Endothelial Growth Medium Research Articles

Autologous human plasma in stem cell culture and cryopreservation in the creation of a tissue-engineered vascular graft

Previous work demonstrated the effectiveness of autologous adipose-derived stem cells (ASCs) as endothelial cell (EC) substitutes in vascular tissue engineering. We further this work toward clinical translation by evaluating ASC function after (1) replacement of fetal bovine serum (FBS) with autologous human plasma (HP) in culture and (2) cryopreservation. Human ASCs and plasma, isolated from periumbilical fat and peripheral blood, respectively, were collected from the same donors. ASCs were differentiated in endothelial growth medium supplemented with FBS (2%) vs HP (2%). Proliferation was measured by growth curves and MTT assay. Endothelial differentiation was measured by quantitative polymerase chain reaction, assessment of acetylated low-density lipoprotein uptake, and cord formation after plating on Matrigel (BD Biosciences, San Jose, Calif). Similar studies were conducted before and after cryopreservation of ASCs and included assessment of cell retention on the luminal surface of a vascular graft. ASCs expanded in HP-supplemented medium showed (1) similar proliferation to FBS-cultured ASCs, (2) consistent differentiation toward an EC lineage (increases in CD31, von Willebrand factor, and CD144 message; acetylated low-density lipoprotein uptake; and cord formation on Matrigel), and (3) retention on the luminal surface after seeding and subsequent flow conditioning. Cryopreservation did not significantly alter ASC viability, proliferation, acquisition of endothelial characteristics, or retention after seeding onto a vascular graft. This study suggests that (1) replacement of FBS with autologous HP--a step necessary for the translation of this technology into human use--does not significantly impair proliferation or endothelial differentiation of ASCs used as EC substitutes and (2) ASCs are tolerant to cryopreservation in terms of maintaining EC characteristics and retention on a vascular graft.

CD34/CD133 enriched bone marrow progenitor cells promote neovascularization of tissue engineered constructs in vivo

Vascularization is critical for 3D tissue engineered constructs. In large size implants the ingrowth of vessels often fails. The purpose of this study was to identify an easily accessible, clinically relevant cell source able to promote neovascularization in engineered implants in vivo and to establish an autologous culture method for these cells.MSCs (mesenchymal stem cells) and an endothelial progenitor containing cell (EPCC) population were obtained from human bone marrow aspirates. The expression of endothelial-markers, uptake of acetylated low density lipoprotein (acLDL) and tube-like structure formation capability of EPCCs were analyzed after expansion in endothelial growth medium or medium supplemented with autologous platelet lysate (PL). EPCCs were co-seeded with MSCs on hydroxyapatite-containing polyurethane scaffolds and then implanted subcutaneously in nude mice.Human EPCCs displayed typical characteristics of endothelial cells including uptake of acLDL and formation of tube-like structures on Matrigel™. In vivo, EPCCs cultured with PL triggered neovascularization. MSC/EPCC interactions promoted the maturation of newly formed luminal structures, which were detected deep within the scaffold and partly perfused, demonstrating a connection with the host vascular system.We demonstrate that this population of cells, isolated in a clinically relevant manner and cultured with autologous growth factors readily promoted neovascularization in tissue engineered constructs in vivo enabling a potential translation into the clinic.

Expression of BCR/ABL fusion gene in circulating endothelial cells from chronic myelogenous leukemia patients and its clinical significance

Several studies have shown that the tumor endothelial cells are different from the normal tissue endothelial cells. These tumor endothelial cells may contribute to tumor neo-vasculogenesis. This study was purposed to analyze the biologic features and determine the expression level of CD133 and BCR/ABL fusion gene in circulating endothelial cells (CEC) isolated from peripheral blood of CML patients, as well as to investigate the role of CEC in disease progression. Mononuclear cells were isolated from peripheral blood by density gradient centrifugation; CEC were sorted by MACS and harvested in the endothelial growth medium. The morphologic features of CEC were observed by microscopy, the cell growth rate was calculated by cell counting, and the cells were identified by immunofluorescence staining for the expression of CD31,CD34,VWF and CD133. The expression of BCR/ABL fusion gene was examined by FISH in 12 CML patients. The results indicated that the isolated CEC displayed the typical cobble-stone morphology. These cells could be identified by the positive immunofluorescence staining for CD31, CD34 and VWF, and showed more increased proliferative potential as compared to that of healthy donors. It was found that the positive rate of CD133 was 31.29% in CML patients, which was significantly different from that of healthy donors (P < 0.05). In 12 CML patients, CEC carried the same chromosome aberration as the leukemia cells (10.77%). Higher expression level of CD133 and BCR/ABL fusion gene positively correlated with progression of disease. It is concluded that the CEC may participate in invasion and angiogenesis in patients with CML and possibly correlate to the spreading and progression of the disease.

