Human Umbilical Vein Endothelial Cells Membrane Research Articles

A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.

Endothelial CLICs (chloride intracellular channel proteins) CLIC1 and CLIC4 are required for the GPCRs (G-protein-coupled receptors) S1PR1 (sphingosine-1-phosphate receptor 1) and S1PR3 to activate the small GTPases Rac1 (Ras-related C3 botulinum toxin substrate 1) and RhoA (Ras homolog family member A). To determine whether CLIC1 and CLIC4 function in additional endothelial GPCR pathways, we evaluated CLIC function in thrombin signaling via the thrombin-regulated PAR1 (protease-activated receptor 1) and downstream effector RhoA. We assessed the ability of CLIC1 and CLIC4 to relocalize to cell membranes in response to thrombin in human umbilical vein endothelial cells (HUVEC). We examined CLIC1 and CLIC4 function in HUVEC by knocking down expression of each CLIC protein and compared thrombin-mediated RhoA or Rac1 activation, ERM (ezrin/radixin/moesin) phosphorylation, and endothelial barrier modulation in control and CLIC knockdown HUVEC. We generated a conditional murine allele of Clic4 and examined PAR1-mediated lung microvascular permeability and retinal angiogenesis in mice with endothelial-specific loss of Clic4. Thrombin promoted relocalization of CLIC4, but not CLIC1, to HUVEC membranes. Knockdown of CLIC4 in HUVEC reduced thrombin-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Knockdown of CLIC1 did not reduce thrombin-mediated RhoA activity but prolonged the RhoA and endothelial barrier response to thrombin. Endothelial-specific deletion of Clic4 in mice reduced lung edema and microvascular permeability induced by PAR1 activating peptide. CLIC4 is a critical effector of endothelial PAR1 signaling and is required to regulate RhoA-mediated endothelial barrier disruption in cultured endothelial cells and murine lung endothelium. CLIC1 was not critical for thrombin-mediated barrier disruption but contributed to the barrier recovery phase after thrombin treatment.

The Complement System Mediates COVID-19 Endothelial Cell Injury, Modifying Cell Permeability

Background Coronavirus Disease 2019 (COVID-19) is a (SARS-CoV-2)-mediated respiratory disease. The severity of COVID-19 has been classified as mild, moderate, severe and critical per the WHO-China Joint Mission. Among a total of 72 314 case records from the Chinese Center for Disease Control and Prevention, approximately 81% of COVID-19 cases were defined as mild, 14% of as severe, and 5% as critical. The overall hospital mortality of COVID-19 cases is approximately 15-20%, but it is up to 40-49% among critical cases requiring intensive care unit admission. The complement system, is a key component of the innate immune response, consists of more than 50 soluble and membrane-bound proteins that provide defense against microbes and mediate inflammatory responses. The activation of the complement system involves a series of enzymatic transformations, where C3b acts as a mediator. The final step of the complement cascade is the formation of a membrane attack complex (MAC, C5b-9) on the plasma membrane (PM) of the cells. Several pathologies including thrombotic, inflammatory, autoimmune and age-related diseases are associated with abnormal complement activation. There is evidence linking the activation of the complement system and endothelial injury to organ damage and complications that increase the risk of mortality in COVID-19. We hypothesize that endothelial cell injury resulting from complement activation contributes to COVID-19-associated vascular injury and organ damage. Aims To identify differences in complement activation associated with mild and severe forms of COVID-19 To determine the role of differential complement activation by COVID-19 patient sera in mediating endothelial cell damage. Methodology Clinical information, and sera from SARS-CoV-2 positive patients with mild and severe COVID-19 were obtained from the Canadian COVID-19 Prospective Cohort Study (CANCOV) to identify complement activation and endothelial cell (EC) injury. Complement activation was determined using a previously established protocol (Noris et al., 2017). Briefly, primary human umbilical vein endothelial cells (HUVECs) were treated with adenosine diphosphate (ADP), followed by treatment with healthy control or COVID-19 patient serum. To confirm the biological activity of complement, we measured the deposition of C3b and C5b-9 on the plasma membrane of HUVECs via immunofluorescence (IF) on cells exposed to healthy or patient sera. In addition, a permeability assay using a transwell model was used to measure the changes in permeability induced by healthy or patient sera via complement activation. Briefly, HUVECs were seeded on the top part of the insert, allowing them to form a monolayer. Monolayer integrity was assessed by imaging cells stained with Hoechst (nuclear stain) and phalloidin (cytoskeleton stain) on the day of the experiment, as well as before and after incubation with healthy or patient sera. Results We were able to detect complement activation/deposition when exposing the cells to COVID-19 sera. As for C3b deposition, cells exposed to patient sera showed no difference compared to those treated with sera from healthy donors. However, we observed increased C5b-9 deposition on the PM of cells treated with patient sera compared to those treated with sera from healthy donors, with further increased deposition when cells are treated with deceased patient sera. We observed a trend demonstrating increased permeability when cells are treated with deceased COVID-19 patient sera compared to healthy donors. Cellular permeability is significantly lower in those cells treated sera from patients with mild or severe COVID-19. Conclusion There is a differential biological activity of complement on endothelial cells depending on the severity of COVID-19, this complement activation leads to an increase in cell permeability. While more research needs to be done, these results contribute to a better understanding of the consequences of complement activation on endothelium in COVID-19.

