Sex-related survival differences in murine cardiomyopathy are associated with differences in TNF-receptor expression.

Epidemiological evidence suggests that the prognosis of heart failure in women is better than in men. In our murine model of dilated cardiomyopathy arising from cardiac-specific overexpression of TNF-alpha, the 6-month survival rate was significantly better in females than in males. Young female transgenic mice exhibited left ventricular wall thickening without dilatation, whereas age-matched male transgenic hearts were markedly dilated. Basal and isoproterenol-stimulated fractional shortening was preserved in female transgenic mice, but not in male transgenic mice. Myocardial expression of proinflammatory cytokines and the extent of myocardial infiltrates were similar in male and female transgenic mice. Myocardial expression of TNF-receptor mRNAs (type I and type II) was significantly higher in male mice in both transgenic and wild-type littermates, whereas sex-specific differences were not observed in either peripheral white blood cells or liver tissue. After TNF-alpha challenge, myocardial but not liver production of ceramide was significantly higher in male than in female mice. Thus, differential expression of myocardial TNF receptors may contribute to sex differences in the severity of congestive heart failure and mortality consequent to cardiac-specific overexpression of TNF-alpha.

Chronic treatment with interleukin-1 beta induces coronary intimal lesions and vasospastic responses in pigs in vivo. The role of platelet-derived growth factor.

Studies in vitro have suggested that inflammatory cytokines may play an important role in the pathogenesis of atherosclerosis. However, little is known about their effects in vivo. Thus, the present study was designed to determine in vivo what histological and functional changes may be induced by chronic treatment with IL-1 beta, one of the major inflammatory cytokines, and also to clarify what mechanisms are involved in those changes. Under aseptic conditions, proximal segments of the left porcine coronary arteries were gently wrapped with cotton mesh absorbing Sepharose beads either with or without recombinant human IL-1 beta. From 1 to 4 wk after the operation, coronary vasospastic responses to intracoronary serotonin or histamine were noted at the IL-1 beta-treated site but not at the control site. Histologically, intimal thickening was greater at the IL-1 beta-treated site than at the control site. Those functional and histological changes induced by the chronic treatment with IL-1 beta were significantly inhibited by the simultaneous treatment with a neutralizing antibody to either IL-1 beta or PDGF. These results indicate that chronic treatment with Il-1 beta induces coronary intimal lesions and vasospastic responses in porcine coronary arteries in vivo and also suggest that these changes are substantially mediated by PDGF.

Tyrosine kinase inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses induced by chronic treatment with interleukin-1 beta in pigs in vivo.

We recently demonstrated that chronic treatment with IL-1 beta induces coronary arteriosclerotic changes and vasospastic responses to autacoids in pigs in vivo and that those responses are importantly mediated by PDGF. The receptors for PDGF and other major growth factors are known to have tyrosine kinase activity. We therefore investigated the effects of a selective tyrosine kinase inhibitor, ST 638, on those responses induced by IL-1 beta in our swine model. Intimal thickening and coronary vasospastic responses to serotonin and histamine were induced at the site of the coronary artery where IL-1 beta was chronically and locally applied. These responses were significantly suppressed in a dose-dependent manner by cotreatment with ST 638. In addition, ST 494, which is an inactive form of ST 638, did not inhibit those responses. The treatment with ST 638 alone did not affect the coronary vasoconstricting responses to the autacoids. Immunoblotting using an antibody to phosphotyrosines confirmed the inhibitory effects of ST 638 on the tyrosine phosphorylations induced by IL-1 beta. These results thus suggest that tyrosine kinase activation may play an important role in mediating the effects of IL-1 beta, while also suggesting that ST 638 has an inhibitory effect on the arteriosclerotic changes and vasospastic responses to autacoids in our swine model in vivo.

Anti-tumor necrosis factor-alpha antibody limits heart failure in a transgenic model.

BACKGROUND: - Tumor necrosis factor (TNF)-alpha has been implicated in the pathophysiology of congestive heart failure. A strain of transgenic mice (TNF1.6) with cardiac-specific overexpression of TNF-alpha develop congestive heart failure. METHODS AND RESULTS: To determine the effect of anti-TNF-alpha therapy in this model, we studied 3 groups: TNF1.6 mice treated with saline, wild-type mice treated with saline, and TNF1.6 mice treated with TNF-alpha neutralizing antibody (cV1q) from 6 to 12 weeks of age. We used echocardiography to compare cardiac hypertrophy, function, and catecholamine response at 12 weeks of age versus baseline (6 weeks). cV1q treatment did not limit cardiac hypertrophy, but it significantly improved basal fractional shortening and responsiveness to beta-adrenergic stimulation, and it limited development of cardiac dilation. CONCLUSIONS: Blockade of TNF-alpha bioactivity by antibody therapy may both preserve cardiac function and partially reverse pathological changes in congestive heart failure.

