Umbilical Cord Endothelial Cells Research Articles

Circulating Hepatocyte Growth Factor Reflects Activation of Vascular Repair in Response to Stress

Background: Acute cardiovascular events arise when the arterial wall fails to repair the damage caused by cardiovascular risk factors. Hepatocyte growth factor (HGF) stimulates growth of vascular cells but has been associated with increased risk of cardiovascular disease (CVD) in epidemiological studies. Here we tested the hypothesis that this association reflects activation of a protective vascular repair response. Methods: Small interfering RNA (siRNA) and recombinant human HGF were used to assess the role of HGF in regulation of human umbilical cord endothelial and coronary artery smooth muscle cell proliferation, migration and viability. Proximity extension assay was used to determine HGF and other biomarkers in 4742 subjects participating in the Malmo Diet and Cancer study and in extracts of 200 human carotid plaques. Findings: HGF promoted migration, proliferation and survival of human endothelial and smooth muscle cells. Human atherosclerotic plaques with a high content of HGF had a more stable phenotype. Cultured vascular cells expressed HGF in response to stress. Plasma HGF correlated with circulating levels of cellular stress biomarkers such as soluble TRAIL receptor-2 and caspase-8. Decreased ability to respond with elevated HGF levels to cellular stress, as assessed by the ratios of HGF to soluble TRAIL receptor-2 and caspase-8, associated with increased risk of cardiovascular events during a follow-up period of 19.4±5.0 years. Interpretations: These findings provide support for a protective role of HGF in CVD and show that subjects with a low ability to express HGF in response to stress have an increased risk of cardiovascular events. Funding Information: The work was supported by the Swedish Society for Medical Research, Emil and Wera Cornell foundation, Hjelt foundation, the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education, Crafoord foundation, the Swedish Medical Society, Diabetes foundation, Swedish Heart and Lung Foundation, Diabetes Research and Wellness foundation, Albert Pahlssons foundation, SUS foundations and funds, Lund University Infrastructure grant ”Malmo population-based cohorts” (STYR 2019/2046) and Lund University Diabetes Center (Swedish Research Council - Strategic Research Area Exodiab Dnr 2009-1039 and the Swedish Foundation for Strategic Research Dnr IRC15-006). Declaration of Interests: None. Ethics Approval Statement: All studies were approved by the respective Regional Ethical Review Boards and conducted in accordance with the Helsinki Declaration. All subjects gave written consent.

NOVEL MIRNAS AS TARGETS OF MESENCHYMAL STEM CELLS-BASED THERAPY FOR TREATMENT OF PREECLAMPSIA

Objective: Preeclampsia is a cardiovascular complication characterised by the new onset of hypertension and proteinuria or other end-organ dysfunction. Currently there are no effective treatments for preeclampsia except delivery of the placenta and the baby, often pre-term. Administration of mesenchymal stem cells (MSCs) has shown therapeutic potential in pre-clinical models of preeclampsia. In this study, we aimed to identify novel placental miRNAs in preeclampsia which could be targeted by MSCs or their extracellular vesicles (MSC-EVs). Design and method: Human MSCs were isolated from abdominal fat tissue during caesarean section and cultured for 48 h. Human umbilical cord endothelial cells (HUVEC) were co-cultured with MSCs from pregnancies with or without preeclampsia (n = 3) up to 90 h and angiogenesis assessed using tubule formation assay in IncuCyteu live cell imaging system. Using separate patient samples, miRNA was isolated from placentae from women with and without preeclampsia (n = 2) and miRNA next generation sequencing (miRNAseq) performed. The expression of two miRNAs identified in miRNAseq (miRNA-183–5p and miRNA-203a-3p) were measured in HUVEC cells exposed to 24 h hypoxia (1%) or normoxia (21%) in the presence of MSC conditioned medium (MSC-CM) or MSC-EVs. Results: MSCs isolated from women with preeclampsia demonstrated impaired pro-angiogenic profile compared to MSCs derived from normotensive pregnancies measured by the network length (mm/m2) of HUVECs following 66 h, 72 h, 78 h, 84 h and 90 h of exposure to MSC-CM (n = 3, p = 0.001). The results of miRNAseq identified a number of aberrantly expressed miRNAs including mir-183–5p (logFC = -2.8, p = 0.001) and mir-203a-3p (logFC = -6.7, p = 0.001). Following exposure of HUVECs to hypoxia, mir-183–5p was significantly increased by approximately 8-fold (p = 0.001) whereas mir-203a-3p showed no change in expression. MSC-CM and MSC-EVs were both capable of ameliorating this increase in mir-183–5p as a result of hypoxia (p = 0.01, MSC-CM; p = 0.001, MSC-EVs). Conclusions: In patients with preeclampsia, MSCs show impaired angiogenic function, potentially leading to inappropriate placental development and angiogenesis in pregnancy. Administration of healthy MSCs or associated MSC-EVs could have a therapeutic potential in the treatment of preeclampsia and the mechanism could involve novel mir-183–5p as a treatment target.