Endothelial differentiation of diabetic adipose-derived stem cells

BackgroundDiabetic (DM) patients frequently lack autologous vascular tissue required for revascularization procedures and dialysis access creation. We have developed a tissue-engineered graft that uses adipose-derived stem cells (ASC) as endothelial cell substitutes. Here, we compare DM versus nondiabetic (NDM) ASC in terms of isolation efficiency, proliferation, commitment toward endothelial lineage, and seeding onto the luminal surface of a graft. MethodsASC were isolated from liposuction specimens of vascular surgery patients. Proliferation was assessed by constructing growth curves over 14 d. ASC were differentiated in endothelial growth medium (EGM2). Endothelial commitment was assessed by measuring endothelial cell-specific gene expression (CD31, von Willebrand factor) and by cord formation on Matrigel. Finally, ASC were seeded onto a vascular scaffold, flow conditioned, and imaged with confocal microscopy. ResultsDiabetes did not alter ASC isolation efficiency (224,028 ± 20,231 cells/g adipose for DM (n = 53) versus 259,345 ± 15,441 cells/g adipose for NDM (n = 145; P = 0.21). Growth curves for DM (n = 6) and NDM (n = 6) also appeared similar. After culture in EGM2, upregulation of CD31 and von Willebrand factor message was observed in NDM; these markers were found within the primary cultures of DM but no upregulation was observed after culture in EGM2. Both groups exhibited similar cord formation on Matrigel and retention to vascular scaffolds. ConclusionsIsolation and proliferation studies suggest that adipose is a promising source of stem cells for tissue engineering in the DM population. The angiogenic potential of DM ASC appears intact; however, differences in acquisition of endothelial cell markers suggest that differentiation may be inhibited or delayed by diabetes.

Acidic preconditioning improves the proangiogenic responses of endothelial colony forming cells.

Acidosis is present in several pathological conditions where vasculogenesis takes place including ischemia, tumor growth and wound healing. We have previously demonstrated that acidosis induces human CD34+ cell apoptosis. Considering that endothelial colony-forming cells (ECFC) are a subpopulation of CD34+ cells and key players in vasculogenesis, in the present study we investigated the effect of acidosis on the survival and functionality of ECFC. Endothelial colony-forming cells obtained by differentiation of human cord blood CD34+ cells in endothelial growth medium-2 for 14-21 days were exposed at pH 7.4, 7.0 or 6.6. We found that acidosis failed to induce ECFC apoptosis and, although an early reduction in proliferation, chemotaxis, wound healing and capillary-like tubule formation was observed, once the medium pH was restored to 7.4, ECFC proliferation and tubulogenesis were augmented. Stromal cell derived factor-1 (SDF1)-driven migration and chemokine receptor type 4 surface expression were also increased. The maximal proangiogenic effect exerted by acidic preconditioning was observed after 6 h at pH 6.6. Furthermore, preconditioned ECFC showed an increased ability to promote tissue revascularization in a murine model of hind limb ischemia. Immunoblotting assays showed that acidosis activated AKT and ERK1/2 and inhibited p38 pathways. Proliferation rises triggered by acidic preconditioning were no longer observed after AKT or ERK1/2 inhibition, whereas p38 suppression not only mimicked but also potentiated the effect of acidosis on ECFC tubule formation abilities. These results demonstrate that acidic preconditioning greatly increases ECFC-mediated angiogenesis in vitro including ECFC proliferation, tubulogenesis and SDF1-driven chemotaxis and is a positive regulator of microvessel formation in vivo.

