Circulating endothelial progenitor cells (EPCs) derived from bone marrow were isolated for the first time in 1997 and characterized. Recent evidence has indicated that EPCs contribute to reendothelialization of injured vessels as well as neovascularization of ischemic lesions and that a decrease in the number of EPCs is an independent predictor of morbidity and mortality of cardiovascular diseases. These finding suggest that EPCs play a major role in the pathogenesis of atherosclerosis and cardiovascular diseases. Interestingly, the number and function of EPCs are regulated by not only various kinds of angiogenic cytokines and cardiovascular risk factors per se but also some interventions, including lifestyle modification (aerobic exercise, body weight loss, and smoking cessation) and pharmacological therapy (e.g., renin-angiotensin system inhibitor, statin, and erythropoietin). It is thought that regulation of the number and function of EPCs directly influences the maintenance and development of atherosclerosis. Therefore, it is clinically important to estimate the degree of EPC bioactivity and to increase the EPC bioactivity by appropriate interventions. In this review, we focus on the relationship between EPCs and cardiovascular risk factors and the role of EPCs in cardiovascular diseases.

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We focused on the therapeutic effect of the stable gastric pentadecapeptide BPC 157 and how its action is related to nitric oxide (NO) in persistent colocutaneous fistula in rats (at 5 cm from anus, colon defect of 5 mm, skin defect of 5 mm); this peptide has been shown to be safe in clinical trials for inflammatory bowel disease (PL14736) and safe for intestinal anstomosis therapy. BPC 157 (10 μg/kg, 10 ng/kg) was applied i) in drinking water until the animals were sacrificed at post-operative day 1, 3, 5, 7, 14, 21, and 28; or ii) once daily intraperitoneally (first application 30 min following surgery, last 24 h before sacrifice) alone or with N^G-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg), L-arginine (200 mg/kg), and their combinations. Sulphasalazine (50 mg/kg) and 6-α-methylprednisolone (1 mg/kg) were given once daily intraperitoneally. BPC 157 accelerated parenterally or perorally the healing of colonic and skin defect, leading to the suitable closure of the fistula, macro/microscopically, biomechanically, and functionally (larger water volume sustained without fistula leaking). L-NAME aggravated the healing failure of colocutaneous fistulas, skin, and colon wounds (L-NAME groups). L-Arginine was effective only with blunted NO generation (L-NAME + L-arginine groups) but not without (L-arginine groups). All of the BPC 157 beneficial effects remained unchanged with blunted NO-generation (L-NAME + BPC 157 groups) and with NO substrate (L-arginine + BPC 157 groups) as well as L-NAME and L-arginine co-administration (L-NAME + L-arginine + BPC 157 groups). Sulphasalazine was only moderately effective, and corticosteroid even had an aggravating effect.

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Periosteum has been demonstrated to contain mesenchymal progenitor cells differentiating to osteoblasts, and both bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) may play important roles in cell-based approaches to bone regeneration. The purpose of this study was to evaluate the feasibility and efficacy of BMP-2 and/or VEGF on periosteal cell differentiation to osteoblasts in vitro and ectopic bone formation in vivo. Human periosteum-derived cells were transfected with BMP-2, VEGF, BMP-2 + VEGF, or vehicle as a control by non-viral gene transfer and then cultured and implanted to nude mice intramuscularly. Real-time polymerase chain reaction analysis of the culture revealed that transgenes for BMP-2 and BMP-2 + VEGF induced more mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than VEGF and vehicle treatments; additionally, alizarin red S staining, alkaline phosphatase staining, and alkaline phosphatase activity were significantly higher in the BMP-2 + VEGF transgene than in the other versions. After implantation, ectopic bone was observed at 4 weeks and greatly increased at 8 weeks in all groups. In particular, the combination of BMP-2 and VEGF formed significantly more bone at 4 weeks, and VEGF transfection resulted in more blood vessels relative to the conditions without VEGF. Thus, VEGF might enhance BMP2-induced bone formation through modulation of angiogenesis.

