Rat Endothelial Cell Line Research Articles

Chronic prostatitis/chronic pelvic pain syndrome impairs erectile function by inducing apoptosis in a rat model of experimental autoimmune prostatitis.

Over the years, numerous epidemiological studies have shown that chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) promotes erectile dysfunction. Nonetheless, the precise underlying mechanism remains to be fully clarified. The objective of this research was to identify crucial signaling pathways responsible for CP/CPPS-induced erectile dysfunction. Thirty 8-week-old male Sprague‒Dawley rats were randomly assigned to either the CP/CPPS model group or the control group. The CP/CPPS rat model was established through subcutaneous injection of a combination of rat prostate protein and Freund's adjuvant. Penile erectile function assessment was conducted 45 days after immunization through electrical stimulation of the cavernous nerve. RNA sequencing of the corpus cavernosum of the penis was then performed using the Kyoto Encyclopedia of Genes and Genomes and protein‒protein interaction network analysis. Western blotting was performed on the cavernous tissue. Cell apoptosis assays, cell counting kit-8 assays, cell cloning assays, and Western blotting were conducted on rat endothelial cells. Erectile function was significantly lower in the CP/CPPS model group than in the control group (p < 0.001). Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that differentially expressed genes were predominantly enriched in the apoptosis pathway. Moreover, an increase in apoptosis in the rat corpus cavernosum, along with a decrease in the protein expression of CD31 (p = 0.0089) and eNOS (p = 0.0069) following CP/CPPS induction, was observed. In a protein‒protein interaction network, Pitx2 was recognized as a central gene. The role of Pitx2 in regulating apoptosis was demonstrated in experiments using rat endothelial cell lines, and it was found to be regulated by the Wnt/β-catenin pathway. This study highlights the occurrence of cavernous endothelial cell apoptosis in CP/CPPS-induced erectile dysfunction, and the potential mechanism of apoptosis may involve inhibition of the Wnt/β-catenin/Pitx2 pathway.

Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines

BackgroundIn vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible MDR1-EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and MDR1-EGFP transduced cells.MethodsMDR1-EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an MDR1-linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and MDR1-EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison.ResultsAll cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4-MDR1-EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and MDR1-EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the MDR1-EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and MDR1-EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport.ConclusionsThe present data indicate that MDR1-EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood–brain barrier.

Dl-3n-butylphthalide reduces oxygen-glucose deprivation-induced endothelial cell damage by increasing PGC-1α.

Animal experiments verified that dl-3-n-butylphthalide (NBP) can protect vascular endothelial cells from ischemic damage and promote vascular proliferation in ischemic stroke treatment, but the underlying mechanism has not been fully clarified. This study aimed to investigate the effects of NBP on peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α) expression in endothelial cells exposed to oxygen-glucose deprivation (OGD) and to clarify the related molecular mechanism. SV40-transformed aortic rat endothelial cell line was cultured and subjected to OGD in the presence or absence of NBP. The cell viability was evaluated by using thiazolyl blue tetrazolium bromide (MTT) method. The cellular endothelial nitric oxide synthase (eNOS) activity was measured by using eNOS activity assay. The nuclear changes were assessed with Hoechst 33342 fluorescent dye. The immunofluorescence analysis and Western blotting assay were conducted to evaluate the protein expression. We found that NBP could significantly prevent endothelial cells from OGD-induced injuries, in terms of cell morphology and cell viability. Both immunofluorescence analysis and Western blot findings confirmed that the NBP treatment further enhanced PGC-1α expression during OGD, which was prevented in the presence of selective endothelial nitric oxide synthetase (eNOS) inhibitor N5-(1-Iminoethyl)-L-ornithine-HCL (L-NIO). Furthermore, we found that NBP could protect the eNOS activity about by 40% during OGD and did not influence the eNOS protein level in the spectrophotometric-based analysis. NBP maintained the endothelial PGC-1α expression via regulating eNOS activity during the exposure to OGD; therefore, it presented its protective function to cell viability and vascular proliferation.

Polyol effects on growth factors and MAPK signaling in rat retinal capillary cells.

