Angiogenic Vascular Endothelial Growth Factor Research Articles

Calreticulin stabilizes vascular endothelial growth factor‐A mRNA via interaction with AU‐rich element at 3′‐UTR

Messenger RNA (mRNA) stability is a post‐transcriptional regulation that determines mRNA fate and influences multiple cellular functions. Previous studies have demonstrated that AU‐rich element (ARE) in 3′‐untranslated region (UTR) of mRNA is critical for mRNA stability via interaction with ARE‐binding proteins (ARE‐BPs). Chaperone protein calreticulin (CRT) has been reported to bind the 3′‐UTR, suggesting that it may act as a novel ARE‐BP. The expression level of CRT positively is correlated with the expression of angiogenic factor vascular endothelial growth factor‐A (VEGF‐A). However, the detail regulatory mechanism of CRT on VEGF‐A expression remains elusive. Therefore, we aimed to investigate whether CRT regulates mRNA stability of VEGF‐A through interaction with ARE. Our results showed that depletion of CRT significantly destabilized VEGF‐A mRNA and subsequently decreased the expression levels of both VEGF‐A RNA and protein in PC3 human prostate cancer cell line. Since several AREs were identified in the mRNA of VEGF‐A, we further explore the relationship between CRT and these AREs. Pull‐down analysis demonstrated that CRT directly interacts with the ARE region of VEGF‐A mRNA. In addition, we constructed luciferase vectors contained the ARE of VEGF‐A and co‐transfected with siRNA of CRT or CRT overexpression vector. Our results demonstrated that CRT interacts with ARE in 3′‐UTR of VEGF‐A mRNA. In summary, this study suggests that CRT stabilizes VEGF‐A mRNA through interacting with ARE at the 3′‐UTR of VEGF‐A mRNA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High mobility group box 1 (HMGB1) acts as an “alarmin” to promote acute myeloid leukaemia progression

ABSTRACTHigh mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1β) production by healthy leukocytes, leading to IL-1β-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.

VEGFR2 is activated by high‐density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia

High-density lipoproteins augment hypoxia-induced angiogenesis by inducing the key angiogenic vascular endothelial growth factor A (VEGFA) and total protein levels of its receptor 2 (VEGFR2). The activation/phosphorylation of VEGFR2 is critical for mediating downstream, angiogenic signaling events. This study aimed to determine whether reconstituted high-density lipoprotein (rHDL) activates VEGFR2 phosphorylation and the downstream signaling events and the importance of VEGFR2 in the proangiogenic effects of rHDL in hypoxia. In vitro, rHDL increased VEGFR2 activation and enhanced phosphorylation of downstream, angiogenic signaling proteins ERK1/2 and p38 MAPK in hypoxia. Incubation with a VEGFR2-neutralizing antibody attenuated rHDL-induced phosphorylation of VEGFR2, ERK1/2, p38 MAPK, and tubule formation. In a murine model of ischemia-driven neovascularization, rHDL infusions enhanced blood perfusion and augmented capillary and arteriolar density. Infusion of a VEGFR2-neutralizing antibody ablated those proangiogenic effects of rHDL. Circulating Sca1+/CXCR4+ angiogenic progenitor cell levels, important for neovascularization in response to ischemia, were higher in rHDL-infused mice 3 d after ischemic induction, but that did not occur in mice that also received the VEGFR2-neutralizing antibody. In summary, VEGFR2 has a key role in the proangiogenic effects of rHDL in hypoxia/ischemia. These findings have therapeutic implications for angiogenic diseases associated with an impaired response to tissue ischemia.-Cannizzo, C. M., Adonopulos, A. A., Solly, E. L., Ridiandries, A., Vanags, L. Z., Mulangala, J., Yuen, S. C. G., Tsatralis, T., Henriquez, R., Robertson, S., Nicholls, S. J., Di Bartolo, B. A., Ng, M. K. C., Lam, Y. T., Bursill, C. A., Tan, J. T. M. VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia.

