Cardiac chymase: pathophysiological role and therapeutic potential of chymase inhibitors.

On release from cardiac mast cells, alpha-chymase converts angiotensin I (Ang I) to Ang II. In addition to Ang II formation, alpha-chymase is capable of activating TGF-beta1 and IL-1beta, forming endothelins consisting of 31 amino acids, degrading endothelin-1, altering lipid metabolism, and degrading the extracellular matrix. Under physiological conditions the role of chymase in the mast cells of the heart is uncertain. In pathological situations, chymase may be secreted and have important effects on the heart. Thus, in animal models of cardiomyopathy, pressure overload, and myocardial infarction, there are increases in both chymase mRNA levels and chymase activity in the heart. In human diseased heart homogenates, alterations in chymase activity have also been reported. These findings have raised the possibility that inhibition of chymase may have a role in the therapy of cardiac disease. The selective chymase inhibitors developed to date include TY-51076, SUN-C8257, BCEAB, NK320, and TEI-E548. These have yet to be tested in humans, but promising results have been obtained in animal models of myocardial infarction, cardiomyopathy, and tachycardia-induced heart failure. It seems likely that orally active inhibitors of chymase could have a place in the treatment of cardiac diseases where injury-induced mast cell degranulation contributes to the pathology.

Evidence for, and importance of, cGMP-independent mechanisms with NO and NO donors on blood vessels and platelets.

In the vasculature it is well established that cGMP is involved in the relaxant response to nitric oxide (NO) and NO donors. However, there is an increasing evidence that alternative/additional pathways that are cGMP-independent may also exist. A key criterion for a response to NO or a NO donor drug to be classified as cGMP-independent is lack of (or incomplete) inhibition by the selective inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). In many blood vessels cGMP-independent mechanisms contribute to the vasorelaxation, and in certain vascular beds cGMP-independent relaxation may be the predominant mechanism of action of NO and NO donors. NO donor drugs that generate NO "spontaneously", like authentic NO (i.e. solutions of NO gas), appear to exhibit a larger component of cGMP-independent vasorelaxation than do those drugs that require bioactivation in the tissue. The long lasting inhibition of responses to vasoconstrictors by S-nitrosothiols, persisting after removal of these NO donors, may be a cGMP-independent process, at least in some vessels. The mechanisms involved in the inhibition of vascular growth by NO and NO donors are predominantly cGMP-independent, as are the mechanisms responsible for the effects of NO donors on apoptosis in vascular smooth muscle and endothelial cells. The ability of NO and NO donors to inhibit platelet aggregation has a significant cGMP-independent component. cGMP-independent pathways are most often, though not exclusively, seen at high concentrations (microM - mM) of NO and NO donors. Hence, in relation to the actions of endogenous NO, these pathways may be particularly important in settings when the inducible isoform of NO-synthase is expressed. Furthermore, cGMP-independent pathways are enhanced in animal models of atherosclerosis and ischaemia. This suggests that it may be possible to target cGMP-independent mechanisms with selected NO donors in disease states.

Nitric oxide donors inhibit 5-hydroxytryptamine (5-HT) uptake by the human 5-HT transporter (SERT).

