Expression and role of mannose receptor/terminal high-mannose type oligosaccharide on osteoclast precursors during osteoclast formation.

Osteoclasts are formed from hematopoietic precursors via cell-cell fusion. We have previously reported that mannose residues are expressed on the outer membranes of monocytes during osteoclast differentiation. In the present study, we have attempted to demonstrate the pattern of expression levels of terminal high-mannose type oligosaccharide and to show that the mannose receptor is expressed on osteoclast precursor cells. Osteoclasts were formed using three different systems, namely mouse bone marrow cell culture, co-culture of mouse spleen cells with stromal cells, and RAW264.7 cell cultures. During osteoclast differentiation, the expression of terminal high-mannose type oligosaccharide gradually increased and then peaked at the stage of fusion in all three systems. Expression of the mannose receptor gradually increased during osteoclast differentiation in bone marrow cells and the co-culture system. In contrast, that in RAW264.7 cells had already been detected in the absence of the soluble receptor activator of NF-kappaB ligand and did not change during osteoclast differentiation. To ascertain whether expression of high-mannose type oligosaccharide is involved in tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation, glycosidase inhibitors were used on RAW264.7 cell culture. Castanospermine, an inhibitor of glucosidase I, inhibited the TRAP-positive MNCs, and deoxymannojirimycin, an inhibitor of alpha-mannosidase I, increased the TRAP-positive MNC formation. These results indicate that the binding of terminal high-mannose and mannose receptor is important for the process of cellular fusion in osteoclast formation.

Inhibitory effects of eicosapentaenoic acid (EPA) on the hypoxia/reoxygenation-induced tyrosine kinase activation in cultured human umbilical vein endothelial cells.

We have previously reported that the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) inhibited the abnormal gap junctional intercellular communication (GJIC) induced by hypoxia/reoxygenation (H/R) via suppressing tyrosine kinase (TK) activation (Zhang et al., Prostaglandins Leukot Essent Fatty Acids, 1999; 61: 33-40). However, the mechanisms by which EPA-inhibited TK activation remained unidentified. In this study we investigated whether reactive oxygen species (ROS) and growth factor-receptor systems would contribute to the H/R-induced TK activation or not. The results showed that H/R-induced ROS production, which reached the peak after 30 min of reoxygenation. Pretreatment with 10 microM EPA significantly inhibited this ROS production. However, the TK inhibitor genistein (10 microM) failed to inhibit the generation of ROS, although it completely inhibited TK activation. On the other hand, the ROS inhibitor DMSO (0.5% v/v) showed little effect on TK activation while it significantly blocked ROS production. Further EPA and genistein, but not DMSO and superoxide dismutase (SOD, 300 U/ml), prevented cells from GJIC injury induced by H/R. Moreover, EPA protected against VEGF-induced reduction in GJIC and phosphorylation of connexin 43. These data suggest that growth factor, but not ROS, might be involved in the EPA-inhibited TK activation induced by H/R.

Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Delta(12,14)-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2.

