Human Umbilical Vein Endothelial Cells Cultures Research Articles

Cell adhesion behavior of poly(l‐lactide‐co‐glycolide)‐poly(1,5‐dioxepan‐2‐one) random multiblock copolymers

AbstractAlthough bioabsorbable polymers are promising implant materials, implanting hydrophobic polymers induces platelet adhesions and subsequent thrombus formation. We recently reported that a multiblock copolymer composed of poly(l‐lactide‐co‐glycolide) (PLGA) and poly(1,5‐dioxepan‐2‐one) (PDXO) exhibited antiplatelet adhesive behavior. In this study, the cell adhesion behaviors of PLGA and PDXO random multiblock copolymers (PLGA−PDXO RMBCs) were examined. All the RMBCs showed antiplatelet adhesion behavior, and the copolymers containing 40 or 51 mol% of the PDXO component showed good antiplatelet adhesive behavior. Good culture behavior was observed for HeLa cells, but human umbilical vein endothelial cells (HUVECs) proliferation was poor on PLGA−PDXO RMBC surfaces. The cell attachment inhibition studies revealed that both integrin‐dependent and integrin‐independent interactions worked for HeLa cell culture, whereas only integrin‐dependent interactions played an important role for HUVEC culture proliferation on copolymer surfaces. The findings of this study revealed the unique cell‐selective adhesion behavior of PDXO containing multiblock polymers—being nonadhesive to platelets and HUVECs, and adhesive to HeLa cells.

Effect of Soybean Extract on sFlt-1 LEVELS in Huvecs Cultures Induced by Preeclampsia Plasma

Preeclampsia is a dangerous pregnancy issue that causes hypertension at ≥20 weeks of gestation. Oxidative stress is known to play a role in the pathophysiology of preeclampsia by raising the activity of soluble fms-like tyrosine kinase 1 (sFlt-1) and causing endothelial dysfunction. As a result, antioxidants are utilized as a therapy in preeclampsia to protect the body from the impacts of free radicals. This study aims to determine the effect of soybean extract on sFlt-1 levels in HUVECs cultures exposed to preeclamptic plasma. The benefit of this study is the consideration for clinicians to provide soybean ethanol extract supplements (Glycine max) to increase the potential of antihypertensive therapy. Human umbilical vein endothelial cell cultures (HUVECs) were used in the study, which was conducted in vitro. HUVECs cultures were exposed to preeclampsia plasma and subsequently treated for 24 hours with various dosages of soybean extract. The enzyme-linked immunosorbent assay (ELISA) measured the sFlt-1 levels in each observation group. The hypothesis was tested using One-Way ANOVA analysis with SPSS version 25 software. This study found a significant difference (p <0,05) between the mean value of the positive control group and the negative control group. A significant difference was also found (p <0,05) in the mean value of sFlt-1 between the positive control group and the treatment group that was given soybean extract at doses of 17,5 ppm. Soybean extract had a significant effect on decreasing sFlt-1 levels in HUVECs cultures exposed to preeclamptic plasma. Thus, soybeans isoflavones have the potential to treat preeclampsia by reducing anti-angiogenic factors.

Sildenafil attenuates oxidative stress and endothelial dysfunction in lead-induced hypertension.

Lead (Pb) reduces NO bioavailability, impairs the antioxidant system, and increases the generation of reactive oxygen species (ROS). Pb-induced oxidative stress may be responsible for the associated endothelial dysfunction. Sildenafil has shown nitric oxide (NO)-independent action, including antioxidant effects. Therefore, we examined the effects of sildenafil on oxidative stress, reductions of NO and endothelial dysfunction in Pb-induced hypertension. Wistar rats were distributed into three groups: Pb, Pb + sildenafil and Sham. Blood pressure and endothelium-dependent vascular function were recorded. We also examined biochemical determinants of lipid peroxidation and antioxidant function. ROS levels, NO metabolites and NO levels in human umbilical vein endothelial cells (HUVECs) were also evaluated. Sildenafil prevents impairment of endothelium-dependent NO-mediated vasodilation and attenuates Pb-induced hypertension, reduces ROS formation, enhances superoxide dismutase (SOD) activity and antioxidant capacity in plasma and increases NO metabolites in plasma and HUVECs culture supernatants, while no changes were found on measurement of NO released from HUVECs incubated with plasma of the Pb and Pb + sildenafil groups compared with the sham group. In conclusion, sildenafil protects against ROS-mediated inactivation of NO, thus preventing endothelial dysfunction and attenuating Pb-induced hypertension, possibly through antioxidant effects.