Can serum progranulin levels be a biomarker following gastric ulcer therapy?

IntroductionProgranulin is a novel growth factor that has several physiological and pathological roles such as cell growth, tumourigenesis, embryogenesis, wound healing, and inflammation.AimTo compare the pre-treatment and post-treatment serum levels of the angiogenic factor vascular endothelial growth factor (VEGF), pro-inflammatory cytokine tumor necrosis factor α (TNF-α), and progranulin in peptic ulcer (PU) patients with a healthy control group.Material and methodsSerum VEGF, TNF-α, and progranulin levels were studied with ELISA in 42 PU patients (antral ulcer (AU): 22, duodenal ulcer (DU): 20) and 15 healthy controls.ResultsThe serum progranulin levels before treatment were 4237.35 ±1091.30 pg/ml in the patients with AU, 4682.64 ±1501.46 pg/ml in the patients with DU, 3055.66 ±626.88 pg/ml in the control group, and 4460 ±1315 pg/ml in the ulcer (AU and DU) group. The serum progranulin levels were 3607.7 ±869.4 pg/ml in the AU group, 4286.5 ±1208.78 pg/ml in the DU group, and 3947.1 ±1094.64 pg/ml in the ulcer group after the treatment. When comparing pre-treatment serum progranulin levels of the AU group, DU group, and ulcer group with the control group there were statistically significant differences (p < 0.001, p < 0.0001, p < 0.0001, respectively).ConclusionsThe disappearance of the difference in terms of post-treatment serum levels of progranulin between the AU group and the control group suggests that serum levels of progranulin can be used as a biomarker of gastric ulcer healing.

Expression of Vascular Endothelial Growth Factor and Microvessel Density in Psoriatic Skin Lesions.

Background:Psoriasis is a common chronic and immune-mediated skin disorder having a significant impact on the patient's quality of life. An analysis of the role of angiogenic vascular endothelial growth factor (VEGF) expression and microvessel density in psoriatic skin lesions may help in better understanding of the disease pathogenesis. The aim of this study was to examine the expression of vascular endothelial growth factor (VEGF) and microvessel density using CD34 antibodies in psoriatic skin lesions by immunohistochemical examination using normal skin of healthy individuals as controls.Materials and Methods:Patients with clinical diagnosis of plaque type of psoriasis (e.g., chronic plaque) (n = 49) were included in the study. 5-mm punch biopsies were taken from the psoriatic skin lesions in these patients. A total of 20 punch biopsies were taken from the control group comprising of 20 healthy volunteers. The biopsies were subjected to histopathological examination for confirmation of diagnosis and grading. Immunohistochemical evaluation was done for the expression of VEGF, and microvessel density was assessed using CD34 and compared with the controls.Results:An increased VEGF expression by keratinocytes (49.80% ± 21.16%) and microvessel density in the papillary dermis (15.302% ± 3.8061%) was observed in patients with psoriasis, which was significantly higher as compared to controls (P < 0.0001). A significant positive correlation was observed between VEGF expression by keratinocytes and the microvessel density in the dermis (r = 0.664, P = 0.01). No significant correlation was observedbetween the histopathological grade of psoriasis and microvessel density, or with the VEGF expression.Conclusion:VEGF expression ascertained to be a significant factor in the pathogenesis of psoriasis.

Effects of thyroid-stimulating hormone on adhesion molecules and pro-inflammatory cytokines secretion in human umbilical vein endothelial cells.

