Human Umbilical Vessel Endothelial Cells Research Articles

Angiogenesis effect of udenafil in a caveolin-1 deficient moyamoya disease model: A pre-clinical animal study

Purpose: Although pathogenic mechanisms of moyamoya disease (MMD) remain unknown, recent studies suggest that it is a caveolae disease. This study evaluated the effect of udenafil, a phosphodiesterase-5 inhibitor, on angiogenesis in in vitro and in vivo MMD models.Methods: Angiogenesis and vessel maturation were assessed in in vitro models, caveolin- 1 (Cav-1) knockdown human umbilical vessel endothelial cells (HUVECs) and coronary artery smooth muscle cells (CASMCs), and in in vivo model of bilateral internal carotid artery occlusion (bICAo). Udenafil was administered (1,3,10, and 30 μM) in cell culture conditions, and functional studies (migration and tube formation assay) were performed and vessel maturation factors and cyclic guanosine monophosphate (cGMP) accumulation were measured.Results: Udenafil (3 and 10 mg/kg) was orally administered once daily for 4 weeks in bICAo rat model, and histological analysis for angiogenesis and vessel maturation was performed. Udenafil increased vessel formation in both Cav-1 knockdown HUVEC and bICAo models without increased migration/proliferation of HUVECs and CASMCs. Udenafil increased CD31+ vessel density and NG2/Col4+ mural cell density in bICAo models. Cav-1 knockdown inhibited accumulation of cGMP, and udenafil treatment restored cGMP levels in Cav-1 knockdown HUVEC models. Vessel maturation factors (angiopoietin- 1 and platelet-derived growth factor receptor-β) and angiogenic factors (endothelial nitric oxide synthase) were increased after treatment with udenafil in vitro.Conclusion: Our results indicate that udenafil reversed cellular levels of cGMP related to Cav-1 deficiency and induced angiogenesis and vessel maturation. Further studies are warranted to confirm the therapeutic effects of this strategy in MMD.

Glucose degradation products in peritoneal dialysis solution impair angiogenesis by dysregulating angiogenetic factors in endothelial and vascular smooth muscle cells.

The accumulation of glucose degradation products (GDPs) during peritoneal dialysis (PD) can lead to immature angiogenesis in the peritoneum. However, the effect of GDPs on angiogenesis, at concentrations observed in dialysate effluent, has not been widely investigated. We do not know how the inflammation observed in PD-related peritonitis affects angiogenesis of the peritoneum. Human umbilical vessel endothelial cells (HUVEC) and human umbilical aortic smooth muscle cells (HUASMC) were used to examine the response to the three main GDPs found in peritoneal dialysate (methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and 5-hydroxymethylfurfural (5-HMF). Supernatant from lipopolysaccharide (LPS)-activated murine macrophage cell lines (RAW 264.7 cells) were used to stimulate angiogenesis in the peritoneum. Changes in the expression of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor B (PDGFB) in HUVEC, and PDGF-receptor beta (PDGF-Rβ) in HUASMC, were examined by real-time PCR, Western blot, and ELISA. In HUVECs, the expression of PDGFB mRNA and protein were decreased by exposure to MGO, 3-DG, and 5-HMF at concentrations observed in dialysate effluent. A subsequent decrease in secreted PDGF-BB was observed. In HUASMCs, MGO and 5-HMF increased the expression of VEGF-A mRNA and protein, while 5-HMF decreased the expression of PDGF-Rβ. VEGF-A is upregulated, and PDGF-Rβ is downregulated, by conditioned medium of LPS-stimulated macrophages in HUASMCs. The GDPs of PD effluent cause an imbalance of angiogenic factors in endothelial cells and vascular smooth muscle cells that may lead to immature angiogenesis in the peritoneum.

