Oenothein B from Eugenia uniflora leaves exerts pro-angiogenic effects by increasing VEGF and TNF-α levels.

Oenothein B (OeB), a dimeric ellagitannin with a macrocyclic structure, is reported to have beneficial effects, including antioxidant, antitumor, antiviral, and antimutagenic effects, on human health. Despite the remarkable properties of OeB, its role in neovascularization process has not yet been evaluated. Thus, this study aimed to evaluate the angiogenic activity of OeB using a chorioallantoic membrane (CAM) assay at different concentrations (6.25, 12.5, and 25 µg/µL), employing digital imaging and histological analysis. Furthermore, to elucidate the mechanisms by which OeB influences angiogenesis, we assessed the levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-α) in CAM using immunohistochemical analysis. All concentrations of OeB significantly increased (p < 0.05) the percentage of vascularization as well as the levels of all the angiogenesis-associated parameters evaluated, indicating the pronounced pro-angiogenic activity of OeB. Our results showed that inflammation was one of the most relevant phenomena observed in CAM histology along with angiogenesis. In addition, a significant increase in VEGF and TNF-α levels was observed in all the CAMs compared to the negative control (p < 0.05). We suggest that OeB may induce the presence of inflammatory cells in CAM, leading to increased VEGF and TNF-α levels that result in the induction of angiogenesis. Therefore, OeB presents a favorable profile that could be further explored for the development of drugs for pro-angiogenic and tissue repair therapies.

Novel sulfonamide-chalcone hybrid stimulates inflammation, angiogenesis and upregulates vascular endothelial growth factor (VEGF) in vivo.

Chalcones and sulfonamides are well-known chemical groups associated with several biological activities such as antibiotic, anti-inflammatory, and antitumor activities. Over the past few decades, a series of sulfonamide-chalcone hybrids have been synthesized and assessed to develop compounds with interesting biological properties for application in disease therapy. In the present study, a new sulfonamide-chalcone hybrid µ - (2,5-dichloro-N-{4-[(3E)-4-(3-nitrophenyl) buta-1,3-dien-2-yl] phenyl} benzene sulfonamide), or simply CL185, was synthesized, and its angiogenic activity was assessed using the chick embryo chorioallantoic membrane (CAM) assay at different concentrations (12.5, 25, and 50 µg/µL). To further investigate the role of CL185 in the angiogenic process, we evaluated the levels of vascular endothelial growth factor (VEGF) in all treated CAMs. The results showed that all concentrations of CL185 significantly increased tissue vascularization (p < 0.05) as well as the parameters associated with angiogenesis, in which inflammation was the most marked phenomenon observed. In all CAMs treated with CL185, VEGF levels were significantly higher than those in the negative control (p < 0.05), and at the highest concentration, VEGF levels were even higher than in the positive control (p < 0.05). The pronounced angiogenic activity displayed by CL185 may be related to the increase in VEGF levels that were stimulated by inflammatory processes observed in our study. Therefore, CL185 presents a favorable profile for the development of drugs that can be used in pro-angiogenic and tissue repair therapies.

Hancornia speciosa serum fraction latex stimulates the angiogenesis and extracellular matrix remodeling processes.

The Hancornia speciosa latex reveals angiogenic, osteogenic, and anti-inflammatory properties, which present its potential for developing of wound healing drugs; however, the latex compounds responsible for angiogenesis remain unknown. One strategy to screen these active compounds is evaluation of latex fractions. This study aimed to obtain different fractions of latex and evaluate its angiogenic activity separately using the chick chorioallantoic membrane (CAM) assay. The serum (SE) fraction was responsible for angiogenesis, which was subject to biochemical characterization and computational simulations in order to understand the contribution of H. speciosa latex in wound healing process. Our results revealed weak antioxidant potential and absence of antimicrobial activity in the SE fraction. Phytochemical analysis identified chlorogenic acids (CGA) as the main compound of SE fraction. CGA bioactivity predictions identify different molecules associated with extracellular matrix (ECM) remodeling, such as metalloproteinases, which also are overexpressed in our CAM assay experiment. Docking simulations revealed the interactions between CGA and matrix metalloproteinase 2. In conclusion, SE latex fraction stimulates angiogenesis and may influence ECM remodeling. These properties may contribute to the wound healing process, and also confirm the widespread use of this plant.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo.

PURPOSE: To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. METHODS: Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. RESULTS: Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. CONCLUSIONS: The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

Effect of autologous transplant of peripheral blood mononuclear cells in combination with proangiogenic factors during experimental revascularization of lower limb ischemia.

