Effects of caffeic acid phenethyl ester on matrix molecules and angiogenetic and anti-angiogenetic factors in gastric cancer cells cultured on different substrates.

Migration, invasion, metastasis and angiogenesis associated with cancer depend on the surrounding microenvironment. Angiogenesis, the growth of new capillaries, is a regulator of cancer growth and a useful target for cancer therapy. We examined matrix protein interactions in a gastric cancer cell culture that was treated with different doses of caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE). We also investigated the relations among the levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), endostatin (ES) and trombospondin-1 (TSP-1). Cytotoxity of CAPE was measured using the 3-(4,5-dmethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We examined the behavior of cells on laminin and collagen I coated surfaces in response to the angiogenic effect of these matrix molecules. We examined the protein alterations of these matrix molecules immunohistochemically and measured the levels of VEGF, MMP-9, ES and TSP-1 using the ELISA test. We showed that application of CAPE to the gastric cancer cell line on tissue culture plastic, laminin and collagen I significantly decreased the VEGF and MMP-9 protein levels. We found that TSP-1 levels were increased significantly in the gastric cancer cells after application of CAPE. The protein levels of gastric cancer cells also were increased significantly when tissue was cultured on laminin and collagen I. Application of CAPE to cells on laminin or collagen I coated surfaces significantly increased all of the proteins except ES. ES levels were increased on the collagen I covered surfaces, but the laminin surface decreased the levels of ES significantly. We demonstrated the beneficial effect of CAPE on a gastric cancer cell line including inhibition of proliferation and induction of some proteins that might be related to decreased angiogenesis.

Protective effect of ACE inhibitors on ischemia-reperfusion-induced arrhythmias in rats: is this effect related to the free radical scavenging action of these drugs?

The antiarrhythmic effects of captopril, a sulphydryl-containing angiotensin converting enzyme (ACE) inhibitor, were compared with those of the nonsulphydryl-containing ACE inhibitor lisinopril and the sulphydryl-containing agent glutathione in an in vivo rat model of coronary artery ligation. To produce arrhythmia, the left main coronary artery was occluded for 7 min, followed by 7 min of reperfusion. Captopril (3 mg kg-1) and lisinopril (0.1, 0.3 or 1 mg kg-1) caused marked decreases in mean arterial blood pressure (BP) and heart rate, whereas glutathione (5 mg kg-1) had no effect on them. The incidence of ventricular tachycardia (VT) on ischemia and reperfusion was significantly reduced by captopril and lisinopril. Captopril and 1 mg kg-1 lisinopril also significantly decreased the number of VEB during occlusion and the duration of VT on reperfusion, respectively. These drugs also attenuated the incidence of reversible ventricular fibrillation (VF) and the number of ventricular ectopic beats (VEB) during reperfusion. However, glutathione only reduced the incidence of VT on reperfusion, significantly. These results suggest that, in this experimental model, ACE inhibitors limit the arrhythmias following ischemia-reperfusion and free radical scavenging action of these drugs does not have a major contributory role in their protective effect.

Evaluation of the alpha-adrenoceptor antagonistic action of berberine in isolated organs.

The selectivity and specificity of berberine (CAS 2086-83-1) for alpha-adrenoceptor subtypes have been studied. alpha 1-Adrenoceptors were investigated in rat and rabbit aorta and alpha 2-adrenoceptors in guinea-pig ileum preparations. Subtype selectivity was then assessed by calculating the selectivity ratios from Ke (equilibrium constant) values. Berberine was found to be more potent for the postsynaptic alpha 1-adrenoceptors than presynaptic alpha 2-adrenoceptors. The selectivity ratios were 1.9 and 3.1 for rabbit and rat aorta, respectively. In the rabbit aorta, responses to norepinephrine and phenylephrine were both inhibited by berberine with lower affinity than that in the rat aorta. The pA2 values for berberine obtained from the norepinephrine and phenylephrine experiments were also found significantly different in rat aorta. However, similar pA2 values were found in rabbit aorta for those agonists.

The role of prostaglandin synthesis stimulation in the protective effect of captopril on ischaemia-reperfusion arrhythmias in rats in vivo.

