Vascular Endothelial Cells In Culture Research Articles

Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model

Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease, caused by Dirofilaria immitis, causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction, and hypoxia. The aim of this study was to analyze whether the excretory/secretory and surface-associated antigens of adult worms interact and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEFG-A (Vascular Endothelial Growth Factor A), as well as DiES+VEFG-A and Cut+VEFG-A were used. The production of VEFG-A and other proangiogenic [soluble VEFGR-2 (sVEFGR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEFGR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non‑stimulated cultures were used as controls. DiES+VEFG-A and Cut+VEFG-A significantly increased the production of VEFG-A and sVEFGR-2, and only Cut+VEFG-A significantly increased the production of VEFGR-1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEFG-A produced a significant increase in viable cell number and significant decrease cell migration, as well as in the organization and number of cell connections. Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.

Effect of somatic antigens of Dirofilaria repens adult worms on angiogenesis, cell proliferation and migration and pseudo-capillary formation in human endothelial cells

BackgroundAngiogenesis is defined as the formation of new vessels by sprouting of endothelial cells from pre-existing vessels in response to stimuli, such as hypoxia or inflammation. Subcutaneous dirofilariasis, caused by Dirofilaria repens, is a zoonotic disease characterized by the formation of subcutaneous nodules with the presence of at least one encapsulated worm, showing perivascular vascularization around it. The aim of this study is to analyze whether the somatic antigen of adult D. repens worms interacts with and modulates the angiogenic mechanism, cell proliferation and migration, and formation of pseudo-capillaries.MethodsThe expression of VEGF-A, VEGFR-1/sFlt, VEGFR-2, mEnd and sEnd in cultures of human vascular endothelial cells stimulated with somatic antigen of adult worms of D. repens (DrSA), vascular endothelial growth factor (VEGF) and DrSA + VEGF were evaluated by using ELISA commercial kits. Cellular viability was analyzed by live cell count, cytotoxicity assays by using a commercial kit, cell proliferation by MTT-based assay, cell migration by wound-healing assay carried out by scratching wounds and capacity of formation of pseudo-capillaries analyzing cell connections and cell groups in Matrigel cell cultures. In all cases unstimulated cultures were used as controls.ResultsDrSA + VEGF significantly increased the expression of VEGF-A, VEGFR-2 and mEndoglin compared to other groups and unstimulated cultures. Moreover, DrSA + VEGF produced cell proliferation and migration and increased the formation of pseudo-capillaries.ConclusionsSomatic antigen of adult D. repens worms activated the proangiogenic mechanism, cell proliferation and cell migration as well as formation of pseudo-capillaries in this in vitro human endothelial cell model. These processes could be related to the survival of adult D. repens in subcutaneous nodules in infected hosts.Graphical

Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits

Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits

Pro-Healing Nanomatrix-Coated Stent Analysis in an In Vitro Vascular Double-Layer System and in a Rabbit Model.

Cardiovascular stent technologies have significantly improved over time. However, their optimal performance remains limited by restenosis, thrombosis, inflammation, and delayed re-endothelialization. Current stent designs primarily target inhibition of neointimal proliferation but do not promote functional arterial healing (pro-healing) in order to restore normal vascular reactivity. The endothelial lining that does develop with current stents appears to have loose intracellular junctions. We have developed a pro-healing nanomatrix coating for stents that enhances healing while limiting neointimal proliferation. This builds on our prior work evaluating the effects of the pro-healing nanomatrix coating on cultures of vascular endothelial cells (ECs), smooth muscle cells (SMCs), monocytes, and platelets. However, when a stent is deployed in an artery, multiple vascular cell types interact, and their interactions affect stent performance. Thus, in our current study, an in vitro vascular double-layer (VDL) system was used to observe stent effects on communication between different vascular cell types. Additionally, we assessed the pro-healing ability and vascular cell interactions after stent deployment in the VDL system and in a rabbit model, evaluating the nanomatrix-coated stent compared to a commercial bare metal stent (BMS) and a drug eluting stent (DES). In vitro results indicated that, in a layered vascular structure, the pro-healing nanomatrix-coated stent could (1) improve endothelialization and endothelial functions, (2) regulate SMC phenotype to reduce SMC proliferation and migration, (3) suppress inflammation through a multifactorial manner, and (4) reduce foam cell formation, extracellular matrix remodeling, and calcification. Consistent with this, in vivo results demonstrated that, compared with commercial BMS and DES, this pro-healing nanomatrix-coated stent enhanced re-endothelialization with negligible restenosis, inflammation, or thrombosis. Thus, these findings indicate the unique pro-healing features of this nanomatrix stent coating with superior efficacy over commercial BMS and DES.