Soluble tumor necrosis factor receptor abrogates myocardial inflammation but not hypertrophy in cytokine-induced cardiomyopathy.

BACKGROUND: Transgenic mice with cardiac-specific overexpression of tumor necrosis factor (TNF)-alpha develop dilated cardiomyopathy. The present study was designed to evaluate therapeutic effects of adenovirus-mediated neutralization of TNF-alpha on this model. METHODS AND RESULTS: An adenovirus encoding the 55-kDa TNF receptor-IgG fusion protein (AdTNFRI) was injected intravenously into 6-week-old transgenic mice, which resulted in high levels of TNFRI in both plasma and myocardium. AdTNFRI did not reverse cardiomegaly but abrogated myocardial inflammation. Furthermore, AdTNFRI blocked the myocardial expression of intercellular adhesion molecule-1 and downstream cytokines, including interleukin-1beta and monocyte chemotactic protein-1. Downregulation of alpha-myosin heavy chain was restored by the treatment, whereas upregulation of beta-myosin heavy chain was not reversed. In contrast, the downregulation of sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban was normalized by AdTNFRI. Echocardiographic measurements showed that left ventricular end-systolic diameter was significantly larger in transgenic mice than in control mice, and this increase was reversed by the AdTNFRI treatment. However, left ventricular wall thickening was not reversed. CONCLUSIONS: These results suggest that anti-TNF therapy may hold promise in the treatment of end-stage heart failure.

Tyrosine kinase inhibitor suppresses the (re) stenotic changes of the coronary artery after balloon injury in pigs.

OBJECTIVE: Restenosis after percutaneous transluminal coronary angioplasty (PTCA) still remains a serious late complication. Many growth factors induced in restenotic lesions may be responsible for restenosis after PTCA. Most of the receptors for such growth factors possess tyrosine kinase activity. This study was designed to determine whether or not a specific tyrosine kinase inhibitor, ST 638, can prevent (re)stenotic changes of the coronary artery after balloon injury. METHODS: A segment of the porcine coronary artery was aseptically wrapped with cotton mesh absorbing either ST 638 or vehicle, followed by balloon injury. Two weeks after the procedure, coronary stenosis and vasoconstricting responses were examined by coronary arteriography and (re)stenotic changes of the coronary artery were histologically examined. Antiphosphotyrosine immunoblotting was also performed to examine the inhibitory effects of ST 638. RESULTS: Coronary arteriography showed the development of mild stenotic lesions at the balloon-injured sites, where hyperconstrictive responses were repeatedly induced by intracoronary serotonin and histamine. Histologically, neointimal formation was noted at the balloon-injured site, where the total vessel area also tended to decrease (geometric remodeling). The treatment with ST 638 suppressed all the hyperconstrictive responses, the neointimal formation, and the geometric remodeling induced by balloon injury. Immunoblotting for phosphotyrosine proteins demonstrated the elevation of proteins at the balloon-injured site, which was suppressed by ST 638. CONCLUSIONS: These results indicate that tyrosine kinases are activated at the balloon-injured site and the inhibition of such kinase activities is effective in reducing both the (re)stenotic changes (neointimal formation and geometric remodeling) and the hyperconstrictive responses of the coronary artery after balloon injury.

The role of fibroblast growth factor-2 in the vascular effects of interleukin-1 beta in porcine coronary arteries in vivo.