Extracellular matrix (ECM)-derived bioinks designed to foster vasculogenesis and neurite outgrowth: Characterization and bioprinting

The field of bioprinting has shown a tremendous development in recent years, focusing on the development of advanced in vitro models and on regeneration approaches. In this scope, the lack of suitable biomaterials that can be efficiently formulated as printable bioinks, while supporting specific cellular events, is currently considered as one of the main limitations in the field. Indeed, extracellular matrix (ECM)-derived biomaterials formulated to enable printability and support cellular response, for instance via integrin binding, are eagerly awaited in the field of bioprinting. Several bioactive laminin sequences, including peptides such as YIGSR and IKVAV, have been identified to promote endothelial cell attachment and/or neurite outgrowth and guidance, respectively. Here, we show the development of two distinct bioinks, designed to foster vasculogenesis or neurogenesis, based on methacrylated collagen and hyaluronic acid (CollMA and HAMA, respectively), both relevant ECM-derived polymers, and on their combination with cysteine-flanked laminin-derived peptides. Using this strategy, it was possible to optimize the bioink printability, by tuning CollMA and HAMA concentration and ratio, and modulate their bioactivity, through adjustments in the cell-active peptide sequence spatial density, without compromising cell viability. We demonstrated that cell-specific bioinks could be customized for the bioprinting of both human umbilical vein cord endothelial cells (HUVECs) or adult rat sensory neurons from the dorsal root ganglia, and could stimulate both vasculogenesis and neurite outgrowth, respectively. This approach holds great potential as it can be tailored to other cellular models, due to its inherent capacity to accommodate different peptide compositions and to generate complex peptide mixtures and/or gradients.

Hypoxia-induced amniotic fluid stem cell secretome augments cardiomyocyte proliferation and enhances cardioprotective effects under hypoxic-ischemic conditions

Secretome derived from human amniotic fluid stem cells (AFSC-S) is rich in soluble bioactive factors (SBF) and offers untapped therapeutic potential for regenerative medicine while avoiding putative cell-related complications. Characterization and optimal generation of AFSC-S remains challenging. We hypothesized that modulation of oxygen conditions during AFSC-S generation enriches SBF and confers enhanced regenerative and cardioprotective effects on cardiovascular cells. We collected secretome at 6-hourly intervals up to 30 h following incubation of AFSC in normoxic (21%O2, nAFSC-S) and hypoxic (1%O2, hAFSC-S) conditions. Proliferation of human adult cardiomyocytes (hCM) and umbilical cord endothelial cells (HUVEC) incubated with nAFSC-S or hAFSC-S were examined following culture in normoxia or hypoxia. Lower AFSC counts and richer protein content in AFSC-S were observed in hypoxia. Characterization of AFSC-S by multiplex immunoassay showed higher concentrations of pro-angiogenic and anti-inflammatory SBF. hCM demonstrated highest proliferation with 30h-hAFSC-S in hypoxic culture. The cardioprotective potential of concentrated 30h-hAFSC-S treatment was demonstrated in a myocardial ischemia–reperfusion injury mouse model by infarct size and cell apoptosis reduction and cell proliferation increase when compared to saline treatment controls. Thus, we project that hypoxic-generated AFSC-S, with higher pro-angiogenic and anti-inflammatory SBF, can be harnessed and refined for tailored regenerative applications in ischemic cardiovascular disease.

The protective effects of memantine against inflammation and impairment of endothelial tube formation induced by oxygen-glucose deprivation/reperfusion.

