Cultures Of Umbilical Vein Endothelial Cells Research Articles

Prolyl carboxypeptidase purified from human placenta: its characterization and identification as an apelin-cleaving enzyme

BackgroundThe proteolytic regulation of peptides involved in feeding behavior is poorly understood. Prolyl carboxypeptidase (PRCP) is particularly known for its role in body weight control by converting the anorexigenic peptide, α-melanocyte-stimulating hormone 1–13 into the inactive form 1–12. The purpose of this study was to characterize purified human PRCP, to investigate its substrate specificity and to discover novel substrates linked to obesity. Pyroglutamated apelin-13, ghrelin, enterostatin and obestatin were investigated since these are feeding-regulating peptides with potential cleaving sites for PRCP. MethodsPRCP was purified from human placenta and identified using western blotting and mass spectrometry. The kinetic parameters of purified and commercially available PRCP for known and potential peptide substrates were determined and compared using a RP-HPLC activity assay, isothermal titration calorimetry and mass spectrometry. ResultsPRCP was purified 575-fold from human placenta and succesfully identified as human lysosomal Pro-X carboxypeptidase. Purified and recombinant PRCP had similar substrate specificity with angiotensin III as the substrate of preference. Pyroglutamated apelin-13 was observed to be a novel substrate for human PRCP in vitro and PRCP-dependent cleavage was shown in a human umbilical vein endothelial cell culture experiment. Other potential substrates e.g. obestatin, ghrelin and enterostatin were not hydrolyzed by PRCP. ConclusionThese results show that placenta is a good source of human PRCP and that PRCP removes the C-terminal phenylalanine from pyroglutamated apelin-13. For the first time, PRCP is identified as an apelin-cleaving enzyme. This finding adds evidence to the hypothesis that PRCP plays a role in energy homeostasis.

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.

Abstract 227: Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma

Abstract Background: The tissue microenvironment plays a major role in tumor development and recurrence. It is known that leptin secreted by cancer and adipose cells can modify the tumor stroma, which is composed of immune, fibroblast, and endothelial cells (EC). Leptin affects both normal and cancer associated fibroblasts. We have previously shown that leptin can modify EC directly via an increase of angiogenic features; which was linked to VEGFR signaling and Notch expression independent of VEGF. It is hypothesized that leptin induces a communication between fibroblast and endothelial cells via induction of Notch signaling, which may be related to exosome production. Methods: Non-expressing Ob-R fibroblasts, and fibroblasts transfected with Ob-R plasmid, were used to investigate leptin's actions on Notch activation. Fibroblasts were challenged with leptin (0.6, 1.2, 6.2 nM) and an inhibitor of Notch signaling activation (DAPT). Fibroblast culture supernatants were added to and human umbilical vein endothelial cells (HUVEC) cultures. Notch proteins and RNA expression and molecular markers of activated fibroblasts and derived exosomes were determined via Western blot, RT-PCR, and multiplex analyses, respectively. EC angiogenic features were also determined. Results: Our findings suggest that leptin induces fibroblast activation, which triggers the expression of Notch and the development of angiogenic features by EC. Conclusions: A novel mechanism is proposed by which leptin promotes tumor stroma development via Notch activation and a crosstalk between fibroblasts and EC. Cancer therapies targeting leptin signaling could be a new strategy for interfering with cellular processes that promotes changes of tumor stroma towards a more aggressive tumor environment. Citation Format: Viola Lanier. Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 227.

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.

Anti-ribosomal-phosphoprotein autoantibodies penetrate to neuronal cells via neuronal growth associated protein, affecting neuronal cells in vitro.