Accumulation of Deleterious Effects in Gastric Epithelial Cells and Vascular Endothelial Cells In Vitro in the Milieu of Helicobacter pylori Components, 7-Ketocholesterol and Acetylsalicylic Acid.

The Gastric pathogen Helicobacter pylori (HP) may influence the development of coronary heart disease (CHD). H. pylori induce reactive oxygen species (ROS), which transform cholesterol to 7-ketocholesterol (7-kCh), a CHD risk factor. Acetylsalicylic acid (ASA)—an Anti-aggregation drug used in CHD patients—may increase gastric bleeding and inflammation. We examined whether H. pylori driven ROS effects in the cell cultures of gastric epithelial cells (AGS) and vascular endothelial cells (HUVEC) progress in the milieu of 7-kCh and ASA. Cell cultures, exposed to 7-kCh or ASA alone or pulsed with the H. pylori antigenic complex—Glycine acid extract (GE), urease (UreA), cytotoxin associated gene A (CagA) protein or lipopolysaccharide (LPS), alone or with 7-kCh and ASA—were examined for ROS, apoptosis, cell integrity, interleukin (IL)-8, the activation of signal transducer, the activator of transcription 3 (STAT3), and wound healing. ASA and 7-kCh alone, and particularly in conjunction with H. pylori components, increased the ROS level and the rate of apoptosis, which was followed by cell disintegration, the activation of STAT3, and IL-8 elevation. AGS cells were unable to undergo wound healing. The cell ROS response to H. pylori components may be elevated by 7-kCh and ASA.

CD47 Binding on Vascular Endothelial Cells Inhibits IL-17-Mediated Leukocyte Adhesion.

To address the conflicting role of thrombospondin (TSP)-1 reported in acute and chronic pathologies, this study investigated the role of TSP-1 in regulating leukocyte recruitment and regulation of VCAM-1 expression using mouse models of uveitis. The spontaneously increased VCAM-1 expression and leukocyte adhesion in retinas of TSP-1-deficient mice suggested a TSP-1-mediated regulation of VCAM-1 expression. In a chronic uveitis model, induced by immunizing wild-type mice with specific interphotoreceptor retinoid-binding protein (IRBP) peptide, topically applied TSP-1-derived CD47-binding peptide significantly reduced the clinical disease course and retinal leukocyte adhesion as compared to the control peptide-treated group. In contrast, in LPS-mediated acute uveitis, TSP-1 deficiency significantly reduced the retinal leukocyte adhesion. The results of our in vitro study, using vascular endothelial cell (EC) cultures, demonstrate that unlike TNF-α, VCAM-1 expression induced by IL-17 is associated with a reduced expression of endogenous TSP-1. Such reduced endogenous TSP-1 expression in IL-17-stimulated ECs helps limit the CD36-mediated increased VCAM-1 expression, while favoring CD47-mediated inhibition of VCAM-1 expression and leukocyte adhesion. Thus, our study identifies TSP-1:CD47 interaction as a molecular pathway that modulates IL-17-mediated VCAM-1 expression, contributing to its anti-inflammatory effect in chronic inflammatory conditions.