Recent studies report that growth factor and signaling changes in rat lenses do not directly result from the presence of diabetes or sorbitol/galactitol (polyol) formation/accumulation, but from secondary osmotic changes associated with the aldose reductase (AR) catalyzed polyol formation. AR is also present in rat retinal pericyte and endothelial cells; however, significant polyol formation only occurs in pericytes and this does not appear to be linked to osmotic changes. The purpose of this study was to determine whether polyol formation and AR activity are similarly linked to growth factor and signaling changes in the rat capillary cells despite the apparent absence of osmotic stress. Conditionally immortalized rat retinal pericyte (TR-rPCT) and endothelial (TR-iBRB) cell lines were cultured on collagen type 1-coated dishes in the DMEM containing 5.5 mM glucose. After 24 h of initial culture, the medium was replaced with a serum-free medium containing 5.5, 25, or 50 mM glucose or galactose with/without the aldose reductase inhibitors (ARIs) AL1576 or tolrestat for periods of up to 48 h. Growth factors and transduction pathways were measured by Western blots using the antibodies against basic FGF, IGF-1, TGF-β, P-ERK1/2, P-SAPK/JNK, and P-Akt. Sorbitol accumulation was only observed in pericytes, while galactitol was present in both pericytes and endothelial cells. Pericytes cultured in high glucose showed increased expression of the growth factors basic FGF, IGF-1, TGF-β, and signaling in P-Akt, P-ERK1/2, and P-SAPK/JNK compared with those cultured in 5.5 mM glucose and these expressions were normalized by the presence of ARIs. Similar results were observed with galactose media. In contrast, endothelial cells cultured in high glucose media showed neither growth factor or signaling changes. In galactose media, endothelial cells showed increased expression of basic FGF, IGF-1, TGF-β, P-ERK1/2, and P-SAPK/JNK, which were only partially reduced by ARIs. Growth factor and MAPK signaling expression in pericytes are linked to the presence of polyols. Pericytes, which readily accumulate sorbitol/galactitol that is inhibited by ARIs, show expression changes similar to those observed in rat lenses. In contrast, endothelial cells only show partial expression changes that are linked to galactitol accumulation.

Mechanism of attenuation of pro-inflammatory Ang II-induced NF-κB activation by genistein in the kidneys of male rats during aging

Angiotensin II (Ang II), a main effector of the renin-angiotensin system, is recognized as a pro-inflammatory mediator on age-related vascular inflammation. Ang II is one of the most important oxidative stress inducer, activates the redox-sensitive transcription factor, nuclear factor-κB (NF-κB) during aging. Genistein, a major component found in isoflavone, has anti-inflammatory activities that are often associated with its anti-oxidative activity. The purpose of this study is to document molecular mechanism of altered Ang II-related NF-κB activation during aging and inhibitory molecular events by genistein regarding to age-related Ang II-induced NF-κB activation. At present, we utilized young (6months old), old (24months old), and genistein-treated (2 and 4mg/kg/day for 10days) old rats. For our current study, we choose to use the kidney and rat endothelial cell line, YPEN-1 because of its vulnerability to age-related oxidative stress and inflammatory responsiveness. The results of the analysis showed that Ang II and AT1 expression increased during aging and that these increases were blunted by treatment with genistein. Furthermore, we investigated the inhibitory effects of genistein on the Ang II-induced redox imbalance in aged rat kidneys. Genistein reduced age-related increases in NF-κB activity and NF-κB-dependent pro-inflammatory genes expression. We also determined genistein attenuated Ang II-induced NF-κB activation through its anti-oxidant activity in YPEN-1 cells. Taken together, our present results show that genistein has potent anti-inflammatory effect resulting in the attenuation of the Ang II-induced NF-κB activation during aging. The most significant new finding from this study is that genistein exerts its anti-Ang II action during aging by suppressive effect of NF-κB activation. Based on these data, genistein may be an anti-Ang II agent that may be used in anti-inflammatory therapies.