Human Placenta Derived Exosomes Enhance Ischemia Repair in a Mouse Model of Hind-Limb Ischemia

Background Ischemic cardiovascular diseases are the major causes of death in the world. Recently, mesenchymal or tissue progenitor cell derived exosomes are being investigated as an emerging therapeutic agent for treating cardiovascular diseases due to their potentials in restoring the damaged vasculature via promoting angiogenesis. Exosome therapy has the advantages in low immunogenicity and stability features comparing to cells 1. Tissue and organ derived exosomes could have a broader functionality than those derived from a homogeneous cell population as they contain multiple cell types. Placenta is a large and highly vascularized organ and placenta exosomes are known to play essential roles during pregnancy in supporting fetus development 2. Placenta is abundant with endothelial cells, epithelial cells, cytotrophoblast, syncytiotrophoblasts and stem cells including both hematopoietic and non-hematopoietic stem cells. It is perceivable that exosomes isolated from a placenta possess the functions of the cells in a placenta. Materials and Methods Full-term human placentas were obtained under the full consent of healthy donors. Placenta tissues were cultured in serum-free DMEM medium supplemented with antibiotics. After culturing 8 to 16 hours, supernatant was harvested, medium was changed every 8 to 12 hours for up to two days. Placenta derived exosomes (pExo) were isolated through sequential centrifugation of the supernatant of the placenta culture and final preparation were suspended in PBS and stored at -80oC. Quantification of pExo was performed with Bicinchoninic Acid protein assay kit. pExo size was determined using NanoSight. MILLIPLEX-MAP human cytokine/chemokine-PX41 was used to analyze the cytokine composition of pExo. In vitro cell migration assays were performed with seeding 1x10e5/mL human umbilical vessel endothelial cells (HUVEC) in basal media (BM) on the top chamber of an 8-um transwell on a 24-well plate with 500uL BM with or without pExo in the bottom chamber. After 6-24 hours of culture, transwells were observed under an inverted microscope after staining. In vitro cell proliferation assay was performed by seeding HUVEC at 2-4x10e3 cells/96-well with a water-soluble tetrazolium salt-based cell viability assay. To evaluate the pro-angiogenic activity of pExo in vivo, a hind limb ischemia (HLI) model in db/db diabetic mice was used. After surgical induction of HLI on Day 0, pExo from two different donors or vehicle (n=15 each group) was injected via i.v. at 100ug/mice or 100uL on Days 1, 6 and 11. The blood flow through hind paws was measured by Speckle Doppler. Results Utilizing the established cultivation and isolation methods, we achieved the average yield of pExo at 327±91 mg per placenta (n=10). pExo has an average size of 118±15 nm (n=10) as determined with nano-particle tracking analysis, consistent with consensus exosome size ranging 50nm to 200nm 1. Flow cytometry and Western blot analyses confirmed that pExo exhibits characteristic markers known for exosomes including CD9, CD63 and CD81. pExo contained abundant IL-8, HGF, FGF2, PDGF-BB, RANTES, MCP-1 and GM-CSF which are known to play roles in supporting angiogenesis and chemotaxis. In co-culture assays, pExo promoted the proliferation of HUVECs by 149±13% (n=5) comparing to basal medium controls. pExo also demonstated chemotactic activities comparable to that of complete HUVEC growth medium in stimulating the migration of HUVECs across membrane of transwell. Chlorpromazine and Pitstop2 abolished pExo-induced transwell-migration of HUVECs, suggesting that HUVECs may uptake pExo through endocytosis. In the HLI study, pExo treated mice showed significantly higher (50% to 250%) blood flow on Days 14, 28 and 35 comparing to the vehicle group. Histology analysis showed pExo treated mice had 40% to 140% higher density of CD34+ capillary on Day 35, suggesting a significant increase of angiogenesis in the pExo treated groups comparing to the vehicle group. Conclusion: In summary, pExo contains proangiogenic cytokines and chemokines and demonstrates the promising pro-angiogenic activities in vitro and in HLI mice model. These results support further development of pExo as a potential therapeutic agent for the treatment of cardiosvascular diseases.

