Cilostazol Inhibits Vascular Smooth Muscle Cell Proliferation and Reactive Oxygen Species Production through Activation of AMP-activated Protein Kinase Induced by Heme Oxygenase-1.

Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cAMP levels and activates protein kinase A, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMP-activated protein kinase (AMPK) activation induced by heme oxygenase-1 (HO-1) is a mediator of the beneficial effects of cilostazol and whether cilostazol may prevent cell proliferation and reactive oxygen species (ROS) production by activating AMPK in VSMC. In the present study, we investigated VSMC with various concentrations of cilostazol. Treatment with cilostazol increased HO-1 expression and phosphorylation of AMPK in a dose- and time-dependent manner. Cilostazol also significantly decreased platelet-derived growth factor (PDGF)-induced VSMC proliferation and ROS production by activating AMPK induced by HO-1. Pharmacological and genetic inhibition of HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and ROS production.These data suggest that cilostazol-induced HO-1 expression and AMPK activation might attenuate PDGF-induced VSMC proliferation and ROS production.

AMPK induces vascular smooth muscle cell senescence via LKB1 dependent pathway.

Vascular cells have a limited lifespan with limited cell proliferation and undergo cellular senescence. The functional changes associated with cellular senescence are thought to contribute to age-related vascular disorders. AMP-activated protein kinase (AMPK) has been discussed in terms of beneficial or harmful effects for aging-related diseases. However, the detailed functional mechanisms of AMPK are largely unclear. An aging model was established by stimulating vascular smooth muscle cell (VSMC) with adriamycin. Adriamycin progressively increased the mRNA and protein expressions of AMPK. The phosphorylation levels of LKB1 and acetyl-CoA carboxylase (ACC), the upstream and downstream of AMPK, were dramatically increased by adriamycin stimulation. The expressions of p53 and p21, which contribute to vascular senescence, were also increased. Inhibition of AMPK diminished senescence-associated ß-galactosidase (SA-ß-gal) staining, and restored VSMC proliferation. Cytosolic translocation of LKB1 by adriamycin could be a mechanism for AMPK activation in senescence. Furthermore, p53 siRNA and p21 siRNA transfection attenuated adriamycin-induced SA-ß-gal staining. These results suggest that LKB1 dependent AMPK activation elicits VSMC senescence and p53-p21 pathway is a mediator of LKB1/AMPK-induced senescence.

Aprotinin Inhibits Vascular Smooth Muscle Cell Inflammation and Proliferation via Induction of HO-1.

Aprotinin is used clinically in cardiopulmonary bypass surgery to reduce transfusion requirements and the inflammatory response. The mechanism of action for the anti-inflammatory effects of aprotinin is still unclear. We examined our hypothesis whether inhibitory effects of aprotinin on cytokine-induced inducible nitric oxide synthase (iNOS) expression (IL-1beta plus TNF-alpha), reactive oxygen species (ROS) generation, and vascular smooth muscle cell (VSMC) proliferation were due to HO-1 induction in rat VSMCs. Aprotinin induced HO-1 protein expression in a dose-dependent manner, which was potentiated during inflammatory condition. Aprotinin reduced cytokine mixture (CM)-induced iNOS expression in a dose dependent manner. Furthermore, aprotinin reduced CM-induced ROS generation, cell proliferation, and phosphorylation of JNK but not of P38 and ERK1/2 kinases. Aprotinin effects were reversed by pre-treatment with the HO-1 inhibitor, tin protoporphyrin IX (SnPPIX). HO-1 is therefore closely involved in inflammatory-stimulated VSMC proliferation through the regulation of ROS generation and JNK phosphorylation. Our results suggest a new molecular basis for aprotinin anti-inflammatory properties.

Heme oxygenase-1 induced by aprotinin inhibits vascular smooth muscle cell proliferation through cell cycle arrest in hypertensive rats.