OBJECTIVE: We recently demonstrated that chronic treatment with interleukin-1 beta (IL-1 beta), a major inflammatory cytokine found in atherosclerotic lesions, induces coronary arteriosclerotic changes and vasospastic responses to serotonin and histamine in pigs in vivo and that those responses are partially mediated by platelet-derived growth factor (PDGF). This study was designed to examine, first, whether the effects of IL-1 beta are also partially mediated by fibroblast growth factor-2 (FGF-2), which is another important growth factor in atherosclerotic lesions, and, secondly, whether chronic treatment with FGF-2 per se also induces histological and functional changes in porcine coronary arteries in vivo. METHODS: Porcine coronary arteries were aseptically wrapped with cotton mesh absorbing IL-1 beta with or without neutralizing antibody to FGF-2. In a separate series of experiments porcine coronary arteries were chronically treated with FGF-2 itself in the same manner. Coronary vascular responses in vivo and histological changes were examined 2 weeks after the operation. RESULTS: Coronary vasospastic responses to serotonin and histamine and neointimal formation were induced at the site of the coronary artery where IL-1 beta was chronically and locally applied. These responses were significantly suppressed by co-treatment with a neutralizing antibody to FGF-2 but not by that with non-immune IgG. Immunostaining revealed the presence of FGF-2 in the endothelial cells, the thickened intima and the media at the IL-1 beta-treated site. Furthermore, chronic treatment with FGF-2 also induced coronary vasospastic responses to serotonin and histamine and neointimal formation. CONCLUSIONS: These results suggest that the vascular effects of IL-1 beta may also be mediated by FGF-2 in our swine model and that chronic treatment with FGF-2 also causes coronary arteriosclerotic changes and vasospastic responses in vivo.

Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats.

The aim of the present study was to investigate the effects of long-term blockade of nitric oxide synthesis with the L-arginine analogue N omega-nitro-L-arginine methyl ester (L-NAME) for 8 weeks on coronary vascular and myocardial structural changes. Four groups of Wistar-Kyoto rats were studied: those with no treatment, those treated with L-NAME 1 g/L (3.7 mmol/L in drinking water), those treated with L-NAME 0.1 g/L (0.37 mmol/L in drinking water), and those treated with L-NAME 1.0 g/L and hydralazine 120 mg/L (0.6 mmol/L in drinking water). After 8 weeks, the heart was excised, and the degrees of structural changes in coronary arteries (wall-to-lumen ratio and perivascular fibrosis), myocardial fibrosis, and myocyte size were quantified by an image analyzer. Chronic inhibition of nitric oxide synthesis increased arterial pressure compared with control animals. Chronic inhibition of nitric oxide synthesis caused significant microvascular remodeling (increased wall-to-lumen ratio and perivascular fibrosis). Cardiac hypertrophy was also observed after chronic inhibition of nitric oxide synthesis. Coadministration of hydralazine prevented arterial hypertension but did not affect microvascular remodeling and cardiac hypertrophy induced by the chronic inhibition of nitric oxide synthesis. In addition, chronic inhibition of nitric oxide synthesis caused scattered lesions of myocardial fibrosis, which was significantly attenuated by cotreatment with hydralazine. These results suggest that long-term blockade of nitric oxide synthesis caused coronary microvascular remodeling and cardiac hypertrophy in rats in vivo by a mechanism other than arterial hypertension. In contrast, arterial hypertension contributed to the development of myocardial fibrosis induced by long-term blockade of nitric oxide synthesis.

Central role of vascular smooth muscle hyperreactivity in coronary hyperconstriction after balloon injury in miniature pigs.

BACKGROUND: Coronary constrictive responses to autacoids become augmented 1 week after balloon injury in our swine model. The present study aimed to elucidate the mechanisms of this effect. METHODS: In 12 hypercholesterolaemic miniature pigs, the coronary constrictive response to serotonin was examined angiographically 1 week after injury. After the angiographic study, organ chamber experiments using excised coronary artery were performed to clarify whether functional changes in endothelial cells or in vascular smooth muscle cells contributed to the hyperconstriction. RESULTS: The coronary constrictive response to serotonin in vivo was significantly greater at the previously injured site than at the non-injured site. The degree of the hyperconstriction at the previously injured site exceeded that predicted from a geometric theory. Histological examination demonstrated that the previously injured site was almost covered with regenerated endothelial cells. In vitro studies demonstrated that serotonin caused significantly greater contraction in coronary artery strips, whether with or without endothelium, from the previously injured site than in those from the non-injured site. Endothelium-dependent relaxation in response to serotonin was comparable at the injured and non-injured sites. CONCLUSIONS: These results suggest that the coronary hyperconstriction response to serotonin 1 week after injury results primarily from hyperreactivity of vascular smooth muscle. Whereas any contribution of endothelial dysfunction or the geometric effect may be minimal.

Platelet activating factor causes hyperconstriction at the inflammatory coronary lesions in pigs in vivo.

