Calf Pulmonary Artery Endothelial Cells Research Articles

Controlled release of vascular endothelial growth factor (VEGF) in Alginate and Hyaluronic acid (ALG − HA) bead system to promote wound healing in punch-induced wound rat model

For wound healing, angiogenesis is one of the main therapeutic factors for recovering the injured tissue. To address this issue, a combination of two different polymers, alginate (ALG) and hyaluronic acid (HA) in 80:20 ratio composition is used to optimize the bead system along with the 5 IU heparin (Hep) by crosslinking into calcium chloride (CaCl2). Encapsulation of Vascular endothelial growth factor (VEGF) in the bead system shows delayed cumulative release in phosphate buffer saline (PBS). For in vitro studies, calf pulmonary artery endothelial (CPAE) cells showed biocompatibility. ALG − HA/VEGF150 improves endothelial Vascular cell adhesion protein 1 (VCAM1) and endothelial nitric oxide synthase (eNOS) expression markers in CPAE cells. In vivo evaluation of the bead system shows around 68% of wound closure 2 weeks post-implantation in 8 mm punch wound models. The treatment group shows decreased epithelial gap between the ends of the wound and neo-epidermal regeneration. ALG − HA/VEGF150 induced significant vascularization, collagen type-1 (Col-1) and fibronectin (FN) development in the in vivo models after 2 weeks of the implantation. Hence, ALG − HA/VEGF150 beads can be used to promote wound healing.

Nanocellulose-based fibres derived from palm oil by-products and their in vitro biocompatibility analysis

Fibres with nanocellulose isolated from oil palm empty fruit bunches (OPEFBs) were produced. Nanocellulose and PVA-nanocellulose fibres were prepared by wet spinning in an acetone coagulation bath without drawing. The addition of nanocellulose was varied from 10% to 30%, to reveal the beneficial effects of nanocellulose content on the properties of produced spun-fibres. Higher concentration of nanocellulose increased the stiffness of spun-fibres. PVA and PVA-bacterial cellulose fibres were also produced as a control and for comparison, respectively. The nanocellulose fibre formed a compact structure, while PVA fibres had hollow structures. The effect of the produced spun-fibres on the biocompatibility of calf pulmonary artery endothelial cells was assayed by an MTT test. Based on the MTT assay the addition of nanocellulose increased the percentage of cell viability of the obtained spun-fibres slightly. These results point towards the use of sustainable sources of nanocellulose as a beneficial and biocompatible fibre material.

A Wearable Iron-Based Implant as an Intramedullary Nail in Tibial Shaft Fracture of Sheep.

A stable repaired fracture is the key factor responsible for the recovery of a damaged bone. The iron-based implant is one of the biodegradable metals that have been proven safe as a fracture fixation device. The objective of our experimental approach was to examine the potential of the iron-based implant as a biodegradable metal in tibia shaft fracture in sheep chronically. The samples used for this experiment were iron-based and stainless steel implants. Each had a diameter of 5 mm. These samples were analyzed through 3 phases which are material characterization, in vitro and in vivo examination. The samples were examined using a scanning electron microscope with energy dispersive spectrometer and X-ray diffraction. Based on the analysis carried out, the samples contained 90,02% and 60,81% Fe for iron-based implant and stainless implant, respectively. Both implants maintained high viability when being in contact with calf pulmonary artery endothelial cells, indicating that both implants had a minimum response to the cell in a hemocytometer and methyl tetrazolium (MTT) assay. The systemic effect of the implants was observed using hematology and blood chemistry examination. Data collection also shows that both implants also had a minimum response to the erythrocytes, leucocytes, blood chemistry, and blood mineral during the period of observation. Therefore, it could be concluded that the iron-based implant is tolerable for a period of 9 months. It also has the potential to be used as a biodegradable orthopedic implant.