Spontaneous hypertensive rats (SHR) are an established model of genetic hypertension. Vascular smooth muscle cells (VSMC) from SHR proliferate faster than those of control rats (Wistar-Kyoto rats; WKY). We tested the hypothesis that induction of heme oxygenase (HO)-1 induced by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats. Aprotinin treatment inhibited VSMC proliferation in SHR more than in normotensive rats. These inhibitory effects were associated with cell cycle arrest in the G1 phase. Tin protoporphyrin IX (SnPPIX) reversed the anti-proliferative effect of aprotinin in VSMC from SHR. The level of cyclin D was higher in VSMC of SHR than those of WKY. Aprotinin treatment downregulated the cell cycle regulator, cyclin D, but upregulated the cyclin-dependent kinase inhibitor, p21, in VSMC of SHR. Aprotinin induced HO-1 in VSMC of SHR, but not in those of control rats. Furthermore, aprotinin-induced HO-1 inhibited VSMC proliferation of SHR. Consistently, VSMC proliferation in SHR was significantly inhibited by transfection with the HO-1 gene. These results indicate that induction of HO-1 by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats.

Isoproterenol inhibits angiotensin II-stimulated proliferation and reactive oxygen species production in vascular smooth muscle cells through heme oxygenase-1.

Heme oxygenase (HO)-1 is a well-known cytoprotectant against oxidative stress and exhibits an antiproliferative effect in vascular smooth muscle cells (VSMCs). The purpose of the present study was to test whether isoproterenol, one of the synthetic catecholamines having beta-adrenergic activity, affected angiotensin II (Ang II)-induced cell proliferation and reactive oxygen species (ROS) production. Also, the presumptive underlying signaling pathways in VSMCs were studied. Aortic VSMCs from 11-week-old male Sprague-Dawley rats were used. Isoproterenol dose-dependently increased HO-1 expression through beta(2)-adrenoceptor (AR) and protein kinase A (PKA) pathway, and isoproterenol concentration-dependently increased beta(2)-AR mRNA expression. Isoproterenol attenuated Ang II-induced cell proliferation, as evidenced by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. This effect of isoproterenol was inhibited by pretreatment of the cells with beta(2)-AR antagonist butoxamine, PKA inhibitor H-89 and HO inhibitor Tin Protoporphyrin IX (SnPP IX), respectively. Isoproterenol inhibited phosphorylation level of Ang II-induced extracellular signal-regulated kinase (ERK1/2). Isoproterenol significantly inhibited Ang II-induced ROS production through the ERK1/2 pathway. These findings suggest that isoproterenol, via induction of HO-1, inhibits Ang II-stimulated proliferation and ROS production in cultured VSMCs.

Hemin inhibits hypertensive rat vascular smooth muscle cell proliferation through regulation of cyclin D and p21.

We tested the hypothesis that HO-1 (heme oxygenase-1) activity varied between vascular smooth muscle cells (VSMC) in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. HO-1 levels were measured under baseline and hemin-stimulated conditions and cell proliferation was monitored. Basal HO-1 levels in untreated cells were lower in SHR compared to WKY rats. Treatment with hemin increased HO-1 mRNA and protein levels in the cells obtained from WKY rats compared to that of SHR rats. However, hemin-treatment showed a greater inhibitory effect on VSMC proliferation in SHR rats than in WKY rats. Tin protoporphyrin IX (SnPPIX) showed a greater reversal of the anti-proliferative effect of hemin on cells from SHR rats than WKY. Similarly, VSMC proliferation from SHR was significantly inhibited in VSMC transfected with the HO-1 gene. These inhibitory effects were associated with cell cycle arrest in the G1 phase. The level of cyclin D, and cyclin dependent kinase inhibitor p21 was higher in SHR cells progressing through the G1 phase. Treatment of the cells with hemin down-regulated the expression of cyclin D and up-regulated that of p21. These results indicate that hemin, an HO-1 inducer, may play a more critical role in VSMC proliferation in SHR than WKY.

NS-398, a selective COX-2 inhibitor, inhibits proliferation of IL-1beta-stimulated vascular smooth muscle cells by induction of HO-1.