Anti-ribosomal-phosphoprotein antibodies (anti-Ribos.P Abs) are detected in 10-45% of NPSLE patients. Intracerebroventricular administration of anti-ribosomal-P Abs induces depression-like behaviour in mice. We aimed to discern the mechanism by which anti-Ribos.P Abs induce behavioural changes in mice. Anti-Ribos.P Abs were exposed to human and rat neuronal cell cultures, as well as to human umbilical vein endothelial cell cultures for a control. The cellular localization of anti-Ribo.P Abs was found by an immunofluorescent technique using a confocal microscope. Identification of the target molecules was undertaken using a cDNA library. Immunohistochemistry and an inhibition assay were carried out to confirm the identity of the target molecules. Neuronal cell proliferation was measured by bromodeoxyuridine, and Akt and Erk expression by immunoblot. Human anti-Ribos.P Abs penetrated into human neuronal cells and rat hippocampal cell cultures in vitro, but not to endothelial cells as examined. Screening a high-content human cDNA-library with anti-Ribos.P Abs identified neuronal growth-associated protein (GAP43) as a target for anti-Ribos.P Abs. Ex vivo anti-Ribos.P Abs bind to mouse brain sections of hippocampus, dentate and amygdala. Anti-Ribos.P Abs brain-binding was prevented by GAP43 protein. Interestingly, GAP43 inhibited in a dose-dependent manner the anti-Ribos.P Abs binding to recombinant-ribosomal-P0, indicating mimicry between the ribosomal-P0 protein and GAP43. Furthermore, anti-Ribos.P Abs reduced neuronal cell proliferation activity in vitro (P < 0.001), whereas GAP43 decreased this inhibitory activity by a factor of 7.6. The last was related to Akt and Erk dephosphorylation. Anti-Ribos.P Abs penetrate neuronal cells in vitro by targeting GAP43. Anti -Ribos.P Abs inhibit neuronal-cell proliferation via inhibition of Akt and Erk. Our data contribute to deciphering the mechanism for anti-Ribos.P Abs' pathogenic activity in NPSLE.

Blockade of CXC chemokine receptor 3 on endothelial cells protects against sepsis-induced acute lung injury

BackgroundCXCR3, a G-protein coupled chemokine receptor, has been shown to play a critical role in recruiting inflammatory cells into lungs in several studies. However, its roles in polymicrobial septic acute lung injury (ALI) is yet unknown. Therefore, the purpose of this study was to elucidate the protective effects of CXCR3 blockade on pulmonary microvascular endothelial cells (PMVECs) in septic ALI and explore potential mechanisms. Materials and methodsALI was induced by polymicrobial sepsis through cecal ligation and puncture surgery. The expression of CXCR3 on pulmonary microvascular endothelial cells was measured 24 h after cecal ligation and puncture surgery. In addition, the protective effects of neutralizing antibody were detected, including protein concentration, inflammation cell counts, lung ​wet-to-dry ratio, and lung damages. In human umbilical vein endothelial cells (HUVECs) culture condition, CXCR3 expression was measured after exposure to tumor necrosis factor-α. The permeability and apoptosis ratio were detected through CXCR3 gene silencing on HUVECs. The p38 mitogen-activated protein kinase (MAPK) was analyzed with Western blot. ResultsCXCR3 expression was upregulated both in vivo and in vitro. After CXCR3 neutralizing antibody administrated intraperitoneally, the protein concentration, inflammatory cell counts in BALF and lung ​wet-to-dry ratio were decreased significantly, as well as the lung tissue damages. In vitro, CXCR3 gene silencing inhibited tumor necrosis factor-α and CXCL10-induced hyperpermeability and apoptosis in HUVECs. In addition, p38 mitogen-activated protein kinase activation was essential for CXCR3-mediated apoptosis. ConclusionsCXCR3 blockade exerts protective effects on ALI at least partly by inhibiting endothelial cells apoptosis and decreasing the leakage of protein-rich fluid and inflammatory cells. Blockade of CXCR3 may be a promising therapeutic strategy for severe sepsis-induced ALI.

Thrombosis recanalization by paeoniflorin through the upregulation of urokinase‑type plasminogen activator via the MAPK signaling pathway.

Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogen-activated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzyme-linked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6-keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, D-dimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the anti-thrombosis pathways.

Paeonol enhances thrombus recanalization by inducing vascular endothelial growth factor165 via ERK1/2 MAPK signaling pathway.