Cell membrane fluidity and ROS resistance define DMSO tolerance of cryopreserved synovial MSCs and HUVECs

ObjectivesSynovial mesenchymal stem cells (MSCs) have high freeze–thaw tolerance, whereas human umbilical vein endothelial cells (HUVECs) have low freezing tolerance. The differences in cell type-specific freeze–thaw tolerance and the mechanisms involved are unclear. This study thus aimed to identify the biological and physical factors involved in the differences in freeze–thaw tolerance between MSCs and HUVECs.Materials and methodsFor biological analysis, MSC and HUVEC viability after freeze-thawing and alteration of gene expression in response to dimethyl sulfoxide (DMSO, a cryoprotectant) were quantitatively evaluated. For physical analysis, the cell membrane fluidity of MSCs and HUVECs before and after DMSO addition was assessed using a histogram for generalized polarization frequency.ResultsHUVECs showed lower live cell rates and higher gene expression alteration related to extracellular vesicles in response to DMSO than MSCs. Fluidity measurements revealed that the HUVEC membrane was highly fluidic and sensitive to DMSO compared to that of MSCs. Addition of CAY10566, an inhibitor of stearoyl-coA desaturase (SCD1) that produces highly fluidic desaturated fatty acids, decreased the fluidity of HUVECs and increased their tolerance to DMSO. The combination of CAY10566 and antioxidant glutathione (GSH) treatment improved HUVEC viability from 57 to 69%. Membrane fluidity alteration may thus contribute to pore-induced DMSO influx into the cytoplasm and reactive oxygen species production, leading to greater cytotoxicity in HUVECs, which have low antioxidant capacity.ConclusionsDifferences in freeze–thaw tolerance originate from differences in the cell membranes with respect to fluidity and antioxidant capacity. These findings provide a basis for analyzing cell biology and membrane-physics to establish appropriate long-term preservation methods aimed at promoting transplantation therapies.

Flagellar Hook Protein FlgE Induces Microvascular Hyperpermeability via Ectopic ATP Synthase β on Endothelial Surface

We previously demonstrated the immunostimulatory efficacy of Pseudomonas aeruginosa flagellar hook protein FlgE on epithelial cells, presumably via ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE–ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both in vitro and in vivo. The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.