BACKGROUND: Although platelet activating factor (PAF) is an important vasoactive substance released from activated leukocytes, platelets and endothelial cells, little is known about its effect at the inflammatory coronary lesions in vivo. OBJECTIVE: To examine the coronary vasomotor responses to PAF at the inflammatory lesions in our swine model with interleukin-1 beta (IL-1 beta) in vivo. METHODS: Under aseptic conditions, the proximal segment of the porcine left coronary artery was dissected and wrapped with cotton mesh absorbing IL-1 beta. Two weeks after the operation, coronary vasomotion in response to intracoronary administration of 0.3 and 1 microgram/kg PAF, 1, 3, and 10 micrograms/kg serotonin, 1, 3 and 10 micrograms/kg histamine, and 5 and 50 micrograms/kg prostaglandin F2 alpha was examined by coronary arteriography. RESULTS: At the IL-1 beta-treated site, PAF, serotonin and histamine, but not prostaglandin F2 alpha, caused hyperconstriction (n = 8). A synergy of the vasoconstricting effects of PAF and serotonin was also noted (n = 6). Administration of TCV-309, a selective PAF receptor antagonist, abolished the hyperconstrictive responses to PAF but not those to other agonists (n = 6). The PAF-induced coronary hyperconstrictions were significantly inhibited by administrations of the protein kinase C inhibitors staurosporine and sphingosine, but not by administrations of ryanodine, thapsigargin, or indomethacin (n = 4 each). CONCLUSIONS: These results indicate that PAF causes hyperconstriction at the inflammatory coronary lesions in vivo by itself as well as in a synergistic manner with serotonin and that the PAF-induced hyperconstrictions are substantially mediated by a protein kinase C-dependent pathway in vivo.

Coronary artery spasm does not depend on the intracellular calcium store but is substantially mediated by the protein kinase C-mediated pathway in a swine model with interleukin-1 beta in vivo.

BACKGROUND: The intracellular mechanism for coronary artery spasm is still unknown. Since the protein kinase C (PKC)-mediated pathway and Ca2+ release from sarcoplasmic reticulum (SR) are important intracellular mechanisms of vascular smooth muscle contraction, we examined the possible role of these two mechanisms in the pathogenesis of coronary spasm in our swine model in vivo. METHODS AND RESULTS: In 25 pigs, interleukin-1 beta (IL-1 beta) was applied chronically to the coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, either intracoronary serotonin or histamine repeatedly induced coronary spasm at the IL-1 beta-treated site. At those spastic sites, phorbol-12, 13-dibutyrate, a PKC-activating phorbol ester, also induced coronary spasm, which was blocked by pretreatment with the PKC inhibitors staurosporine and sphingosine. Serotonin- and histamine-induced coronary spasm was also significantly inhibited by pretreatment with staurosporine, sphingosine, or nifedipine (an L-type Ca2+ channel antagonist) but not by ryanodine (an inhibitor of Ca(2+)-induced Ca2+ release from SR) or thapsigargin (an inhibitor of Ca(2+)-ATPase of SR). Bay K 8644 (an L-type Ca2+ channel agonist) also induced coronary spasm at the IL-1 beta-treated site, which was significantly inhibited by pretreatment with staurosporine, sphingosine, and nifedipine. In contrast, coronary vasoconstriction induced by prostaglandin F2 alpha was not affected by pretreatment with staurosporine or sphingosine but was significantly inhibited by pretreatment with ryanodine, thapsigargin, or nifedipine. CONCLUSIONS: These results suggest that (1) PKC activation largely accounts for the serotonin- and histamine-induced coronary spasm; (2) at the spastic site, the calcium influx through L-type Ca2+ channels may be augmented via the PKC-mediated pathway; and (3) the Ca2+ release from the SR into the cytosol may not play a primary role in coronary spasm.

Disappearance of coronary artery-ventricular fistulas after a radical operation for tetralogy of Fallot.

A 42-year-old Japanese man was diagnosed as tetralogy of Fallot (TOF). Coronary arteriography demonstrated coronary artery-ventricular fistulas (left coronary artery to left ventricle, right coronary artery to right ventricle). A radical operation for TOF was performed, including a patch closure of the ventricular septal defect, right ventricular muscle resection and a patch enlargement of the right ventricular outflow tract. After the operation, coronary arteriography showed a marked decrease in the shunt flow through the coronary artery-ventricular fistulas. Six months later, the patient was diagnosed as postoperative constrictive pericarditis, while the fistulas had almost completely disappeared. After pericardiectomy, when all hemodynamic variables were normal, the fistulas did not reappear. This is the first report of TOF complicated with multiple coronary artery-ventricular fistulas which completely disappeared after a radical operation. It is conceivable that in our case coronary artery-ventricular fistulas partially compensated for reduced arterial O2 saturation and disappeared after surgical correction with an improvement in hemodynamic variables.