1. The aim was to test the hypothesis that nitric oxide (NO) donor drugs can inhibit the 5-hydroxytryptamine (5-HT) transporter, SERT. 2. The NO donors, MAHMA/NO (a NONOate; (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)-amino]]diazen-1-ium-1,2-diolate), SIN-1 (a sydnonimine; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride), FK409 (an oxime; (+/-)-(4-ethyl-2E-(hydroxyimino)-5-nitro-3E-hexenamide)) and peroxynitrite, but not Angeli's salt (source of nitroxyl anion) or sodium nitrite, caused concentration-dependent inhibition of the specific uptake of [3H]-5-HT in COS-7 cells expressing human SERT. 3. Superoxide dismutase (150 U ml(-1)) plus catalase (1200 U ml(-1)), used to remove superoxide and hence prevent peroxynitrite formation, prevented the inhibitory effect of SIN-1 (which generates superoxide) but not of MAHMA/NO or FK409. 4 The inhibitory effects of the NO donors were not affected by the free radical scavenger, hydroxocobalamin (1 mM) or the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 3 microM). 5. L-Cysteine (1 mM; source of excess thiol residues) abolished or markedly reduced the inhibitory effects of MAHMA/NO, SIN-1, FK409 and peroxynitrite. 6. It is concluded that inhibition of SERT by the NO donors cannot be attributed exclusively to NO free radical nor to nitroxyl anion. It does not involve guanosine-3',5'-cyclic monophosphate, but may involve nitrosation of cysteine residues on the SERT protein. Peroxynitrite mediates the effect of SIN-1, but not the other drugs. 7. Data in mice with hypoxic pulmonary hypertension suggest that SERT inhibitors may attenuate pulmonary vascular remodelling. Thus, NO donors may be useful in pulmonary hypertension, not only as vasodilators, but also because they inhibit SERT, provided they display this effect in vivo at appropriate doses.

Proteinase-activated receptor (PAR)-mediated vasorelaxation in pulmonary arteries from normotensive and hypoxic pulmonary hypertensive rats.

Proteinase-activated receptor (PAR)-mediated vasorelaxant responses were examined in main pulmonary artery preparations from control rats and rats exposed to hypoxia (10% oxygen) for 1 or 4 weeks to induce pulmonary hypertension. Trypsin and the PAR-2 peptide, SLIGRL, relaxed phenylephrine precontracted preparations, with maximum responses the same as the maximum response to acetylcholine. Responses to trypsin and SLIGRL were abolished by endothelium removal or a nitric oxide (NO) synthase inhibitor, and were, therefore, due to release of endothelium-derived NO. In pulmonary arteries from rats exposed to hypoxia for 1 week, the potencies and maximal responses for acetylcholine and trypsin were markedly reduced compared with data in control rats, but these values were restored to normal in arteries from 4-week hypoxic rats. In contrast, the potency of SLIGRL was unchanged in arteries from either group of hypoxic rats. The data for trypsin and acetylcholine are consistent with previous findings for a variety of endothelium-dependent vasodilators and reflect an impairment of endothelial function in early hypoxic pulmonary hypertension. The data for SLIGRL emphasise that one cannot necessarily predict changes in PAR-2-mediated endothelium-dependent vasorelaxation in disease states from data with the classical endothelium-dependent vasodilator, acetylcholine.

Vascular chymase: pathophysiological role and therapeutic potential of inhibition.

Chymase is a chymotrypsin-like serine protease secreted from mast cells. Mammalian chymases are classified into two subgroups (alpha and beta) according to structure and substrate specificity; human chymase is an alpha-chymase. An important action of chymase is the ACE-independent conversion of Ang I to Ang II, but chymase also degrades the extracellular matrix, activates TGF-beta1 and IL-1beta, forms 31-amino acid endothelins and is involved in lipid metabolism. Under physiological conditions, the role of chymase in blood vessels is uncertain. In pathological situations, however, chymase may be important. In animal models of hypertension and atherosclerosis, chymase may be involved in lipid deposition and intimal and smooth muscle hyperplasia, at least in some vessels. In addition, chymase has pro-angiogenic properties. In human diseased blood vessels (e.g. atherosclerotic and aneurysmal aorta; remodeled pulmonary blood vessels), there are increases in chymase-containing mast cells and/or in chymase-dependent conversion of Ang I to Ang II. These findings have raised the possibility that inhibition of chymase may have a role in the therapy of vascular disease. The effects of chymase can theoretically be attenuated either by reducing availability of the enzyme, with a mast cell stabiliser, or alternatively with specific chymase inhibitors. The mast cell stabiliser, tranilast, was shown to be beneficial in animal models of atherosclerosis, where a prevention protocol was used, but was not effective in clinical trials where it was administered after angioplasty. Chymase inhibitors could have the advantage of being effective even if used after injury. Several orally active inhibitors, including SUN-C8257, BCEAB, NK3201 and TEI-E548, are now available. These have yet to be tested in humans, but promising results have been obtained in animal models of atherosclerosis and angiogenesis. It is concluded that orally active inhibitors of chymase may have a place in the treatment of vascular diseases where injury-induced mast cell degranulation contributes to the pathology.