Mechanism of Ang II involvement in activation of NF-κB through phosphorylation of p65 during aging

Angiotensin II (Ang II), a major effector of the renin-angiotensin system, is now recognized as a pro-inflammatory mediator. This Ang II signaling, which causes transcription of pro-inflammatory genes, is regulated through nuclear factor-κB (NF-κB). At present, the molecular mechanisms underlying the effect of aging on Ang II signaling and NF-κB activation are not fully understood. The purpose of this study was to document altered molecular events involved in age-related changes in Ang II signaling and NF-κB activation. Experimentations were carried out using kidney tissues from Fischer 344 rats at 6, 12, 18, and 24months of age, and the rat endothelial cell line, YPEN-1 for the detailed molecular work. Results show that increases in Ang II and Ang II type 1 receptor during aging were accompanied by the generation of reactive species. Increased Ang II activated NF-κB by phosphorylating IκBα and p65. Increased phosphorylation of p65 at Ser 536 was mediated by the enhanced phosphorylation of IκB kinase αβ, while phosphorylation site Ser 276 of p65 was mediated by upregulated mitogen-activated and stress-activated protein kinase-1. These altered molecular events in aged animals were partly verified by experiments using YPEN-1 cells. Collectively, our findings provide molecular insights into the pro-inflammatory actions of Ang II, actions that influence the phosphorylation of p65-mediated NF-κB activation during aging. Our study demonstrates the age-related pleiotropic nature of the physiologically important Ang II can change into a deleterious culprit that contributes to an increased incidence of many chronic diseases such as atherosclerosis, diabetes, and dementia.

Response of Rat Retinal Capillary Pericytes and Endothelial Cells to Glucose

The purpose of this study was to investigate the effects of hyperglycemia, its fluctuations, and glucose starvation on the expression of glucose-regulated protein 78/binding immunoglobulin protein (GRP78/BiP), one of the most commonly used markers of endoplasmic reticulum stress, in rat capillary pericytes and endothelial cells cultured separately and together. Conditionally immortalized rat retinal pericyte and endothelial cell lines were cultured in dishes coated with collagen type I in Dulbecco's modified Eagle's medium containing 5.5 mM glucose. For cocultures, pericytes and endothelial cells were seeded together on rat tail collagen type I-coated cell culture plates. After 24 h of initial culture, the medium was replaced with serum-free medium containing 0-100 mM glucose for periods of up to 72 h. GRP78/BiP, caspase-3, and nuclear factor-κB expression were investigated using western blots. No significant increase in GRP78/BiP expression was observed when pericytes, endothelial cells, or cocultures were exposed to either 25, 50, or 100 mM glucose for 48 h compared with the control level of 5.5 mM glucose. Similarly, no change in expression of GRP78/BiP was observed when media glucose levels were reduced from either 5.5 or 25 to 1 mM. GRP78/BiP expression significantly increased when cells were cultured for 24 h in glucose-deprived medium. This was accompanied by a time-dependent increase in the expression of caspase-3 and nuclear factor-κB. In diabetic retinopathy, hyperglycemia has been reported to induce apoptosis in retinal capillary vascular cells, but these studies suggest that the apoptosis is not linked to the expression of GRP78/BiP, one of the most commonly used markers of endoplasmic reticulum stress. However, GRP78/BiP-linked apoptosis may play a role in vascular changes associated with retinal ischemia/reperfusion.

Uptake of folates by rat and human brain endothelial cell lines

The blood–brain barrier (BBB) is essential for central nervous system homeostasis. Folates are essential for the biosynthesis of purines, pyrimidines and methionine. Our aim was to characterize 3H‐folic acid (3H‐FA) and 14C‐methyltetrahydrofolic acid (14C‐MTHF) uptake by rat (RBE4) and human (hCMEC/D3) BBB endothelial cell lines. Uptake of 3H‐FA and 14C‐MTHF by RBE4 cells was time‐dependent and linear for the first 2 min of incubation. On the other hand, uptake of these substances by hCMEC/D3 cells showed less time‐dependency. Further experiments were performed in RBE4 cells. Uptake of 3H‐FA was found to be (1) stimulated at an acidic and at an alkaline pH, (2) Na+‐dependent and stimulated when F‐ substituted for Cl−, (3) energy‐independent, (4) inhibited by premetrexed (PTX) and stimulated by cytochalasin D (cyt D) and (5) not affected by MTHF, DIDS, SITS, methotrexate (MTX), monensin (MON) and FA. Uptake of 14C‐MTHF was found to be (1) pH‐independent, (2) Na+, Cl− and energy‐independent, (3) inhibited by PTX and MTX and stimulated by cyt D and (4) not affected by FA, DIDS, SITS, MON and MTHF. RT‐PCR analysis demonstrates mRNA expression of RFC (reduced folate carrier), but neither of FRα (folate receptor α) nor of PCFT (proton‐coupled folate transporter) in RBE4 cells and mRNA expression of both RFC and PCFT, but not of FRα, in hCMEC/D3 cells. Human and rat BBB cells show little capacity for folates uptake.