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Oridonin, a diterpenoid isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, the growth-inhibitory activity of oridonin for L929 cells was exerted in a time-and dose-dependent manner. After treatment with oridonin for 24 h, L929 cells underwent both apoptosis and necrosis as measured by an lactate dehydrogenase (LDH) activity-based assay. A rapid generation of reactive oxygen species (ROS) was triggered by oridonin, and subsequently up-regulation of phospho-p53 (ser 15) expression and an increased expression ratio of Bax/Bcl-2 was observed. Furthermore, there was a significant fall in mitochondrial membrane potential (MMP) and increase in caspase-3 activity after exposure to oridonin for 24 h. Surprisingly, the pan-caspase inhibitor z-VAD-fmk and caspase3 inhibitor z-DEVD-fmk rendered L929 cells more sensitive to oridonin, rather than preventing oridonin-induced cell death. Oridonin and z-VAD-fmk co-treatment not only resulted in an even higher ROS production, but also made a more significant reduction in the MMP. Pretreatment of ROS scavenger N-acetylcysteine (NAC) led to a complete inhibition of oridonin-induced cell death, intracellular ROS generation, and MMP collapse. NAC treatment also reversed the potentiation of cell death by the pan-caspase inhibitor z-VAD-fmk. Taken together, these observations showed that oridonin-induced cell death in L929 cells involved intracellular ROS generation, activation of phospho-p53 (ser 15), and up-regulation of the Bax/Bcl-2 ratio; and the augmented cell death by z-VAD-fmk was dependent on an increased ROS production.

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We investigated the antidiabetic effects of E3030, which is a potent dual activator of peroxisome proliferator-activated receptor (PPAR) α and PPARγ, in an animal model of diabetes, C57BL/KsJ-db/db mice (db/db mice), and the lipidemic effects of E3030 in beagle dogs, whose PPARα and PPARγ transactivation responses to E3030 were similar to those of humans. E3030 activated human PPARα, mouse PPARα, dog PPARα, human PPARγ, mouse PPARγ, and dog PPARγ with EC₅₀ values of 65, 920, 87, 34, 73, and 34 nM, respectively, in the chimeric GAL4-PPAR receptor transactivation reporter assay. In db/db mice orally administered E3030 decreased blood glucose, triglyceride (TG), non-esterified fatty acids (NEFA), and insulin levels and increased blood adiponectin levels during a 14-day experimental period. Significant effects on blood glucose and adiponectin levels were observed at a dose of 3 mg/kg or greater. Furthermore, significant effects on blood TG, NEFA, and insulin levels were observed at doses of 1 mg/kg or more. An oral glucose tolerance test (OGTT) performed on Day 15 showed that E3030 at 3 mg/kg improved glucose tolerance in this model. Fourteen days of oral treatment with E3030 at a dose of 0.03 mg/kg or greater showed remarkable TG- and non high-density lipoprotein (non-HDL) cholesterol–lowering effects in beagle dogs. These results were similar to those observed for the PPARα agonist fenofibrate. E3030 also reduced apo C-III levels on Days 7 and 14, and elevated lipoprotein lipase (LPL) levels on Day 15. These results indicate that the TG- and non-HDL cholesterol-lowering actions of E3030 involve combined effects on reduction of apo C-III and elevation of LPL, resulting in increased lipolysis. The experimental results in animals suggest that E3030 has potential for use in the treatment of various aspects of metabolic dysfunction in type 2 diabetes, including dyslipidemia, hyperglycemia, hyperinsulinemia, and impaired glucose disposal.

