Effects of selective TNFR1 inhibition or TNFR2 stimulation, compared to non-selective TNF inhibition, on (neuro)inflammation and behavior after myocardial infarction in male mice.

BACKGROUND: Myocardial infarction (MI) coinciding with depression worsens prognosis. Although Tumor Necrosis Factor alpha (TNF) is recognized to play a role in both conditions, the therapeutic potential of TNF inhibition is disappointing. TNF activates two receptors, TNFR1 and TNFR2, associated with opposite effects. Therefore, anti-inflammatory treatment with specific TNF receptor interference was compared to non-specific TNF inhibition regarding effects on heart, (neuro)inflammation, brain and behavior in mice with MI. METHODS: Male C57BL/6 mice were subjected to MI or sham surgery. One hour later, MI mice were randomized to either non-specific TNF inhibition by Enbrel, specific TNFR1 antagonist-, or specific TNFR2 agonist treatment until the end of the protocol. Control sham and MI mice received saline. Behavioral evaluation was obtained day 10-14 after surgery. Eighteen days post-surgery, cardiac function was measured and mice were sacrificed. Blood and tissue samples were collected for analyses of (neuro)inflammation. RESULTS: MI mice displayed left ventricular dysfunction, without heart failure, (neuro) inflammation or depressive-like behavior. Both receptor-specific interventions, but not Enbrel, doubled early post-MI mortality. TNFR2 agonist treatment improved left ventricular function and caused hyper-ramification of microglia, with no effect on depressive-like behavior. In contrast, TNFR1 antagonist treatment was associated with enhanced (neuro)inflammation: more plasma eosinophils and monocytes; increased plasma Lcn2 and hippocampal microglia and astrocyte activation. Moreover, increased baseline heart rate, with reduced beta-adrenergic responsiveness indicated sympathetic activation, and coincided with reduced exploratory behavior in the open field. Enbrel did not affect neuroinflammation nor behavior. CONCLUSION: Early receptor interventions, but not non-specific TNF inhibition, increased mortality. Apart from this undesired effect, the general beneficial profile after TNFR2 stimulation, rather than the unfavourable effects of TNFR1 inhibition, would render TNFR2 stimulation preferable over non-specific TNF inhibition in MI with comorbid depression. However, follow-up studies regarding optimal timing and dosing are needed.

The Wnt/Frizzled pathway as a therapeutic target for cardiac hypertrophy: where do we stand?

Cardiac hypertrophy is an enlargement of the heart muscle in response to wall stress. This hypertrophic response often leads to heart failure. In recent years, several studies have shown the involvement of Wnt signalling in hypertrophic growth. In this review, the role of Wnt signalling and the possibilities for therapeutic interventions are discussed. In healthy adult heart tissue, Wnt signalling is very low. However, under pathological condition such as hypertension, Wnt signalling is activated. In recent years, it has become clear that both ß-catenin-dependent signalling and ß-catenin-independent signalling are involved in hypertrophic growth. Several studies, both in vitro and in vivo, have shown that genetic interventions in Wnt signalling at different levels resulted in an attenuated or diminished hypertrophic response. Therefore, inhibition of Wnt signalling could provide a new therapeutic strategy for cardiac hypertrophy, but further research on the Wnts and Frizzleds involved in the different forms of cardiac hypertrophy will be needed to achieve this goal.

Dynamics of cardiac wound healing following myocardial infarction: observations in genetically altered mice.

Recent improvements in the clinical management of acute myocardial infarction (MI) have resulted in a dramatic decrease in mortality because of this condition. This implies that more patients enter the process of infarct healing. This is a highly complex cascade of events which, although studied for decades, is still not completely understood. An increasing number of genetically altered mice can now be studied in a mouse model of MI, to investigate the contribution of the product of the targeted gene to the infarct healing process. In this review, we will discuss the defects in infarct healing that have been observed in null mutants for plasminogen, urokinase-type plasminogen activator (u-PA), matrix metalloproteinases (MMPS), thrombospondin-2 and dishevelled-1. These studies provide new insights in the infarct healing process itself, but may also help to define new diagnostic and therapeutic targets in humans suffering from MI.

Overexpression of components of the Frizzled-Dishevelled cascade results in apoptotic cell death, mediated by beta-catenin.

