Vascular Tube Formation Research Articles

Improvement of Stability and Efficacy of C16Y Therapeutic Peptide via Molecular Self-Assembly into Tumor-Responsive Nanoformulation.

Peptide therapeutics hold great promise for the treatment of cancer due to low toxicity, high specificity, and ease of synthesis and modification. However, the unfavorable pharmacokinetic parameters strictly limit their therapeutic efficacy and clinical translation. Here, we tailor-designed an amphiphilic chimeric peptide through conjugation of functional 3-diethylaminopropyl isothiocyanate (DEAP) molecules to a short antitumor peptide, C16Y. The ultimate DEAP-C16Y peptides self-assembled into spherical nanostructures at physiologic conditions, which dissociated to release individual peptide molecules in weakly acidic tumors. DEAP-C16Y peptides showed negligible cytotoxicity but impaired vascular endothelial cell migration and tubule formation by inactivation of the focal adhesion kinase and PI3K-Akt pathways, as well as tumor cell invasion by decreasing invadopodia formation. Compared with C16Y, the systemically administered DEAP-C16Y nanostructures exhibited superior stability, thereby allowing prolonged treatment interval and resulting in significant decreases in microvessel density, tumor growth, and distant metastasis formation in orthotopic mammary tumor models. Through encapsulation of hydrophobic doxorubicin, DEAP-C16Y nanostructure served as a smart carrier to achieve targeted drug delivery and combination therapy. Our study, for the first time, demonstrates that a simple nanoformulation using a functional antitumor peptide as the building block can show innate antitumor activity and also provide a nanoplatform for combination therapy, opening a new avenue for the design of antitumor nanotherapeutics.

Anti-angiogenic effect of ALS-L1023, an extract of Melissa officinalis L., on experimental choroidal neovascularization in mice.

The effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) leaves, on experimental choroidal neovascularization (CNV) in mice was evaluated. C57BL/6 mice were given either vehicle or ALS-L1023 daily via oral gavage for 3 weeks (days 0-21). CNV was induced by rupturing Bruch's membrane using laser photocoagulation (day 7). Two weeks after laser injury (day 21), the CNV lesions were evaluated by an examination of choroidal flat mounts using fluorescein-labelled dextran, immunofluorescence staining with isolectin B4 and fluorescence angiography. The effects of ALS-L1023 on endothelial cell tube formation and the expression of phosphorylated extracellular signal-regulated kinase 1/2 were evaluated using human umbilical vein endothelial cells. The extent of CNV was reduced by ALS-L1023. Mice treated with 100 and 200 mg/kg/day of the material exhibited 44.3 and 68.1% reductions in the extent of CNV lesions, respectively, compared to the vehicle group (P < 0.001). The size of the isolectin B4-labelled area was also significantly decreased in the ALS-L1023-treated groups (P < 0.001). On fluorescein angiography, ALS-L1023-treated mice exhibited significantly less leakage of fluorescent material than did vehicle-treated mice. ALS-L1023 decreased vascular endothelial growth factor-induced human umbilical vein endothelial cell tube formation in a dose-dependent manner. The expression of phosphorylated extracellular signal-regulated kinase 1/2 was suppressed by ALS-L1023. The laser-induced CNV in mice can be inhibited by ALS-L1023. Therefore, it may have therapeutic potential for the treatment of diseases involving CNV.

Akt2 knock-down reveals its contribution to human lung cancer cell proliferation, growth, motility, invasion and endothelial cell tube formation.

The Akt/PKB serine/threonine protein kinase consists of three isoforms: Akt-1, −2 and −3. Their overexpression has been detected in human cancers, but their roles in cancer progression are unclear. We investigated the impact of specific silencing of Akt1 and Akt2 on human lung cancer cell proliferation, colony growth, motility, and invasion in vitro as well as tumor growth in vivo using human Non-Small Cell Lung Cancer cells LNM35, and on the vascular tube formation using HUVEC cells. Although silencing of Akt1 decreased cellular invasion at least in part via COX-2 inhibition, it had almost no effect on cell motility, proliferation, colony formation, and angiogenesis. Transient as well as stable silencing of Akt2 resulted in a strong inhibition of Rb phosphorylation associated with a decrease in cellular proliferation and colony formation, leading to the inhibition of tumor growth in the xenograft model. Silencing of Akt2 also reduced cellular motility and invasion in vitro, presumably via COX-2 inhibition. Moreover, silencing of Akt2 in the HUVEC cells resulted in the inhibition of their spontaneous angiogenic phenotype. Altogether, these results indicate that Akt2 plays an important role in lung cancer progression and can be a promising target for lung cancer therapy.