Atherosclerosis is a multifactorial disorder, which affects the arterial wall. It has been reported that, hypothyroidism and thyroid hormone deficiency are related to cardiovascular disorders. Also, endothelial dysfunction plays an essential role in the development of atherosclerosis. We aimed to evaluate the effects of thyroid-stimulating hormone (TSH) on pro-inflammatory tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), angiogenic vascular endothelial growth factor (VEGF) and leukocyte adhesion, intercellular adhesion molecule 1 (ICAM-1) and E-selectin in human umbilical vein endothelial cells (HUVECs). In this study, HUVEC cells were treated with 1 and 2 μM of TSH in different treatment times. The gene and protein expression of ICAM-1, VEGF, and E-selectin were performed by real-time polymerase chain reaction and western blotting, respectively. Likewise, TNF-α and IL-6 protein levels were determined by the ELISA method. VEGF, ICAM-1, and E-selectin as endothelial dysfunction markers and also, TNF-α and IL-6 as pro-inflammatory cytokines were detectable in HUVEC. Besides, the results of this study revealed that TSH treatment down-regulates TNF-α and IL-6. Evaluating the gene and protein expression data revealed the upregulation of ICAM-1, E-selectin, and VEGF in TSH treated cases in different periods of exposure. Considering the multiple actions of TSH, it could be concluded that TSH plays a controversial role in atherogenesis by anti-inflammatory effects and on the other side, angiogenesis and leukocyte adhesion induction which is related to vascular cell proliferation.

Prevascularization of natural nanofibrous extracellular matrix for engineering completely biological three-dimensional prevascularized tissues for diverse applications.

Self-sustainability after implantation is one of the critical obstacles facing large engineered tissues. A preformed functional vascular network provides an effective solution for solving the mass transportation problem. With the support of mural cells, endothelial cells (ECs) can form microvessels within engineered tissues. As an important mural cell, human mesenchymal stem cells (hMSCs) not only stabilize the engineered microvessel network, but also preserve their multi-potency when grown under optimal culture conditions. A prevascularized hMSC/extracellular matrix (ECM) sheet fabricated by the combination of hMSCs, ECs and a naturally derived nanofibrous ECM scaffold offers great opportunity for engineering mechanically strong and completely biological three-dimensional prevascularized tissues. The objective of this study was to create a prevascularized hMSC/ECM sheet by co-culturing ECs and hMSCs on a nanofibrous ECM scaffold. Physiologically low oxygen (2% O2 ) was introduced during the 7day hMSC culture to preserve the stemness of hMSCs and thereby their capability to secrete angiogenic factors. The ECs were then included to form microvessels under normal oxygen (20% O2 ) for up to 7days. The results showed that a branched and mature vascular network was formed in the co-culture condition. Angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) were significantly increased by low-oxygen culture of hMSCs, which further stabilized and supported the maturation of microvessels. A differentiation assay of the prevascularized ECM scaffold demonstrated a retained hMSC multi-potency in the hypoxia cultured samples. The prevascularized hMSC/ECM sheet holds great promise for engineering three-dimensional prevascularized tissues for diverse applications.

Therapeutic Angiogenesis via Solar Cell-Facilitated Electrical Stimulation.

Cell therapy has been suggested as a treatment modality for ischemic diseases, but the poor survival and engraftment of implanted cells limit its therapeutic efficacy. To overcome such limitation, we used electrical stimulation (ES) derived from a wearable solar cell for inducing angiogenesis in ischemic tissue. ES enhanced the secretion of angiogenic growth factors and the migration of mesenchymal stem cells (MSCs), myoblasts, endothelial progenitor cells, and endothelial cells in vitro. In a mouse ischemic hindlimb model, ES generated by a solar cell and applied to the ischemic region promoted migration of MSCs toward the ischemic site and upregulated expression of angiogenic paracrine factors (vascular endothelial, basic fibroblast, and hepatocyte growth factors; and stromal cell-derived factor-1α). Importantly, solar cell-generated ES promoted the formation of capillaries and arterioles at the ischemic region, attenuated muscle necrosis and fibrosis, and eventually prevented loss of the ischemic limb. Solar cell ES therapy showed higher angiogenic efficacy than conventional MSC therapy. This study shows the feasibility of using solar cell ES as a novel treatment for therapeutic angiogenesis.