Frizzled (fz) functions as a 7-transmembrane receptor in the Frizzled-Dishevelled signal transduction cascade. It is involved in architectural control of development in species as divergent as Drosophila and vertebrates. Regulation of multicellular architecture requires control of cell alignment, but also involves an equilibrium among cell proliferation, differentiation, and apoptosis. Recently, modulation of the Frizzled-Dishevelled (Dvl) cascade has been related to apoptosis. However, the role of beta-catenin, a second messenger in the Frizzled-Dishevelled cascade, in programmed cell death is a matter of debate. To elucidate the role of this cascade in apoptosis, we studied the effect of overexpression of fz1, fz2, dvl1, and beta-catenin. The signal transduction pathway and the involvement of beta-catenin were further investigated by using different inhibitors. These experiments were performed in different cell types: COS7, 293, and PC12. Overexpression of fz1, fz2, and dvl1 induced apoptosis in COS7 and 293 cells. beta-Catenin appears to be the mediator for this process since beta-catenin overexpression as well as lithium and valproate induced apoptosis. In contrast, lithium treatment did not result in apoptosis in PC12 cells. We conclude that different components of the Frizzled-Dishevelled cascade can induce apoptosis, but that this effect is dependent on the cell type.

Frizzled 2 is transiently expressed in neural crest-containing areas during development of the heart and great arteries in the mouse.

Frizzled 2 acts as a 7-transmembrane receptor in the Wnt-Dishevelled signal transduction cascade. Among others, this cascade has been associated with neural crest cell proliferation and early migration during development in mammals. The genes for some components of this cascade are located in chromosomal regions that are deleted in human syndromes associated with neural crest cell defects, like DiGeorge and Velo-Cardio-Facial Syndrome. These syndromes are often accompanied by abnormalities in cardiac morphology. Furthermore, we have reported in previous studies the upregulation of the tissue polarity gene frizzled 2 in myofibroblasts during their migration into the necrotic area after myocardial infarction in the adult heart. It is known that genes that are upregulated during cardiac remodeling due to pathology often play a role during development. To investigate whether frizzled 2 can be associated with the process of cardiac morphogenesis we studied its expression in the thoracic arterial system and heart of mouse embryo's of 10, 12, 14, 16 and 18 days after conception by means of in situ hybridization. At day 10 after conception signal could be found in the pharyngeal arches and arch arteries. The outflow tract, the ascending aorta and the pulmonary trunk were positive for frizzled 2 from day 12 on. This expression decreased with time and at day 18 only some signal could be detected in the aorta and pulmonary trunk. In contrast, in coronary and pulmonary arteries no expression was observed at any time point. Minor myocardial expression was observed in the ventricular septum at days 12 and 14. Atrial expression, although considerably lower than ventricular expression, could be detected somewhat later at days 14 and 16. Our results indicate that there is transient expression of frizzled 2 in areas that are invested by neural crest cells. This expression is downregulated upon neural crest cell differentiation. The frizzled 2 expression supports a role for the Wnt-frizzled pathway in neural crest-related disorders.

Beta-catenin, an inducer of uncontrolled cell proliferation and migration in malignancies, is localized in the cytoplasm of vascular endothelium during neovascularization after myocardial infarction.

Beta-catenin is a protein involved in cell-cell adhesion and proliferation. In neoplastic diseases, defects in the regulation of the cellular beta-catenin content and cytoplasmic accumulation of the protein contribute to the uncontrolled cell proliferation and migration. Whether beta-catenin plays a role in the controlled proliferative and migratory responses to injury, eg, of vascular endothelial cells during neovascularization after myocardial infarction (MI), is not known. In the present study, we examined the localization of beta-catenin in the infarcted rat heart at different time points after MI. Cytoplasmic beta-catenin was observed in the endothelial cells of the newly formed and pre-existing blood vessels in the infarct area in the first week after MI, but not in the uninjured parts of the heart and not at later time points. Adenomatous polyposis coli (APC) protein was also detected; interaction of APC with beta-catenin has been reported to be critical in epithelial tube formation in vitro. Moreover, the expression of dishevelled-1, an upstream regulatory molecule of the cellular beta-catenin content, was observed in vascular endothelial cells in the infarct area. These findings suggest a role for the beta-catenin-APC complex in the proliferation and migration of vascular endothelial cells during neovascularization of the infarct area.

A homologue of Drosophila tissue polarity gene frizzled is expressed in migrating myofibroblasts in the infarcted rat heart.

