In Vitro Angiogenesis Inhibition and Endothelial Cell Growth and Morphology

A co-culture assay with human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) was used to study whether selected angiogenesis inhibitors were able to inhibit differentiation and network formation of HUVECs in vitro. The effect of the inhibitors was determined by the morphology and the calculated percentage area covered by HUVECs. Neutralizing VEGF with avastin and polyclonal goat anti-VEGF antibody and inhibiting VEGFR2 with sorafenib and vatalanib resulted in the formation of HUVEC clusters of variable sizes as a result of inhibited EC differentiation. Furthermore, numerous inhibitors of the VEGF signaling pathways were tested for their effect on the growth and differentiation of HUVECs. The effects of these inhibitors did not reveal a cluster morphology, either individually or when combined to block VEGFR2 downstream pathways. Only the addition of N-methyl-p-bromolevamisole revealed a similar morphology as when targeting VEGF and VEGFR2, meaning it may have an inhibitory influence directly on VEGFR signaling. Additionally, several nuclear receptor ligands and miscellaneous compounds that might affect EC growth and differentiation were tested, but only dexamethasone gave rise to cluster formation similarly to VEGF-neutralizing compounds. These results point to a link between angiogenesis, HUVEC differentiation and glucocorticoid receptor activation.     

Curcuminoids Inhibit Angiogenic Behaviors of Human Umbilical Vein Endothelial Cells via Endoglin/Smad1 Signaling

Background: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib^®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. Methods: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. Results: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. Conclusions: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.     

Verifiable Hypotheses for Thymosin β4-Dependent and -Independent Angiogenic Induction of Trichinella spiralis-Triggered Nurse Cell Formation

Trichinella spiralis has been reported to induce angiogenesis for nutrient supply and waste disposal by the induction of the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation. However, the action mechanism to induce VEGF in nurse cells by T. spiralis is not known. Hypoxia in nurse cells was suggested as a possible mechanism; however, the presence of hypoxic conditions in infected muscle or nurse cells and whether hypoxia indeed induces the expression of VEGF and subsequent angiogenesis in the infected muscle are both a matter of debate. Our recent studies have shown that thymosin β4, a potent VEGF inducing protein, is expressed in the very early stages of T. spiralis muscle infection suggesting the induction of VEGF in early stage nurse cells. Nevertheless, we now show that hypoxic conditions were not detected in any nurse cell stage but were detected only in the accumulated inflammatory cells. These studies propose that induction of angiogenesis by VEGF in T. spiralis-infected nurse cells was mediated by thymosin β4 and is unrelated to hypoxic conditions.     

Formyl Peptide Receptor 2 Alleviates Hepatic Fibrosis in Liver Cirrhosis by Vascular Remodeling

Hexapeptide WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met), a ligand of formyl peptide receptor 2, exhibits anti-inflammatory and angiogenic properties in disease models. However, the therapeutic effects of WKYMVm on hepatic fibrosis have not been evaluated to date. Therefore, we investigated whether WKYMVm exerts antifibrotic effects and induces vascular regeneration in a rat model of bile duct ligation (BDL). The antifibrotic and angiogenic effects of WKYMVm on liver regeneration in the BDL rat model were analyzed using biochemical assays, qRT-PCR, western blotting, immunofluorescence, and immunohistochemistry. To determine the effects of WKYMVm on hepatic fibrosis and angiogenesis in vitro, we measured the expression levels of fibrotic factors in hepatic stellate cells (HSCs) and angiogenic factors in human umbilical vein endothelial cells (HUVECs). WKYMVm attenuated the expression of collagen type I (Col I) and α-smooth muscle actin (α-SMA) and significantly increased the levels of angiogenetic factors in the BDL model (p < 0.05). WKYMVm reduced fibrotic marker expression in transforming growth factor (TGF)-β-induced HSCs and promoted angiogenic activity through tube formation in 5-Fluorouracil (FU)-treated HUVECs (p < 0.05). Also, WKYMVm administration enhanced hepatocyte proliferation in BDL rats (p < 0.05). The WKYMVm alleviates hepatic fibrosis by inhibiting HSC activation and promotes hepatic regeneration via vascular remodeling. These data suggest that the WKYMVm may be a new therapeutic agent for liver fibrosis.     