Paeonol (2'-hydroxy-4'-methoxyacetophenone) is the major active compound of Mautan cortex and has been demonstrated to inhibit platelet aggregation in previous studies. The current study aimed to elucidate the underlying molecular mechanism of paeonol in recanalizing thrombi. The presence of indicators of prothrombotic state (PTS) in the serum of the model animals were determined by enzyme‑linked immunosorbent assay (ELISA) assay and the cytotoxicity of paeonol on human umbilical vein endothelial cell (HUVEC) cultures was estimated by 3‑(4,5 dimethylthiazol‑2‑yl)-2,5-diphenyltetrazolium bromide assay. The possible underlying signaling pathway involved in the interaction between paeonol and vascular endothelial growth factor165 (VEGF165) was investigated using western blotting. The levels of 6‑keto‑prostaglandinF1α, fibronectin, and VEGF165 in serum were significantly upregulated by the treatment of paeonol while the levels of fibrinogen, D‑dimer, and thromboxaneB2 were significantly downregulated (P<0.05). With increased paeonol concentration, the cell viability of HUVECs gradually decreased. The results of the western blot analysis demonstrated that paeonol increased the expression levels of phosphorylated‑extracellular signal‑regulated kinase (ERK1/2) and VEGF165 but had no marked effect on the expression level of ERK1/2. Paeonol has the potential to improve PTS and recanalize thrombi in animal models, which may be by the upregulation of VEGF165 via the ERK1/2 mitogen activated protein kinase signaling pathway. However, this positive effect depended on the concentration of paeonol used, an unsuitably high concentration of the compound exerted negative effects on the anti‑thrombosis signaling pathways.

Anti-angiogenic effects of ethanolic extract of Artemisia sieberi compared to its active substance, artemisinin

Angiogenesis plays a key role in tumor growth, invasion and metastasis of cancer diseases and therefore, the inhibition of angiogenesis can provide an important therapeutic approach in cancer diseases. This study was designed to compare the anti-angiogenic activities of the ethanolic extract of Artemisia sieberi Besser, Asteraceae, and its active substance, artemisinin in both in vitro and in vivo models. To compare cytotoxicity level of ethanolic extract of A. sieberi with artemisinin, different concentrations (1–100μg/ml) were tested using MTT assay on human umbilical vein endothelial cells. The anti-angiogenic properties of serial concentrations of ethanolic extract of A. sieberi and artemisinin were examined on human umbilical vein endothelial cells using a three-dimensional angiogenesis assay (in vitro model) and in the chick chorioallantoic membrane assay as in vivo model. The effects of ethanolic extract of A. sieberi and artemisinin were also tested on the expression of VEGFR-1, VEGFR-2 and CD34 genes using real-time PCR. Ethanolic extract of A. sieberi and artemisinin significantly (p<0.001) inhibited the angiogenesis in the human umbilical vein endothelial cells culture whilst the ethanolic extract of A. sieberi showed higher effect in a concentration-dependent fashion (p<0.001). The chick chorioallantoic membrane angiogenesis was also completely inhibited by ethanolic extract of A. sieberi at concentration of 33ng/100μl/egg. The gene expression analysis showed that the ethanolic extract of A. sieberi and artemisinin reduced the transcription of VEGFR-1, VEGFR-2 and CD34 genes in a concentration-dependent manner. This study demonstrated that the ethanolic extract of A. sieberi is strongly able to inhibit the angiogenesis in human umbilical vein endothelial cells and chick chorioallantoic membrane models compared to the artemisinin.

Cellular and molecular effects of metronomic vinorelbine and 4-O-deacetylvinorelbine on human umbilical vein endothelial cells.

