James E. Clark

Interactions of Mast Cell Tryptase with Thrombin Receptors and PAR-2

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Cardioprotective Actions by a Water-Soluble Carbon Monoxide–Releasing Molecule

&NA; Carbon monoxide, which is generated in mammals during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator. Transition metal carbonyls have been recently shown to function as carbon monoxide‐releasing molecules (CO‐RMs) and to elicit distinct pharmacological activities in biological systems. In the present study, we report that a water‐soluble form of CO‐RM promotes cardioprotection in vitro and in vivo. Specifically, we found that tricarbonylchloro(glycinato)ruthenium(II) (CORM‐3) is stable in water at acidic pH but in physiological buffers rapidly liberates CO in solution. Cardiac cells pretreated with CORM‐3 (10 to 50 &mgr;mol/L) become more resistant to the damage caused by hypoxia‐reoxygenation and oxidative stress. In addition, isolated hearts reperfused in the presence of CORM‐3 (10 &mgr;mol/L) after an ischemic event displayed a significant recovery in myocardial performance and a marked and significant reduction in cardiac muscle damage and infarct size. The cardioprotective effects mediated by CORM‐3 in cardiac cells and isolated hearts were totally abolished by 5‐hydroxydecanoic acid, an inhibitor of mitochondrial ATP‐dependent potassium channels. Predictably, cardioprotection is lost when CORM‐3 is replaced by an inactive form (iCORM‐3) that is incapable of liberating CO. Using a model of cardiac allograft rejection in mice, we also found that treatment of recipients with CORM‐3 but not iCORM‐3 considerably prolonged the survival rate of transplanted hearts. These data corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO and highlight the bioactivity and potential therapeutic features of CO‐RMs in the mitigation of cardiac dysfunction. The full text of this article is available online at http://www.circresaha.org. (Circ Res. 2003;93:e2‐e8.)

Thiol Compounds Interact with Nitric Oxide in Regulating Heme Oxygenase-1 Induction in Endothelial Cells INVOLVEMENT OF SUPEROXIDE AND PEROXYNITRITE ANIONS

Thiols are very important antioxidants that protect cells against oxidative insults. Recently, a different and new physiological role has been defined for these compounds because of their involvement in nitric oxide (NO) binding and transport in biological systems. In view of these characteristics, we examined the effect of thiols and NO on the expression of the inducible form of heme oxygenase (HO-1), a stress protein that degrades heme to carbon monoxide and biliverdin. Cultured bovine aortic endothelial cells exposed to the NO donors sodium nitroprusside (SNP) andS-nitroso-N-acetylpenicillamine (SNAP) resulted in increased heme oxygenase activity and HO-1 expression. Co-incubation with N-acetylcysteine, a precursor of glutathione synthesis, significantly attenuated heme oxygenase induction by SNP and SNAP, and a reduction in heme oxygenase activity was also observed when cells were preincubated with N-acetylcysteine for 16 h prior to exposure to NO donors. This effect appears to be associated with NO stabilization by thiols through the formation ofS-nitrosothiols. Hydroxocobalamin, a specific NO scavenger, significantly decreased endothelial heme oxygenase activity, indicating a direct involvement of NO released by NO donors to regulate the expression of this stress protein. Moreover, superoxide anion (O·̄2) and its reaction product with NO, peroxynitrite (ONOO−), were found to partially contribute to the observed NO-mediated activation of endothelial heme oxygenase. Thus, we suggest the existence of a dynamic equilibrium among free NO, O·̄2, and endogenous glutathione, which might constitute an interactive signaling mechanism modulating stress and adaptive responses in tissues.

Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1

1 The contribution of haeme oxygenase‐derived carbon monoxide (CO) to the regulation of vascular tone in thoracic aorta was investigated following induction of the inducible isoform of haeme oxygenase (HO‐1). 2 Isometric smooth muscle contractions were recorded in isolated rat aortic ring preparations. Rings were incubated in the presence of the nitric oxide (NO) donor S‐nitroso‐N‐acetyl penicillamine (SNAP, 500 μm) for 1 h, then repetitively washed and maintained for a further 4 h prior to producing a concentration‐response curve to phenylephrine (PE, 1–3000 nm). 3 Treatment with SNAP resulted in increased mRNA and protein expression of aortic HO‐1 and was associated with a significant suppression of the contractile response to PE (P<0.05 vs control). Immunohistochemical staining procedures revealed marked HO‐1 expression in the endothelial layer and, to a lesser extent, in smooth muscle cells. 4 Induction of HO‐1 in SNAP‐treated rings was associated with a higher 14CO release compared to control, as measured by scintillation counting after incubation of aortas with [2‐14C]‐L‐glycine, the precursor of haeme. Guanosine 3′,5′‐monophosphate (cyclic GMP) content was also greatly enhanced in aortas expressing high levels of HO‐1. 5 Incubation of aortic rings with the NO synthase inhibitor, NG‐monomethyl‐L‐arginine (100 μm), significantly (P<0.05) increased the contractile response to PE in controls but failed to restore PE‐mediated contractility in SNAP‐treated rings. In contrast, the selective inhibitor of haeme oxygenase, tin protoporphyrin IX (SnPP‐IX, 10 μm), restored the pressor response to PE in SNAP‐treated rings whilst markedly reducing CO and cyclic GMP production. 6 We conclude that up‐regulation of the HO‐1/CO pathway significantly contributes to the suppression of aortic contractility to PE. This effect appears to be mediated by the elevation of cyclic GMP levels and can be reversed by inhibition of the haeme oxygenase pathway.

Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule

Carbon monoxide (CO), one of the end products of heme catabolism by heme oxygenase, possesses antihypertensive and vasodilatory characteristics. We have recently discovered that certain transition metal carbonyls are capable of releasing CO in biological fluids and modulate physiological functions via the delivery of CO. Because the initial compounds identified were not water soluble, we have synthesized new CO‐releasing molecules that are chemically modified to allow solubility in water. The aim of this study was to assess the vasoactive properties of tricarbonylchloro(glycinato)ruthenium(II) (CORM‐3) in vitro and in vivo. CORM‐3 produced a concentration‐dependent relaxation in vessels precontracted with phenylephrine, exerting significant vasodilatation starting at concentrations of 25–50 μM. Inactive CORM‐3, which does not release CO, did not affect vascular tone. Blockers of ATP‐dependent potassium channels (glibenclamide) or guanylate cyclase activity (ODQ) considerably reduced CORM‐3‐dependent relaxation, confirming that potassium channels activation and cGMP partly mediate the vasoactive properties of CO. In fact, increased levels of cGMP were detected in aortas following CORM‐3 stimulation. The in vitro and in vivo vasorelaxant activities of CORM‐3 were further enhanced in the presence of YC‐1, a benzylindazole derivative which is known to sensitize guanylate cyclase to activation by CO. Interestingly, inhibiting nitric oxide production or removing the endothelium significantly decreased vasodilatation by CORM‐3, suggesting that factors produced by the endothelium influence CORM‐3 vascular activities. These results, together with our previous findings on the cardioprotective functions of CORM‐3, indicate that this molecule is an excellent prototype of water‐soluble CO carriers for studying the pharmacological and biological features of CO.

Bioactivity and Pharmacological Actions of Carbon Monoxide-Releasing Molecules

Carbon monoxide (CO) is a resourceful gas as recent advances in the area of cell signaling are revealing an unexpected physiological role for CO in the cardiovascular, immune and nervous systems. Transition metal carbonyls have been lately discovered to function as CO-releasing molecules (CO-RMs) and elicit distinct pharmacological activities in biological systems. Studies currently ongoing in our laboratories are investigating both the chemical and bioactive features of a series of water-soluble CO-RMs and their specific utilization as vasoactive mediators, anti-inflammatory agents and inhibitors of cellular and tissue damage. The data presented in this review corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO in mammals and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cellular and organ dysfunction.

Peroxynitrite induces haem oxygenase-1 in vascular endothelial cells: a link to apoptosis.