Recent studies have demonstrated that bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vivo, and in vitro, and that peroxisome proliferator-activated receptor gamma (PPAR gamma) plays a central role in the control of adipocyte differentiation. In the present study, we treated a murine stromal cell line (TMS-14) with a cocktail of dexamethasone, insulin and glucose (DIG cocktail), which caused the cells to convert to fat-laden cells with adipocyte-like morphology. We also exposed TMS-14 cells to DIG cocktail followed by 15-deoxy Delta(12,14)-prostaglandin J2 (15d-PGJ2), a ligand of PPAR gamma, interleukin- 11 (IL-11), 9-cis retinoic acid (9-cis RA) and vitamin K2. 15d-PGJ2 enhanced DIG cocktail-induced adipogenesis, whereas IL-11, 9-cis RA and vitamin K2 each inhibited adipogenesis induced by DIG cocktail. The gene expressions of four adipogenesis markers, PPAR gamma 2, adipocyte P2 (aP2), adipocyte determination and differentiation factor 1 (ADD1), and fatty acid synthase (FAS) were enhanced by DIG cocktail and these expressions were more enhanced by 15d-PGJ2, in contrast they were attenuated by 9-cis RA. IL-11 also attenuated the adipogenesis markers except ADD1. Western blotting showed that 15d-PGJ2 enhanced the levels of PPAR gamma, C/EBP alpha and RXR alpha proteins, while IL-11 and 9-cis RA decreased the level of PPAR gamma protein, but not C/EBP alpha protein and vitamin K2 decreased the level of C/EBP alpha protein. We also tested the effect of 15d-PGJ2 on osteoblastogenesis, using TMS-12 cells, another stromal cell clone from the same mouse, which differentiate into osteoblasts spontaneously. 15d-PGJ2 did not affect osteoblastogenesis, as detected by von Kossa staining and Cbfa-1 gene expression. These data indicate that 15d-PGJ2 enhances the expression of both PPAR gamma and C/EBP alpha and as a result it stimulates adipogenesis in murine bone marrow cells.

Novel mechanism for age-related macular degeneration: an equilibrium shift between the angiogenesis factors VEGF and PEDF.

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.

Connexin43 suppresses proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of p27.

Many lines of evidence indicate that connexin genes expressing gap junction (GJ) proteins inhibit tumor cell proliferation. However, the precise molecular mechanisms remain unclear. In this study, we show that overexpression of connexin43 (Cx43) suppressed proliferation of human osteosarcoma U2OS cells through inhibition of the cell cycle transition from G1 to S phase. This inhibition was attributed to a significant accumulation of the hypophosphorylated retinoblastoma (Rb) protein, which was causally related to decreases in the kinase activities of cyclin-dependent kinases (CDKs) 2 and 4. Enforced Cx43 expression markedly increased the level of the CDK inhibitor p27. This increase resulted from an increased synthesis and a reduced degradation of the p27 proteins, but not influence of the p27 mRNA. Moreover, we show that the Cx43-modulated GJ function was the main contributor to the elevation in p27 levels, in which cAMP was involved. These data suggest that Cx43 appears to inhibit proliferation of U2OS cells by increasing the levels of p27 proteins via post-transcriptional regulatory mechanisms.

Inhibition of phosphatidylinositol-3 kinase/Akt or mitogen-activated protein kinase signaling sensitizes endothelial cells to TNF-alpha cytotoxicity.

Bovine carotid artery endothelial (BAE) cells are resistant to tumor necrosis factor-alpha (TNF), like most other cells. We examined if mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI3) kinase/Akt pathways are involved in this effect. In BAE cells, TNF activates MAP kinase in a MAP kinase kinase 1 (MEK1) manner and Akt in PI3-kinase-dependent manner. Pretreatment with either the MEK1 inhibitor U0126 or PI3-kinase inhibitor LY294002 sensitized BAE cells to TNF-induced apoptosis. Neither U0126 nor LY294002 pretreatment affected TNF-induced activation of NF-kappaB, suggesting that the MAP kinase or PI3-kinase/Akt-mediated anti-apoptotic effect induced by TNF was not relevant to NF-kappaB activation. Both MAP kinase and PI3-kinase/Akt -mediated signaling could prevent cytochrome c release and mitochondrial transmembrane potential (Deltapsi) decrease. PI3-kinase/Akt signaling attenuated caspase-8 activity, whereas MAP kinase signaling impaired caspase-9 activity. These results suggest that TNF-induced MAP kinase and PI3-kinase/Akt signaling play important roles in protecting BAE cells from TNF cytotoxicity.

A narrow window for rescuing cells by the inhibition of calcium influx and the importance of influx route in rat cortical neuronal cell death induced by glutamate.