Antiangiogenic activity of herboxidiene via downregulation of vascular endothelial growth factor receptor-2 and hypoxia-inducible factor-1α.

Antiangiogenesis is now thought of as one of the most important approaches for anticancer therapy. In this study, we determined the antiangiogenic property of herboxidiene, a polyketide natural product. Herboxidiene effectively inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) at concentrations not exhibiting cytotoxicity. Furthermore, the natural product significantly suppressed vascular endothelial growth factor-induced invasion and tube formation in HUVECs as well as neovascularization of the chorioallantoic membrane in developing chick embryos. We also identified an association between the antiangiogenic activity of herboxidiene and the downregulation of both the phosphorylation of VEGF receptor 2 (KDR/Flk-1) and the expression of hypoxia-inducible factor-1α at the transcriptional level. These results suggest that herboxidiene functions as a potential antiangiogenic agent and may be applicable for anticancer therapy by targeting tumor angiogenesis.

Abstract 3: Human CD34+ Cells Deficient in miR-377 Attenuates Cardiac Dysfunction and Repair After Ischemia-reperfusion Injury

microRNAs (miRNA/miR) dysregulation has been implicated in cardiac remodeling after injury or stress, however its effects on Human CD34 + cells (hCD34 + ) biology and function, particularly in the context of cell-based therapy for cardiomyopathy is not fully understood. miRNA array data analysis indicates that miR-377 is a potential interest. pre-miR-377 transfection in EPCs inhibits their migration and vascular tube formation ability in HUVECs. Furthermore, hCD34 + cells treated with miR-377 mimic showed decrease in expression of STK35 (a novel serine/threonine kinase). Moreover, STK35 is predicted as a potential target gene of miR-377 by computational analysis. Interestingly, in a relevant mouse model of ischemia reperfusion, intramyocardial transplantation of miR-377-silenced hCD34 + cells promotes neovascularization leading to improvement in myocardial function and repair. Echocardiography showed LV function was significantly improved in mice receiving miR-377-silenced hCD34 + cells compared to control-miR-transfected hCD34 + cells. Taken together, these data suggest that inhibiting miR-377 in hCD34 + cells promotes their angiogenic ability after transplantation into ischemic myocardial tissue, potentially through activation of STK35 signaling.

Downregulation of BDNF Expression by PKC and by TNF-α in Human Endothelial Cells

Brain-derived neurotrophic factor (BDNF) is a neurotrophin best characterized for its survival and differentiative effects on neurons. Recent studies demonstrated that BDNF and its receptors are also expressed in the peripheral vasculature, where it stimulates angiogenesis and promotes the survival of endothelial cells. This study was designed to investigate the angiogenic effects of BDNF and its expressional regulation by tumor necrosis factor (TNF-α) and protein kinase C (PKC) in endothelial cells. In the Matrigel angiogenesis assay, BDNF-stimulated vascular tube formation of human umbilical vein endothelial cells (HUVEC) was completely blocked by an inhibition of the TrkB receptor, but only partially inhibited by the inhibition of the p75^NTR signaling. Treatment of HUVEC and HUVEC-derived EA.hy 926 cells with TNF-α resulted in a downregulation of BDNF expression, which could be prevented by the TNFR1 antagonist WP9QY. BDNF downregulation by TNF-α was associated with decreased angiogenic activity of HUVEC. The effect of TNF-α on BDNF expression could not be abolished by the inhibition of PKC. Treatment of HUVEC and EA.hy 926 cells with PKC-activating phorbol esters (phorbol-12-myristate-13-acetate, PMA or phorbol-12,13-dibutyrate) resulted in a downregulation of BDNF expression, whereas the inactive 4α-phorbol-12,13-didecanoate was without effect. PMA had no significant effect on BDNF mRNA stability and the downregulation of BDNF mRNA expression by PKC activation was likely a transcriptional event. BDNF downregulation by PMA could be prevented by PKC inhibitors Gö 6983 and rottlerin, but not by Gö 6976. Thus, a Gö 6983/rottlerin-sensitive PKC isoform is likely to be responsible for PMA-induced BDNF downregulation.