BCG immune activation reduces growth and angiogenesis in an in vitro model of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancers worldwide and is associated with poor survival and significant treatment morbidity. The immune profile in patients with HNSCC is immunosuppressive and presents cytokine-mediated adaptive immune responses, triggered apoptosis of T cells, and alterations in antigen processing machinery. Bacille Calmette-Guerin (BCG) immunotherapy has been used successfully as a treatment for several types of cancer. In the present study, we sought to determine the antitumor effect of soluble mediators from peripheral blood mononuclear immune cells (PBMCs) activated with BCG vaccine in a three-dimensional coculture model of HNSCC growth using FaDu hypopharynx carcinoma squamous cells. BCG activation of PBMCs led to an increase in CD4+ and CD8+ lymphocyte subsets concomitant with an elevation in the levels of the antitumor cytokines IL-6, TNF-α and IFN-γ, and a EGFR in FaDu cells. In addition, coculture with BCG-activated PBMCs reduced FaDu proliferation and increased cytotoxicity and apoptosis in parallel with an increase in caspase-3 activity and p53 expression. Finally, conditioned medium from BCG-activated PBMCs reduced the levels of the angiogenic factors vascular endothelial growth factor and angiopoietin-2 produced by human aortic endothelial cells (HAECs), and inhibited their proliferation and differentiation into capillary-like structures. Taken together, these results demonstrate that BCG vaccination induces antitumor responses in an HNSCC in vitro model and suggest that the BCG vaccine could be an effective alternative therapy for the treatment of HNSCC.

Abstract 5023: Disruption of Monocarboxylate transporter-4 Basigin interaction as an effective strategy to inhibit hypoxic response, tumor growth and vascularization, and stem cell phenotype in human glioblastoma in vitro and in vivo

Abstract Monocarboxylate transporters, constitute a family (SLC16) of proton-linked plasma membrane transporters that carry molecules containing a single carboxylate group across biological membranes. Basigin (CD147), is involved in many physiological functions during early stages of development and in cancer. Basigin has been shown to be required for functional plasma membrane expression of Monocarboxylate transporter-1 and Monocarboxylate transporter-4. Using a cell-based screening assay, we identified acriflavine, a small molecule that inhibits the binding between Basigin and Monocarboxylate transporters in vitro and in vivo. Surface Plasmon Resonance analysis confirmed direct binding of acriflavine to Basigin’s immunoglobulin extracellular domain with a low binding constant (kD) of 0.16µM. Acriflavine inhibits normoxic growth of glioma stem cells in vitro and this activity is augmented by hypoxia or by expression of oxygen-stable mutant forms of HIF-1α or HIF-2α. Treatment of mice bearing established glioma stem cell-derived xenografts resulted in inhibition of tumor growth. Acriflavine treatment inhibited intratumoral expression of the angiogenic cytokine vascular endothelial growth factor and tumor vascularization. Our work shows that disruption of monocarboxylate transporter binding to Basigin is an effective approach to target glioma stem cells. Citation Format: Raffaella Spina, Dillon M. Voss, Kah Suan Lim, Constance J. Jeffery, Eli E. Bar. Disruption of Monocarboxylate transporter-4 Basigin interaction as an effective strategy to inhibit hypoxic response, tumor growth and vascularization, and stem cell phenotype in human glioblastoma in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2017-5023

Angiogenesis in Dermatology – Insights of Molecular Mechanisms and Latest Developments

Angiogenesis is the growth of new blood vessels from pre-existing vessels. It is a biological process essential in physiological wound healing or pathological inflammation and tumor growth, which underlies a complex interplay of stimulating and inhibiting signals. Extracellular matrix, cells of innate and adaptive immunity and endothelial cells itself are a major source of angiogenic factors that activate or inhibit specific receptors and consequently influence intracellular signaling pathways.Most inflammatory and neoplastic diseases in dermatology are characterized by excessive angiogenesis, such as psoriasis, atopic dermatitis, as well as melanoma, non-melanoma skin cancer, but also benign vascular neoplasia. In this article we describe current knowledge of angiogenesis and its most relevant mechanisms in different dermatological disorders with particular emphasis on the angiogenic factors (vascular endothelial growth factor) and angiopoietins as a target of current and future directions of anti-angiogenic therapy.

