Release Of Angiogenic Growth Factors Research Articles

Development of tissue-engineered tracheal scaffold with refined mechanical properties and vascularisation for tracheal regeneration.

Introduction: Attempted tracheal replacement efforts thus far have had very little success. Major limiting factors have been the inability to efficiently re-vascularise and mimic the mechanical properties of native tissue. The major objective of this study was to optimise a previously developed collagen-hyaluronic acid scaffold (CHyA-B), which has shown to facilitate the growth of respiratory cells in distinct regions, as a potential tracheal replacement device. Methods: A biodegradable thermoplastic polymer was 3D-printed into different designs and underwent multi-modal mechanical assessment. The 3D-printed constructs were incorporated into the CHyA-B scaffolds and subjected to in vitro and ex vivo vascularisation. Results: The polymeric backbone provided sufficient strength to the CHyA-B scaffold, with yield loads of 1.31-5.17N/mm and flexural moduli of 0.13-0.26MPa. Angiogenic growth factor release (VEGF and bFGF) and angiogenic gene upregulation (KDR, TEK-2 and ANG-1) was detected in composite scaffolds and remained sustainable up to 14 days. Confocal microscopy and histological sectioning confirmed the presence of infiltrating blood vessel throughout composite scaffolds both in vitro and ex vivo. Discussion: By addressing both the mechanical and physiological requirements of tracheal scaffolds, this work has begun to pave the way for a new therapeutic option for large tracheal defects.

Current Application of Beta-Tricalcium Phosphate in Bone Repair and Its Mechanism to Regulate Osteogenesis

Large segmental bone loss and bone resection due to trauma and/or the presence of tumors and cysts often results in a delay in healing or non-union. Currently, the bone autograft is the most frequently used strategy to manage large bone loss. Nevertheless, autograft harvesting has limitations, namely sourcing of autograft material, the requirement of an invasive procedure, and susceptibility to infection. These disadvantages can result in complications and the development of a bone substitute materials offers a potential alternative to overcome these shortcomings. Among the biomaterials under consideration to date, beta-tricalcium phosphate (β-TCP) has emerged as a promising material for bone regeneration applications due to its osteoconductivity and osteoinductivity properties as well as its superior degradation in vivo. However, current evidence suggests the use β-TCP can in fact delay bone healing and mechanisms for this observation are yet to be comprehensively investigated. In this review, we introduce the broad application of β-TCP in tissue engineering and discuss the different approaches that β-TCP scaffolds are customized, including physical modification (e.g., pore size, porosity and roughness) and the incorporation of metal ions, other materials (e.g., bioactive glass) and stem cells (e.g., mesenchymal stem cells). 3D and 4D printed β-TCP-based scaffolds have also been reviewed. We subsequently discuss how β-TCP can regulate osteogenic processes to aid bone repair/healing, namely osteogenic differentiation of mesenchymal stem cells, formation of blood vessels, release of angiogenic growth factors, and blood clot formation. By way of this review, a deeper understanding of the basic mechanisms of β-TCP for bone repair will be achieved which will aid in the optimization of strategies to promote bone repair and regeneration.

Tissues with Patterned Vessels or Protein Release Induce Vascular Chemotaxis in an In Vitro Platform.