Abstract TP475: Effect of Udenafil, a Phosphodiesterase-5 Inhibitor, on Angiogenesis in a Cav-1 Deficient Moyamoya Disease Model

Background: Although pathogenic mechanisms of moyamoya disease remain unknown, recent studies suggest that it is a caveolae disease. This study evaluated the effect of udenafil, a phosphodiesterase-5 inhibitor, on vessel maturation in in vitro and in vivo moyamoya disease models. Methods: Angiogenesis and vessel maturation were assessed in in vitro models, caveolin-1 (Cav-1) knockdown human umbilical vessel endothelial cells (HUVECs) and coronary artery smooth muscle cells (CASMCs), and in in vivo model of bilateral internal carotid artery occlusion (bICAo). Udenafil was administered (1,3,10 and 30 μM) in cell culture conditions, and functional studies (migration and tube formation assay) were performed and vessel maturation factors and cyclic guanosine monophosphate (cGMP) accumulation were measured. Udenafil (3 and 10 mg/kg) was orally administered once daily for 4 weeks in bICAo rat model, and histological analysis for angiogenesis and vessel maturation was performed. Results: Udenafil increased vessel formation in both Cav-1 knockdown HUVEC and bICAo models without increased migration/proliferation of HUVECs and CASMCs. Udenafil increased CD31+ vessel density and NG2/Col4+ mural cell density in bICAo models. Cav-1 knockdown inhibited accumulation of cGMP, and udenafil treatment restored cGMP levels in Cav-1 knockdown HUVEC models. Vessel maturation factors (angiopoietin-1 and platelet-derived growth factor receptor-β) and angiogenic factors (endothelial nitric oxide synthase) were increased after treatment with udenafil in vitro . Conclusion: Our results indicate that udenafil reversed cellular levels of cGMP related to Cav-1 deficiency and induced angiogenesis and vessel maturation. Further studies are warranted to confirm the therapeutic effects of this strategy in moyamoya disease.

Tumor angiogenesis of SCLC inhibited by decreased expression of FMOD via downregulating angiogenic factors of endothelial cells.

Fibromodulin (FMOD), an ECM small leucine-rich proteoglycan (SLRP), was reported to promote angiogenesis not only during wound healing, but also in optical and cutaneous angiogenesis-dependent diseases. However, whether it plays important roles in tumor angiogenesis remains unclear. To explore the role of FMOD in tumor angiogenesis of human small cell lung cancer (SCLC), initially the study analyzed the relationship of FMOD expression in cancer tissues of SCLC with clinical characteristics. The analysis revealed that the positive FMOD expression was significantly associated with extensive stage of SCLC and higher vascular density. In mouse models, xenograft tumors developed with FMOD-silenced H446 cells (H446-shFMOD) exhibited slowed growth rate, decreased microvessel density, and reduced blood perfusion related to that of controls (H446-shCON). Additionally, compared with that of controls, the decreased secretion of FMOD in conditioned medium (CM) from H446-shFMOD inhibited proliferation, migration, and invasion of human umbilical vessel endothelial cells (HUVECs). Moreover, the decreased secretion of FMOD downregulated the expression of VEGF, TGF-β1, FGF-2, and PDGF-B in HUVECs. The findings strongly suggested that the autocrine FMOD of cancer cells may promote tumor angiogenesis of SCLC by upregulating the expression of angiogenic factors that act in concert to facilitate the angiogenic phenotype of endothelial cells as a proangiogenic factor. Therefore, silencing FMOD may be a potentially clinical therapy for repressing tumor angiogenesis.