Acute Antioxidant and Cytoprotective Effects of Caffeine in the Model of Neurovascular Injury by Oxidative Stress

Caffeine is commonly used in the neonatal intensive care units for respiratory disorders, but the effects of this medication in the neonatal cerebral circulation remain largely unexplored. We used newborn pigs as a clinically relevant model of the neonatal human brain to investigate the effects of caffeine on cerebral arterioles and primary neurovascular cells. First, we examined dose‐dependent cerebrovascular and systemic effects of water‐soluble anhydrous caffeine in anesthetized newborn pigs equipped with closed cranial windows. Caffeine administered systemically at therapeutic concentrations (20‐40 mg/kg i.v.) had no significant effects on diameter of pial arterioles, mean arterial blood pressure (MABP), and heart rate within 10‐60 min after the injection. When used at a higher concentration (70 mg/kg i.v.), caffeine produced dilation of pial arterioles (15‐20%) accompanied by an increased heart rate, without any effect on MABP. Topical application of caffeine (10‐5‐10‐3M) under the cranial window produced a dose‐dependent dilation of pial cerebral arterioles (15‐30%) that was fully reversed by a brief flushing with artificial cerebrospinal fluid. These in vivo data indicate that cerebral vascular cells are potential targets for caffeine. Next, we tested the hypothesis that caffeine exhibits cytoprotective properties in cerebral circulation during oxidative stress conditions. We used an in vitro model of acute oxidative stress‐induced neurovascular injury caused by excitotoxic and inflammatory conditions concomitant with epileptic seizures, asphyxia, and cerebral ischemia. Endothelial cells and cortical astrocytes are the key components of the neurovascular unit that are most vulnerable to oxidative stress injury. Primary cultures of cerebral vascular endothelial cells (CVEC) and cortical astrocytes from newborn pigs responded to pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) and excitotoxic glutamate (1‐2 mM) by a surge in reactive oxygen species (ROS) production leading to apoptosis. NADPH oxidase is the major ROS‐generating system in the neurovascular unit during inflammation and excitotoxicity. We examined the acute effects of caffeine on oxidative stress‐induced neurovascular cell death caused by TNF‐alpha and excitotoxic glutamate. Caffeine (0.1‐10 µM) inhibited NADPH oxidase activation, reduced overall ROS generation, and attenuated major key events of apoptosis (caspase‐3 activation, DNA fragmentation, and loss of cell‐cell contacts) in CVEC and astrocytes exposed to TNF‐a and glutamate. These data demonstrate, for the first time, that caffeine exhibits acute antioxidant and cytoprotective effects in cerebral vascular endothelial cells and astrocytes. Overall, endothelial and astrocytic components of the neurovascular unit represent target cells for caffeine cytoprotective therapy in the neonatal cerebral circulation during oxidative stress.

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior.

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte–derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.

Study on the Mechanism of Platelet-Released Clusterins Inducing Restenosis after Carotid Endarterectomy by Activating TLR3/NF-κb p65 Signaling Pathway.

This study aimed to explore the role of clusterin released by platelet aggregation in restenosis after carotid endarterectomy. 35 patients who underwent carotid endarterectomy due to carotid artery stenosis were enrolled in this study. They were admitted to the Third Affiliated Hospital of Qiqihar Medical University from January 2018 to January 2019. All the patients were divided into two groups: the restenosis group and the nonrestenosis group, according to the follow-up results within 12 months. Peripheral blood was collected on the first day, 6 months, and 12 months after operation. The expression of CLU in serum of plasma and platelet culture medium was detected by an ELISA experiment. The vascular endothelial cells were cultured in vitro with 100 ng/mL of human recombinant CLU added to the medium. Cell proliferation, migration, and invasion were detected by CCK8, scratch, and Transwell invasion tests. The expression level of TLR3 and NF-κb p65 proteins in cells was detected by western blot. TLR3 knockout plasmids in vascular endothelial cell lines were transfected. Cell proliferation and migration were detected by CCK8 and the scratch assay. The CLU content in peripheral blood plasma and supernatant of platelet culture medium was significantly higher in the restenosis group than that of the control group (p=0.003) 6 months after operation (p=0.047) and 12 months after operation (p=0.011). When CLU was added to vascular endothelial cell culture medium, the proliferation and migration were significantly enhanced. The TLR3/NF-κb p65 protein expression level in cells also significantly increased. After the transfection of TLR3 knockout plasmids into vascular endothelial cell lines, CLU cannot promote the proliferation and migration of vascular endothelial cells. Platelet-released clusterin can induce vascular endothelial cell proliferation and migration by activating the TLR3/NF-kb p65 signaling pathway, leading to carotid artery restenosis after carotid endarterectomy.