A rescue window for glutamate-insulted cells with regard to Ca(2+) influx was first investigated. The addition of EGTA, an impermeable calcium chelator to the culture within 5 min after the beginning of glutamate stimulation potently suppressed the neuronal cell death examined at 22 h. The effect of EGTA on rescuing cells decreased with the time delay of its addition to the system. MK-801, an antagonist of N-methyl-D-asparate (NMDA) receptor channels also inhibited the neuronal cell death in a similar manner to EGTA, suggesting Ca(2+) influx up to 5 min after the insult determined the fate of cells. But we also demonstrated that the elevated intracellular Ca(2+) did not always induce neurotoxicity. High concentration of potassium chloride plus FPL64176, an agonist of L-type Ca(2+) channels did not induce neuronal cell death, even though their combination elicited equal elevation of intracellular Ca(2+) to that by toxic concentration of glutamate, demonstrating that locally elevated intracellular Ca(2+) around NMDA receptors is important in the induction of neuronal cell death.

Downregulation of lipopolysaccharide-induced intercellular adhesion molecule-1 expression via EP2/EP4 receptors by prostaglandin E2 in human fibroblasts.

In the present study, the effect of prostaglandin E2 (PGE2) on intercellular adhesion molecule-1 (ICAM-1) expression in human gingival fibroblasts (HGF) stimulated with lipopolysaccharides (LPS) was investigated. LPS were isolated from periodontopathic bacteria, Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) and Porphyromonas gingivalis (P. gingivalis), by the phenol-water method and Escherichia coli (E. coli) LPS was used as a control. PGE2 significantly inhibited A. actinomycetemcomitans-, P. gingivalis- and E. coli-LPS-induced ICAM-1 expression. Next, of four PGE2 receptor subtypes (EP1, EP2, EP3 and EP4), we examined which subtype(s) was involved in inhibition of LPS-elicited ICAM-1 expression by PGE2. Eleven-deoxy-PGE1, a selective EP2/EP4 agonist, and butaprost, a selective EP2 agonist, attenuated A. actinomycetemcomitans-, P. gingivalis- and E. coli-LPS-elicited ICAM-1 expression, although butaprost was less potent than PGE2 and 11-deoxy-PGE1. Sulprostone, an EP1/EP3 agonist, and ONO-AP-324, an EP3 agonist, was inert to the LPS-elicited ICAM-1 expression. Furthermore, dibutyryl cAMP, a cAMP analogue, and forskolin, an adenylate cyclase activator, downregulated A. actinomycetemcomitans-, P. gingivalis- and E. coli-LPS-elicited ICAM-1 expression in HGF. Our data suggest that PGE2 downregulates A. actinomycetemcomitans- and P. gingivalis-LPS-induced ICAM-1 expression in HGF, via EP2/EP4 receptors by cAMP-dependent signaling pathways. The cAMP-elevating agents such as EP2/EP4 receptor activators may serve to control inflammatory and immune responses in periodontal disease.

Pharmacological characterization of cepharanthin in chronic idiopathic thrombocytopenic purpura.

Cepharanthin, a bisbenzylisoquinoline (biscolaurine) alkaloid drug, has been reported to improve the symptoms of intractable or steroid-resistant chronic idiopathic thrombocytopenic purpura (ITP). To clarify the mechanism by which the cepharanthin is beneficial to ITP, we examined the effects of cepharanthin on thrombocytopenia in (NZW x BXSB) F1 (W/B F1) mice and on the formation of colony forming unit of megakaryocyte (CFU-MK) derived from human CD34-positive progenitor cells. The decrease in platelet numbers in W/B F1 was diminished by the administration of 5 mg/kg cepharanthin for 6 weeks as well as by 2 mg/kg prednisolone. Furthermore, the administration of over 0.2 mg/kg cepharanthin enhanced the therapeutic effect of prednisolone. From the data in this animal model, it is suggested that cepharanthin may prolong the platelet lifespan. The treatment of CD34-positive progenitor cells isolated from cord blood with cepharanthin (over 5 x 10(-10)g/ml) caused an increase in the formation of CFU-MK induced by the cocktail of thrombopoietin, interleukin (IL)-6 and IL-3. The addition of 0.1% normal human serum dramatically increased the number of CFU-MK. In contrast, the serum isolated from patients with ITP at the same concentration decreased the number of CFU-MK. However, the simultaneous addition of 5 x 10(-8)g/ml cepharanthin recovered the number of CFU-MK to the level induced by normal serum. These findings indicate that cepharanthin has the potent therapeutic activity not only on the platelet destruction process, but also on the platelet production process of thrombocytopenia in chronic ITP.