Myocardial infarction results in the formation of granulation tissue in the injured ventricular wall. This tissue contains myofibroblasts in highly organized arrays; their contractile properties may help to prevent the infarct area from dilatation. The mechanisms that control myofibroblast alignment are unknown. We found that myofibroblasts express a homologue of Drosophila tissue polarity gene frizzled (fz2) when migrating into the granulation tissue. The expression is decreased after the cells have aligned. This suggests that fz2 is involved in the spatial control of cardiac wound repair after infarction, possibly through intra- and intercellular transmission of polarity signals as in developing Drosophila. Mutations in the fz2 gene may impair myofibroblast alignment in the infarct area, thereby resulting in ventricular dilatation and aneurism following infarction.

Increased expression of a homologue of drosophila tissue polarity gene "frizzled" in left ventricular hypertrophy in the rat, as identified by subtractive hybridization.

The molecular mechanisms that govern the development of left ventricular hypertrophy are not fully elucidated. We performed a subtractive hybridization procedure to identify genes controlling this adaptive process. Using this approach, we isolated a rat homologue of Drosophila tissue polarity gene "frizzled" 2 (fz-2). The expression of this gene was quantified by competitive reverse transcriptase polymerase chain reactions. The expression was higher in hypertrophic left ventricles at all time points tested, reaching statistical significance at days 1 and 10. We conclude that the fz-2 gene, a highly conserved gene for which a role in intra- and intercellular communication has been described, may be involved in the spatial control of ventricular remodeling.

beta-Adrenergic relaxation in mesenteric resistance arteries of spontaneously hypertensive and Wistar-Kyoto rats: the role of precontraction and intracellular Ca2+.

An attenuated beta-adrenergic vasodilation of small arteries may help explain the increased peripheral resistance in hypertension. To investigate this, we compared the isoprenaline-induced relaxation of mesenteric resistance arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) using a small vessel myograph. The arteries had similar diameters, but the contractile force induced by cumulative addition of K+ (10-130 mM) was 1.3-fold higher for the SHR. The beta-adrenoceptor-mediated relaxation of arteries, precontracted with 40 mM K+, was significantly less in SHR (41 +/- 3%, n = 11) than in WKY (56 +/- 3%, n = 15, p = 0.003), and the pD2 value for isoprenaline was significantly lower in SHR (7.13 +/- 0.09 vs. 7.41 +/- 0.07, p = 0.02). In contrast, when precontracted with phenylephrine (PE, alpha 1-adrenoceptor agonist, 3-10 microM), isoprenaline relaxation was almost complete in both SHR and WKY, and the pD2 value for isoprenaline did not differ between strains. Forskolin induced complete relaxation of both precontractions. Because the beta-adrenergic relaxation of the mesenteric resistance arteries was attenuated only after K(+)-precontraction, we conclude that alterations in this precontracting mechanism in SHR rather than a defect in the beta-adrenoceptor system may provide an explanation for the decreased relaxation in these vessels. Intracellular Ca2+ measurements and a review of the literature support this conclusion.

Forskolin-stimulated adenylyl cyclase activity is decreased but beta 2-adrenoceptor function is unchanged in primary hypertension.

beta 2-Adrenoceptor function may be decreased in primary hypertension, resulting in increased peripheral resistance. To study the beta 2-adrenoceptor function, we used circulating mononuclear leukocytes (MNL) as a model system. Twenty untreated hypertensive subjects [(HT) 10 men and 10 women] and 20 age- and sex-matched healthy normotensive (NT) volunteers were studied. The beta 2-adrenoceptor density was not significantly different between HT and NT, but the dissociation constants for the high- and low-affinity agonist binding states, studied by isoprenaline competition assays, were significantly higher in HT. Stimulation of adenylyl cyclase with isoprenaline (10 microM, beta 2-adrenoceptor-mediated stimulation) was not significantly different between the two groups. Forskolin-mediated direct stimulation of adenylyl cyclase was significantly higher in women than in men. For both sexes, the forskolin-induced cyclic AMP production was lower in the HT group, reaching statistical significance in the men. No major abnormalities were observed in beta 2-adrenoceptor function in mononuclear leukocytes. The putative relation between the decreased forskolin-mediated adenylyl cyclase activity and primary hypertension requires further study.

Adrenoceptors on blood cells in patients with primary hypertension: correlation with blood pressure and related variables.