Association of 5-aminolevulinic acid with intraoperative hypotension in malignant glioma surgery

BackgroundUse of 5-aminolevulinic acid for photodynamic malignant tumor diagnosis reportedly causes intraoperative hypotension (systolic blood pressure < 70 mmHg) during urologic surgery. However, its association with intraoperative hypotension in malignant glioma surgery and underlying mechanisms has not yet been elucidated.. This study aimed to investigate whether 5-aminolevulinic acid administration is associated with intraoperative hypotension in malignant glioma surgery and explore the mechanisms of 5-aminolevulinic acid-induced hypotension in vitro. MethodsIn this retrospective multicenter cohort study, we investigated intracellular nitric oxide as a candidate mediator of hypotension in response to 5-aminolevulinic acid in vitro in human umbilical vein endothelial cell cultures. ResultsOf 142 patients, 94 underwent 5-aminolevulinic acid-guided surgery. Systolic blood pressure was significantly lower throughout surgery with 5-aminolevulinic acid administration. 5-Aminolevulinic acid administration was an independent risk factor for intraoperative hypotension according to multivariable logistic regression analysis (89% vs. 56%; odds ratio = 6.72, 95% confidence interval [2.05–22.1], P = 002). In subgroup analysis of the 5-aminolevulinic acid group, increasing age and use of renin-angiotensin system inhibitors had a synergistic effect with 5-aminolevulinic acid on decreased blood pressure. In the vascular endothelial cell culture study, 5-aminolevulinic acid induced a significant increase in intracellular nitric oxide generation. Conclusions5-Aminolevulinic acid administration was associated with intraoperative hypotension in malignant glioma surgery, with increasing age and use of renin-angiotensin system inhibitors boosting the blood pressure-lowering effect of 5-aminolevulinic acid. According to in vitro results, the low blood pressure induced by 5-aminolevulinic acid may be mediated by a nitric oxide increase in vascular endothelial cells.

The Application of a Bone Marrow Mesenchymal Stem Cell Membrane in the Vascularization of a Decellularized Tracheal Scaffold.

Airway stenosis is a common problem in the neonatal intensive care unit (NICU) and pediatric intensive care unit (PICU). A tissue-engineered trachea is a new therapeutic method and a research hotspot. Successful vascularization is the key to the application of a tissue-engineered trachea. However, successful vascularization studies lack a complete description. In this study, it was assumed that rabbit bone marrow mesenchymal stem cells were obtained and induced by ascorbic acid to detect the tissue structure, ultrastructure, and gene expression of the extracellular matrix. A vascular endothelial cell culture medium was added in vitro to induce the vascularization of the stem cell sheet (SCS), and the immunohistochemistry and gene expression of vascular endothelial cell markers were detected. At the same time, vascular growth-related factors were added and detected during SCS construction. After the SCS and decellularized tracheal (DT) were constructed, a tetrandrine allograft was performed to observe its vascularization potential. We established the architecture and identified rabbit bone marrow mesenchymal stem cell membranes by 14 days of ascorbic acid, studied the role of a vascularized membrane in inducing bone marrow mesenchymal stem cells by in vitro ascorbic acid, and assessed the role of combining the stem cell membranes and noncellular tracheal scaffolds in vivo. Fourteen experiments confirmed that cell membranes promote angiogenesis at gene level. The results of 21-day in vitro experiments showed that the composite tissue-engineered trachea had strong angiogenesis. In vivo experiments show that a composite tissue-engineered trachea has strong potential for angiogenesis. It promotes the understanding of diseases of airway stenosis and tissue-engineered tracheal regeneration in newborns and small infants.