Effect of Monocyte-Like THP-1 Cells on the Formation of Vascular Tubes by EA.Hy926s Endothelial Cells in the Presence of Cytokines.

The interaction of endothelial cells with cells of the microenvironment, including monocytes/ macrophages, and extracellular matrix during angiogenesis is controlled by cytokines. The stimulating effect bFGF, IL-8, and VEGF on the formation of capillary-like structures by endothelial cells was demonstrated in both monoculture and in co-culture with THP-1 cells; in the latter case, the effects of bFGF and VEGF were more pronounced. IL-8 reduced branching of vascular tubes in co-culture in comparison with monoculture of endothelial cells. Placental growth factor PlGF had no effect of tube formation by endothelial cells in monoculture, but in co-culture with THP-1 cells this cytokine in high concentrations exhibited proangiogenic activity. TGFb inhibited the formation of vascular tubes by endothelial cells and its antiangiogenic potential was more pronounced in co-culture with THP-1 cells.

Body protective compound-157 enhances alkali-burn wound healing in vivo and promotes proliferation, migration, and angiogenesis in vitro.

Chemical burns take up a high proportion of burns admissions and can penetrate deep into tissues. Various reagents have been applied in the treatment of skin chemical burns; however, no optimal reagent for skin chemical burns currently exists. The present study investigated the effect of topical body protective compound (BPC)-157 treatment on skin wound healing, using an alkali burn rat model. Topical treatment with BPC-157 was shown to accelerate wound closure following an alkali burn. Histological examination of skin sections with hematoxylin–eosin and Masson staining showed better granulation tissue formation, reepithelialization, dermal remodeling, and a higher extent of collagen deposition when compared to the model control group on the 18th day postwounding. BPC-157 could promote vascular endothelial growth factor expression in wounded skin tissues. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell cycle analysis demonstrated that BPC-157 enhanced the proliferation of human umbilical vein endothelial cells (HUVECs). Transwell assay and wound healing assay showed that BPC-157 significantly promoted migration of HUVECs. We also observed that BPC-157 upregulated the expression of VEGF-a and accelerated vascular tube formation in vitro. Moreover, further studies suggested that BPC-157 regulated the phosphorylation level of extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as its downstream targets, including c-Fos, c-Jun, and Egr-1, which are key molecules involved in cell growth, migration, and angiogenesis. Altogether, our results indicated that BPC-157 treatment may accelerate wound healing in a model of alkali burn-induced skin injury. The therapeutic mechanism may be associated with accelerated granulation tissue formation, reepithelialization, dermal remodeling, and collagen deposition through ERK1/2 signaling pathway.

Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells.

The level of vascular endothelial growth inhibitor (VEGI) has been reported to be negatively associated with neovascularization in malignant tumors. The soluble form of VEGI is a potent anti-angiogenic factor due to its effects in inhibiting endothelial cell proliferation. This inhibition is mediated by death receptor 3 (DR3), which contains a death domain in its cytoplasmic tail capable of inducing apoptosis that can be subsequently blocked by decoy receptor 3 (DcR3). We investigated the effects of sodium valproate (VPA) and trichostatin A (TSA), histone deacetylase inhibitors, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Consequently, treatment with VPA and TSA increased the VEGI and DR3 expression levels without inducing DcR3 production in the OS cell lines. In contrast, the effect on the HMVE cells was limited, with no evidence of growth inhibition or an increase in the DR3 and DcR3 expression. However, VPA-induced soluble VEGI in the OS cell culture medium markedly inhibited the vascular tube formation of HMVE cells, while VEGI overexpression resulted in enhanced OS cell death. Taken together, the HDAC inhibitor has anti-angiogenesis and antitumor activities that mediate soluble VEGI/DR3-induced apoptosis via both autocrine and paracrine pathways. This study indicates that the HDAC inhibitor may be exploited as a therapeutic strategy modulating the soluble VEGI/DR3 pathway in osteosarcoma patients.

