Human Umbilical Vein Endothelial Cells Cultures Research Articles

Uric Acid Impairs Insulin Signaling by Promoting Enpp1 Binding to Insulin Receptor in Human Umbilical Vein Endothelial Cells.

High levels of uric acid (UA) are associated with type-2 diabetes and cardiovascular disease. Recent pieces of evidence attributed to UA a causative role in the appearance of diabetes and vascular damage. However, the molecular mechanisms by which UA induces these alterations have not been completely elucidated so far. Among the mechanisms underlying insulin resistance, it was reported the role of a transmembrane glycoprotein, named either ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) or plasma cell antigen 1, which is able to inhibit the function of insulin receptor (IR) and it is overexpressed in insulin-resistant subjects. In keeping with this, we stimulated human umbilical vein endothelial cells (HUVECs) with insulin and UA to investigate the effects of UA on insulin signaling pathway, testing the hypothesis that UA can interfere with insulin signaling by the activation of ENPP1. Cultures of HUVECs were stimulated with insulin, UA and the urate transporter SLC22A12 (URAT1) inhibitor probenecid. Akt and endothelial nitric oxide synthase (eNOS) phosphorylation levels were investigated by immunoblotting. ENPP1 binding to IR and its tyrosine phosphorylation levels were tested by immunoprecipitation and immunoblotting. UA inhibited insulin-induced Akt/eNOS axis. Moreover, UA induced ENPP1 binding to IR that resulted in an impairment of insulin signaling cascade. Probenecid reverted UA effects, suggesting that UA intracellular uptake is required for its action. In endothelial cells, UA directly interferes with insulin signaling pathway at receptor level, through ENPP1 recruitment. This evidence suggests a new molecular model of UA-induced insulin resistance and vascular damage.

Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).

Maternal hypertension and feto-placental growth restriction is reversed by sildenafil: Evidence of independent effects of circulating nitric oxide levels

Sildenafil has shown nitric oxide (NO)-independent pleiotropic effects, however the mechanisms involved are unclear. We investigated the protective effects of sildenafil against hypertension in pregnancy and feto-placental growth restriction induced by NO inhibition, and if sodium nitrite-derived NO formation influences sildenafil effects. We evaluated the plasmatic levels of NO metabolites, cyclic guanosine monophosphate (cGMP), oxidative stress and myeloperoxidase, which are involved in endothelial dysfunction during hypertension in pregnancy. Also, we performed in vitro experiments to examine cell viability and NO synthesis in human umbilical vein endothelial cells (HUVECs) cultures incubated with plasma from healthy or hypertensive pregnant rats treated (or not) with both drugs, either alone or in association. Sildenafil blunted hypertension in pregnancy and protected against feto-placental growth restriction induced by NO inhibition and these effects of sildenafil alone were similar to those presented by its association with sodium nitrite. Protective effects of sildenafil were observed even with low plasmatic NO levels and were not followed by increases in cGMP levels. Also, sildenafil, but not sodium nitrite, blunted the increases in myeloperoxidase activity. Both drugs (isolated or in association) presented antioxidant effects. Plasma from hypertensive pregnant rats treated with sildenafil, but not sodium nitrite alone, increased the viability of HUVECs. NO synthesis in HUVECs cultures was increased with plasma from rats treated with both drugs. We conclude that sildenafil effects are not dependent of circulating NO levels in hypertension and feto-placental growth restriction. These findings may reflect a protection against myeloperoxidase and pro-oxidant activation in hypertension in pregnancy.

New Approach to Cryopreservation of Primary Noncultivated Human Umbilical Vein Endothelium in Biobanking.