Acute myocardial infarction (AMI) is one of the leading causes of death and disability. The dysregulation of cardiac endothelial cells plays a significant role in the pathogenesis of AMI. In the present study, we investigated the potential of memantine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist used in the treatment of Alzheimer’s disease, to mitigate the effects of ischemia-reperfusion injury in the peripheral vasculature using human umbilical cord endothelial cells (HUVECs). Previous studies have identified anti-inflammatory and antioxidant effects of memantine, but the effects of memantine on angiogenesis and microtubule formation have not been fully elucidated. Our findings indicate that pretreatment with memantine significantly reduced the expression of interleukin (IL)-6 and IL-8, which are both serum markers if AMI severity. We also demonstrate that memantine could prevent mitochondrial dysfunction and oxidative stress by rescuing mitochondrial membrane potential and reducing the production of reactive oxygen species (ROS) by NADPH oxidase-4 (NOX-4). Importantly, memantine also promoted the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant signaling pathway. Importantly, memantine pretreatment improved cell viability and prevented the decrease in microtubule formation induced by OGD/R. Through a phosphoinositide-3-kinase (PI3K) inhibition experiment, we determined that the PI3K/protein kinase B (Akt) pathway is essential for the effects of memantine on angiogenesis. Together, our findings suggest a potential role for memantine in the prevention and treatment of AMI.

Nanostructured Titanium for Improved Endothelial Biocompatibility and Reduced Platelet Adhesion in Stent Applications

Although coronary stents have improved the early and long-term consequences of arterial lesions, the prevention of restenosis and late stent thrombosis is key to prevent a new obstruction of the vessel. Here we aimed at improving the tissue response to stents through surface modification. For that purpose, we used two different approaches, the use of nanostructuration by electrochemical anodization and the addition of a quercitrin (QR) coating to the Ti surface. Four surfaces (Ti, NN, TiQR and NNQR) were characterized by atomic force microscopy, scanning electronic microscopy and contact angle analysis and QR content was evaluated by fluorescent staining. Cell adhesion, cytotoxicity, metabolic activity and nitric oxide (NO) production was evaluated on primary human umbilical cord endothelial cells (HUVECs). Platelet adhesion, hemolysis rate and Staphylococcus epidermidis CECT 4184 adhesion at 30 min were analyzed. Nanostructuration induced an increase on surface roughness, and QR coating decreased the contact angle. All surfaces were biocompatible, with no hemolysis rate and lower platelet adhesion was found in NN surfaces. Finally, S. epidermidis adhesion was lower on TiQR surfaces compared to Ti. In conclusion, our results suggest that NN structuration could improve biocompatibility of bare metal stents on endothelial cells and reduce platelet adhesion. Moreover, QR coating could reduce bacterial adhesion.

Interactome of miRNAs and transcriptome of human umbilical cord endothelial cells exposed to short-term simulated microgravity

Adaptation of humans in low gravity conditions is a matter of utmost importance when efforts are on to a gigantic leap in human space expeditions for tourism and formation of space colonies. In this connection, cardiovascular adaptation in low gravity is a critical component of human space exploration. Deep high-throughput sequencing approach allowed us to analyze the miRNA and mRNA expression profiles in human umbilical cord vein endothelial cells (HUVEC), cultured under gravity (G), and stimulated microgravity (MG) achieved with a clinostat. The present study identified totally 1870 miRNAs differentially expressed in HUVEC under MG condition when compared to the cells subjected to unitary G conditions. The functional association of identified miRNAs targeting specific mRNAs revealed that miRNAs, hsa-mir-496, hsa-mir-151a, hsa-miR-296-3p, hsa-mir-148a, hsa-miR-365b-5p, hsa-miR-3687, hsa-mir-454, hsa-miR-155-5p, and hsa-miR-145-5p differentially regulated the genes involved in cell adhesion, angiogenesis, cell cycle, JAK-STAT signaling, MAPK signaling, nitric oxide signaling, VEGF signaling, and wound healing pathways. Further, the q-PCR based experimental studies of upregulated and downregulated miRNA and mRNAs demonstrate that the above reported miRNAs influence the cell proliferation and vascular functions of the HUVEC in MG conditions effectively. Consensus on the interactome results indicates restricted fluctuations in the transcriptome of the HUVEC exposed to short-term MG that could lead to higher levels of endothelial functions like angiogenesis and vascular patterning.

Heme oxygenase-1 gene modified human placental mesenchymal stem cells promote placental angiogenesis and spiral artery remodeling by improving the balance of angiogenic factors in vitro