Human uterine myometrial smooth muscle cell proliferation and vascular endothelial growth-factor production in response to platelet-derived growth factor.

It has been recognized that tissue-specific growth factors and angiogenic factors play important roles in the growth of tumors and in the tissue-repair system. In uterine myometrial smooth muscle cells, it has also been reported that the platelet-derived growth factor (PDGF) binds to PDGF receptors and stimulates proliferation. In this paper, we examine whether or not PDGF is able to stimulate production of vascular endothelial growth factor (VEGF) in cultured human myometrial smooth muscle cells. PDGF treatment enhanced immunoreactive VEGF production as well as cell proliferation. Production of VEGF121 and VEGF165 in the cells was detected by reverse transcription-polymerase chain reaction analysis, but the PDGF treatment did not change the ratio of VEGF165 to VEGF121. The effect of PDGF on cell proliferation leveled off at 10 ng/ml, whereas its effect on VEGF production continued to increase linearly at concentrations above 10 ng/ml. Upon treatment of the cells with antibody against VEGF, the cell proliferation increased linearly even at PDGF concentrations above 10 ng/ml. The enhanced [3H]thymidine incorporation by PDGF was abolished by either mitogen-activated protein kinase kinase (MAPKK) inhibitor or protein kinase C (PKC) inhibitor. In contrast, VEGF production was abolished by MAPKK inhibitor, but not by PKC inhibitor. These results indicate that PDGF stimulates both cell proliferation and VEGF production in partly different signal pathways, and thus PDGF might play a role in the physiology and pathology of the myometrium.

Role of SERCA2b in mobilization of nuclear Ca2+ in HeLa cells.

Ca2+ liberation from the endoplasmic reticulum activates sarco-endoplasmic reticulum ATPase (SERCA) to return Ca2+ to storage. We explored the role of SERCA in dynamic changes of intranuclear Ca2+ in single HeLa cells. Application of forskolin, as an activator of SERCA, caused the phosphorylation of SERCA2b but not SERCA3 on serine residues, which increased the rate of Ca2+ uptake. Forskolin also induced the changes of Ca2+ movement pattern in the nucleus when cells were stimulated with the Ca2+-releasing agents, histamine or A23187. Immunofluorescence staining showed that SERCA2b was densely populated on special parts of the nuclear envelope, but SERCA3 only existed in endoplasmic reticulum. Injection of an anti-SERCA2 antibody into the cytoplasm blocked the rise in the nuclear Ca2+ concentration ([Ca2+]n). However, injection of an anti-SERCA3 antibody did not affect the initiation of Ca2+ oscillations in the nucleus. Our data suggest that the activated-SERCA2b elevates the rate of uptake of free Ca2+ into stores along the nuclear envelope, which might support and maintain the nuclear Ca2+ homeostasis.

Eicosapentaenoic acid protects endothelial cell function injured by hypoxia/reoxygenation.

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.

Induction of hepatocyte growth factor (HGF) in rat microglial cells by prostaglandin E(2).