Characterization of uptake of folates by rat and human blood–brain barrier endothelial cells

This study aimed to characterize (3)H-folic acid ((3)H-FA) and (14)C-methyltetrahydrofolic acid ((14)C-MTHF) uptake by rat (RBE4) and human (hCMEC/D3) blood-brain barrier (BBB) endothelial cell lines. Uptake of (3)H-FA and (14)C-MTHF by RBE4 cells was time dependent and linear for the first 2 min of incubation; uptake by hCMEC/D3 cells showed a less marked time-dependency and a greater experimental variability. So, further experiments were performed in RBE4 cells only. Uptake of (3)H-FA was stimulated at acidic and alkaline pH, Na(+) dependent, stimulated when F(-) substituted for Cl(-), energy independent, inhibited by premetrexed, stimulated by cytochalasin D, and unaffected by MTHF, DIDS, SITS, methotrexate, monensin, and FA. Uptake of (14)C-MTHF was found to be pH-, Na(+)-, Cl(-)- and energy independent, inhibited by premetrexed and methotrexate, stimulated by cytochalasin D, and unaffected by FA, DIDS, SITS, monensin, and MTHF. RT-PCR analysis showed mRNA expression of reduced folate transporter (RFC), but neither of FRalpha nor of proton-coupled folate transporter (PCFT) in RBE4 cells, and mRNA expression of RFC and PCFT, but not of FRalpha, in hCMEC/D3 cells. In conclusion, both human and rat BBB endothelial cells show little capacity for (3)H-FA and (14)C-MTHF apical uptake. Hence, these cell lines do not appear to be a good model to study the transport of folates at the BBB.

Kaempferol modulates pro-inflammatory NF-κB activation by suppressing advanced glycation endproducts-induced NADPH oxidase

Advanced glycation endproducts (AGE) are oxidative products formed from the reaction between carbohydrates and a free amino group of proteins that are provoked by reactive species (RS). It is also known that AGE enhance the generation of RS and that the binding of AGE to a specific AGE receptor (RAGE) induces the activation of the redox-sensitive, pro-inflammatory transcription factor, nuclear factor-kappa B (NF-kB). In this current study, we investigated the anti-oxidative effects of short-term kaempferol supplementation on the age-related formation of AGE and the binding activity of RAGE in aged rat kidney. We further investigated the suppressive action of kaempferol against AGE's ability to stimulate activation of pro-inflammatory NF-kB and its molecular mechanisms. For this study, we utilized young (6 months old), old (24 months old), and kaempferol-fed (2 and 4 mg/kg/day for 10 days) old rats. In addition, for the molecular work, the rat endothelial cell line, YPEN-1 was used. The results show that AGE and RAGE were increased during aging and that these increases were blunted by kaempferol. In addition, dietary kaempferol reduced age-related increases in NF-kappaB activity and NF-kB-dependant pro-inflammatory gene activity. The most significant new finding from this study is that kaempferol supplementation prevented age-related NF-kappaB activation by suppressing AGE-induced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Taken together, our results demonstrated that dietary kaempferol exerts its anti-oxidative and anti-inflammatory actions by modulating the age-related NF-kappaB signaling cascade and its pro-inflammatory genes by suppressing AGE-induced NADPH oxidase activation. Based on these data, dietary kaempferol is proposed as a possible anti-AGE agent that may have the potential for use in anti-inflammation therapies.