It is widely accepted that endothelial dysfunction (ED) is a common feature and a risk factor for cardiovascular diseases and metabolic disorders. Cultures of human umbilical vein endothelial cells (HUVECs) are routinely used in cell-based models to study in vitro molecular and cellular mechanisms of development of different aspects of ED. The methods of the HUVEC extraction and expansion are well developed and standardized. However, when large collections of samples are needed for certain projects, or when samples from a rare population of patients should be collected for future experimental use, HUVEC samples should be transferred to a biobank to be saved in liquid nitrogen for a long period of time until the required collection is completed. This scenario is not always convenient since it requires a lot of effort, a large quantity of expensive culture reagents with limited expiration periods, and sometimes special facilities and well-trained cell biologists among the biobank staff. In this project, we evaluated a method of HUVEC cryopreservation, where the stage of cell culturing and expansion before the transfer of samples to the biobank is eliminated. A total of 55 samples of umbilical cord (UC) were obtained from women immediately after delivery. A primary endothelium pellet derived from 17 UC samples was isolated, frozen, and placed in long-term storage in a liquid nitrogen freezer. Other samples were used to obtain HUVEC cultures. We have demonstrated that cryopreservation of primary endothelium pellets from UC veins without culturing and expansion steps does not affect the physiological features of HUVECs. This new approach would improve the efficiency of biobanking logistics, especially in the case of banking of large collections of endothelial samples.

Proangiogenic functions of an RGD-SLAY-containing osteopontin icosamer peptide in HUVECs and in the postischemic brain

Osteopontin (OPN) is a phosphorylated glycoprotein secreted into body fluids by various cell types. OPN contains arginine-glycine-aspartate (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs that bind to several integrins and mediate a wide range of cellular processes. In the present study, the proangiogenic effects of a 20-amino-acid OPN peptide (OPNpt20) containing RGD and SLAY motifs were examined in human umbilical vein endothelial cells (HUVECs) and in a rat focal cerebral ischemia model. OPNpt20 exerted robust proangiogenic effects in HUVECs by promoting proliferation, migration and tube formation. These effects were significantly reduced in OPNpt20-RAA (RGD->RAA)-treated cells, but only slightly reduced in OPNpt20-SLAA (SLAY->SLAA)-treated cells. Interestingly, a mutant peptide without both motifs failed to induce these proangiogenic processes, indicating that the RGD motif is crucial and that SLAY also has a role. In OPNpt20-treated HUVEC cultures, AKT and ERK signaling pathways were activated, but activation of these pathways and tube formation were suppressed by anti-αvβ3 antibody, indicating that OPNpt20 stimulates angiogenesis via the αvβ3-integrin/AKT and ERK pathways. The proangiogenic function of OPNpt20 was further confirmed in a rat middle cerebral artery occlusion model. Total vessel length and vessel densities were markedly greater in OPNpt20-treated ischemic brains, accompanied by induction of proangiogenic markers. Together, these results demonstrate that the 20-amino-acid OPN peptide containing RGD and SLAY motifs exerts proangiogenic effects, wherein both motifs have important roles, and these effects appear to contribute to the neuroprotective effects of this peptide in the postischemic brain.

The Effect of Vitamin C towards Endothelial Dysfunction in CdCl2-induced HUVEC Culture

Cardiovascular diseases are the leading cause of death globally which usually begins with endothelial dysfunction. This vascular abnormality is potentially caused by one kind of heavy metal called cadmium (Cd). Vitamin C, as an antioxidant, can serve as the free radical scavenger. This research aimed to determine the effect of vitamin C on the viability of endothelial cells and endothelial dysfunction through nitric oxide (NO) levels and malondialdehyde (MDA) in human umbilical vein endothelial cells (HUVEC) induced with CdCl2. HUVEC was induced by 24.154 µg/L CdCl2. Three treatment groups of CdCl2-induced HUVEC were employed, each given vitamin C concentrations of 50, 100, and 200 µM, respectively. Cell viability was assessed by MTT assay. Levels of NO and MDA were determined by with the calorimetric test. Cell viability was increased significantly in treatment group receiving vitamin C 100 and 200 µM. However, there were no significant differences on NO and MDA levels in vitamin C treatment groups compared to control. In conclusion, vitamin C increases endothelial cell viability and NO level, and reduces MDA levels in CdCl2-induced HUVEC. This study implies the importance of vitamin C supplementation to prevent cardiovascular diseases.