A multiplexed immuno-sensor for on-line and automated monitoring of tissue culture protein biomarkers

Frequent on-line and automated monitoring of multiple protein biomarkers level secreted in the culture media during tissue growth is essential for the successful development of Tissue Engineering and Regenerative Medicine (TERM) products. Here, we present a low-cost, rapid, reliable, and integrable anion-exchange membrane-(AEM) based multiplexed sensing platform for this application. Unlike the gold-standard manual ELISA test, incubation/wash steps are optimized for each target and precisely metered in microfluidic chips to enhance selectivity. Unlike optical detection and unreliable visual detection for the ELISA test, which require standardization for every usage, the AEM ion current signal also offers robustness, endowed by the pH and ionic strength control capability of the ion-selective membrane, such that a universal standard curve can be used to calibrate all runs. The electrical signal is enhanced by highly charged silica nanoparticle reporters, which also act as hydrodynamic shear amplifiers to enhance selectivity during wash. This AEM-based sensing platform is tested with vascular protein biomarkers, Endothelin-1 (ET-1), Angiogenin (ANG) and Placental Growth Factor (PlGF). The limit of detection and three-decade dynamic range are comparable to ELISA assay but with a significantly reduced assay time of 1 h vs 7 h, due to the elimination of calibration and blocking steps. Optimized protocol for each target renders the detection highly reliable with more than 98% confidence. The multiplexed detection capability of the platform is also demonstrated by simultaneous detection of ET-1, ANG and PlGF in 40 μl of the vascular endothelial cell culture supernatants using three-membrane AEM sensor and the performance is validated against ELISA.

Tandem mass tag-based proteomic analysis reveals cathepsin-mediated anti-autophagic and pro-apoptotic effects under proliferative diabetic retinopathy

Proliferative diabetic retinopathy (PDR) is a severe complication of diabetes and can cause blindness. However, the available therapeutic modalities to PDR have unsatisfactory efficacies and incur adverse effects, which is due to the paucity in the understanding of pathogenic mechanisms responsible for the disease. In this study, tandem mass tag labeling technology combined with liquid chromatography and tandem mass spectrometry were utilized to identify differentially expressed proteins in vitreous humor of patients with rhegmatogenous retinal detachment and PDR. The data are available via ProteomeXchange with identifier PXD021788. Afterwards, the downregulated protein expression of Cathepsin B, D, and L was verified in vitreous and serum of another cohort. The gene expression profiling of the 3 cathepsins was confirmed in blood cells of an extra cohort. Furthermore, in high glucose (HG)-treated retinal vascular endothelial cell cultures recapitulating the cathepsin expression patterns, Cathepsin B or D downregulation mediated the HG-induced anti-autophagic and pro-apoptotic effects, thereby may contribute to vascular lesions under hyperglycemia. This study demonstrates previously undescribed expression patterns of cathepsins, reveals a novel cathepsin-involved pathogenic mechanism under PDR, and sheds light on potential therapeutic targets to this debilitating retinal disease.

Abstract 13295: Novel Experimental Therapeutics for COVID-19 Derived From a Nitric Oxide Donor

Urea transport protein B, the product of the gene SLC14A1 , facilitates transport of urea, water and urea analogues across cell membranes. SLC14A1 mRNA is overexpressed in human vascular endothelial cells in culture under hypoxic (1% oxygen) conditions compared with normoxia. This leads to transport of urea out of the endothelial cell and likely contributes to the reduction in eNOS (endothelial nitric oxide synthase) pathway activity in hypoxia. NO has antiviral activity. Novel compounds were developed by binding a urea-like moiety to the backbone of the generic agent isosorbide mononitrate, a well-known NO donor, to combat vascular endothelial dysfunction in COVID-19, a disease characterized by systemic hypoxia and inflammation due to SARS-CoV-2 infection. A study of drug-protein interactions was undertaken using in silico modelling. Novel compounds were studied against 9 key SARS-CoV-2 targets using Maestro, Schrödinger Suite software (Glide docking). Docking scores and intermolecular interactions within the target’s key binding amino acid residues were studied to compare investigational compounds and known antivirals. Several novel agents tested had a better Glide Score (a prediction of ligand affinity) against the papain-like protease (PL pro ) of SARS-CoV-2 compared with known antiviral drugs. PL pro is considered to be a primary target for therapeutic inhibition of the SARS viruses. The candidate compounds CR-305, CR-607, CR-510 and CR-605 were all superior to Remdesivir, GS-441524, Lopinavir, Boceprevir, and Ribavirin. Given the known direct antiviral action of NO and evidence of specific binding of these compounds to the PL pro of SARS-CoV-2 based on the in silico results, we conclude there is a high likelihood these novel compounds will prove to be of therapeutic value against COVID-19. CR-305 appears to have a higher affinity to SARS-CoV-2 than other antivirals as it sits firmly in the PL pro catalytic pocket and makes the most of key interactions with the catalytic pocket residues: Gly163, Asp164, Gln271 and Tyr264. These data call for a new focus on these novel antiviral agents as they appear to bind with an increased avidity to PL pro (compared with other known antivirals) while targeting delivery of NO to the SARS-CoV-2 virus in COVID-19.