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Kv1.5 is considered to be a potential molecular target for treatment of atrial fibrillation or flutter. Disopyramide is widely used in the treatment of atrial flutter and/or atrial fibrillation. The present study was undertaken to characterize the effects of disopyramide on currents mediated by Kv1.5 channels and to determine the putative binding site involved in the inhibitory effects of disopyramide. Experiments were carried out on wild-type and site directed mutated hKv1.5 channels expressed on HEK 293 cells using the patch-clamp technique. Disopyramide acting from the cytoplasmic side of the membrane produced blocking effects on Kv1.5 that exhibited several features typical of an open channel blocker. Ala-scanning mutagenesis of the Kv1.5 pore domain combined with macroscopic current analysis suggested that disopyramide interacted only with the Val512 residue that faces to the central cavity of the channel. Mutation of this key residue to Ala caused marked change in the IC₅₀ of disopyramide (22-fold). The single interaction between disopyramide and Val512 in the PVP region is able to change the mechanism of channel closure, reproducing the “foot-in-the-door” phenomenon.

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Patients with Parkinson’s disease (PD) often have attenuated baroreflex function, which may occur before the onset of PD-associated movement disorders. The aim of the present study was to test whether impaired arterial baroreflex (ABR) function could contribute to the pathogenesis of PD. 6-Hydroxydopamine (8 μg in 4 μl) was microinjected into the left substantia nigra of rats to establish unilateral PD models, and bilateral PD models were established in rats by administration of rotenone by osmotic minipump for four weeks, at a dose of 2.5 mg · kg⁻¹ · day⁻¹. An ABR dysfunction model was obtained by performing sinoaortic denervation (SAD). Hemodynamic variables were determined in conscious rats. PD-like symptoms and dopamine content in corpus striatum (CS) were also assessed. 6-Hydroxydopamine and rotenone treatment and SAD were associated with enhanced blood pressure variability (BPV) and blunted baroreflex sensitivity (BRS). Rotenone, but not SAD, significantly reduced dopamine content in the CS, induced catalepsy, and inhibited rearing and exploratory behavior. SAD before the administration of rotenone did not aggravate the rotenone-induced dopaminergic lesion. Our findings do not support the presumption that ABR dysfunction contributes to the pathogenesis of PD in rats.

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We have characterized the modulation of adenylyl cyclase (AC) activity by ligands of dopaminergic receptors in rat striatal homogenate and compared the results with receptor-ligand binding affinities. Despite the fact that rat striatum contains high level of both dopamine D₁ and D₂ receptors, only the D₁-specific AC activation by agonists could be determined. All D₁-receptor agonists (dopamine, dihydrexidine, and A 77636) used were able to increase cAMP accumulation in a concentration-dependent manner, while D₁-receptor antagonists (SCH23390, SKF83566, and butaclamol) blocked the effects induced by the aforementioned agonists. At the same time, the D₂-receptor agonist quinpirole and antagonist sulpiride had no effect on cAMP accumulation in striatal homogenate neither on the basal level nor on the activated level of AC, while inhibited [³H]raclopride binding to these membranes. Comparing the ligands of the D₁ receptor in modulating the activity of AC and displacing D₁-receptor–specific radioligand [³H]SCH23390 binding revealed that the ligands modulate both of these processes with similar affinities. It indicates that under given experimental conditions, only dopamine D₁-receptor–mediated stimulation of AC activity can be measured in membrane homogenate of rat striatum, while dopamine D₂-receptor effects remain fully hidden.

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Ca²⁺ is a pivotal signal in human pulmonary artery smooth muscle cells (PASMCs) proliferation. Capacitative Ca²⁺ entry (CCE) via the store-operated channel (SOC), which encoded by the transient receptor potential (TRP) gene, is an important mechanism for regulating intracellular Ca²⁺ concentration ([Ca²⁺]_i) in PASMCs. Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been proposed as a therapeutic tool to treat or prevent pulmonary arterial hypertension (PAH); however, the mechanism of its antiproliferative effect on PASMCs remains unclear. This study was designed to investigate the possible antiproliferative mechanism of sildenafil on human PASMCs, namely, its effect on the Ca²⁺-signal pathway. Cultured normal PASMCs were treated with endothelin-1 (ET-1) or ET-1 plus sildenafil separately. Cell number and viability were determined with a hemocytometer or MTT assay. [Ca²⁺]_i was measured by loading PASMCs with fura 2-AM. Expression of the TRPC1 gene and protein was detected by RT-PCR and Western blot, respectively. The results show that sildenafil dose-dependently inhibited the proliferation of PASMCs, the enhancement of basal [Ca²⁺]_i level, increase of CCE, and upregulation of TRPC expression induced by ET-1. These results suggest that sildenafil potently inhibits ET-1–induced PASMCs proliferation and downregulation of CCE and TRPC expression may be responsible for its antiproliferative effect.