Anticancer effects of adenovirus-mediated calreticulin and melanoma-associated antigen 3 expression on non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is highly prevalent and needs novel therapies. Melanoma-associated antigen 3 (MAGE-A3) is a lung cancer antigen and calreticulin (CALR) can modulate immune responses. Our previous study has shown that up-regulated MAGE-A3 and CALR expression inhibits the proliferation and invasion of glioma cells. In this study, we examined the effect of adenovirus (Ad)-mediated MAGE-A3 and/or CALR expression on the proliferation, invasion, and apoptosis of human NSCLC cells and on the vascular tube formation of human endothelial cells as well as on dendritic cell (DC) activation and induced CD8(+) cytotoxic T lymphocyte (CTL) activity in vitro. We found that low levels of CALR and MAGE-A3 were expressed by A549 cells, but only very low CALR was expressed by DC. Up-regulated CALR and MAGE-A3 expression by infection with Ad-CALR/MAGE-A3 significantly inhibited the proliferation and invasion, but promoted the apoptosis of A549 cells. Up-regulated CALR and MAGE-A3 expression significantly inhibited cyclin D1 expression and the AKT, ERK1/2 and NF-κB expression and phosphorylation in A549 cells. Up-regulated CALR expression inhibited the tube formation in human endothelial cells. Up-regulated CALR and MAGE-A3 expression synergistically enhanced classical DC activation by enhancing IL-12, but reducing IL-10 secretion. Furthermore, CTLs induced by up-regulated CALR and MAGE-A3 expressing DCs synergistically triggered A549 cell apoptosis, which was abrogated by treatment with anti-HLA I, but not anti-HLA II antibodies. Moreover, CTLs induced by CALR and MAGE-A3-expressing DCs had a higher frequency of A549-specific IFN-γ-secreting T cells. Our data indicated that up-regulated CALR and MAGE-A3 expression inhibited the carcinogenesis of NSCLC by modulating the AKT, ERK MAPK and NF-κB signaling and enhanced classical DC activation and MAGE-A3-specific CTL cytotoxicity. Therefore, our findings may provide new insights in understanding the role of CALR in modulating antigen-specific T cell immunity and may aid in the design of new therapies for NSCLC.

Characterization of endothelial colony-forming cells from peripheral blood samples of adult horses

To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. Jugular venous blood samples from 24 adult horses. Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates. Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry. ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.

Cilostazol improves high glucose-induced impaired angiogenesis in human endothelial progenitor cells and vascular endothelial cells as well as enhances vasculoangiogenesis in hyperglycemic mice mediated by the adenosine monophosphate-activated protein kinase pathway

Cilostazol is an antiplatelet agent with vasodilatory effects that works by increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP). This study investigated the effects of cilostazol in preventing high glucose (HG)-induced impaired angiogenesis and examined the potential mechanisms involving activation of AMP-activated protein kinase (AMPK). Assays for colony formation, adhesion, proliferation, migration, and vascular tube formation were used to determine the effect of cilostazol in HG-treated endothelial progenitor cells (EPCs) or human umbilical vein endothelial cells (HUVECs). Animal-based assays were performed in hyperglycemic ICR mice undergoing hind limb ischemia. An immnunoblotting assay was used to identify the expression and activation of signaling molecules invitro and invivo. Cilostazol treatment significantly restored endothelial function in EPCs and HUVECs through activation of AMPK/acetyl-coenzyme A carboxylase (ACC)-dependent pathways and cAMP/protein kinase A (PKA)-dependent pathways. Recovery of blood flow in the ischemic hind limb and the population of circulating CD34(+) cells were significantly improved in cilostazol-treated mice, and these effects were abolished by local AMPK knockdown. Cilostazol increased the phosphorylation of AMPK/ACC and Akt/endothelial nitric oxide synthase signaling molecules in parallel with or downstream of the cAMP/PKA-dependent signaling pathway invitro and invivo. Cilostazol prevents HG-induced endothelial dysfunction in EPCs and HUVECs and enhances angiogenesis in hyperglycemic mice by interactions with a broad signaling network, including activation of AMPK/ACC and probably cAMP/PKA pathways.