Electrospun Fiber-Coated Human Amniotic Membrane: A Potential Angioinductive Scaffold for Ischemic Tissue Repair

Cardiac patch implantation helps maximize the paracrine function of grafted cells and serves as a reservoir of soluble proangiogenic factors required for the neovascularization of infarcted hearts. We have previously fabricated a cardiac patch, EF-HAM, composed of a human amniotic membrane (HAM) coated with aligned PLGA electrospun fibers (EF). In this study, we aimed to evaluate the biocompatibility and angiogenic effects of EF-HAM scaffolds with varying fiber thicknesses on the paracrine behavior of skeletal muscle cells (SkM). Conditioned media (CM) obtained from SkM-seeded HAM and EF-HAM scaffolds were subjected to multiplex analysis of angiogenic factors and tested on HUVECs for endothelial cell viability, migration, and tube formation analyses. All three different groups of EF-HAM scaffolds demonstrated excellent biocompatibility with SkM. CM derived from SkM-seeded EF-HAM 7 min scaffolds contained significantly elevated levels of proangiogenic factors, including angiopoietin-1, IL-8, and VEGF-C compared to plain CM, which was obtained from SkM cultured on the plain surface. CM obtained from all SkM-seeded EF-HAM scaffolds significantly increased the viability of HUVECs compared to plain CM after five days of culture. However, only EF-HAM 7 min CM induced a higher migration capacity in HUVECs and formed a longer and more elaborate capillary-like network on Matrigel compared with plain CM. Surface roughness and wettability of EF-HAM 7 min scaffolds might have influenced the proportion of skeletal myoblasts and fibroblasts growing on the scaffolds and subsequently potentiated the angiogenic paracrine function of SkM. This study demonstrated the angioinductive properties of EF-HAM composite scaffold and its potential applications in the repair and regeneration of ischemic tissues.     

Expression of Connexin43 Stimulates Endothelial Angiogenesis Independently of Gap Junctional Communication In Vitro

Connexins (Cx) form gap junctions (GJ) and allow for intercellular communication. However, these proteins also modulate gene expression, growth, and cell migration. The downregulation of Cx43 impairs endothelial cell migration and angiogenetic potential. Conversely, endothelial Cx43 expression is upregulated in an in vivo angiogenesis model relying on hemodynamic forces. We studied the effects of Cx43 expression on tube formation and proliferation in HUVECs and examined its dependency on GJ communication. Expectedly, intercellular communication assessed by dye transfer was linked to Cx43 expression levels in HUVECs and was sensitive to a GJ blockade by the Cx43 mimetic peptide Gap27. The proliferation of HUVECs was not affected by Cx43 overexpression using Cx43 cDNA transfection, siRNA-mediated knockdown of Cx43, or the inhibition of GJ compared to the controls (transfection of an empty vector, scrambled siRNA, and the solvent). In contrast, endothelial tube and sprout formation in HUVECs was minimized after Cx43 knockdown and significantly enhanced after Cx43 overexpression. This was not affected by a GJ blockade (Gap27). We conclude that Cx43 expression positively modulates the angiogenic potential of endothelial cells independent of GJ communication. Since proliferation remained unaffected, we suggest that Cx43 protein may modulate endothelial cell migration, thereby supporting angiogenesis. The modulation of Cx43 expression may represent an exploitable principle for angiogenesis induction in clinical therapy.     

可溶性血管内皮生长因子受体2片段的表达及其对血管内皮细胞增殖的影响

目的探讨可溶性血管内皮生长因子受体2(sKDR)抑制血管内皮细胞增殖及在血管生成中的作用。方法提取脐静脉内皮细胞(HUVEC)总RNA,扩增KDR基因膜外1~4结构域,构建原核表达载体pQE40-KDR,转化E.coli M15,经IPTG诱导表达,镍离子柱亲和层析纯化后复性,用Western blot检测sKDR蛋白的表达,MTT比色法和鸡胚尿囊膜(CAM)试验分别检测其对HUVEC增殖的影响及其对血管生成的作用。结果经RT-PCR扩增得到了1150 bp左右的sKDR片段,并在pQE40原核表达系统中表达了sKDR蛋白,以包涵体形式存在。纯化后蛋白电泳呈现相对分子质量50000左右的单一条带,纯化蛋白占总蛋白的98%,蛋白含量为80μg/ml。Western blot证实其为重组sKDR蛋白。MTT检测结果显示,sKDR可抑制血管内皮生长因子(VEGF)刺激的HUVEC增殖,并阻滞VEGF诱导的CAM血管增生。结论已成功构建sKDR原核表达载体,并在大肠杆菌M15中获得表达,纯化的sKDR片段具有与VEGF结合的生物学功能,有望成为基因治疗肿瘤血管形成的理想靶点。     

Linoleic Acid Attenuates Denervation-Induced Skeletal Muscle Atrophy in Mice through Regulation of Reactive Oxygen Species-Dependent Signaling

