMPCs can be enhanced, which may be used as a new therapeutic approach to improve the efficiency of cell-based therapy for cardiac regeneration by enhancing the formation of new vasculature.

Coating Decellularized Equine Carotid Arteries with CCN1 Improves Cellular Repopulation, Local Biocompatibility, and Immune Response in Sheep

Decellularized equine carotid arteries (dEAC) are potential alternatives to alloplastic vascular grafts although there are certain limitations in biocompatibility and immunogenicity. Here, dEAC were coated with the matricellular protein CCN1 and evaluated in vitro for its cytotoxic and angiogenic effects and in vivo for cellular repopulation, local biocompatibility, neovascularization, and immunogenicity in a sheep model. CCN1 coating resulted in nontoxic matrices not compromising viability of L929 fibroblasts and endothelial cells (ECs) assessed by WST-8 assay. Functionality of CCN1 was maintained as it induced typical changes in fibroblast morphology and MMP3 secretion. For in vivo testing, dEAC±CCN1 (n=3 each) and polytetrafluoroethylene (PTFE) protheses serving as controls (n=6) were implanted as cervical arteriovenous shunts. After 14 weeks, grafts were harvested and evaluated immunohistologically. PTFE grafts showed a patency rate of only 33% and lacked cellular repopulation. Both groups of bioartificial grafts were completely patent and repopulated with ECs and smooth muscle cells (SMCs). However, whereas dEAC contained only patch-like aggregates of SMCs and a partial luminal lining with ECs, CCN1-coated grafts showed multiple layers of SMCs and a complete endothelialization. Likewise, CCN1 coating reduced leukocyte infiltration and fibrosis and supported neovascularization. In addition, in a three-dimensional assay, CCN1 coating increased vascular tube formation in apposition to the matrix 1.6-fold. Graft-specific serum antibodies were increased by CCN1 up to 6 weeks after implantation (0.89±0.03 vs. 1.08±0.04), but were significantly reduced after 14 weeks (0.85±0.04 vs. 0.69±0.02). Likewise, restimulated lymphocyte proliferation was significantly lower after 14 weeks (1.78±0.09 vs. 1.32±0.09-fold of unstimulated). Thus, CCN1 coating of biological scaffolds improves local biocompatibility and accelerates scaffold remodeling by enhancing cellular repopulation and immunologic tolerance, making it a promising tool for generation of bioartificial vascular prostheses.

Synergy between Kaposi's sarcoma-associated herpesvirus (KSHV) vIL-6 and HIV-1 Nef protein in promotion of angiogenesis and oncogenesis: role of the AKT signaling pathway

Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma (KS), which is the most common AIDS-associated malignancy. KS is characterized by neovascularization and spindle cell proliferation. The interaction between HIV-1 and KSHV has a central role in promoting the aggressive manifestations of KS in AIDS patients; however, the pathogenesis underlying AIDS-related KS (AIDS-KS) remains unknown. Herein, we examined the potential of HIV-1 negative factor (Nef) to impact KSHV viral interleukin-6 (vIL-6)-induced angiogenesis and tumorigenesis. In vitro experiments showed that exogenous Nef penetrated vIL-6-expressing endothelial cells. Both internalized and ectopic expression of Nef in endothelial cells and fibroblasts synergized with vIL-6 to promote vascular tube formation and cell proliferation. Using a chicken chorioallantoic membrane (CAM) model, we demonstrated that Nef synergistically promotes vIL-6-induced angiogenesis and tumorigenesis. Animal experiments further showed that Nef facilitates vIL-6-induced angiogenesis and tumor formation in athymic nu/nu mice. Mechanistic studies indicated that Nef synergizes with vIL-6 to enhance angiogenesis and tumorigenesis by activating the AKT pathway in the CAM model, as well as nude mice. LY294002, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K), significantly impaired the ability of Nef to promote vIL-6-induced tumorigenesis in an allograft model of nude mice. Our data provide first-line evidence that Nef may contribute to the pathogenesis underlying AIDS-KS in synergy with vIL-6. These novel findings also suggest that targeting the PI3K/AKT signal may be a potentially effective therapeutic approach in AIDS-KS patients.

Abstract 1615: Inhibition of vascular endothelial cell tube formation by zinc nanoparticles.