Decreased Expression of Angiopoetin 1 on Perivascular Mesenchymal Stem Cells from Ssc Patients Induces an Anti Angiogenetic Effect, when Co-cultured with Endothelial Cells

Introduction: The Angiopoietin (Ang)/Tie2 system plays crucial roles in vascular functions, regulating endothelialpericyte interaction and promoting vascular stabilization. We assessed if an impaired cross-talk, in Systemic Sclerosis (SSc), between endothelial cells (ECs) and perivascular mesenchymal stem cells (MSCs) may affect the normal interaction among Ang1, Ang2 and Tie2 thus contributing of the impaired angiogenesis in SSc. Methods: We investigated Ang1, Ang2 and their receptor performing co-cultures with ECs and bone marrow MSCs obtained from patients and healthy controls (HC). After 48 hours, cells were sorted and analysed for molecular assays. Furthermore, we investigated, by ELISA assay, the proteins released in the supernatants. Finally, we silenced Ang-1 expression in HC-MSCs by siRNA-Ang1. Results: At molecular level, SSc-MSCs, cultured alone, expressed lower amount of Ang1 when compared to HC-MSCs. After co-culture, a significant decreased of Ang1 mRNA levels was observed in the SSc-MSCs/SSc-ECs. On the contrary, SSc-ECs expressed higher levels of Ang2 and Tie2 in each co-culture condition, when compared to the expressions of cells cultured alone. The WB and ELISA assays mirrored the results observed in gene expression. HC-MSCs transfected with siRNA-Ang1 lacked the ability to support the formation of tube like structure. Conclusions: In this work we provided evidence that an imbalance of Ang1/Ang2 molecules and a decreased expression of their receptor, Tie2, during ECs-perivascular MSCs interplay, may modulate vessel stability, and vascular tube formation, thus contributing to the angiogenic alteration observed during SSc.

Vascularization mediated by mesenchymal stem cells from bone marrow and adipose tissue: a comparison.

Tissue-engineered constructs are promising to overcome shortage of organ donors and to reconstruct at least parts of injured or diseased tissues or organs. However, oxygen and nutrient supply are limiting factors in many tissues, especially after implantation into the host. Therefore, the development of a vascular system prior to implantation appears crucial. To develop a functional vascular system, different cell types that interact with each other need to be co-cultured to simulate a physiological environment in vitro. This review provides an overview and a comparison of the current knowledge of co-cultures of human endothelial cells (ECs) with human adipose tissue-derived stem/stromal cells (ASCs) or bone marrow-mesenchymal stem cells (BMSCs) in three dimensional (3D) hydrogel matrices. Mesenchymal stem cells (MSCs), BMSCs or ASCs, have been shown to enhance vascular tube formation of ECs and to provide a stabilizing function in addition to growth factor delivery and permeability control for ECs. Although phenotypically similar, MSCs from different tissues promote tubulogenesis through distinct mechanisms. In this report, we describe differences and similarities regarding molecular interactions in order to investigate which of these two cell types displays more favorable characteristics to be used in clinical applications. Our comparative study shows that ASCs as well as BMSCs are both promising cell types to induce vascularization with ECs in vitro and consequently are promising candidates to support in vivo vascularization.

Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment.