Engineered tissues designed for translational applications in regenerative medicine require vascular networks to deliver oxygen and nutrients rapidly to the implanted cells. A limiting factor of in vivo translation is the rapid and successful inosculation, or connection, of host and implanted vascular networks and subsequent perfusion of the implant. An approach gaining favor in vascular tissue engineering is to provide instructive cues from the engineered tissue to enhance host vascular penetration and connection with the implant. Here, we use a novel in vitro platform based on the aortic ring assay to evaluate the impact of patterned, endothelialized vessels or growth factor release from engineered constructs on preinosculative vascular cell outgrowth from surrogate host tissue in a controlled, defined environment, and introduce robust tools for evaluating vascular morphogenesis and chemotaxis. We demonstrate the creation of engineered vessels at the arteriole scale, which develop basement membrane, exhibit tight junctions, and actively sprout into the surrounding bulk hydrogel. Vessel-containing constructs are co-cultured adjacent to rodent aortic rings, and the resulting heterocellular outgrowth is quantified. Cells originating from the aortic ring migrate preferentially toward constructs containing engineered vessels with 1.5-fold faster outgrowth kinetics, 2.5-fold increased cellular density, and 1.6-fold greater network formation versus control (no endothelial cells and growth factor-reduced culture medium). Growth factor release from constructs with nonendothelialized channels and in reduced factor medium equivalently stimulates sustained vascular outgrowth distance, cellular density, and network formation, akin to engineered vessels in endothelial growth medium 2 (EGM-2) medium. In conclusion, we show that three-dimensional endothelialized patterned vessels or growth factor release stimulate a robust, host-derived vascular cell chemotactic response at early time points critical for instructive angiogenic cues. Further, we developed robust, unbiased tools to quantify metrics of vascular morphogenesis and preinosculative heterocellular outgrowth from rat aortic rings and demonstrated the utility of our complex, controlled environment, heterocellular in vitro platform. Impact statement Using a novel in vitro platform, we show that engineered constructs with patterned vessels or angiogenic growth factor release, two methods of instructing host revascularization responses, equivalently improve early host-derived vascular outgrowth. Our platform leverages the aortic ring assay in a tissue engineering context to study preinosculative vascular cell chemotaxis from surrogate host vascular cells in response to paracrine cues from co-cultured engineered tissues using robust, open-source quantification tools. Our accessible and flexible platform enables translationally focused studies in revascularization using implantable therapeutics containing prepatterned vessels with greater environmental control than in vivo studies to advance vascular tissue engineering.

Tumor Microenvironment: Extracellular Matrix Alterations Influence Tumor Progression.

The tumor microenvironment (TME) is composed of various cell types embedded in an altered extracellular matrix (ECM). ECM not only serves as a support for tumor cell but also regulates cell–cell or cell–matrix cross-talks. Alterations in ECM may be induced by hypoxia and acidosis, by oxygen free radicals generated by infiltrating inflammatory cells or by tumor- or stromal cell-secreted proteases. A poorer diagnosis for patients is often associated with ECM alterations. Tumor ECM proteome, also named cancer matrisome, is strongly altered, and different ECM protein signatures may be defined to serve as prognostic biomarkers. Collagen network reorganization facilitates tumor cell invasion. Proteoglycan expression and location are modified in the TME and affect cell invasion and metastatic dissemination. ECM macromolecule degradation by proteases may induce the release of angiogenic growth factors but also the release of proteoglycan-derived or ECM protein fragments, named matrikines or matricryptins. This review will focus on current knowledge and new insights in ECM alterations, degradation, and reticulation through cross-linking enzymes and on the role of ECM fragments in the control of cancer progression and their potential use as biomarkers in cancer diagnosis and prognosis.

Modulating Heparanase Activity: Tuning Sulfation Pattern and Glycosidic Linkage of Oligosaccharides.

Heparanase cleaves polymeric heparan sulfate (HS) molecules into smaller oligosaccharides, allowing for release of angiogenic growth factors promoting tumor development and autoreactive immune cells to reach the insulin-producing β cells. Interaction of heparanase with HS chains is regulated by specific substrate sulfation sequences. We have synthesized 11 trisaccharides that are highly tunable in structure and sulfation pattern, allowing us to determine how heparanase recognizes HS substrate and selects a favorable cleavage site. Our study shows that (1) N-SO3- at +1 subsite and 6-O-SO3- at -2 subsite of trisaccharides are critical for heparanase recognition, (2) addition of 2-O-SO3- at the -1 subsite and of 3-O-SO3- to GlcN unit is not advantageous, and (3) the anomeric configuration (α or β) at the reducing end is crucial in controlling heparanase activity. Our study also illustrates that the α-trisaccharide having N- and 6-O-SO3- at -2 and +1 subsites inhibited heparanase and was resistant toward hydrolysis.