Abstract 2654: Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein

Abstract Mantle Cell Lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma that frequently metastasizes. Lymphoma dissemination into distant sites is the primary cause of cancer mortality. Lymphoma metastasis requires development of supporting vasculature in which the interaction between malignant B cells and the vasculature or lymphatics is critical to tumor cell growth and dissemination. Human umbilical vein endothelial cells (HUVEC) or human lymphatic endothelial cells (HLEC) cultured on Matrigel, in vitro, assemble into networks of capillary like structures (CLS) or lymphatic like structures (LLS). When Mino or Jeko-1 MCL cells are co-cultured with vascular or lymphatic cells they migrate toward and anneal to nascent CLS or LLS. We constructed a novel antibody fusion protein, αCD20-IgG1-huEndoP125A, to inhibit such interactions. αCD20-IgG1-huEndoP125A links an αCD20 human IgG1 targeting domain and a mutant version of human endostatin, huEndoP125A, with enhanced anti-angiogenic properties. αCD20-IgG1-huEndoP125A completely inhibits CLS or LLS formation and prevents the alignment and annealing of MCL to CLS or LLS. αCD20-IgG1-huEndoP125A also significantly reduces both endothelial and MCL migration and invasion in vitro. Cell migration and trafficking is tightly regulated by chemokine interactions with corresponding chemokine receptors. We assayed for the presence of chemokines using a dot blot chemokine array. Jeko-1 and HUVEC cells were cultured on Matrigel, alone or together in co-culture, and either treated with αCD20-IgG1-huEndoP125A or left untreated. CXCL12 was expressed at high levels in HUVEC cultures undergoing CLS formation and expression was further increased in HUVEC and MCL co-cultures. HUVEC or HUVEC/MCL co-cultures treated with αCD20-IgG1-huEndoP125A showed markedly decreased CXCL12 expression. Flow cytometry showed that corresponding chemokine receptor CXCR4 was upregulated on MCL cells compared to normal B cells. CXCR4 was also upregulated on HUVEC undergoing CLS formation, and further increased on HUVEC in co-culture with MCL cells. αCD20-IgG1-huEndoP125A inhibited HUVEC CLS formation, reduced MCL migration to and alignment with nascent CLS or LLS, downregulated CXCL12 levels in HUVEC or coculture supernatants, and reduced CXCR4 expression on both HUVEC and MCL cells. Murine 38c13-huCD20 lymphoma cells were implanted s.c. into C3H mice and mice were treated i.p. with either PBS, Rituximab, or equimolar αCD20-IgG1-huEndoP125A. Mice treated with αCD20-IgG1-huEndoP125A showed significantly reduced tumor growth compared to mice treated with PBS or rituximab. αCD20-IgG1-huEndoP125A ability to inhibit angiogenesis, lymphangiogenesis, and lymphoma association with angiogenic or lymphatic vasculature may be a potent means of preventing lymphoma growth and metastasis. Citation Format: Christian Elledge, Seung-Uon Shin, Rathin Das, Yu Zhang, Hyun-Mi Cho, Sundaram Ramakrishnan, Ankita Sankar, Joseph Rosenblatt. Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2654.

In vitro vascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture

The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge of in vitro vascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineered in vitro tissue constructs.

Perfusion culture of endothelial cells under shear stress on microporous membrane in a pressure-driven microphysiological system

This paper reports perfusion culture of human umbilical vein endothelial cells (HUVECs) on a microporous membrane in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. We designed fluidic culture unit with microporous membrane to culture HUVECs under fluidic shear stress and constructed a perfusion culture model in the PD-MPS platform. Four fluidic culture units were arranged in the microplate-sized device, which enables four-throughput assay for characterization of HUVECs under flow. Medium flow was generated above and below the membrane by sequential pneumatic pressure to apply physiological shear stress to HUVECs. HUVECs exhibited aligned morphology to the direction of the flow with shear stress of 11.5-17.7dyn/cm2 under the flow condition, while they randomly aligned under static culture condition in a 6 well plate. We also observed 3.3- and 5.0-fold increase in the expression levels of the thrombomodulin and endothelial nitric oxide synthase mRNAs, respectively, under the flow condition in the PD-MPS compared to the static culture in 6 well plate. We also observed actin filament aligned to the direction of flow in HUVECs cultured under the flow condition.