Interleukin-34 (IL-34) was recently characterized as the M-CSF "twin" cytokine, regulating the proliferation/differentiation/survival of myeloid cells. The implication of M-CSF in oncology was initially suspected by the reduced metastatic dissemination in knock-out mice, due to angiogenesis impairment. Based on this observation, our work studied the involvement of IL-34 in the pathogenesis of osteosarcoma. The in vivo effects of IL-34 were assessed on tissue vasculature and macrophage infiltration in a murine preclinical model based on a paratibial inoculation of human osteosarcoma cells overexpressing or not IL-34 or M-CSF. In vitro investigations using endothelial cell precursors and mature HUVEC cells were performed to analyse the involvement of IL-34 in angiogenesis and myeloid cell adhesion. The data revealed that IL-34 overexpression was associated with the progression of osteosarcoma (tumor growth, lung metastases) and an increase of neo-angiogenesis. In vitro analyses demonstrated that IL-34 stimulated endothelial cell proliferation and vascular cord formation. Pre-treatment of endothelial cells by chondroitinases/heparinases reduced the formation of vascular tubes and abolished the associated cell signalling. In addition, IL-34 increased the in vivo recruitment of M2 tumor-associated macrophages into the tumor tissue. IL-34 increased in vitro monocyte/CD34(+) cell adhesion to activated HUVEC monolayers under physiological shear stress conditions. This work also demonstrates that IL-34 is expressed by osteosarcoma cells, is regulated by TNF-α, IL-1β, and contributes to osteosarcoma growth by increasing the neo-angiogenesis and the recruitment of M2 macrophages. By promoting new vessel formation and extravasation of immune cells, IL-34 may play a key role in tumor development and inflammatory diseases.

Synergistic effects of extracellular matrix rigidity and immobilized vascular endothelial growth factor on vascular tube formation and cell morphology of human umbilical vein endothelial cells

Tube formation by endothelial cells (EC) is a one of the key factors involved in initial revascularization during the tissue regeneration process. Several previous studies suggested that EC tube formation can be regulated by the stiffness of the culture substrate and immobilization of vascular endothelial growth factor (VEGF). However, the roles of substrate stiffness and chemical signals involved in network formation are unclear. In this study, the effects of substrate rigidity and immobilized VEGF concentration on EC tube formation were examined. Human umbilical vein endothelial cells (HUVEC) were cultured on rigidity-controlled polyacrylamide (PAAM) gels coated by L-DOPA (3,4-dihydroxy-L-phenylalanine), or L-DOPA-Hep (heparin). The HUVECs were cultured on various amounts of immobilized VEGF on the L-DOPA-Hep-coated PAAM (5, 10, and 25 ng). We found that HUVECs formed more tube-like structures when they were cultured on the more rigid substrates, from 1.5 kPa to 144.1 kPa compared to the soft substrate. We also compared various amounts of VEGF immobilized on various rigidity substrates to determine if there was an effect on the tube area of HUVEC. Moreover, we determined an appropriate concentration of VEGF immobilized with various rigidity substrates for vascular tube formation. These findings suggest that there is a synergistic effect of substrate rigidity and chemical signals. Thus, it is possible to combine therapies to synergistically promote an in vitro model of revascularization.

TRPM7 regulates vascular endothelial cell adhesion and tube formation.

Transient receptor potential melastatin 7 (TRPM7) is a nonselective cation channel with an α-kinase domain in its COOH terminal, known to play a role in diverse physiological and pathological processes such as Mg2+ homeostasis, cell proliferation, and hypoxic neuronal injury. Increasing evidence suggests that TRPM7 contributes to the physiology/pathology of vascular systems. For example, we recently demonstrated that silencing TRPM7 promotes growth and proliferation and protects against hyperglycemia-induced injury in human umbilical vein endothelial cells (HUVECs). Here we investigated the potential effects of TRPM7 on morphology, adhesion, migration, and tube formation of vascular endothelial cells and the potential underlying mechanism. We showed that inhibition of TRPM7 function in HUVECs by silencing TRPM7 decreases the density of TRPM7-like current and cell surface area and inhibits cell adhesion to Matrigel. Silencing TRPM7 also promotes cell migration, wound healing, and tube formation. Further studies showed that the extracellular signal-regulated kinase (ERK) pathway is involved in the change of cell morphology and the increase in HUVEC migration induced by TRPM7 silencing. We also demonstrated that silencing TRPM7 enhances the phosphorylation of myosin light chain (MLC) in HUVECs, which might be involved in the enhancement of cell contractility and motility. Collectively, our data suggest that the TRPM7 channel negatively regulates the function of vascular endothelial cells. Further studies on the underlying mechanism may facilitate the development of the TRPM7 channel as a target for the therapeutic intervention of vascular diseases.