Polymer scaffolds for vascular tissue engineering fabricated by combined electrospinning and hot embossing

Although tissue engineering has shown great advances in recent years, creating proper mechanical properties and cell growth microenvironments is still challenging. In this study, electrospun nanofibrous membranes were hot embossed to develop three-dimensional hierarchical micro/nanostructures that load and culture human umbilical-vein endothelial cells (HUVECs). The hot-embossed membranes exhibited not only superior mechanical properties (the tensile strength was 7.01 ± 0.18 MPa and the tensile modulus was 166.91 ± 15.54 MPa), but also better cell viability evaluated through a CCK-8 assay and fluorescent dye. The grating and well arrays of the micropatterned fibre mats encouraged the HUVECs to proliferate. Therefore, the approach proposed in this paper—combined electrospinning and hot embossing—has bright prospects in biomedical applications for the use of polymer scaffold in tissue engineering.

Protective, repairing and fibrinolytic effects of rivaroxaban on vascular endothelium.

Rivaroxaban, a direct inhibitor of activated factor X (FXa), is the only new oral anticoagulant approved for secondary prevention after acute coronary syndrome. Our objective was to identify the possible molecular mechanisms of rivaroxaban that contribute to endothelial function. Cell viability and growth of human umbilical vein endothelial cells (HUVEC) were registered. Gene expression studies comparing the effects of rivaroxaban and FXa were conducted by a selective RNA array and confirmed by protein quantification. Wound-healing experiments on HUVEC, platelet adhesion, enzymatic activity, and cell-based assays for fibrin formation were performed with rivaroxaban. Rivaroxaban (50nM) only altered (>2 fold change) the expression of matrix metallopeptidase 2 and urokinase plasminogen activator (u-PA), but counteracted the FXa (9nM)-induced upregulation of several pro-inflammatory genes (P<0.05) and FXa-enhanced platelet adhesion over HUVEC. Rivaroxaban increased u-PA protein expression in HUVEC supernatants and enhanced u-PA activity (up to 4IUng-1 of u-PA). Rivaroxaban (1nM-1μM) showed a significant and dose-dependent positive effect on HUVEC growth that was inhibited by BC-11-hydroxibromide, an inhibitor of u-PA. Healing properties after a wound on HUVEC cultures, and fibrinolytic properties were also shown by rivaroxaban. Both effects were reversed by BC-11-hydroxibromide. Rivaroxaban enhanced viability, growth and migration of HUVEC, mainly by u-PA activation and upregulation, which also participate in the rivaroxaban-induced fibrinolytic activity at endothelial level. Rivaroxaban also protected from the pro-inflammatory effects of FXa on HUVEC. Altogether may improve endothelial functionality and could contribute to the cardiovascular benefits of rivaroxaban.

Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury.

Cerebral ischemia is a leading cause of death and disability. A previous study indicated that remote ischemic postconditioning (RIP) in the treatment of cerebral ischemia reduces ischemia/reperfusion (I/R) injury. However, the underlying mechanism is not well understood. In the present study, the authors hypothesized that the protective effect of RIP on neurological damage is mediated by exosomes that are released by endothelial cells in femoral arteries. To test this, right middle cerebral artery occlusion/reperfusion with RIP was performed in rats. In addition, an I/R injury cell model was tested that included human umbilical vein endothelial cells (HUVECs) and SH-SY5Y cells. Both the in vivo and in vitro models were examined for injury. Markers of exosomes (CD63, HSP70 and TSG101) were assessed by immunohistochemistry, western blot analysis and flow cytometry. Exosomes were extracted from both animal serum and HUVEC culture medium and identified by electron microscopy. They investigated the role of endothelial cell-derived exosomes in the proliferation, apoptosis, cell cycle, migration and invasion of I/R-injured SH-SY5Y cells. In addition, apoptosis-related molecules caspase-3, Bax and Bcl-2 were detected. RIP was determined to increase the number of exosomes and the expression levels of CD63, HSP70 and TSG101 in plasma, but not in brain hippocampal tissue. The size of exosomes released after I/R in HUVECs was similar to the size of exosomes released in rats subjected to RIP. Endothelial cell-derived exosomes partly suppressed the I/R-induced cell cycle arrest and apoptosis, and inhibited cell proliferation, migration and invasion in SH-SY5Y nerve cells. Endothelial cell-derived exosomes directly protect nerve cells against I/R injury, and are responsible for the protective role of RIP in I/R.