Peroxynitrite (ONOO-) is a potent oxidizing agent generated by the interaction of nitric oxide (NO) and the superoxide anion. In physiological solution, ONOO- rapidly decomposes to a hydroxyl radical, one of the most reactive free radicals, and nitrogen dioxide, another species able to cause oxidative damage. In the present study we investigated the effect of ONOO- on the expression of haem oxygenase-1 (HO-1), an inducible protein that is highly up-regulated by oxidative stress. Exposure of bovine aortic endothelial cells to ONOO- (250-1000 microM) produced a concentration-dependent increase in haem oxygenase activity and HO-1 protein expression. This effect was completely abolished by the ONOO- scavengers uric acid and N-acetylcysteine, and partly attenuated by 1,3-dimethyl-2-thiourea, a scavenger of hydroxyl radicals. ONOO- also produced a concentration-dependent increase in apoptosis and cytotoxicity, which were considerably decreased by uric acid and N-acetylcysteine. A 70% decrease in apoptosis was observed when cells were exposed to ONOO- in the presence of 10 microM tin protoporphyrin IX (SnPPIX), an inhibitor of haem oxygenase activity. When SnPPIX was added 5 min after ONOO-, apoptosis decreased by only 40%, which suggests that an interaction between ONOO- and the protoporphyrin occurs in our system. Increased haem oxygenase activity by pretreatment of cells with haemin resulted in elevated bilirubin production and was associated with a substantial decrease (35%) in ONOO--mediated apoptosis. These results indicate the ability of ONOO- to modulate the expression of the stress protein HO-1 and suggest that the haem oxygenase pathway contributes to protection against the cytotoxic action of ONOO-.

Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2–deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti–MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

Glycogen Synthase Kinase-3 Inactivation Is Not Required for Ischemic Preconditioning or Postconditioning in the Mouse

The inactivation of glycogen synthase kinase-3β (GSK-3β) is proposed as the event integrating protective pathways initiated by preconditioning and other interventions. The inactivation of GSK-3 is thought to decrease the probability of opening of the mitochondrial permeability transition pore. The aim of this study was to verify the role of GSK-3 using a targeted mouse line lacking the critical N-terminal serine within GSK-3β (Ser9) and the highly homologous GSK-3α (Ser21), which when phosphorylated results in kinase inactivation. Postconditioning with 10 cycles of 5 seconds of reperfusion/5 seconds of ischemia and preconditioning with 6 cycles of 4 minutes of ischemia/6 minutes of reperfusion, similarly reduced infarction of the isolated perfused mouse heart in response to 30 minutes of global ischemia and 120 minutes of reperfusion. Preconditioning caused noticeable inactivating phosphorylation of GSK-3. However, both preconditioning and postconditioning still protected hearts of homozygous GSK-3 double knockin mice. Moreover, direct pharmacological inhibition of GSK-3 catalytic activity with structurally diverse inhibitors before or after ischemia failed to recapitulate conditioning protection. Nonetheless, cyclosporin A, a direct mitochondrial permeability transition pore inhibitor, reduced infarction in hearts from both wild-type and homozygous GSK-3 double knockin mice. Furthermore, in adult cardiac myocytes from GSK-3 double knockin mice, insulin exposure was still as effective as cyclosporin A in delaying mitochondrial permeability transition pore opening. Our results, which include a novel genetic approach, suggest that the inhibition of GSK-3 is unlikely to be the key determinant of cardioprotective signaling in either preconditioning or postconditioning in the mouse.

Thymosin β4 facilitates epicardial neovascularization of the injured adult heart

Ischemic heart disease complicated by coronary artery occlusion causes myocardial infarction (MI), which is the major cause of morbidity and mortality in humans (http://www.who.int/cardiovascular_diseases/resources/atlas/en/index.html). After MI the human heart has an impaired capacity to regenerate and, despite the high prevalence of cardiovascular disease worldwide, there is currently only limited insight into how to stimulate repair of the injured adult heart from its component parts. Efficient cardiac regeneration requires the replacement of lost cardiomyocytes, formation of new coronary blood vessels, and appropriate modulation of inflammation to prevent maladaptive remodeling, fibrosis/scarring, and consequent cardiac dysfunction. Here we show that thymosin β4 (Tβ4) promotes new vasculature in both the intact and injured mammalian heart. We demonstrate that limited EPDC‐derived endothelial‐restricted neovascularization constitutes suboptimal “endogenous repair,” following injury, which is significantly augmented by Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage.