Endothelial Cell Growth Medium Research Articles

Isolation, culture and identification of bone marrow-derived endothelial progenitor cells in mice

Objective To explore a practical and feasible method for isolation, culture and identification of mouse bone marrow endothelial progenitor cells(EPCs). Methods Bone marrow-derived mononuclear cells isolated by density gradient centrifugation were cultured in endothelial cell growth medium-2 MV medium. Growth and morphological changes of the cells were observed under inverted microscopy. Cell proliferation was observed by cell counting kit-8 assay. Surface markers of the EPCs were detected by flow cytometry. Angiogenic tube formation was determined by Matrigel tube formation assay. Fluores cein isothiocyanat e-ulex europaeus agglutinin-1(FITC-UEA-1) binding and Dil-Ac-LDL uptake capabilities were observed by fluorescent microscopy. Results In the early stage, the cells were round and spindle-shaped after induced culture for 4 days. After 7 days, the cells grew in colony arrangement and gradually increased in number. After 14 days, the cells were differently shaped, such as short shuttle and triangle. After 21 days, the typical paving stone appearance of the cells was observed. The cells were positive for endothelial markers in flow cytometry: CD34+ (84.3%), vascular endothelial growth factor receptor 2+ (74.1%), but CD45+ (4.04%). The cells were capable of forming capillary-like tubes, up-taking Dil-Ac-LDL and binding FITC-UEA-1 in Matrigels. Conclusions A reliable method for isolation, culture and identification of mouse bone marrow EPCs may be improved on the basis of previous experiences. Since the EPCs obtained by this method may be capable of good proliferation, large in number, and stable in biological characteristics, they can serve as ideal seed cells for related subsequent studies. Key words: Bone marrow; Cell culture techniques; Monocytes; Endothelial progenitor cells; Cell identification

The Role of Human Umbilical Vein Endothelial Cells in Osteogenic Differentiation of Dental Follicle-Derived Stem Cells in In Vitro Co-cultures.

Angiogenesis and vascularization are essential for the growth and survival of most tissues. Engineered bone tissue requires an active blood vessel network for survival and integration with mature host tissue. Angiogenesis also has an effect on cell growth and differentiation in vitro. However, the effect of angiogenic factors on osteoprogenitor cell differentiation remains unclear. We studied the effects of human umbilical vein endothelial cells (HUVECs) on osteogenic differentiation of dental follicle-derived stem cells (DFSCs) in vitro by co-culturing DFSCs and HUVECs. Cell viability, based on metabolic activity and DNA content, was highest for co-cultures with a DFSC/HUVEC ratio of 50:50 in a 1:1 mixture of mesenchymal stem cell growth medium and endothelial cell growth medium. Osteoblastic and angiogenic phenotypes were enhanced in co-cultures with a DFSC/HUVEC ratio of 50:50 compared with DFSC monocultures. Increased expression of angiogenic phenotypes and vascular endothelial growth factor (VEGF) levels were observed over time in both 50:50 DFSC/HUVEC co-cultures and DFSC monocultures during culture period. Our results showed that increased angiogenic activity in DFSC/HUVEC co-cultures may stimulate osteoblast maturation of DFSCs. Therefore, the secretion of angiogenic factors from HUVECs may play a role in the osteogenic differentiation of DFSCs.

Myricetin Inhibits Angiogenesis by Inducing Apoptosis and Suppressing PI3K/Akt/mTOR Signaling in Endothelial Cells.

BackgroundMyricetin has been shown to possess potential antiangiogenic effects in endothelial cells. However, the underlying mechanisms are not fully understood. Therefore, we evaluated its antiangiogenic effects in human umbilical vascular endothelial cells (HUVECs).MethodsHUVECs were cultured in endothelial cell growth medium-2 to induce proliferation and angiogenesis and treated with different doses of myricetin (0.25, 0.5, and 1 μM) for 24 hours. Cell proliferation was analyzed by the MTT and lactate dehydrogenase release assays; angiogenesis was determined by the tube formation assay. In addition, cell signaling pathways related to angiogenesis were investigated by Western blotting.ResultsMyricetin induced apoptosis and procaspase-3 cleavage though the induction of reactive oxygen species (ROS). It significantly inhibited cell migration, tube formation, and PI3K/Akt/mTOR signaling in HUVECs.ConclusionsMyricetin exerts antiangiogenic effects by inducing ROS-mediated apoptosis and inhibiting PI3K/Akt/mTOR signaling in HUVECs.