Tyrosine kinase inhibitor markedly suppresses the development of coronary lesions induced by long-term treatment with platelet-derived growth factor in pigs in vivo.

Platelet-derived growth factor (PDGF) plays an important role in the development of coronary atherosclerosis. However, it remains to be examined what morphologic and functional changes are induced in vivo by the long-term treatment with PDGF itself or what pharmacologic interventions could suppress those changes in vivo. Our study was designed to address these points. We examined the effects of long-term treatment with PDGF on the porcine coronary artery in vivo. Under aseptic conditions, the proximal segments of the left porcine coronary artery were gently wrapped with cotton mesh absorbing sepharose beads either with or without recombinant human PDGF-AA or -BB. Two weeks after the operation, coronary hyperconstrictions to intracoronary serotonin or histamine were noted at the sites treated with PDGF-AA or -BB. Histologically, neointimal formation and geometric remodeling (reduction of the total vessel area) were noted at the PDGF-treated sites. These functional and histologic changes of the coronary artery induced by PDGF were markedly inhibited by cotreatment with ST 638, a specific inhibitor of tyrosine kinases. A Western blot analysis showed that ST 638 markedly suppressed the PDGF-induced tyrosine phosphorylations in the coronary segment. These results indicate that long-term treatment with PDGF induces neointimal formation, geometric remodeling, and vasospastic responses in vivo, for all of which, activation of tyrosine kinases is substantially involved.

Altered serotonin receptor subtypes mediate coronary microvascular hyperreactivity in pigs with chronic inhibition of nitric oxide synthesis.

BACKGROUND: We previously reported that chronic inhibition of nitric oxide synthesis by administration of N omega-nitro-L-arginine methyl ester (L-NAME) causes microvascular hyperreactivity to 5-hydroxytryptamine (5-HT) and vascular structural changes in pigs in vivo. In the present study, we investigated the relative contributions of 5-HT receptor subtypes to microvascular hyper-reactivity in this animal model. METHODS AND RESULTS: Coronary vasomotor response was studied in 16 pigs treated with oral L-NAME for 4 weeks (L group) and in 11 control pigs (C group). Intracoronary administration of 5-HT at 30 micrograms/kg decreased coronary blood flow (CBF) in the two groups. The decrease in CBF by 5-HT was greater (P < .01) in the L group than in the C group. The decrease in CBF by 5-HT in the C group was blocked completely by pretreatment with ketanserin, a 5-HT2 antagonist. In contrast, the augmented decrease in CBF by 5-HT in the L group was only partly inhibited by ketanserin alone and was blocked completely by ketanserin and methiothepin, a 5-HT1/5-HT2 antagonist. The decrease in CBF caused by prostaglandin F2 alpha and the increase in CBF caused by nitroglycerin were comparable between the two groups and were not affected by the 5-HT antagonists. CONCLUSIONS: These results suggest that the 5-HT-induced microvascular hyperreactivity may be mediated by relative changes in affinity for 5-HT receptors or de novo expression of 5-HT1 receptors in microvascular smooth muscle cells in our animal model.

Inflammatory cytokines cause coronary arteriosclerosis-like changes and alterations in the smooth-muscle phenotypes in pigs.

We recently developed a porcine model in which chronic, local treatment with interleukin-1 beta (IL-1 beta) causes coronary arteriosclerosis-like changes and hyperconstrictive responses. This study was designed to examine whether or not other major inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) might also cause similar coronary responses and whether those responses are associated with alterations in the smooth-muscle phenotypes. A segment of the porcine coronary artery was aseptically wrapped with cotton mesh, absorbing IL-1 beta, TNF-alpha, and IL-1 alpha. Two weeks after the operation, coronary arteriography showed the development of mild stenotic lesions at the cytokine-treated sites, where hyperconstrictive responses were repeatedly induced by intracoronary serotonin or histamine. Histologically mild intimal thickening was noted at those cytokine-treated sites. Immunostaining and immunoblotting demonstrated that all three myosin heavy chain isoforms, SM1, SM2 (smooth-muscle type), and SMemb (nonmuscle type), were noted in the normal coronary segments, whereas in the segments treated with inflammatory cytokines, SM1 and SM2 were markedly reduced, and only SMemb was noted. These results indicate that inflammatory cytokines all have a similar ability to induce coronary arteriosclerosis-like changes and hyperconstrictive responses, which are associated with alterations in smooth-muscle phenotypes toward dedifferentiation.

Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy.

Myocardial fibrosis caused by maladaptive extracellular matrix (ECM) remodeling is implicated in the dysfunction of the failing heart. Matrix metalloproteinases (MMPs) regulate ECM remodeling, and are regulated by cytokines. Transgenic mice with cardiac-specific overexpression of tumor necrosis factor alpha (TNF-alpha) (TNF1.6) develop heart failure. We hypothesized that modulation of TNF-alpha and/or MMP activity might alter the myocardial ECM remodeling process and the development of heart failure. To test this hypothesis, we took advantage of the TNF1.6 mice and studied soluble and total collagens and collagen type profiling by using hydroxyproline quantification, Sircol collagen assay, Northern blot analysis, and immunohistochemistry and studied myocardial function by using echocardiography. Progressive ventricular hypertrophy and dilation in the TNF1.6 mice were accompanied by a significant increase in MMP-2 and MMP-9 activity, an increase in collagen synthesis, deposition, and denaturation, and a decrease in undenatured collagens. In young TNF1.6 mice, these changes in the ECM were associated with marked diastolic dysfunction as demonstrated by significantly reduced transmitral Doppler echocardiographic E/A wave ratio. Anti-TNF-alpha treatment with adenoviral vector expressing soluble TNF-alpha receptor type I attenuated both MMP-2 and MMP-9 activity, prevented further collagen synthesis, deposition and denaturation, and preserved myocardial diastolic function in young, but not old, TNF1.6 mice. The results suggest a critical role of TNF-alpha and MMPs in myocardial matrix remodeling and functional regulation and support the hypothesis that both TNF-alpha and MMPs may serve as potential therapeutic targets in the treatment of heart failure.

Overexpression of tumor necrosis factor- alpha activates both anti- and pro-apoptotic pathways in the myocardium.

We have previously reported that mice with cardiac-specific overexpression of tumor necrosis factor (TNF)- alpha develop myocardial inflammation, cardiac hypertrophy, and dilated cardiomyopathy. TNF- alpha is reported to induce apoptosis in cultured cardiac myocytes. To investigate the role of apoptosis in this transgenic model, wild-type controls (WT) and transgenic mice (TG) at the age of 1, 8, and 40 weeks were analyzed. Increased incidence of apoptosis in TG was indicated by DNA laddering. TUNEL assays revealed that the frequencies of apoptotic cells were increased in the TG myocardium at all ages. However, as revealed by histochemical and immunofluorescent methods, most of the apoptotic cells appeared to be non-myocytes even in the mice with overt congestive heart failure. To elucidate the signaling pathways responsible for TNF- alpha induced apoptosis, expression of apoptosis-related genes were evaluated by multi-probe RNase protection assays. Transcripts for death-domain-related proteins, including TNFR1, Fas, FADD, TRADD, and RIP, were constitutively expressed in WT and upregulated in the TG myocardium. Expression of caspase-1 through -8 was also enhanced in TG. While both anti- and pro-apoptotic Bcl-2 family genes were constitutively expressed in WT, TNF- alpha overexpression strongly induced anti-apoptotic A1 in the myocardium. Furthermore, TNF- alpha overexpression activated NF- kappa B, a mediator of anti-apoptotic pathways, in the myocardium. Thus, overexpression of TNF- alpha activated both anti- and pro-apoptotic pathways in the myocardium, resulting in an increase of apoptosis, primarily in non-myocytes. These results suggest that TNF- alpha by itself is not sufficient to induce apoptosis in cardiac myocytes in vivo.

Effects of soluble TNF receptor treatment on lipopolysaccharide-induced myocardial cytokine expression.