Osteoprotegerin Is a New Regulator of Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

Osteoprotegerin (OPG) is a novel regulator of endothelial cell function, angiogenesis, and vasculogenesis. We correlated expression levels of OPG with those of the angiogenic and inflammatory factors vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1/CCL2) in proliferative diabetic retinopathy (PDR). We also examined expression of OPG in retinas from diabetic rats and diabetic patients and measured production of OPG by human retinal microvascular endothelial cells (HRMEC) and investigated its angiogenic activity. Vitreous samples from 47 PDR and 28 nondiabetic patients, epiretinal membranes from 14 patients with PDR, human retinas (10 from diabetic patients and 10 from nondiabetic subjects), and rat retinas and HRMEC were studied by using enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, Western blot analysis, and RT-PCR. In vitro and in vivo angiogenesis assays were performed. We showed a significant increase in the expression of OPG, VEGF, and MCP-1/CCL2 in a comparison between vitreous samples from PDR patients and those from nondiabetic controls. Significant positive correlations were found between levels of OPG and levels of VEGF and MCP-1/CCL2. In epiretinal membranes, OPG was expressed in vascular endothelial cells and stromal cells. Significant increases of OPG mRNA and protein were detected in the retinas from diabetic patients. The proinflammatory cytokines TNF-α and IL-1β, but not VEGF, MCP-1/CCL2 or thrombin, induced upregulation of OPG in HRMEC. Osteoprotegerin induced ERK1/2 and Akt phosphorylation in HRMEC and stimulated their migration. Osteoprotegerin potentiated the angiogenic effect of VEGF in the in vivo protein gelatin plug assay. These results suggest that OPG is involved in PDR angiogenesis.

Age-related modulation of angiogenesis-regulating factors in the swine meniscus.

An in‐depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti‐angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve‐CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro‐cartilaginous differentiation. Expression of VEGF and Ve‐CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.

The Role of Mitochondria-Associated Reactive Oxygen Species in the Amyloid β Induced Production of Angiogenic Factors b y ARPE-19 Cells.

This study aimed to investigate the mechanisms whereby Amyloidbeta (Aβ) induces the production of angiogenic factors by a human retinal pigment epithelial cell line (ARPE-19) cells. ARPE-19 cells obtained from the American Type Culture Collection (ATCC) were utilized in this study. The expression level of vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and complement activation fragments C3a and C5a were measured by Real-time quantitative PCR (RT-PCR) and Enzyme-linked immunosorbent assay (ELISA). The production of mitochondria-associated reactive oxygen species (ROS) was measured by flow cytometry. The expression of VEGF, IL-8, MCP-1, C3a and C5a was significantly increased in Aβ-treated ARPE-19 cells. Mitochondria-associated ROS production was also significantly increased when exposed to Aβ. Inhibition of mitochondrial ROS with Diphenyleneiodonium chloride (DPI) markedly decreased the Aβ induced production of VEGF, IL-8, MCP-1, C3a and C5a by ARPE-19 cells. Anti-C3a or anti-C5a neutralizing antibodies did not have a detectable influence on the secretion of VEGF, IL-8 and MCP-1 by ARPE-19 cells upon stimulation with Aβ. Our results support the hypothesis that Aβ is involved in the pathogenesis of choroidal neovascularization (CNV) formation by promoting the production of the angiogenic cytokines VEGF, IL-8 and MCP-1 by RPE cells. Mitochondrial ROS was shown to play a role in the regulation of Aβ induced expression of these cytokines.

Vascular Endothelial Growth Factor Isoform-B Stimulates Neurovascular Repair After Ischemic Stroke by Promoting the Function of Pericytes via Vascular Endothelial Growth Factor Receptor-1.