The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H2O2 on von Willebrand Factor Exocytosis

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel–Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel–Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

Activation of Piezo1 by ultrasonic stimulation and its effect on the permeability of human umbilical vein endothelial cells

The acoustic radiation forces produced by ultrasonic stimulation induce shear stress on objects in the acoustic field. Piezo1, a mechanosensitive ion channel protein that is expressed on the plasma membranes of vertebrate cells, can sense shear stress and transduce it into downstream signaling. In this study, we examined the sensitivity of Piezo1 to ultrasonic stimulation and assessed its downstream biological functions in human umbilical vein endothelial cells (HUVECs). Ultrasonic stimulation using a stimulation power of 0.2 W and a frequency of 1 MHz for 10 s did not induce cell damage. However, ultrasonic stimulation induced an influx of calcium ions, which increased with an increase in the stimulation duration. Knockdown of Piezo1 protein decreased the influx of calcium ions during ultrasonic stimulation, which demonstrated that Piezo1 may be activated by the shear stress produced by ultrasonic stimulation. The influx of calcium ions in response to ultrasonic stimulation could be modulated by the Piezo1 protein level. Additionally, ultrasonic stimulation reduced the levels of downstream factors such as MLCK and ATP, which are involved in the Ca2+/CaM/MLCK pathway, by suppressing Piezo1. As the Ca2+/CaM/MLCK pathway influences the permeability of the cell membrane, the internalization of FITC-Dextran into cells under ultrasonic stimulation was validated. Ultrasonic stimulation was demonstrated to promote the increase in cell permeability, and the suppression of Piezo1 was shown to induce the decrease in cell permeability. Therefore, this study shows that ultrasonic stimulation may modulate the permeability of the membrane of HUVECs by modulating the expression of Piezo1 protein.

Reparative effect of mesenchymal stromal cells on endothelial cells after ischemic and inflammatory injury

Background & Aim Renal endothelium is damaged by ischemia reperfusion injury (IRI) during transplantation as a result of the hypoxic metabolism and the resumption of oxygen supply after reperfusion. It has been shown that mesenchymal stromal cells (MSC) are effective in enhancing kidney function after IRI. However, the cellular targets and mechanisms of action behind MSC effects are still poorly understood. Therefore, our objective was to evaluate the role of MSC in repairing injured endothelium as it is the first tissue with which they interact after infusion and to determine the specific interactions involved in this process. The role of physical communication and cytokine secretion in the reparative action of MSC was studied as well as its molecular mechanisms. Methods, Results & Conclusion Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxic and inflammatory insult (TNF-a). This increased the expression of activation molecules such as CD54, CD62e and HLA-II on HUVEC's cell surface. Consequently, an increase of almost 20% in the adherence of MSC to HUVEC was observed in comparison to untreated HUVEC. Moreover, an increase of around 20% in the migratory capacity of MSC towards injured HUVEC was observed. MSC showed a 15 to 20% increase in their ability to transmigrate through injured HUVEC monolayers. Once MSC were in contact with HUVEC, the oxidative stress of HUVEC decreased as observed by a decrease in reactive oxygen species levels, their proliferative status improved and MSC restored the angiogenic potential to levels close to those from healthy HUVEC. To determine whether physical interaction or soluble factors released by MSC are fundamental for MSC regenerative capacity, a Boyden chamber system experiment was performed and we observed that both were required to achieve the full reparative effect of MSC. Moreover, we observed an increase in the expression of CD44 and CD29 adhesion molecules on MSC membrane after co-culture with injured HUVEC, as well as their ligands CD62e and CD106 on HUVEC membrane. We conclude that MSC are able to repair the injury caused by ischemic and inflammatory injury on HUVEC via a mechanism that requires both physical and paracrine interaction between MSC and HUVEC. We are currently studying the specific effector molecules involved in MSC and HUVEC cell-to-cell interaction which will allow targeted modification to improve the regenerative effects of MSC therapy.

Improved Antitumor Efficacy of Combined Vaccine Based on the Induced HUVECs and DC-CT26 Against Colorectal Carcinoma.