Abstract Tumor angiogenesis, new blood vessel formation induced by cancer cells, is a rate-limiting step in cancer growth and metastasis, and is therefore an excellent target for therapy. Currently, the anticancer drugs based on anti-angiogenic strategies have limited efficacy, and often engender inherent or acquired resistance. Indeed, there is growing evidence to suggest that anti-angiogenic treatment of cancer using current inhibitors may trigger more invasive and metastatic tumors due to the fact that when one angiogenic signaling pathway is blocked, new signaling pathways are triggered. We have tackled the problem from a different angle by targeting the copper ion rather than the many angiogenesis inducing biomolecules. Copper is a co-factor for more than a dozen key angiogenic promoters essential for cancer angiogenesis. As such, depletion of copper should inactivate multiple angiogenic signaling pathways. Specifically, we have developed a nanoparticle-based drug to selectively sequester copper ions in order to disrupt tumor angiogenesis, resulting in inhibition of tumor growth and metastasis. Cellular uptake of nanoparticles (NPS) was documented by confocal microscopy of HuVEC and cancer cell lines treated with carboxyfluorescein tagged NPS. Copper levels were measured by atomic absorption spectrophotometry of lysates of CuCl2 treated cells, after 8-hr treatment with NPS or control diluent. Following treatment of HepG2 cells with copper depleting NPS, we observed that copper levels dropped from 402 fg/cell to undetectable levels. Although NPS significantly reduced copper levels of CuCl2 treated HepG2 cells, cell viability measured by trypan blue exclusion and MTT assays was > 89% (for 50 uM NPS). Similar viability was seen for the three cell lines tested. Using an in vitro model system for angiogenesis, we have examined induction of tube formation by human Vascular Endothelial cells (HuVEC) cultured on basement membrane extracts. Following induction by fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) treatment, outgrowth and branching of HuVEC cells was measured in the presence or absence of NPS. We observed that the copper depleting NPS suppressed FGF2 induction of tube formation and branching by HuVEC cells. Overall our results have demonstrated that these novel zinc NPS: (i) are highly effective copper depleting agents able to accumulate in endothelial cells; (ii) are non-toxic to vascular endothelial and cancer cells, and (iii) inhibit endothelial cell tube formation in vitro. We expect these novel copper-depleting agents will significantly impact tumor angiogenesis in vivo and dramatically enhance cancer therapy in the future. Citation Format: Vindya S. Perera, Haiwa Wu, Liu D. Yang, Songping D. Huang, Gail C. Fraizer. Inhibition of vascular endothelial cell tube formation by zinc nanoparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1615. doi:10.1158/1538-7445.AM2013-1615

Abstract 3097: microRNA-21 inhibitor potentiates anti-tumor effect of radiation therapy in vitro and in vivo.

Abstract microRNA-21 (miR-21) plays significant roles in carcinogenesis and is highly expressed in diverse human cancers. In this study, we evaluated the potential of targeting miRNA-21 to overcome the radioresistance of human cancer cells having an activated PI3K-Akt signaling and also aimed to elucidate the mechanisms of radiosensitization, and the effect on epithelial- mesenchymal transition (EMT). A panel of human cancer cells (U251, U87, A549, MDA-MB-468, SKBR-3 cells) were transfected with miR-21 led to up-regulation of PI3K-Akt signaling and increased radioresistance compared with the cancer cells that were treated with nonspecific control. In contrast, a specific inhibitor of miR-21, an antipeptide nucleic acid (PNA) attenuated PI3K-Akt signaling and radiosensitized a panel of human cancer cells we tested. Inhibition of miR-21 was associated with persistent γH2AX foci formation and down-regulation of p-DNA-PKcs. While ectopic overexpression of miR-21 exhibited higher invasion and migration potential and increased vascular tube formation, inhibitor of miR-21 decreased these typical features of EMT. Treatment of anti-miR-21 significantly decreased tumor burden in nude mice bearing intracranial U251 xenografts compared to control as measured by bioluminescence. Combined treatment of anti-miR-21 and radiation further decreased tumor burden compared to each treatment alone. In summary, miR-21 is an important onco-miR which confers radioresistance and diverse features of EMT. Inhibition of miR-21 could be a potential strategy for improving the efficacy of radiation therapy via unique modulation of PI3K-Akt signaling and EMT. Citation Format: Bong Jun Cho, Eun Jung Choi, Hans H. Kim, David J. Lee, Sun Ha Chun, In-Ah Kim. microRNA-21 inhibitor potentiates anti-tumor effect of radiation therapy in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3097. doi:10.1158/1538-7445.AM2013-3097