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.

Phenotypic differences in early outgrowth angiogenic cells based on in vitro cultivation.

Bone marrow-derived early outgrowth cells play an important role in endothelial repair. In vitro isolation techniques have identified two distinct morphological early outgrowth cell populations, but it is still unknown whether they present some functional phenotypic differences. Accordingly, the aim of the present study was to determine whether there are phenotypic differences in cellular function between two putative early outgrowth cells in culture. Peripheral blood samples were collected from 18 healthy adults. Thereafter, mononuclear cells were isolated by Ficoll density-gradient centrifugation and plated on 6-well plates coated with human fibronectin. After 2 and 7days, respectively, non-adherent cells (NAC) and adherent cells (AC) underwent functional assays in order to measure the migratory capacity (Boyden chamber), angiogenic growth factor release (ELISA) and apoptosis (TUNEL). Migration to both VEGF (517 ± 74 vs. 273 ± 74AU) and SDF-1 (517 ± 68 vs. 232 ± 68AU) were approximately twofold higher (P < 0.05) in the NAC when compared to AC. Release of angiogenic factors, granulocyte colony-stimulating and hepatocyte growth factor, were not different between cell types. Apoptotic response to staurosporine was significantly lower in NAC (20 ± 32 vs. 125 ± 32%). In summary, NAC and AC demonstrated functional phenotypic differences in migratory capacity and apoptotic susceptibility, which makes it difficult to compare these two early outgrowth cell populations in literature.

Injectable shear-thinning hydrogels for delivering osteogenic and angiogenic cells and growth factors.

Bone nonunion may occur when the fracture is unstable, or blood supply is impeded. To provide an effective treatment for the healing of nonunion defects, we introduce an injectable osteogenic hydrogel that can deliver cells and vasculogenic growth factors. We used a silicate-based shear-thinning hydrogel (STH) to engineer an injectable scaffold and incorporated polycaprolactone (PCL) nanoparticles that entrap and release vasculogenic growth factors in a controlled manner. By adjusting the solid composition of gelatin and silicate nanoplatelets in the STH, we defined optimal conditions that enable injection of STHs, which can deliver cells and growth factors. Different types of STHs could be simultaneously injected into 3D constructs through a single extrusion head composed of multiple syringes and needles, while maintaining their engineered structure in a continuous manner. The injected STHs were also capable of filling any irregularly shaped defects in bone. Osteogenic cells and endothelial cells were encapsulated in STHs with and without vasculogenic growth factors, respectively, and when co-cultured, their growth and differentiation were significantly enhanced compared to cells grown in monoculture. This study introduces an initial step of developing a new platform of shape-tunable materials with controlled release of angiogenic growth factors by utilizing PCL nanoparticles.

Effects of two calcium silicate cements on cell viability, angiogenic growth factor release and related gene expression in stem cells from the apical papilla.

To evaluate the effects of two types of calcium silicate cements on viability, angiogenic growth factor release, and angiogenic and inflammation-related gene expression in human stem cells from the apical papilla (SCAP). SCAPs were grown for 7days with either ProRoot mineral trioxide aggregate (MTA) or Biodentine (BD). Cell viability and media concentrations of vascular endothelial growth factor (VEGF/VEGFA) and angiopoietin 1 (ANGPT1) were measured. The expression of genes related to angiogenic potential and inflammatory response was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). One-way and two-way analyses of variance with multiple comparisons Tukey's test were performed (P<0.05). Cells in contact with either cement were associated with increased cell viability compared with the no-treatment group at day 1 but there were no differences amongst groups at days 3 and 7. Exposure to either cement significantly increased VEGF concentrations at day 3; however, ANGPT-1 levels decreased significantly compared with the no-treatment group at day 3. Exposure to MTA and BD stimulated expression of VEGFA and FIGF/VEGFD. Furthermore, exposure to both cements significantly decreased the mRNA levels of ANGPT1 and FGF2 relative to the no-treatment group. Both MTA and BD stimulated the expression of angiogenic genes and release of VEGF, inducing similar expression patterns; however, they appeared to inhibit the expression of specific genes, including ANGPT1 and FGF2.