Metronomic oral vinorelbine (VRL; Navelbine) was shown in clinical trials to yield sustainable antitumor activity possibly through antiangiogenic mechanisms. We investigated the effects of protracted low-dose VRL on human umbilical vein endothelial cells, compared with a conventional chemotherapy model. Human umbilical vein endothelial cell cultures were treated with different concentrations of VRL (0.001 nmol/l to 1 mmol/l) for 4, 24 and 96 h. The effects of different drug concentrations on cell growth, cell cycle, apoptosis and expression of the angiogenesis-modulating genes interleukin-8, cyclooxygenase-2, CD36 and peroxisome proliferator-activated receptor γ were assessed using the metronomic or conventional chemotherapy model. Apoptosis and cell-cycle effects were assessed by flow cytometry. Gene expression was measured at the transcript level by quantitative reverse transcriptase-PCR, protein expression by immunoblotting and levels of proteins secreted in the cell medium by enzyme-linked immunosorbent assay. Activation of the nuclear factor-κB pathway was investigated by immunoblot analysis of cytosolic and nuclear protein extracts. The half-maximal inhibitory concentrations (IC50) of VRL at 96 h were four orders lower compared with those after a 24-h exposure (1.23 nmol/l vs. 32 mmol/l for VRL). Drug concentrations at high nanomolar levels and above, which are relevant to conventional pulsatile dosing of VRL, induced a dose-dependent and nuclear factor-κB-related increase in proangiogenic interleukin-8 and cyclooxygenase-2 and a decrease in the thrombospondin-1 receptor CD36 and peroxisome proliferator-activated receptor γ at mRNA and protein levels. In contrast, the opposite was evident with protracted picomolar to low nanomolar concentrations (metronomic dosing). Our data provide experimental support for metronomic VRL by showing that a protracted low dose outperforms pulsed high-dose administration in inducing antiangiogenic effects in proliferating human endothelial cells.

Electrospun pure chitosan nanofibrous mats with high structural stability for dura mater regeneration

Event Abstract Back to Event Electrospun pure chitosan nanofibrous mats with high structural stability for dura mater regeneration Yuandong Dou1, Xianchang Sun1, Gang Guo1*, Jiahuan Dong1*, Meijiao Lu1* and Wencai Zhang1 1 Yantai Zhenghai Bio-tech Co., Ltd., China Introduction: Dura defect is a commonly encountered problem in spine- and neurosurgery. Numerous materials have been exploited as dural substitutes including autologous tissue and xenogenic materials, yet the majority still cannot meet the whole clinical requirements in dura repair[1]. Chitosan has been intensively explored as a suitable functional material for tissue regeneration. However, the successful fabrication of pure chitosan nanofibers with good structure stability through electrospinning still remains a great challenge[2]. Here, we reported the feasible preparation of pure electrospun chitosan nanofibers and their utilization for effective reconstruction of dura defect. The structural and functional performances of the chitosan dura substitutes were systemically investigated. Materials and Methods: Chitosan powders were added into the mixed solvents of hexafluoro-2-propanol and acetic acid (2 vol.%) with a 1.25% (w/v) concentration. The electrospinning process was conducted at feeding rate of 3.0 ml/h, voltage of 12.5 Kv and tip-to-collector distance of 10 cm. Structural stability evaluation was carried out through soaking in PBS for 24 h. In vitro cytocompatibility was evaluated through human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVEC) culture on chitosan nanofibrous mats. The cerebrospinal fluid leakage prevention and dura regeneration study were conducted through a rabbit duraplasty model. Results and Discussion: As can be seen from Figure 1, there are no obvious differences of fibrous microstructure and dimension size (insets of Fig. 1b, d) between the as-spun and soaked samples, indicating excellent structural integrity in aqueous solutions. The electrospun pure chitosan nanofibers were usually prepared using trifluroacetic acid (TFA) as solvents[3]. However, the resultant chitosan nanofibrous mats showed poor structural stability and would completely dissolve as soon as they contact neutral aqueous solutions because of the water-soluble salts formation between chitosan molecules and TFA[4]. The combination and proportion of HFP and acetic acid used in this study avoided the drawbacks of using TFA and assured the successful fabrication of pure stable chitosan nanofibers. Figure 1. SEM morphology of the as-spun chitosan nanofibers (a, b) and after 24 h soaking in PBS (c, d). Insets of b, d are the corresponding appearance photo, scale bars represent 1cm. As the main functional cells during the wound healing process, HDF and HUVEC attachment and proliferation were well supported when cultured on pure chitosan nanofibers, suggesting excellent in vitro cytocompatibility. The CSF leakage prevention operation showed no leakage of cranial methylene blue solution from the defect area covered by the chitosan nanofibers. The reconstruction results of chitosan nanofibrous substitutes on dura defect were shown in Figure 2. A freshly generated neodura-like connective tissue was found beneath the chitosan implants at the defect area after 8 weeks implantation. The histological results of the newly formed tissue showed a dense characteristic with a thick layer. The arrangement of constructed collagen fibrils within the newly formed neodura was in a highly ordered manner, which is almost the same as those of natural dura mater (Figure 2d). Figure 2. a) Chitosan nanofibers onlaid over the right durotomy site, the left site was set as blank. b) the circle indicates a newly formed neodura beneath the implants at week 8. c) Histological image of the neodura (ND) around the implanted chitosan nanofibers (CHN). d) TEM images of newly formed neodura, inset is the incised natural rabbit dura mater. Conclusion: Pure chitosan nanofibers with excellent structural stability were successfully prepared through electrospinning process without any post-treatments. This nanofibrous dura substitutes showed excellent in vitro cytocompatibility as to HDFs and HUVEC. Moreover, chitosan nanofibers displayed overall excellent performance in CSF leakage prevention and dura reconstruction, making them ideal candidates for dura substitutes.

