Margot C. LaPointe

A nitric oxide donor induces neurogenesis and reduces functional deficits after stroke in rats.

The adult rodent brain is capable of generating neuronal progenitor cells in the subventricular zone, and in the dentate gyrus of the hippocampus, throughout the life of the animal. Signals that regulate progenitor cell proliferation, differentiation, and migration are not well known. We report that administration of a nitric oxide donor, (Z)‐1‐[N‐(2‐aminoethyl)‐N‐(2‐ammonioethyl) aminio]diazen‐1‐ium‐1,2‐diolate (DETA/NONOate), to young adult rats significantly increases cell proliferation and migration in the subventricular zone and the dentate gyrus. Treatment with DETA/NONOate also increases neurogenesis in the dentate gyrus. Furthermore, administration of DETA/NONOate to rats subjected to embolic middle cerebral artery occlusion significantly increases cell proliferation and migration in the subventricular zone and the dentate gyrus, and these rats exhibit significant improvements of neurological outcome during recovery from ischemic stroke. Administration of DETA/NONOate significantly increases cortical levels of guanosine monophosphate both in ischemic and nonischemic rats, supporting the role of nitric oxide in promoting cell proliferation and neurogenesis. Thus, our data indicate that nitric oxide is involved in the regulation of progenitor cells and neurogenesis in the adult brain. This suggests that nitric oxide delivered to the brain well after stroke may have therapeutic benefits.

Interleukin-1β Regulation of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Involves the p42/44 and p38 MAPK Signaling Pathways in Cardiac Myocytes

The genes encoding inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2, also known as prostaglandin-endoperoxide synthase-2) are induced in many types of cells in response to proinflammatory cytokines. We have previously shown that interleukin-1beta (IL) stimulates iNOS and COX-2 mRNA in cardiac myocytes. Because IL has been shown to activate mitogen-activated protein kinase (MAPK) signaling pathways in many different cells, we tested whether the p42/44 and p38 MAPK pathways were involved in IL stimulation of iNOS and COX-2, using a specific inhibitor of p42/44 activation, PD98059 (PD), and the p38 inhibitor SB205380 (SB). Nitrites were measured using the Griess reagent, prostaglandin PGE2 by an enzyme immunoassay, iNOS and COX-2 protein by Western blot analysis, and iNOS mRNA by Northern blot analysis. Tested separately, the p38 kinase and MAPK inhibitors partially reduced IL stimulation of nitrite, iNOS protein, and iNOS mRNA; used together, they completely abolished the effect of IL. SB and PD inhibited IL-stimulated COX-2 protein by 60% and 80%, respectively, and IL-stimulated COX-2 protein was totally prevented by the combination of inhibitors. PGE2 production was inhibited more than 99% by either drug alone, suggesting a posttranslational effect on enzyme activity. To test whether this posttranslational effect involved the cytosolic phospholipase A2 (cPLA2) isoform, Western blots were probed for cPLA2 protein. Results indicated that IL stimulated cPLA2 activity and synthesis, which was inhibited by SB but not PD. These data indicate that (1) IL induction of iNOS synthesis depends on both the p42/44 and p38 signaling pathways, acting primarily at the level of transcriptional regulation; and (2) IL regulation of COX-2 synthesis involves the p42/44 and p38 signaling pathways, with an additional level of regulation occurring posttranslationally, perhaps at the level of activation of the cPLA2 isoform, which may be involved in intracellular signaling, as well as regulation of arachidonic acid release for COX-2 activity.

Tadalafil, a long-acting type 5 phosphodiesterase isoenzyme inhibitor, improves neurological functional recovery in a rat model of embolic stroke

Sildenafil, a type 5 phosphodiesterase isoenzyme (PDE5) inhibitor with a short half-life, increases brain cyclic guanosine monophosphate (cGMP) levels and improves neurological functional recovery when administered after stroke. In the present study, we investigated the effects of tadalafil (Cialis), a long acting PDE5 inhibitor, on brain cGMP levels, neurogenesis, angiogenesis, and neurological function during stroke recovery in a rat model of embolic stroke. Male Wistar rats (n=28) were subjected to embolic middle cerebral artery (MCA) occlusion. Tadalafil was orally administered every 48 h at a dose of 2 mg/kg or 10 mg/kg for 6 consecutive days starting 24 h after stroke onset. Control animals received the equivalent volume of saline at the same time points. For mitotic labeling, bromodeoxyuridine (BrdU, 100 mg/kg) was administered twice a day at 5, 6, and 7 days after stroke. ELISA assays were performed to evaluate the specificity of the effect of tadalafil on cGMP. Treatment with tadalafil at a dose of 2 or 10 mg/kg significantly improved neurological functional recovery compared with saline-treated rats. In addition, tadalafil treatment increased cerebral vascular density and the percentage of BrdU-positive endothelial cells around the ischemic boundary compared with saline-treated rats. Moreover, tadalafil-treated rats showed greater ipsilateral SVZ cell proliferation than saline-treated rats. However, treatment with tadalafil did not reduce infarct volume when compared to the saline group. Tadalafil selectively increased cGMP but not cyclic adenosine monophosphate (cAMP) in brain. Our data demonstrate that treatment of ischemic stroke with tadalafil improved functional recovery, which was associated with increases of brain cGMP levels and enhancement of angiogenesis and neurogenesis.