Gestational diabetes mellitus is associated with increased pro-migratory activation of vascular endothelial growth factor receptor 2 and reduced expression of vascular endothelial growth factor receptor 1.

Placentas from gestational diabetes mellitus (GDM) are often hypervascularized; however, participation of vascular endothelial growth factor (VEGF) and its receptors in this placental adaptation is unclear. We aimed to test whether changes in phosphorylation of tyrosine 951 or tyrosine 1175 (pY951 or pY1175) of the vascular endothelial growth factor receptor 2 (KDR) are associated with the proangiogenic state observed in placentas from GDM. We obtained placental samples from women with normal pregnancies (n = 24) or GDM (n = 18). We measured the relative expression of markers for endothelial cell number (CD31, CD34), VEGF, vascular endothelial growth factor receptor 1 (Flt-1), KDR, pY951 and pY1175 of KDR in placental homogenate. Immunohistochemistry of placental blood vessels were performed using CD34. Proliferation and migration of human umbilical vein endothelial cells (HUVEC) obtained from normal pregnancy and GDM were determined in absence or presence of conditioned medium (CM) harvested from GDM or normoglycemic HUVEC cultures. GDM was associated with more CD31 and CD34 protein compared to normal pregnancy. High number, but reduced area of placental blood vessels was found in GDM. Reduced Flt-1 levels (mRNA and protein) are associated with reduced KDR mRNA, but higher KDR protein levels in placentas from GDM. No significant changes in Y951-or Y1175-phosphorylation of KDR in placentas from GDM were found. GDM did not alter proliferation of HUVECs, but enhanced migration. Conditioned medium harvested from GDM HUVEC cultures enhanced KDR protein amount, tube formation capacity and cell migration in HUVEC isolated from normoglycemic pregnancies. The data indicate that GDM is associated with reduced expression of Flt-1 but high pro-migratory activation of KDR reflecting a proangiogenic state in GDM.

Total flavones of abelmoschus manihot enhances angiogenic ability both in vitro and in vivo.

Angiogenesis is a process of new blood vessel formation from pre-existing vessels. It is a normal and vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. Total flavones of Abelmoschus manihot (TFA) are the major constituents of the traditional Chinese herb Abelmoschus manihot L. Medic. The aim of this study is to investigate the effect of TFA on angiogenic ability using human umbilical vein endothelial cells (HUVECs) in vitro and chick chorioallantoic membrane (CAM) in vivo. HUVECs were treated with TFA at different concentrations. Cell viability, cell cycle progression, cell apoptosis, cell migration and tubular formation were investigated. The expression of vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR, VEGFR-2) was examined by immunohistochemistry to identify mechanism of action of TFA. CAM model was used to evaluate the effect of TFA on angiogenesis in vivo. Our results showed that TFA promoted HUVECs proliferation in a dose- and time-dependent manner. It increased HUVECs migratory ability and the number of tubular structure, promoted vessel formation in HUVECs culture and CAM model. Furthermore, TFA treatment resulted in a decrease in cell apoptosis and enhanced the expression of VEGF and KDR. Taken together, TFA, as the major active component isolated from the traditional Chinese herb Abelmoschus manihot L. Medic, could enhance angiogenic ability of HUVECs in vitro and CAM in vivo. TFA may be used in the treatment of wound healing and ischemic/reperfusion injuries.

LPS, Oleuropein and Blueberry extracts affect the survival, morphology and Phosphoinositide signalling in stimulated human endothelial cells.

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes' expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.