Effect of Low-Dose Paclitaxel and Docetaxel on Endothelial Progenitor Cells

Objective: Bone marrow (BM)-derived endothelial progenitor cells (EPC) play an important role in neovascularization and tumor growth. It has been reported that docetaxel and paclitaxel inhibit angiogenesis, but the effect of docetaxel and paclitaxel on EPC-induced neovascularization has not been examined. We aimed to clarify the cytotoxic and inhibitory effects of these drugs on EPC. Methods: The effects of drugs on growth, tube formation, and migration of EPC were analyzed in vitro using a rat BM-derived EPC cell line (TR-BME). Fluorescence-labeled TR-BME cells were injected into tumor-bearing rats and accumulation at the tumor site was analyzed by fluorescence-activated cell sorting (FACS). Results: In in vitro cytotoxicity assays of these drugs in TR-BME, rat endothelial cell line TR-BBB and rat tumor cell line Walker 256, the IC₅₀ values for TR-BME were higher than those for TR-BBB or Walker 256. Both drugs inhibited tube formation and migration of TR-BME at lower concentrations than the cytotoxic IC₅₀. In vivo studies showed that a low dose of both drugs inhibited EPC accumulation at the tumor site in tumor-bearing rats, as determined by FACS, and caused a decrease in microvessel density. Conclusion: Docetaxel and paclitaxel directly inhibited EPC-initiated vasculogenesis at low (non-cytotoxic) concentrations, causing suppression of tumor growth.

Expression of VACM-1/cul5 mutant in endothelial cells induces MAPK phosphorylation and maspin degradation and converts cells to the angiogenic phenotype

Vasopressin-activated calcium mobilizing receptor (VACM-1) is a member of the cullin gene family involved in ubiquitin–proteosome dependent regulation of cellular functions. Expression of VACM-1 cDNA in cos-1 cells in vitro decreases basal cAMP levels and inhibits growth. The expression of S730AVACM-1 mutant cDNA, which removes PKA-dependent phosphorylation site in the VACM-1 cDNA sequence, reverses this phenotype. Since the expression of VACM-1 protein in vivo localizes largely to the vascular endothelial cells, in this study, we examined the effects of S730AVACM-1 cDNA expression on cellular signaling in the rat endothelial cell line RAMEC. Our results indicate that expression of S730AVACM-1 cDNA in RAMEC promotes cellular proliferation and induces angiogenic growth patterns. Western blot analyses indicate that S730AVACM-1 cDNA transfected cells express increased levels of Nedd8 modified VACM-1 and have higher levels of phosphorylated MAPK protein when compared to controls. Furthermore, expression of S730AVACM-1 cDNA induces translocation of the endogenous early response gene, egr-1, to the nucleus and leads to morphological changes that involve actin rearrangement. Finally, expression of S730AVACM-1 cDNA in RAMEC decreases concentration of maspin, a putative anti-angiogenic factor with a tumor suppressor activity. These results show that VACM-1 protein regulates endothelial cell growth and may modulate angiogenesis by a mechanism that involves MAPK phosphorylation, nuclear localization of egr-1, maspin expression, and actin polymerization.

Altered Expression of Basement Membrane-related Molecules in Rat Brain Pericyte, Endothelial, and Astrocyte Cell Lines after Transforming Growth Factor-β1 Treatment

The basement membrane at the blood-brain barrier (BBB) plays important roles in maintaining the structure and function of capillary vessels. The BBB is constructed from endothelial cells, astrocytes and pericytes, but their interactions in the formation or maintenance of basement membrane have not been established. Transforming growth factor-beta1 (TGF-beta1) is known to increase fibronectin in brain capillary basement membrane with deposition of beta-amyloid. We previously reported that the mRNA level of alpha-smooth muscle actin in a brain capillary pericyte cell line TR-PCT1 was increased by treatment with TGF-beta1. In this study, expression of mRNAs encoding basement membrane-related molecules in TR-PCT1, a rat endothelial cell line TR-BBB13, and a type 2 astrocyte cell line TR-AST4 was evaluated by RT-PCR. The effects of TGF-beta1 on expression of basement membrane-related genes in these cell lines were also examined. Fibronectin, MMP-9, tPA, TIMP-1, and PAI-l in TR-PCT1 were higher than in TR-BBB13 and TR-AST4. In TR-PCT1 treated with TGF-beta1, collagen type IV, PAI-1, and MMP-9 were increased, and TIMP-2 was reduced. The change in PAI-1 mRNA was faster than those in MMP-9, TIMP-2, collagen type IV mRNAs. These results suggest that pericytes may be key cells in the maintenance of the basement membrane at the BBB.