Anti-CENP-B and anti-TOPO-1-containing sera from systemic sclerosis-related diseases with Raynaud's phenomenon induce vascular endothelial cell senescence not via classical p53-p21 pathway.

Raynaud's phenomenon (RP) is the earliest and most common clinical manifestation in patients with systemic sclerosis (SSc) and its related diseases containing anti-TOPO-1 and/or anti-CENP-B autoantibodies in the sera. However, the cause-effect relationship between the two autoantibodies and RP remains elucidation. Sera containing anti-CENP-B and anti-TOPO-1 autoantibodies were obtained from SSc-related diseases manifesting RP. The polyclonal auto-antibodies were purified from pooled sera by affinity chromatography. Mouse monoclonal anti-CENP-B and anti-TOPO-1 were purchased. Calf pulmonary arterial endothelial cells (CPAE) were incubated with 40% patient sera, purified polyclonal antibodies or mouse monoclonal antibodies for 1-6days. The vascular endothelial biomarkers von Willebrand factor (vWF), thrombomodulin (CD141) and 6-keto-prostaglandin F1α (6-keto-PGF1α), cell viability marker ATP, and cell necrosis/lysis marker LDH in the culture supernatants were measured by ELISA. The cell senescence biomarker β-galactosidase and telomere content in the cells were stained by the respective kit. The classical p53-p21 senescence pathway was detected by Western blot. We found that 40% anti-CENP-B or anti-TOPO-1-containing sera without heat-inactivation and mouse monoclonal antibodies suppressed 6-keto-PGF1α production, increased β-galactosidase, and decreased relative telomere content. The cell senescence effects were proved not via p53-p21 pathway. The pathognomonic anti-CENP-B and anti-TOPO-1 autoantibodies in SSc-related diseases accelerate vascular endothelial cell senescence and functional impairment inducing RP. The real signaling pathway for autoantibody-induced cell senescence remains exploration.

Treatment with a JNK inhibitor increases, whereas treatment with a p38 inhibitor decreases, H2O2-induced calf pulmonary arterial endothelial cell death.

Oxidative stress induces apoptosis in endothelial cells (ECs). Reactive oxygen species (ROS) promote cell death by regulating the activity of various mitogen-activated protein kinases (MAPKs) in ECs. The present study investigated the effects of MAPK inhibitors on cell survival and glutathione (GSH) levels upon H2O2 treatment in calf pulmonary artery ECs (CPAECs). H2O2 treatment inhibited the growth and induced the death of CPAECs, as well as causing GSH depletion and the loss of mitochondrial membrane potential (MMP). While treatment with the MEK or JNK inhibitor impaired the growth of H2O2-treated CPAECs, treatment with the p38 inhibitor attenuated this inhibition of growth. Additionally, JNK inhibitor treatment increased the proportion of sub-G1 phase cells in H2O2-treated CPAECs and further decreased the MMP. However, treatment with a p38 inhibitor reversed the effects of H2O2 treatment on cell growth and the MMP. Similarly, JNK inhibitor treatment further increased, whereas p38 inhibitor treatment decreased, the proportion of GSH-depleted cells in H2O2-treated CPAECs. Each of the MAPK inhibitors affected cell survival, and ROS or GSH levels differently in H2O2-untreated, control CPAECs. The data suggest that the exposure of CPAECs to H2O2 caused the cell growth inhibition and cell death through GSH depletion. Furthermore, JNK inhibitor treatment further enhanced, whereas p38 inhibitors attenuated, these effects. Thus, the results of the present study suggest a specific protective role for the p38 inhibitor, and not the JNK inhibitor, against H2O2-induced cell growth inhibition and cell death.

Gallic acid inhibits the growth of calf pulmonary arterial endothelial cells through cell death and glutathione depletion.