Isolation of Rickettsia amblyommatis in HUVEC line.

Rickettsia amblyommatis, formerly named Rickettsia amblyommii and ‘Candidatus Rickettsia amblyommii’ is an intracellular bacterium belonging to the spotted fever group Rickettsia. It is highly prevalent in Amblyomma americanum and in other Amblyomma spp. throughout the Western Hemisphere. R. amblyommatis has been cultivated in chicken fibroblast, primary embryonated chicken eggs, Vero cells and arthropod-derived cells. Because of the affinity of rickettsiae to invade vascular endothelial cells, we tried to isolate R. amblyommatis from a nymph of Amblyomma cajennense s.l. collected in Saltillo (Coahulia, Mexico) using human umbilical vein endothelial cells (HUVEC). One tick half was analysed by ompA PCR and was found to be positive for R. amblyommatis. The other half was selected for in vitro culture of Rickettsia spp. It was triturated in 1 mL of endothelial cell growth medium with 1% antibiotic–antimycotic solution, and the homogenate was inoculated into a HUVEC line. Culture was maintained at 33°C in endothelial cell growth medium plus 2 mM l-glutamine and 2% fetal calf serum, with 5% CO2. The medium was changed weekly. Culture was checked by Gimenez stain for Rickettsia-like intracellular organisms. After 48 days of incubation, Rickettsia-like organisms were observed in HUVEC. PCR assays and sequencing of ompA gene in the culture suspension showed 100% identity with R. amblyommatis. This isolate was successfully established in HUVEC, and it has been deposited in the collection of the Center of Rickettsioses and Arthropod-Borne Diseases, Infectious Diseases Department, Hospital San Pedro–Center of Biomedical Research from La Rioja, Logroño, Spain. The HUVEC line is a useful tool for the isolation of R. amblyommatis.

Intratracheal transplantation of endothelial progenitor cells attenuates smoking-induced COPD in mice.

BackgroundEndothelial progenitor cells (EPCs) might play a protective role in COPD. The aim of this study was to investigate whether intratracheal allogeneic transplantation of bone-marrow-derived EPCs would attenuate the development of smoking-induced COPD in mice.MethodsIsolated mononuclear cells from the bone marrow of C57BL/6J mice were cultured in endothelial cell growth medium-2 for 10 days, yielding EPCs. A murine model of COPD was established by passive 90-day exposure of cigarette smoke. On day 30, EPCs or phosphate-buffered saline alone was administered into the trachea. On day 90, EPCs or 30 μL phosphate-buffered saline alone was administered into the trachea, and on day 120, inflammatory cells, antioxidant activity, apoptosis, matrix metalloproteinase (MMP)-2, and MMP-9 were measured.ResultsAfter EPC treatment, the lung function of the mice had improved compared with the untreated mice. Mean linear intercept and destructive index were reduced in the EPCs-treated group compared with the untreated group. In addition, the EPCs-treated mice exhibited less antioxidant activity in bronchoalveolar lavage fluid compared with the untreated mice. Moreover, decreased activities of MMP-2, MMP-9, and TUNEL-positive cells in lung tissues were detected in EPCs-treated mice.ConclusionIntratracheal transplantation of EPCs attenuated the development of pulmonary emphysema and lung function disorder probably by alleviating inflammatory infiltration, decelerating apoptosis, inhibiting proteolytic enzyme activity, and improving antioxidant activity.

