Fibronectin-coated Dishes Research Articles

Grain and bean lysates improve function of endothelial progenitor cells from human peripheral blood: involvement of the endogenous antioxidant defenses.

Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs), the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG), a Triticum Sativum grain powder, and Lady Joy (LJ), a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H2O2). Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2)-like 2)/ARE (antioxidant response element) activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35–0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35–0.7 mg/ml protect EPCs viability against H2O2-induced injury. LG 0.7 and LJ 0.35–0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H2O2. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H2O2, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H2O2-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2) expression; upon H2O2 exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H2O2 exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a protective effect of lysates on EPCs exposed to oxidative stress through the involvement of antioxidant systems. Lisosan G seems to activate the Nrf-2/ARE pathways.

Green tea extract protects endothelial progenitor cells from oxidative insult through reduction of intracellular reactive oxygen species activity.

Many studies have reported that tea consumption decreases cardiovascular risk, but the mechanisms remain unclear. Green tea is known to have potent antioxidant and free radical scavenging activities. This study aimed to investigate whether green tea extract (GTE) can protect endothelial progenitors cells (EPCs) against oxidative stress through antioxidant mechanisms. Mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation with Ficoll. The cells were then plated on fibronectin-coated culture dishes. After 7 days of culture, EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptake and lectin binding. EPCs were further identified by assessing the expression of CD34/45, CD133, and KDR. EPCs were then treated with hydrogen peroxide (H2O2) at doses of 50, 100, 200 µM and incubated with or without GTE (25 µg/ml). The intracellular reactive oxygen species (ROS) levels were detected by flow cytometry using a 2',7'-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. GTE ameliorated the cell viability of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 25.47, 22.52, and 11.96% higher than controls, respectively. GTE also decreased the intracellular ROS levels of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 84.24, 92.27, and 93.72% compared to controls, respectively. GTE improves cell viability by reducing the intracellular ROS accumulation in H2O2-induced EPCs.

Nicotine promotes late endothelial progenitor cells functional activity in a PI 3-kinase-dependent manner.

Recent studies have shown that endothelial progenitor cells (EPCs) participated in angiogenic effects of nicotine and nicotine dose dependently increased the functional activity of early EPCs. The effects of nicotine on late EPCs remain to be determined. Therefore, we investigated whether nicotine had influences on the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood and characterized. Late EPCs of 3-5 passages were treated for 32 h with either vehicle or nicotine. The proliferative, migratory, and in vitro vasculogenesis activities of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in matrigel, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Nicotine enhanced proliferative, migratory, adhesive, and in vitro vasculogenesis capacities of late EPCs. These effects were significantly reduced in the presence of phosphatidylinositol (PI) 3-kinase inhibitor.

Dextran induces differentiation of circulating endothelial progenitor cells.

Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating‐circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin‐coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)‐R1/2, VE‐cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF‐R1/2, VE‐cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium‐related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran‐treated EPCs; however, those of hematopoietic‐ and antiangiogenic‐related transcription factors TAL1, RUNX1, c‐MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran‐exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines.

Lipopolysaccharide Impaired the Functional Activity of Endothelial Colony-Forming Cells

Background: Recent studies have shown that endothelial progenitor cells (EPCs) contribute to lung repair after lipopolysaccharide (LPS)-induced lung injury and infusion of LPS decreased early EPCs in human peripheral blood. However, the effects of LPS on endothelial colony-forming cells (ECFCs) remain to be determined. Objective: To investigate possible effects of LPS on the functional activity of ECFCs. Methods: ECFCs were isolated from human umbilical cord blood and characterized. ECFCs at passages 3-5 were treated for 24 h with either LPS or vehicle control. Their viability, migration and in vitro vasculogenesis activity were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, modified Boyden chamber and in vitro angiogenesis assays, respectively. ECFC adhesion was assessed by replating cells on fibronectin-coated dishes and subsequent counting of adherent cells. Results: Incubation with LPS dose-dependently inhibited the viable, migratory, adhesive and in vitro vasculogenesis capacity of ECFCs. Conclusion: LPS impaired the functional activity of ECFCs.