Fabrication of succinate-alginate xerogel films for in vitro coupling of osteogenesis and neovascularization

The osseointegration of metallic implants is reliant on a cascade of molecular interactions and the delivery of macromolecules to the implant environment that occurs before substantial bone formation. Early blood vessel formation is a requisite first step in the healing timeline for osteoid formation, where vascular development can be accelerated as a result of controlled hypoxic conditioning. In this study, alginate-derived xerogel films containing varied concentrations of disodium succinate salt which has been shown to induce pseudohypoxia (short-term hypoxic effects while maintaining an oxygenated environment) were developed. Xerogels were characterized for their morphology, succinate release over time and cellular response with osteoblast-mimicking Saos-2 and human umbilical vein endothelial cells (HUVEC). Scanning electron microscopy revealed a multiscale topography that may favour osseointegration and alamarBlue assays indicated no cytotoxic effects during in vitro proliferation of Saos-2 cells. pH measurements of eluted succinate reach 95 % of peak value after 7 h of immersion for all gels containing 10 mM of succinate or less, and 60 % within the first 40 min. In vitro exposure of HUVECs to succinate-conditioned media increased the net concentration of total proteins measured by bicinchoninic acid (BCA) assay and maintains stable vascular endothelial growth factor (VEGF) and extracellular platelet-derived growth factor (PDGF) for vessel formation through comparison of enzyme-linked immunosorbent assays (ELISAs) of the culture media and cell lysate. Tube formation assays also showed a sustained increase in tube diameter across the first 48 h of HUVEC culture when succinate concentrations of 1 and 10 μM in the xerogel. Overall, the succinate-alginate films serve as a prospective organic coating for bone-interfacing implant materials which may induce temporary pseudohypoxic conditions favourable for early angiogenesis and bone regeneration in vivo at succinate concentrations of 1 or 10 μM.

Induction of Periodontal Ligament–like Cells by Coculture of Dental Pulp Cells, Dedifferentiated Cells Generated from Epithelial Cell Rests of Malassez, and Umbilical Vein Endothelial Cells

IntroductionApart from the epithelial cell rests of Malassez (ERMs), dental pulp (DP) contains the same types of mesenchymal cells as the periodontal ligament (PDL). ERMs may affect the characteristics of the mesenchymal cells in the PDL. The aim of this study was to examine whether DP cells cultured with ERMs and human umbilical vein endothelial cells (HUVECs) could transform into PDL-like cells. MethodsProgenitor-dedifferentiated into stem-like cells (Pro-DSLCs) were produced by the induction of ERMs with 5-Azacytidine and valproic acid. DP cells were cultured in mesenchymal stem cell medium for 1 week under the following conditions: DP cells alone (controls); PDL cells alone; coculture of DP cells and ERMs (DP + ERM) or Pro-DSLCs (DP + Pro-DSLC); and coculture of DP cells, HUVECs, and ERMs (DP + ERM + HUVEC) or Pro-DSLCs (DP + Pro-DSLC + HUVEC). Quantitative real-time reverse transcription polymerase chain reaction, quantitative methylation-specific polymerase chain reaction, and flow cytometry were performed. ResultsThe expression levels of PDL-related markers Msx1, Msx2, Ncam1, Postn, and S100a4 and mesenchymal stem cell–positive markers Cd29, Cd90, and Cd105 were significantly higher in the PDL cells and DP + Pro-DSLC + HUVEC cultures than in the controls (P < .05). The DNA methylation levels of Msx1 and Cd29 in the PDL cells and the DP + Pro-DSLC + HUVEC culture were significantly lower than in the controls (P < .01). We found a significant increase in the number of cells stained with MSX1 (P < .05) and CD29 (P < .01) in the DP + Pro-DSLC + HUVEC culture than in the controls. ConclusionsCoculture of DP cells with Pro-DSLCs and HUVECs induced their transformation into PDL-like cells. This method may prove to be useful for periodontal regeneration via tissue engineering.