Molecular regulation of the brain natriuretic peptide gene

After brain natriuretic peptide (BNP) was isolated in 1988, rapid progress was made in cloning its cDNA and gene, facilitating studies of tissue-specific expression and molecular regulation of gene expression. This review focuses on the molecular determinants of regulation of the rat and human BNP genes, including signaling pathways that impact on changes in gene expression and cis regulatory elements responsive to these signaling pathways. For both rat and human genes, elements in the proximal promoter (-124 to -80), including GATA, MCAT, and AP-1-like, have been shown to contribute to basal and inducible regulation. More distal elements in the human BNP gene respond to calcium signals (an NF-AT site at -927), thyroid hormone (a thyroid-responsive element at -1000), and mechanical stretch (shear stress-responsive elements at -652 and -162). Understanding how BNP is regulated by signaling molecules that are activated in the hypertrophied and ischemic heart should be useful in understanding the underlying pathology. This may lead to therapeutic strategies that prevent hypertrophy while allowing for the beneficial effects of BNP production.

Angiotensin II-induced hypertrophy of adult rat cardiomyocytes is blocked by nitric oxide

The aim of the present study was to test the hypothesis that bradykinin-stimulated release of nitric oxide (NO) and/or prostacyclin from endothelium blocks myocyte hypertrophy in vitro. Angiotensin II increased [3H]phenylalanine incorporation by 21 ± 2% in myocytes cocultured with endothelial cells; this was abolished by bradykinin in the presence of endothelial cells. Bradykinin increased cytosolic concentrations of cGMP by 29 ± 4% in myocytes cocultured with endothelial cells. This was abolished by inhibition of NO synthase and by a cyclooxygenase inhibitor. Angiotensin II also increased [3H]phenylalanine incorporation by 28 ± 3% in myocytes cultured in the absence of endothelial cells. This effect of angiotensin II in monoculture was abolished by donors of NO but not by bradykinin. Neither the stable analog of prostacyclin (iloprost) nor the prostacyclin second messanger analog 8-bromo-cAMP (8-BrcAMP) blocked the effect of angiotensin II. Furthermore, 8-BrcAMP and iloprost individually increased [3H]phenylalanine incorporation. The antihypertrophic effects of bradykinin are critically dependent on endothelium-derived NO.The aim of the present study was to test the hypothesis that bradykinin-stimulated release of nitric oxide (NO) and/or prostacyclin from endothelium blocks myocyte hypertrophy in vitro. Angiotensin II increased [3H]phenylalanine incorporation by 21 +/- 2% in myocytes cocultured with endothelial cells; this was abolished by bradykinin in the presence of endothelial cells. Bradykinin increased cytosolic concentrations of cGMP by 29 +/- 4% in myocytes cocultured with endothelial cells. This was abolished by inhibition of NO synthase and by a cyclooxygenase inhibitor. Angiotensin II also increased [3H]phenylalanine incorporation by 28 +/- 3% in myocytes cultured in the absence of endothelial cells. This effect of angiotensin II in monoculture was abolished by donors of NO but not by bradykinin. Neither the stable analog of prostacyclin (iloprost) nor the prostacyclin second messanger analog 8-bromo-cAMP (8-BrcAMP) blocked the effect of angiotensin II. Furthermore, 8-BrcAMP and iloprost individually increased [3H]phenylalanine incorporation. The antihypertrophic effects of bradykinin are critically dependent on endothelium-derived NO.