Hepatocyte growth factor (HGF) is a multifunctional protein that exerts trophic effects on neural cells. HGF is expressed in normal brains and increased after brain injury. Recent studies suggest that neurons and astrocytes are the main producers of HGF in the brain. Here we report that microglia also produce HGF both in vitro and in vivo. Treatment of cultured microglia with prostaglandin E(2) (PGE(2)), one of the major inflammatory mediators in the brain, induced significant production of HGF, and this induction was suppressed by pretreatment with the adenylate cyclase inhibitor SQ22536, suggesting that the induction of HGF by PGE(2) in microglia proceeds via a cAMP-mediated pathway. We further investigated whether microglia also produce HGF in vivo under the pathological condition of cerebral ischemia. We found that HGF expression was increased after permanent occlusion of the middle cerebral artery (MCA), and double immunohistochemical staining revealed that the most of HGF-positive cells were microglia. PGE(2) level was increased 8 hr after start of MCA occlusion, and this enhancement is in parallel with the increase in HGF expression, suggesting that PGE(2) not only may induce HGF production in microglia in vitro but may also be an inducer in vivo.

Human oral squamous cell carcinoma cell lines promote angiogenesis via expression of vascular endothelial growth factor and upregulation of KDR/flk-1 expression in endothelial cells.

Angiogenesis is an important phenomenon for the growth and metastasis of solid tumors. The present study examined the characterization of angiogenic factors produced by human oral squamous cell carcinoma (oral SCC) cell lines established from lymph node metastatic tumors and primary tumor in different patients. The conditioned medium of HSC3 with the strongest metastatic ability among the examined lines enhanced a tube-forming activity of bovine carotid artery endothelial (BAE) cells in collagen gel cultures. The treatment of HSC3 with anti-vascular endothelial growth factor (VEGF) antibody or anti-basic fibroblast growth factor (bFGF) antibody, either alone or in combination, attenuated the activity of urokinase-type plasminogen activator (uPA) in the endothelial cells stimulated by the conditioned medium of HSC3. In contrast, neither anti-interleukin-8 (IL-8) antibody nor anti-hepatocyte growth factor (HGF beta) antibody affected uPA activity in the endothelial cells. Among these HSC cell lines, HSC3 secreted VEGF with the highest (1.92 +/- 0.24 ng/10(6) cells/24 h) level and bFGF. The level of bFGF secreted by HSC3 was lower than that secreted by BAE cells. Other oral SCC cell lines secreted lower levels of VEGF and undetectable levels of bFGF. By reverse transcriptase-polymerase chain reaction analysis of mRNA the production of VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206 in these cell lines was able to be detected. Moreover, the conditioned medium of HSC3 enhanced the tyrosine phosphorylation and expression of kinase insert domain-containing receptor (KDR/flk-1) in the endothelial cells. These results suggest that oral SCC promotes angiogenesis via expression of VEGF and upregulation of their receptor KDR/flk-1 expression in endothelial cells.

Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow.

In vitro osteoclast differentiation is supported by stromal cells. In order to isolate a stromal cell line that can support osteoclast differentiation, 22 cell lines were cloned from mouse bone marrow. One of these clones, TMS-14, is a line of preadipocytes that supports osteoclast-like cell formation without any bone resorbing factors; and another, TMS-12, is a line of preosteoblasts that supports osteoclast-like cell formation with bone resorbing factors such as prostaglandin E(2)(PGE(2)). The difference of these two lines for osteoclast formation was not related with their abilities of PGE(2)production, but with the expression of osteoclast differentiation factor (ODF, also called OPGL, RANKL, and TRANCE), which detected with RT-PCR, in both cell lines. In TMS-14 cells, ODF mRNA was detected with or without PGE(2). In TMS-12 cells, ODF expression was detected in the PGE(2)-treated cells alone. When TMS-14 cells were induced to undergo adipogenic differentiation in response to treatment with thiazolidinedione, a ligand and activator of peroxisome proliferator-activated receptor gamma (PPARgamma), the ability of TMS-14 cells to support osteoclast-like cell formation was prevented in the presence or absence of 1,25(OH)(2)D(3). The gene expression of ODF in TMS-14 cells was also inhibited by treatment with thiazolidinedione. These results suggest that adipogenesis in bone marrow cells is related to the ability to support osteoclast differentiation. This is the first report of a cloned stromal cell line that can support osteoclastogenesis without the treatment with any osteotropic factors. Furthermore, this murine clonal preadipose cell line may be useful for studying senescence-dependent osteoporosis.