Outgrowing endothelial and smooth muscle cells for tissue engineering approaches

In recent years, circulating progenitors of endothelial cells and smooth muscle cells were identified in the peripheral blood. In our study, we evaluated the utilization of both cell types isolated and differentiated from peripheral porcine blood in terms for their use for tissue engineering purposes. By means of density gradient centrifugation, the monocyte fraction from porcine blood was separated, split, and cultivated with specific culture media with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 for the differentiation of endothelial cells or smooth muscle cells. Obtained cells were characterized at an early stage of cultivation before the first passage and a late stage (fourth passage) on the basis of the expression of the antigens CD31, CD34, CD45, nitric oxide synthase, and the contractile filaments smooth-muscle alpha-actin (sm-alpha-actin) and smoothelin. Functional characterization was done based on the secretion of nitric oxide, the formation of a coherent monolayer on polytetrafluoroethylene, and capillary sprouting. During cultivation in both endothelial cell growth medium-2 and smooth muscle cell growth medium-2, substantially two types of cells grew out: early outgrown CD45-positive cells, which disappeared during further cultivation, and in 85% (n = 17/20) of cultures cultivated with endothelial cell growth medium-2 colony-forming late outgrowth endothelial cells. During cultivation with smooth muscle cell growth medium-2 in 80% (n = 16/20) of isolations colony-forming late outgrowth smooth muscle cells entered the stage. Cultivation with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 had selective effect on the late outgrown cells to that effect that the number of CD31-positive cells increased from 34.8% ± 13% to 83.9% ± 8% in cultures cultivated with endothelial cell growth medium-2 and the number of sm-α-actin+ cells increased from 52.6% ± 18% to 88% ± 5% in cultures cultivated with smooth muscle cell growth medium-2, respectively. Functional analyses revealed significantly higher levels of nitric oxide secretion, endothelialization capacity, and capillary formation in not expanded cultures cultivated with endothelial cell growth medium-2 in comparison to later stages of cultivation and mature aortic cells. Blood seems to be a reliable and feasible source for the isolation of both endothelial and smooth muscle cells for application in tissue engineering approaches. Whereas, early co-cultures of early and late outgrowth cells provide functional advantages, the differentiation of cells can be directed selectively by the used culture medium for the expansion of highly proliferative late outgrowth endothelial cells and late outgrowth smooth muscle cells, respectively.

Isolation and Primary Culture of Mouse Aortic Endothelial Cells.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

Differentiation of nerve-derived adult pluripotent stem cells into osteoblastic and endothelial cells

Background ContextStem cell-involved tissue engineering has gained dramatic attention as a therapeutic strategy for tissue regeneration including bone repair. However, the currently available possibilities to use embryonic stem cells and induced pluripotent stem cells (iPCs) face potential ethical issues, as well as risks of malignant transformation and immune rejection. Recently identified peripheral nerve-derived adult pluripotent stem cells (NEDAPS) that quickly proliferate after exposure to bone morphogenetic protein-2 (BMP-2) or nerve trauma and exhibit many embryonic stem cell characteristics may provide an attractive source cells for a variety of regenerative therapies. PurposeThe study aimed to examine the differentiation potential of the NEDAPS cells into osteoblastic cells and endothelial cells. Study Design/SettingAn in vitro investigation was undertaken to induce mouse NEDAPS cells into the phenotypes of osteoblastic and endothelial cells. MethodsNEDAPS cells were isolated from low-dose BMP-2-exposed mouse sciatic nerves by collagenase and trypsin extraction. The cells were cultured in a stem cell maintenance medium, and the expression of KLF4, Sox2, c-Myc, and Oct4 before differentiation was confirmed. The cells were then subcultured in a complete osteogenic cell induction medium or endothelial cell growth medium, respectively, at 37°C and 5%CO2 atmosphere. Histologic, morphologic, and molecular assessments were performed 7 days later. ResultsThe cells propagated in complete osteogenic medium for 7 days showed strong staining for type I collagen and alkaline phosphatase, suggesting the structural and functional properties of the osteoblastic cells. Further, real-time polymerase chain reaction (RT-PCR) revealed a significant expression of the osteoblast markers osteocalcin, osteopontin, and type I collagen. Similarly, the cells in endothelial growth medium were successfully differentiated into cobblestone-shaped endothelial cells expressing vascular endothelial growth factor (VEGF) receptors Flk-1 and Flt-1 demonstrated by RT-PCR. ConclusionsNEDAPS cells are readily induced to osteoblastic and endothelial cells, suggesting therapeutic potential for bone repair and other regenerative therapies.

Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells

In this study, adipose-derived mesenchymal stem cells (ADSCs) were isolated from adipose tissues of rats. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by the results of oil red O staining, alkaline phosphatase (ALP), and alcian blue staining, respectively. ADSCs at a density of 1×10(6)/cm(2) were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 d. The cell sheets were positive with von kossa staining. The transmission electron microscopy (TEM) result showed that needle-like calcium salt crystals were deposited on the ECM. These results suggested that the osteogenic cell sheets may have potential osteogenesis ability. ADSCs at a density of 1×10(6)/cm(2) were cultured in the endothelial cell growth medium-2 and the endothelial cell sheets can be formed after 16 d of culture. The TEM image confirmed that the Weibel-Palade corpuscle was seen in the cells. The expression of CD31 was positive, suggesting that the endothelial cell sheets may have a strong ability to form blood vessels. In this study, two types of cell sheets with the potential abilities of osteogenesis and blood vessels formation were obtained by induced culture of ADSCs in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defects.

Abstract 1388A: Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system

Abstract Angiogenesis is a hallmark of most cancers, and is thus an attractive target for the treatment of cancer. One of the easiest screening and target validation strategies for anti-angiogenic target identification involves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation in Corning's Matrigel product. The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector™ Technology. In the current study we used the Lonza 4D-Nucleofector™ X-Unit to transfect single-donor Clonetics™ HUVEC cultured in EGM™-2 Endothelial Cell Growth Media. Cells were transfected with siRNA directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Transfection conditions were fine-tuned using pmaxGFP™ vector, and it was shown that the Nucleofection™ process had no deleterious effect on the tube formation potential of HUVEC on Corning's Matrigel product. Efficient knock-down of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency was observed 24 hours after transfection. VEGFR2-siRNA transfected cells demonstrated significant inhibition of tube formation on Corning's Growth Factor Reduced Matrigel product in comparison to control samples. Since the efficient knock-down of the VEGFR2 protein in Clonetics™ HUVEC using the Nucleofector™ Technology can be demonstrated at time points as early as 24 hours after transfection, this is an efficient approach for target identification, validation and screening of siRNA based anti-angiogenesis therapeutics for cancer treatment. Citation Format: Srinivasan Kokatam, Kanchan Tiwari, Jenny Schroeder, Andrea Toell, Lubna Hussain, Preeti Kapoor. Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1388A. doi:10.1158/1538-7445.AM2015-1388A

Transplanted endothelial progenitor cell improves lung structure in neonatal rats exposed to hyperoxia