Effect of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline on DNA and Collagen Synthesis in Rat Cardiac Fibroblasts

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell entry into the S phase of the cell cycle and is normally present in human plasma. Ac-SDKP is exclusively hydrolyzed by ACE, and its plasma concentration is increased 5-fold after ACE inhibition in humans. We examined the effect of 0.05 to 100 nmol/L Ac-SDKP on 24-hour 3H-thymidine incorporation (DNA synthesis) by cardiac fibroblasts both in the absence and presence of 5% FCS. Captopril (1 &mgr;mol/L) was added in all cases to prevent the degradation of Ac-SDKP. Treatment of cardiac fibroblasts with 5% FCS increased thymidine incorporation from a control value of 12 469±594 to 24 598±1051 cpm (P <0.001). Cotreatment with 1 nmol/L Ac-SDKP reduced stimulation to control levels (10 373±200 cpm, P <0.001). We measured hydroxyproline content and incorporation of 3H-proline into collagenous fibroblast proteins and found that Ac-SDKP blocked endothelin-1 (10−8 mol/L)–induced collagen synthesis in a biphasic and dose-dependent manner, causing inhibition at low doses, whereas high doses had little or no effect. It also blunted the activity of p44/p42 mitogen-activated protein kinase in a biphasic and dose-dependent manner in serum-stimulated fibroblasts, suggesting that the inhibitory effect of DNA and collagen synthesis may depend in part on blocking mitogen-activated protein kinase activity. Participation of p44/p42 in collagen synthesis was confirmed, because a specific inhibitor for p44/p42 activation (PD 98059, 25 &mgr;mol/L) was able to block endothelin-1–induced collagen synthesis, similar to the effect of Ac-SDKP. The fact that Ac-SDKP inhibits DNA and collagen synthesis in cardiac fibroblasts suggests that it may be an important endogenous regulator of fibroblast proliferation and collagen synthesis in the heart. Ac-SDKP may participate in the cardioprotective effect of ACE inhibitors by limiting fibroblast proliferation (and hence collagen production), and therefore it would reduce fibrosis in patients with hypertension.

Interleukin-1β Regulation of the Human Brain Natriuretic Peptide Promoter Involves Ras-, Rac-, and p38 Kinase–Dependent Pathways in Cardiac Myocytes

Because both the brain natriuretic peptide (BNP) gene and the cytokine interleukin-1beta (IL-1beta) are induced in the infarcted myocardium, localized production of IL-1beta may regulate the BNP gene. We tested whether (1) IL-1beta regulates the human BNP promoter, (2) cis elements in the proximal promoter respond to IL-1beta, and (3) mitogen-activated protein kinase (MAPK) signaling pathways [p42/44, c-jun (JNK) and p38 kinase] are involved. We transferred the hBNP promoter coupled to a luciferase reporter gene or constructs with mutations in the proximal promoter GATA and M-CAT elements into neonatal rat ventricular myocytes and treated the cells with IL-1beta for 24 hours. IL-1beta-stimulated hBNP luciferase activity was eliminated by pretreatment with the transcription inhibitor actinomycin D. Both the p38 kinase inhibitor SB205380 (SB) and cotransfection of a dominant-negative mutant of p38 kinase reduced IL-1beta stimulation of the hBNP promoter. Dominant-negative mutants of Ras and Rac inhibited IL-1beta-stimulated hBNP luciferase activity by 64% and 90%, respectively. Constitutively active forms of Rac and MKK6, the immediate upstream activator of p38, were stimulatory; however, only the effect of MKK6 was inhibited by SB. Neither the p42/44 nor the JNK pathway was involved in the action of IL-1beta. Both IL-1beta and MKK6 activation of the hBNP promoter were partially reduced when the promoter contained a mutated M-CAT element. In summary, (1) IL-1beta is a transcriptional activator of the hBNP promoter; (2) IL-1beta acts through a Ras-dependent pathway not coupled to activation of p42/44 MAPK or JNK; (3) IL-1beta acts through a Rac-dependent pathway, but the downstream effector is not known; and (4) IL-1beta activation of p38 kinase is partially involved in regulation of the hBNP promoter, targeting the proximal M-CAT element.

Reduction of myocardial infarct size by inhibition of inducible nitric oxide synthase.

The inducible nitric oxide synthase isoform (iNOS) is upregulated by cytokines and endotoxins in many types of cells, including cardiac myocytes. Nitric oxide (NO) induced by cytokines can be cytotoxic, and has been implicated in the pathophysiology of myocardial infarction, cardiomyopathy, and septic shock. To examine the role of iNOS in the ischemic myocardium, we studied: 1) the time course of expression of iNOS mRNA after myocardial infarction (MI) in male Sprague-Dawley rat hearts and expression of iNOS protein in the infarcted region; 2) whether hypoxia in vitro is a potential mediator of the induction of iNOS mRNA; and 3) whether inhibition of iNOS by two different selective inhibitors (aminoguanidine and S-methylisothiourea sulfate) in vivo influences infarct size. Myocardial infarction was induced by ligation of the left anterior descending coronary artery (LAD), and tissue was collected at selected times thereafter from both ligated and sham-operated rats. iNOS mRNA was induced in the infarcted region of the left ventricle for 7 days; iNOS protein was also detected in the infarcted area. We next tested whether hypoxia would induce iNOS in vitro. In cultured neonatal ventricular myocytes, iNOS mRNA was slightly induced by 6 to 24 h of hypoxia; however, iNOS protein was only detected when the cytokine interleukin-1beta was present. To study whether iNOS activity contributed to myocardial damage (eg, infarct size), we administered the first dose of the NOS inhibitors 24 h before LAD occlusion and then a second dose after surgery. Inhibition of iNOS activity with aminoguanidine reduced infarct size by 20% but had no effect on infiltration by neutrophils, whereas the more selective inhibitor S-methylisothiourea sulfate reduced infarct size by 41%. These data suggest that NO derived from the iNOS isoform contributes to some of the myocardial injury following MI, possibly by causing myocardial cell death in areas bordering the ischemic region of the heart.