In vitro study on the inhibitory effect of ketamine to LPS-induced apoptosis in HUVEC culture

ObjectiveTo determine the effect of lipopolysaccharide (LPS) exposure and treatment with ketamine to the levels of nitric oxide, intracellular calcium and caspase-3 of human umbilical vein endothelial cell culture. MethodsThis research used the human umbilical vein endothelial cell culture with LPS induction. ELISA was conducted to measure the levels of apoptosis markers such as nitric oxide, intracellular calcium and caspase-3. ResultsThe results showed significant elevation of all parameters after LPS induction. Ketamine addition resulted in various effects. Measurement of intracellular calcium and caspase-3 showed similar result, in which the inhibitory effect of ketamine was more effective when added at the same time as LPS. Vice versa, in nitric oxide measurement, the highest inhibitory effect of ketamine was obtained when added 6 h after LPS induction. ConclusionsAt least in part, ketamine was suggested to have inhibitory effect in apoptosis mechanism.

The effect of ketamine on the lipopolysaccharide-induced inflammation in in vitro culture of HUVEC

ABSTRACT Objective To report the effect of ketamine treatment on inflammatory cytokines induced by lipopolysaccharide. Methods Human umbilical vein endothelial cells culture was used in this research. The measurement of inflammatory cytokines was performed by using ELISA. Results The results showed various levels of inflammatory cytokines. The nuclear factor-κB results were unstable, but interleukin 6 and tumor necrosis factor-α showed similar results. The expressions of interleukin 6 and tumor necrosis factor-α decreased in all observation times. Conclusions In general, our results of human umbilical vein endothelial cells culture were supported by other researches related with the effect of ketamine in suppressing inflammatory effect induced by lipopolysaccharide. Ketamine could suppress inflammatory cytokines in all observation times.

Novel mechanism of gene transfection by low-energy shock wave.

Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression

Context: Bevacizumab (BEV) is a monoclonal antibody to vascular endothelial growth factor (VEGF) that ameliorates atheroma progression by inhibiting neovascularization.Objective: We aimed to determine whether BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US) and whether the released BEV inhibits VEGF expression by endothelial cells in vitro.Materials and methods: BEV-ELIP samples were subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 min. We assessed release of BEV with a direct ELISA and with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method. Human umbilical vein endothelial cell (HUVEC) cultures were stimulated to express VEGF by 10 nM phorbol-12-myristate 13-acetate (PMA). Cell-associated VEGF levels were determined using a cell-based ELISA.Results: Overall, US caused an additional 100 µg of BEV to be released or exposed per BEV-ELIP aliquot within 60 min BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US. Also, US-treated BEV-ELIP inhibited HUVEC proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US.Discussion and conclusion: We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody.

Effects of PTEN on the longevity of cultured human umbilical vein endothelial cells: the role of antioxidants.