An injectable silk sericin hydrogel promotes cardiac functional recovery after ischemic myocardial infarction

Acute myocardial infarction (MI) leads to morbidity and mortality due to cardiac dysfunction. Here we identify sericin, a silk-derived protein, as an injectable therapeutic biomaterial for the minimally invasive MI repair. For the first time, sericin prepared in the form of an injectable hydrogel has been utilized for cardiac tissue engineering and its therapeutical outcomes evaluated in a mouse MI model. The injection of this sericin hydrogel into MI area reduces scar formation and infarct size, increases wall thickness and neovascularization, and inhibits the MI-induced inflammatory responses and apoptosis, thereby leading to a significant functional improvement. The potential therapeutical mechanisms have been further analyzed in vitro. Our results indicate that sericin downregulates pro-inflammatory cytokines (TNF-α and IL-18) and chemokine (CCL2) and reduces TNF-α expression by suppressing the TLR4-MAPK/NF-κB pathways. Moreover, sericin exhibits angiogenic activity by promoting migration and tubular formation of human umbilical vessel endothelial cells (HUVECs). Also, sericin stimulates VEGFa expression via activating ERK phosphorylation. Further, sericin protects endothelial cells and cardiomyocytes from apoptosis by inhibiting the activation of caspase 3. Together, these diverse biochemical activities of sericin protein lead to a significant recovery of cardiac function. This work represents the first study reporting sericin as an effective therapeutic biomaterial for ischemic myocardial repair in vivo. Statement of SignificanceIntramyocardial biomaterial injection is thought to be a potential therapeutic approach to improve cardiac performance after ischemic myocardial infarction. In this study, we report the successful fabrication and in vivo application of an injectable sericin hydrogel for ischemic heart disease. We for the first time show that the injection of in situ forming crosslinked sericin hydrogel promotes heart functional recovery accompanied with reduced inflammatory responses, attenuated apoptosis and increased microvessel density in the infarcted hearts. Further, we reveal that the improvement in those aspects is ascribed to sericin protein’s functional bioactivities that are comprehensively uncovered in this study. Thus, we identify sericin, a natural protein, as a biomaterial suitable for myocardial repair and demonstrate that the in vivo application of this injectable sericin hydrogel can be an effective strategy for treating MI.

Human Endothelial Cell Transport Bovine Extracellular Vesicles

MicroRNAs play essential roles in gene regulation; the majority of microRNAs in body fluids is encapsulated in extracellular vesicles (EVs), thereby conferring protection against degradation and a pathway for cellular transport by endocytosis. Our previous studies suggest that 1) humans absorb EV-encapsulated microRNAs from cow's milk, 2) milk microRNAs are delivered to peripheral tissues where they alter the expression of human genes, and 3) endogenous synthesis of microRNAs does not compensate for dietary microRNA depletion in mice. Ongoing studies suggest that EV-encapsulated microRNAs cross the intestinal mucosa by processes involving endocytosis and exocytosis. Here we tested the hypothesis that endothelial cells in blood vessels also transport milk EVs. Transport was assessed using labeled exosomes from bovine milk, i.e., the quantitatively most important fraction of EVs in milk, and human umbilical vessel endothelial cells (HUVEC). The uptake of exosomes was assessed at timed intervals and was line...

Anti-inflammatory mechanism of tocilizumab, a humanized anti-IL-6R antibody: effect on the expression of chemokine and adhesion molecule