Stromal cells cultivated from the choroid of human eyes display a mesenchymal stromal cell (MSC) phenotype and inhibit the proliferation of choroidal vascular endothelial cells in vitro

Mesenchymal stromal cells (MSC), with progenitor cell and immunological properties, have been cultivated from numerous vascularized tissues including bone marrow, adipose tissue and the corneal-limbus of the eye. After observing mesenchymal cells as contaminants in primary cultures of vascular endothelial cells derived from the choroidal tunic of the human eye, we investigated whether the choroid might also provide a source of cultured MSC. Moreover, we examined the effect of the choroidal stromal cells (Ch-SC) on the proliferation of freshly isolated choroidal vascular endothelial cells (ChVEC) in vitro. The phenotype of cultures established from five choroidal tissue donors was examined by flow cytometry and immunocytochemistry. The potential for mesenchymal cell differentiation was examined in parallel with MSC established from human bone marrow. Additional cultures were growth-arrested by treatment with mitomycin-C, before being tested as a potential feeder layer for ChVEC. The five unique cultures established from choroidal stroma displayed a phenotype consistent with the accepted definition for MSC (CD34−, CD45−, HLA-DR-, CD73+, CD90+, and CD105+), including the capacity for mesenchymal differentiation when cultivated under osteogenic, adipogenic and chondrogenic conditions. Growth-arrested Ch-SC inhibited the proliferation of ChVEC derived from five separate donors. Cultures of Ch-SC secreted approximately 40-fold higher concentrations of the anti-angiogenic factor pigment epithelium derived factor (PEDF/serpin F1) compared to the pro-angiogenic factor, vascular endothelial growth factor (VEGF), regardless of normal or growth-arrested state. Our results provide first evidence of a resident MSC cell type within the choroid and encourage investigation of new mechanisms for altering the growth of ChVEC.

Inhibitory effect of paeonol on aortic endothelial inflammation in atherosclerotic rats by up-regulation of caveolin-1 expression and suppression of NF-κB pathway

To explore whether paeonol can play an anti-atherosclerotic role by regulating the expression of aortic caveolin-1 and affecting NF-κB pathway, so as to inhibit the inflammatory response of vascular endothelium in atherosclerotic rats. The atherosclerotic model of rats was induced by high-fat diet and vitamin D_2. The primary culture of vascular endothelial cells(VECs) was carried out by tissue block pre-digestion and adherent method. The injury model of VECs was induced by lipopolysaccharide(LPS), and filipin, a small concave protein inhibitor, was added for control. HE staining was used to observe pathological changes of aorta. TNF-α, IL-6 and VCAM-1 were detected by ELISA. Western blot assay was used to detect the protein expression levels of caveolin-1 and p65 in aorta and VECs. The results showed that as compared with model group, paeonol significantly reduced aortic plaque area and lesion degree in rats, decreased the level of serum TNF-α, IL-6 and VCAM-1 in the rats and enhanced the relative expression level of caveolin-1, decreased p65 expression conversely(P&lt;0.05 or P&lt;0.01). In vitro, as compared to model group, paeonol obviously improved cell morphology, decreased the secretion of TNF-α, IL-6 and VCAM-1 in VECs, increased caveolin-1 expression, and decreased p65 protein expression(P&lt;0.05 or P&lt;0.01). Furthermore, filipin could reverse the effect of paeonol on expression of inflammatory factors and proteins(P&lt;0.05 or P&lt;0.01). According to the results, it was found that paeonol could play the role of anti-atherosclerosis by up-regulating the expression of caveolin-1 and inhibiting the activation of NF-κB pathway to reduce vascular inflammation in atherosclerotic rats.