Telmisartan promotes proliferation and differentiation of endothelial progenitor cells via activation of Akt

Background Numerous studies have demonstrated that the peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in regulating endothelial progenitor cells (EPC) function. Telmisartan, as a partial agonist of PPARγ, may have an effect on the regulation of EPC functions. The purpose of this study was to investigate the effects of telmisartan on EPC proliferation and differentiation. Methods Peripheral blood derived mononuclear cells containing EPC were isolated from healthy volunteers and then cultured on fibronectin-coated dishes in the presence or absence of telmisartan. The proliferative activity of EPC was determined by colony forming units (CFU) and MTT assay. The migratory activity of EPC was assessed by transwell assay. The expression of endothelial cells (EC) markers, including vascular endothelial cadherin (VE-cadherin), von Willebrand factor (vWF) and endothelial nitric oxide synthase (eNOS), were measured by Western blotting analysis. Results Morphological analysis revealed that telmisartan significantly increased the proliferation of EPC and the number of endothelial cell colony forming units. Telmisartan could enhance the expression of the makers of mature EC, including VE-cadherin, vWF, and eNOS, which indicated telmisartan could stimulate EPC to differentiate into mature EC. Telmisartan increased the phosphorylation of Akt in EPC. The inhibition of Akt activation significantly attenuated the effect of telmisartan on EPC functions, suggesting that Akt is involved in the stimulatory effect of telmisartan on EPC differentiation. Conclusions The results of this study demonstrate that telmisartan promotes EPC functions via activation of Akt.

Effects of lycopene on number and function of human peripheral blood endothelial progenitor cells cultivated with high glucose.

BACKGROUND/OBJECTIVESThe objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs.MATERIALS/METHODSMononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and 50 µg/mL of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK.RESULTSThe proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and 50 µg/mL of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury.CONCLUSIONSLycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.

Chronic Exposure to Testosterone Increases Expression of Transient Outward Current in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Cardiomyocytes (CM)

Background The transient outward potassium current (I to ) plays a prominent role in the development of cardiac arrhythmias associated with the J-wave syndromes, including Brugada and early repolarization syndromes. The J-wave syndromes are strongly male-dominated, presumably due to higher expression of I to in the right ventricular epicardium of male vs female dogs. In this study we tested the hypothesis that the male androgen testosterone underlies these gender differences by examining its influence on expression of I to in hiPSC-derived cardiomyocytes. Methods and Results Embryoid bodies (EBs) were made from a human iPS cell line reprogrammed with Oct4, Nanog, Lin28, and Sox2 using a serum-free differentiation protocol supplemented with growth factors for selective cardiac differentiation. The beating clusters were microdissected from the entire EBs, enzymatically dissociated, and plated on fibronectin-coated dishes. Cells were treated with 1 µM testosterone for 4 weeks. I to was measured using whole-cell patch-clamp techniques at 36oC. I to density was significantly greater in testosterone-treated hiPSC CMs compared to controls (18.7 ± 3.2 pA/pF vs 11.8 ± 1.6 pA/pF, 1 μM testosterone, P P = .036). Steady state (SS) inactivation was unchanged. Expression of the I to -associated gene transcript (KCND3, Kv4.3) was 547.51% greater ( P to from inactivation and a depolarized MDP (–65.4 ± 10.3 mV) due to a deficient I K1 . Conclusions Our results suggest that testosterone is capable of increasing expression of I to , at least in part by upregulating expression of Kv4.3 mRNA. These results provide insights into the gender-dependent expression of I to and may explain the marked gender differences observed in the manifestation of life-threatening arrhythmias associated with the J-wave syndromes. [Supported by NYSTEM contract # C026424.]

Study on MR Imaging of Endothelial Progenitor Cells Labeled with the Complex of Super Paramagnetic Iron Oxide and Transfection

To explore the characteristics of magnetic resonance（MR）imaging of the rat endothelial progenitor cells（EPCs）labeled with superparamagnetic iron oxide（SPIO）. Total mononuclear cells (MNCs) were isolated from SD rat peripheral blood by ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. Attached cells were collected after 7 days cultured. EPCs were indentified by the laser confocal microscope and were counted in the inverted fluorescence microscope. EPCs were incubated with Fe2O3-arginine for 24 h, and the cells underwent MR imaging with three sequences (T1 WI, T2 WI, T2*WI). The results showed that the effective rate of labeled EPCs was 96%, and the survival rate of cells was 95%. The signal intensity on MRI was significantly decreased in labeled EPCs compared with unlabeled cells. EPCs labeled with SPIO can be sensitively displayed by the MR imaging.