We studied the influence of IL-6 on chemokine production from peripheral blood mononuclear cells (PBMC), fibroblastic synovial cells and human umbilical vessel endothelial cells (HUVEC). Moreover, we examined the effect of IL-6 on the adhesion of U937 cells to HUVEC. For chemokine production, PBMC, fibroblastic synovial cells and HUVEC were cultured with IL-6 or IL-6 + soluble IL-6R (sIL-6R) for 24 h and then the production of MCP-1 and IL-8 were measured in supernatants. IL-6 and IL-6 + sIL-6R induced production of both MCP-1 and IL-8 in PBMC and synovial cells, respectively. In HUVEC, IL-6 + sIL-6R induced MCP-1 production, but inhibited IL-8 production. For adhesion molecule expression, the production of soluble form of adhesion molecules in HUVEC culture supernatant were measured by ELISA and the expression of adhesion molecules on cell surface were examined by flow cytometry analysis. Soluble ICAM-1 was detectable in IL-6 + sIL-6R-treated HUVEC and IL-6 + sIL-6R-induced ICAM-1 expression on cell membrane of HUVEC. In addition, U937 cells were added to HUVEC, which were pre-treated with IL-6 + sIL-6R for 24 h, and 3 h later attached U937 cells were counted. The adhesion of U937 cells to HUVEC was augmented when HUVEC was pretreated by IL-6 + sIL-6R. This adhesion was suppressed by anti-ICAM-1 antibody and anti-IL-6R antibody, but not by antibodies against VCAM-1 or E-selectin. In conclusion, IL-6 signaling plays an important role in inflammatory cell migration by increasing the rate of cell adhesion and by inducing chemokine production in inflamed joints.

Gene Expression Profiles by Microarray Analysis during Matrigel-induced Tube Formation in a Human Extravillous Trophoblast Cell line: Comparison with Endothelial Cells

Endovascular differentiation of extravillous cytotrophoblasts (EVT) during placentogenesis induces remodeling of spiral arteries that increases blood flow toward the intravillous space and is required for maintaining pregnancy. To address the molecular mechanisms involved in this differentiation, we investigated the gene expression profile during matrigel-induced tube formation in TCL1 cells, a human immortalized EVT cell line, and HUV-EC-C, human umbilical vessel endothelial cells, and compared their profiles. The numbers of genes that showed significant up-regulation (>3-fold expression at both 3 and 6 h, and/or >5-fold expression at either 3 or 6 h) during tube formation and significant down-regulation (0.33-fold expression at both 3 and 6 h, and/or less than 0.2-fold expression at either 3 or 6 h), were 969 and 659 in TCL1, respectively. In HUV-EC-C, the numbers of genes that showed significant up-regulation and down-regulation were 86 and 65, respectively. Only 73 of 1628 genes that showed significant expression changes in TCL1 were common with HUV-EC-C. The genes showing significant expression change specifically in TCL1 were associated with cellular, metabolisms, proliferation, anti-apoptosis, proteolysis adhesion, and some known to be involved in EVT differentiation or related to angiogenesis. The gene expression profile in EVT during tube formation is very different from that of endothelial cells. Further investigations based on the current data may help to elucidate mechanisms of normal and abnormal placentogenesis.

In vivo inhibition of angiogenesis by a soluble form of melanotransferrin

Melanotransferrin (MTf), the membrane-bound human melanoma antigen p97, binds to plasminogen and stimulates its activation, thus regulating a crucial step involved in angiogenesis. In our study, a truncated soluble form of MTf (sMTf) inhibits, in a dose-dependent manner, the in vitro tubulogenesis of human umbilical vessel endothelial cells (HUVEC) induced by media conditioned with MTf-expressing cells. Following these results, we used the in vivo Matrigel plug angiogenesis assay to investigate whether truncated sMTf could inhibit neovascularization in mice. We found that basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and MTf-expressing cells strongly stimulate in vivo Matrigel neovascularization. However, subcutaneous (s.c.) administration of truncated sMTf inhibits both bFGF- and VEGF- as well as human melanoma SK-Mel-28-induced angiogenesis. This inhibition was dependent on the truncated sMTf concentration and reached maximal inhibition at 40 mg/kg/week. Furthermore, we found that a single s.c. injection of truncated sMTf into nude mice at 5 mg/kg produced a maximal blood concentration of approximately 40 nM, which is comparable with the level required to inhibit in vitro HUVEC tubulogenesis. Overall, our results strongly suggest that s.c. administration of truncated sMTf may provide a novel therapeutic strategy for the treatment of angiogenesis-related disorders.