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Epoxyeicosatrienoic acids (EETs), including 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET, are produced by cytochrome P450 (P450) such as CYP2C8 and 2C9; and they are hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. Particular interest in the epoxygenase reaction has developed because of the potent biological activities (modulation of vascular tone and anti-inflammatory activity, etc.) attributed to EETs. We focused on a new biological function of EETs and DHETs, which induce vascular endothelial growth factor (VEGF) and erythropoietin (EPO) under hypoxia. Human hepatoma cells, Hep3B, and human umbilical artery endothelial cells (HUAEC) were used in this study. An inhibitor of phospholipase A₂, methyl arachidonyl fluorophosphonate (MAFP), and inhibitors of P450s inhibited the VEGF and EPO induction of HUAEC and Hep3B, respectively, under hypoxia. Overexpression of CYP2C8 in Hep3B induced EPO and VEGF under hypoxia. Sulfaphenazole, an inhibitor of CYP2C8/2C9 suppressed luciferase promoter activity with the hypoxia response element (HRE) of VEGF in HUAEC. Exogenous 11,12-EET and 14,15-DHET induced reporter activity in HUAEC and Hep3B cells concomitant with increased levels of hypoxia-inducible factor-1α (HIF-1α), which is a key factor in the hypoxia response, but 11,12-DHET and 14,15-EET did not. These results suggested that EETs and DHETs play an important role in the hypoxia response of cells.

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The effects of goshuyuto and chotosan, traditional Japanese medicines, on collagen-induced platelet aggregation were examined using guinea-pig blood. Goshuyuto at the concentration of 1,000 μg/mL inhibited collagen-induced platelet hyper-aggregation to the same degree as aspirin at the concentration of 100 μmol/L, but chotosan did not. Goshuyuto is composed of four medicinal herbs. Of them, aqueous extracts of Evodiae Fructus and Zingiberis Rhizoma inhibited platelet aggregation, but aqueous extracts of Zizyphi Fructus and Ginseng Radix did not. Two components of Zingiberis Rhizoma, 6-shogaol and 6-gingerol, also inhibited platelet aggregation. These results suggest that Evodiae Fructus and Zingiberis Rhizoma may play important roles in the anti-aggregation effects of goshuyuto and that 6-shogaol and 6-gingerol are among the active ingredients. Therefore, goshuyuto may ameliorate migraine by preventing the hyper-aggregation of platelets in migraine with aura.

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Vascular endothelium regulates vascular tone by releasing endothelium-derived vasoactive substances. We performed this study to characterize the inhibitory effect of the endothelium on vasoconstrictor stimuli in rat mesenteric vascular beds. Changes in perfusion pressure induced by continuous perfusion of Krebs solution containing methoxamine (α₁-adrenoceptor agonist) or high KCl were measured over 180 min. In preparations with intact endothelium, methoxamine-induced vasoconstriction was time-dependently decreased to cause 60% – 80% reduction of the initial vasoconstriction level, while no reduction was observed in high-KCl–induced vasoconstriction. Endothelium removal significantly blunted the time-dependent reduction of methoxamine-induced vasoconstriction without affecting high-KCl–induced vasoconstriction. Neither a nitric oxide synthase inhibitor (L-NAME) nor indomethacin (cyclooxygenase inhibitor) altered the time-dependent reduction of vasoconstriction. High KCl, K⁺-channel inhibitors tetraethylammonium and apamin plus charybdotoxin, and 18α-glycyrrhetinic acid (18α-GA, a gap-junction inhibitor) significantly inhibited the time-dependent reduction of methoxamine-induced vasoconstriction. In preconstricted preparations, bolus injection of acetylcholine and Ca²⁺-ionophore A23187 (A23187) evoked a sharp vasodilation, which was inhibited by endothelium removal, high KCl and tetraethylammonium, but not indomethacin, L-NAME, or 18α-GA. However, 18α-GA plus L-NAME inhibited vasodilation induced by A23187, but not acetylcholine. These findings suggest that endothelium-derived hyperpolarizing factor (EDHF) via gap junctions mainly counteracts vasoconstriction induced by methoxamine in mesenteric resistance arteries.