Gallic acid (GA) exhibits a number of cellular effects, including apoptosis, which is associated with oxidative stress. The present study investigated the effects of GA on calf pulmonary arterial endothelial cell (CPAEC) growth and death, along with the levels of reactive oxygen species (ROS) and glutathione (GSH). GA treatment inhibited the growth of CPAECs at 24h, and the half‑maximal inhibitory concentration (IC50) value of GA was ~30µM. GA treatment also induced cell death, which was accompanied by a loss of mitochondrial membrane potential (ΔѰm). GA treatment in CPAECs resulted in decreased ROS levels, including O2•‑, whereas the number of GSH‑depleted cells increased. Neither a pan‑caspase inhibitor (benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone) nor buthionine sulfoximine treatment affected GA‑induced cell growth inhibition, cell death, ROS and GSH levels in CPAECs, whereas co‑treatment with N‑acetyl‑cysteine (NAC) resulted in enhanced cell growth inhibition, cell death and ΔѰm loss in these cells. Although NAC treatment did not significantly influence ROS levels in GA‑treated CPAECs, it significantly enhanced GSH depletion in these cells. In conclusion, GA inhibited the growth of CPAECs via cell death, which was associated with GSH depletion rather than alterations to ROS levels.

Tissue Specificity: SOCE: Implications for Ca2+ Handling in Endothelial Cells.

Many cellular functions of the vascular endothelium are regulated by fine-tuned global and local, microdomain-confined changes of cytosolic free Ca2+ ([Ca2+]i). Vasoactive agonist-induced stimulation of vascular endothelial cells (VECs) typically induces Ca2+ release through IP3 receptor Ca2+ release channels embedded in the membrane of the endoplasmic reticulum (ER) Ca2+ store, followed by Ca2+ entry from the extracellular space elicited by Ca2+ store depletion and referred to as capacitative or store-operated Ca2+ entry (SOCE). In vascular endothelial cells, SOCE is graded with the degree of store depletion and controlled locally in the subcellular microdomain where depletion occurs. SOCE provides distinct Ca2+ signals that selectively control specific endothelial functions: in calf pulmonary artery endothelial cells, the SOCE Ca2+ signal drives nitric oxide (an endothelium-derived relaxing factor of the vascular smooth muscle) production and controls activation and nuclear translocation of the transcription factor NFAT. Both cellular events are not affected by Ca2+ signals of comparable magnitude arising directly from Ca2+ release from intracellular stores, clearly indicating that SOCE regulates specific Ca2+-dependent cellular tasks by a unique and exclusive mechanism. This review discusses the mechanisms of intracellular Ca2+ regulation in vascular endothelial cells and the role of store-operated Ca2+ entry for endothelium-dependent smooth muscle relaxation and nitric oxide signaling, endothelial oxidative stress response, and excitation-transcription coupling in the vascular endothelium.

Rocuronium Bromide Inhibits Inflammation and Pain by Suppressing Nitric Oxide Production and Enhancing Prostaglandin E2 Synthesis in Endothelial Cells.

PurposeRocuronium bromide is a nondepolarizing neuromuscular blocking drug and has been used as an adjunct for relaxation or paralysis of the skeletal muscles, facilitation of endotracheal intubation, and improving surgical conditions during general anesthesia. However, intravenous injection of rocuronium bromide induces injection pain or withdrawal movement. The exact mechanism of rocuronium bromide-induced injection pain or withdrawal movement is not yet understood. We investigated whether rocuronium bromide treatment is involved in the induction of inflammation and pain in vascular endothelial cells.MethodsFor this study, calf pulmonary artery endothelial (CPAE) cells were used, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Western blot, nitric oxide detection, and prostaglandin E2 immunoassay were conducted.ResultsRocuronium bromide treatment inhibited endothelial nitric oxide synthase and suppressed nitric oxide production in CPAE cells. Rocuronium bromide activated cyclooxygenase-2, inducible nitric oxide synthase and increased prostaglandin E2 synthesis in CPAE cells.ConclusionsRocuronium bromide induced inflammation and pain in CPAE cells. Suppressing nitric oxide production and enhancing prostaglandin E2 synthesis might be associated with rocuronium bromide-induced injection pain or withdrawal movement.