Coadministration of adipose-derived stem cells and control-released basic fibroblast growth factor facilitates angiogenesis in a murine ischemic hind limb model

Adipose-derived stem cells (ASCs) have angiogenic potential owing to their differentiation into endothelial cells and their release of angiogenic growth factors to elicit paracrine effects. In addition, control-released basic fibroblast growth factor (bFGF) sustained with a gelatin hydrogel also supports effective angiogenesis. We sought to determine if coadministration of ASCs and control-released bFGF into murine ischemic limbs facilitates angiogenesis. Levels of growth factors in the conditioned media of ASCs cultured with or without control-released bFGF were measured by enzyme-linked immunosorbent assays. A murine ischemic hind limb model was generated and intramuscularly injected with the following: gelatin hydrogel (group 1), a high number of ASCs (group 2), control-released bFGF (group 3), a small number of ASCs and control-released bFGF (group 4), and a high number of ASCs and control-released bFGF (group 5). Macroscopic and microscopic vascular changes were evaluated until day 7 by laser Doppler perfusion imaging and histologic analyses, respectively. Secretion of hepatocyte growth factor, vascular endothelial growth factor, and transforming growth factor-β1 was enhanced by control-released bFGF. Vascular improvement was achieved in groups 4 and 5 according to laser Doppler perfusion imaging. Hematoxylin and eosin staining and CD31 immunohistochemical staining demonstrated an increase in the vascular density, vessel diameter, and thickness of vessel walls in groups 4 and 5. Cells positively stained for CD146, α-smooth muscle actin, and transforming growth factor-β1 were observed around vessel walls in groups 4 and5. These findings suggest that coadministration of ASCs and control-released bFGF facilitates angiogenesis in terms of vessel maturation in a murine ischemic hind limb model.

Protective Factors of the Gastric and Duodenal Mucosa: An Overview.

The structural and functional integrity of the gastric and duodenal mucosa represents equilibrium between aggressive factors and protective mechanisms. Mucus-buffers-phospholipid layer as pre-epithelial barrier, enhanced by prostaglandins and epidermal growth factor, remains a vanguard of mucosal protection. It maintains a neutral pH at the surface epithelial luminal interface, facing luminal pH dropping to 1.0, i.e., hydrogen ion concentration gradient equal 1,000,000. The surface epithelial cells, elaborating mucins, buffers, phospholipids, prostaglandins, trefoil peptides, peptide growth factor and their receptors, heat shock proteins, cathelicidins, and β-defensins form the second line of defense. Endothelium exerts mucosal protection through production of potent vasodilators like nitric oxide and prostacyclins and through release of angiogenic growth factors, securing adequate blood flow and representing the third and an ultimate line of mucosal protection. This microcirculation is instrumental for supply of oxygen, nitric oxide, hydrogen sulfide and removal of ad hoc generated toxic substances as well as for continuous mucosal cell renewal from progenitor cells, secured by growth factors accompanied by survivin preventing early apoptosis.

Vascular protective effects of Angiotensin Receptor Blockers: Beyond Blood pressure.