Muscle atrophy is a major muscle disease, the symptoms of which include decreased muscle volume leading to insufficient muscular support during exercise. One cause of muscle atrophy is the induction of oxidative stress by reactive oxygen species (ROS). This study aimed to identify the antioxidant mechanism of linoleic acid (LA) in muscle atrophy caused by oxidative stress. H₂O₂ has been used to induce oxidative stress in myoblasts in vitro. C2C12 myoblasts treated with H₂O₂ exhibited decreased viability and increased ROS synthesis. However, with LA treatment, the cells tended to recover from oxidative effects similar to those of the control groups. At the molecular level, the expression of superoxide dismutase 1 (SOD1), Bax, heat shock protein 70 (HSP70), and phosphorylated forkhead box protein O1 was increased by oxidative stress, causing apoptosis. LA treatment suppressed these changes. In addition, the expression of MuRF1 and Atrogin-1/MAFbx mRNA increased under oxidative stress but not in the LA-treated group. Sciatic denervation of C57BL/6 mice manifested as atrophy of the skeletal muscle in micro-computed tomography (micro-CT). The protein expression levels of SOD1, HSP70, and MuRF1 did not differ between the atrophied muscle tissues and C2C12 myoblasts under oxidative stress. With LA treatment, muscle atrophy recovered and protein expression was restored to levels similar to those in the control. Therefore, this study suggests that LA may be a candidate substance for preventing muscle atrophy.     

VEGF Regulation of Angiogenic Factors via Inflammatory Signaling in Myeloproliferative Neoplasms

Background: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. Aims: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. Results: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors—endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)—in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. Conclusion: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition.     

Modeling In Vitro Osteoarthritis Phenotypes in a Vascularized Bone Model Based on a Bone-Marrow Derived Mesenchymal Cell Line and Endothelial Cells

The subchondral bone and its associated vasculature play an important role in the onset of osteoarthritis (OA). Integration of different aspects of the OA environment into multi-cellular and complex human, in vitro models is therefore needed to properly represent the pathology. In this study, we exploited a mesenchymal stromal cell line/endothelial cell co-culture to produce an in vitro human model of vascularized osteogenic tissue. A cocktail of inflammatory cytokines, or conditioned medium from mechanically-induced OA engineered microcartilage, was administered to this vascularized bone model to mimic the inflamed OA environment, hypothesizing that these treatments could induce the onset of specific pathological traits. Exposure to the inflammatory factors led to increased network formation by endothelial cells, reminiscent of the abnormal angiogenesis found in OA subchondral bone, demineralization of the constructs, and increased collagen production, signs of OA related bone sclerosis. Furthermore, inflammation led to augmented expression of osteogenic (alkaline phosphatase (ALP) and osteocalcin (OCN)) and angiogenic (vascular endothelial growth factor (VEGF)) genes. The treatment, with a conditioned medium from the mechanically-induced OA engineered microcartilage, also caused increased demineralization and expression of ALP, OCN, ADAMTS5, and VEGF; however, changes in network formation by endothelial cells were not observed in this second case, suggesting a possible different mechanism of action in inducing OA-like phenotypes. We propose that this vascularized bone model could represent a first step for the in vitro study of bone changes under OA mimicking conditions and possibly serve as a tool in testing anti-OA drugs.     

Extracellular Environment-Controlled Angiogenesis,and Potential Application for Peripheral Nerve Regeneration

Endothelial cells acquire different phenotypes to establish functional vascular networks. Vascular endothelial growth factor (VEGF) signaling induces endothelial proliferation, migration, and survival to regulate vascular development, which leads to the construction of a vascular plexuses with a regular morphology. The spatiotemporal localization of angiogenic factors and the extracellular matrix play fundamental roles in ensuring the proper regulation of angiogenesis. This review article highlights how and what kinds of extracellular environmental molecules regulate angiogenesis. Close interactions between the vascular and neural systems involve shared molecular mechanisms to coordinate developmental and regenerative processes. This review article focuses on current knowledge about the roles of angiogenesis in peripheral nerve regeneration and the latest therapeutic strategies for the treatment of peripheral nerve injury.     

