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Mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) has been reported to enhance wound healing. Exosomes contain nucleic acids, proteins, and lipids, and function as an intercellular communication vehicle for mediating some paracrine effects. However, the function of MSC-derived exosomes (MSC-exo) remains elusive. In this study, we isolated human placenta MSC (PlaMSC)-derived exosomes (PlaMSC-exo) and examined their function in vitro. PlaMSCs were isolated from human term placenta using enzymatic digestion. PlaMSC-exo were prepared from the conditioned medium of PlaMSC (PlaMSC-CM) by ultracentrifugation. The expression of stemness-related genes, such as OCT4 and NANOG, in normal adult human dermal fibroblasts (NHDF) after incubation with PlaMSC-exo was measured by real-time reverse transcriptase PCR analysis (real-time PCR). The effect of PlaMSC-exo on OCT4 transcription activity was assessed using Oct4-EGFP reporter mice-derived dermal fibroblasts. The stimulating effects of PlaMSC-exo on osteoblastic and adipocyte-differentiation of NHDF were evaluated by alkaline phosphatase (ALP), and Alizarin red S- and oil red O-staining, respectively. The expression of osteoblast- and adipocyte-related genes was also assessed by real-time PCR. The treatment of NHDF with PlaMSC-exo significantly upregulated OCT4 and NANOG mRNA expression. PlaMSC-exo also enhanced OCT4 transcription. The NHDF treated with PlaMSC-exo exhibited osteoblastic and adipocyte-differentiation in osteogenic and adipogenic induction media. PlaMSC-exo increase the expression of OCT4 and NANOG mRNA in fibroblasts. As a result, PlaMSC-exo influence the differentiation competence of fibroblasts to both osteoblastic and adipocyte-differentiation. It shows a new feature of MSCs and the possibility of clinical application of MSC-exo. J. Cell. Biochem. 117: 1658-1670, 2016. © 2015 Wiley Periodicals, Inc.

Background: Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. Methods: PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte-like functions of PDLSCs were examined in co-culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. Results: Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146- and NG2-positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary-like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary-like structure. Conclusions: PDLSCs possess pericyte-like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell–based periodontal therapy. J Periodontol 2013;84:1425-1433.

We hypothesized that gene expression in placenta and umbilical vessels are affected by intrauterine environment and some of the expression in umbilical vessels originating from the fetus could reflect fetal condition of these complicated pregnancies. Expression of angiogenesis-related factors and inflammatory cytokines were examined in placenta and umbilical vessels to clarify the effects of intrauterine environment of pregnancies complicated by preeclampsia and chorioamnionitis/funisitis. Forty-six preterm cesarean section deliveries were classified into three groups based on maternal condition during prenatal monitoring: preeclampsia (PE) (n = 11), chorioamnionitis/funisitis (CAM) (n = 8), and preterm control (PC) (n = 27). Angiogenesis-related factors and inflammatory cytokines in placentas, umbilical arteries and umbilical veins were analyzed by RT-PCR and immunohistochemistry. We demonstrated that Ang-2, Tie-2, and Dll4 increase in the placentas of PE compared to PC for the first time, and we confirmed the findings of previous reports showing the high expression of HIF-1α, sFlt-1, endoglin, leptin, and AT1R. Expression of angiogenesis-related factors, including HIF-1α, VEGF, angiopoietin, and TGF-β systems, and inflammatory cytokines, such as TNF-α and IL-6, increased in umbilical vessels of PE. Umbilical veins of CAM showed a higher Dll4 level than did PC. In preeclampsia, abnormal expressions of angiogenesis-related factors related to lifestyle diseases in adulthood were seen in the placenta and umbilical vessels as compared to PC. Chorioamnionitis/funisitis showed only upregulation of DII4 in umbilical veins.

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth-anchoring structure. Periodontal ligament is a unique non-mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)-positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over-expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast-related cells, but it also regulates cementoblast commitment in periodontal ligament cells.

The epithelial–mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-β induced EMT and that long-term exposure to TGF-β elicited the epithelial–myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-β induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-β-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-β and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-β and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.

Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated from Escherichia coli (the PURE system) in the presence of liposomes. Connexin-43 (Cx43), which is a water-insoluble integral membrane protein that forms a hexameric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. The addition of liposomes following protein expression (post-translational presence of liposomes) did not lead to the integration of Cx43 into the liposome membranes. The amount of integrated Cx43 increased as the liposome concentration increased. The presence of liposomes did not influence the total amount of synthesized Cx43. The Cx43 integrated into the liposome membranes formed open membrane pores. These results indicate that the liposomes act in a chaperone-like manner by preventing Cx43 from aggregating in solution, because of integration into the bilayer, and also by functionalization of the integrated Cx43 in the membrane. This is the first report that cell-free-synthesized water-insoluble membrane protein is directly integrated with a uniform orientation as a functional oligomer into liposome membranes. This simple proteoliposome preparation procedure should be a valuable approach for structural and functional studies of membrane proteins. Structured digital abstract • MINT-7900670: Cx-43 (uniprotkb:P08050) and Cx-43 (uniprotkb:P08050) bind (MI:0407) by cross-linking study (MI:0030)

Increasing evidence points to a link between histone deacetylases (HDACs) and tumorigenesis. Although several HDAC inhibitors have been tested in clinical trials for cancer therapies, the mechanisms of HDAC activation in tumors remain unknown. In this study, we investigated the pathway of HDAC activation in the context of hypoxia and inflammation, common features of solid tumors. In HeLa cells, hypoxia was a more potent activator of HDAC than IL-1beta. As HDAC protein expression did not change during treatment, we hypothesized that hypoxia regulated HDAC activity through post-translational modification. We observed that hypoxia induced HDAC1 and HDAC2 protein phosphorylation both in the presence and absence of IL-1beta. Using TBB, an inhibitor of protein kinase CK2, we showed that CK2 was required for hypoxia-induced HDAC activation. We also observed that CK2 activity was induced by hypoxia but not by IL-1beta alone. While CK2beta subunits were retained in the cytoplasm upon hypoxic treatment, CK2alpha and CK2alpha' subunits were shuttled to the nucleus, where HDAC1 and HDAC2 are predominantly localized. Knockdown of catalytic and regulatory subunits of CK2 revealed that formation of heterotetramic complex was not required for HDAC phosphorylation. von Hippel-Lindau protein (pVHL) inactivation and hypoxia inducible factor-1alpha (HIF-1alpha) activation are associated with tumor growth and vasculogenesis. Use of Apicidin (an HDAC inhibitor) and TBB revealed that CK2-dependent HDAC activation contributed to pVHL downregulation and HIF-1alpha stabilization under hypoxia. Our findings that CK2 may be a key mediator for HDAC activation under hypoxia support the future application of CK2 inhibitors in cancer therapy.

To clarify the mechanism of vascular endothelial cell injury induced by activated leukocytes, we examined the effects of antibodies against adhesion molecules on the injury and on the intracellular peroxide level in endothelial cells. Treatment of leukocytes with phorbol myristate acetate (PMA) caused significant increases in the expression of adhesion molecules, CD11a, CD11b, CD11c, and CD18, on the surface of the leukocytes. Monoclonal antibodies against CD11a, CD11b and CD18, and ICAM-1, an adhesion molecule in the side of endothelial cells, abolished significantly the endothelial cell injury induced by PMA-stimulated leukocytes. These antibodies affected neither the production of active oxygen species by the leukocytes nor the rate of adhesion of leukocytes to endothelial cells. These data indicated that adhesion through CD11/CD18-ICAM-1 is necessary for leukocytes to induce endothelial cell injury. To investigate the phenomenon that occurred after the specific adhesion, the change in the intracellular peroxide level was measured using fluorescence of 2,7-dichlorofluorescein diacetate. The fluorescence intensity of the endothelial cells exposed to PMA-stimulated leukocytes increased with time up to 15 minutes, although neither PMA alone nor unstimulated leukocytes alone showed such activity at all. The monoclonal antibodies against CD11a, CD11b, CD18, and ICAM-1 also showed inhibitory effects on the increase in intracellular fluorescence intensity of the endothelial cells exposed to PMA-stimulated leukocytes. In contrast, CD11c could block neither the cell injury nor the increase in intracellular fluorescence in endothelial cells exposed to PMA-stimulated leukocytes. Thus, the addition of PMA-stimulated leukocytes to an endothelial cell monolayer caused a significant increase in the intracellular peroxide level in the endothelial cells after 15 minutes and severe endothelial cell injury after 5 hours. Both the early increase in peroxide production and late cell lysis were abolished by specific antibodies against CD11a, CD11b, CD18, and ICAM-1, but not CD11c. There seems to be a close relationship between the early and late events. Both events were only partially blocked by catalase (approximately 40%), but almost completely abolished by deferoxamine, a chelator of ferrous ions, suggesting that hydroxyl radicals produced in endothelial cells themselves from xanthine oxidase may injure the cells from their inside. Therefore, the effect of allopurinol, a specific inhibitor of xanthine oxidase, was examined. Pretreatment of endothelial cells with allopurinol caused significant but not complete inhibition (approximately 60%) of both the early and the late events, suggesting that influx of hydrogen peroxide may also be important.(ABSTRACT TRUNCATED AT 400 WORDS)