Angiogenesis is essential for the development, growth, and metastasis of solid tumors. Vaccination with viable human umbilical vein endothelial cells (HUVECs) has been used for antitumor angiogenesis. However, the limited immune response induced by HUVECs hinders their clinical application. In the present study, we found that HUVECs induced by a tumor microenvironment using the supernatant of murine CT26 colorectal cancer cells exerted a better antiangiogenic effect than HUVECs themselves. The inhibitory effect on tumor growth in the induced HUVEC group was significantly better than that of the HUVEC group, and the induced HUVEC group showed a strong inhibition in CD31-positive microvessel density in the tumor tissues. Moreover, the level of anti-induced HUVEC membrane protein antibody in mouse serum was profoundly higher in the induced HUVEC group than in the HUVEC group. Based on this, the antitumor effect of a vaccine with a combination of induced HUVECs and dendritic cell-loading CT26 antigen (DC-CT26) was evaluated. Notably, the microvessel density of tumor specimens was significantly lower in the combined vaccine group than in the control groups. Furthermore, the spleen index, the killing effect of cytotoxic T lymphocytes (CTLs), and the concentration of interferon-γ in the serum were enhanced in the combined vaccine group. Based on these results, the combined vaccine targeting both tumor angiogenesis and tumor cells may be an attractive and effective cancer immunotherapy strategy.

A monoclonal antibody derived by inoculation of human umbilical vein endothelial cells is a potential inhibitor of acetylcholine receptor-linked vasorelaxation.

In a previous study, we produced antibodies from rats immunized with human umbilical vein endothelial cells (HUVECs) and determined the vascular function of the monoclonal antibodies. However, unanswered question remains still about their role in vascular function. The current study explored vasoreactivity, in particular, focusing on the vascular contractility of a functional antibody against proteins expressed on the plasma membrane of HUVECs developed in a previous study. Among the antibodies developed, A-7 significantly attenuated endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to sodium nitroprusside or histamine. In addition, the A-7 antibody did not affect norepinephrine-stimulated contraction in both endothelium-intact and -denuded aorta. Immunocytochemical and immunoblotting analyses showed that A-7 attenuated ACh-increased expression of ACh receptor on the plasma membrane of HUVECs. These findings suggest that the monoclonal A-7 antibody may act as an inhibitor of endothelium-dependent vasorelaxation, probably in part via downregulation of ACh receptor expression.

Long noncoding RNA MEG3 prevents vascular endothelial cell senescence by impairing miR-128-dependent Girdin downregulation.

Long noncoding RNAs (lncRNAs) are commonly associated with various biological functions, in which the function of lncRNA maternally expressed gene 3 (MEG3) has been identified in various cancers. Strikingly, an association between MEG3 with microRNAs (miRNAs), mRNAs, and proteins has been reported. This study investigates the role of MEG3 in vascular endothelial cell (VEC) senescence. Expression of Girdin and miR-128 was monitored in the blood vessel samples of young and old mice/healthy volunteers, along with the measurement of human umbilical vein endothelial cells (HUVECs). The relationship between MEG3/Girdin and miR-128 was determined and verified. Loss- and gain-of-function approaches were applied to analyze the regulatory effects of MEG3 on platelet phagocytosis and lipoprotein oxidation of HUVEC membrane. In addition, the effect of MEG3 on HUVEC senescence was evaluated by detection of the reactive oxygen species, telomerase activity, and telomere length. To further analyze the MEG3-mediated regulatory mechanism, miR-128 upregulation and inhibition were introduced into the HUVECs. Downregulated Girdin and upregulated miR-128 were found in the blood vessels of old individuals and old mice, as well as in senescent HUVECs. MEG3 downregulation was found to be capable of inhibiting Girdin but enhancing miR-128 expression. It was also indicated to inhibit platelet phagocytosis and reduce telomerase activity and telomere length, while enhancing lipoprotein oxidation and reactive oxygen species production, which ultimately contributed in preventing and protecting HUEVCs from senescence. These findings provide evidence supporting that MEG3 leads to miR-128 downregulation and Girdin upregulation, which promotes platelet phagocytosis, thus protecting VECs from senescence.

The Anti-inflammatory Effects of Short Chain Fatty Acids on Lipopolysaccharide- or Tumor Necrosis Factor α-Stimulated Endothelial Cells via Activation of GPR41/43 and Inhibition of HDACs.