Tumor necrosis factor (TNF)-alpha plays a key role in the pathogenesis of septic shock syndrome, and myocardial TNF-alpha expression may contribute to this pathophysiology. We examined the myocardial expression of TNF-alpha-related cytokines and chemokines in mice exposed to lipopolysaccharide (LPS) and tested the effects of anti-TNF therapy on myocardial cytokine expression. Cytokine mRNA levels were measured by RNase protection assay, and protein levels in the plasma and myocardium were assessed by enzyme-linked immunosorbent assays. LPS (4 microg/g body wt ip) induced marked cytokine expression, including TNF-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein (MCP)-1, in both the plasma and myocardium. Pretreatment with adenovirus-mediated TNF receptor fusion protein (AdTNFR1; 10(9) plaque-forming units iv) decreased plasma cytokine levels. In contrast, whereas myocardial IL-1beta expression was also suppressed, expression of IL-6 and MCP-1 was not inhibited by AdTNFR1. In summary, anti-TNF treatment differentially altered the cytokine expression in the plasma and myocardium during endotoxemia. Inability to block myocardial expression of IL-6 and MCP-1 suggests a possible mechanism for the failure of anti-TNF therapies in the treatment of endotoxin shock.

Chronic inhibition of endothelium-derived nitric oxide synthesis causes coronary microvascular structural changes and hyperreactivity to serotonin in pigs.

BACKGROUND: Endothelium-derived nitric oxide (NO) is believed to regulate myocardial perfusion and structural changes in the vascular wall. Our objective was to determine whether chronic inhibition of NO synthesis causes structural and functional changes in coronary arteries. METHODS AND RESULTS: Coronary vasomotor response was studied in pigs before and after chronic oral administration of the NO synthesis antagonist N omega-nitro-L-arginine methyl ester (L-NAME) 30 mg.kg-1.d-1 for 2 weeks. Chronic L-NAME treatment increased (P < .01) arterial pressure but did not alter baseline coronary blood flow (CBF), epicardial coronary diameter, or heart rate. Chronic L-NAME treatment augmented (P < .01) the decrease in CBF in response to intracoronary serotonin (30 micrograms/kg) from 5 +/- 14% to 40 +/- 5% but did not alter the CBF response to prostaglandin F2 alpha. The serotonin-induced decrease in CBF after acute L-NAME administration was still less before (1.3 +/- 0.4%) than after chronic L-NAME treatment (51 +/- 6%). Chronic L-NAME treatment attenuated the increase in CBF with bradykinin (100 ng/kg) but did not alter the CBF response to nitroglycerin (10 micrograms/kg). Compared with intact pigs without L-NAME treatment, L-NAME-treated pigs had significant thickening of the media in the microvessels (diameter, < 300 microns) but not in the large epicardial vessels. Chronic intracoronary infusion of L-NAME at 3 mg.kg-1.d-1 for 2 weeks, which did not produce arterial hypertension, caused similar microvascular medial thickening. CONCLUSIONS: These results indicate that chronic administration of L-NAME caused coronary microvascular structural changes and hyperreactivity to serotonin in pigs in vivo, suggesting an important role of defective NO synthesis in coronary microvascular disorders.

Vasculoprotective role of inducible nitric oxide synthase at inflammatory coronary lesions induced by chronic treatment with interleukin-1beta in pigs in vivo.

BACKGROUND: We recently developed a porcine model in which chronic, local treatment with interleukin-1beta (IL-1beta) causes coronary arteriosclerotic changes and hyperconstrictive responses. Inflammatory cytokines are known to induce inducible NO synthase (iNOS) in the vascular smooth muscle. This study was designed to examine whether or not the production of NO by iNOS has a protective or deleterious effect on the coronary artery in vivo. METHODS AND RESULTS: A segment of the porcine coronary artery was aseptically wrapped with cotton mesh absorbing IL-1beta suspension. We inhibited both eNOS and iNOS activity by cotreatment with L-NAME (a nonspecific inhibitor of NOS) and iNOS activity alone by aminoguanidine (a selective inhibitor of iNOS). Immunostaining showed that iNOS was absent in the normal coronary artery, whereas it was highly expressed 1 day after the application of IL-1beta and thereafter downregulated until 14 days. In contrast, eNOS was well maintained throughout the study period. Two weeks after the operation, hyperconstrictive responses to intracoronary serotonin and neointimal formation were noted at the IL-1beta-treated site, and both responses were significantly greater at the site cotreated with either L-NAME or aminoguanidine. CONCLUSIONS: These results indicate that iNOS is transiently induced in vivo in response to local inflammation and that NO produced by iNOS exerts an inhibitory effect against the cytokine-induced proliferative/vasospastic changes of the coronary artery in vivo.