CCL14 exacerbates intraplaque vulnerability by promoting neovascularization in the human carotid plaque

ObjectiveTo examine the role of CCL14 in the neovascularization process and vulnerability progression within carotid plaques by investigating the mechanism of CCL14 regulation of VEGF-A. MethodsWe first performed histological analysis and immunohistochemical staining of human carotid plaque tissue to detect the expression of CCL14, JAK2, STAT3 and VEGF-A. We next examined the protein expression of CCL14, VEGF-A, JAK2, STAT3, and phosphorylation of JAK2 and STAT3 in human carotid atherosclerotic plaques by Western blotting. Finally, we performed in vitro culture of human umbilical vein endothelial cells (HUVEC). In the tube formation assay of HUVEC, we added CCL14 siRNA or VEGF-A siRNA to the culture medium using lentiviral transfection to knock down CCL14 or VEGF-A and grouped them for control assays, and detected the changes in the expression of the above proteins using Western blotting. ResultsHistological and Western blotting analysis of human carotid plaque samples showed that the expression of CCL14 and VEGF-A was higher in the vulnerable plaques than in stable plaques. In the in vitro cultures of HUVEC, CCL14 was found to increase the number and length of intercellularly generated tubular structures. CCL14 increases VEGF-A expression via activating JAK2/STAT3 signaling. ConclusionIn the human carotid plaques, CCL14 promotes angiogenesis by upregulation of VEGF-A via JAK2/STAT3 pathway and thus drives the progression of carotid plaques vulnerability.

874-P: Potential Anti-Inflammatory Effects of Canagliflozin in Tumour Necrosis Factor-α and Interferon-ϒ-Stimulated Endothelial Cells

Background: This study investigates the potential direct anti-inflammatory effects of the SGLT2 inhibitors (canagliflozin, C; dapagliflozin D; or empagliflozin, E) on cultured endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) or Human cardio microvascular endothelial cells (HCMECs) were pre-incubated in the presence or absence of C (0.5μM, 1μM, and 10μM) , D (0.5μM, 1μM, and 10μM) and E (0.5μM, 1μM, and 10μM) for 1 hr and NaCl (150 mM) for 24 hr. The cells were then stimulated with TNF-α (ng/mL) and IFN-Υ (ng/mL) for 24 hrs. Expression of IL-6 and was analysed using Quantikine ELISA kits. The expression of SGLT2 mRNA was assessed using RT-PCR in cDNA prepared from two independent cultures of HUVECs and HCMECs using cDNA from human kidney cortex as a positive control. Results: TNF-α and IFN-Υ and significantly induced expression of IL-6 in both HUVECs and HCMECs compared to controls (p = &amp;lt;0.0001) . The effects were significantly inhibited by canagliflozin 0.1μM, 1μM, and 10μM (p=0.0099, p&amp;lt; 0.0001, and p&amp;lt;0.0001, respectively) in HUVECs. In HCMECs, C, (0.5μM, 1μM, and 10μM) significantly inhibit the TNF-α and IFN-Υ IL-6 secretion (p&amp;lt; 0.0001, p&amp;lt; 0.0001, and p&amp;lt;0.0001, respectively) . No significant effect of D and E was observed. High NaCl had no influence on C (0.5μM, 1μM, and 10μM) inhibition of TNF-α and IFN-Υ induced of IL-6 secretion, and high salt alone had no influence on the secretion of IL-6 secretion. SGLT2 mRNA levels were below the detection level in HUVECs and HCMECs alone and on high glucose and TNF-α and IFN-Υ conditions. Conclusion: This result indicates that SGLT2-I directly inhibits endothelial pro-inflammatory cytokines secretions. This striking anti-inflammatory of the SGLT2 inhibitor class merits further mechanistic research. Disclosure A.S.Alshnbari: None. R.N.Khan: None. I.R.Idris: None.

Aneurysm-on-a-Chip: Setting Flow Parameters for Microfluidic Endothelial Cultures Based on Computational Fluid Dynamics Modeling of Intracranial Aneurysms.

Intracranial aneurysms are pouch-like extrusions from the vessels at the base of the brain which can rupture and cause a subarachnoid hemorrhage. The pathophysiological mechanism of aneurysm formation is thought to be a consequence of blood flow (hemodynamic) induced changes on the endothelium. In this study, the results of a personalized aneurysm-on-a-chip model using patient-specific flow parameters and patient-specific cells are presented. CT imaging was used to calculate CFD parameters using an immersed boundary method. A microfluidic device either cultured with human umbilical vein endothelial cells (HUVECs) or human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) was used. Both types of endothelial cells were exposed for 24 h to either 0.03 Pa or 1.5 Pa shear stress, corresponding to regions of low shear and high shear in the computational aneurysm model, respectively. As a control, both cell types were also cultured under static conditions for 24 h as a control. Both HUVEC and hiPSC-EC cultures presented as confluent monolayers with no particular cell alignment in static or low shear conditions. Under high shear conditions HUVEC elongated and aligned in the direction of the flow. HiPSC-EC exhibited reduced cell numbers, monolayer gap formation and cells with aberrant, spread-out morphology. Future research should focus on hiPSC-EC stabilization to allow personalized intracranial aneurysm models.

Impact of maternal diabetes exposure on soluble adhesion molecules in the offspring

Background and aimsSoluble adhesion molecules are associated with cardiovascular disease and increased in individuals with diabetes. This study assesses the impact of diabetes exposure in utero on the abundance of circulating adhesion molecules in cord serum and soluble adhesion molecules released from human umbilical vein endothelial cells (HUVEC) exposed to high glucose concentrations. Methods and resultsWomen with and without diabetes were recruited. DM was diagnosed based on the American Diabetes Association criteria. Primary cultures of HUVEC were cultured in 5 mM and 25 mM glucose with 25 mM mannitol osmotic control. The soluble adhesion molecules, intracellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and E-selectin were measured by ELISA in the cord blood serum and conditioned HUVEC media. The mothers with DM were older with higher BMI (p = 0.027 and 0.008, respectively). In a fully adjusted model, VCAM was significantly increased in the cord serum of infants born to mothers with diabetes (p = 0.046), but ICAM and E-selectin were not different. ICAM was also significantly correlated with maternal HbA1c (r2 = 0.16, p = 0.004) and cord serum non-esterified fatty acids (r2 = 0.08, p = 0.013). From the HUVEC media, the abundance of adhesion molecules was not different based on DM or high glucose exposure; however, VCAM abundance in the HUVEC supernatant was significantly correlated with ICAM (r2 = 0.27, p = 0.010) and cord serum c-peptide (R2 = 0.19, p = 0.043). ConclusionsAlterations in soluble adhesion molecule abundance in infants exposed to the diabetic milieu of pregnancy may reflect early alterations in vascular function predicting future cardiovascular disease.

α-Mangostin Alleviated HIF-1α-Mediated Angiogenesis in Rats With Adjuvant-Induced Arthritis by Suppressing Aerobic Glycolysis.

A previously validated anti-rheumatic compound α-mangostin (MAN) shows significant metabolism regulatory effects. The current study aimed to clarify whether this property contributed to its inhibition on synovial angiogenesis. Male wistar rats with adjuvant-induced arthritis (AIA) were orally treated by MAN for 32 days. Afterwards, biochemical parameters and cytokines in plasma were determined by corresponding kits, and glycometabolism-related metabolites were further accurately quantified by LC-MS method. Anti-angiogenic effects of MAN were preliminarily assessed by joints based-immunohistochemical examination and matrigel plug assay. Obtained results were then validated by experiments in vitro. AIA-caused increase in circulating transforming growth factor beta, interleukin 6, hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in blood and local HIF-1α/VEGF expression in joints was abrogated by MAN treatment, and pannus formation within matrigel plugs implanted in AIA rats was inhibited too. Scratch and transwell assays revealed the inhibitory effects of MAN on human umbilical vein endothelial cells (HUVECs) migration. Furthermore, MAN inhibited tubule formation capability of HUVECs and growth potential of rat arterial ring-derived endothelial cells in vitro. Meanwhile, MAN eased oxidative stress, and altered glucose metabolism in vivo. Glycolysis-related metabolites including glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglyceric acid and phosphoenolpyruvic acid in AIA rats were decreased by MAN, while the impaired pyruvate-synthesizing capability of lactate dehydrogenase (LDH) was recovered. Consistently, MAN restored lipopolysaccharide-elicited changes on levels of glucose and LDH in HUVECs culture system, and exerted similar effects with LDH inhibitor stiripentol on glycometabolism and VEGF production as well as tubule formation capability of HUVECs. These evidences show that MAN treatment inhibited aerobic glycolysis in AIA rats, which consequently eased inflammation-related hypoxia, and hampered pathological neovascularization.

Uncaria tomentosa extract (AC-11) improves pregnancy hypertension together with suppression of sFlt-1 and sEng.

Disruption of well-controlled reproductive functions leads to pregnancy complications such as hypertensive disorders of pregnancy (HDP). Uncaria tomentosa (Wild), known as cat's claw, is widely used for the treatment of a various types of health problems; AC-11 (AC-11®, hot-water extract of U. tomentosa) is unique phytochemical compound and has potential roles as anti-inflammatory or anti-oxidant processes. We investigated whether AC-11 has a protective effect on pathogenesis of HDP in vivo and production of anti-angiogenic factors (sFlt-1 and sEng, major factors for the onset of HDP) in in vitro. Non-pregnant or pregnant mice were administered AC-11 (4mg/mL), then, angiotensin II (Ang II) was subcutaneously infused to increase blood pressure. Human placental tissues or human umbilical vein endothelial cells (HUVECs) were incubated with or without AC-11. Treatment with AC-11 significantly reduced blood pressure induced by Ang II infusion. The population of CD8+T cells, the ratio of CD8/CD4, and plasma interleukin-6 levels were increased by Ang II infusion, and were decreased by AC-11 both in pregnant and non-pregnant mice. In pregnant mice, plasma levels of sFlt-1 and sEng were decreased by AC-11. In in vitro cell culture of HUVECs or placental tissue culture, treatment with AC-11 significantly inhibited secretion of sFlt-1 and sEng. We suggest a novel role of AC-11 in regulating blood pressure by controlling the balance of T cell population and inflammatory cytokine production both in non-pregnant and pregnant conditions. In addition, AC-11 inhibits HDP-related factors, including sFlt-1 and sEng, suggesting that AC-11 may useful for relieving HDP.

Congxin Lunzhi Fang improves aortic atherosclerosis and regulates ACE2 expression in ApoE-/- mice

To investigate the effect of Congxin Lunzhi Fang (CXLZF) for improving atherosclerosis (AS) and explore its therapeutic mechanism. Thirty ApoE-/- mice were fed on high-fat diet (HFD) to establish AS models, which were randomized equally into model group, low-, medium- and high-dose CXLZF groups (daily dose 4.5, 9, and 18 g/kg, respectively), and simvastatin (5 mg/kg daily) group, with 6 male C57BL/6 mice fed a normal diet as the control group. The mice in the control and model groups were gavaged with normal saline, and those in the treatment groups were gavaged with the indicated drugs for a treatment course of 12 weeks. The changes of atherosclerotic plaque and liver pathologies were observed using oil-red O, HE and Masson staining, and blood lipid profiles and serum levels of glucose, Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) of the mice were examined. The protein expressions of ACE2 and E-selectin were determined with Western blotting. Drug-containing serum was collected from SD rats gavaged with CXLZF for treatment of human umbilical vein endothelial cells (HUVECs) with oxidized low-density lipoprotein (ox-LDL)-induced injury, and the protein expression of ACE2 in the cells was determined. In ApoE-/- mice, CXLZF at all the 3 doses significantly reduced the plaque area and increased the collagen content in atherosclerotic plaque, indicating improved plaque stability. CXLZF treatment, especially at the high dose, also significantly reduced the levels of serum lipids (P < 0.05). CXLZF significantly lowered serum ALT and AST levels, mitigated hepatocyte steatosis (P < 0.05), increased the protein expression of ACE2 and down-regulated E-selectin expression (P < 0.05) in the mice. In HUVEC cultures, CXLZFcontaining serum (5%, 10%, and 20%) significantly up-regulated the cellular expression of ACE2 protein (P < 0.05). CXLZF relieves aortic atherosclerosis in ApoE-/- mice possibly by regulating ACE2 protein.