Attenuation of ischaemia-reperfusion induced arrhythmias by several angiotensin converting enzyme (ACE) inhibitors, such as captopril, has been demonstrated. The role of prostaglandin synthesis stimulation in this protective effect of ACE inhibition was evaluated in an in vivo rat model. To produce arrhythmia, the left main coronary artery was occluded for 7 min, followed by 7 min of reperfusion. Captopril (3 mg kg-1) and a prostaglandin synthesis inhibitor, indomethacin (2 mg kg-1) alone or together were administered by intravenous (i.v.) injection 10 min before occlusion. Captopril reduced the incidence of ventricular tachycardia (VT) and the number of ventricular ectopic beats (VEB) on ischaemia and reperfusion as well as the incidence of reversible ventricular fibrillation (VF) on reperfusion. These protective effects of captopril against ischaemia-reperfusion-induced arrhythmias were prevented by indomethacin. Captopril also caused a sustained decrease of preocclusion values in the arterial blood pressure (BP) and heart rate (HR), whereas in the presence of indomethacin, captopril had no significant effect on either HR or arterial BP values except the heart rate value just before occlusion. Indomethacin alone did not affect either the severity of arrhythmias or the haemodynamic parameters. These results suggest that, in this experimental model, the protective effects of ACE inhibitors on the arrhythmias following ischaemia-reperfusion are mediated by the stimulation of prostaglandin synthesis and the haemodynamic effects of these drugs may have a contributory role in their protective effect.

Effects of m-cholinoceptor antagonists on the neuromuscular junction.

The effects of four m-cholinoceptor antagonists and d-tubocurarine on carbachol- or nicotine-induced contractions of frog rectus abdominus muscle were investigated. The n-cholinoceptor-mediated contractile responses of frog rectus abdominis muscles were inhibited competitively by d-tubocurarine whereas m-cholinoceptor antagonists inhibited them noncompetitively. Antagonistic potencies for m-cholinoceptor antagonists, assessed by pD'2 values, indicated a rank order of potency which was 4-DAMP > AF-DX 116 > atropine > pirenzepine. The antagonistic potencies of m-cholinoceptor antagonists on electrically induced twitch responses of rat phrenic nerve-diaphragm preparation were also examined. These responses were blocked by 4-DAMP, but pirenzepine had no effect. However, they were enhanced by AF-DX 116 or atropine. Inhibitory action of 4-DAMP in this preparation was suggested to be due to noncompetitive blockade of postjunctional n-cholinoceptors.

Effects of captopril on ischaemia-reperfusion-induced arrhythmias in an in vivo rat model.

The antiarrhythmic effects of captopril, an angiotensin converting enzyme (ACE) inhibitor, were investigated in an in vivo rat model of coronary artery ligation. Captopril (0.3-3 mg kg-1) or saline were administered by intravenously 10 min before coronary ischaemia. The left main coronary artery was then occluded for 7 min, followed by 7 min of reperfusion. Captopril caused a marked decrease in mean arterial blood pressure which was transient at 0.3 and 1 mg kg-1, and at doses of 1 and 3 mg kg-1, it produced marked bradycardia. The incidence of ventricular tachycardia (VT) on ischaemia was significantly reduced the captopril at a dose of 3 mg kg-1 only and on reperfusion at doses of 1 and 3 mg kg-1. At the same doses, captopril significantly reduced the mean duration of ventricular fibrillation (VF) on reperfusion. The incidence of mortality resulting from reperfusion-induced irreversible VF in the control group decreased from 42.9% to 14.3% (NS), 21.4% (NS) and 7.7% (P < 0.05) in captopril at 0.3, 1 and 3 mg kg-1, respectively. Our results indicate that captopril appears to limit the arrhythmias following reperfusion and this may be due in part to the antiischemic effect associated with bradycardia and vasodepression.

Inhibitory muscarinic cholinoceptors on postganglionic sympathetic nerves in the guinea pig isolated atrium are of the M3 subtype.

Pre- and postjunctional effects of four muscarinic (m)-cholinoceptor antagonists were investigated against carbachol in isolated spontaneously beating guinea pig atria which were also exposed to brief periods of electrical field stimulation. The cholinoceptor agonist carbachol concentration-dependently inhibited the contraction rate of the atria with an EC50 value of 92.3 +/- 20.6 nmol/l. The m-cholinoceptor antagonists atropine, 4-DAMP (M1- and M3-selective), AF-DX 116 (M2-selective) and pirenzepine (M1-selective) shifted the concentration-response curves of carbachol to the right without modifying the maximal responses yielding pA2 values of 8.98, 8.37, 7.44 and 6.79, respectively. Electrical field stimulation increased the concentration rate of the atria which was potentiated by atropine or 4-DAMP while AF-DX 116 or pirenzepine had no significant effect. Therefore, on the basis of the findings with 4-DAMP, it is concluded that prejunctional m-cholinoceptors on the sympathetic nerve endings in guinea pig atria are inhibitory and are of the M3 subtype.

