Comparing the angiogenic potency of naive marrow stromal cells and Notch-transfected marrow stromal cells.
ABSTRACT: Angiogenesis 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.Angiogenic 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.Conditioned 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.These data indicate that SB623 cell-secreted angiogenic factors promoted several aspects of angiogenesis, which likely contribute to promoting recovery in the injured brain.
Project description:Hamacanthins, bis (indole) alkaloids, are found in a few marine sponges, including Spongosorites sp. Hamacanthins have been shown to possess cytotoxic, antibacterial and antifungal activities. However, the precise mechanism for the biological activities of hamacanthins has not yet been elucidated. In the present study, the anti-angiogenic effects of 6"-debromohamacanthin A (DBHA), an active component of isolated hamacanthins, were evaluated in cultured human umbilical vascular endothelial cells (HUVEC) and endothelial-like cells differentiated from mouse embryonic stem (mES) cells. DBHA significantly inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation, migration and tube formation in the HUVEC. DBHA also suppressed the capillary-like structure formation and the expression of platelet endothelial cell adhesion molecule (PECAM), an endothelial biomarker, in mES cell-derived endothelial-like cells. To further understand the precise molecular mechanism of action, VEGF-mediated signaling pathways were analyzed in HUVEC cells and mES cell-derived endothelial-like cells. DBHA suppressed the VEGF-induced expression of MAPKs (p38, ERK and SAPK/JNK) and the PI3K/AKT/mTOR signaling pathway. In addition, DBHA inhibited microvessel sprouting in mES/EB-derived embryoid bodies. In an ex vivo model, DBHA also suppressed the microvessel sprouting of mouse aortic rings. The findings suggest for the first time that DBHA inhibits angiogenesis by targeting the vascular endothelial growth factor receptor 2 (VEGFR2)-mediated PI3K/AKT/mTOR signaling pathway in endothelial cells.
Project description:Mesenchymal stem cells (MSCs) have been found to benefit patients with a variety of ischemic diseases via promoting angiogenesis. It is also well established that exosomes secreted from MSCs deliver bioactive molecules, including microRNAs (miRs) to recipient cells. Therefore, we hypothesized that exosomes secreted from MSCs deliver miRs into endothelial cells and mediate angiogenesis. The pro-angiogenic stimulatory capacity of exosomes was investigated using tube-like structure formation and spheroid-based sprouting of human umbilical vein endothelial cells (HUVECs), and in vivo Matrigel plug assay. The secretion of pro-angiogenic miRs (pro-angiomiRs) from MSCs into culture medium and transfer of the miRs to HUVECs were confirmed using real-time quantitative PCR. Supplementation of the exosome secretion blocker GW4869 (10 ?M) reduced the pro-angiomiRs in the MSC-derived conditioned medium (CdMMSC). Addition of exosomes isolated from CdMMSC could directly 1) promote HUVEC tube-like structure formation in vitro; 2) mobilize endothelial cells into Matrigel plug subcutaneously transplanted into mice; and 3) increase blood flow inside Matrigel plug. Fluorescence tracking showed that the exosomes were internalized rapidly by HUVECs causing an upregulated expression of pro-angiomiRs in HUVECs. Loss-and-gain function of the pro-angiomiRs (e.g., miR-30b) in MSCs significantly altered the pro-angiogenic properties of these MSC-derived exosomes, which could be associated with the regulation of their targets in HUVECs. These results suggest that exosomal transfer of pro-angiogenic miRs plays an important role in MSC mediated angiogenesis and stem cell-to-endothelial cell communication.
Project description:BACKGROUND: Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. METHODS: Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. RESULTS: Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. CONCLUSION: The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.
Project description:Angiogenesis is an effective target in cancer control. The antiangiogenic efficacy and associated mechanisms of acacetin, a plant flavone, are poorly known. In the present study, acacetin inhibited growth and survival (up to 92%; P < 0.001), and capillary-like tube formation on Matrigel (up to 98%; P < 0.001) by human umbilical vein endothelial cells (HUVEC) in regular condition, as well as VEGF-induced and tumor cells conditioned medium-stimulated growth conditions. It caused retraction and disintegration of preformed capillary networks (up to 91%; P < 0.001). HUVEC migration and invasion were suppressed by 68% to 100% (P < 0.001). Acacetin inhibited Stat-1 (Tyr701) and Stat-3 (Tyr705) phosphorylation, and downregulated proangiogenic factors including VEGF, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), matrix metalloproteinase-2 (MMP-2), and basic fibroblast growth factor (bFGF) in HUVEC. It also suppressed nuclear localization of pStat-3 (Tyr705). Acacetin strongly inhibited capillary sprouting and networking from rat aortic rings and fertilized chicken egg chorioallantoic membrane (CAM; ?71%; P < 0.001). Furthermore, it suppressed angiogenesis in Matrigel plugs implanted in Swiss albino mice. Acacetin also inhibited tyrosine phosphorylation of Stat-1 and -3, and expression of VEGF in cancer cells. Overall, acacetin inhibits Stat signaling and suppresses angiogenesis in vitro, ex vivo, and in vivo, and therefore, it could be a potential agent to inhibit tumor angiogenesis and growth.
