ECM625 Sigma-AldrichIn Vitro Angiogenesis Assay Kit

Connexins (Cx) are suggested to play important roles in growth and differentiation. Aim of our study was to investigate the role of endothelial Cx in the angiogenic process. Several parameters of angiogenesis were assessed in 18 h Matrigel in vitro angiogenesis assays with human umbilical vein endothelial cells (HUVEC). Prior to culture on Matrigel cells were treated with nicotine or the gap junction inhibitor palmitoleic acid (PA), or siRNA-knock-down of either Cx37, Cx40 or Cx43 was performed. Changes in Cx expression and their effects on gap-junctional communication were investigated using immunofluorescence microscopy, Western blot and Lucifer Yellow dye transfer. Knock-down of each Cx-isoform significantly reduced the amount of specific Cx protein in HUVEC. Cx-knock-down as well as treatment with PA impaired intercellular communication via gap junctions and diminished significantly the number of capillary branches. Knock-down of Cx43 and Cx40 or treatment with PA reduced complexity pattern in the angiogenesis assay. Nicotine significantly reduced expression of Cx43 and Cx37 as well as average length of capillary branches, number of branches and pattern in the Matrigel assay. We can conclude that connexins are involved in angiogenesis, in particular in branch formation. This can partly explain the changes in angiogenesis seen under nicotine treatment.

For tissue engineering applications, effective bone regeneration requires rapid neo-vascularization of implanted grafts to ensure the survival of cells in the early post-implantation phase. Incorporation of autologous endothelial progenitor cells (EPCs) for the promotion of primitive vascular network formation ex vivo has offered great promise for improved graft survival, enhanced rate of vascularization and bone regeneration in vivo. For clinical usage, identification of an optimal EPC isolation source from the patient is critical. We have, for the first time, characterized and directly compared EPCs from rabbit peripheral blood and bone marrow (PB-EPCs and BM-EPCs, respectively). PB-EPCs outperformed BM-EPCs on all measures. PB-EPCs displayed typical endothelial cell markers, such as CD31, as well as high angiogenic potential in three-dimensional extracellular matrix in vitro. Furthermore, PB-EPCs cultured simultaneously with mesenchymal stem cells, displayed significantly enhanced expression levels of key osteogenic and vascular markers, including alkaline phosphatase, bone morphogenetic protein 2, and vascular endothelial growth factor. On the contrary, putative BM-EPCs did not express CD31, and instead, expressed key smooth muscle markers. BM-EPCs further failed to display vasculogenic activity. Hence, the highly angiogenic PB-derived EPCs may serve as an ideal cell population for enhanced vascularization and success of engineered bone tissue. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1507-1515, 2012.

Two new polyketides: 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1) and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen- 4-one (2), together with eleven known compounds: 4-hydroxy-2-[(3,4-methylenedioxy- phenyl)tridecanoyl] cyclohexane-1,3-dione (3), oleiferinone (4), 4-hydroxy-2-[(3,4- methylenedioxyphenyl)undecanoyl]cyclohexane-1,3-dione (5), 4-hydroxy-2-[(11-phenyl- undecanoyl)cyclohexane-1,3-dione (6), proctorione C (7), surinone C (8), 5-hydroxy- 7,8,4'-trimethoxyflavone (9), 5-hydroxy-7,8,3',4'-tetramethoxyflavone (10), 5-hydroxy- 7,3',4'-trimethoxyflavone (11), 5,8-dihydroxy-7,3',4'-trimethoxyflavone (12) and cepharanone B (13) were isolated from the whole plant of Peperomia dindygulensis Miq. Their structures were elucidated by spectroscopic methods, including 2D-NMR techniques. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cell (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.

Angiogenesis is central to wound healing and tumor growth. Postoperative (postop) plasma from weeks 2 and 3 after minimally invasive colorectal resection (MICR) stimulates endothelial cell (EC) migration (MIG), invasion (INV), and proliferation (all vital to angiogenesis) compared with preoperative (preop) plasma results and may promote postop tumor growth. The purpose of this study was to determine whether plasma from open colorectal resection (OCR) patients has similar proangiogenic EC effects in vitro.

Traditional bone regeneration strategies relied on supplementation of biomaterials constructs with stem or progenitor cells or growth factors. By contrast, cell homing strategies employ chemokines to mobilize stem or progenitor cells from host bone marrow and tissue niches to injured sites. Although silica-based biomaterials exhibit osteogenic and angiogenic potentials, they lack cell homing capability. Stromal cell-derived factor-1 (SDF-1) plays a pivotal role in mobilization and homing of stem cells to injured tissues. In this work, we demonstrated that 3-dimensional collagen scaffolds infiltrated with intrafibrillar silica are biodegradable and highly biocompatible. They exhibit improved compressive stress-strain responses and toughness over nonsilicified collagen scaffolds. They are osteoconductive and up-regulate expressions of osteogenesis- and angiogenesis-related genes more significantly than nonsilicified collagen scaffolds. In addition, these scaffolds reversibly bind SDF-1α for sustained release of this chemokine, which exhibits in vitro cell homing characteristics. When implanted subcutaneously in an in vivo mouse model, SDF-1α-loaded silicified collagen scaffolds stimulate the formation of ectopic bone and blood capillaries within the scaffold and abrogate the need for cell seeding or supplementation of osteogenic and angiogenic growth factors. Intrafibrillar-silicified collagen scaffolds with sustained SDF-1α release represent a less costly and complex alternative to contemporary cell seeding approaches and provide new therapeutic options for in situ hard tissue regeneration.-Niu, L.-N., Jiao, K., Qi, Y.-P., Nikonov, S., Yiu, C. K. Y., Arola, D. D., Gong, S.-Q., El-Marakby, A., Carrilho, M. R. O., Hamrick, M. W., Hargreaves, K. M., Diogenes, A., Chen, J.-H., Pashley, D. H., Tay, F. R. Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration.