Regulation of angiogenesis by controlling VEGF receptor.

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.

Differential effects of nitric oxide on the activity of prostaglandin endoperoxide H synthase-1 and -2 in vascular endothelial cells.

A number of studies have demonstrated that prostacyclin and nitric oxide (NO) regulate blood pressure, blood flow and platelet aggregation. In this paper, we have examined the possible relationship between NO and prostaglandin endoperoxide H synthase (PGHS)-1 and -2 activities in cultured bovine aortic endothelial cells. In the non-activated condition endothelial cells expressed PGHS-1 activity alone. When these cells were pretreated with aspirin to inactivate their PGHS-1 and then activated by serum and phorbol ester (TPA) for 6 h, the cells expressed PGHS-2 activity alone. The PGHS activity was assessed by the generation of 6-ketoprostaglandin F1alpha (6-ketoPGF1alpha), a stable metabolite of prostacyclin, after the treatment of these cells with arachidonic acid. The simultaneous addition of NOC-7, a NO donor, with arachidonic acid did not affect the production of 6-ketoPGF1alpha in PGHS-1 expressed cells, but attenuated it in PGHS-2-expressed cells. The inhibitory effect of NOC-7 on PGHS-2 activity was dose dependent, and the different effects of NOC-7 on the activities of PGHS isozymes were also observed in other NO donors. To confirm the different effect of NO on PGHS isozymes demonstrated in the cultured endothelial cells, we carried out an ex vivo perfusion assay in aorta isolated from normal and lipopolysaccharide (LPS)-treated rats. In the aortae isolated from normal rats, where dominant expression of PGHS-1 was expected, the NO donor did not affect the PGHS activity, while in aortae isolated from LPS-treated rats, where PGHS-2 was dominantly expressed, the NO donor dramatically inhibited the PGHS activity, suggesting that NO suppressed PGHS-2 activity alone. The inhibitory effect of NO on PGHS-2 activity was not mediated by cyclic GMP (cGMP), since (a) methylene blue, an inhibitor of soluble guanylate cyclase did not abolish the inhibitory effect of the NO donor on PGHS-2 activity, and (b) 8-Br-cGMP, a permeable cGMP analogue, failed to mimic the effect of NO donors. These data suggest that the effect of NO on prostacyclin production in endothelial cells was dependent on the expression rate of PGHS-1 and PGHS-2 in the cells.

Screening of anti-hypoxia/reoxygenation agents by an in vitro model. Part 1: Natural inhibitors for protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells.

Protein tyrosine kinase (PTK) signaling pathways play important roles in ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) injuries. Inhibition of PTK activation can protect against I/R- or H/R-induced damages. As one part of our work for seeking bioactive compounds from natural sources against I/R or H/R, in the present study we examined the effects of 54 compounds purified from various traditional Chinese herbs on H/R-induced PTK activation by means of an in vitro H/R model in cultured human umbilical vein endothelial cells (HUVEC). The results demonstrated that an increase in PTK activation was induced after 2 h of reoxygenation. Compounds 2 (macrostemososide A), 3 (laxogenin-3-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-arabinopyra nosyl- (1-->6)-beta-D-glucopyranoside), 4 (chinenoside II), 7 (ginsenoside-Rd), 52 (icariin), 53 (icariside), and 54 (icaritin) showed relatively obvious inhibition on this H/R-induced PTK activation. Compounds 5 (beta-sitosterol) and 6 (daucosterine), especially 5, completely blocked such an increased activation of PTK induced by H/R. On the contrary, compound 29 (isocumarine) significantly promoted PTK activation further. Moreover, the effects of these compounds on PTK activation were dose-dependent.