Platelet inhibitory effects of the nitric oxide donor drug MAHMA NONOate in vivo in rats.

The platelet inhibitory effects of the nitric oxide (NO) donor drug MAHMA NONOate ((Z-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen-1-ium-1,2-diolate) were examined in anaesthetised rats and compared with those of S-nitrosoglutathione (GSNO; an S-nitrosothiol). Bolus administration of the aggregating agent ADP dose-dependently reduced the number of circulating free platelets. Intravenous infusions of MAHMA NONOate (3-30 nmol/kg/min) dose-dependently inhibited the effect of 0.3 micromol/kg ADP. MAHMA NONOate was approximately 10-fold more potent than GSNO. MAHMA NONOate (0.3-10 nmol/kg/min) also reduced systemic artery pressure and was again 10-fold more potent than GSNO. Thus MAHMA NONOate has both platelet inhibitory and vasodepressor effects in vivo. The dose ranges for these two effects overlapped, although blood pressure was affected at slightly lower doses. The platelet inhibitory effects compared favourably with those of GSNO, even though NONO-ates generate free radical NO which, in theory, could have been scavenged by haemoglobin. Therefore platelet inhibition may be a useful therapeutic property of NONOates.

Potentiation of 5-hydroxytryptamine (5-HT) responses by a 5-HT uptake inhibitor in pulmonary and systemic vessels: effects of exposing rats to hypoxia.

The aim was to determine whether uptake of 5-hydroxytryptamine (5-HT) by the 5-HT transporter (SERT) modulates contractile responses to 5-HT in rat pulmonary arteries and whether this modulation is altered by exposure of rats to chronic hypoxia (10% oxygen; 8 h/day; 5 days). The effects of the SERT inhibitor, citalopram (100 nM), on contractions to 5-HT were determined in isolated ring preparations of pulmonary artery (intralobar and main) and compared with data obtained in systemic arteries. In intralobar pulmonary arteries citalopram produced a potentiation (viz. an increase in potency, pEC(50)) of 5-HT. The potentiation was endothelium-dependent in preparations from normoxic rats but endothelium-independent in preparations from hypoxic rats. In main pulmonary artery endothelium-independent potentiation was seen in preparations from hypoxic rats but no potentiation occurred in preparations from normoxic rats. In systemic arteries, citalopram caused endothelium-independent potentiation in aorta but no potentiation in mesenteric arteries; there were no differences between hypoxic and normoxic rats. It is concluded that SERT can influence the concentration of 5-HT in the vicinity of the vasoconstrictor receptors in pulmonary arteries. The data suggest that in pulmonary arteries from hypoxic rats, unlike normoxic rats, the SERT responsible for this effect is not in the endothelium and, hence, is probably in the smooth muscle. The data are compatible with reports that, in the pulmonary circulation, hypoxia induces/up-regulates SERT, and hence increases 5-HT uptake, in vascular smooth muscle. The findings may have implications in relation to the suggested use of SERT inhibitors in the treatment of pulmonary hypertension.

Alterations in pulmonary vascular function in rats exposed to intermittent hypoxia.