Development of dynamic well plate system for cell culture with mechanical stimulus of shear stress and magnetic field

Investigating the effects of various physical stimuli on cells is important for improving the efficiency of tissue repair and regeneration. In this research, we developed a dynamic well plate system by integrating the advantages of conventional well plates and a bioreactor to provide simultaneous physical stimuli of shear stress and a static magnetic field. The dynamic well plate involving perfusion of culture medium can control hydrodynamic shear while retaining the inherent simplicity of conventional well plates. The specific well plate cover was designed to load shear stress on cells during cultivation and was built to fit over a standard six-well plate. Additionally, to investigate the effects of a magnetic field on cell proliferation, a static neodymium magnet was placed beneath each well. To assess the system developed, calf pulmonary artery endothelial (CPAE) cells were cultured using the developed system. CPAE cells under hydrodynamic shear stress conditions were elongated and aligned in the direction of the flow and the magnetic field enhanced CPAE cell proliferation. Simultaneous application of a magnetic field and shear flow in CPAE cell cultivation allowed the development of optimized culture conditions, initially for cell proliferation and then for functional expression, such as cell shape changes.

Immune modulation of glycosaminoglycan derived from P. lewisi in TNF-α stimulated cells.

Poecilocoris lewisi (Korean name: "Kwangdaenolinjae") is a red-striped gold stink bug (insect) which has been used as a crude drug in traditional medicine of East Asia and Korea. In this study, ethanol extract and glycosaminoglycan from P. lewisi (Pl GAG), as an active substance among its components, were investigated for their potential anti-inflammatory actions. They were found to be a potent inducer of nitric oxide (NO) production from calf pulmonary artery endothelial (CPAE) cells and a stimulator of endothelial nitric oxide synthase in a dose-dependent manner. The anti-inflammatory activities were also evaluated by determining the level of adhesion molecules related to atherogenesis and pro-inflammatory cytokines, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), secretory phospholipase A2, and prostaglandin E2, stimulated by tumor necrosis factor (TNF)-α in human umbilical vein endothelial cells (HUVEC). They also showed inhibitory effects on vascular endothelial growth factor (VEGF) production in HUVECs. Matrix metalloproteinases (MMP-2 and 9) were also inhibited by treatment with this extract or glycosaminoglycan. Furthermore, this GAG showed cytotoxicity against CT-26 colon cancer cells whereas having no cytotoxicity in CHO normal cells. Monosaccharide (amino, acidic, neutral monosaccharides) composition of used GAG was characterized by trimethylsilylated GC-MS analysis method.

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies.

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm2. The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm2) than on surfaces with a higher concentration of FGF-2 (120 ng/cm2).

Evidence for the Involvement of MMP14 in MMP2 Processing and Recruitment in Exosomes of Corneal Fibroblasts.

Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechanisms are poorly understood. In this study, we investigated exosomal transport of MMP14 and its target, MMP2, from corneal fibroblasts to vascular endothelial cells as a possible mechanism governing MMP14 activity in corneal angiogenesis. We isolated MMP14-containing exosomes from corneal fibroblasts by sucrose density gradient and evaluated exosome content and purity by Western blot analysis. We then investigated exosome transport in vitro from corneal fibroblasts to two populations of vascular endothelial cells, human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAECs). Western blot analysis and gelatin zymography were used to determine levels of MMP14 and MMP2, respectively, in exosomal fractions derived from cultured wild-type, MMP14 enzymatic domain-deficient (MMP14Δexon4), and MMP14-null corneal fibroblasts. Matrix metalloproteinase 14-containing exosomes isolated from corneal fibroblasts were readily taken up in vitro by HUVECs and CPAECs. We found that MMP14 was enriched in exosomal fractions of cultured corneal fibroblasts. Moreover, loss of the MMP14 enzymatic domain resulted in accumulation of pro-MMP2 protein in exosomes, whereas MMP2 was nearly undetectable in exosomes of MMP14-null fibroblasts. Our results indicate that exosomes secreted by corneal fibroblasts can transport proteins, including MMP14, to vascular endothelial cells. In addition, recruitment of MMP2 into corneal fibroblast exosomes is an active process that depends, at least in part, on the presence of MMP14. The role of exosomal MMP14 transport in corneal angiogenesis has important implications for therapeutic applications targeting angiogenic processes in the cornea.