Effect of Ketone Body β-Hydroxybutyrate to Attenuate Inflammation-Induced Mitochondrial Oxidative Stress in Vascular Endothelial Cells

To investigate the regulatory function and mechanism of β-hydroxybutyrate (β-OHB), a ketone body, on the mitochondrial oxidative stress of inflammatory human umbilical vein endothelial cells (HUVECs). Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) were used to induce macrophages to release proinflammatory factors, and the culture supernatant was collected as a macrophage-conditioned medium (MCM) to culture HUVECs. A total of 7 groups of cells were used in the study: ①control group, or normal cultured HUVECs; ②MCM group, or the MCM-cultured HUVECs; groups ③ to ⑦ were all HUVECs co-cultured with different reagents, including ③MCM+β-OHB group, ④MCM+N-acetylcysteine (NAC) group, ⑤MCM+β-OHB+NAC group, ⑥MCM+β-OHB+histone deacetylase agonist ITSA1 group, and ⑦MCM+β-OHB+histone deacetylase inhibitor Entinostat group. MitoSOX immunofluorescence staining was conducted to analyzes the mitochondrial superoxide levels, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was performed to examine the mRNA expression of antioxidant genes, and Seahorse mitochondrial energy analyzer was used to measure mitochondrial aerobic respiration capacity. Compared with the control group, mitochondrial superoxide production was significantly increased in the MCM cultured HUVECs cells, while β-OHB treatment significantly inhibited mitochondrial superoxide production, which was accompanied by an increase in the mRNA expression of antioxidant genes, and significant increase in the basal mitochondrial oxygen consumption rate and respiratory reserve capacity. NAC treatment did not further enhance the protective effect of β-OHB on mitochondrial functions. In addition, ITSA1 treatment could completely offset the antioxidant and mitochondrial protective effects of β-OHB, and these stated effects were still maintained after Entinostat treatment. The ketone body β-OHB attenuates the mitochondrial oxidative stress of vascular endothelial cells through activating the antioxidant pathway and inhibiting histone deacetylase activity.

154-LB: Impact of Maternal Diabetes Exposure on Soluble Adhesion Molecules

Soluble adhesion molecules are associated with cardiovascular disease and increased in individuals with diabetes. The current study assesses the impact of diabetes exposure in utero on the abundance of circulating adhesion molecules in cord serum as well as soluble adhesion molecules released from human umbilical vein endothelial cells (HUVEC) upon exposure to high levels of glucose. Women with and without diabetes (DM; gestational or type 2) were recruited at delivery. DM was diagnosed based on the criteria by the American Diabetes Association. Primary cultures of HUVEC were established in M199 medium with growth supplements. For the high glucose treatment, medium was adjusted to a final glucose concentration of 25mM with media collected 48 hours post treatment. The soluble adhesion molecules intracellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and E-selectin were measured by ELISA in the cord blood serum and the conditioned HUVEC media. Serum adhesion molecules were measured in 42 infants born to mothers with normal glycemia and 35 infants of mothers with DM. The mothers with DM were older with higher BMI (p=0.027 and 0.008, respectively). In a fully adjusted model, VCAM was significantly increased in the cord serum of infants born to mothers with diabetes (p=0.046), but ICAM and E-selectin were not different. ICAM was significantly correlated with E-selectin (r2=0.25, p&amp;lt;0.001), but not VCAM. ICAM was also significantly correlated with maternal HbA1c (r2=0.16, p=0.004) and cord serum non-esterified fatty acids (r2=0.08, p=0.013). From the HUVEC, the abundance of adhesion molecules was not different based on DM or high glucose exposure; however, VCAM abundance in the HUVEC supernatant was significantly correlated with ICAM (r2=0.27, p=0.010) and cord serum insulin (r2=0.19, p=0.042). Alterations in soluble adhesion molecule abundance in infants exposed to the diabetic milieu of pregnancy may reflect early alterations in vascular function predicting future cardiovascular disease. Disclosure S. B. Landreth: None. A. M. Teague: None. M. Jensen: None. S. Gulati: None. J. B. Tryggestad: None. Funding National Institutes of Health (1K23DK106533)

Effects of bioactive glass on proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells

OBJECTIVE To investigate the effects of phytic acid derived bioactive P2O5-SiO2-CaO gel-glasses (PSC) on the proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. METHODS HUVECs were cultured in PSC extracts, which were prepared with endothelial cell medium (ECM) at a gradient concentration of 0.01, 0.1, 1 and 2 g/L. Cells cultured in ECM were used as the control. The effect of PSC on HUVECs proliferation was assessed on the 1st, 3rd, 5th, 7th and 10th days with (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide assay (MTT), and the optimum PSC concentration for HUVECs proliferation was used in the following experiments. The subsequent experiments were divided into two groups. The experimental group used PSC extracts to culture HUVECs (PSC group) and the control group used ECM to culture HUVECs (ECM group). Gene expression of angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), was detected on the 2nd, 4th and 7th days by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). The morphology and number of tubules formation were observed at the 4th and 10th hours. Image J software was used for counting and quantitative analysis. RESULTS The results of MTT assay showed that 0.1 g/L PSC group had the most significant effect on promoting HUVECs proliferation. The optical density values of 0.1 g/L PSC group on the 5th and 7th days were significantly higher than those of the other PSC groups and the control group (P < 0.05). The result of real-time RT-PCR showed that 0.1 g/L PSC extract up-regulated the mRNA expression of VEGF and bFGF significantly (P < 0.05). On the 4th day, the gene expressions of VEGF and bFGF in PSC group were 1.59 and 1.45 times higher than those in ECM group respectively, and on the 7th day, the gene levels of VEGF and bFGF in PSC group were 1.98 and 1.37 times higher than those in ECM group respectively. The tubule formation assay showed that the maturity and density of the tubules in 0.1 g/L PSC group was much better than that in the ECM group at the 10th hour. The quantitative analysis by Image J indicated that the tubules number in PSC group (29.63±2.29) was higher than in the ECM group (20.13±2.36), with statistical significance (P < 0.05). CONCLUSION PSC showed significant promoting effects on HUVECs' proliferation, differentiation and angiogenesis in vitro.

Effects of vitamin D-induced supernatant of placental explants from preeclamptic women on oxidative stress and nitric oxide bioavailability in human umbilical vein endothelial cells.

The study evaluated the effect of the supernatant of placental explants from preeclamptic (PE) and normotensive (NT) pregnant women after tissue treatment with or without vitamin D (VD) on oxidative stress and nitric oxide (NO) bioavailability in human umbilical vein endothelial cells (HUVEC). Placental explants were prepared from eight NT and eight PE women, and supernatants were obtained after incubation with or without hydrogen peroxide (H2O2) and/or VD. HUVEC were cultured for 24 h with supernatants, and the following parameters were analyzed in HUVEC cultures: NO, nitrate (NO3 -), and nitrite (NO2 -) levels, lipid peroxidation, and intracellular reactive oxygen species (ROS). Results showed that the production of NO3 -, NO2 -, malondialdehyde (MDA), and ROS were significantly higher in HUVEC treated with explant supernatant from PE compared to NT pregnant women, while the supernatant of PE explants treated with VD led to a decrease in these parameters. A significantly high production of NO was detected in HUVEC cultured with control supernatant of NT group, and in cultures treated with supernatant of PE explants treated with VD. Taken together, these results demonstrated that cultures of placental explants from PE women with VD treatment generated a supernatant that decreased oxidative stress and increased the bioavailability of NO in endothelial cells.

AF cell derived exosomes regulate endothelial cell migration and inflammation: Implications for vascularization in intervertebral disc degeneration

AimsThe intervertebral disc is the largest avascular organ of the body. Vascularization of the disc has been typically regarded as a pathological feature of intervertebral disc degeneration (IDD). However, the underlying mechanism of vascularization in IDD is still unclear. The current study aimed to investigate the role of AF cell derived exosome (AF-exo) in the interaction with human umbilical vein endothelial cells (HUVECs) and its potential role in the regulation of vascularization in IDD. Main methodsHuman AF tissues were obtained from patients with IDD and idiopathic scoliosis. The AF-exo were isolated and identified by transmission electron microscopy (TEM), nanoparticle trafficking analysis (NTA) and Western blotting. Then, the AF-exo were used for HUVECs cultures. The migration of HUVECs was observed in 2D and 3D cultures. The inflammatory phenotype of HUVECs was examined by Real-time PCR and enzyme-linked immunosorbent assay (ELISA). Additionally, apoptosis of HUVECs were analyzed by flow cytometry. Key findingsHere, we for the first time found that AF cells could secrete AF-exo and that the AF-exo could be phagocytosed by HUVECs. Additionally, we found that degenerated AF-exo exerted pro-vascularization effect on HUVECs by promoting cell migration (in 2D and 3D cultures) and inflammatory factor expression including IL-6, TNF-α, MMP-3, MMP-13 and VEGF, whereas the application of non-degenerated AF-exo demonstrated inverse effects. SignificanceThese results showed that AF-exo is an essential regulator mediating intercellular communication between AF cells and HUVECs, suggesting its important role in vascularization in the intervertebral disc.