Isolation Methods and Culture Conditions of Human Umbilical Vein Endothelial Cells from Malaysian Women

Human umbilical vein endothelial cell (HUVEC) isolated from umbilical cord is widely used as endothelial cell model. However, HUVEC has been characteristically hard to maintain and showed molecular heterogeneity depending on the umbilical cord donors. Commercial HUVEC is commonly derived from European and Caucasian population which have different molecular characteristics from Asian women. This study aimed to optimize the isolation and culture condition of HUVEC using combinations of growth factors and extracellular matrix components so that the isolated HUVEC will purely represent the population under study. Umbilical cords were obtained from women post-labour. Different incubation times and digestive enzymes were used during endothelial cells isolation process. The culture conditions were optimized based on the coating materials and the media supplements. The results showed that 0.1% collagenase for 40 min incubation was the optimal isolation condition of HUVEC. HUVEC grown in 0.2% gelatin coated plate with 10% heat-inactivated fetal calf serum showed higher proliferative capacity and reduced cell death compared to other conditions (p<0.05). The results generated from this study provide a basic protocol of HUVEC isolation and culture conditions in order to generate working endothelial cell populations purely represent the Malaysian population.

Endothelial cells microparticle-associated protein disulfide isomerase promotes platelet activation in metabolic syndrome.

BackgroundMetabolic syndrome (MetS) is a common challenge in the world, and the platelet activation is enhanced in MetS patients. However, the fundamental mechanism that underlies platelet activation in MetS remains incompletely understood. Endothelial cells are damaged seriously in MetS patients, then they release more endothelial microparticles (EMPs). After all, whether the EMPs participate in platelet activation is still obscure. If they were, how did they work?ResultsWe demonstrated that the levels of EMPs, PMPs (platelet derived microparticles) and microparticle-carried-PDI activity increased in MetS patients. IR endothelial cells released more EMPs, the EMP-PDI was more activated. EMPs can enhance the activation of CD62P, GPIIb/IIIa and platelet aggregation and this process can be partly inhibited by PDI inhibitor such as RL90 and rutin. Activated platelets stimulated by EMPs expressed more PDI on cytoplasm and released more PMPs.Materials and MethodsWe obtained plasma from 23 MetS patients and 8 normal healthy controls. First we built insulin resistance (IR) model of human umbilical vein endothelial cells (HUVECs), and then we separated EMPs from HUVECs culture medium and used these EMPs to stimulate platelets. Levels of microparticles, P-selectin(CD62P), Glycoprotein IIb/IIIa (GPIIb/IIIa) were detected by flow cytometry and levels of EMPs were detected by enzyme-linked immunosorbent assay (ELISA). The protein disulfide isomerase (PDI) activity was detected by insulin transhydrogenase assay. Platelet aggregation was assessed by turbidimetry.ConclusionEMPs can promote the activation of GPIIb/IIIa in platelets and platelet aggregation by the PDI which is carried on the surface of EMPs.

Multilayered Hollow Tubes as Blood Vessel Substitutes.

The available therapies for cardiovascular pathologies often require the replacement of diseased vascular grafts. However, the current blood vessel substitutes are unsuitable for small-diameter blood vessel replacements. Herein, we propose the creation of multilayered hollow tubes as blood vessel substitutes. Hollow tubes were obtained by building-up multilayers of marine-derived polysaccharides (i.e., chitosan and alginate) on sacrificial tubular templates using layer-by-layer technology and template leaching. A cross-linking degree of ≈59% was achieved using genipin, which is reflected in an increase of the mechanical properties and a decrease of the water uptake. To further improve the cell adhesive properties of the multilayers, fibronectin (FN) was immobilized on the surface of the hollow tubes. The in vitro biological performance of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) was assessed. In addition, to perform the culture of HUVECs on the inner side and the HASMCs on the outer side of the tubes, an in-house developed apparatus was created that allowed us to feed cells with their respective culture medium. The developed hollow tubes were shown to be a suitable structure to promote cell adhesion, spreading, and proliferation. It is our belief that the creation of these functional structures will open a new research field in order to develop innovative multilayered tubular structures for cardiovascular TE applications.