Apicularen A and the known vacuolar-type (H(+))-ATPase (V-ATPase) inhibitor bafilomycin A(1) induced apoptosis of RAW 264.7 cells, while apicularen B, an N-acetyl-glucosamine glycoside of apicularen A, was far less effective. Apicularen A inhibited vital staining with acridine orange of the intracellular organelles of RAW 264.7 cells, inhibited the ATP-dependent proton transport into inside-out microsome vesicles, and inhibited the bafilomycin A(1)-sensitive ATP hydrolysis. The IC(50) values of the proton transport were 0.58 nM for apicularen A, 13 nM for apicularen B, and 0.95 nM for bafilomycin A(1). Furthermore, apicularen A inhibited the bafilomycin A(1)-sensitive ATP hydrolysis more potently than apicularen B. F-ATPase and P-ATPase were not inhibited by apicularen A. We concluded that apicularen A inhibits V-ATPase, and thus induces apoptosis in RAW 264.7 cells.

The endothelial cells cultured in collagen gel caused upregulation of KDR expression, which resulted in an increase in tube formation. Endothelial cells exposed to high glucose (33 mmol/l) for 30 days increased the tube formation induced by VEGF, but not by serum and bFGF. Immunohistochemical study showed that KDR expression was upregulated by the high-glucose treatment. The endothelial cells treated with 0.5-5 micrograms/ml eicosapentaenoic acid (EPA, 20:5, n-3) for 48 h displayed a dose-dependent suppression of tube formation, VEGF-induced proliferation, and activation of p42/p44 MAP kinase but not bFGF-induced ones. Pretreatment with arachidonic acid (20:4, n-6) and docosahexaenoic acid (22:6, n-3) did not show such effects. The expression of KDR was downregulated by the EPA pretreatment. The bone is the richest tissue in microvessel networks except for the liver. Osteoblasts produced VEGF and some factor(s) that could induce KDR upregulation in endothelial cells and could enhance tube formation. These results lead to the speculation that the regulation of KDR expression as well as VEGF production is deeply involved in angiogenesis under various conditions.

Eicosapentaenoic acid (EPA) may protect against atherosclerosis by improving lipid metabolism and modulating vascular cell function. Ischemia/ reperfusion injury is one risk factor for atherosclerosis. We investigated if EPA could improve hypoxia/reoxygenation (H/R)-induced endothelial cell dysfunction of gap junctional intercellular communication (GJIC). GJIC in human umbilical vascular endothelial cells (HUVECs) was measured using a photobleaching technique. Results demonstrated that H (24h)/R 2h) induced a GJIC reduction in HUVECs; however, it was inhibited by EPA pretreatment. H/R produced reactive oxygen species, but it was not affected by EPA, and it contributed little to GJIC dysfunction. By contrast, tyrosine kinase activated by H/R was inhibited by EPA pretreatment, and tyrosine kinase inhibitors also abolished H/R-induced GJIC reduction. The protective effects of EPA on the H/R-induced GJIC reduction was also observed in cells treated with tyrosine phosphatase inhibitor. These data indicate the EPA improves H/R-induced endothelial dysfunction through inhibition of tyrosine kinase activation, and it could lead to prevention of progression and/or initiation of atherosclerosis.