Ageing is a major cause of illness, disease and mortality, mainly due to the shortening of telomeres, resulting in cells undergoing senescence and apoptosis. Increasing autophagy and the levels of antioxidants removes oxidants that cause DNA and telomere damage, thus reducing the rate at which telomeres shorten, resulting in a longer cellular lifespan. Phosphatase and tensin homolog (PTEN) has been shown to increase the lifespan of organisms by upregulating pathways involved in DNA damage repair, autophagy/antioxidants. The aim of this study was to investigate the effects of the overexpression of PTEN on the longevity of human cell cultures by examining the increase in antioxidant potential. Human umbilical vein endothelial cell (HUVEC) cultures were transfected with PTEN plasmids using lipofectamine. An assay was performed to quantify the protein levels of PTEN and the antioxidant potential of the cell cultures. The cell cultures were maintained until senescence occurred in order to determine longevity. The results of each assay were then compared and correlated with each other and with the longevity of the cells. The transfected cultures showed a significant increase in PTEN protein levels, total antioxidant potential and longevity (all P-values <0.001) compared with the non-transfected cell cultures. The correlation coefficient between cell longevity and PTEN levels was 0.8727; and the correlation coefficient between cell longevity and antioxidant potential was 0.6564. The successful transfection of PTEN led to an increase in PTEN levels, antioxidant potential and an increased cellular longevity. This study demonstrates that there is a potential for PTEN to be used to extend human longevity. This can lay the foundation for further studies to be carried out on humans involving PTEN and longevity.

A Cisplatin Derivative that Inhibits Collagen Fibril‐Formation in vitro

Using an in vitro random screening of small-molecule compounds, we discovered cis-diamminedichloroplatinum(II) (cisplatin), an anticancer agent, as a potential inhibitor of collagen fibril-formation. The inhibitory effect was found only when cisplatin was dissolved in dimethylsulphoxide (DMSO), indicating that the active species were cisplatin derivatives formed in the DMSO solution. The cisplatin derivatives inhibited the formation of collagen fibrils in vitro without affecting the triple-helical conformation of the collagen molecules. Incubation with the cisplatin solution in DMSO also inhibited in situ deposition of collagen fibrils in a human umbilical vein endothelial cell (HUVEC) culture. In addition, the derivatization of cisplatin in DMSO abolished the cytotoxicity of the original compound. The platinum complex was further revealed to interact with specific sites on the collagen triple helix, and the binding sites were suggested to contain His and/or Met residues. Mass spectrometry analysis of the cisplatin solution in DMSO and a structure-activity relationship study strongly suggested that the active compound is [Pt(NH3 )2 (Cl)(DMSO)](+) . This platinum complex will be useful for investigating molecular mechanisms of collagen self-assembly and for drug development for the treatment of fibrotic diseases.