Project description:Angiogenesis is important in cancer progression and can be influenced by tumor-associated myofibroblasts. We addressed the hypothesis that glucocorticoids indirectly affect angiogenesis by altering the release of pro-angiogenic factors from colon cancer-derived myofibroblasts. Our study shows that glucocorticoids reduced prostanoids, urokinase-type plasminogen activator (uPA) and angiopoietin-like protein-2 (ANGPTL2) levels, but increased angiogenin (ANG) in supernatant from human CT5.3hTERT colon cancer-derived myofibroblasts. Conditioned medium from solvent- (CMS) and dexamethasone (Dex)-treated (CMD) myofibroblasts increased human umbilical vein endothelial cell (HUVEC) proliferation, but did not affect expression of pro-angiogenic factors or tube-like structure formation (by HUVECs or human aortic ECs). In a HUVEC scratch assay CMS-induced acceleration of wound healing was blunted by CMD treatment. Moreover, CMS-induced neovessel growth in mouse aortic rings ex vivo was also blunted using CMD. The latter effect could be ascribed to both Dex-driven reduction of secreted factors and potential residual Dex present in CMD (indicated using a dexamethasone-spiked CMS control). A similar control in the scratch assay, however, revealed that altered levels of factors in the CMD, and not potential residual Dex, were responsible for decreased wound closure. In conclusion, our results suggest that glucocorticoids indirectly alter endothelial cell function during tumor development in vivo.
Project description:<h4>Background</h4>Myeloid cells have been associated with physiological and pathological angiogenesis, but their exact functions in these processes remain poorly defined. Monocyte-derived tissue macrophages of the CNS, or microglial cells, invade the mammalian retina before it becomes vascularized. Recent studies correlate the presence of microglia in the developing CNS with vascular network formation, but it is not clear whether the effect is directly caused by microglia and their contact with the endothelium.<h4>Methodology/principal findings</h4>We combined in vivo studies of the developing mouse retina with in vitro studies using the aortic ring model to address the role of microglia in developmental angiogenesis. Our in vivo analyses are consistent with previous findings that microglia are present at sites of endothelial tip-cell anastomosis, and genetic ablation of microglia caused a sparser vascular network associated with reduced number of filopodia-bearing sprouts. Addition of microglia in the aortic ring model was sufficient to stimulate vessel sprouting. The effect was independent of physical contact between microglia and endothelial cells, and could be partly mimicked using microglial cell-conditioned medium. Addition of VEGF-A promoted angiogenic sprouts of different morphology in comparison with the microglial cells, and inhibition of VEGF-A did not affect the microglia-induced angiogenic response, arguing that the proangiogenic factor(s) released by microglia is distinct from VEGF-A. Finally, microglia exhibited oriented migration towards the vessels in the aortic ring cultures.<h4>Conclusions/significance</h4>Microglia stimulate vessel sprouting in the aortic ring cultures via a soluble microglial-derived product(s), rather than direct contact with endothelial cells. The observed migration of microglia towards the growing sprouts suggests that their position near endothelial tip-cells could result from attractive cues secreted by the vessels. Our data reveals a two-way communication between microglia and vessels that depends on soluble factors and should extend the understanding of how microglia promote vascular network formation.
Project description:Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates.
Project description:Among the three nonmuscle myosin 2 (NM2) paralogs, NM 2A and 2B, but not 2C, are detected in endothelial cells. To study the role of NM2 in vascular formation, we ablate NM2 in endothelial cells in mice. Ablating NM2A, but not NM2B, results in reduced blood vessel coverage and increased vascular branching in the developing mouse skin and coronary vasculature. NM2B becomes essential for vascular formation when NM2A expression is limited. Mice ablated for NM2B and one allele of NM2A develop vascular abnormalities similar to those in NM2A ablated mice. Using the embryoid body angiogenic sprouting assay in collagen gels reveals that NM2A is required for persistent angiogenic sprouting by stabilizing the endothelial cell cortex, and thereby preventing excessive branching and ensuring persistent migration of the endothelial sprouts. Mechanistically, NM2 promotes focal adhesion formation and cortical protrusion retraction during angiogenic sprouting. Further studies demonstrate the critical role of Rho kinase-activated NM2 signaling in the regulation of angiogenic sprouting in vitro and in vivo.
Project description:Most antiangiogenic therapies currently being evaluated in clinical trials target the vascular endothelial growth factor pathway; however, the tumor vasculature can acquire resistance to vascular endothelial growth factor-targeted therapy by shifting to other angiogenesis mechanisms. Insulin-like growth factor binding protein-3 (IGFBP-3) has been reported to suppress tumor growth and angiogenesis by both IGF-dependent and IGF-independent mechanisms; however, understanding of its IGF-independent mechanisms is limited. We observed that IGFBP-3 blocked tumor angiogenesis and growth in non-small cell lung cancer and head and neck squamous cell carcinoma. Conditioned media from an IGFBP-3-treated non-small cell lung cancer cell line displayed a significantly decreased capacity to induce HUVEC proliferation and aortic sprouting. In cancer cells, IGFBP-3 directly interacted with Erk1/2, leading to inactivation of Erk1/2 and Elk-1, and suppressed transcription of early growth response protein 1 and its target genes, basic fibroblast growth factor and platelet-derived growth factor. These data suggest that IGF-independent Erk1/2 inactivation and decreased IGFBP-3-induced Egr-1 expression block the autocrine and paracrine loops of angiogenic factors in vascular endothelial and cancer cells. Together, these findings provide a molecular framework of IGFBP-3's IGF-independent antiangiogenic antitumor activities. Future studies are needed for development of IGFBP-3 as a new line of antiangiogengic cancer drug.
Project description: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.