We investigated whether NS-398, a selective inhibitor of COX-2, induces HO-1 in IL-1beta-stimulated vascular smooth muscle cells (VSMC). NS-398 reduced the production of PGE(2) without modulation of expression of COX-2 in IL-1beta-stimulated VSMC. NS-398 increased HO-1 mRNA and protein in a dose-dependent manner, but inhibited proliferation of IL-1beta-stimulated VSMC. Furthermore, SnPPIX, a HO-1 inhibitor, reversed the effects of NS-398 on PGE(2) production, suggesting that COX-2 activity can be affected by HO-1. Hemin, a HO-1 inducer, also reduced the production of PGE(2) and proliferation of IL-1beta-stimulated VSMC. CORM-2, a CO-releasing molecule, but not bilirubin inhibited proliferation of IL-1beta-stimulated VSMC. NS-398 inhibited proliferation of IL-1beta-stimulated VSMC in a HbO(2)-sensitive manner. In conclusion, NS-398 inhibits proliferation of IL-1beta-stimulated VSMC by HO-1-derived CO. Thus, NS-398 may facilitate the healing process of vessels in vascular inflammatory disorders such as atherosclerosis.

Kinetic Changes of COX-2 Expression during Reperfusion Period after Ischemic Preconditioning Play a Role in Protection Against Ischemic Damage in Rat Brain.

A brief ischemic insult induces significant protection against subsequent massive ischemic events. The molecular mechanisms known as preconditioning (PC)-induced ischemic tolerance are not completely understood. We investigated whether kinetic changes of cyclooxygenase (COX)-2 during reperfusion time-periods after PC were related to ischemic tolerance. Rats were given PC by occlusion of middle cerebral artery (MCAO) for 10 min and sacrificed after the indicated time-periods of reperfusion (1, 2, 4, 8, 12, 18 or 24 h). In PC-treated rats, focal ischemia was induced by occlusion of MCA for 24 h and brain infarct volume was then studied to determine whether different reperfusion time influenced the damage. We report that the most significant protection against focal ischemia was obtained in rats with 8 h reperfusion after PC. Administration of indomethacin (10 mg/kg, oral) or rofecoxib (5 mg/kg, oral) 48 h prior to PC counteracted the effect of PC. Immunohistochemical analysis showed that COX-2 and HO-1 protein were induced in PC-treated rat brain, which was significantly inhibited by rofecoxib. Taken together, we concluded that the kinetic changes of COX-2 expression during the reperfusion period after PC might be partly responsible for ischemic tolerance.

Upregulation of PBR mRNA expression in human neuroblastoma cells by flavonoids.

To investigate the putative mediation of peripheral benzodiazepine receptor (PBR) in the cytotoxicity of flavonoids, in this study, modulatory effects of several flavonoids on the lipid peroxide (LPO) production and PBR mRNA expression of human neuroblastoma cells were observed. Elevated levels of peroxidated products in cancer cells may activate pro-apoptotic and anti-proliferative signaling pathways. Treatment of 10(-6) M 4'-chlorodiazepam and PK 11195 ligands of the PBR for 6 days enhanced the generation of LPO of the human neuroblastoma cells. Several flavonoids, well-known cytotoxic substances, potentiated the enhancement of LPO production by PBR ligands. Treatment of 10(-6) M flavonoids for 6 days elevated the expression of PBR mRNA in cells. These findings indicate that the potential of flavonoids to induce apoptosis in cancer cells is strongly associated with their PBR-inducing properties, thereby providing a new mechanism by which polyphenolic compounds may exert their cancer-preventive and anti-neoplastic effects.

Attenuation of extracellular acidic pH-induced cyclooxygenase-2 expression by nitric oxide.

Corneal endothelial cells play an important role in maintaining the transparency and ionic balance of the cornea. Inflammation causes many changes in the intracellular and extracellular environment of the cornea, including acidosis. We examined the relationship between changes in extracellular pH and expression of cyclooxygenase-2 in cultured bovine corneal endothelial cells. When extracellular pH ([pH]o) was reduced to pH 6.4, COX-2 mRNA increased, with a peak at 2 h. This was blocked by pretreatment with actinomycin D and incubation with spermine NONOate (SPER/NO, a nitric oxide donor). Exposure to the H+ ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), also raised COX-2 mRNA levels. CCCP-induced COX-2 mRNA expression was also reduced by SPER/NO. These results were confirmed immuno-cytochemically. These data demonstrate that COX-2 expression is stimulated by the lowering of extracellular pH that could result from bacterial infection, and that this is countered by over-production of nitric oxide, which could also result from bacterial infection.