Peripheral blood mononuclear cells (PBMCs) contain a cell fraction of mononuclear progenitor cells (MPCs), which own significant angiogenic potential. Autologous transplant of PBMC and/or platelet-rich plasma (PRP) promotes endothelial cells differentiation in experimental lower limb ischemia, which is considered a safe and effective strategy to support revascularization, either in animal models or clinical trials. In addition, thrombin has been proposed to enrich biological scaffolds, hence increasing MPC viability after intramuscular administration, whereas proangiogenic mediators such as vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-α), inhibitor of the plasminogen activator-1 (PAI-1), and chemokine (CXCL1; GRO-α) participate in the endothelial response to ischemia, through their proangiogenic effects over endothelial cells proliferation, survival, migration, endothelial integrity maintenance, and physiologic vascular response to injury. In the present study, we describe the effect of autologous PBMCs transplant and PRP, either with or without thrombin, over proangiogenic mediators (measured by enzyme-linked immunosorbent assay) and revascularization response (angiographic vascular pattern at 30 days after vascular occlusion) in a rat model of lower limb ischemia. The group treated with PBMC + PRP significantly induced PAI-1, an effect that was prevented by the addition of thrombin. Furthermore, treatment with PBMC + PRP + thrombin resulted in the induction of VEGF. GRO-α showed a sensitive induction of all proangiogenic mediators. All treatments significantly stimulated revascularization, according to angiographic assessment, whereas higher effect was observed with PBMC + PRP treatment (p < .0001). In conclusion, autologous PBMC transplant stimulates revascularization during experimental ischemia of the lower limb, whereas particular effects over proangiogenic and fibrinolytic mediators may be attributed to PBMCs and its combination with PRP and thrombin.

Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements.

OBJECTIVE: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). METHODOLOGY: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. RESULTS: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). CONCLUSION: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Wortmannin targeting phosphatidylinositol 3-kinase suppresses angiogenic factors in shear-stressed endothelial cells.

Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.

Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements

Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

TNF-alpha increases angiogenic potential in a co-culture system of dental pulp cells and endothelial cells.

We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Effects of benzalkonium chloride and cyclosporine applied topically to rabbit conjunctiva: a histomorphometric study / Efeitos do uso tópico do conservante cloreto de benzalcônio e ciclosporina na conjuntiva de coelhos: estudo histomorfométrico

ABSTRACT Purpose: Chronic instillation of benzalkonium chloride, a preservative, has inflammatory effects on the ocular surface. However, addition of the anti-inflammatory agent cyclosporine to a therapeutic protocol may mitigate these effects. This study compared the toxic effects of a 0.1% benzalkonium chloride solution and the possible protective effect of 0.05% cyclosporine when applied topically to the rabbit conjunctiva. Methods: Fifteen age- and weight-matched, female New Zealand white rabbits were categorized into three groups and treated for 30 consecutive days. Group 1, 2, and 3 - benzalkonium chloride received 0.1% every 24 h, 0.05% cyclosporine every 6 h, and both treatments, respectively. In each rabbit, the left eye was subjected to treatment and the right eye was a control. The rabbits were euthanized at after the experiment. Goblet cells and blood vessels were then enumerated in conjunctival tissues stained with periodic acid-Schiff and hematoxylin-eosin, respectively. Differences between treated and untreated eyes and between groups were compared using the t-test and analysis of variance. Results: Benzalkonium chloride treatment, with and without cyclosporine, significantly reduced (p≤0.05) in the number of goblet cells in treatment eyes compared with that in respective control eyes. Alternatively, adding cyclosporine to benzalkonium chloride did not prevent the loss of conjunctival goblet cells, and a significant reduction in the number of goblet cells was noted. Benzalkonium chloride-induced significant increase in the number of new blood vessels was mitigated significantly by the addition of cyclosporine. Conclusion: This study demonstrated the magnitude of conjunctival injury caused by chronic instillation of benzalkonium chloride. Although cyclosporine did not mitigate the effects on goblet cells, its addition minimized inflammatory angiogenesis induced by benzalkonium chloride.

RESUMO Objetivo: A instilação crônica de cloreto de benzal­cônio, um conservante, tem efeitos inflamatórios na superfície ocular. No entanto, a adição do agente anti-inflamatório ciclosporina a um protocolo terapêutico pode atenuar esses efeitos. Este estudo comparou os efeitos tóxicos de uma solução de cloreto de benzalcônio a 0,1% e o possível efeito protetor de ciclosporina a 0,05% quando aplicado topicamente à conjuntiva de coelho. Métodos: Quinze coelhos fêmeas brancos da raça Nova Zelândia, pareados por idade e peso, foram categorizados em três grupos e tratados por 30 dias consecutivos. Os grupos 1, 2 e 3 - receberam cloreto de benzalcônio 0,1% a cada 24h, ciclosporina a 0,005% a cada 6h e ambos os tratamentos, respectivamente. Em cada coelho, o olho esquerdo foi submetido a tratamento e o olho direito foi controle. Os coelhos foram submetidos à eutanásia após o experimento. Células caliciformes e vasos sanguíneos foram então enumerados em tecidos conjuntivais corados com ácido periódico-Schiff e hematoxilina-eosina, respectivamente. As diferenças entre os olhos tratados e não tratados e entre os grupos foram comparadas usando o teste t e análise de variância. Resultados: O tratamento com cloreto de benzalcônio, com e sem ciclosporina, reduziu significativamente (p£0,05) o número de células caliciformes nos olhos tratados em comparação com os olhos controle correspondentes. Alternativamente, a adição de ciclosporina ao cloreto de benzalcônio não impediu a perda de células caliciformes conjuntivais, e foi observada uma redução significativa no número de células caliciformes. O aumento significativo induzido pelo cloreto de benzalcônio no número de novos vasos sanguíneos foi significativamente mitigado pela adição da ciclosporina. Conclusão: Este estudo demonstrou a magnitude da lesão conjuntival resultante da instilação crônica de cloreto de benzalcônio. Embora a ciclosporina não tenha atenuado os efeitos nas células caliciformes, sua adição minimizou a angiogênese inflamatória induzida pelo cloreto de benzalcônio.

Effects of benzalkonium chloride and cyclosporine applied topically to rabbit conjunctiva: a histomorphometric study.

PURPOSE: Chronic instillation of benzalkonium chloride, a preservative, has inflammatory effects on the ocular surface. However, addition of the anti-inflammatory agent cyclosporine to a therapeutic protocol may mitigate these effects. This study compared the toxic effects of a 0.1% benzalkonium chloride solution and the possible protective effect of 0.05% cyclosporine when applied topically to the rabbit conjunctiva. METHODS: Fifteen age- and weight-matched, female New Zealand white rabbits were categorized into three groups and treated for 30 consecutive days. Group 1, 2, and 3 - benzalkonium chloride received 0.1% every 24 h, 0.05% cyclosporine every 6 h, and both treatments, respectively. In each rabbit, the left eye was subjected to treatment and the right eye was a control. The rabbits were euthanized at after the experiment. Goblet cells and blood vessels were then enumerated in conjunctival tissues stained with periodic acid-Schiff and hematoxylin-eosin, respectively. Differences between treated and untreated eyes and between groups were compared using the t-test and analysis of variance. RESULTS: Benzalkonium chloride treatment, with and without cyclosporine, significantly reduced (p≤0.05) in the number of goblet cells in treatment eyes compared with that in respective control eyes. Alternatively, adding cyclosporine to benzalkonium chloride did not prevent the loss of conjunctival goblet cells, and a significant reduction in the number of goblet cells was noted. Benzalkonium chloride-induced significant increase in the number of new blood vessels was mitigated significantly by the addition of cyclosporine. CONCLUSION: This study demonstrated the magnitude of conjunctival injury caused by chronic instillation of benzalkonium chloride. Although cyclosporine did not mitigate the effects on goblet cells, its addition minimized inflammatory angiogenesis induced by benzalkonium chloride.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Mast Cell Protease 7 Promotes Angiogenesis by Degradation of Integrin Subunits.

Previous studies from our laboratory have shown that during angiogenesis in vitro, rmMCP-7 (recombinant mouse mast cell protease-7) stimulates endothelial cell spreading and induces their penetration into the matrix. The ability of rmMCP-7 to induce angiogenesis in vivo was assessed in the present study using a directed in vivo angiogenesis assay (DIVAA™). Vessel invasion of the angioreactor was observed in the presence of rmMCP-7 but was not seen in the control. Since integrins are involved in endothelial cell migration, the relationship between rmMCP-7 and integrins during angiogenesis was investigated. Incubation with rmMCP-7 resulted in a reduction in the levels of integrin subunits αv and ß1 on SVEC4-10 endothelial cells during angiogenesis in vitro. Furthermore, the degradation of integrin subunits occurs both through the direct action of rmMCP-7 and indirectly via the ubiquitin/proteasome system. Even in the presence of a proteasome inhibitor, incubation of endothelial cells with rmMCP-7 induced cell migration and tube formation as well as the beginning of loop formation. These data indicate that the direct degradation of the integrin subunits by rmMCP-7 is sufficient to initiate angiogenesis. The results demonstrate, for the first time, that mMCP-7 acts in angiogenesis through integrin degradation.

Pro-Fibrogenic and Anti-Inflammatory Potential of a Polyphenol-Enriched Fraction from Annona crassiflora in Skin Repair.

A polyphenol-enriched fraction from Annona crassiflora fruit peel (PEF-Ac) containing chlorogenic acid, epi-catechin, procyanidins B2 and C1, quercetin-glucoside, kaempferol, and caffeoyl-glucoside was investigated for its anti-inflammatory, pro-angiogenic, and profibrogenic potential in the healing of cutaneous wounds. Four wounds were performed on the back of C57 mice and the lesions were treated with the vehicle (Vaseline and lanolin) and PEF-Ac at concentrations of 2%, 4%, and 6% for 4 and 7 d. Neutrophils and macrophages activities were evaluated indirectly by the activity of myeloperoxidase and N-acetyl-ß-D-glycosaminidase, angiogenesis was evaluated by hemoglobin dosing and vessel count in histological sections, and collagen deposition was assessed from histological sections stained with picrosirius red. PEF-Ac demonstrated anti-inflammatory activity, with reduced activities of neutrophil and macrophage in the cutaneous wounds. In addition, there was an increase in the synthesis of types I and III collagen, as well as in the percentage of wound closure, mainly after 4 d of treatment. On the other hand, PEF-Ac did not present an effective pro-angiogenic activity. A. crassiflora fruit peel showed anti-inflammatory and profibrogenic properties, indicating a promising natural source of bioactive molecules for treatment of cutaneous wounds.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo

Abstract Purpose To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. Methods Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. Results Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. Conclusions The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

TNF-alpha increases angiogenic potential in a co-culture system of dental pulp cells and endothelial cells

Abstract We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Induction by innate defence regulator peptide 1018 of pro-angiogenic molecules and endothelial cell migration in a high glucose environment.

Synthetic innate defence regulator (IDR) peptides such as IDR-1018 modulate immunity to promote key protective functions including chemotaxis, wound healing, and anti-infective activity, while suppressing pro-inflammatory responses to non-pathological levels. Here we demonstrated that IDR-1018 induced, by up to 75-fold, pro-angiogenic VEGF-165 in keratinocytes but suppressed this isoform in endothelial cells. It also induced early angiogenin and prolonged anti-inflammatory TGFß expression on endothelial cells, while suppressing early pro-inflammatory IL-1ß expression levels. IDR-1018 also down-regulated the hypoxia induced transcription factor HIF-1α in both keratinocytes and endothelial cells. Consistent with these data, in an in vitro wound healing scratch assay, IDR-1018 induced migration of endothelial cells under conditions of hypoxia while in epithelial cells migration increased only under conditions of normoxia.

Pre-culture in endothelial growth medium enhances the angiogenic properties of adipose-derived stem/stromal cells.

Considerable progress has been made on the development of adipose-derived stem/stromal cells (ASCs) as pro-angiogenic therapeutic tools. However, variable clinical results highlight the need for devising strategies to enhance their therapeutic efficacy. Since ASCs proliferate and stabilize newly formed vessels during the angiogenic phase of adipose tissue formation, we hypothesized that mimicking an angiogenic milieu during culture of ASCs would enhance their capacity to support endothelial cell survival and angiogenesis. To test this, we compared the effect of an endothelial growth medium (EGM-2) and conventional media (αMEM) on the progenitor and angiogenic properties of ASCs. ASCs cultured in EGM-2 (ASC-EGM) displayed the highest clonogenic efficiency, proliferative potential and multilineage potential. After co-culture under growth factor starvation, only ASC-EGM attenuated luciferase-expressing human umbilical vein endothelial cells (HUVECluc) apoptosis and supported the formation of endothelial cords in a dose-dependent manner. These effects were recapitulated by the conditioned medium of ASC-EGM, which displayed a 100-fold higher expression of hepatocyte growth factor in comparison with ASC-αMEM. Next, HUVECluc and ASCs were co-transplanted subcutaneously into immunodeficient mice, and the survival of HUVECluc was monitored by bioluminescent imaging. After 60 days, the survival of HUVECluc transplanted alone was equivalent to that of HUVECluc co-transplanted with ASC-αMEM (15.0 ± 0.7 vs. 13.0 ± 0.5%). Strikingly, co-transplantation with ASC-EGM increased HUVECluc survival to 105.0 ± 3.5%, and the resulting organoids displayed functional vasculature with the highest human-derived vascular area. These findings demonstrate that pre-conditioning of ASCs in endothelial growth medium augment their pro-angiogenic properties and could enhance their therapeutic efficacy against ischemic diseases.