Abstract 332: Cell-Free Conditioned Media From Endothelial Progenitor Cells Enhance Endothelial Angiogenesis, Migration and Survival: Paracrine Implications for Cell Therapy in Pulmonary Arterial Hypertension

Background: Endothelial progenitor cells (EPCs) have been shown to be effective in the prevention of pulmonary arterial hypertension (PAH) in preclinical models. Our group has also demonstrated that xenotransplantation of human early EPCs in the rat monocrotaline (MCT) model prevented pulmonary arterial hypertension, despite the near complete loss of human cells from the lung after even 24 hours. Therefore, we hypothesize that the therapeutic effects of EPCs in PAH are mediated by a paracrine mechanism, which in turn, may enhance endothelial repair and integrity. Methods: EPCs were derived from human peripheral blood mononuclear cells obtained via leukapheresis by adherence to fibronectin and growth in endothelial growth medium (EGM-2MV). 24 hour conditioned medium from Day 6 EPCs (EPC-CM) was collected and concentrated by ultrafiltration prior to in vitro and in vivo studies. PAH was induced by a single intraperitoneal injection of (MCT) in nude athymic rats (150-200 g). EPC (5x106/kg) or EPC-CM from an equivalent number of cells were administered via jugular vein at day 3 after MCT and hemodynamics and echocardiography were assessed at day 24. Results: Incubation of human umbilical vein endothelial cells (ECs) with EPC-CM enhanced Matrigel capillary-like network formation by approximately 2-fold compared with serum-free media (p&lt;0.05). EPC-CM increased migration in the scratch wound-healing assay compared to control media (4-fold, p&lt;0.001). EPC-CM also significantly improved survival of HUVECs after serum deprivation (p&lt;0.05) in a dose-dependent manner. Injection of EPCs resulted in decreased right ventricular systolic pressure (p&lt;0.05) and improvement in pulmonary artery acceleration time (+28%, p&lt;0.05). However, infusion of an equivalent amount of EPC-CM as well as a 10-fold increased dose of EPC-CM failed to prevent PAH. Conclusions: Therefore, EPC-CM enhanced in vitro EC angiogenesis, migration, and survival, consistent with potential of a paracrine mechanism. However, in an in vivo preclinical model, EPC conditioned media did not prevent PAH, suggesting that intact cells act through mechanisms that may involve cell-cell signaling.

Neuron-derived Neurotrophic Factor Functions as a Novel Modulator That Enhances Endothelial Cell Function and Revascularization Processes

Strategies to stimulate revascularization are valuable for cardiovascular diseases. Here we identify neuron-derived neurotrophic factor (NDNF)/epidermacan as a secreted molecule that is up-regulated in endothelial cells in ischemic limbs of mice. NDNF was secreted from cultured human endothelial cells, and its secretion was stimulated by hypoxia. NDNF promoted endothelial cell network formation and survival in vitro through activation of Akt/endothelial NOS (eNOS) signaling involving integrin αvβ3. Conversely, siRNA-mediated knockdown of NDNF in endothelial cells led to reduction of cellular responses and basal Akt signaling. Intramuscular overexpression of NDNF led to enhanced blood flow recovery and capillary density in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of Akt and eNOS. The stimulatory actions of NDNF on perfusion recovery in ischemic muscles of mice were abolished by eNOS deficiency or NOS inhibition. Furthermore, siRNA-mediated reduction of NDNF in muscles of mice resulted in reduction of perfusion recovery and phosphorylation of Akt and eNOS in response to ischemia. Our data indicate that NDNF acts as an endogenous modulator that promotes endothelial cell function and ischemia-induced revascularization through eNOS-dependent mechanisms. Thus, NDNF can represent a therapeutic target for the manipulation of ischemic vascular disorders.