IntroductionAbnormal placental vascular development is a possible cause of preeclampsia. Mesenchymal stem cell (MSC)-based therapy is a promising approach for tissue repair and angiogenesis. Further, heme oxygenase-1 (HO-1) has beneficial effects on the angiogenic balance during pregnancy. We explored the effects of HO-1 overexpression on placental vascularization using human placenta-derived MSCs (hPMSCs).Methods: hPMSCs were isolated from term placenta, and the HO-1 gene was transfected with a lentivirus. Proliferation, migration, and apoptosis of hPMSCs and HO-hPMSCs were examined using CCK8 assay, trans-well assay, and flow cytometry, respectively. Paracrine secretion of the angiogenesis factors VEGF and PlGF, as well as the anti-angiogenesis factors sFlt-1 and sEng, from hPMSC/HO-hPMSCs was measured by qRT-PCR and ELISA. Human umbilical cord endothelial cells and a villus-decidua co-culture were treated with conditioned media to study the effect of HO-1-hPMSCs on tube formation and villus vascular remodeling. ResultsHO-1 significantly improved the proliferation and migration of hPMSCs. Additionally, HO-1 reduced hPMSCs apoptosis. The levels of VEGF were increased in HO-1-hPMSCs, whereas those of sFlt-1 decreased. Tube formation assays showed that the conditioned media from HO-1-hPMSCs resulted in more branching points than those from the controls. The villus-decidua co-culture system confirmed that HO-1-hPMSCs are conducive to angiogenesis and vascular remodeling. DiscussionHO-1-modified hPMSCs improve placental vascularization by promoting a balance of pro- and anti- angiogenesis factors, which is worthy of further study as an alternative treatment for preeclampsia.

Cotransplantation of human umbilical cord mesenchymal stem cells and endothelial cells for angiogenesis and pulp regeneration in vivo

AimsTo explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration and the possibility of cotransplantation hUCMSCs and endothelial cells (ECs) for angiogenesis and pulp regeneration in vivo. Materials and methodshUCMSCs and human umbilical vein endothelial cells (HUVECs) were cocultured for matrigel angiogenesis assay in vitro and Matrigel plug assay in vivo. Next, we used the transwell coculture system to coculture hUCMSCs and HUVECs in vitro for RNA- sequencing (RNA-seq). Last, encapsulated hUCMSCs and HUVECs in scaffolds were injected into the root segments, and transplanted into immunodeficient mice for dental pulp regeneration. Key findingsIn vitro Matrigel angiogenesis assay and in vivo Matrigel plug assay indicated that cocultured hUCMSCs and HUVECs promote vascular formation of HUVECs, especially in 1:5 (hUCMSCs:HUVECs) coculture group. The RNA-seq result indicated that cocultured HUVECs exhibited high Hif-1 signaling pathway activity. We performed the cell transfection assay to knock down HIF1A-AS2 in HUVECs and then coculture with hUCMSCs, and the expression of VEGFA, HIF1A and PECAM1 were reduced. In pulp regeneration assay, Cotransplantation of hUCMSCs and HUVECs (1,5) group showed pulp-like tissue regeneration. SignificanceCocultured hUCMSCs and HUVECs can promote vascular formation of HUVECs, and the optimal coculture ration is 1:5 (hUCMSCs:HUVECs). hUCMSCs promote angiogenesis of HUVECs through the long noncoding RNA HIF1A-AS2-activation of the Hif-1 signaling pathway. Cotransplantation of hUCMSCs and HUVECs can regenerate dental pulp-like tissue in vivo.

Significance of Soluble CD93 in Type 2 Diabetes as a Biomarker for Diabetic Nephropathy: Integrated Results from Human and Rodent Studies.

Cluster of differentiation 93 (CD93) is a glycoprotein expressed in activated endothelial cells. The extracellular portion of CD93 can be secreted as a soluble form (sCD93) under inflammatory conditions. As diabetic nephropathy (DN) is a well-known inflammatory disease, we hypothesized that sCD93 would be a new biomarker for DN. We prospectively enrolled 97 patients with type 2 diabetes and evaluated the association between serum sCD93 and DN prevalence. The association between CD93 and development of DN was investigated using human umbilical cord endothelial cells (HUVECs) in vitro and diabetic db/db mice in vivo. Subjects with higher sCD93 levels had a lower estimated glomerular filtration rate (eGFR). The sCD93 level was an independent determinant of both the albumin-to-creatinine ratio (ACR) and the eGFR. The risk of prevalent DN was higher in the high sCD93 group (adjusted odds ratio 7.212, 95% confidence interval 1.244–41.796, p = 0.028). In vitro, CD93 was highly expressed in HUVECs and both CD93 expression and secretion were upregulated after lipopolysaccharides (LPS) stimulation. In vivo, peritoneal and urine sCD93 levels and the renal glomerular expression of CD93 were significantly higher in the db/db mice than in the control db/m+ mice. These results suggest the potential of sCD93 as a candidate biomarker associated with DN.