OBJECTIVE: Alterations in platelet alpha 2-adrenoceptor and mononuclear leucocyte beta 2-adrenoceptor characteristics in primary hypertension have been extensively studied. The results of the reports have not been consistent, possibly because of the small number of subjects in most of the studies. We therefore studied the blood-cell adrenoceptor characteristics in a relatively large group of primary hypertensive and normotensive subjects. DESIGN: Platelet alpha 2-adrenoceptor characteristics were compared in 65 hypertensive and 51 normotensive subjects. Mononuclear leucocyte beta 2-adrenoceptor characteristics were compared in 72 hypertensives and 67 normotensives. Untreated hypertensive subjects were selected from the outpatient clinic and the normotensive controls were recruited by a newspaper announcement. METHODS: Platelets and mononuclear leucocytes were isolated from blood samples obtained after at least 10 min supine rest. The alpha 2- and beta 2-adrenoceptor characteristics were determined with [3H]-rauwolscine and [125I]-(-1)cyanopindolol, respectively. Correlations between the adrenoceptor characteristics and clinical parameters of the subjects were studied. RESULTS: No differences in alpha 2- or beta 2-adrenoceptor densities were observed between the two groups. However, a significantly lower equilibrium dissociation constant for [3H]-rauwolscine was observed in the hypertensive group. The correlations between the adrenoceptor characteristics and clinical parameters were weak and mostly not statistically significant. The results were compared with the most relevant studies in the literature. CONCLUSIONS: From our study and the literature, we conclude that blood-cell adrenoceptor characteristics are unchanged in primary hypertension.

Effect of NG-monomethyl-L-arginine on the beta-adrenoceptor-mediated relaxation of rat mesenteric resistance arteries.

beta-Adrenoceptors are present on vascular smooth muscle and on endothelium. We investigated whether the endothelial beta-adrenoceptors induce relaxation of rat mesenteric resistance arteries by stimulation of endothelium-derived relaxing factor (EDRF) release. To this end, the relaxation was studied in the presence and absence of 100 microM NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of the production of EDRF. The maximal relaxation with isoprenaline, expressed as a percentage of the precontraction, was 44.0 +/- 4.0% (n = 12) in the L-NMMA treated group and 58.0 +/- 2.6% (n = 13) in the untreated group, a statistically significant difference (P = 0.008). However, the precontraction with 40 mM K+ tended to be higher in the presence of L-NMMA. The pD2-value for isoprenaline was not significantly changed by the L-NMMA treatment. We conclude that the isoprenaline-mediated relaxation of mesenteric resistance arteries is inhibited by L-NMMA, but that this effect can at least in part be ascribed to an inhibition of baseline EDRF-release.

Characterization of muscarinic receptors in rat kidney.

Muscarinic receptors in mammalian kidney seem to be involved in diuresis. In this study we give a detailed characterization of receptors in rat kidney. Specific binding of [3H](-)-quinuclidinylbenzilate ([3H]QNB) to membranes of rat kidney cortex was saturable and of high affinity. A dissociation constant of 0.063 +/- 0.003 nM and a receptor density of 1.46 +/- 0.07 pmol/g wet weight were obtained. The dissociation kinetics could be best described by assuming a mono-exponential function (k-1 = (0.52 +/- 0.1) x 10(-4) s-1). The binding of [3H]QNB reached a maximum in 60 min at 0.6 nM at 37 degrees C. Competition experiments with the enantiomers of benzetimide confirmed the muscarinic nature of the [3H]QNB binding sites. The inhibition constants of pirenzepine (0.23 +/- 0.02 microM), (+-)-hexahydrosiladifenidol (0.040 +/- 0.002 microM), AF-DX 116 (1.45 +/- 0.07 microM), methoctramine (1.67 +/- 0.02 microM) and gallamine (78 +/- 3 microM) classified this receptor as an M3 receptor. Inhibition of [3H]QNB binding by the agonists methylfurtrethonium, arecoline, isoarecoline methiodide, arecaidine propargyl ester and McN-A-343 displayed monophasic inhibition curves. With (+/-)-cis-2-methyl-4-dimethylaminomethyl-1,3- dioxolane methiodide in two out of four experiments a small (11%) population of high affinity agonist sites could be detected. The potassium sparing diuretic amiloride inhibited [3H]QNB binding (36 +/- 3 microM). Although in a way related to the amiloride binding site, the muscarinic receptors in rat kidney are unlikely to be the primary target of diuretic action of this drug.

Does treatment with beta-adrenergic blocking agents cause a decrease in beta 2-adrenoceptor affinity?