Background and Aim: Previously, we found that short chain fatty acids (SCFA) inhibit LPS or TNFα-induced endothelial inflammatory responses and excessive vascular cell adhesion molecule-1 (VCAM-1) expression, two important steps in the development of atherosclerosis. However, the mechanisms involved are still unclear. We hypothesized that the effects of SCFA are associated with activation of G-protein coupled receptor 41/43 (GPR41/43) and/or inhibition of histone deacetylases (HDACs).Methods: The expression and location of GPR41/43 and HDAC3 in human umbilical vein endothelial cells (HUVEC) were confirmed. HUVEC were pre-incubated with acetate, butyrate or propionate alone or in combination with GLPG0974 (GLPG, antagonist of GPR43) or β-hydroxybutyrate (SHB, antagonist of GPR41) and then exposed to LPS or TNFα. Interleukin (IL)-6 and IL-8 levels and VCAM-1 expression were measured. HDAC activity was measured after treatment with butyrate, propionate and trichostatin A (TSA, HDAC inhibitor). The peripheral blood mononuclear cell (PBMC) adhesive level was also determined after TSA treatment.Results: GPR41/43 were expressed on the membrane of HUVEC and HDAC3 was located in cytoplasm and nucleus. The GLPG and/or SHB treatments restored the inhibitory effects of acetate on IL-6 and IL-8 production and the inhibitory effects of butyrate or propionate on IL-6 production, but not on IL-8. In contrast, GLPG and/or SHB treatments did not affect the inhibitory effects of butyrate or propionate on TNFα-induced VCAM-1 expression. TSA showed similar effects on IL-8 production and VCAM-1 expression as butyrate and propionate. In addition, TSA significantly inhibited the adhesion of PBMC to an endothelial monolayer.Conclusion: Activation of GPR41/43 mediates the effects of acetate on IL-6 and IL-8 production and the effects of butyrate and propionate on IL-6 production. Furthermore, inhibition of HDACs mediates the effects of butyrate and propionate on IL-8 production, VCAM-1 expression, and PBMC adhesion to an endothelial monolayer. These data indicate the beneficial roles of SCFA in preventing vascular inflammation and relevant diseases by activation of GPR41/43 and inhibition of HDACs.

Toll-like receptor 2 and dectin-1 function as promising biomarker for Aspergillus fumigatus infection.

In recent years, along with the wide application of organ transplantation and immunosuppressive agents, as well as the abuse of broad spectrum antibiotics, the incidence of invasive fungal infections has been increasing gradually. The present study aimed to identify novel biomarkers in cells infected with Aspergillus fumigatus. Human umbilical vein endothelial cells (HUVECs) were infected with Aspergillus fumigatus and then harvested at different time-points (0, 1, 2, 4 and 6 h). The expression Toll-like receptor 2 (TLR2) and dectin-1 expression were examined using flow cytometry and western blotting, and fluorescence-based microscopy was used to evaluate their distribution. The results indicated that TLR2 and dectin-1 protein levels were localized on the surface of HUVECs, and that dectin-1 was distributed on HUVEC membranes as observed under confocal microscope. Immunofluorescence assay result revealed that the optical intensity of dectin-1 in the Aspergillus fumigatus-infected group was significantly increased at 0, 1 and 2 h compared with the control group (P<0.05). However, the optical intensity of TLR2 in the Aspergillus fumigatus-infected group was markedly decreased between 0 and 6 h, as compared with the control group (P<0.05). Western blot analysis indicated that dectin-1 expression was significantly increased and TLR2 expression was significantly decreased at 0, 1 and 2 h post infection in the Aspergillus fumigatus-infected group compared with the control group. Furthermore, the expression of TLR2 was also negatively correlated with the concentration of Aspergillus fumigatus. In conclusion, upon infection of cells with Aspergillus fumigatus, TLR2 and dectin-1 expression levels were significantly altered. Therefore, TLR2 and dectin-1 levels may function as promising biomarkers for the treatment or diagnosis of Aspergillus fumigatus infection.

The Disulfide Bond of the Peptide Thanatin Is Dispensible for Its Antimicrobial Activity In Vivo and In Vitro.