Regulatory role of E-NTPase/NTPDase in fat/CD36-mediated fatty acid uptake.

Fatty acid translocase (FAT)/CD36-mediated long-chain fatty acid uptake in human umbilical vessel endothelial cells is associated with as yet uncharacterized translocase activity. The molecular mechanism of its function is not yet understood. Numerous attempts to purify rat cardiac sarcolemmal E-NTPase (an integral membrane protein also referred to as ecto-Ca(2+)/Mg(2+)ATPase) have revealed a complete amino acid sequence identity for FAT/CD36 protein. The most striking observation is that purified CD36 from human platelets shows significant E-NTPase activity. In view of recent progress in understanding CD36 functional properties, an attempt is made in this article to illustrate the point that association of E-NTPase (possibly extracellular Ca(2+)/Mg(2+)nucleotide triphosphate diphosphohydrolase) activity with CD36 may be of potential functional significance.

Ultrastructure of human umbilical vessels in pre-eclampsia

Objective: Our objective in this study was to investigate the ultrastructure of endothelial and muscle cells of human umbilical vessels in both normal and pre-eclamptic pregnancies. Methods: Ten umbilical cords from pre-eclamptic (36, 38 and 40 weeks) and four from normal pregnancies (40 weeks) were collected immediately after vaginal deliveries. Umbilical veins and arteries were isolated and fixed in phosphate-buffered 2.5% glutaraldehyde solution (pH 7.2) for 4 h and postfixed with 1% osmium tetroxide at 4°C for 2 h. The sections were embedded in Araldit CY 212. Ultrathin sections were stained with uranyl acetate, examined and photographed. Results: Human umbilical vessel endothelial cells showed ultrastructural changes in pre-eclamptic patients. Weibel–Palade bodies and some organelles such as rough endoplasmic reticulum were found in increased numbers in venous endothelial cells. Accumulations of granular material were detected under the venous endothelium. Conclusion: The endothelial and muscle cells of the umbilical vessels from pregnancies complicated by pre-eclampsia showed morphological changes.

Ultrastructure of human umbilical vessels in pre-eclampsia

Objective: Our objective in this study was to investigate the ultrastructure of endothelial and muscle cells of human umbilical vessels in both normal and pre-eclamptic pregnancies.Methods:Ten umbilical cords from pre-eclamptic (36, 38 and 40 weeks) and four from normal pregnancies (40 weeks) were collected immediately after vaginal deliveries. Umbilical veins and arteries were isolated andfixed in phosphate-buffered 2.5% glutaraldehyde solution (pH 7.2) for 4 h and postfixed with 1% osmium tetroxide at 4°C for 2 h. The sections were embedded in Araldit CY 212. Ultrathin sections werestained with uranyl acetate, examined and photographed.Results: Human umbilical vessel endothelial cells showed ultrastructural changes in pre-eclamptic patients. Weibel–Palade bodiesand some organelles such as rough endoplasmic reticulum were found in increased numbers in venous endothelial cells. Accumulations of granular material were detected under the venous endothelium.Conclusion:The endothelial and muscle cells of the umbilical vessels from pregnancies complicated by pre-eclampsia showed morphological changes.

Colocalization of vasoactive substances in the endothelial cells of human umbilical vessels.

Human umbilical vessels are unique in lacking any innervation; thus endothelial cells may play the major role in local control and regulation of the blood flow. In the present study, we examined ultrathin sections of cultured human umbilical vein endothelial cells and tissue preparations of umbilical vein and artery, immunostained by the post-embedding colloidal gold double-labelling technique. We observed colocalization of atrial natriuretic peptide and neuropeptide Y, as well as colocalization of atrial natriuretic peptide and neuropeptide Y with other vasoactive substances, namely, vasoactive intestinal peptide, substance P, calcitonin gene-related peptide and arginine vasopressin. The functional significance of the colocalization of these vasoactive substances in the human umbilical vessel endothelial cells is discussed.