AT1R blockers (ARBs) represent a major class of antihypertensive medications. They are considered first line treatment for essential hypertension. Moreover, ARBs are the cornerstone treatment for other cardiovascular diseases especially in patients with diabetic and renal comorbidities. Clinical and experimental evidence have documented the beneficial actions of ARBs beyond the blood pressure lowering effect. Ischemic diseases such as stroke and proliferative retinopathy are characterized by hypoxia-driven release of angiogenic growth factors [2] . However, revascularization of the ischemic areas is inadequate, resulting in impaired neuro-vascular function. ARBs have been shown to exhibit vascular protective and pro- or anti-angiogenic effects depending on the tissue/cell type and disease condition under study [3] . Our group has demonstrated the vascular protective effects of ARBs and candesartan, in particular, in models of ischemic stroke and retinopathy. The positive impact of candesartan was mainly via enhancing the proangiogenic state and stimulation of reparative angiogenesis. This commentary aims to highlight the recently identified pathways that took place as result of directly blocking AT1 receptor or indirectly by possible activation of AT2 receptor in the context of the published literature.

Adipose tissue-derived microvascular fragments from aged donors exhibit an impaired vascularisation capacity.

Adipose tissue-derived microvascular fragments are promising vascularisation units for applications in the field of tissue engineering. Elderly patients are the major future target population of such applications due to an increasing human life expectancy. Therefore, we herein investigated the effect of aging on the fragments' vascularisation capacity. Microvascular fragments were isolated from epididymal fat pads of adult (8 months) and aged (16 months) C57BL/6 donor mice. These fragments were seeded onto porous polyurethane scaffolds, which were implanted into dorsal skinfold chambers to study their vascularisation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with fragments from aged donors exhibited a significantly lower functional microvessel density and intravascular blood flow velocity. This was associated with an impaired vessel maturation, as indicated by vessel wall irregularities, constantly elevated diameters and a lower fraction of CD31/α-smooth muscle actin double positive microvessels in the implants' border and centre zones. Additional in vitro analyses revealed that microvascular fragments from adult and aged donors do not differ in their stem cell content as well as in their release of angiogenic growth factors, survival and proliferative activity under hypoxic conditions. However, fragments from aged donors exhibit a significantly lower number of matrix metalloproteinase -9-positive perivascular cells. Taken together, these findings demonstrate that aging is a crucial determinant for the vascularisation capacity of isolated microvascular fragments.

The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs.

The zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72h post-fertilization, we observed that green fluorescent protein-positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells.

Influence of Dietary Fat Intake on Endothelial Progenitor Cell Function (LB678)

High dietary fat intake has been linked to promoting cardiovascular disease and increasing the risk of heart attack. Bone‐marrow derived circulating endothelial progenitor cells (EPCs) play an important role in repairing and maintaining the health of the cardiovascular system. Circulating EPCs home to sites of vascular damage to initiate a repair response. Declines in circulating EPC function has been linked to greater risk of cardiovascular disease and vascular events. It is currently unknown whether habitual consumption of a diet high in fat is associated with impairments in EPC function. Cells with phenotypic EPC characteristics were isolated from peripheral blood samples collected from 26 middle‐aged and older adults: 13 who habitually consumed a diet high in fat (蠅35% of total calories); 13 who habitually consumed a diet lower in fat (&lt;35% of total calories). All subjects were sedentary and free of cardiometabolic disease. Diet was assessed using 4‐day food records and analyzed using a nutritional software. EPC functional characteristics were assessed by the following: colony formation (in vitro colony forming assay); migration (modified Boyden chamber); release of angiogenic growth factors (ELISA); and apoptosis (active intracellular caspase‐3). There were no significant differences between the high and lower fat groups in EPC colony formation (11±3 vs 8±2) and migration (1297±114 vs 1035±124 RFUs). Basal and phytohemagglutinin (PHA; 10 µg/mL) stimulated release of vascular endothelial growth factor (21.5±2.1 to 84.1±14.3 vs 22.9±1.4 to 95.6±14.1 ng/mL) and granulocyte colony stimulating factor (34.0±6.3 to 1246.9±283.6 vs 38.0±6.6 to 873.7±143.1 ng/mL). Moreover, there was no difference in unstimulated (0.3±0.1 vs 0.3±0.1 ng/mL) and stimulated (2.6±0.3 and 2.5±0.3 ng/mL) active caspase‐3 concentrations between the high and lower fat groups. In conclusion, regular consumption of a diet high in fat (蠅35% of calories) does not appear to adversely influence circulating EPC function in middle‐aged and older adults. Impaired EPC function may not contribute to the increased cardiovascular risk associated with a high fat diet.