Background: As periodontal tissues are constantly exposed to mechanical stress during mastication, the relationship between mechanical stimulation and biochemical phenomena has been extensively investigated. Objectives: The aim of the present study was to assess the change in the production of angiogenic regulators produced by human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF), cultured on a flexible substrate, before and after application of cyclic tensile stretching. Materials and methods: Both cell types were stretched in a Flexercell Strain Unit to 7, 14 and 21% elongation, at a frequency of 12 cycles/min. Medium cultured with HGF or HPLF was examined by enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF), Western blotting of pigment epithelium-derived factor (PEDF) and in vitro angiogenesis assay. The residual cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for both VEGF and PEDF mRNA expression. Results: Stretching increased the VEGF mRNA level and VEGF secretion in both HGF and HPLF. The concentration of VEGF in the conditioned medium of the stretched HPLF was almost the same as that of stretched HGF. In the in vitro angiogenesis assay, the conditioned medium of HPLF after stretching showed a dramatic increase in tube formation. In contrast, stretched HGF did not show enhanced tube formation, despite the increase in VEGF secretion by stretched HGF. The mRNA levels of PEDF, an inhibitor of angiogenesis, were higher in HGF than HPLF. The protein level of PEDF in HGF was also higher than that in HPLF. Conclusion: These findings suggest that under mechanical stress HPLF promotes angiogenesis via expression of VEGF, whereas under the same conditions angiogenesis is not promoted in HGF, due to the expression of PEDF.

Megakaryocyte differentiation is composed of three distinct stages: formation of erythromegakaryocytic progenitor cells, maturation of megakaryocytes and production of platelets. We have developed a liquid culture system for megakaryocyte terminal differentiation from haematopoietic stem cells into proplatelets. In this system, CD34+ cells isolated from human cord blood, differentiated to CD41+ cells, were classified either as propidium iodide (PI)+ cells (large) or PI- cells (small) by fluorescence-activated cell sorting analysis on the late-stage CD41+ cells. Transmission electron microscopy showed that the cultured small cells were morphologically identical to platelets isolated from normal peripheral blood. Moreover, the number of differentiated cells that were CD42b-positive attained an approximately 60-fold expansion over that of the primary CD34+ cells in this culture system. Furthermore, gene expression of megakaryocytopoietic transcriptional factors, GATA-1 and NF-E2, and several megakaryocytic markers such as glycoprotein (GP)IIb and thromboxane synthase was observed in the individual differentiation stage. Treatment with fibrinogen, a ligand of GPIIb/IIIa, increased the number of CD41+/PI+ cells, but treatment in the late stage suppressed CD41+/PI- cell formation, suggesting that fibrinogen promotes megakaryocytopoiesis, but not thrombopoiesis. We conclude that this liquid culture system using human CD34+ cells may be used to mimic the physiological development from haematopoietic stem cells into megakaryocytes, as well as promote subsequent thrombopoiesis.

Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.

We investigated gene expression profiles of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in differentiated and non-differentiated retinal pigment epithelial (RPE) cells during oxidative stress. Human RPE cells were grown in culture on laminin-coated flasks to obtain differentiated features. Cells cultured on plastic were used as non-differentiated controls. After confluence, hydrogen peroxide (H2O2) was added for 48 h, then, total RNA was extracted and used for RT-PCR and Northern blot analysis. Medium conditioned by RPE was used for ELISA, Western blotting, and in vitro angiogenesis assay. As a result, differentiated RPE cells expressed significantly higher levels of VEGF protein, as compared to their non-differentiated counterparts. The expression pattern remained consistent even after cellular exposure to H2O2. Conversely, while elevated levels of PEDF transcript and protein were seen in differentiated RPE cells, compared to non-differentiated cells, a marked decrease at both PEDF mRNA and protein levels was seen after treatment with H2O2. Moreover, this decrease in PEDF expression was dosage dependent. In in vitro angiogenesis assay, conditioned medium from differentiated human RPE cells after exposure to H2O2 showed a dramatic increase in tubular formation and migratory activity of microvascular endothelial cells. These data suggest that, in physiological conditions, a critical balance between PEDF and VEGF exists, and PEDF may counteract the angiogenic potential of VEGF. Under oxidative stress, PEDF decreases disrupting this balance. This equilibrium shift may be significant in promoting a pathological condition of RPE cells and contributing to choroidal neovascularization in age-related macular degeneration.