Objective To study the effect of transplanted endothelial progenitor cell (EPC) on hyperoxia-induced lung injury in neonatal rats. Methods Rat bone marrow mononuclear cells were cultured in endothelial cell growth medium to obtain EPCs, which were identified by morphology, phagocytosis and CD34+ analyses. Sixty neonatal Sprague-Dawley rats were allowed to acclimate in room air for 24 h after birth, and were then divided into four groups (15 per group), including the air group, the hyperoxia group, the EPCs transplantation group and the Nω-nitro-L-arginine methyl ester (L-NAME) intervention group. Neoborn rats in the Air and Hyperoxia groups were fed in the room air or hyperoxia (85% oxygen) for 28 days. For rats in transplantation group were exposed continuously to hyperoxia for 28 days, and got an EPC (1×105 cells) injection on the 21st day. Rats in Intervention group were exposed continuously to hyperoxia for 28 days, got an EPC (1×105 cells) injection on the 21st day, and a daily injection of L-NAME from day 21 to day 28, with a daily dose of 20 mg/kg. Levels of circulating CD34+ cells and serum VEGF expression were detected. Specimens from lung tissues were analyzed by immunohistochemistry or immunofluorescence. The expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and eNOS were detected by real-time polymerase chain reaction and Western-blotting. NO production were detected by nitrate reductase assay. One-way ANOVA and Bonferroni test were used for statistical analysis. Results (1) The cultured cells had a typical cobblestone appearance; double positive cell binding of fluorescein Ulex Europaeus agglutinin-1 and uptake of Dil-labeled acetylated low density lipoprotein accounted for approximately 85% of the total number of cells. CD34+ cells accounted for 68.2%-72.4% of total cultured cells. (2) Circulating CD34+ cells in the air group, hyperoxia group, EPC transplantation group and L-NAME intervention group were (1.91±0.34)%, (1.06±0.10)%, (1.47±0.06)% and (0.77±0.11)% (F=32.710, P=0.000). The number of circulating CD34+ cells in the hyperoxia group was lower than the air group, in the EPC transplantation group the number of these cells was higher than the hyperoxia group, and in the L-NAME intervention group the number of these cells was lower than that in the EPC transplantation group, and the differences between these two groups were statistically significant (P<0.05, respectively). Serum VEGF in the four groups was (7.90±2.72), (6.38±0.72), (14.00±1.66) and (11.70±1.91) pg/ml, respectively. The difference between the four groups was statistically significant (F=22.809, P=0.000), and serum VEGF in the EPC transplantation group was higher than that in the hyperoxia group (P<0.05). (3) Transplanted EPCs could engraft in pulmonary vascular endothelium and alveolar interstitium, and L-NAME intervention significantly reduced the engraftment of EPCs in the lungs (10.7±0.47 / field vs 16.95±0.5 /field, t=17.820, P=0.000). (4) There were significant differences in the radial alveolar count (RAC) and number of microvessels between the four groups (F=859.580 or 211.150, P=0.000, respectively). RAC and the number of microvessels in the hyperoxia group were less than those in the air group (7.98±0.23 vs 13.12±0.20, 3.98±0.42 vs 9.50±0.22, P<0.05, respectively). The number of microvessels in the EPC transplantation group was 5.40±0.41, being higher than that in the hyperoxia group (P<0.05). (5) VEGF mRNA in lungs in the hyperoxia group was lower than that in the air group (0.23±0.16 vs 1.05±0.33, P<0.05); in the EPC transplantation group, VEGF mRNA was higher than that in the hyperoxia group (0.69±0.09 vs 0.23±0.16, P<0.05); and in the L-NAME intervention group, VEGF mRNA was lower than that in the EPC transplantation group (0.31±0.08 vs 0.69±0.09, P<0.05). VEGF protein in the lungs in the hyperoxia group was lower than the air group (0.52±0.01 vs 0.82±0.01, P<0.05), and was higher in the EPC transplantation group than the hyperoxia group (0.58±0.05 vs 0.52±2501, P<0.05). VEGFR2 mRNA in the hyperoxia group was lower than the air group (0.35±0.13 vs 1.07±0.45, P<0.05). eNOS mRNA in the hyperoxia group was lower than the air group (0.46±0.10 vs 1.05±0.36, P<0.05). eNOS protein in the hyperoxia group was lower than the air group (0.32±0.01 vs 0.51±0.03, P<0.05), and was higher in the EPC transplantation group than the hyperoxia group (0.86±0.02 vs 0.32±0.01, P<0.05). Conclusion Transplanted EPC can engraft in the lung tissue, improving alveolar and pulmonary vascular development, which may be associated with upregulation of the expression of eNOS and VEGF in lung. Key words: Endothelial progenitor cells; Stem cell transplantation; Bronchopulmonary dysplasia; Vascular endothelial growth factor A; Nitric Oxide Synthase; Rats

Characterization of endothelial colony-forming cells from peripheral blood samples of adult horses

To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. Jugular venous blood samples from 24 adult horses. Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates. Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry. ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.

Optimization of in vitro cultivation strategies for human adipocyte derived stem cells

With adipose-derived stem cells being in the focus of research in regenerative medicine, the need arises for fast reliable cultivation protocols. We have tested the cultivation of human adipose-derived stem cells in endothelial cell growth medium prior to induction and differentiation, against the long-established use of DMEM/F12 medium-based cultivation protocols. We found that cultivation in endothelial cell growth medium not only accelerates growth before induction and differentiation, but also allows shorter induction and differentiation times than those following precultivation with DMEM/F12 medium with regard to the formation of mature adipocytes and to the viability undifferentiated cells. These results were first observed morphologically but could be confirmed by performing adiponectin ELISA and cell proliferation assays.

In vitro study on regeneration of periodontal tissue microvasculature using human dedifferentiated fat cells.