Induction of endothelial cell proliferation and von Willebrand factor expression and secretion by leukemic plasma of patients with chronic lymphocytic leukemia before and after inhibition of NF-κB.

Although certain evidence has indicated a role for angiogenesis in the pathophysiology of hematopoietic malignancies, its role in chronic lymphocytic leukemia (CLL) prognosis is yet to be defined. To our knowledge, the effects of CLL plasma on cell culture have not been addressed. Therefore, we investigated the effects of CLL plasma on cell cycle regulation and von Willebrand factor (vWF) secretion, and expression in human umbilical vein endothelial cell cultures (HUVECs). Since nuclear factor-kappa B (NF-κB) transcription factor has been a therapeutic target for treatment of cancer, we inhibited NF-κB using small interfering RNA to clarify if there is a role for this factor in probable effects. The cells were treated with the plasma of patients with CLL. Subsequently, cell cycle phase distribution, vWF secretion, expression, and storage were detected using ELISA, flow cytometry, and immunohistochemical staining. In addition, NF-κB was inhibited using small interfering RNA. Plasma treatment promoted cell cycle progression by decreasing the cell number in G1 phase, while increasing the cell number in S phase and G2M phase. A significant increase of vWF expression, secretion, and storage was found, associated with the vWF levels of patients' plasma. We found that induction of cell cycle promotion, but not vWF expression and secretion, was partially suppressed by this inhibition. We found that endothelial cell cycle and vWF expression and secretion affected by CLL plasma and NF-κB play a role in the former. These findings would be useful for understanding the prognostic importance of plasma angiogenic factor levels in CLL.

Dual-function synthetic peptide derived from BMP4 for highly efficient tumor targeting and antiangiogenesis.

Angiogenesis plays a critical role in the growth and metastasis of cancer, and growth factors released from cancer promote blood-vessel formation in the tumor microenvironment. The angiogenesis is accelerated via interactions of growth factors with the high-affinity receptors on cancer cells. In particular, heparan sulfate proteoglycans (HSPGs) on the surface of cancer cells have been shown to be important in many aspects of determining a tumor’s phenotype and development. Specifically, the regulation of the interactions between HSPGs and growth factors results in changes in tumor progression. A peptide with heparin-binding (HBP) activity has been developed and synthesized to inhibit tumor growth via the prevention of angiogenesis. We hypothesized that HBP could inhibit the interaction of growth factors and HSPGs on the surface of cancer cells, decrease paracrine signaling in endothelial cells (ECs), and finally decrease angiogenesis in the tumor microenvironment. In this study, we found that HBP had antiangiogenic effects in vitro and in vivo. The conditioned media obtained from a breast cancer cell line treated with HBP were used to culture human umbilical vein ECs (HUVECs) to evaluate the antiangiogenic effect of HBP on ECs. HBP effectively inhibited the migration, invasion, and tube formation of HUVECs in vitro. In addition, the expressions of angiogenesis-mediating factors, including ERK, FAK, and Akt, were considerably decreased. HBP also decreased the levels of invasive factors, including MMP2 and MMP9, secreted by the HUVECs. We demonstrated significant suppression of tumor growth in a breast cancer xenograft model and enhanced distribution of HBP at the site of tumors. Taken together, our results show that HBP has antiangiogenic effects on ECs, and suggest that it may serve as a potential antitumor agent through control of the tumor microenvironment.

Dimethyl sulfoxide attenuates nitric oxide generation via modulation of cationic amino acid transporter-1 in human umbilical vein endothelial cells