Higher propensity of Wharton's jelly derived mesenchymal stromal cells towards neuronal lineage in comparison to those derived from adipose and bone marrow

Mesenchymal stromal cells (MSCs) derived from different tissue sources are capable of differentiating into neural and glial cell types. However, the efficiency of differentiation varies between MSCs derived from different tissues. We compared the efficiency of neural progenitor population generation between adipose (AD), bone marrow (BM) and Wharton's jelly (WJ) derived MSCs. MSCs isolated from the three sources were induced to form primary neurospheres using epidermal growth factor (20 ng/mL) and bFGF (20 ng/mL). The self-renewal potential of the primary neurospheres was assessed by secondary neurosphere assay. Primary neurospheres were differentiated to neuronal lineage on fibronectin-coated dishes. The neurospheres and the resulting differentiated cells were characterized by immunocytochemistry and the RT-PCR analyses. We have also investigated the secretome profile of neuronal-related growth factors using Ray biotech cytokine array. The results show that MSCs from the three sources can be induced to generate neurospheres and they expressed neural progenitor markers nestin, Sox2 and Pax6 transcription factors. When differentiated on fibronectin coated dishes in mitogen free culture conditions, the primary spheres from all three sources were able to generate neuron/glial - like cells which expressed Nfl, Map2 and GFAP with varied efficiency. Self-renewal potential of these progenitors was determined by secondary sphere formation. WJ- and BM-derived neurospheres were able to self-renew, while AD derived progenitors failed to do so. Comparison of the secretome profile suggested that WJ derived MSCs secrete more neurotrophic factors. The data suggest that human WJ derived MSCs can be induced to make neural progenitors with higher efficiency compared to BM and AD derived MSCs.

Ochratoxin A Inhibits Mouse Embryonic Development by Activating a Mitochondrion-Dependent Apoptotic Signaling Pathway

Ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish) or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

242 EFFECTS OF CHILDHOOD OBESITY ON THE ENDOTHELIAL FUNCTION AND FUNCTIONAL PROPERTIES OF EARLY OUTGROWTH ENDOTHELIAL PROGENITOR CELLS

Background: The prevalence of cardiovascular risk factors has been increasing among children/adolescents in connection to rapidly increased childhood obesity. Given that endothelial dysfunction is an early event in the development of atherosclerosis and endothelial progenitor cells (EPCs) are important in vascular repair, we wanted to investigate the impact of childhood obesity on the endothelial function and functional properties of EPCs. Methods: We examined 20 overweight/obese (BMI: 33.7±6 kg/m2, Age: 19.2±2.1) and 10 normal weight young people (BMI: 21.8±1.7 kg/m2, Age: 18.5±1.4). These subjects were identified in a longitudinal study that began 5 years ago and remained in their original weight group. EPCs were obtained by culturing Ficoll-density isolated peripheral mononuclear cells on fibronectin-coated dishes. The number of early outgrowth EPCs and their adhesive and migratory capacity were analyzed. The endothelial function was assessed using reactive hyperemia (RH) peripheral arterial tonometry (Itamar). Global fitting was used to compare RH-response curves (GraphPad 5). Results: Compared with controls, overweight/obese subjects had lower RH-response (Figure) and lower number of early outgrowth EPCs (0.33±0.16 vs 0.47±0.2 million cells, p=0.039). However, EPCs expanded from overweight/obese had similar adhesive and migratory capacity as controls. The overweight/obese had higher levels of insulin (12.1±6.7 vs 6.9±2.4 mU/L, p=0.02), systolic blood pressure (121±9 vs 109±9 mmHg, p=0.001) and diastolic blood pressure (72±7 vs 63±5 mmHg, p=0.002) than the controls.Conclusion: Childhood obesity impaired endothelial function and reduced the number of early outgrowth EPCs already in young age.

Altered Angiogenesis in Caveolin-1 Gene–Deficient Mice Is Restored by Ablation of Endothelial Nitric Oxide Synthase