Abstract 18134: Cardiomyocyte Overexpression of the alpha1A-Adrenergic Receptor in the Rat Protects Post-Infarct Heart Failure through Angiogenesis and the MEK-ERK Pathway

α1A-Adrenergic receptors (α1A-AR) are important for regulating cardiac contractility, but whether they regulate coronary vessels is not known. To examine this we investigated the effects of chronic, complete coronary artery occlusion (CAO) in rats with cardiac myocyte-restricted transgenic (TG) α1A-AR overexpression, since protection of the heart with complete CAO requires increased blood flow. 4 weeks after permanent left anterior descending CAO TG rats displayed improved contractility vs. non-transgenic littermates (NTL) (n=8/group, p&lt;0.05): 23% higher in LV dP/dt (7188±167mmHg/s), 13% higher in LV ejection fraction (57±0.9) and 23% lower in end-systolic wall stress (LVSWS) (79±6dynes/cm2) compared to NTLs. The scar size at 4 wks after CAO was significantly smaller in TG rats (18±2%) compared to NTL rats (28±3%), p&lt;0.05, but more importantly there were increased blood vessels within the scar in the TG rat hearts suggesting angiogenesis. This was confirmed using colored microspheres to measure coronary blood flow, which was higher in area at risk in TGs (1.4±0.2ml/min/g) as compared to NTLs (0.5±0.08ml/min/g), p&lt;0.05. This increased blood flow was further supported by a 41% increase in arterial density within the ischemic zone compared to NTLs at 2 weeks after CAO. In addition, capillary density was also found to be significantly increased, by 18% in the remote zone, compared to 2109 caps/mm2 in NTL. Utilizing an In vitro model, treatment with 25μM A61603 for 48 hours in cultured NTL neonatal myocytes resulted in an increase in VEGF-A secretion into the culture media and 26% increase in vascular tubule formation in HUVECs by a paracrine mechanism coinciding with an increased VEGF-A concentration compared to vehicle treated myocytes. Since VEGF is linked to the MEK-ERK signaling pathway, we blocked this mechanism by chronically administering the MEK blocker, U0126, sc. In these rats LV function was no longer different after 4 wks permanent CAO in TG and WTL treated with the blocker. Therefore, overexpression of cardiac myocyte specific α1A-AR in rats protected against cardiac dysfunction and remodeling associated with chronic myocardial infarction through the MEK-ERK pathway and via angiogenesis with a VEGF-A related paracrine mechanism.

Abstract 17399: Glutathione-S-Transferase P Regulates Neovascularization in the Remodeling Heart via Effects on Endothelial Progenitor Cells and Reparative Monocytes