Thanatin (THA) displays potent antibiotic activity, especially against extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli both in vitro and in vivo, with minimal hemolytic toxicity and satisfactory stability in plasma. However, the high cost of thanatin significantly limits its development and clinical application. To reduce the cost of peptide synthesis, a formulation of cyclic thanatin (C-thanatin) called linear thanatin (L-thanatin) was synthesized and its activity was evaluated in vivo and in vitro Results showed that C-thanatin and L-thanatin MICs did not differ against eight Gram-negative and two Gram-positive bacterial strains. Furthermore, the survival rates of ESBL-producing-E. coli-infected mice were consistent after C-thanatin or L-thanatin treatment at 5 or 10 mg/kg of body weight. Neither C-thanatin nor L-thanatin showed toxicity for human red blood cells (hRBCs) and human umbilical vein endothelial cells (HUVECs) at a concentration as high as 256 μg/ml. Results of circular dichroism spectroscopy indicated that the secondary structure of L-thanatin is extremely similar to that of C-thanatin. Membrane permeabilization and depolarization assays showed that C-thanatin and L-thanatin have similar abilities to permeabilize the outer and inner membranes and to induce membrane depolarization in ESBL-producing E. coli However, neither of them caused significant HUVEC membrane permeability. These findings indicate that the two peptides have similar effects on bacterial cell membranes and that the disulfide bond in thanatin is not essential for its antimicrobial activities in vivo and in vitro L-thanatin is thus a promising low-cost peptide candidate for treating ESBL-producing E. coli infections.

Abstract 14619: Ultrasound-stimulated Microbubbles for Drug/gene Delivery: Microbubble-generated Shear Stress Induces Pores in Cellular Membranes

Background: Ultrasound (US)-stimulated microbubbles (MBs) are emerging as novel target-specific non-viral gene/drug delivery vectors for treatment of vascular disease, e.g. to deliver VEGF plasmid DNA to promote arteriogenesis. The nature of the MB-target cell interactions that facilitate nucleic acid delivery across cell membranes, and hence strategies to optimize the efficiency of this platform, are poorly understood. The objective of this study was to gain mechanistic insight into MB-mediated increases in cell membrane permeability. Methods: Phospholipid coated MBs were allowed to lay adjacent to cultured human umbilical vein endothelial cells (HUVEC) at 37C. Propidium iodide (PI) was put into the media before US exposure to assess membrane permeability (sonoporation). Individual MB-cell pairs (n=351) were exposed to a single US pulse at 0.5, 1, or 2 MHz, with a duration of 4, 8 or 16 μs and pressures 0.1-0.8 MPa. Ultrafast microscopy recorded MB oscillations (Fig. 1a); a second camera simultaneously recorded fluorescence (PI uptake, Fig. 1b). US-induced MB radius change (excursion) was measured and used to calculate shear stress. HUVEC membranes were fluorescently labeled to visualize pore features. Results: Absolute MB excursion distance and associated shear stress were strong threshold indicators of sonoporation (PI uptake). For an 8 μs pulse, the oscillation (shear stress) threshold above which HUVECs porate was 3.9, 2.6, and 1.4 μm (7.8, 14.5, 22.7 kPa) at 0.5, 1 and 2 MHz respectively. At a given US frequency, increases in pulse duration decreased the required excursion and shear stress. Cell membrane imaging showed that MBs can induce apical and basal pores (sub-μm to μm sizes) that reseal (Fig. 1c). Conclusions: Sonoporation is related to absolute MB excursion distance and its associated shear stress and can occur with various US parameters and MB sizes. This data has implications on intelligent US-MB design for their use as payload delivery vehicles.