Regulation of tumor angiogenesis by the microtubule-binding protein CLIP-170

Angiogenesis, the expansion of preexisting blood vessels, is a complex process required for tumor growth and metastasis. Although current antiangiogenic strategies have shown promising results in several cancer types, identification of additional antiangiogenic targets is required to improve the therapeutic response. Herein, we show that the microtubule-binding protein CLIP-170 (cytoplasmic linker protein of 170 kDa) is highly expressed in breast tumor samples and correlates positively with blood vessel density. Depletion of CLIP-170 significantly impaired vascular endothelial tube formation and sprouting in vitro and inhibited breast tumor growth in mice by decreasing tumor vascularization. Our data further show that CLIP-170 is important for the migration but not the proliferation of vascular endothelial cells. In addition, CLIP-170 promotes the polarization of endothelial cells in response to the angiogenic stimulus. These findings thus demonstrate a critical role for CLIP-170 in tumor angiogenesis and suggest its potential as a novel antiangiogenic target.

Suppression of renal cell carcinoma growth in vivo by forced expression of vascular endothelial growth inhibitor

Vascular endothelial growth inhibitor (VEGI) has been associated with tumor-related vasculature in certain malignancies. However, its implication in renal cell carcinoma (RCC), an angiogenesis-dependent tumor, remains unknown. In the present study, we investigated the role played by VEGI in RCC. The expression of VEGI was examined in human renal tissue and RCC cell lines using immunohistochemical staining and RT-PCR, respectively. The biological impact of modifying the expression of VEGI in RCC cells was evaluated using in vitro and in vivo models. We show that VEGI mRNA is expressed in a wide variety of human RCC cell lines, all of normal renal and most of RCC tissue specimens. VEGI protein expression was observed in normal renal tubular epithelial cells, but was decreased or absent in RCC specimens, particularly in tumors with high grade. Moreover, forced expression of VEGI led to an inhibition of vascular endothelial tube formation, decrease in the motility and adhesion of RCC cells in vitro. Interestingly, forced expression of VEGI had no bearing on growth, apoptosis and invasive capacity of RCC cells. However, tumor growth was reduced in xenograft models. Immunohistochemical staining showed that microvessel density decreased in VEGI forced expression xenograft tumor samples. Taken together, our findings showed that the expression of VEGI is decreased in RCC, particularly in tumors with higher grade. Together with its inhibitory effect on cellular motility, adhesion, vascular endothelial tube formation and tumor growth in vivo, this suggests that VEGI functions mainly through inhibition of angiogenesis and is a negative regulator of aggressiveness during the development and progression of RCC.

Comparing the angiogenic potency of naïve marrow stromal cells and Notch-transfected marrow stromal cells

BackgroundAngiogenesis is a critical part of the endogenous repair process in brain injury and disease, and requires at least two sequential steps. First, angiogenic sprouting of endothelial cells occurs, which entails the initial proliferation of endothelial cells and remodeling of the surrounding extracellular matrix. Second, vessel stabilization is necessary to prevent vascular regression, which relies on vascular smooth muscle recruitment to surround the young vessels. Marrow stromal cells (MSCs) have been shown to promote revascularization after hindlimb ischemia, cardiac ischemia, and stroke. SB623 cells are derived from marrow stromal cells by transfection with a Notch1 intracellular domain (NICD)-expressing plasmid and are known to elicit functional improvement in experimental stroke. These cells are currently used in human clinical testing for treatment of chronic stroke. In the current study, the angiogenic property of SB623 cells was investigated using cell-based assays.MethodsAngiogenic paracrine factors secreted by SB623 cells and the parental MSCs were identified using the Qantibody Human Angiogenesis Array. To measure the angiogenic activity of conditioned medium from SB623 cells and MSCs, endothelial tube formation in the human umbilical vein endothelial cell (HUVEC) assay and endothelial cell sprouting and branching in the rodent aortic ring assay were quantified. To validate the angiogenic contribution of VEGF in conditioned medium, endothelial cells and aortic rings were treated with SU5416, which inhibits VEGFR2 at low dose.ResultsConditioned medium from SB623 cells promoted survival and proliferation of endothelial cells under serum-deprived conditions and supports HUVEC vascular tube formation. In a rodent aortic ring assay, there was enhanced endothelial sprouting and branching in response to SB623-derived conditioned medium. SU5416 treatment partially reversed the effect of conditioned medium on endothelial cell survival and proliferation while completely abrogate HUVEC tube formation and endothelial cell sprouting and branching in aortic ring assays.ConclusionsThese data indicate that SB623 cell-secreted angiogenic factors promoted several aspects of angiogenesis, which likely contribute to promoting recovery in the injured brain.