Effects of Gold Nanoparticles on Capillary Tube Formation with Special Reference to the Alteration of Pericyte Morphology and Function

Pericytes are perivascular cells that wrap around endothelial cells to form wall of capillaries and post‐capillary venules. These cells are primary importance in physiological and pathological processes, especially, involved in angiogenesis. At present, gold nanoparticles (AuNPs) were used for anti‐angiogenesis by inhibiting the activity of endothelial cell proliferation, migration, and tube formation. However, the effects of gold nanoparticles on the alteration of pericyte morphology and theirs function were not reported. Therefore, in this study, 20 nm‐AuNPs were applied to human placenta pericyte monoculture with concentration of 30 ppm. Morphology of pericytes was observed by transmission electron microscopy and cell migration was evaluated by transwell migration assay. Real‐time qRT‐PCR was used to determine Ki‐67, as a proliferation marker, and PDGFR‐β mRNA expression. Moreover, these AuNPs 30 ppm‐treated pericytes were co‐cultured with human umbilical cord endothelial cells on Matrigel® to investigate the 3D tube formation. The results revealed that AuNPs significantly suppressed migration, proliferation, and PDGFR‐β mRNA expression of pericytes. The ultrastructure of pericytes showed AuNPs in several organelles, i.e., swollen or damaged mitochondria, engulfed late endosomes and lysosomes. Moreover, the number of capillary tube formations was essentially reduced and we found that a number of pericytes on these capillary tubes were round shape and did not extend the processes along the tube, which in turn increased incomplete tube formation compared with the control. In conclusion, AuNPs could effect on pericyte biology including proliferation, migration, morphology, and tube formation property. Moreover, the results highlighted the potential application of AuNPs in anti‐angiogenesis by pericyte‐targeted therapy.Support or Funding InformationThe Ratchadaphiseksompotch Fund, Faculty of Medicine, Chulalongkorn University, grant number RA61/100 and Overseas Academic Presentation Scholarship for Graduate Students from the Graduate School, Chulalongkorn University

Comparative Study on Effect of Mesenchymal Stem Cells and Endothelial Progenitor Cells on Treatment of Experimental CCL4 Induced Liver Fibrosis

Abstract Background Bone marrow mesenchymal stem cells (BM-MSCs) and human umbilical cord endothelial progenitor cells (UC-EPCs) have several benefits for liver regeneration. We speculate huge impacts of rat BM-MSCs and UC-EPCs on reversal of hepatic injury and improvement of liver function in liver fibrosis induced by carbon tetrachloride (CCl4). Methods Forty adult rats were divided into 4 groups; control group, CCl4 group, CCl4/BM-MSCs group and CCl4/UCEPCs group. Blood samples were driven from rats at 1, 2 and 3months to measure serum concentration of albumin and alanine aminotransferase (ALT). Quantitative expression of HGF,TGF-β, MMP-2, and VEGF were assessed by polymerase chain reaction. Histological examination of the liver tissue was performed. α-SMA immunohistochemistry to identify the myoepithelial cells (MECs) and Ki-67 to identify cell prolifration immunohistochemistry are detected in groups injected with cells to confirm cells regeneration. Results Regarding liver function, there was elevating albumin (P < 0.05) and reducing ALT (P < 0.05) concentrations in groups treated with BM-MSCs and UC-EPCs effect compared to untreated CCL4 group. Concerning gene expression, UC-EPCs treated group have significantly higher MMP-2 and VEGF (P < 0.01) genes expression than BM-MSCs treated group. Furthermore, UC-EPCs were more valuable than BM-MSCs in increasing gene expression of HGF (P < 0.05) and immunohistochemistry of α-SMA and Ki-67 (P < 0.01). BM-MSCs have significantly lower TGF- β (P < 0.00) compared to UC-EPCs. Conclusion This study highlighted on liver regeneration role of both human UC-EPCs and BM-MSCs in liver fibrosis by different signaling mechanistic.