Impaired fasting blood glucose and circulating endothelial progenitor cell function (1072.4)

Impaired fasting blood glucose (IFG: 100‐125 mg/dL) is a risk factor for type 2 diabetes and confers cardiovascular risk. Functional impairment of circulating endothelial progenitor cells (EPCs) is linked to atherogenesis. We tested the hypothesis that IFG is associated with EPC dysfunction. Peripheral blood samples were collected from 22 adults (19 M/3 F): 10 normal fasting glucose (NFG; age: 57±3 yr; BMI: 29.7±1.4 kg/m2; glucose: 90±1 mg/dL) and 12 IFG (59±1 yr; 28.6±1.1 kg/m2; 104±1 mg/dL). Cells with phenotypic putative EPC characteristics were isolated from peripheral blood mononuclear cells and EPC clonogenic (colony forming unit [CFU] assay) and migratory (Boyden chamber) capacity as well as apoptotic susceptibility (intracellular active caspase‐3 concentrations) and angiogenic growth factor release (ELISA) were determined. EPC colony forming capacity was ~60% lower (p&lt;0.05) in the IFG (10±3) vs NFG (23±6) group. Basal (0.31±0.1 vs 0.21±0.1 ng/mL) and staurosporine‐stimulated (2.9±0.3 vs 2.1±0.3 ng/mL) active caspase‐3 levels were markedly (~45%; p&lt;0.05) higher in EPCs from IFG compared with NFG adults. There were no significant differences in EPC migration or release of angiogenic factors. These results suggest that impaired fasting glucose adversely affects the ability of EPCs to form colonies and resist apoptosis, potentially contributing to the increased cardiovascular risk with hyperglycemia.

Phenotypic differences in putative EPCs based on in vitro cultivation (855.8)

Circulating endothelial progenitor cells (EPCs) have been extensively studied over the past decade. Isolation techniques have differed in the literature based on whether the non‐adherent or adherent cells from fibronectin‐coated plates are studied. We determined whether there are phenotypic differences in cellular function between putative EPCs derived from the adherent or non‐adherent mononuclear cell population. Peripheral blood samples were collected from 15 healthy adults. Mononuclear cells were isolated by Ficoll density‐gradient centrifugation and plated on 6‐well plates coated with human fibronectin. After 2 days, non‐adherent cells were collected; in a separate culture, adherent cells were removed and collected after 7 days. Migratory capacity (Boyden chamber), angiogenic growth factor release (ELISA) and apoptosis (TUNEL) were assessed in both cell types. Migration to both VEGF (505+86 vs 254+ 55 AU) and SDF‐1 (498+83 vs 231+46 AU) were ~2‐fold higher (P&lt;0.05) in the non‐adherent vs adherent cells. Release of angiogenic factors: granulocyte colony‐stimulating factor and hepatocyte growth factor were not different between cell types. Apoptotic response to staurosporine was significantly lower (~60%) in non‐adherent cells. In summary, non‐adherent and adherent cultured putative EPCs demonstrate functional phenotypic differences in migratory capacity and apoptotic susceptibility.

Application of strontium-doped calcium polyphosphate scaffold on angiogenesis for bone tissue engineering