Role of vitamin D in endothelial repair mechanisms in systemic lupus erythematosus

BackgroundPatients with systemic lupus erythematosus (SLE) have an increased prevalence of cardiovascular disease (CVD). Vitamin D deficiency is common in SLE and is an independent risk factor for CVD in the general population. We have previously shown that low vitamin D is associated with arterial stiffness in patients with SLE. Myeloid angiogenic cells (MACs) have an important role in endothelial repair in animal models. Preliminary studies have shown impaired function of these cells in SLE. Myeloid cells respond to vitamin D in vitro and express functional vitamin D receptors. We aimed to assess whether vitamin D has beneficial effects on MAC function and thus endothelial repair. MethodsPeripheral blood mononuclear cells (PBMCs) from vitamin D deficient (<20 ng/mL) patients with SLE, or vitamin D replete (>20 ng/mL) healthy controls, were cultured for 7 days on fibronectin in endothelial growth media with or without 1,25(OH)2D3 (0·1–100 nM). For survival studies, the number of cells able to take up LDL was counted in random fields. Surface CD marker expression (CD14, CD68, CD86, CCR7, CD206) in MACs was determined with real-time quantitative PCR. Angiogenic factor secretion was measured with a Bio-Plex human angiogenesis suspension array (Bio-Rad Laboratories, CA, USA). Angiogenic function of MACs was determined with human aortic endothelial cells in a Matrigel angiogenesis model. Data were analysed using linear regression, paired t tests, and one-way ANOVA where appropriate. FindingsMACs expressed high levels of CD206, and low CCR7, compared with PBMCs, consistent with an M2 macrophage phenotype. Phagocytosis was demonstrated by MAC uptake of fluorescein isothiocyanate-labelled beads. MAC secreted interleukin 8 (mean 13·9 ug/mL, SD 20·1), vascular endothelial growth factor (37·2 pg/mL, 26·1), hepatocyte growth factor (1414 pg/mL, 568), leptin (209 pg/mL, 144), and platelet-derived growth factor (564 pg/mL, 365) but not granulocyte-colony stimulating factor or angiopoetin. 1,25(OH)2D3 (0·1–100 nM) increased the number of MACs from patients with SLE after 8 days in those with a low number (<150 per field) at baseline (p=0·037) in a dose-dependent manner. This effect was replicated in healthy MACs treated with 0·1 ng/mL interferon-alpha2b (p≤0·001). Expression of all macrophage markers was reduced by 1,25(OH)2D3 (10 nM) but the M2 marker CD206 was preferentially reduced compared with M1 markers CCR7 (p=0·008) and CD86 (p=0·043). Conditioned media from vitamin D (10 nM) treated MACs increased endothelial tubule network density in the angiogenesis model (mean relative density 1·43 [SD 0·12] vs 1·66 [0·15], p=0·012) whereas vitamin D alone did not (0·96 [0·10], p=0·582). Vitamin D did not, however, increase the concentration of any of the angiogenic factors measured in the Bio-Plex array. InterpretationResults from this study suggest that MACs are a population of M2 macrophages with angiogenic capacity in vitro, and that vitamin D both increases the number of MACs and changes the surface maker profile in SLE. The angiogenic capacity of MACs was increased by vitamin D ex vivo, but this was not due to changes in expression of common angiogenic factors. Further work will identify the mechanism by which vitamin D might augment endothelial repair in patients with SLE. FundingUK Medical Research Council.

Hydromorphone attenuates intercellular adhesion molecule-1 expressions induced by lipopolysaccharide on HCT-116 human colon cancer cells.

Resently, Min et al. [1] showed that morphine attenuated the stimulant effect of lipopolysaccharide (LPS). LPS is especially abundant in the colon. LPS stimulates the synthesis and secretion of various cytokines and other pro-inflammatory mediators such as interleukin-1β (IL-1β), IL-8, IL-6, and vascular endothelial growth factor [2]. These cytokines induced endothelial adhesion molecule expressions through a nuclear factor κB (NF-κB)- dependent pathway. LPS has been shown to be a stimulant of vascular metastatic processes as well,especially in colon cancer. Min et al. [1] also demonstrated that the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM- 1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were attenuated on human umbilical vascular endothelial cells (HUVECs) when LPS was co-administered with morphine. However, the adhesion ratio of cancer cells to HUVECs was not demonstrated at that time. Hydromorphone is a semi-synthetic opioid that is a hydrogenated ketone of morphine. Hydromorphone was selected in this study because it had a longer duration of action and less intense adverse effects such as pruritus and nausea than morphine [3,4]. Due to these potential advantages, hydromorphone is widely used in cancer patients for relief of pain. Therefore, in this study, we investigated the adhesion ratio of colon cancer cells to LPS-stimulated HUVECs and whether co-administration with hydromorphone attenuated the adhesion of colon cancer cells to HUVECs. Four different growth conditions were used in this study: (1) HUVECs incubated in endothelial growth medium without growth factors (the control group), (2) HUVECs incubated with the supernatant from LPS-stimulated HCT 116 cells (the LPS-SUP group), (3) HUVECs incubated with the supernatant from HCT 116 cells that were not stimulated with LPS (the SUP group), and (4) hydromorphone co-administered to HCT 116 cells during stimulation with LPS (the HYDRO + LPS-SUP group). The LPS-SUP, the HYDRO + LPS-SUP groups were prepared following a previously published experimental protocol (Fig. 1A) [1]. Hydromorphone was supplemented at three different concentrations, 0.1, 1, and 10 µM [5]. The surface expression of adhesion molecules was measured by flow cytometry (Facsflow from BD Biosciences, San Jose, CA, USA) by using an ICAM-1 specific fluorescein isothiocyanate-labeled antibody. And we demonstrated that the adhesion ratios of HCT 116 cancer cells to HUVECs by using carboxy-flourescein HCT 116 cancer cells. Adhesions with HUVECs were counted by optical microscopy. As result, the adhesion ratios of HCT 116 cancer cells to HUVECs were significantly lower in the HYDRO + LPSSUP group than in the other groups (Fig. 1B). Therefore, we concluded hydromorphone might reduce the adhesion of cancer cells to the vascular wall by reducing the expression of adhesion molecules on endothelial cells. Fig. 1 (A) Experimental protocol. For the control group, HUVECs were incubated in growth factor-free endothelial medium. For the LPS-Sup group, HUVECs were incubated with the supernatant of LPSstimulated human HCT 116 colon carcinoma cells. For the HYDRO + LPS-SUP ... In this study, we found evidence, which suggests that hydromorphone may have anti-tumor effects similar to morphine. Considering the benefits of hydromorphone over morphine, these results show that hydromorphone might be the opioid analog of choice for treatment of cancer pain. Although this study has the limitation of being an in vitro analysis, we concluded that hydromorphone might reduce LPS-stimulated metastasis in colon cancer by attenuating the expression of ICAM-1 on endothelial cells. In the future, we plan to evaluate the anti-metastatic effect of hydromorphone using both in vivo and in clinical approaches.

132 Viability of human umbilical vein endothelial cells ( HUVEC) in the presence of permeating and non-permeating cryoprotectants after interrupted cooling

Graded freezing (interrupted slow cooling without hold time) and two-step freezing (interrupted rapid cooling with hold time) are two types of interrupted cooling protocols used to investigate freeze-thaw responses of cells and the actions of cryoprotectants. Graded freezing can be used to investigate progressive cell injury during slow cooling and two-step freezing can be used to examine cell injury during interrupted rapid cooling with hold time. Cell response using graded freezing has been studied using fast osmotically-responding cells such as fibroblasts or TF-1 cells but not with a slow osmotically-responding cell such as human umbilical vein endothelial cells (HUVEC). Cell response using two-step freezing has been studied for a variety of cell types in the presence or absence of permeating cryoprotectants, however, for only one cell type (fast osmotically-responding fibroblasts) has the effect of two-step freezing with both permeating dimethyl sulfoxide (Me 2 SO) and non-permeating hydroxyethyl starch (HES) been studied. In this study, HUVEC were subjected to graded and two-step freezing after which samples were rapidly thawed and assessed for cell viability using a dual fluorescence nucleic acid stain consisting of Syto®13 and ethidium bromide. HUVEC membrane integrity was measured throughout the interrupted-cooling profiles in (i) cryoprotectant-free endothelial growth medium (EGM), and measured throughout the two-step freezing protocol in the presence of (ii) permeating cryoprotectant (5% Me 2 S0), and (iii) non-permeating cryoprotectant (5% HES). Graded freezing resulted in higher viabilities of HUVEC after plunge-thaw compared to two-step freezing, opposite to what was observed with fast-osmotically responding TF-1 cells. Two-step freezing in the presence of 5% Me 2 SO resulted in higher viabilities of HUVEC after plunge-thaw as compared to the same cooling profile in 5% HES or EGM. Two-step freezing with HES did not result in higher viability of HUVEC than without cryoprotectant, whereas fibroblasts have higher reported viability with 5% HES than without cryoprotectant. Source of funding: University of Alberta, travel awards: Cryo2007 fund and Bell McLeod Education Fund; CIHR MOP 86492, and OGBF INO 126778 from the INMHA and NSERC of Canada; JAW Elliott holds a Canada Research Chair in Thermodynamics. Conflict of interest: None declared. asultani@ualberta.ca