ICAM-1 Signal Transduction in Cells Stimulated with Neutrophil Elastase

Neutrophil elastase, which enhances intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells, plays an important role in ischemia/reperfusion injury. Here, we investigated signal transduction of ICAM-1 expression in endothelial cells stimulated by neutrophil elastase. Pretreatment of animals with the neutrophil elastase inhibitor, ONO-5046.Na significantly decreased the number of neutrophils or Mac-1(+) (CD11b/CD18) cells in ischemic liver lobes after reperfusion. ICAM-1 expression in the rat endothelial cell line (WK-5) was significantly upregulated after stimulation with neutrophil elastase, but this reaction was inhibited by the neutrophil elastase inhibitor ONO-5046.Na. ICAM-1 mRNA expression, which is induced by neutrophil elastase in a dose-dependent manner, was repressed by the alpha1-protease inhibitor. ICAM-1 expression, stimulated by neutrophil elastase, was partially reduced by a diacylglycerol kinase inhibitor and protein kinase C inhibitor, but was completely inhibited by a phospholipase C inhibitor, cytosolic Ca(2+) chelator, calmodulin antagonist, and nuclear transcription factor kappa B inhibitor. Binding of (125)I-neutrophil elastase to WK-5 cells was competitively inhibited by the addition of unlabeled neutrophil elastase. The neutrophil elastase inhibitor significantly reduces ICAM-1 expression and Mac-1(+) cell accumulation in ischemic liver lobes after reperfusion. Neutrophil elastase stimulates ICAM-1 expression in endothelial cells by intracellular signal transduction via activation of diacylglycerol kinase, protein kinase C, phospholipase C, Ca(2+)-calmodulin, and nuclear transcription factor kappa B.

Gene therapy for experimental brain tumors using a xenogenic cell line engineered to secrete hIL-2.

Local delivery of cytokines has been shown to have a potent anti-tumor activity against a wide range of malignant brain tumors. In this study, we examined the feasibility and efficacy of using a rat endothelial cell line (NTC-121) transfected with the human interleukin-2 (IL-2) gene in treating experimental murine CNS tumors. The NTC-121 cells were injected intracranially in C57BL/6 mice (N = 10/group) along with non-irradiated, non-transfected B16/F10 (wild type) melanoma cells. Sixty percent of mice treated with IL-2 (p < 0.001 vs. control) were long-term survivors (LTS) of > 120 days. Control animals that received only wild type cells had a median survival of 18 days (range 15-20). Histopathological examination of brains from animals sacrificed at different times showed no tumor growth in the non-irradiated NTC-121 group, moderate (1-2 mm) tumor growth in the irradiated group, and gross tumor invasion (>2 mm) and tissue necrosis in the control group. Moreover, animals treated with IL-2 showed an accumulation of CD8+ T cells around the site of the injected tumor. The use of a xenogenic cell line to deliver hIL-2 stimulates a strong immunologic cytotoxic anti-tumor response that leads to significant prolongation of survival in mice challenged with the B16/F10 intracranial melanoma tumor. Our findings demonstrate that the use of a xenogenic cell line can provide a potent vehicle for the delivery of gene therapy and may therefore represent a new approach for brain tumor therapy.

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4–5%, p<0.05). Immunofluorescence showed a sizable (39%) and significant ( p<0.01) enhancement of P-selectin expression at the lowest concentration of ethamsylate tested (1 μM), with maximal enhancement of P-selectin expression (75–90%) at 10 μM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance.