Vascular endothelial cells (EC), communicating with one another across gap junctions, are usually made dysfunctional by hypoxia and reoxygenation (H/R); however, very limited information exists regarding the effects of H/R on the endothelial gap junctions. We investigated whether H/R interferes with endothelial gap junctional intercellular communication (GJIC). After human umbilical vein EC had grown to confluence, they were exposed to hypoxia (pO2 < 0.1%) for 12–16 h and then returned to normal atmospheric conditions for reoxygenation. At 0-, 2-, 4-, 6-h reoxygenation, GJIC was detected by means of a fluorescence recovery after a photobleaching technique. The results demonstrated that a GJIC reduction (about 20% less than that under normoxia) was induced after 2 h of reoxygenation; after 4 h of reoxygenation, it began to recover (to about 10% less than that under normoxia); and after 6 h of reoxygenation, GJIC was restored to the normal level. Calphostin C (1 × 10−7 mol/l), a specific protein kinase C inhibitor, partially inhibited the reduction in GJIC (resulting in a level about 10% less than that under normoxia), whereas the tyrosine kinase inhibitor genistein (10 µmol/L) completely blocked the reduction in GJIC. Vanadate (1.5 mmol/l), a tyrosine phosphatase inhibitor, amplified the inhibitory effect of H/R on GJIC (to about 40% less than that under normoxia). Immunofluorescence and immunoprecipitation showed that 2-h reoxygenation significantly stimulated tyrosine protein phosphorylation, and this phosphorylation event was obviously enhanced by vanadate. The results of Western blotting showed that the gap junctional protein connexin 43 (Cx43) was phosphorylated by H/R; moreover, immunoprecipitation demonstrated that 2-h reoxygenation induced a prominent increase of tyrosine phosphorylation of Cx43 compared with that under normoxia. These data indicate that H/R induces a transient endothelial GJIC dysfunction through the activation of tyrosine kinase and phosphorylation of tyrosine residues of Cx43. J. Cell. Physiol. 180:305–313, 1999. © 1999 Wiley-Liss, Inc.

We examined the role of ion movement in staurosporine-induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by propidium iodide assay. Treatment of the endothelial cells with staurosporine (10 nmol/l-1 micromol/l) for 6 h induced nuclear fragmentation in a dose-dependent manner. Staurosporine (1 micromol/l) elicited apoptosis in 70.5+/-1.5% of cells. Concomitant treatment of endothelial cells with 1 mmol/l of 4, 4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a chloride-bicarbonate exchange blocker, completely inhibited staurosporine-induced apoptosis. Other ion transporter inhibitors such as dimethyl amiloride and anthracene-9 carboxylic acid were less effective inhibitors of staurosporine-induced apoptosis of endothelial cells. DIDS prevented staurosporine-induced apoptosis of endothelial cells as well as cardiomyocytes. Next, we determined whether chloride ions or bicarbonate are involved in apoptosis. Incubation with a chloride ion removal buffer did not inhibit staurosporine-induced apoptosis of endothelial cells. However, endothelial cell apoptosis was completely suppressed by an inhibitor of caspase, benzyloxycarbonyl-Asp-CH(2)-O(C)O-dichlorobenzene (zD-dcb, 50 micromol/l). Staurosporine (1 micromol/l) increased the intracellular pH of endothelial cells, and DIDS (1 mmol/l), but not a caspase inhibitor, inhibited this increase in pH caused by staurosporine. Our findings suggest that endothelial cell apoptosis induced by staurosporine may be associated with the Cl(-)and bicarbonate (HCO-3) ions. Thus, Cl(-)efflux from cells or HCO-3 influx to cells (which increases pH) may play an important role in signal transduction leading events such as activation of caspase in staurosporine-induced apoptosis.