Caveolin-1 is an essential structural protein of caveolae, specialized plasma membrane organelles highly abundant in endothelial cells, where they regulate multiple functions including angiogenesis. Caveolin-1 exerts a tonic inhibition of endothelial nitric oxide synthase (eNOS) activity. Accordingly, caveolin-1 gene-disrupted mice have enhanced eNOS activity as well as increased systemic nitric oxide (NO) levels. We hypothesized that excess eNOS activity, secondary to caveolin deficiency, would mediate the decreased angiogenesis observed in caveolin-1 gene-disrupted mice. We tested tumor angiogenesis in mice lacking either one or both proteins, using in vitro, ex vivo, and in vivo assays. We show that endothelial cell migration, tube formation, cell sprouting from aortic rings, tumor growth, and angiogenesis are all significantly impaired in both caveolin-1-null and eNOS-null mice. We further show that these parameters were either partially or fully restored in double knockout mice that lack both caveolin-1 and eNOS. Furthermore, the effects of genetic ablation of eNOS are mimicked by the administration of the NOS inhibitor N-nitro-L-arginine methyl ester hydrochloride (L-NAME), including the reversal of the caveolin-1-null mouse angiogenic phenotype. This study is the first to demonstrate the detrimental effects of unregulated eNOS activity on angiogenesis, and shows that impaired tumor angiogenesis in caveolin-1-null mice is, at least in part, the result of enhanced eNOS activity.

New method to differentiate human peripheral blood monocytes into insulin producing cells: Human hematosphere culture

Strategy to differentiate stem cells into insulin producing cells (IPCs) in vitro has been a promising one to get cell source of β-cell replacement therapy for diabetes. It has been suggested that islets and neurons share features and nestin-positive cells could differentiate into IPCs. We have recently developed a three-dimensional culture system using human peripheral blood cells named as blood-born hematosphere (BBHS). Here we showed that most of BBHS were composed of nestin-positive cells. Under the four-stage differentiation protocol for IPCs, we plated nestin-positive BBHS onto fibronectin-coated dish. These cells form islet-like clusters and most of them expressed insulin. Pancreatic specific genes were turned on, such as transcription factors (Pdx-1, Ngn3 and Nkx6.1), genes related to endocrine function (Glut-2 and PC2) or β cell function (Kir6.2, SUR1). Furthermore islet differentiation was confirmed by dithizone (DTZ) staining to detect zinc ion which binds insulin protein within the cells. Finally, IPCs derived from BBHS showed capability to secrete insulin in response to glucose stimulation. Taken together, our novel protocol successfully induced islet-like human insulin producing cells out of BBHS. This strategy of ex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatment of diabetes.

Effects of urotensin II on functional activity of late endothelial progenitor cells

Urotensin II (UII) is a potent vasoactive cyclic peptide which has multiple effects on the cardiovascular system. However, the effects of UII on late endothelial progenitor cells (EPCs) are still unclear. The aim of the present study is to investigate whether UII influences the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood by Ficoll density gradient centrifugation and treated with UII (10−10, 10−9, 10−8, 10−7 and 10−6M), or vehicle control. Expression of urotensin II receptor (UT) in late EPCs was confirmed by indirect immunofluorescence staining. Late EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell chamber assay, and matrigel tube formation assay. Late EPCs adhesive assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Incubation with UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited the proliferative activity of late EPCs. Furthermore, these UII-mediated effects on late EPCs were attenuated by pretreatment with the UT antagonist urantide. These findings indicate that UII may exert multiple effects on functional activity of late EPCs through UT.

Enhanced chondrogenic marker expression of human mesenchymal stem cells by interaction with both TGF‐β3 and hyaluronic acid

This study was designed to evaluate the additive effects of transforming growth factor-beta3 (TGF-β3) and hyaluronic acid (HA) on chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The hMSCs were cultured on collagen type I-, HA-, or fibronectin-coated cell culture dishes with or without TGF-β3 added to the culture medium. Four weeks after cell culture, chondrogenic differentiation of hMSCs was determined by evaluating the expression of cartilage-specific markers using real-time polymerase chain reaction, immunocytochemistry, and Western blot analysis. hMSCs cultured on HA-coated dishes with TGF-β3 supplementation revealed a prominent increase in collagen type II, aggrecan, and Sox9. When hMSCs were cultured without TGF-β3 supplementation, only hMSCs cultured on HA-coated dishes showed prominent expression of the cartilage-specific markers. This study shows that chondrogenic differentiation of hMSCs can be enhanced additively by interactions with both a specific cell-adhesion matrix and a soluble growth factor.

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.