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We examined the mechanism of thrombin on proliferation of synovial fibroblasts obtained from rheumatoid arthritis (RA). Thrombin concentration-dependently induced proliferation of synovial fibroblasts. Proliferation in response to thrombin (10 U/ml) was completely blocked by hirudin. TP367 and TP508, peptides corresponding to 2 noncatalytic regions of thrombin, failed to induce cell proliferation. Thrombin did not induce the production of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) in synovial fibroblasts. Expression of proteinase-activated receptor (PAR)-1 and PAR-3 mRNAs was observed in synovial fibroblasts. Thrombin and PAR-1 agonist peptide (AP), but not PAR-3 AP, induced intracellular calcium mobilization. PAR-1 AP induced cell proliferation whereas PAR-3 AP and PAR-4 AP had no effect on proliferation. Pertussis toxin (PTX), a Giα protein inhibitor; wortmannin, a PI (phosphatidylinositol) 3-kinase inhibitor; and PD98059, a specific MEK [mitogen-activated protein (MAK) kinase kinase] inhibitor, inhibited the thrombin-induced cell proliferation. Furthermore, the proliferation of synovial fibroblasts was suppressed by U-73122, a PLC (phospholipase C) inhibitor; 2-APB, an antagonist of InsP3 (inositol 1,4,5-triphosphate) receptor; and GF-109203X, a PKC (protein kinase C) inhibitor. These results suggest that thrombin induces the proliferation of RA synovial fibroblasts through the activation of PAR-1, leading to the PTX-sensitive G proteins – PI3 kinase pathway and PTX-insensitive G proteins – PLC (InsP3 receptor) Ca²⁺-PKC branch.

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We have reported that facilitation of central histaminergic activity prevents the development of ischemia-induced brain injury. Since cerebral edema is a major cause of brain damage, we studied effects on brain edema of postischemic administration of L-histidine, a precursor of histamine, and thioperamide, a histamine H₃–receptor antagonist, both of which enhance central histaminergic activity. Focal cerebral ischemia for 2 h was provoked by transient occlusion of the right middle cerebral artery in rats, and the water content and infarct size were determined 24 h after reperfusion. Changes in the extracellular concentration of histamine were examined in the striatum by a microdialysis procedure, and effects of these compounds were evaluated. Repeated administration of L-histidine (1000 mg/kg × 2, i.p.), immediately and 6 h after reperfusion, reduced the increase in the water contents in ischemic regions. Simultaneous administration of thioperamide (5 mg/kg, s.c.) with L-histidine (1000 mg/kg, i.p.) completely prevented edema formation and alleviated brain infarction, although a single dose of L-histidine, immediately after reperfusion, showed no benefits. The striatal histamine level was gradually increased after reperfusion as well as during ischemia. Simultaneous administration of thioperamide with L-histidine markedly increased the brain histamine concentration, and the value increased up to 230% of that in the saline group 5 – 6 h after reperfusion. L-Histidine alone did not affect the increase in the histamine output after ischemia. These findings suggest that further activation of the central histaminergic system after initiation of cerebral ischemia prevents development of ischemia-induced brain edema.