Multifunction Sr doped microporous coating on pure magnesium of antibacterial,osteogenic and angiogenic activities

Strontium (Sr) ions were added to porous magnesium (Mg) oxide with silicon and fluorine by microarc oxidation (MAO) to improve its osteogenic and pro-angiogenic properties. First, pure Mg was oxidized by MAO, and Sr was added by electrolysis. The surface of the resulting Sr coating was characterized by SEM, EDS, and EDS mapping. The release of Sr ions was monitored by ICP-OES. The antibacterial property of the coating was assessed against Staphylococcus aureus. The effect of Sr coating on osteogenesis was tested in MC3T3-E1 cell line by performing cell adhesion and proliferation tests, alkaline phosphatase (ALP) activity detection, cell morphology characterization, alizarin red staining, and osteogenic-related gene expression analysis. Finally, HUVECs cells were used to test the effect of Sr coating on angiogenesis through cell migration and tube formation assays, VEGF quantification, chicken embryo chorioallantoic membranes (CAM) test, and angiogenic-related gene expression analysis. The results showed that Sr coating had a hierarchical microstructure with a microporous structure evenly covered with nano-grains and that the Sr elements from the coating were released slowly and continuously. Sr coating had effective antibacterial properties and promoted cell adhesion, proliferation, ALP release, calcium nodule formation, and upregulated osteogenic gene expression. Moreover, the coating could promote migration, tube formation, VEGF expression, and angiogenic gene upregulation in endothelial cells. Sr coating also enhanced angiogenesis of CAM. This study supports that Sr coating on Mg- MAO enhances osteogenesis and angiogenesis.     

Tetrandrine Inhibits Skeletal Muscle Differentiation by Blocking Autophagic Flux

Tetrandrine is well known to act as a calcium channel blocker. It is a potential candidate for a tumor chemotherapy drug without toxicity. Tetrandrine inhibits cancer cell proliferation and induces cell death through apoptosis and autophagy. As cancer patients usually experience complications with sarcopenia or muscle injury, we thus assessed the effects of tetrandrine on skeletal muscle cells. We report in this study that a low dose of tetrandrine (less than 5 μM) does not affect the proliferation of C2C12 myoblasts, but significantly inhibits myogenic differentiation. Consistently, tetrandrine inhibited muscle regeneration after BaCl₂-induced injury. Mechanistic experiments showed that tetrandrine decreased the p-mTOR level and increased the levels of LC3 and SQSTM1/p62 during differentiation. Ad-mRFP-GFP-LC3B transfection experiments revealed that the lysosomal quenching of GFP signals was suppressed by tetrandrine. Furthermore, the levels of DNM1L/Drp1, PPARGA1 and cytochrome C (Cyto C), as well as caspase 3 activation and ROS production, were decreased following tetrandrine administration, indicating that the mitochondrial network signaling was inhibited. Our results indicate that tetrandrine has dual effects on autophagic flux in myoblasts during differentiation, activation in the early stage and blockade in the late stage. The ultimate blocking of autophagic flux by tetrandrine led to the disruption of mitochondria remodeling and inhibition of myogenic differentiation. The inhibitory effects of tetrandrine on skeletal muscle differentiation may limit its application in advanced cancer patients. Thus, great attention should be paid to the clinical use of tetrandrine for cancer therapy.     

辛二酰苯胺氧肟酸对人脐静脉内皮细胞增殖及血管形成的影响

目的探讨组蛋白去乙酰化酶抑制剂(Histone deacetylases inhibitor,HDACi)辛二酰苯胺氧肟酸(Suberoy-lanilide hydroxamic acid,SAHA)对人脐静脉内皮细胞(Human umbilical vein endothelial cell,HUVEC)增殖及血管形成能力的影响。方法收集处于对数生长期的HUVEC,以不同浓度的SAHA分别处理24和48 h,另设不加SAHA的对照组,采用CCK-8法检测细胞的增殖活力,并计算增殖抑制率和半数抑制浓度(IC50)。采用流式细胞术检测经15μmol/L SAHA处理48 h的HUVEC凋亡和细胞周期,基质胶体外血管生成试验检测HUVEC的体外成管能力,Western blot法检测HUVEC细胞周期及凋亡相关蛋白的表达水平。结果随着SAHA浓度的增加及作用时间的延长,其对HUVEC增殖的抑制作用增强,SAHA浓度高于80μmol/L时,抑制率增加不明显,24和48 h的IC50值分别为60.53和30.49μmol/L;经15μmol/L SAHA处理48 h,与对照组相比,HUVEC的凋亡率明显增加(P<0.001),S期细胞比例明显升高(P<0.001),G0/G1期比例明显降低(P<0.001),体外成管能力明显下降,P21、caspase-3激活型、caspase-9酶原和激活型蛋白的表达水平均明显升高(P<0.001)。结论 SAHA能够抑制HUVEC增殖及体外血管形成能力,并使P21、caspase-3和caspase-9蛋白水平上调,为肿瘤的治疗提供了一种新的思路。     