PPARγ Inhibition of Cyclooxygenase-2, PGE2 Synthase, and Inducible Nitric Oxide Synthase in Cardiac Myocytes

Abstract—Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. They regulate lipid metabolism, glucose homeostasis, cell proliferation, and differentiation and modulate inflammatory responses. We examined whether PPAR&ggr; is functional in cultured neonatal ventricular myocytes and studied its role in inflammation. Western blots revealed PPAR&ggr; in myocytes. When myocytes were transfected with a PPAR response element reporter plasmid (PPRE-TK-luciferase), the PPAR&ggr; activator 15-deoxy-&Dgr;12,14-prostaglandin J2 (15dPGJ2) increased promoter activity, whereas cotransfection of a dominant negative PPAR&ggr; inhibited it. To determine the role of 15dPGJ2 in expression of proinflammatory genes, we tested its effect on interleukin-1&bgr; induction of cyclooxygenase-2 (COX-2). 15dPGJ2 decreased interleukin-1&bgr; stimulation of COX-2 by 40% and PGE2 production by 73%. We next questioned whether 15dPGJ2 was modulating the expression of inducible prostaglandin E2 synthase (PGES) and found that it completely blocked interleukin-1&bgr; induction of PGES. Use of a second PPAR&ggr; agonist, troglitazone, and the selective PPAR&ggr; antagonist GW9662 demonstrated that the effects seen were PPAR&ggr;-dependent. In addition, we found that 15dPGJ2 blocked interleukin-1&bgr; stimulation of inducible nitric oxide synthase (iNOS). We concluded that 15dPGJ2 may play an anti-inflammatory role in a PPAR&ggr;-dependent manner, decreasing COX-2, PGES, and PGE2 production, as well as iNOS expression.

Role of Ca2+-Independent Phospholipase A2 in the Regulation of Inducible Nitric Oxide Synthase in Cardiac Myocytes

We have previously shown that the regulation by interleukin-1beta (IL-1beta) of inducible nitric oxide synthase (iNOS) involves phospholipase A(2) (PLA(2)) metabolites in neonatal ventricular myocytes. Based on studies in which ONO-RS-082 is used to inhibit secretory PLA(2) and methyl arachidonyl fluorophosphonate is used to inhibit cytosolic PLA(2), our data suggest that a secretory PLA(2) metabolite was involved in the regulation by IL-1beta of iNOS. In addition, a third PLA(2) isoform, which is Ca(2+) independent (iPLA(2)), has also been detected in cardiac myocytes and shown to be regulated by cytokines. We tested whether iPLA(2) metabolites are involved in the regulation by IL-1beta of iNOS with the use of bromoenol lactone (BEL), a specific and irreversible inhibitor of iPLA(2). For this, we measured IL-1beta-stimulated nitrite (NOx) production with use of the Griess reagent, prostaglandin E(2) (PGE(2)) production with use of an enzyme immunoassay, and arachidonic acid release in the presence and absence of BEL. We also detected iNOS and iPLA(2) proteins by Western blotting. Treatment with IL-1beta (5 ng/mL) for 24 hours stimulated NOx production by 8-fold and iNOS protein levels by at least 10-fold. In addition, arachidonic acid release was increased by 1.6-fold and PGE(2) production was increased by 300-fold. When neonatal ventricular myocytes were treated with 10 micromol/L BEL, both IL-1beta-stimulated PGE(2) production and arachidonic acid release were inhibited. BEL inhibited IL-1beta-stimulated NOx production and iNOS protein by 88% and 93%, respectively. Lysophosphatidic acid, but not arachidonic acid or lysophosphatidylcholine, stimulated iNOS expression. Our results indicate that an iPLA(2) metabolite, perhaps lysophosphatidic acid, may be involved in the IL-1beta-signaling pathway, regulating the synthesis of iNOS.