The biological effects and mechanisms of calcitonin gene-related peptide on human endothelial cell

Background: Calcitonin gene-related peptide (CGRP) is a neuropeptide distributed in bone tissue involved in bone remodeling. Previously we demonstrated that CGRP can promote proliferation and migration of endothelial cells, relating to the expression of vascular endothelial growth factor (VEGF) and focal adhesion kinase (FAK). Methods: CGRP1 receptor expression in human umbilical vein endothelial cells (HUVECs) was examined by immunofluorescence microscopy and real-time PCR. Tube formation was measured by a Matrigel tube formation assay. VEGF protein and mRNA levels were quantified by ELISA and real-time PCR, respectively. The expression of VEGF receptor 1 (FLT1) and VEGF receptor 2 (KDR) were measured by real-time PCR and immunoblotting assays. Results: CGRP significantly induced vascular tube formation of outgrowth HUVECs in a Matrigel. The expression of FLT and KDR were significantly increased by CGRP, and CGRP enhanced the expression of CGRP1 receptors. Compared to the known angiogenesis regulator VEGF165, CGRP had an equal or stronger effect on migration and tube formation, but not on proliferation of endothelial cells. The upregulation of calcitonin receptor-like receptor (CRLR), FAK, VEGF and its two main receptors (FLT1, KDR) by CGRP was also more pronounced than that obtained by VEGF165. Conclusion: It is concluded that CGRP is a strong proangiogenic growth factor, thereby contributing to bone development and remodeling by promoting angiogenesis.

Synthesis and evaluation of nicotinamide derivative as anti-angiogenic agents

Previously, we have found that BRN-103, a nicotinamide derivative, inhibits vascular endothelial growth factor (VEGF)-mediated angiogenesis signaling in human endothelial cells. During our continuous efforts to identify more potent anti-angiogenic agents, we synthesized various nicotinamide derivatives and evaluated their anti-angiogenic effects. We found that 2-{1-[1-(6-chloro-5-fluoropyrimidin-4-yl)ethyl]piperidin-4-ylamino}-N-(3-chlorophenyl) pyridine-3-carboxamide (BRN-250) significantly inhibited human umbilical vascular endothelial cells (HUVECs) proliferation, migration, tube formation, and microvessel growth in a concentration range of 10–100nM. Furthermore, BRN-250 inhibited the VEGF-induced phosphorylation and intracellular tyrosine kinase activity of VEGF receptor 2 (VEGFR2) and the activation of its downstream AKT pathway. Taken together, these findings suggest that BRN-250 be considered a potential lead compound for cancer therapy.

Inhibitory Effect of Adenoviral Vector-Mediated Delivery of p21WAF1/CIP1 on Retinal Vascular Endothelial Cell Proliferation and Tube Formation in Cultured Rhesus Monkey Cells (RF/6A)