OR14-06 Inhibition of Protein Kinase C-beta2 Phosphorylation Restores Nuclear Factor-Kappa B Activation and Improves Peripheral Arterial Disease in Diabetes

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessel outside the heart and most commonly affects the lower extremities. Diabetes (DM) accelerates the course and severity of PAD. Studies have shown that vascular endothelial cell NF-κB activity is required for post ischemic adaptation in experimental PAD. To better understand how DM contributes to PAD severity, we investigated the role of DM hyperglycemia in the activation of NF-κB under ischemic conditions. Induced ischemia in human vascular endothelial cell (HUVEC) cultures increased components of both canonical and non-canonical NF-κB pathways in the nucleus (p65 1.0 ± 0.1 vs 1.5 ± 0.2, p< 0.05, RelB 1.0 ± 0.1 vs 1.5 ± 0.2, p<0.01). Similarly, HUVEC acutely exposed to high glucose (HG, 25 mM) activated both canonical (IκB-α degradation, normal vs. HG 1.25 ± 0.02 vs 0.9 ± 0.0, p<0.05) and non-canonical NF-κB (p100 degradation, normal vs HG 0.021±0.001 vs 0.016±0.000, p<0.05) pathways. Prolonged exposure (3 days) of HUVEC to high glucose before ischemia resulted in impaired NF-κB activation as evident from decreased IκB phosphorylation (pIκB/IκB in normal glucose and ischemia 1.56 ± 0.22 vs 1.12 ± 0.35, p<0.01). To understand the signaling pathways underlying the ischemic activation of the NF-κB pathway, we used an array of antibodies to phosphoproteins involved in the inflammatory pathway. Compared to the lysates from cells grown in normal glucose, the lysates from cells grown in prolonged high glucose had dramatically increased phosphorylation of PKC-β2 (PKC-β2pSer661, 8-fold increase). To test whether this increase in PKC-β2pSer66 impairs NF-κB activation by ischemia, we treated HUVECS with prolonged high glucose exposure and ruboxystaurin (Rbx) (20 nM), an inhibitor of PKC-β2 phosphorylation, prior to ischemic exposure. Immunoblotting results confirmed that inhibition of PKC-β2 phosphorylation enhanced the ischemia induced NF-κB activation in HUVEC in this condition. We then tested the effect of Rbx on PKC-β2 phosphorylation and NF-κB activation in vivo in Akita mice, a model for type 1 diabetes. Consistent with our in vitro findings, in experimental PAD, NF-κB activity in the ischemic hind limb of Akita mice was significantly lower than those of the wild type (WT) mice as measured by IκB-α degradation (WT ischemic vs Akita ischemic; 0.04 ± 0.03 vs 0.10 ± 0.04 p<0.05). However, treatment of Akita mice with Rbx increased NF-κB activation in the ischemic hind limb (Akita ischemic 0.10 ± 0.04 vs ischemic+ Rbx 0.05 ± 0.02, p<0.05). Moreover, compared to the WT mice, the untreated Akita mice showed an impaired perfusion in the ischemic limbs (% perfusion recovery, WT vs Akita; 80.1 ± 10.3 vs 55.7 ± 10.1, p<0.05, n=5-8) that was improved in Rbx treated Akita mice (96.3 ± 2.3, p<0.01). Thus, hyperglycemic conditions increase PKC-β2pSer66 in endothelial cells attenuating salutary NF-κB activation contributing to poor PAD outcomes in DM.