In vivo studies on angiogenic activity of two designer self-assembling peptide scaffold hydrogels in the chicken embryo chorioallantoic membrane

The rapid promotion of angiogenesis is critical for tissue engineering and regenerative medicine. The angiogenic activity of tissue-engineered scaffolds has already been the major criterion for choosing and designing ideal biological materials. We here report systematic in vivo studies on the angiogenic activity of two functionalized self-assembling peptides PRG (Ac-(RADA)(4)GPRGDSGYRGDS-CONH(2)) and KLT (Ac-(RADA)(4)G(4)KLTWQELYQLKYKGI-CONH(2)) using the chicken embryo chorioallantoic membrane (CAM) assay. 3D migration/sprouting bead assays showed that the two functional motifs PRGDSGYRGDS and KLTWQELYQLKYKGI improved the bioactivities of the self-assembling peptide RADA16-I (Ac-(RADA)(4)-CONH(2)) dramatically and provided ideal synthetic microenvironments for endothelial cell migration and cordlike structure sprout formation. A CAM assay was carried out to assess the efficiency of various peptide scaffolds in inducing capillary invasion in vivo. Among these three peptide scaffolds, the functionalized peptide scaffold RAD/KLT presented a significantly better angiogenic activity inducing CAM tissue invasion and new capillary vessel formation within the scaffolds in the absence of VEGF. With the addition of VEGF, more newly formed vessel lumen could be observed in all peptide scaffolds. Our results suggested that the functionalized peptide scaffolds had satisfactory angiogenic properties, and may also have wide potential applications in tissue regeneration.     

SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that the succinate–SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate–SUCNR1 axis and placental angiogenesis. In our in vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.     

Hydrogel‐mediated formation of living cartilage template for endochondral initiation

The use of hydrogel in cartilage tissue engineering is especially popular due to its high hydrophilic property which is similar to native cartilage matrix. Alginate hydrogel was used as a transient scaffold material to facilitate chondrocyte proliferation into a three‐dimensional scaffold‐free living hyaline cartilaginous graft (LhCG). As LhCG is purely cell‐based and has a marked resemblance to native hyaline cartilage, it served as an excellent in vitro platform for studying the endochondral ossification pathway. Due to the complexity of events involved throughout endochondral ossification, this study only focuses on early stages of the process where it involves chondrocyte hypertrophy and blood vessel invasion. Human umbilical vein endothelial cells (HUVECs) were selected as the target cells for possible endothelialization in the LhCG template. They were seeded onto the LhCG construct and subjected to vascular endothelial growth factor (VEGF) treatment. Results suggested that VEGF is indeed a potent driving force for initiation of the endochondral pathway. It alone is sufficient to induce hypertrophy in chondrocytes and the corresponding expression of osteogenic genes with or without the presence of HUVECs in the LhCG template. On the other hand, the effect of HUVECs in the LhCG system was less evident. It is hypothesized that this is attributed to the preservation of anti‐angiogenic properties in primary chondrocytes from the LhCG construct, inhibiting HUVECs from endothelialization in the LhCG+HUVEC construct. Based on the outcome from this study, it is recommended that hypertrophy in chondrocytes should be induced prior to endothelial cell introduction so that the microenvironment will be altered to favor angiogenesis within the cartilaginous template. © 2013 Society of Chemical Industry     

Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

Resistin is a novel hormone that is secreted by human adipocytes and mononuclear cells and is associated with obesity, insulin resistance and inflammation. Recently, resistin has been postulated to play a role in angiogenesis. Here, we investigated the hypothesis that resistin regulates ovary carcinoma production of vascular endothelial growth factor (VEGF) and the angiogenic processes. We found that in human ovarian epithelial carcinoma cells (HO-8910), resistin (10–150 ng/mL) enhanced both VEGF protein and mRNA expression in a time- and concentration-dependent manner, as well as promoter activity. Furthermore, resistin enhanced DNA-binding activity of Sp1 with VEGF promoter in a PI3K/Akt-dependent manner. PI3K/Akt activated by resistin led to increasing interaction with Sp1, triggering a progressive phosphorylation of Sp1 on Thr453 and Thr739, resulting in the upregulation of VEGF expression. In an in vitro angiogenesis system for endothelial cells (EA.hy926) co-cultured with HO-8910 cells, we observed that the addition of resistin stimulated endothelial cell tube formation, which could be abolished by VEGF neutralizing antibody. Our findings suggest that the PI3K/Akt-Sp1 pathway is involved in resistin-induced VEGF expression in HO-8910 cells and indicates that antiangiogenesis therapy may be beneficial treatment against ovarian epithelial carcinoma, especially in obese patients.