Screening of anti-hypoxia/reoxygenation agents by an in vitro method. Part 2: Inhibition of tyrosine kinase activation prevented hypoxia/reoxygenation-induced injury in endothelial gap junctional intercellular communication.

In this study, we demonstrated that hypoxia/reoxygenation (H/R) induced an injury in gap junctional intercellular communication (GJIC) after 2 h of reoxygenation in cultured HUVEC. Free radical scavenger (DMSO) and antioxidant (SOD) did not prevent this GJIC injury at all. Protein kinase C inhibitor (calphostin C) partly blocked this injury. However, the protein tyrosine kinase (PTK) inhibitor genistein completely inhibited this GJIC injury. Compounds 1 [laxogenin-3-O-alpha-L-arabinosyl-(1-->6)- beta-D-glucopyranoside], 2 (macrostemososide A), 3 [laxogenin-3-O-beta-D-xylopyranosyl-(1-->4)-alpha- L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside], 4 (chinenoside II), 5 (beta-sitosterol), 6 (daucosterine), 7 (ginsenoside-Rd), 29 (isocumarine), 52 (icariin), 53 (icariside), and 54 (icaritin), which showed obvious influence on H/R-induced PTK activation as stated in Part 1 (except 1), were explored for their effects on GJIC. The results showed that compounds 2-7 and 52-57 partly protected H/R-induced GJIC injury. Compounds 5 and 6 (especially 5), which showed the strongest inhibitory effects on PTK activation, completely blocked H/R-provoked GJIC injury. Compound 1, which did not influence PTK activation, failed to prevent this GJIC injury. In contrast, compound 29, which significantly promoted PTK activation, enhanced this H/R-induced GJIC injury further. Western blotting of connexin 43, an important gap junctional protein for modulating GJIC in HUVEC, revealed that interference with the gap junctional protein might be the most direct mechanism for compounds 2, 5, 29, and 53 to affect H/R-injured GJIC.

Simvastatin enhanced sodium nitroprusside-induced apoptosis of smooth muscle cells. An involvement of geranylgeraniol.

A decrease in serum cholesterol is one of the most beneficial effects in anti-atherogenesis. Nitric oxide is also an anti-atherogenic substance, inducing vasodilation and inhibits proliferation of smooth muscle cells (SMC). Therefore, we examined sodium nitroprusside (SNP)-induced apoptosis of vascular SMC with respect to cholesterol metabolism. Cultured vascular SMC from bovine carotid arteries and rat aorta were used. Apoptosis was determined by propidium iodide assay. Treatment of the SMC with SNP(100 micromol/l-1 mmol/l ) for 6 h induced a little nuclear fragmentation. SNP (1 mmol/l ) elicited apoptosis in 4.4+/-2.2% of cells. Pretreatment of SMC with simvastatin (1 microg/ml, 2 days), a hydroxymethylglutaryl Coenzyme A (HMG CoA) reductase inhibitor, synergistically enhanced SNP-induced apoptosis (% apoptosis =15. 9+/-3.3%). Either mevalonate (100 micromol/l) or geranylgeraniol (30 micromol/l) recovered the simvastatin (1 microg/ml)-enhanced SMC apoptosis induced by SNP. Neither squalene (10 mmol/l) nor farnesol (30 micromol/l) had a recovery effect on the simvastatin-enhanced SMC apoptosis induced by SNP. Pretreatment with simvastatin (1 microg/ml) reduced total cholesterol content in SMC. Mevalonate (100 micromol/l) restored a decrease in total cholesterol content. However, incubation with LDL deficient serum did not enhance SNP-induced apoptosis of SMC, although treatment with LDL deficient serum decreased the total cholesterol content in SMC. These data suggested that decrease in HMG CoA reductase metabolites, especially geranylgeraniol might enhance the SNP-induced apoptosis of SMC, and that, apoptosis was not involved in a decrease in cholesterol of SMC.