CD14 glycoprotein expressed in vascular smooth muscle cells.

The inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells is an important factor for pathogenesis of septic shock or multiple organ dysfunction syndrome. The mechanisms of iNOS expression in such conditions are partly known. This study tried to clarify the signal transduction of lipopolysaccharide (LPS) single stimulation that induces iNOS mRNA and protein in vascular smooth muscle cells (VSMC). VSMC were primarily cultured from rat aorta. The concentrations of nitrite in culture media were measured by the Griess reaction. Western blottings and immunoreaction for iNOS, nuclear factor kappaB (NFkappaB) p65, and CD14 protein were performed. mRNAs of iNOS and tumor necrosis factor (TNF) alpha were analyzed by RT-PCR. Genistein inhibited LPS induced early phase nitrite production, while pyrrolidine dithiocarbamate (PDTC) inhibited nitrite production at a late phase. PDTC significantly reduced NFkappaB p65 and iNOS protein expression by LPS. TNFalpha mRNA expression by LPS was not detected in VSMC. Membranous CD14 glycoprotein was detected in VSMC and soluble CD14 glycoprotein was not detected in fetal bovine serum added in culture media. These results suggest that CD14 glycoprotein is present on the cell membranes of VSMC, a non-myelomonocyte lineage, acting as an LPS receptor. Activations of tyrosine kinase and NFkappaB p65 are essential for iNOS expression by LPS single stimulation, while TNFalpha is not a concern to iNOS expression in VSMC.

Potentiated modulation of pregnolone on GABAA receptors in behaviorally stressed borderline-hypertensive rats.

The modulatory effects of behavioral stress on [(3)H]flunitrazepam, an agonist for the central-type benzodiazepine receptor binding to the GABA(A)-benzodiazepine receptor complex, in borderline hypertensive rats (BHR) were examined. In repeatedly immobilized (for 2 weeks, for 2 h/d) BHR, enhancement of [(3)H]flunitrazepam binding to the receptor was observed to be potentiated. The percent enhancement of [(3)H]flunitrazepam binding in BHR was higher than that in normotensive control Wistar-Kyoto rats. Pregnanolone, a neuroactive steroid that has been reported to be a putative endogenous modulator in the stress response, concentration dependently enhanced [(3)H]flunitrazepam binding to the receptor. Enhancement of [(3)H]flunitrazepam binding was observed to be potentiated by the same immobilized stress, and the EC(50) values of pregnanolone in BHR was significantly lower than those in controls and E(max) values were higher. From the above results, it can be concluded that neural modulation to behavioral stress, especially in GABAergic neurotransmission, is exaggerated in BHR. We propose strain-specific differences of stress reactivity as an important pathogenetic factor in psychosomatic disorders including stress-induced hypertension. This is supported by reports showing exaggerated cardiovascular and symathoadrenal responses to stress in BHR.

Modulation of radioligand binding to the GABA(A)-benzodiazepine receptor complex by a new component from Cyperus rotundus.

Four sesquiterpenes, beta-selinene, isocurcumenol, nootkatone and aristolone and one triterpene, oleanolic acid were isolated from the ethylacetate fraction of the rhizomes of Cyperus rotundus and tested for their ability to modulate gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor function by radioligand binding assays using rat cerebrocortical membranes. Among these compounds, only isocurcumenol, one of the newly identified constituents of this plant, was found to inhibit [3H]Ro15-1788 binding and enhance [3H]flunitrazepam binding in the presence of GABA. These results suggest that isocurcumenol may serve as a benzodiazepine receptor agonist and allosterically modulate GABAergic neurotransmission via enhancement of endogenous receptor ligand binding.