Investigating membrane and mitochondrial cryobiological responses of HUVEC using interrupted cooling protocols

The success of cryopreservation protocols is largely based on membrane integrity assessments after thawing, since membrane integrity can be considered to give an upper limit in assessment of cell viability and the plasma membrane is considered to be a primary site of cryoinjury. However, the exposure of cells to conditions associated with low temperatures can induce injury to cellular structure and function that may not be readily identified by membrane integrity alone. Interrupted cooling protocols (including interrupted slow cooling without a hold time (graded freezing), and interrupted rapid cooling with a hold time (two-step freezing)), can yield important information about cryoinjury by separating the damage that occurs upon cooling to (and possibly holding at) a critical intermediate temperature range from the damage that occurs upon plunging to the storage temperature (liquid nitrogen). In this study, we used interrupted cooling protocols in the absence of cryoprotectant to investigate the progression of damage to human umbilical vein endothelial cells (HUVEC), comparing an assessment of membrane integrity with a mitochondrial polarization assay. Additionally, the membrane integrity response of HUVEC to interrupted cooling was investigated as a function of cooling rate (for interrupted slow cooling) and hold time (for interrupted rapid cooling). A key finding of this work was that under slow cooling conditions which resulted in a large number of membrane intact cells immediately post thaw, mitochondria are predominantly in a non-functional depolarized state. This study, the first to look directly at mitochondrial polarization throughout interrupted cooling profiles and a detailed study of HUVEC response, highlights the complexity of the progression of cell damage, as the pattern and extent of cell injury throughout the preservation process differs by injury site.

Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways.

Melatonin Suppresses Hypoxia-Induced Migration of HUVECs via Inhibition of ERK/Rac1 Activation

Melatonin, a naturally-occurring hormone, possesses antioxidant properties and ameliorates vascular endothelial dysfunction. In this study, we evaluate the impact of melatonin on the migratory capability of human umbilical vein endothelial cells (HUVECs) to hypoxia and further investigate whether ERK/Rac1 signaling is involved in this process. Here, we found that melatonin inhibited hypoxia-stimulated hypoxia-inducible factor-1α (HIF-1α) expression and cell migration in a dose-dependent manner. Mechanistically, melatonin inhibited Rac1 activation and suppressed the co-localized Rac1 and F-actin on the membrane of HUVECs under hypoxic condition. In addition, the blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1-T17N suppressed HIF-1α expression and cell migration in response to hypoxia, as well, but constitutive activation of Rac1 mutant Rac1-V12 restored HIF-1α expression, preventing the inhibition of melatonin on cell migration. Furthermore, the anti-Rac1 effect of melatonin in HUVECs appeared to be associated with its inhibition of ERK phosphorylation, but not that of the PI3k/Akt signaling pathway. Taken together, our work indicates that melatonin exerts an anti-migratory effect on hypoxic HUVECs by blocking ERK/Rac1 activation and subsequent HIF-1α upregulation.

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

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

Buffalo Colostrum β-lactoglobulin Inhibits VEGF-Induced Angiogenesis by Interacting with G protein-Coupled Receptor Kinase

β-lactoglobulin (β-lg), a major whey protein was purified and characterised from buffalo colostrum. The in silico analysis of the tryptic peptides based on LC-CID-MS/MS facilitated the identification of protein as β-lg. The sequences IIVTQ f[1-5] and LSFNPTQLEEQCHV f(149-162) of m/z 933(+) and 851(2+) were found to match N- and C-extreme of β-lg while IDALNENK f(84-91) and TPEVDDEALEKFDK f(125-138) sequences deduced for m/z 916(+) and 818(2+) were in compliance to buffalo milk β-lg. Considering the sequence similarity of β-lg to glycodelin, a proven angiogenic protein, similar role for β-lg from buffalo colostrum (BLG-col) was examined. Interestingly, BLG-col exhibited anti-angiogenic activity by potently inhibiting cell proliferation, micro-vessel sprouting, cell migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but having varied effect on Ehrlich ascites tumor cells, MCF-7, MDA-MB 435 and MDA-MB 231 cell lines. The anti-angiogenic potential of BLG-col was found to be vascular endothelial growth factor mediated. The immunolocalisation of BLG-col on the cell surface of HUVECs evidenced using FITC-labelled β-lg antibody indicated its extra-cellular binding. Furthermore, BLG-col interacting HUVEC membrane protein (64kDa) was detected by immunoblot and its identity was established by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, which showed peptide sequence homology to G protein-coupled receptor kinase 4.