CD166+CD34+ Cells Exhibit Marked Functional Differences During Fetal and Adult Life

ALCAM/CD166 is expressed from the onset of hematopoiesis in the yolk sac and in a variety of hematopoietic tissues throughout ontogeny. Both hematopoietic and stromal cells in the AGM region, fetal liver, and fetal and adult marrow express this molecule. CD166 double knockout mice are viable and fertile, without any major blood defects, but their microenvironment and hematopoietic stem cells (HSC) exhibit deficiencies in their ability to support and engraft long-term, respectively. In order to further study the role of CD166 in hematopoiesis, we characterized, during ontogeny, the origin, function, and sub-populations of CD166+ cells in different hematopoietic organs. To this end, we used flow cytometry, confocal microscopy, and colony-forming assays to analyze human fetal liver (FL) at 18 and 20 gestational weeks (gw), bone marrow (BM) from 10 to 20gw, and adult BM. Flow cytometric analysis of FL at 18 and 20gw demonstrated that although 3±1% of liver cells at this age were CD166+, less than 1% were endothelial CD166+CD34+ cells, and no hematopoietic CD166+CD34+CD45+ cells were detected. In fetal BM, CD166+ cells emerged after 15gw, expressed Flk-1 and CD34, and their percentage increased progressively with gestational age. Flow cytometric analysis at 20gw showed that 1.5±1% of cells were CD166+CD34+, of which 93±0.5% were CD45+. Human adult BM contained 2±0.5% of CD166+CD34+ cells, of which only 66±0.6% were CD45+. In order to functionally characterize CD166+CD34+ cells from adult and fetal BM (20gw), we plated these cells in mesenchymal cell growth medium (MSCGM), endothelial growth medium (EGM-2), and complete methylcellulose (MC). MSCGM and EGM-2 did not support growth and expansion of fetal or adult CD166+CD34+ cells. Quantification of the hematopoietic colony-forming potential of these cells demonstrated that fetal CD166+CD34+ generated/1000 cells: 8±1 Blast; 27±2 CFU-Mix; 51±10 CFU-GM; and 0 BFU-E, while adult CD166+CD34+ gave rise to 7 Blast; 17 CFU-Mix; 36 CFU-GM; and 10 BFU, demonstrating differences in the hematopoietic potential of these cells. Furthermore, at day 12 of MC culture, adherent stromal cells were detected underneath MC, but only in cultures from fetal BM. Characterization of these cells by flow cytometry showed that more than 90±2% of these cells were CD166+CD9+, and 30±5% were CD146+. Furthermore, these stromal CD166+ cells did not express CD34, CD45, CD31, CD209, or CD6. Immunostaining demonstrated that the CD166+CD146+ cells expressed osteopontin and Stro-1. A CD41+CD68+ population of cells was also found.In conclusion, we found that, during ontogeny, expression of CD166 in FL is not associated with hematopoietic cells. In the BM, expression of CD166 is associated with CD34 and Flk2, and its expression on HSC commences later in gestation, suggesting that these cells either arise in the BM, or that CD166 expression is triggered at a certain time point in gestation, probably associated with rapid proliferation of HSC during this time period. Furthermore, we demonstrated that CD34+CD166+ cells from 20gw fetal BM contain hematopoietic and stromal cell populations, while adult BM-derived CD34+ CD166+ cells are exclusively hematopoietic. Disclosures:No relevant conflicts of interest to declare.

Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton’s jelly-derived mesenchymal stem cells

IntroductionHuman Wharton’s jelly (WJ) has become a preferred source of mesenchymal stem cells (MSCs) whose clinical applications are limited by the use of adequate xeno-free (XF), in vitro manipulation conditions. Therefore, the objective of our study was to characterize WJ-derived MSCs (WJ-MSCs), isolated by different methods and cultured in a commercially available, MSC XF medium, not least of all by investigating their endothelial differentiation capacity.MethodsWJ explants and enzymatically dissociated WJ cells were cultured in a defined, XF medium for MSCs. Adherent cells at passages 2 and 5 were characterized as MSCs by flow cytometry, MTT, real-time quantitative reverse transcription PCR, and functional multipotent differentiation assays. The endothelial differentiation capacity of MSCs isolated and expanded until passage 2 in the MSC XF medium, and then subcultured for five passages in a commercially available endothelial growth medium (group A), was assessed over serial passages, as compared to adherent WJ-derived cells isolated and expanded for five consecutive passages in the endothelial medium (group B).ResultsThe MSC phenotype of WJ explant- and pellet-derived cells, isolated and expanded in the MSC XF medium, was proven based on the expression of CD44/CD73/CD90/CD105 surface markers and osteo-/adipo-/chondrogenic multipotent differentiation potential, which differed according to the isolation method and/or passage number. Upon exposure to endothelial differentiation cues, cells belonging to group A did not exhibit endothelial cell characteristics over serial passages; by contrast, WJ pellet-derived cells belonging to group B expressed endothelial characteristics at gene, protein and functional levels, potentially due to culture conditions favoring the isolation of other stem/progenitor cell types than MSCs, able to give rise to an endothelial progeny.ConclusionsThe use of defined, MSC XF media for isolation and expansion of human WJ-MSCs is a prerequisite for the establishment of their real endothelial differentiation capacity, as candidates for clinical therapy applications. Thus, the standardization of WJ-MSCs isolation and culture expansion techniques in defined, MSC XF media, for their accurate characterization, would be a priority in the stem cell research field.

AMP-dependent Kinase Inhibits Oxidative Stress-induced Caveolin-1 Phosphorylation and Endocytosis by Suppressing the Dissociation between c-Abl and Prdx1 Proteins in Endothelial Cells

Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H2O2 exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

IntroductionEndothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37°C for up to 20h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4°C regardless of the preservation solution. At temperatures above 20°C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20°C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20°C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20°C. Only ECGM maintains HUVEC viability at temperatures above 20°C.

THU0067 Endothelial-to-Mesenchymal Transition in Cultured Human Dermal Microvascular Endothelial Cells: Effects of Endothelin-1

BackgroundEndothelial/microvascular damage and myofibroblast activation are considered early events in the development of connective tissue diseases, such as systemic sclerosis (SSc) (1). Recent studies showed that vascular endothelial cells may...

206 ASSESSMENT OF GAP JUNCTION COMMUNICATION BETWEEN HUMAN UMBILICAL ENDOTHELIAL CELLS AND MONOCYTES IN RESPONSE TO TUMOUR NECROSIS FACTOR (TNF-Α)

IntroductionAtherosclerosis is a chronic inflammatory disease characterised by accumulation of monocytic cells and lipids within the sub-endothelial space. This is facilitated by monocyte (MO) trans-endothelial (EC) migration early in atherosclerosis...

Decreased Telomerase Activity Sensitizes the Resistance Vasculature to Stress‐Induced Endothelial Dysfunction

Telomerase, an established modulator of senescence and tissue aging is traditionally described as a nuclear enzyme crucial in telomere maintenance. Recently, telomerase has been shown to co‐localize with mitochondria and to be a novel regulator of mitochondrial derived reactive oxygen species (mtROS). Previous data show that decreased that telomerase activity (TA) increases mtROS production in human and mouse resistance vessels. We hypothesized that TA has an endothelial protective role under conditions of external stress. Mouse mesenteric arteries (~200 μm) were isolated and incubated in endothelial growth medium with a subconstrictor dose of angiotensin II (A II; 10 nM) or vehicle (V) with and without telomerase inhibitor BIRB 1532 (BIRB; 10 μm) for 15–20 h. Flow‐mediated dilation (FMD) was studied in vitro (pressure gradient 0–100 cm H2O). Values expressed as % Max dilation at 50 cmH2O ± SEM; * p &lt; 0.05 vs. control one way ANOVA RM. A II caused a trend toward a decrease in FMD (V 57.0 ±9.2 vs. A II 42.8±13.6; n≥4). BIRB did not decrease FMD, but changed the mediator from NO to mtROS (catalase and rotenone inhibitable 1.5*±6.8; 11.9*±11.1). Co‐incubation with A II significantly reduced FMD (BIRB 59.1±10.6 vs. + A II 16.3*±7.4 n≥4). These data indicate that telomerase protects the microvascular endothelium from mtROS‐induced vascular dysfunction under stress.