Poly(ethylene oxide) brushes prepared by the “grafting to” method as a platform for the assessment of cell receptor–ligand binding

Poly(ethylene oxide) (PEO) with terminal alkyne and amino groups was grafted to a poly(glycidyl methacrylate) (PGMA) anchoring layer and the PEO/PGMA coatings were investigated as a non-fouling platform for the assessment of ligand–cell receptor interactions. The PEO/PGMA coatings deposited on Si/SiO2 substrate were stable in phosphate buffered saline over a period of 8days if the thickness of the PEO was less than 30nm. The stability of the coating could be enhanced by an additional layer of 3-mercaptopropyltrimethoxysilane between the substrate and the PGMA layer. The grafted layers were shown to efficiently suppress nonspecific protein adsorption and cell adhesion. Based on the theoretical ligand-binding capacity, protein adsorption and stability data, the optimum thickness range of the PEO layer is 10–20nm. The binding of an arginine–glycine–aspartic acid (RGD) ligand using azide–alkyne click chemistry demonstrated that the ligand surface density is controllable in the range from 100pmol/cm2 up to the capacity of the grafted layer of 102pmol/cm2 by varying the ligand concentration in the reaction mixture. Calf pulmonary artery endothelial cells adhered to and spread on ligand modified layers proportionally to the ligand surface density, thus demonstrating the applicability of these “grafted to” PEO brushes as a platform for cell receptor–ligand engagement studies.

Providencia alcalifaciens causes barrier dysfunction and apoptosis in tissue cell culture: potent role of lipopolysaccharides on diarrheagenicity

Providencia alcalifaciens is a member of the Enterobacteriaceae family that occasionally causes diarrheagenic illness in humans via the intake of contaminated foods. Despite the epidemiological importance of P. alcalifaciens, little is known about its pathobiology. Here we report that P. alcalifaciens causes barrier dysfunction in Caco-2 cell monolayers and induces apoptosis in calf pulmonary artery endothelial cells. P. alcalifaciens infection caused a 30% reduction in transepithelial resistance in Caco-2 cell monolayers, which was greater than that for cells infected with Shigella flexneri or non-pathogenic Escherichia coli. As with viable bacteria, bacterial lysates treated with heat, benzonase or proteinase, but not with polymixin B, were also involved in the cellular response. TLR4 antibody neutralisation significantly restored the P. alcalifaciens-induced transepithelial resistance reduction in Caco-2 cells, suggesting that lipopolysaccharides (LPSs) might play a central role in this cellular response. Western blotting further indicated that P. alcalifaciens LPSs reduced occludin levels, whereas LPSs from Shigella or E. coli did not. Although the viability of Caco-2 cells was not altered significantly, the calf pulmonary artery endothelial cell line was highly sensitive to P. alcalifaciens infection. This sensitivity was indeed dependent on LPS, which induced rapid apoptosis. Together, these data show that P. alcalifaciens LPSs participate in epithelial barrier dysfunction and endothelial apoptosis. The findings give insight into the LPS-dependent cell signal events affecting diarrheagenicity during infection with P. alcalifaciens.

Anti-inflammatory Effect of Bumblebee Alcohol Extracts in CFA-Induced Rat Edema.