The effect of beta-adrenoceptor antagonists (BAAs) differing in lipophilicity and partial agonist activity (PAA), and a full agonist, on the dissociation constant for [125I]-(-)- iodocyanopindolol binding to beta 2-adrenoceptors (KD) has been investigated. Twelve healthy, normotensive male volunteers (mean age 22.3 y) were treated with different BAAs according to a cross-over design. The drugs used were propranolol (highly lipophilic BAA, no PAA), pindolol (moderately lipophilic BAA, strong PAA), dilevalol (highly lipophilic BAA, weak PAA) and salbutamol (full agonist). Before and after a single dose and an 8 day course of one of the drugs, blood pressure and the beta 2-adrenoceptor characteristics of mononuclear leukocytes (MNL) were determined. Between the treatment periods, there was a washout interval of 14 days. All BAAs decreased the blood pressure, but only propranolol lowered heart rate. Treatment with salbutamol decreased the diastolic and increased the systolic blood pressure and heart rate. Three hours after the single dose of any of the BAAs, a more than 2-fold increase in KD was observed, and the increase became larger after 8 days of administration (up to 3.7-fold increase). In contrast, no effect on KD was observed after treatment with salbutamol. BAAs with PAA and salbutamol induced a 30% decrease in beta 2-adrenoceptor density. It is concluded that treatment with BAAs, irrespective their lipophilicity or PAA, induces a decrease in the affinity of MNL beta 2-adrenoceptors for antagonists. This phenomenon may help to explain the contradictory relationship between the kinetics and dynamics of BAAs.

Lambda-crystallin, a major rabbit lens protein, is related to hydroxyacyl-coenzyme A dehydrogenases.

It has recently been discovered that several lens proteins in birds and lower vertebrates are active enzymes or enzyme-related proteins (Wistow, G., Mulders, J. W. M., and de Jong, W. W. (1987) Nature 326, 622-624; Wistow, G., and Piatigorsky, J. (1987) Science 236, 1554-1556). We report here a novel lens protein, designated as lambda-crystallin, that occurs in rabbit and hare. It constitutes 7-8% of the total lens protein and has a subunit molecular mass of 35 kDa. Sequencing of cDNA clones encoding rabbit lambda-crystallin revealed 30% homology (at the amino acid sequence level) with L-3-hydroxyacyl-CoA dehydrogenase from pig mitochondria and 26% homology with enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase from rat peroxisomes. Also, the presence of a putative beta-alpha-beta nucleotide-binding fold and low levels of non-lens expression are indicative of some enzymatic function for lambda-crystallin (or highly related sequences) in non-lens tissues. lambda-Crystallin thus represents the first example of an enzyme-related crystallin in lenses from mammalian species. The recruitment of enzymes as lens structural proteins apparently is an evolutionary strategy which has been applied independently in different lineages.

Autoradiographic visualization of muscarinic receptors in human bronchi.

To visualize muscarinic receptors in human bronchi, the stripping film method was used which permits direct autoradiographic localization of tissue labeling. Cryostate sections of human bronchi were fixed in 0.5% glutaraldehyde in Krebs-Ringer buffer, pH 7.0 for 30 min at 0 degrees C, washed in Krebs-Ringer buffer for 20 min at 0 degrees C and incubated with (-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] for 90 min at 37 degrees C. Specific (-)-[3H]QNB binding to tissue sections was saturable (receptor density of 0.14 +/- 0.03 fmol/tissue section) and of high affinity (Kd of 40 +/- 9 pM). For autoradiography, labeled tissue sections were covered with stripping film and exposed for 5 months. Muscarinic receptors in human bronchi were located predominantly in submucosal glands and parasympathetic ganglia. There was less labeling in smooth muscle cells and nerve bundles. Epithelium and blood vessels located within the bronchial wall were devoid of specific labeling.

Autoradiographic visualization of muscarinic receptors in pulmonary nerves and ganglia.

We investigated autoradiographically the distribution of muscarinic receptors in bovine airways using (-)-[3H]quinuclidinyl benzilate as radioligand. The autoradiographs demonstrated the presence of muscarinic receptors in smooth muscle as well as neuronal muscarinic receptors in pulmonary nerves and ganglia. It is reasonable to believe that the neuronal muscarinic receptors participate in the regulation of neurotransmitter release at the peripheral nerve terminals innervating the bronchial smooth muscle.