The capacity to induce rapid vascular ingrowth during new bone formation is an important feature of biomaterials that are to be used for bone regeneration. Akermanite, a Ca-, Mg- and Si-containing bioceramic, has been demonstrated to be osteoinductive and to promote bone repair. This study further demonstrates the ability of akermanite to promote angiogenesis and investigates the mechanism of this behavior. The akermanite ion extract predominantly caused Si-ion-stimulated proliferation of human aortic endothelial cells. The Si ion in the extract was the most important component for the effect and the most effective concentration was found to be 0.6-2 μg ml(-1). In this range of Si ion concentration, the stimulating effect of the ceramic ion extract was demonstrated by the morphology of cells at the primary, interim and late stages during in vitro angiogenesis using ECMatrix™. The akermanite ion extract up-regulated the expression of genes encoding the receptors of proangiogenic cytokines and also increased the expression level of genes encoding the proangiogenic downstream cytokines, such as nitric oxide synthase and nitric oxide synthesis. Akermanite implanted in rabbit femoral condyle model promoted neovascularization after 8 and 16 weeks of implantation, which further confirmed its stimulation effect on angiogenesis in vivo. These results indicate that akermanite ceramic, an appropriate Si ion concentration source, could induce angiogenesis through increasing gene expression of proangiogenic cytokine receptors and up-regulated downstream signaling. To our knowledge, akermanite ceramic is the first Si-containing ceramic demonstrated to be capable of inducing angiogenesis during bone regeneration.

HPV-16 infection may play an important role in the development of non-small cell lung cancer (NSCLC) among never-smokers. Due to the critical role of angiogenesis in NSCLC development, we describe here the effect of HPV-16 oncoproteins on angiogenesis in NSCLC and the underlying mechanisms. We found that overexpression of HPV-16 E6 and E7 oncoproteins in NSCLC cells significantly promoted angiogenesis both in vitro and in vivo, and correspondingly, an enhanced expression of HIF-1α and VEGF, important pro-angiogenic factors in tumor angiogenesis. Meanwhile, overexpression of HPV-16 oncoproteins also led to HIF-1α-dependent increases in the secretion of several other pro-angiogenic factors, including IL-8. Our findings suggest that HPV-16 oncoproteins contribute to the development of NSCLC possibly by promoting HIF-1α/VEGF-mediated tumor angiogenesis.Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Two new secolignans, peperomins G and H (1 and 2, resp.), were isolated from the whole plant of Peperomia dindygulensis, together with five known secolignans, peperomin A (3), peperomin E (4), peperomin B (5), 2,3-trans-2-methyl-3-{(3-hydroxy-4,5-dimethoxyphenyl)[5-methoxy-3,4-(methylenedioxy)phenyl]methyl}butyrolactone (6), 2,3-cis-2-(hydroxymethyl)-3-{bis[5-methoxy-3,4-(methylenedioxy)phenyl]methyl}butyrolactone (7). Their structures and configurations were elucidated by spectroscopic methods including 2D-NMR techniques. Antiangiogenic effects of all compounds were evaluated using human umbilical vein endothelial cells (HUVEC) proliferation and tube-formation tests, with compounds 4 and 5 being active in the bioassay. Compounds 4 and 5 induced obvious cell toxicity to HUVEC with IC(50) values of 1.64±0.19 and 8.44±0.4 μM, respectively. Compounds 4 and 5 also exhibited significant HUVEC tube formation-inhibiting activity with IC(50) values of 3.13±0.09 and 6.24±0.12 μM, respectively.

The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar-Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair.

Sonic hedgehog (SHH) expression is tightly regulated throughout development. In the adult, aberrant expression of SHH is associated with the onset and progression of pancreatic cancer, as evidenced by increased levels of expression in premalignant and malignant lesions of the pancreas. We investigated the hypothesis that SHH, secreted from pancreatic tumors, functions in a paracrine manner to influence the biological condition of mesenchymal and endothelial cells. Orthotopic implantation of a pancreatic tumor cell line expressing SHH (Capan-2) and a transformed primary cell line that overexpresses SHH (T-HPNE.SHH) were used to show that overexpression of SHH increased primary tumor size and metastasis. Treatment with a neutralizing antibody, 5E1, decreased primary tumor volume and inhibited metastasis. Lyve-1+ vessels and stromal fibroblasts in tumors expressed primary cilium and showed localization of the receptor Smoothened to the primary cilium, providing evidence of active SHH signaling through this pathway. Although primary cilia are present on normal ductal cells of the pancreas, we did not observe primary cilium on the ductal tumor cells, suggesting decreased autocrine signaling through pathways mediated by the primary cilium in pancreatic cancer. These data support the hypothesis that SHH, secreted from pancreatic epithelia, is critical in establishing and regulating the tumor microenvironment and thereby contributes to progression of pancreatic cancer.