In this study, we prepared alcohol extracts of the larva, pupa, queen, and cocoon (clony) of B. ignitus, B. terrestris, and B. h. sapporoensis, and tested the anti-inflammatory activity of the extracts by using a rat model of adjuvant-induced edema. The extracts derived from the queen of B. ignitus, the queen of B. terrestris, and the cocoon of B. ignitus decreased hind paw edema after 1 day of i.p. administration. These extracts also induced vasorelaxation and NO production in calf pulmonary artery endothelial cells. These results suggest that bumblebee alcohol extracts has anti-inflammatory and vasorelaxant properties.

The effects of exogenous H2O2 on cell death, reactive oxygen species and glutathione levels in calf pulmonary artery and human umbilical vein endothelial cells

Enhanced oxidative stress contributes to endothelial dysfunction via the apoptotic induction of endothelial cells (ECs). However, the precise molecular mechanisms underlying its important effect remain unclear. Here, we evaluated the effects of exogenous hydrogen peroxide (H(2)O(2)) on cell growth and death in ECs such as calf pulmonary artery endothelial cells (CPAECs) and human umbilical vein endothelial cells (HUVECs) and investigated its mechanism of action in CPAECs. H(2)O(2) inhibited the growth of CPAECs and HUVECs at 24 h with IC50 of approximately 20 and 300 µM, respectively. H(2)O(2) induced cell death in both ECs, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). H(2)O(2)-induced CPAEC death occurred via apoptosis, demonstrated by Annexin V-staining cells and Z-VAD (a pan-caspase inhibitor) treatment. H(2)O(2) increased superoxide anion levels in HUVECs but not in CPAECs. Treatment with 30 µM H(2)O(2) significantly decreased the activities of superoxide dismutases and catalase in CPAECs. H(2)O(2) induced glutathione (GSH) depletion in both ECs. Z-VAD and N-acetyl cysteine (NAC; a well-known antioxidant) attenuated apoptotic cell death and GSH depletion in H(2)O(2)-treated CPAECs. In conclusion, H(2)O(2) induced growth inhibition and death in ECs via GSH depletion. HUVECs were relatively resistant to H(2)O(2) compared with CPAECs. H(2)O(2)-induced CPAEC apoptosis required the activation of various caspases.

Characterization and optimization of vascular endothelial growth factor165 (rhVEGF165) expression in Escherichia coli

Vascular endothelial growth factors165 (VEGF165) is the most potent and widely used pro-angiogenic factor. Here we determined optimal culture condition of recombinant human VEGF165 (rhVEGF165) in Escherichia coli (E. coli). rhVEGF165 expression was the highest in 0.25% of l-arabinose induction concentration, at 20°C induction temperature, and for 5h induction time under the control of araBAD promoter using pBADHisA vector. In biological activity test, rhVEGF165 significantly increased the proliferative activity of CPAE cells (p<0.001) and upregulated the expressions of endothelial cell growth-related genes, such as platelet endothelial cell adhesion molecule (PECAM-1), endothelial-specific receptor tyrosine kinase (TEK), kinase insert domain protein receptor (KDR), and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1) in calf pulmonary artery endothelial (CPAE) cells.

Platycodon grandiflorum root attenuates vascular endothelial cell injury by oxidized low-density lipoprotein and prevents high-fat diet–induced dyslipidemia in mice by up-regulating antioxidant proteins