Abstract 2077: Interactions between sulfiredoxin, S100A4 and non-muscle myosin IIA regulate cancer cell adhesion and motility

Abstract Sulfiredoxin (Srx) is an antioxidant protein that reduces over-oxidized catalytic cysteine residues of peroxiredoxins I-IV and catalyzes reduction of some S-glutathionylated proteins. We previously showed that Srx plays a role in cell migration and interacts with non-muscle myosin IIA (NM IIA; Bowers et al AACR 2010). The purpose of the present study was to extend our knowledge of the effect of Srx on cell adhesion and migration. Immunoprecipitation of Srx from non-small lung cancer (A549 cells) revealed a number of Srx-interacting proteins including NM IIA and S100A4. NM IIA plays a central role in the control of cell adhesion and migration, and S100A4 is a key regulator of NM IIA activity that is linked to cancer metastases. Adhesion assays showed that A549 cells over-expressing Srx have reduced adhesion to fibronectin-coated dishes and decreased levels of Tyr397-phosphorylated focal adhesion kinase (p-FAK). Conversely, Srx knockdown by shRNA increased A549 cell adhesion to fibronectin and p-FAK levels. Srx over-expression in A549 cells increased actin stress fiber formation and altered focal adhesion formation. Depletion of Srx in A549 cells resulted in decreased actin stress fiber formation and also altered sites of cell:extracellular matrix adhesion. In silico molecular modeling of an Srx-S100A4 homodimer complex showed close proximity (∼13Å) of the single Cys99 of Srx to the Cys85 of S100A4. The calculated binding constant for the Srx-S100A4 interaction indicated reasonable affinity (KD∼ 62 nM). This affinity was not dependent upon disulfide bonding, since mutation of Srx residue Cys99 to Ser resulted in increased affinity (KD ∼22 nM). In vitro S-glutathionylation of Cys85 of S100A4 markedly increased its affinity to Srx (KD∼4 nM), but had no effect with the inactive Srx C99S mutant. Srx over-expression produced a doubling of the basal cellular level of free Ca2+ suggesting that the protein:protein interactions altered the capacity of S100A4 to bind Ca2+. Our data show that Srx may play important role in S100A4-mediated cancer cell cytoskeletal remodeling and motility and could potentially be a target for development of specific anticancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2011-2077

The effect of rAAV2-PEDF on proliferation of human retinal capillary endothelium cells

To culture human retinal capillary endothelium cells (HRCECs) in vitro and explore the effect of rAAV2-PEDF on proliferation of HRCEs. Retinas were digested by 2.5% trypsin and 0.1% collagenase I in order. The isolated cells were cultured on fibronectin-coated dishes in media of human endothelial-sFM basal growth medium (HE-SFM BGM) with 10% fetal bovine serum, insulin-transferrin-selenium (ITS) and endothelial cell growth factor (ECGF). The cultured cells were identified by anti-factor VIII related antigen though immunohistochemistry stain. The effect of hypoxia induced by CoCl2 on proliferation of HRCECs was assessed by MTT assay. After rAAV2-PEDF were transfected into HRCECs, the EGPF positive cells were observed by laser confocal scanning microscopy, the protein expression of PEDF were detected by Western blot, and the proliferation of HRCECs were checked by MTT assay. Flow cytometry was used to analyze the apoptosis of HRCECs. Cultured HRCECs attached in the bottom of dishes in 48 h - 72 h and grew to confluence in 2 weeks after seeding. HRCECs were with a positive brown staining for factor VIII. EGPF positive cells were seen under laser confocal scanning microscopy after 48 h of rAAV2-EGFP transfection. The expression level of PEDF protein was higher in experimental group than in control group. The results of MTT assay showed the numeric value OA was 0.085 ± 0.021 in normal group and 0.166 ± 0.024 in hypoxia group (t = 3.938, P < 0.05). In normal oxygen condition, the numeric value OA was 0.171 ± 0.011 in normal control group, 0.178 ± 0.016 in rAAV2-EGFP treated group, and 0.169 ± 0.017 in rAAV2-PEDF treated group (F = 0.01, P > 0.05). In hypoxia condition, the numeric value OA was 0.166 ± 0.013 in CoCl(2) treated group, 0.155 ± 0.012 in CoCl(2) + rAAV2-EGFP treated group, and 0.116 ± 0.015 in CoCl(2) + rAAV2-PEDF treated group. In normal oxygen condition, the ratio of apoptosis was 2.3% in normal control group, and 3.3% in rAAV2-EGFP treated group, and 1.7% in rAAV2-PEDF treated group. In hypoxia condition, the ratio of apoptosis was 3.6% in CoCl(2) treated group, 6.7% in CoCl(2) + rAAV2-EGFP treated group, and 36.4% in CoCl(2) + rAAV2-PEDF treated group. PEDF gene can stably express in HRCECs after rAAV2-PEDF transfection and can obviously inhibit proliferation of HRCECs in hypoxia.