Ischemic stroke triggers endogenous angiogenic mechanisms, which correlates with longer survival in patients. As such, promoting angiogenesis appears to be a promising approach. Experimental studies investigated mostly the potent angiogenic factor vascular endothelial growth factor isoform-A (VEGF-A). However, VEGF-A increases the risk of destabilizing the brain microvasculature, thus hindering the translation of its usage in clinics. An attractive alternative VEGF isoform-B (VEGF-B) was recently reported to act as a survival factor rather than a potent angiogenic factor. In this study, we investigated the therapeutic potential of VEGF-B in ischemic stroke using different in vivo and in vitro approaches. We showed that the delayed intranasal administration of VEGF-B reduced neuronal damage and inflammation. Unexpectedly, VEGF-B stimulated the formation of stable brain microvasculature within the injured region by promoting the interaction between endothelial cells and pericytes. Our data indicate that the effects of VEGF-B were mediated via its specific receptor VEGF receptor-1 (VEGFR-1) that is predominately expressed in brain pericytes. Importantly, VEGF-B promoted the survival of pericytes, and not brain endothelial cells, by inducing expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and the main protein involved in energy homeostasis AMP-activated protein kinase α (AMPKα). Moreover, we showed that VEGF-B stimulated the pericytic release of factors stimulating a "reparative angiogenesis" that does not compromise microvasculature stability. Our study unraveled hitherto unknown role of VEGF-B/VEGFR-1 signaling in regulating the function of pericytes. Furthermore, our findings suggest that brain microvasculature stabilization via VEGF-B constitutes a safe therapeutic approach for ischemic stroke.

Sulfated Hyaluronan Alters Endothelial Cell Activation in Vitro by Controlling the Biological Activity of the Angiogenic Factors Vascular Endothelial Growth Factor-A and Tissue Inhibitor of Metalloproteinase-3

Several pathologic conditions such as rheumatoid arthritis, ocular neovascularization, cancer, or atherosclerosis are often associated with abnormal angiogenesis, which requires innovative biomaterial-based treatment options to control the activity of angiogenic factors. Here, we studied how sulfated hyaluronan (sHA) and oversulfated chondroitin sulfate derivatives as potential components of functional biomaterials modulate vascular endothelial growth factor-A (VEGF-A) signaling and endothelial cell activity in vitro. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an effective angiogenesis inhibitor, exerts its activity by competing with VEGF-A for binding to VEGF receptor-2 (VEGFR-2). However, even though TIMP-3 and VEGF-A are known to interact with glycosaminoglycans (GAGs), the potential role and mechanism by which GAGs alter the VEGF-A/TIMP-3 regulated VEGFR-2 signaling remains unclear. Combining surface plasmon resonance, immunobiochemical analysis, and molecular modeling, we demonstrate the simultaneous binding of VEGF-A and TIMP-3 to sHA-coated surfaces and identified a novel mechanism by which sulfated GAG derivatives control angiogenesis: GAG derivatives block the binding of VEGF-A and TIMP-3 to VEGFR-2 thereby reducing their biological activity in a defined, sulfation-dependent manner. This effect was stronger for sulfated GAG derivatives than for native GAGs. The simultaneous formation of TIMP-3/sHA complexes partially rescues the sHA inhibited VEGF-A/VEGFR-2 signaling and endothelial cell activation. These results provide novel insights into the regulation of angiogenic factors by GAG derivatives and highlight the potential of sHA derivatives for the treatment of diseases associated with increased VEGF-A and VEGFR-2 levels.

Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel\xae, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product