Effects of chronic ethanol consumption on alpha-adrenergic-induced contractions and endothelium-dependent relaxations in rat thoracic aorta.

The effects of chronic oral administration of ethanol (7.2% daily during 24 weeks) on the contractions induced by phenylephrine (Phe) and the endothelium-dependent relaxation responses to acetylcholine (ACh) were studied in rat thoracic aorta. Ethanol pretreatment significantly attenuated the contractile responses to Phe, resulting in parallel shift of the concentration-response curve to the right. EC(50)values of Phe were 64.6+/-11.2 and 95.5+/-8.5 nmol l(-1)in control and ethanol-fed rats, respectively. On the other hand, either calcium-induced contractions or relaxation responses to ACh and sodium nitroprusside were similar in the vessels of the control and ethanol-treated rats. These results suggest that chronic ethanol ingestion significantly attenuates the alpha(1)-adrenergic-induced contractions but does not affect the relaxation responses mediated by nitric oxide in rat aortic rings.

Acute host-mediated endothelial injury after adenoviral gene transfer in normal rabbit arteries: impact on transgene expression and endothelial function.

Acute injury after adenoviral vascular gene transfer remains incompletely characterized. Here, we describe the early response (< or =days) in 52 New Zealand White rabbits undergoing gene transfer (beta-galactosidase or empty vector) or sham procedures to both carotid arteries. After gene transfer, arteries were either left in vivo for 1 hour to 3 days (in vivo arteries) or were excised immediately after gene transfer and cultured (ex vivo arteries). Within 1 hour, in vivo arteries receiving infectious titers of > or = 4X10(9) plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in beta-galactosidase expression through 2X10(10) pfu/mL, whereas in in vivo arteries, titers above 4X10(9) pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time- and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1X10(11) pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury.

In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-Fed rabbits.

BACKGROUND: The vascular endothelium is anatomically intact but functionally abnormal in preatherosclerotic states, and an early deficit in the bioavailability of nitric oxide (NO) or related molecules has been described in both humans and animal models. We hypothesized that the targeted gene transfer of NO synthase (NOS) isoforms might ameliorate or reverse the deficit. METHODS AND RESULTS: We constructed a recombinant adenovirus, Ad.nNOS, that expresses the neuronal isoform of NOS (nNOS) and used it for in vivo endovascular gene transfer to carotid arteries (CA) from normal and cholesterol-fed rabbits. Vessels were harvested 3 days after gene transfer. In CA from normal rabbits, Ad.nNOS generated high levels of functional nNOS protein predominantly in endothelial cells and increased vascular NOS activity by 3.4-fold relative to sham-infected control CA. Ad.nNOS gene transfer also significantly enhanced endothelium-dependent vascular relaxation to acetylcholine; at 3 micromol/L acetylcholine, Ad.nNOS-treated arteries showed an 86+/-4% reduction in precontracted tension, whereas control CA showed a 47+/-6% reduction in tension. Contraction in response to phenylephrine and relaxation in response to nitroprusside were unaffected in both control and Ad.nNOS-treated CA. To determine the effect of Ad.nNOS in atherosclerotic arteries, 10 male New Zealand White rabbits maintained on a 1% cholesterol diet for 10 to 12 weeks underwent gene transfer according to the same protocol used in normal rabbits. Ad.nNOS-treated arteries showed a 2-fold increase in NADPH-diaphorase staining intensity relative to sham-infected and Ad. betaGal-treated arteries. The CA from cholesterol-fed rabbits showed impaired acetylcholine-induced relaxation, but this abnormality was almost entirely corrected by Ad.nNOS gene transfer. CONCLUSIONS: In vivo adenovirus-mediated endovascular delivery of nNOS markedly enhances vascular NOS activity and can favorably influence endothelial physiology in the intact and atherosclerotic vessel wall.