Vasoactive agents were examined in arteries from control rats and rats exposed to intermittent hypoxia (10% oxygen; 8 h/day) for 3, 5 or 20 days. Hypoxic rats developed right ventricular hypertrophy after 5 days, but became pulmonary hypertensive (elevated right ventricular systolic pressure; RVSP) only after 20 days. In pulmonary arteries (main and intralobar), responses to acetylcholine and ionomycin (endothelium-dependent vasodilators) were reduced after 20 and 5 days of intermittent hypoxia, whereas contractions to 5-hydroxytryptamine (5-HT) were enhanced (potency increase >10-fold) after 20, 5 and 3 days. Contractions to endothelin-1 and a thromboxane-mimetic, but not Ca(2+), were also increased. No changes in vascular function occurred in aorta. Since changes in pulmonary vascular function preceded the increase in RVSP they do not result from, but may contribute to, the development of hypoxia-induced pulmonary hypertension. If similar changes occur in humans, they may be important in conditions characterised by intermittent, as opposed to continuous, hypoxia.

S-nitrosocaptopril: acute in-vivo pulmonary vasodepressor effects in pulmonary hypertensive rats.

The effects of S-nitrosocaptopril (SNOcap), administered either intravenously or by oral gavage, on pulmonary artery pressure (PAP) were examined in anaesthetised normotensive rats and rats with hypoxic pulmonary hypertension (10% oxygen for 1 week). Mean PAP (MPAP) values in hypoxic and normoxic rats were (mmHg) 26 +/- 1.7 and 15 +/- 1.1, respectively. When given intravenously, 1 mg kg(-1) SNOcap reduced MPAP by 28 and 32% in hypoxic and normoxic rats, respectively. The effects of 2 mg kg(-1) were no greater than those of 1 mg kg(-1). Pulmonary vasodepressor responses reached equilibrium in 1.7 +/- 0.18 min following intravenous administration. When given orally 30 min before the measurement of PAP, 30 mg kg(-1), but not 10 mg kg(-1), significantly reduced MPAP in hypoxic rats to 17 +/- 1.5 mmHg. These in-vivo data are consistent with previous in-vitro data showing that SNOcap has direct pulmonary vasorelaxant properties in both large and small pulmonary arteries and also show that SNOcap causes pulmonary vasodepression in the setting of pulmonary hypertension. Since SNOcap also inhibits pulmonary vascular angiotensin converting enzyme (ACE) in pulmonary blood vessels (previous study), it would be an interesting drug with which to assess the benefits of direct pulmonary vasodilatation combined with ACE inhibition (which attenuates pulmonary vascular remodelling) in a long-term study in pulmonary hypertension.

Will chymase inhibitors be the next major development for the treatment of cardiovascular disorders?

Chymase is contained in the secretory granules of mast cells. In addition to the synthesis of angiotensin II, chymase is involved in transforming growth factor-beta activation and cleaves Type I procollagen to produce collagen. NK301 and BCEAB are orally-active inhibitors of chymase. NK301 was tested in a dog model of vascular intimal hyperplasia after balloon injury and shown to reduce the increased chymase activity in the injured arteries and prevent intimal thickening. In a hamster model of cardiac fibrosis associated with cardiomyopathy, BCEAB reduced the increased cardiac chymase activity in cardiomyopathy and reduced fibrosis. Chymase inhibitors may be an important development for the treatment of cardiovascular injury associated with mast cell degranulation.

S-nitrosocaptopril: in vitro characterization of pulmonary vascular effects in rats.