Dimethyl sulfoxide (DMSO) is a solvent that is commonly used in medicine. Conflicting data exist as to its effects on endothelial function. Endothelial cell dysfunction (ECD) is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1), the specific arginine transporter for eNOS, has been shown to modulate eNOS activity. We hypothesize that DMSO inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. We studied the effect of DMSO on arginine transport, NO2/NO3 generation as an index of NO production, as well as CAT-1 and Protein Kinase C alpha (PKC-α) (CAT-1 inhibitor) protein expression in human umbilical vein endothelial cell cultures (HUVECs). DMSO 2.5% and 3.5% (v/v) significantly attenuated arginine transport, a phenomenon which was prevented by co-incubation with l-arginine (1 mM). The aforementioned findings were accompanied by a decrease in NO2/NO3 generation. DMSO significantly increased the abundance of phosphorylated CAT-1 (the inactive form) and phosphorylated PKC-α protein, an effect that was attenuated by l-arginine. GO 6976 (PKC-α antagonist) prevented the decrease in arginine transport caused by DMSO. DMSO also induced profound transient morphological changes in HUVECs' structure but these were not related to its effect on arginine transport. In conclusion, DMSO inhibits NO generation by endothelial cells through modulation of CAT-1 activity.

Artesunate inhibits type I interferon-induced production of macrophage migration inhibitory factor in patients with systemic lupus erythematosus.

Macrophage migration inhibitory factor (MIF) is a key regulator of both atherosclerosis and systemic lupus erythematosus (SLE), yet factors leading to its overproduction remain unclear. To explore regulation of MIF in SLE, we studied effects and potential mechanisms of type I interferon (IFN) and artesunate (ART), an antimalarial agent extracted from Chinese herbs, on levels of MIF. Serum and peripheral blood cells from SLE patients and healthy controls were measured for MIF levels by ELISA and type I IFN-inducible gene expressions by real-time PCR, respectively, and assessed for associations by Spearman correlation. ART was added to human umbilical vein endothelial cell (HUVEC) cultures with or without prior IFNα-1b stimulation and to SLE peripheral blood mononuclear cell (PBMC) cultures. Protein levels of STATs and phosphorylated (p-) STATs in HUVECs were determined by Western blotting. Serum MIF levels were elevated in SLE patients and positively associated with disease activity (r = 0.86, p < 0.0001), accumulated damage (r = 0.34, p < 0.05), and IFN scores in SLE PBMCs (r = 0.74, p = 0.0002). The addition of IFNα-1b promoted MIF production in a time- and dose-dependent manner in HUVEC cultures. ART could inhibit expressions of IFN-inducible genes (LY6E and ISG15) in both HUVEC and SLE PBMC cultures, and suppress MIF production and over-expression of p-STAT1, but not p-STAT3 or STAT5, induced by IFNα-1b stimulation. IFNγ-induced expression of p-STAT1 in HUVECs was not inhibited by ART. MIF could be regulated by type I IFN in SLE patients. ART counteracts the effect of IFNα to inhibit MIF production by blocking STAT1 phosphorylation and thus may have therapeutic potential for SLE-associated atherosclerosis.

Human endothelial cells hollow fiber membrane bioreactor as a model of the blood vessel for in vitro studies.

Human endothelial cells are used in experimental models for studying in vitro pathophysiological mechanisms of different diseases. We developed an original bioreactor, which can simulate human blood vessel, with capillary polysulfone membranes covered with the human umbilical vein endothelial cells (HUVECs) and we characterized its properties. The elaborated cell seeding and culturing procedures ensured formation of a confluent cell monolayer on the inside surface of capillaries within 24h of culturing under the shear stress of 6.6 dyn/cm(2). The optimal density of cells to be seeded was 60,000 cells/cm(2). Labeling HUVECs with carboxyfluorescein succinimidyl ester (CFSE) did not influence cells' metabolism. Flow cytometry-based analysis of HUVECs stained with CFSE demonstrated that in a presence of the shear stress cells' proliferation was much inhibited (after 72h proliferation index was equal to 1.9 and 6.2 for cultures with and without shear stress, respectively) and the monolayer was formed mainly due to migration and spreading of cells that were physiologically elongated in a direction of the flow. Monitoring of cells' metabolism showed that HUVECs cultured in a presence of the shear stress preferred anaerobic metabolism and they consumed 1.5 times more glucose and produced 2.3 times more lactate than the cells cultured under static conditions. Daily von Willebrand factor production by HUVECs was near 2 times higher in a presence of the shear stress. The developed model can be used for at least 3days in target studies under conditions mimicking the in vivo state more closely than the static HUVEC cultures.