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Recently it has been suggested that resveratrol relaxes different isolated arteries. The present study addressed the question whether different ion channels are involved in the endothelium-independent mechanism of vasodilatation induced by resveratrol. For that purpose, we tested the action of resveratrol on the rat mesenteric artery without endothelium. Resveratrol induced concentration-dependent relaxation of rat mesenteric artery. Among the K⁺-channel blockers, 4-amynopiridine (4-AP) moderately antagonized the resveratrol-induced relaxation, while glibenclamide, tetraethylammonium chloride, charybdotoxin, margatoxin, and barium chloride did not inhibit resveratrol-induced vasorelaxation. In rings, precontracted with 100 mM K⁺, the relaxant responses to resveratrol were highly significantly shifted to the right compared to those obtained in rings precontracted with phenylephrine, but resveratrol-induced maximal relaxation was only slightly affected. In order to minimize the influence of K⁺ channels and voltage-gated Ca²⁺ channels (VGCCs) in vascular smooth muscle, the third contraction was made by 100 mM K⁺ in the presence of nifedipine. The relaxant response to resveratrol was abolished. Thus, the mechanism of vasorelaxation induced by resveratrol probably involves activation of 4-AP-sensitive K⁺ channels. Its ability to completely relax the mesenteric artery precontracted with K⁺-rich solution suggests that K⁺ channel–independent mechanism(s) are involved in its vasorelaxant effect. It seems that interaction with VGCCs plays a part in this K⁺ channel–independent effect of resveratrol.

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The cholestane amide conjugate MCC-257 has been shown to augment the effects of nerve growth factor (NGF) on cell survival and on tyrosine phosphorylation of the TrkA receptor in PC12 cells. Recent findings suggest that signaling pathways downstream of Trk are regulated independently. We describe here our finding that the NGF-induced phosphorylation of both ERK and Akt are accelerated by MCC-257. Analysis of the common features of the augmented pathways suggests that TrkA is most likely to be the primary target of MCC-257 and that both ERK and Akt may be involved in the cellular effects of this compound.

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It has been suggested that a new type of large-conductance Ca²⁺-activated K⁺ (BK) channel is distributed in the inner mitochondrial membrane (mitoK_Ca channel) and that its opening may attenuate ischemic cardiac injury. We examined effects of 12,14-dichlorodehydroabietic acid (diCl-DHAA), a novel BK-channel opener, on rat cardiac myocytes and mitochondria. Application of diCl-DHAA concentration-dependently reduced Ca²⁺ overload in isolated mitochondria, activated mitoK_Ca channels in inside-out patches of mitochondrial membrane, facilitated flavoprotein-oxidization in myocytes, and increased cellular viability under simulated ischemia. In conclusion, diCl-DHAA directly opens mitoK_Ca channels, prevents Ca²⁺ influx into matrix, and reduces ischemic injury in cardiac myocytes.

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In the present study, we examined the effects of calcium-channel antagonists on marble-burying behavior, which is an animal model of obsessive-compulsive disorder. Amlodipine (10 mg/kg, i.p.), cilnidipine (10 mg/kg, i.p.), nilvadipine (3 and 10 mg/kg, i.p.), and flunarizine (30 mg/kg, i.p.) significantly inhibited marble-burying behavior in mice. These results suggest that calcium channels may be involved in the marble-burying behavior.

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Intestinal drug efflux proteins play a major role in the pharmacokinetics of many drugs. We assessed diurnal rhythmicity in the expression of ten major drug transporters. We acquired male Sprague-Dawley rats and harvested jejunal mucosa at 3-h intervals across a 24-h period. qPCR was performed for ten transporters: Mdr1, Mdr3, Mrp1 – 3, Mct1, Brcp, Pept1, Octn2, and Oatp-b. Rhythmicity was assessed with the cosinor procedure. Diurnal rhythmicity was observed for Mdr1, Mct1, Mrp2, Pept1, and Bcrp (1.6 – 5.4-fold-changes). Acrophases occurred during fasting hours. We conclude that many drug transporters display profound diurnal rhythms in transcription, which may underlie diurnal rhythms in drug pharmacokinetics.

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