Integrated On-Chip 3D Vascular Network Culture under Hypoxia.

We developed a portable device made of poly(dimethylsiloxane) (PDMS)/polymethylmethacrylate (PMMA) for long-term 3D cell culture of vascular endothelial cells for the development of a vascular network and evaluated the device under different transitions between normoxia and hypoxia with good optical accessibility. The combination of a nested reservoir device and a bicarbonate/ascorbate buffer system accomplished on-chip incubation with 4.91 ± 0.86% pO2 and 5.19 ± 1.70% pCO2 for up to 10 days. Seventy-two hours of normoxic incubation preceding hypoxic culture increased the cell viability, network formation, and size and stability of the resulting lumens compared with those completely maintained in normoxia for the same total duration. We employed different parameters of the network (e.g., total mesh area, total length, number of branches, among others) for the comparison of different oxygen treatments in the device. The differential effect of hypoxic conditions based on the maturity of the vessels may be used as an external factor to improve vascular development in vitro.

Cannabidiol (CBD) Inhibited Rhodamine-123 Efflux in Cultured Vascular Endothelial Cells and Astrocytes Under Hypoxic Conditions.

Despite the constant development of new antiepileptic drugs (AEDs), more than 30% of patients develop refractory epilepsy (RE) characterized by a multidrug-resistant (MDR) phenotype. The “transporters hypothesis” indicates that the mechanism of this MDR phenotype is the overexpression of ABC transporters such as P-glycoprotein (P-gp) in the neurovascular unit cells, limiting access of the AEDs to the brain. Recent clinical trials and basic studies have shown encouraging results for the use of cannabinoids in RE, although its mechanisms of action are still not fully understood. Here, we have employed astrocytes and vascular endothelial cell cultures subjected to hypoxia, to test the effect of cannabidiol (CBD) on the P-gp-dependent Rhodamine-123 (Rho-123) efflux. Results show that during hypoxia, intracellular Rho-123 accumulation after CBD treatment is similar to that induced by the P-gp inhibitor Tariquidar (Tq). Noteworthy, this inhibition is like that registered in non-hypoxia conditions. Additionally, docking studies predicted that CBD could behave as a P-gp substrate by the interaction with several residues in the α-helix of the P-gp transmembrane domain. Overall, these findings suggest a direct effect of CBD on the Rho-123 P-gp-dependent efflux activity, which might explain why the CBD add-on treatment regimen in RE patients results in a significant reduction in seizure frequency.

Poly(l-lactide-co-caprolactone)/tussah silk fibroin nanofiber vascular scaffolds with small diameter fabricated by core-spun electrospinning technology

A biomimetic vascular scaffold with excellent mechanical properties and biocompatibility is urgently needed, and designing such a scaffold is a challenge. The majority of vascular scaffolds have satisfactory biocompatibility, but poor mechanical properties. Herein, biodegradable poly(l-lactide-co-caprolactone) (PLCL) with biomimetic mechanical properties was used to prepare small-diameter (< 1.5 mm) PLCL/tussah silk fibroin (TSF) nanofiber vascular scaffolds grafted with TSF using the core-spun electrospinning technology. The morphology, biocompatibility, and mechanical properties of the PLCL/TSF nanofiber vascular scaffolds were characterized. The diameter of PLCL/TSF nanofibers was almost unchanged after grafting TSF with an average diameter of 408 ± 13 nm and a reduced water contact angle of 56 ± 8°. The protein adsorption was twice that of pure PLCL nanofiber vascular scaffolds. Remarkably, PLCL/TSF nanofiber vascular scaffolds displayed excellent mechanical properties. Their radial tensile strength and axial Young’s modulus were twice and more than three times those of traditional nanofiber vascular scaffolds, respectively. The biological properties of PLCL/TSF nanofiber vascular scaffolds were evaluated by in vitro culture of vascular endothelial cells. The scaffolds effectively promoted vascular endothelial cell adhesion and proliferation. The developed PLCL/TSF nanofiber vascular scaffolds are potentially valuable for use as readily available vascular grafts.