The key factor for regenerating large segmental bone defects through bone tissue engineering is angiogenesis in scaffolds. Attempts to overcome this problem, it is a good strategy to develop a new scaffold with bioactivity to induce angiogenesis in bone tissue engineering. In our previous research, the ability of strontium-doped calcium polyphosphate (SCPP) to stimulate the release of angiogenic growth factors from cultured osteoblasts was studied. This study was performed to determine the ability of SCPP to induce angiogenesis within in vitro co-culture model of human umbilical vein endothelial cells (HUVEC) and osteoblasts co-cultured. The bioactivity of developed scaffolds to induce angiogenesis in vivo was also researched in this paper. Co-cultured model has been developed in vitro and then cultured with SCPP scaffold as well as calcium polyphosphate (CPP) scaffold and hydroxylapatite (HA) scaffold. The results showed that the optimal ratio of HUVEC and osteoblasts co-cultured model for in vitro angiogenesis was 5:1. The model could maintain for more than 35days when cultured with the scaffold and show the best activity at 21st day. Compared with those in CPP and HA scaffold, the formation of tube-like structure and the expression of platelet endothelial cell adhesion molecule in co-cultured model is better in SCPP scaffold. The in vivo immunohistochemistry staining for VEGF also showed that SCPP had a potential to promote the formation of angiogenesis and the regeneration of bone. SCPP scaffold could be served as a potential biomaterial with stimulating angiogenesis in bone tissue engineering and bone repair.

Activated Blood Coagulation Factor X (FXa) Induces Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

To determine the transcriptional regulation of the blood coagulation factor X (FX) in cultured human retinal pigment epithelial (RPE) cells, and whether the effects of FXa on the chemotaxis and expression of angiogenic growth factors are mediated by autocrine growth factor signaling. Alterations in gene expression and secretion of growth factors were determined by real-time RT-PCR and ELISA, respectively. Cellular proliferation and chemotaxis were investigated with a bromodeoxyuridine immunoassay and a Boyden chamber assay, respectively. The gene expression of FX in RPE cells was increased by hypoxia and prostaglandin E(2), and decreased by blood serum, FXa, thrombin, transforming growth factor beta (TGF-β1), and platelet-derived growth factor (PDGF). The serum-induced downregulation of FX was mediated by thrombin and TGF-β signaling. FXa induced chemotaxis of RPE cells via activation of the p38 mitogen-activated protein kinase signal transduction pathway. FXa also induced expression of vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF), as well as release of VEGF, bFGF, and TGF-β1 from RPE cells. The stimulatory effects of FXa on the expression of growth factors and secretion of VEGF were prevented by inhibition of the TGF-β activin receptor-like kinase, but not by the thrombin inhibitor hirudin. FXa induced phosphorylation of ERK1/2, p38, and Akt proteins. FXa induces chemotaxis of RPE cells, as well as expression and release of angiogenic growth factors from RPE cells, including VEGF. The effects of FXa on the expression and secretion of VEGF are mediated by autocrine/paracrine TGF-β signaling.

MicroRNA-214 inhibits angiogenesis by targeting Quaking and reducing angiogenic growth factor release

Angiogenesis is a critical component of many pathological conditions in adult tissues and is essential for embryonic development. MicroRNAs are indispensable for normal vascular development, but their exact role in regulating angiogenesis remains unresolved. Previously, we have observed that miR-214 is differentially expressed in compensatory arteriogenesis. Here, we investigated the potential role of miR-214 in the process of angiogenesis. miR-214 is expressed in all major vascular cell types, and modulation of miR-214 levels in endothelial cells significantly affected tubular sprouting. In vivo silencing of miR-214 enhanced the formation of a perfused vascular network in implanted Matrigel plugs and retinal developmental angiogenesis in mice. miR-214 directly targets Quaking, a protein critical for vascular development. Quaking knockdown reduced pro-angiogenic growth factor expression and inhibited endothelial cell sprouting similar to miR-214 overexpression. In accordance, silencing of miR-214 increased the secretion of pro-angiogenic growth factors, including vascular endothelial growth factor, and enhanced the pro-angiogenic action of the endothelial cell-derived conditioned medium, whereas miR-214 overexpression had the opposite effect. Here, we report a novel role for miR-214 in regulating angiogenesis and identify Quaking as a direct target of miR-214. The anti-angiogenic effect of miR-214 is mediated through the down-regulation of Quaking and pro-angiogenic growth factor expression. This study presents miR-214 as a potential important target for pro- or anti-angiogenic therapies.