Hyperglycemia-induced endothelial dysfunction is alleviated by thioredoxin mimetic peptides through the restoration of VEGFR-2-induced responses and improved cell survival

BackgroundDiabetes mellitus is an important cardiovascular risk factor characterized by elevated plasma glucose levels. High glucose (HG) negatively influences endothelial cell (EC) function, which is characterized by the inability of ECs to respond to vascular endothelial growth factor (VEGF-A) stimulation. We aimed to identify potential strategies to improve EC function in diabetes. Methods and resultsHuman umbilical cord endothelial cells (HUVECs) were subjected to hyperglycemic milieu by exposing cells to HG together with glucose metabolite, methylglyoxal (MG) in vitro. Hyperglycemic cells showed reduced chemotactic responses towards VEGF-A as revealed by Boyden chamber migration assays, indicating the development of “VEGF resistance” phenotype. Furthermore, HG/MG-exposed cells were defective in their general migratory and proliferative responses and were in a pro-apoptotic state. Mechanistically, the exposure to HG/MG resulted in reactive oxygen species (ROS) accumulation which is secondary to the impairment of thioredoxin (Trx) activity in these cells. Pharmacological and genetic targeting of Trx recapitulated VEGF resistance. Functional supplementation of Trx using thioredoxin mimetic peptides (TMP) reversed the HG/MG-induced ROS generation, improved the migration, proliferation, survival and restored VEGF-A-induced chemotaxis and sprouting angiogenesis of hyperglycemic ECs. Importantly, TMP treatment reduced ROS accumulation and improved VEGF-A responses of placental arterial endothelial cells isolated from gestational diabetes mellitus patients. ConclusionsOur findings suggest a putative role for Trx in modulating EC function and its functional impairment in HG conditions contribute to EC dysfunction. Supplementation of TMP could be used as a novel strategy to improve endothelial cell function in diabetes.

Abstract 708: Platelet Derived Growth Factor-Beta Communication Axis is Critical for Mediating Pericyte-Endothelial Cell Interactions and Angiogenic Support in Combination Therapy of Human Umbilical Cord Perivascular Cells and Endothelial Progenitor Cells

Introduction: First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVC) have higher cardiovascular regenerative potential compared to older sources of MSC. We designed a cell combination therapy with FTM HUCPVC and endothelial progenitor cells (EPC) to enhance angiogenesis after MI. However, the mechanism of PVC-EPC interaction is unclear. Hypothesis: Platelet derived growth factor beta receptor (PDGFB-R) expression of HUCPVC mediates homing and integration with endothelial networks and required for supporting neovasculature. Methods: Expression of pericyte markers PDGFB-R and CD146 of xeno-free expanded (HPL 2.5%) FTM and term HUCPVC was analyzed by flow cytometry. PDGFB-driven trans-ECM migration was assessed by Corning Biocoat Invasion assay with 1-100 ng/ml PDGFB. siRNA for PDGFB-R, CD146 and scrambled control was used with lipofectamine for 72 hours. Silencing was confirmed by qPCR and flow cytometry. 10% HPL was used for PDGFB-R desensitization. HUCPVC of all treatment groups were co-cultured with EPC and rat aortic rings on Matrigel TM . Cell migration, endothelial adhesion and network properties were quantified. Statistics: one-way ANOVA N=5 Results: PDGFB-R and CD146 expression and endothelial network augmenting function of FTM HUCPVC was significantly higher than of term HUCPVC. PDGFB dependent migration of FTM HUCPVC was disabled by both ligand (<10%) and siRNA-based (<5%) downregulation of PDGFB-R. Fluorescence microscopy (24 h) showed limited homing and endothelial interactions of PDGFB-R-reduced FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC had significantly decreased network growth and nodes ( p <0.0001) compared to control groups (day3). In EPC co-cultures, PDGFB-R-reduced FTM HUCPVC had limited endothelial interactions and coverage compared to control FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC treated networks had significantly thinner ( p <0.001) and shorter tubules ( p <0.001) compared to control FTM HUCPVC groups. Down-regulation of C146 did not alter FTM HUCPVC pericyte properties. Conclusions: PDGFB-R is crucial for the homing and endothelial support of HUCPVC. This interaction is critical in vascular cell combination therapies and PDGFB-R expression can be a novel criterion for cardiovascular cell therapy.

Role of vitamin D in cell-cell interaction of fetal endothelial progenitor cells and umbilical cord endothelial cells in a preeclampsia-like model.