(1) On rat isolated pulmonary arteries, vasorelaxation by S-nitrosocaptopril (SNOcap) was compared with S-nitrosoglutathione (GSNO) and nitroprusside, and inhibition by SNOcap of contractions to angiotensin I was compared with the angiotensin converting enzyme (ACE) inhibitor, captopril. (2) SNOcap was equipotent as a vasorelaxant on main (i.d. 2-3 mm) and intralobar (i.d. 600 micro m) pulmonary arteries (pIC(50) values: 5.00 and 4.85, respectively). Vasorelaxant responses reached equilibrium rapidly (2-3 min). (3) Pulmonary vasorelaxant responses to SNOcap, like GSNO, were (i) partially inhibited by the soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4) oxadiazolo(4,3-a)-quinoxalin-1-one; 3 micro M) whereas responses to nitroprusside were abolished and (ii) potentiated by hydroxocobalamin (HCOB; NO. free radical scavenger; 100 micro M) whereas responses to nitroprusside were inhibited. (4) The relative potencies for pulmonary vasorelaxation compared with inhibition of platelet aggregation were: SNOcap 7 : 1; GSNO 25 : 1; nitroprusside >2000 : 1. (5) SNOcap, like captopril, concentration-dependently and time-dependently increased the EC(50) for angiotensin I but not angiotensin II. The dependence on incubation time was independent of the presence of tissue but differed for SNOcap and captopril. This difference reflected the slow dissociation of SNOcap and instability of captopril, and precluded a valid comparison of the potency of the two drugs. After prolonged incubation (>/=5.6 h) SNOcap was more effective than captopril. (6) Thus, in pulmonary arteries SNOcap (i) possesses NO donor properties characteristic of S-nitrosothiols but different from nitroprusside and (ii) inhibits ACE at least as effectively as captopril. These properties suggest that SNOcap could be valuable in the treatment of pulmonary hypertension.

5-Hydroxytryptamine and platelets: uptake and aggregation in hypoxic pulmonary hypertensive rats.

Pulmonary hypertension is associated with various alterations in 5-hydroxytryptamine (5-HT) physiology. In this study in platelets from hypoxic pulmonary hypertensive rats (10% O(2); 1 week) and normoxic rats (room air), (i) initial rates of specific [3H]5-HT uptake were measured and (ii) potentiation of collagen- and ADP-induced aggregation by 5-HT was quantified. The platelet count was almost halved in hypoxic rats. In uptake experiments, there was a decrease in 5-HT uptake in platelets from hypoxic compared with normoxic rats, due to a 36% reduction in the maximal initial rate of uptake. The aggregation experiments showed that 5-HT (1-100 microM) increased the magnitude of responses to collagen and the duration of responses to ADP, but there was no difference between hypoxic and normoxic rats. Abnormalities in platelet function may conceivably lead to increases in plasma 5-HT levels in hypoxic pulmonary hypertension, but are unlikely to aggravate pulmonary thromboembolism.

Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate.

1. Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-N-methyl-N-[6-(N-methylammoniohexyl)amino]diazen-1-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and to investigate the possible role of activation of sarco-endoplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2-10 micro g ml(-1)) and adenosine diphosphate (ADP; 2 micro M) in platelet rich plasma. It was (i). more effective at inhibiting aggregation induced by collagen than by ADP, and (ii). less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3. ODQ (10 micro M) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4. The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole; 1-100 micro M), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1-1 mM), caused minimal inhibition. 5. On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 micro M present) were abolished by thapsigargin (200 nM). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6. Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7. Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension.

OBJECTIVE: To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. METHODS: Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50-150 and 151-420 microm) were determined in Vegfb knockout mice (Vegfb(-/-); n=17) and corresponding wild-type mice (Vegfb(+/+); n=17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. RESULTS: In Vegfb(+/+) mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb(+/+) mice; P<0.001), (ii) right ventricular hypertrophy (a 66% increase in RV/[LV+S]; P<0.001) and (iii) pulmonary vascular remodelling (a 27-36% increase in pulmonary arterial medial thickness; P<0.05). In contrast, in Vegfb(-/-) mice hypoxia did not cause any increase in either right ventricular systolic pressure or pulmonary arterial medial thickness; also right ventricular hypertrophy (41% increase in RV/[LV+S]; P<0.001) was less pronounced (P<0.05) than in Vegfb(+/+) mice. CONCLUSION: Vegf-B may have a role in the development of chronic hypoxic pulmonary hypertension in mice by contributing to pulmonary vascular remodelling. If so, the effect of Vegf-B appears to be different from that of Vegf-A which is reported to protect against, rather than contribute to, hypoxia-induced pulmonary vascular remodelling.