Objective: To investigate the inhibitory effect(s) of adenovirus (Ad) vector-mediated delivery of p21WAF1/CIP1 (Ad-p21) on proliferation and tube formation in Rhesus monkey choroid-retina vascular endothelial cells (RF/6A).Methods: In vitro-cultured RF/6A cells were divided into three groups: phosphate-buffered saline (PBS), Ad-p21-transfected, and negative control. Plasmid vectors were transfected via Ad-p21. The mRNA and protein expressions of p21 and cyclin-dependent kinase (CDK)2 in RF/6A cells were measured by reverse transcription-PCR (RT-PCR) and western blot analyses. Cell-cycle distributions were analyzed by flow cytometry. Matrigel was used as a matrix for endothelial cell tube formation.Results: Expressions of p21 mRNA and protein were greater, and expressions of CDK2 mRNA and protein lower, in the Ad-p21-transfected group than in either the PBS or negative control groups. Cell-cycle distribution analysis indicated that the proportion of G0/G1 cells was significantly higher in the Ad-p21 transfected group than in either the PBS or negative control groups (p = 0.000). There were significantly fewer endothelial cell tubes in the Ad-p21-transfected group than in either the PBS or negative control groups (p = 0.004).Conclusions: Ad-p21 inhibits RF/6A cell proliferation and tube formation. The underlying mechanism to account for this may be that overexpression of p21 arrests the cell-cycle transition from the G1- to the S-phase via inhibition of CDK2 activity.

Identification of DEAD-box RNA Helicase 6 (DDX6) as a Cellular Modulator of Vascular Endothelial Growth Factor Expression under Hypoxia*

Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5'-untranslated region (5'-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5'-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5'-UTR and 3'-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5'-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.

Spindle Shaped Human Mesenchymal Stem/Stromal Cells from Amniotic Fluid Promote Neovascularization

Human amniotic fluid obtained at amniocentesis, when cultured, generates at least two morphologically distinct mesenchymal stem/stromal cell (MSC) subsets. Of these, the spindle shaped amniotic fluid MSCs (SS-AF-MSCs) contain multipotent cells with enhanced adipogenic, osteogenic and chondrogenic capacity. Here, we demonstrate, for the first time, the capacity of these SS-AF-MSCs to support neovascularization by umbilical cord blood (UCB) endothelial colony forming cell (ECFC) derived cells in both in vitro and in vivo models. Interestingly, although the kinetics of vascular tubule formation in vitro were similar when the supporting SS-AF-MSCs were compared with the best vasculogenic supportive batches of bone marrow MSCs (BMSCs) or human dermal fibroblasts (hDFs), SS-AF-MSCs supported vascular tubule formation in vivo more effectively than BMSCs. In NOD/SCID mice, the human vessels inosculated with murine vessels demonstrating their functionality. Proteome profiler array analyses revealed both common and distinct secretion profiles of angiogenic factors by the SS-AF-MSCs as opposed to the hDFs and BMSCs. Thus, SS-AF-MSCs, which are considered to be less mature developmentally than adult BMSCs, and intermediate between adult and embryonic stem cells in their potentiality, have the additional and very interesting potential of supporting increased neovascularisation, further enhancing their promise as vehicles for tissue repair and regeneration.

HIV-1 Tat Promotes Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) vIL-6-Induced Angiogenesis and Tumorigenesis by Regulating PI3K/PTEN/AKT/GSK-3β Signaling Pathway

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is etiologically associated with KS, the most common AIDS-related malignancy. KS is characterized by vast angiogenesis and hyperproliferative spindle cells. We have previously reported that HIV-1 Tat can trigger KSHV reactivation and accelerate Kaposin A-induced tumorigenesis. Here, we explored Tat promotion of KSHV vIL-6-induced angiogenesis and tumorigenesis. Tat promotes vIL-6-induced cell proliferation, cellular transformation, vascular tube formation and VEGF production in culture. Tat enhances vIL-6-induced angiogenesis and tumorigenesis of fibroblasts and human endothelial cells in a chicken chorioallantoic membrane (CAM) model. In an allograft model, Tat promotes vIL-6-induced tumorigenesis and expression of CD31, CD34, SMA, VEGF, b-FGF, and cyclin D1. Mechanistic studies indicated Tat activates PI3K and AKT, and inactivates PTEN and GSK-3β in vIL-6 expressing cells. LY294002, a specific inhibitor of PI3K, effectively impaired Tat’s promotion of vIL-6-induced tumorigenesis. Together, these results provide the first evidence that Tat might contribute to KS pathogenesis by synergizing with vIL-6, and identify PI3K/AKT pathway as a potential therapeutic target in AIDS-related KS patients.