Maternal endothelial dysfunction is a cental feature of preeclampsia (PE), a hypertensive disorder of pregnancy. Factors in the maternal circulation are thought to contribute to this endothelial dysfunction. Although understudied, factors in the fetal circulation may influence fetal endothelial cell interactions with endothelial progenitor cells as critical steps in placental angiogenesis. We hypothesize that cell-cell interactions that are important for pregnancy health are impaired by fetal serum from PE pregnancies and that 1,25(OH)2-vitamin D3 attenuates the negative effects of this serum on cell function. We tested the ability of fetal cord blood-derived endothelial progenitor cells [endothelial colony-forming cells (ECFCs)] to invade into established monolayers and capillary tubule-like structures of human fetal umbilical venous endothelial cells (HUVECs), while in the presence/absence of fetal cord serum from uncomplicated or PE pregnancies, and tested the ability of 1,25(OH)2-vitamin D3 to modulate the serum-mediated effects. PE cord serum reduced the invasion of fetal ECFCs into HUVEC monolayers or tubule networks. Vitamin D attenuated these effects of PE fetal serum on endothelial functional properties. Immunocytochemical studies revealed involvement of VE-cadherin contacts in interactions between ECFCs and mature fetal endothelial cells. PE cord serum reduces the ability of fetal endothelial progenitor cells to incorporate into fetal endothelial cell networks. Physiologic concentrations of vitamin D reverse these PE serum-mediated effects. These data appear consistent with lines of evidence that vitamin D has antipreeclampsia effects.

537-P: sCD93 Is a Potential Biomarker for Diabetic Nephropathy

Objective: CD93 is a glycoprotein that is known to enhance phagocytosis. The extracellular portion of CD93 can be enzymatically cleaved and secreted to the serum as a soluble form (sCD93). Diabetic nephropathy (DN) is a well-known inflammatory disease that commonly progresses to end stage renal disease (ESRD) in diabetes. Although albuminuria and estimated glomerular filtration rate (eGFR) is currently used for the diagnosis and the prognostication of DN, the prognostic value of the two parameters is modest at best. Here, we hypothesized that sCD93 would be a new biomarker for DN. Methods: We registered 97 human subjects with type 2 diabetes and evaluated the association between serum sCD93 and DN. The underlying mechanism of CD93 regulation was elucidated using human umbilical cord endothelial cell (HUVEC) in vitro and diabetic db/db mice in vivo. Results: The group with higher sCD93 has lower eGFR level and higher prevalence of CKD defined by eGFR. The log(sCD93) was significantly associated with albumin-to-creatinine ratio (ACR) (r=0.229, p=0.028) and the sCD93 level was an independent determinant of ACR and eGFR. The odds ratio of having DN was higher in the high serum sCD93 group even after adjusting for other covariates (adjusted odds ratio 7.283, 95% CI 1.255-42.268, p=0.027). In vitro, CD93 was highly expressed in HUVEC and both CD93 expression and sCD93 secretion was upregulated after LPS stimulation. In db/db mice, sCD93 level was much higher in the peritoneal fluid compared to the db/m mice. The db/db mice showed significantly higher CD93 expression in the renal glomerulus compared to that of the db/m mice suggesting that the elevated sCD93 according to the degree of DN progression might be associated with upregulated expression of CD93 in glomerulus. Conclusion: Based on these human and rodent data, we conclude that sCD93 could be a candidate molecule associated with DN. Disclosure S. Lee: None. H. Park: None. M. Lee: None. A. Choi: None. J. Park: None. C. Ahn: None. S. Lee: None. S. Kang: None. Funding Korean National Research Foundation (2014R1A1A3051221)

Omega-3 fatty acids suppress Fusobacterium nucleatum-induced placental inflammation originating from maternal endothelial cells.

Fusobacterium nucleatum is an oral anaerobe prevalent in intrauterine infection associated with a wide spectrum of adverse pregnancy outcomes. We demonstrate here that F. nucleatum triggers placental inflammation through maternal, rather than paternal, TLR4-mediated signaling. Elimination of TLR4 from maternal endothelial cells alleviated placental inflammation and reduced fetal and neonatal death, while elimination of TLR4 in the hematopoietic cells had no effect. The placental inflammatory response followed a spatiotemporal pattern, with NF-κB activation observed first in the maternal endothelial cells and then in the decidual cells surrounding the endothelium, followed by induction of inflammatory cytokines and chemokines. Supplementation of pregnant mice with fish oil as a source of omega-3 fatty acids suppressed placental inflammation, reduced F. nucleatum proliferation in the placenta, and increased fetal and neonatal survival. In vitro analysis illustrates that omega-3 fatty acids inhibit bacterial-induced inflammatory responses from human umbilical cord endothelial cells. Our study therefore reveals a mechanism by which microbial infections affect pregnancy and identifies a prophylactic therapy to protect against intrauterine infections.

Cellular Clearance and Biological Activity of Calciprotein Particles Depend on Their Maturation State and Crystallinity.