Human chorionic gonadotropin controls luteal vascular permeability via vascular endothelial growth factor by down-regulation of a cascade of adhesion proteins

To study the functional interactions of junctional proteins acting as regulators of vascular permeability in the human corpus luteum. We investigated the role of vascular endothelial (VE)-cadherin, nectin 2, and claudin 5 as controllers of vascular endothelial cell permeability. Performing immunohistochemical dual staining, we colocalized the above-mentioned proteins in the human corpus luteum. Not applicable. Not applicable. Not applicable. Using a granulosa-endothelial coculture system, we revealed that hCG-treatment down-regulates VE-cadherin, nectin 2, and claudin 5 in endothelial cells via vascular endothelial growth factor (VEGFA). Furthermore, the interaction of VE-cadherin, nectin 2, and claudin 5 was investigated by silencing these proteins that perform siRNA knockdown. Interestingly, knockdown of VE-cadherin and claudin 5 induced a decrease of the respective other protein. This down-regulation was associated with changed rates of vascular permeability: hCG induced a VEGFA-dependent down-regulation of VE-cadherin, nectin 2, and claudin 5, which increased the endothelial permeability in the coculture system. Furthermore, knockdown of VE-cadherin, nectin-2, and claudin 5 also resulted in a consecutive increase of endothelial permeability for each different protein. These results demonstrate for the first time that VE-cadherin, nectin 2, and claudin 5 are involved in the regulation of vascular permeability in a mutually interacting manner, which indicates their prominent role for the functionality of the human corpus luteum.

Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.

Astaxanthin Decreases Inflammatory Biomarkers Associated with Cardiovascular Disease in Human Umbilical Vein Endothelial Cells

Oxidative stress and inflammation are strongly linked to the development of cardiovascular disease (CVD). As a potent antioxidant, astaxanthin may downregulate inflammation-associated factors involved in endothelial dysfunction and CVD progression. We studied the possible protective effects of astaxanthin against endothelial dysfunction in human umbilical vein endothelial cells (HUVEC) induced with hydrogen peroxide (H2O2). Cells were pre-incubated with 0, 0.01, 0.1, or 1.0 μmol/L astaxanthin for 48 h, then oxidative stress induced with 100 μmol/L H2O2 overnight. Uptake kinetics of astaxanthin showed a time-dependent uptake of astaxanthin (1.0 μmol/L) by HUVEC over the 48 h incubation period. Oxidative stress induction with H2O2 in HUVEC decreased intracellular antioxidant activity and increased the production of inflammatory biomarkers and reactive oxygen species (ROS). In contrast, pre-incubation of HUVEC with astaxanthin prior to H2O2 stress increased (P < 0.05) superoxide dismutase activity and decreased ROS, prostaglandin E2, leukotriene B4, NO, IL-8, and IFN- production. Pre-treatment with astaxanthin also downregulated the transcriptional activation of NF-κB and activator protein-1, thereby inhibiting downstream production of inflammatory mediators and cytokines. Therefore, astaxanthin protects against factors initiated by H2O2 addition, likely by scavenging ROS required for transcriptional activation and inhibiting the production of inflammatory biomarkers involved in endothelial dysfunction and CVD. Abbreviations: AP-1: activator protein-1; CVD: cardiovascular disease; CS: culture supernatant; EGM-2: endothelial growth medium-2; GPx: glutathione peroxidase; H2O2: hydrogen peroxide; HUVEC: human umbilical vein endothelial cell; LTB4: leukotriene B4; PGE2: prostaglandin E2; ROS: reactive oxygen species; SOD: superoxide dismutase; THF: tetrahydrofuran.