Human dedifferentiated fat cells (HDFATs) may be a new cell type suitable for regenerative therapies. The aim of this study is to assess the potential of HDFATs for vascular regeneration of periodontal tissue. To do this, HDFATs and human gingival endothelial cells (HGECs) were cocultivated, and vascular regeneration was examined in vitro. HDFATs were isolated from subcutaneous adipose tissue, and HGECs were isolated from gingival cells using anti-cluster of differentiation 31 antibody-coated magnetic beads. HDFATs were cocultured with HGECs in microvascular endothelial cell growth medium-2 (EGM-2MV) for 7 days. Expression of endothelial cell (EC) markers, the formation of capillary-like tubes, and the expression of pericyte markers were determined. HDFATs, cultured in EGM-2MV or cocultured with HGECs, expressed EC markers. HDFATs in both conditions initiated tube formation within 5 hours of seeding and formed extensive capillary-like structures within 12 hours. These structures disintegrated within 24 hours when cells were cultured in EGM-2MV alone, whereas cocultured HDFATs maintained tubes for >24 hours. Cocultured HDFATs significantly increased expression of pericyte markers, a cell type associated with microvasculature. HDFATs possess the ability to express EC markers, and coculture with HGECs promotes differentiation into pericytes involved in the maturation and stabilization of the microvasculature.

Whole bone marrow cell culture: A convenient protocol for the in vitro expansion of endothelial progenitor cells.

The number and function of endothelial progenitor cells (EPCs) may be a predictive factor for the severity and outcome of cardiovascular disease. However, the manipulation of bone marrow mononuclear cell (BMMC) cultures for EPCs is an elaborate and difficult procedure in small experimental animals. The present study aimed to assess the feasibility of whole bone marrow cell (WBMC) culture for expanding EPCs in small experimental animals. C57BL/6 mice (age, 3–4 weeks; weight, 9.47±0.76 g) were used as the experimental animals, and WBMCs were isolated from the femora and tibiae and cultured in endothelial cell growth medium-2. A BMMC culture for EPCs was used as a control. EPC growth, phenotype and functions were assessed in vitro and in vivo. The results demonstrated that EPCs were easily obtained from a WBMC culture in vitro. The cells exhibited similar growth and biological characteristics when compared with the EPCs derived from the traditional BMMC culture system. Thus, the cells were able to simultaneously bind to lectin and cause phagocytosis of acetylated-low density lipoproteins. In addition, the cells exhibited high expression levels of cluster of differentiation 34 and fetal liver kinase 1, and possessed similar functional properties to BMMC-derived EPCs, including vascular network formation, proliferation, adhesion and migration abilities in vitro. Thus, WBMC-derived EPCs can improve the outcome of pulmonary vascular disease when transplanted into a monocrotaline-induced pulmonary hypertension mouse model. The results of the present study indicated that the WBMC culture system is a more convenient and effective method of obtaining and expanding EPCs compared with BMMC culture, with the advantage of a simplified procedure.

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.

MicroRNA-200C and -150 play an important role in endothelial cell differentiation and vasculogenesis by targeting transcription repressor ZEB1

To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigel-coated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation. CD146(+) cells were isolated from differentiating hES cells and expanded in vitro. The in vitro expanded CD146(+) cells were positive for EC markers, capable of Ac-LDL uptake, lectin binding, and the formation of vascular structures in vitro and in vivo. miRNA gain/loss-of-function analyses revealed that miR-150 and miR-200c were crucial in EC differentiation. Transcriptional repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the communal target gene of miRNA-200C and -150, and inhibition of ZEB1 was required for miRNA-200C or -150 mediated EC gene expressions. Moreover, we demonstrated that ZEB1 could transcriptionally repress EC gene expression through direct binding to promoters of EC genes. Finally, we also demonstrated that miRNA-200c and -150 played an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or -150 in developing chick embryos, and blocking ZEB1 signaling in CD146-positive cells could rescue the inhibitory effects of miR-200c inhibiton in in vivo vasculogenesis. Our findings revealed that miR-150 and miR-200c play an important role in human endothelial lineage specification and chick embryonic vasculogenesis by targeting ZEB1.

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...

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.