Glutathione- S -transferase P (GSTP) modulates c-Jun N-terminal kinase (JNK) activation and the proliferation of bone marrow (BM) myeloid progenitors; however, its effects on endothelial progenitor cells (EPCs) and neovascularization are unknown. Hypothesis: GSTP promotes EPC function in a JNK-dependent manner, thereby supporting neovascularization in heart failure (HF). GSTP-/- mice exhibited significantly (p&lt;0.01 vs. wild type [WT]) reduced circulating and BM EPCs, increased pro-inflammatory F4/80 + CD11b + Gr-1 hi monocytes, diminished BM EPC function (proliferation, migration, and vascular tube formation), and increased BM JNK phosphorylation. These effects were reversed upon treatment with the JNK inhibitor SP600125. HF was induced by left coronary ligation in WT and GSTP-/- mice (sham control). GSTP-/- HF mice exhibited (p&lt;0.05 vs. WT HF): 1) fewer circulating and BM EPCs and increased pro-inflammatory monocytes; 2) exaggerated left ventricular (LV) remodeling and inflammation; and 3) reduced myocardial capillary density. Next, chimeric mice were generated via BM ablation in WT mice and reconstitution with either GSTP-/- (GSTP-/-c) or WT (WTc) BM. GSTP-/-c and WTc mice underwent coronary ligation or sham operation (n = 8-15/group). As compared with WTc HF, GSTP-/-c HF hearts had significant (p &lt; 0.01): 1) exaggeration of LV remodeling and dysfunction; 2) increases in remote zone fibrosis; 3) reductions in capillary density and VEGF-A, -B, and -C gene expression; and 4) augmentation of interleukin (IL)-1β and IL-6 expression. GSTP-/-c mice also exhibited (p &lt; 0.01 vs. WTc mice) increased BM JNK activation, reduced circulating EPCs, increased pro-inflammatory CD11b + Ly6C hi monocytes, but comparable Ly6C low monocytes at baseline. Moreover, at 3 and 7 d post-ligation, GSTP-/-c HF mice had fewer circulating EPCs and reparative Ly6C low monocytes, but significantly higher Ly6C hi monocytes. Conclusion: GSTP promotes EPC function and reparative monocyte mobilization, and suppresses inflammation, thereby improving angiogenic gene expression, neovascularization and LV remodeling after infarction. Hence, human GSTP polymorphisms, which are frequent, may impact cardiac reparative capacity in patients with myocardial infarction.

Abstract 1550: Differential angiogenic roles of serum amyloid A 1 (SAA1) isoforms in esophageal squamous cell carcinoma (ESCC)

Abstract Esophageal Caner (EC), a highly metastatic and fatal cancer, is ranked the eighth in mortality rate in Hong Kong cancer patients (Hong Kong Cancer Registry, Hospital Authority, 2010). Esophageal Squamous Cell Carcinoma (ESCC) is the predominant type comprising more than 90% of EC. Using a functional complementation approach, SAA1 was identified as one of the tumor suppressor gene candidates. SAA1 is located at chromosome 11p15.1 and is expressed as a secretary protein in liver, human cultured smooth muscle cells, monocyte-macrophage cell lines, and in histologically-normal human epithelial tissues. Genetic polymorphisms of SAA1 have been identified as a risk factor of diseases such as amyloidosis. Three SAA1 isoforms with two single nucleotide polymorphisms at exon three (SAA1.1, 1.3, and 1.5) were observed in the ESCC patients and healthy individuals. The SAA proteins contain the functional YIGSR-like and RGD-like motifs, proteins with these motifs can inhibit angiogenesis, cell adhesion to ECM, growth and metastasis. To understand the anti-tumorigenic and anti-angiogenic roles of the three SAA1 isoforms in ESCC progression, both recombinant proteins and secreted proteins from the conditioned media of lentiviral-infected ESCC cell lines were used for functional assays. For the vascular endothelial cell tube formation assay, the treatments with SAA1.1 and 1.3 proteins showed suppression of tube formation, whereas no significant effects could be observed in the treatment with the SAA1.5. Suppression of cell proliferation and induction of cell death were observed when the endothelial cells were cultured with SAA1.1 and 1.3 proteins. To further elucidate the differential effects among the three SAA1 isoforms in anti-angiogenesis, the cytoskeleton arrangement of the vascular endothelial cells was studied. The SAA1.1 and SAA1.3 proteins could abolish the endothelial cell adhesion by disturbing the formation of stress fibers and focal adhesions. As a conclusion, the present data has shown the variation in anti-angiogenic potential of the three SAA1 isoforms. We acknowledge the financial support of the Food and Health Bureau of the Hong Kong Special Administration Region, China, grant number HMRF 01120886 to HLL. Citation Format: ON YING MAN, Maria Lung, Hong Lok Lung. Differential angiogenic roles of serum amyloid A 1 (SAA1) isoforms in esophageal squamous cell carcinoma (ESCC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1550. doi:10.1158/1538-7445.AM2014-1550