Interleukin-1β-induced type IIA secreted phospholipase A2gene expression and extracellular activityin rat vascular endothelial cells

Two phospholipase A2(PLA2) isoforms, secretory and cytosolic, have been implicated in inflammation. Secretory type IIA PLA2(sPLA2-IIA), which hydrolyzes fatty acids bound at the sn-2 position of glycerophospholipids, has been detected universally in a variety of mammalian tissues and cells. The expression of the sPLA2-IIA gene and its extracellular activity were shown to be regulated by different factors such as hypoxia, cytokines and phorbol esters. In the present study, we examined the effects of interleukin-1β (IL-1β) on the expression of the 14kDa sPLA2-IIA, determined using reverse transcription polymerase chain reaction and radiometricEscherichia coli enzyme assay in primary cultures of rat endothelial cells and in two different rat endothelial cell lines (SVAREC and RBE4). These experiments revealed that IL-1β induces sPLA2-IIa gene expression and secretion of the enzyme in endothelial cells in a dose- and time-dependent manner. The cAMP-elevator forskolin did not augment the cytokine-induced elevation of sPLA2-IIa enzyme activity but significantly increased the IL-1β-stimulated sPLA2-IIa mRNA contents in endothelial cells.

Occludin modulates organization of perijunctional circumferential actin in rat endothelial cells.

The tight junction is not a constitutional junctional apparatus in endothelial cells, but develops in a particular lineage of endothelia, such as the capillary endothelia in the brain and retina, and thus is considered to be pivotal for the maintenance of the blood-tissue barrier. Occludin is an integral membrane component of tight junctions, but the role of occludin in the endothelial cell function remains to be elucidated. We have cloned and transfected rat full-length occludin cDNA into a rat endothelial cell line (RLE) that expressed only a trace amount of occludin with no fine circumferential actin bundles at the cell border in native conditions. Occludin was expressed at the cell border of RLE cells, and circumferential fine actin bundles developed in close relation to the sites of occludin localization. Even under subconfluent culture conditions, fine circumferential actin bundles were formed at the sites where occludin-positive cell-cell contact was achieved. In immunoelectron microscopy, occludin was localized at distinct areas of the plasma membrane, always in association with the cytoplasmic actin filaments. On the other hand, actin bundles were not seen in occludin-negative juxtaposing plasma membranes. Collectively, these data strongly suggested a possible determinant function of occludin for the organization of actin in endothelial cells.

A comparative study of MMP and TIMP-2 production in rat and human foetal CNS vascular endothelial cell lines

A comparative study of MMP and TIMP-2 production in rat and human foetal CNS vascular endothelial cell lines

Neutrophil elastase enhances intercellular adhesion molecule-1 expression.

Elastase released from activated neutrophils is an important mediator of inflammatory tissue damage. We investigated the effect of human neutrophil elastase (NE) inhibitor (ONO-5046) on reperfusion injury after pancreaticoduodenal transplantation in rats by measuring the expression of intercellular adhesion molecule-1 (ICAM-1). Additional in vitro experiments were conducted to investigate the effect of NE on ICAM-1 mRNA transcription in a rat endothelial cell line (WK-5) and human umbilical vein endothelial cells (HUVEC). In an in vivo experiment, male Wistar rats were transplanted with syngeneic pancreaticoduodenal grafts. An NE inhibitor, ONO-5046, was injected intravenously 5 min before vascular clamping and immediately after reperfusion at a dose of 10 mg/kg. ICAM-1 expression was determined by immunostaining and Northern analysis. In in vitro experiments, the effects of NE and chemical agents on ICAM-1 mRNA transcripts were determined in WK-5 cells and HUVEC. Pretreatment with ONO-5046 decreased ICAM-1 immunostaining in the pancreatic graft and inhibited the increase in ICAM-1 mRNA levels in grafts after reperfusion. ICAM-1 mRNA levels in WK-5 cells and HUVEC showed stimulation by NE, while ONO-5046 inhibited this increase. Calcium ionophore (A23187) augmented NE stimulation of ICAM-1 mRNA levels in these cells. In contrast, a phospholipase C inhibitor (U73122) blunted NE induction of ICAM-1 mRNA, and either calcium chelator (TMB-8) or a nuclear factor-kappa B inhibitor (pyrrolidine dithiocarbamate) completely inhibited induction. These results indicate that NE stimulates ICAM-1 expression in pancreatic grafts via intracellular Ca2+ influx and a phospholipase C signal transduction.