Eicosapentaenoic acid (EPA; 20:5, n-3) can restrain tumor growth and metastasis in vivo; however, the mechanism of its antitumor effect is still not fully understood. Angiogenesis is a crucial process for tumor growth and metastasis and inhibition of tumor angiogenesis can suppress tumor growth and metastasis in vivo. Vascular endothelial growth factor (VEGF) is an important angiogenic factor. In this study, we investigated the mechanisms of the inhibitory effect of EPA on VEGF-induced proliferation of bovine carotid artery endothelial (BAE) cells. BAE cells, treated with 0-5 microg/ml EPA for 48 h, displayed a dose-dependent suppression to VEGF (0.2 nM)-induced proliferation. Similar inhibitory effect was not found in BAE cells treated with arachidonic acid (AA; 20:4, n-6), or docasahexaenoic acid (DHA; 22:5, n-3). In contrast to its effect on VEGF-induced proliferation, EPA had no inhibition to basic fibroblast growth factor (bFGF, 0.2 nM)-induced proliferation in BAE cells. Both VEGF and bFGF activated mitogen-activated protein (MAP) kinase in BAE cells; however, EPA selectively inhibited VEGF-induced, but not bFGF-induced activation of MAP kinase. Flk-1 expression was inhibited dose-dependently in EPA-treated cells, whereas Flt-1 expression was increased in EPA treated cells. This in vitro inhibitory effect by EPA on Flk-1 receptor expression provides indirect evidence that one of the mechanisms of EPA for antitumor action in vivo maybe related to its antiangiogenic action.

Recent studies on ischemic brain disease in vivo and glutamate excitotoxicity in vitro suggest that apoptosis may play a role in excitotoxic neuronal death. To examine the possible involvement of apoptosis in glutamate excitotoxicity, we studied an early process of morphological changes in rat cortical neurons exposed to 1 mM glutamate. Observations under Nomarski optics combined with a digital image processor revealed a rapid change in the nucleus followed by a cellular swelling. The nucleus increased in granularity and swelled in 5 min, then became liquefied in 30 min. The cell body swelled slowly in 15-45 min. These changes could be prevented by treatment of the neuron with MK-801 (dizocilpine maleate), a blocker of N-methyl-D-aspartate (NMDA) receptor-coupled ion channel. However, treatment of the neurons with N(G)-nitro-L-arginine (N-NORG), a nitric oxide synthase inhibitor, had no significant effect. Use of the in situ end-labeling technique for the demonstration of free 3'-hydroxyl ends revealed that DNA fragmentation took place within 1 hr after glutamate exposure. A change in intracellular Ca(2+) concentration was examined with fluo-3 under a confocal laser microscope. Application of 1 mM glutamate induced rapid Ca transients in the nucleus as well as in the cytoplasm. Both of these Ca responses were blocked by MK-801. These results indicate that glutamate excitotoxicity in the brain neuron does not fulfill morphological criteria of apoptosis, but suggest that the nuclear disintegration associated with DNA fragmentation is involved as a leading step in glutamate excitotoxicity.

We previously have described a quantitative angiogenesisin vitro model, in which endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures. Using this model, the effect of eicosapentaenoic acid (20∶5n−3) on tube formation was investigated. When the endothelial cells isolated from bovine carotid artery were treated for 2 days with 5 μg/mL of arachidonic acid (20∶4n−6), eicosapentaenoic acid or docosahexaenoic acid (22∶6n−3), these polyunsaturated fatty acids were extensively incorporated into cellular phospholipids. The content of arachidonic, eicosapentaenoic and docosahexaenoic acid increased from 9.58% to 23.29%, from 0.98% to 11.76% and from 6.88% to 18.40%, respectively. When the eicosapentaenoic acid-treated cells were cultured between collagen gels, the tube-forming ability of the cells was markedly inhibited. The inhibition was dose-dependent between 1.0 and 5.0 μg/mL of eicosapentaenoic acid. At 5.0 μg/mL of eicosapentaenoic acid the inhibition reached 76%. By contrast, arachidonic acid increased tube formation, and docosahexaenoic acid had no effect. To elucidate the mechanism of eicosapentaenoic acid induced inhibition ofin vitro tube formation, we examined the effect of the acid on the proliferation of endothelial cells. Eicosapentaenoic acid at any dose (