The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis.

Saliva is a key factor that contributes to the high efficiency of wound healing in the oral mucosa. This is not only attributed to physical cues but also to the presence of specific peptides in the saliva, such as histatins. Histatin-1 is a 38 aa antimicrobial peptide, highly enriched in human saliva, which has been previously reported to promote the migration of oral keratinocytes and fibroblasts in vitro However, the participation of histatin-1 in other crucial events required for wound healing, such as angiogenesis, is unknown. Here we demonstrate that histatin-1 promotes angiogenesis, as shown in vivo, using the chick chorioallantoic membrane model, and by an in vitro tube formation assay, using both human primary cultured endothelial cells (HUVECs) and the EA.hy926 cell line. Specifically, histatin-1 promoted endothelial cell adhesion and spreading onto fibronectin, as well as endothelial cell migration in the wound closure and Boyden chamber assays. These actions required the activation of the Ras and Rab interactor 2 (RIN2)/Rab5/Rac1 signaling axis, as histatin-1 increased the recruitment of RIN2, a Rab5-guanine nucleotide exchange factor (GEF) to early endosomes, leading to sequential Rab5/Rac1 activation. Accordingly, interfering with either Rab5 or Rac1 activities prevented histatin-1-dependent endothelial cell migration. Finally, by immunodepletion assays, we showed that salivary histatin-1 is required for the promigratory effects of saliva on endothelial cells. In conclusion, we report that salivary histatin-1 is a novel proangiogenic factor that may contribute to oral wound healing.-Torres, P., Díaz, J., Arce, M., Silva, P., Mendoza, P., Lois, P., Molina-Berríos, A., Owen, G. I., Palma, V., Torres, V. A. The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis.

Improved in vitro angiogenic behavior on anodized titanium dioxide nanotubes.

BACKGROUND: Neovascularization over dental implants is an imperative requisite to achieve successful osseointegration onto implanted materials. The aim of this study was to investigate the effects on in vitro angiogenesis of anodized 70 nm diameter TiO2 nanotubes (NTs) on Ti6Al4V alloy synthesized and disinfected by means of a novel, facile, antibacterial and cost-effective method using super oxidized water (SOW). We also evaluated the role of the surface roughness and chemical composition of materials of materials on angiogenesis. METHODS: The Ti6Al4V alloy and a commercially pure Ti were anodized using a solution constituted by SOW and fluoride as electrolyte. An acid-etched Ti6Al4V was evaluated to compare the effect of micro-surface roughness. Mirror-polished materials were used as control. Morphology, roughness, chemistry and wettability were assessed by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy, atomic force microscopy, energy dispersive X-ray spectroscopy (EDX) and using a professional digital camera. Bovine coronary artery endothelial cells (BCAECs) were seeded over the experimental surfaces for several incubation times. Cellular adhesion, proliferation and monolayer formation were evaluated by means of SEM. BCAEC viability, actin stress fibers and vinculin cellular organization, as well as the angiogenic receptors vascular endothelial growth factor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) were measured using fluorescence microscopy. RESULTS: The anodization process significantly increased the roughness, wettability and thickness of the oxidized coating. EDX analysis demonstrated an increased oxygen (O) and decreased carbon (C) content on the NTs of both materials. Endothelial behavior was solidly supported and improved by the NTs (without significant differences between Ti and alloy), showing that endothelial viability, adhesion, proliferation, actin arrangement with vinculin expression and monolayer development were evidently stimulated on the nanostructured surface, also leading to increased activation of VEGFR2 and eNOS on Ti6Al4V-NTs compared to the control Ti6Al4V alloy. Although the rougher alloy promoted BCAECs viability and proliferation, filopodia formation was poor. CONCLUSION: The in vitro results suggest that 70 nm diameter NTs manufactured by anodization and cleaned using SOW promotes in vitro endothelial activity, which may improve in vivo angiogenesis supporting a faster clinical osseointegration process.