We hypothesized that a Platycodon grandiflorum root (PG) ethyl acetate extract (PGEA) would help reduce the vascular cell injury caused by oxidized low-density lipoprotein (oxLDL) and prevent high-fat (HF) diet-induced dyslipidemia and oxidative stress by up-regulating antioxidant proteins. We investigated the protective effects of PGEA against vascular endothelial cell injury induced by oxLDL and dyslipidemia induced by an HF diet, and the mechanisms underlying these effects were studied. The protective effects of PGEA were investigated with respect to calf pulmonary arterial endothelial (CPAE) cell viability and the lactate dehydrogenase release during oxLDL treatment. The in vivo effects of PGEA were examined using C57BL/6 mice, which were fed an HF diet for 9 weeks. The HF diet was supplemented with 0, 25, or 75 mg/kg PGEA during the last 4 weeks of the experimental period. Histologic analyses of hepatic lipid accumulation were performed. The changes in antioxidant protein levels induced by PGEA, which protects against HF diet-induced oxidative stress, were measured using a proteomics approach. We found that PGEA exhibited antioxidant activity. In CPAE cells, PGEA inhibited both oxLDL-induced cell death and lactate dehydrogenase release. In the HF diet-induced obese mice that received PGEA, we observed significantly reduced plasma and hepatic lipid levels, demonstrating that PGEA has beneficial effects on hyperlipidemia. In addition, we found that PGEA caused the up-regulation of antioxidant proteins. These findings suggest that the antioxidant effects of PGEA may protect against oxidative stress-related diseases.

Combination of nitric oxide stimulation with high-dose 18F-FDG promotes apoptosis and enhances radiation therapy of endothelial cells

High-dose (18)F-FDG can provide targeted nuclear therapy of cancer. Endothelial cell injury is a key determinant of tumor response to radiotherapy. Here, we tested the hypothesis that activation of endothelial cell glycolytic metabolism with nitric oxide can enhance the therapeutic effect of high-dose (18)F-FDG. Calf pulmonary artery endothelial (CPAE) cells were treated with graded doses of (18)F-FDG. Glycolysis was stimulated by 24 h of exposure to the nitric oxide donor, sodium nitroprusside (SNP). Cell viability was assessed by MTT and clonogenic assays. Apoptosis was evaluated by ELISA of cytosolic DNA fragments and Western blots of cleaved caspase-3. SNP stimulation (0.1 and 1 mM) augmented CPAE cell (18)F-FDG uptake to 2.6- and 4.6-fold of controls without adverse effects. Treatment with 333 μCi/ml (18)F-FDG alone reduced viable cell number to 35.4% of controls by Day 3. Combining 0.1 mM SNP stimulation significantly enhanced the killing effect, reducing cell numbers to 19.2% and 39.2% of controls by 333 and 167 μCi/ml of (18)F-FDG, respectively. (18)F-FDG also suppressed clonogenic survival to 80.8% and 43.2% of controls by 83 and 167 μCi/ml, which was again intensified by SNP to 59.7% and 21.1% of controls. The cytotoxic effect of (18)F-FDG was attributed to induction of apoptosis as shown by increased cytosolic fragmented DNA and cleaved caspase-3 levels (26.4% and 30.7% increases by 167 μCi/ml). Combining SNP stimulation significantly increased both of these levels to 1.8-fold of control cells. High-dose (18)F-FDG combined with nitric oxide-stimulated glycolysis is an effective method to inhibit endothelial cell survival and promote apoptosis. These results suggest a potential role of this strategy for targeted radiotherapy of angiogenic vasculature.

MG132, a proteasome inhibitor-induced calf pulmonary arterial endothelial cell growth and death, are changed by MAPK inhibitors

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK inhibitors on MG132-treated calf pulmonary artery endothelial cells (CPAECs) in relation to cell death, ROS, and glutathione (GSH). MG132 inhibited the growth of CPAEC and also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). MG132 increased ROS levels and GSH-depleted cell numbers in CPAEC. Treatment with MAPK (MEK, JNK, and p38) inhibitors showed a slight enhancement of cell-growth inhibition by MG132. All the MAPK inhibitors decreased cell death by MG132. Especially, the JNK inhibitor showed a strong effect. They all did not affect ROS levels and GSH depletion in MG132-treated CPAEC, but increased ROS and GSH levels in MG132-untreated CPAEC. In conclusion, MG132 induced apoptosis in CPAEC, which was accompanied by ROS increase and GSH depletion. The changes of MG132-induced CPAEC growth inhibition and death by MAPK inhibitors were not tightly correlated to ROS and GSH levels.