BackgroundMesenchymal stromal cells (MSCs) have emerged as a more beneficial alternative to conventional therapy and may offer a potential cure for unmet medical needs. MSCs are known to possess strong immunomodulatory and anti-inflammatory properties. Moreover, they promote angiogenesis and tissue regeneration through the secretion of trophic factors. For these reasons, the past decade witnessed a sharp increase in the number of clinical trials conducted with stem cells for various vascular diseases requiring angiogenesis. In this study, we evaluated the in vitro angiogenic potency of Stempeucel®, which is an allogeneic pooled human bone marrow-derived mesenchymal stromal cell (phBMMSC) product. We previously established the safety of Stempeucel® in our pre-clinical studies, and clinical trials conducted for critical limb ischaemia and acute myocardial infarction.MethodsBecause the proposed mechanism of action of phBMMSCs is mainly through the secretion of pro-angiogenic cytokines, we developed a surrogate potency assay by screening various batches of large-scale expanded phBMMSCs for the expression of angiogenic factors and cytokines through gene expression and growth factor analyses, followed by in vitro functional assays.ResultsThe well characterized angiogenic vascular endothelial growth factor (VEGF) was selected and quantified in twenty six manufactured batches of phBMMSCs to establish consistency following the United States Food and Drug Administration recommendations. According to recommendations 21 CFR 211.165(e) and 211.194(a)(2), we also established and documented the specificity and reproducibility of the test methods employed through validation. Moreover, we also attempted to elucidate the mechanism of action of the cell population to ensure appropriate biological activity. The functional role of VEGF has been established through in vitro angiogenic assays and a dose-dependent correlation was observed with in vitro functional results.ConclusionsThe data generated from this study suggest the selection of VEGF as a single surrogate marker to test the angiogenic potency of phBMMSCs. Our study reports the quantification of VEGF in twenty six batches of large-scale manufactured phBMMSCs, and a concentration-dependent correlation of secreted VEGF to endothelial cell functions of migration, proliferation and tube formation, in the conditioned medium obtained from nine phBMMSC batches. To our cognizance, this is the first study in which a single angiogenic factor (VEGF) has been qualified as a surrogate potency marker through all three in vitro functional assays to determine the angiogenic potency of the phBMMSC population.

Angiogenesis in Dermatology – Insights of Molecular Mechanisms and Latest Developments

Angiogenesis is the growth of new blood vessels from pre-existing vessels. It is a biological process essential in physiological wound healing or pathological inflammation and tumor growth, which underlies a complex interplay of stimulating and inhibiting signals. Extracellular matrix, cells of innate and adaptive immunity and endothelial cells itself are a major source of angiogenic factors that activate or inhibit specific receptors and consequently influence intracellular signaling pathways.Most inflammatory and neoplastic diseases in dermatology are characterized by excessive angiogenesis, such as psoriasis, atopic dermatitis, as well as melanoma, non-melanoma skin cancer, but also benign vascular neoplasia. In this article we describe current knowledge of angiogenesis and its most relevant mechanisms in different dermatological disorders with particular emphasis on the angiogenic factors (vascular endothelial growth factor) and angiopoietins as a target of current and future directions of anti-angiogenic therapy.

Improved in vitro angiogenic behavior on anodized titanium dioxide nanotubes

BackgroundNeovascularization 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.MethodsThe 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.ResultsThe 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.ConclusionThe 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.

Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study

Adipose tissue-derived stem cells (ASCs) are known to be able to promote repair of injured tissue via paracrine factors. However, the effect of cell density and inflammatory cytokines on the paracrine ability of ASCs remains largely unknown. To investigate these effects, ASCs were cultured in 8000 cells/cm2, 20,000 cells/cm2, 50,000 cells/cm2, and 400,000 cells/cm2 with and without 10 or 20 ng/ml tumor necrosis factor alpha (TNFα) and 25 or 50 ng/ml interferon gamma (IFNγ). ASC-sheets formed at 400,000 cells/cm2 after 48 h of culture. With increasing concentrations of TNFα and IFNγ, ASC-sheets with 400,000 cells/cm2 had increased production of angiogenic factors Vascular Endothelial Growth Factor and Fibroblast Growth Factor and decreased expression of pro-inflammatory genes TNFA and Prostaglandin Synthase 2 (PTGS2) compared to lower density ASCs. Moreover, the conditioned medium of ASC-sheets with 400,000 cells/cm2 stimulated with the low concentration of TNFα and IFNγ enhanced endothelial cell proliferation and fibroblast migration. These results suggest that a high cell density enhances ASC paracrine function might beneficial for wound repair, especially in pro-inflammatory conditions.