Background: The liver-derived plasma protein fetuin-A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes saturated mineral solutions by forming colloidal protein-mineral complexes called calciprotein particles (CPP). CPP are initially spherical, amorphous and soft, and are referred to as primary CPP. These particles spontaneously convert into secondary CPP, which are larger, oblongate, more crystalline, and less soluble. CPP mediate excess mineral transport and clearance from circulation.Methods: We studied by intravital two-photon microscopy the clearance of primary vs. secondary CPP by injecting i.v. synthetic fluorescent CPP in mice. We analyzed CPP organ distribution and identified CPP endocytosing cells by immunofluorescence. Cellular clearance was studied using bone marrow-derived mouse wildtype and scavenger receptor A (SRA)-deficient macrophages, as well as human umbilical cord endothelial cells (HUVEC), monocyte-derived macrophages (hMDM), and human aortic endothelial cells (haEC). We employed mouse wildtype and mutant immortalized macrophages to analyze CPP-induced inflammasome activation and cytokine secretion.Results: In live mice, only primary CPP were rapidly cleared by liver sinusoidal endothelial cells (LSEC), whereas primary and secondary CPP were cleared by Kupffer cells. Scavenger receptor A (SRA)-deficient bone marrow macrophages endocytosed secondary CPP less well than did wildtype macrophages. In contrast, primary CPP endocytosis did not depend on the presence of SRA, suggesting involvement of an alternative clearance pathway. CPP triggered TLR4 dependent TNFα and IL-1β secretion in cultured macrophages. Calcium content-matched primary CPP caused twice more IL-1β secretion than did secondary CPP, which was associated with increased calcium-dependent inflammasome activation, suggesting that intracellular CPP dissolution and calcium overload may cause this inflammation.Conclusions: Secondary CPP are endocytosed by macrophages in liver and spleen via SRA. In contrast, our results suggest that primary CPP are cleared by LSEC via an alternative pathway. CPP induced TLR4-dependent TNFα and inflammasome-dependent IL-1β secretion in macrophages suggesting that inflammation and calcification may be considered consequences of prolonged CPP presence and clearance.

Synergistic Effect of Umbilical Cord Mesenchymal Stem Cells and Endothelial Colony-Forming Cells on Angiogenesis in Diabetic Mice

Synergistic Effect of Umbilical Cord Mesenchymal Stem Cells and Endothelial Colony-Forming Cells on Angiogenesis in Diabetic Mice

ZEB1 REGULATES MULTIPLE ASPECTS OF ENDOTHELIAL QUIESCENCE DURING VASCULAR REMODELLING

Generation of new blood vessels is essential for vascular remodeling in exercise to provide greater blood flow to developing muscles. As blood vessels grow, the sprouting endothelial tips need to recognise each other, and move from a motile, angiogenic phenotype to a maturing, quiescent phenotype,, form tubes and allow blood flow to occur and regulate their development. They grow in response to cytokine signalling such as VEGF and other angiogenic factors, but they mature as a consequence of cell contact and flow. As they do so, they change their metabolic state from growing, glycolytic, to quiescent oxidative metabolism. The mechanisms behind the transcriptional control of this phenotype are unknown, but recent work has demonstrated the dynamic interplay between ‘quiescence’ factors (such as FOXO1 expression) and expression of glycolytic enzymes such as PFKFB3 through c‐MYC. We investigated potential drivers of this process and identified Zeb1 (a transcription factor with known functions in cancer progression) to be expressed by quiescent (non‐angiogenic) endothelial cells and reduced in angiogenic endothelial cells in the developing murine retina. In vitro analyses demonstrates that Zeb1 is expressed by confluent monolayers of Human Vein Umbilical Cord Endothelial cells (HUVECs) but is downregulated in actively growing cells. Zeb1 knockdown promotes an angiogenic EC phenotype: including enhanced PFKFB3, c‐myc and VEGFR2 and reduced FOXO1, Tie2 and VE‐cadherin expression. Consistently, Zeb1 knockdown EC were metabolically more active (both through oxidative and glycolytic pathways). Furthermore, loss of Zeb1 promoted EC migration and proliferation in vitro. In vivo shRNA knockdown of Zeb1 led to altered vessel formation and hypercellularity. In conclusion, Zeb1 appears to be linked to key quiescence regulating signaling pathways and furthermore regulates aspects of these pathways. Zeb1 expression appears to be a critical component for endothelial cells to generate functional vasculatures that can support enhanced muscle blood flow, by its control of FOXO1, PFKFB3 and Myc.Support or Funding InformationThis work was supported by the British Heart Foundation (grant PG/11/67/29067), the University of Nottingham Cancer Research Priority Area, and the Medical Research Council Newton Fund (MR/N01247X/1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.