ADIPONECTIN LIMITS MONOCYTIC MICROPARTICLE-INDUCED INFLAMMASOME ACTIVATION AND ENDOTHELIAL DYSFUNCTION

Monocytic microparticles (mono-MPs) are small vesicles (100-1000 nm) that are shed from monocytes following cellular activation or apoptosis. Levels of mono-MPs are heightened in the inflammatory state and mono-MPs have been reported to play a fundamental role at all stages of the atherothrombotic process. Alterations in monocyte-endothelial cell (EC) interaction mediate immune activation and propagate inflammation within the developing atheroma. Adiponectin is an anti-inflammatory adipokine that primes monocyte differentiation into anti-inflammatory M2 macrophages vs. pro-inflammatory M1 macrophages and inhibits EC activation thereby retarding plaque development. The goal of this work was to uncover the mechanistic root underlying the pathophysiological and clinical benefits of adiponectin. Specifically, we hypothesized that mono-MPs trigger NLRP3 inflammasome-dependent EC activation and adiponectin inhibits this phenomenon, in part, through limiting mono-MP production and activation. Mono-MPs were characterized using electron microscopy, flow cytometry and NanoSight microscopy. Exosomes were excluded based on size and absence of the MP-specific marker phosphatidylyserine. Studies were conducted with THP-1 monocytes and peritoneal monocytes isolated from adiponectin knockout (Adipoq-/-) mice. We evaluated the effects of lipopolysaccharide (LPS; 5 μg/ml, 24 h) and recombinant adiponectin (10 μg/ml) on mono-MP production, expression of the NLRP3 inflammasome and its components, and key downstream inflammatory factors. We also studied how MPs alter the expression of critical adhesion molecules and inflammatory transcription factors in human umbilical vein endothelial cell (HUVEC) cultures. Inflammasome activation was examined in mono-MPs derived from THP-1 cells. Imaging analysis confirmed that the mono-MPs isolated were sized 100-1000 nm and expressed phosphatidylyserine. Absence of exosomes in the final mono-MP samples was further confirmed by a lack of CD63 expression. LPS concentration-dependently increased MP generation by THP-1 monocytes. Adiponectin suppressed MP release in untreated and LPS-stimulated monocytes. LPS-treated mono-MPs exhibited a marked upregulation in the NLRP3 inflammasome and its components - ASC, caspase-1, pro-IL1β and IL1β. Adiponectin attenuated these changes. Mono-MP-HUVEC co-cultures revealed significant increases in markers of endothelial activation (ICAM-1, VCAM-1) and inflammatory transcription factors (p-IkB, p-NFkB and p-ERK-1). Adiponectin inhibited these outcomes. Compared to monocytes from Adipoq+/+ mice, those from Adipoq-/- littermates showed a ∼6-fold greater production of MPs (Cd11b+/Ly6Chi/AnnexinV+) following LPS stimulation. We report a novel mechanism through which mono-MPs sustain NLRP3 inflammasome activation and endothelial dysfunction. These data reveal a previously unknown atheroprotective mechanism for adiponectin that may account for the inverse association observed between circulating adiponectin levels and the risk of diabetes and obesity.

In vitro and in vivo anti-angiogenic effects of hydroxyurea

Hydroxyurea (HU), or hydroxycarbamide, is used for the treatment of some myeloproliferative and neoplastic diseases, and is currently the only drug approved by the FDA for use in sickle cell disease (SCD). Despite the relative success of HU therapy for SCD, a genetic disorder of the hemoglobin β chain that results in red-cell sickling, hemolysis, vascular inflammation and recurrent vasoocclusion, the exact mechanisms by which HU actuates remain unclear. We hypothesized that HU may modulate endothelial angiogenic processes, with important consequences for vascular inflammation. The effects of HU (50–200μM; 17–24h) on endothelial cell functions associated with key steps of angiogenesis were evaluated using human umbilical vein endothelial cell (HUVEC) cultures. Expression profiles of the HIF1A gene and the miRNAs 221 and 222, involved in endothelial function, were also determined in HUVECs following HU administration and the direct in vivo antiangiogenic effects of HU were assessed using a mouse Matrigel-plug neovascularization assay. Following incubation with HU, HUVECs exhibited high cell viability, but displayed a significant 75% inhibition in the rate of capillary-like-structure formation, and significant decreases in proliferative and invasive capacities. Furthermore, HU significantly decreased HIF1A expression, and induced the expression of miRNA 221, while downregulating miRNA 222. In vivo, HU reduced vascular endothelial growth factor (VEGF)-induced vascular development in Matrigel implants over 7days. Findings indicate that HU is able to inhibit vessel assembly, a crucial angiogenic process, both in vitro and in vivo, and suggest that some of HU's therapeutic effects may occur through novel vascular mechanisms.

Endogenous controls in human umbilical vein endothelial cells under metabolic and oxidative stress

Background Gene expression studies on the effect of hypoxia and hyperglycemia using human umbilical vein endothelial cell (HUVEC) cultures are of particular interest in cardiovascular disease and diabetes. Normalization of gene expression data refers to the comparison of expression values using endogenous control that is steady across independent experimental conditions, a crucial step for gene expression studies. The endogenous controls for experiments involving hyperglycemia, hypoxia and a combination of the two have not been identified before in HUVEC. Our objective was to identify endogenous controls that are stable under oxidative (hypoxia) and metabolic stress (hyperglycemia) in HUVEC.