Soluble epoxide hydrolase deficiency attenuates neointima formation in the femoral cuff model of hyperlipidemic mice.

OBJECTIVE: Epoxyeicosatrienoic acids (EETs) have antiinflammatory effects and are required for normal endothelial function. The soluble epoxide hydrolase (sEH) metabolizes EETs to their less active diols. We hypothesized that knockout and inhibition of sEH prevents neointima formation in hyperlipidemic ApoE(-/-) mice. METHODS AND RESULTS: Inhibition of sEH by 12-(3-adamantan-1-yl-ureido) dodecanoic acid or knockout of the enzyme significantly increased plasma EET levels. sEH activity was detectable in femoral and carotid arteries. sEH knockout or inhibition resulted in a significant reduction of neointima formation in the femoral artery cuff model but not following carotid artery ligation. Although macrophage infiltration occurred abundantly at the site of cuff placement in both sEH(+/+) and sEH(-/-), the expression of proinflammatory genes was significantly reduced in femoral arteries from sEH(-/-) mice. Moreover, an in vivo 5-bromo-2'-deoxyuridine assay revealed that smooth muscle cell proliferation at the site of cuff placement was attenuated in sEH knockout and sEH inhibitor-treated animals. CONCLUSION: These observations suggest that inhibition of sEH prevents vascular remodeling in an inflammatory model but not in a blood flow-dependent model of neointima formation.

Inhibition of the soluble epoxide hydrolase attenuates monocrotaline-induced pulmonary hypertension in rats.

OBJECTIVES: The soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to their less active dihydroxy derivatives. Because EETs have antiinflammatory properties, we determined whether or not inhibition of sEH attenuates disease development in the monocrotaline model of pulmonary hypertension in rats. METHODS: sEH inhibition was achieved using 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (25 mg/l) and cis- 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (5 mg/l) administered via drinking water starting 3 days prior to monocrotaline injection (60 mg/kg). RESULTS: Monocrotaline induced the development of progressive pulmonary hypertension. sEH inhibition increased the plasma ratio of EETs to DHETs and attenuated the monocrotaline-induced increase in pulmonary artery medial wall thickness as well as the degree of vascular muscularization. Moreover, right ventricular pressure was significantly lower in the group treated with sEH inhibitors. Pulmonary sEH protein expression and sEH activity, as well as pulmonary cytochrome P450 epoxygenase activity were all impaired in monocrotaline-treated rats as compared with control animals. sEH inhibitors, however, increased the plasma ratio of EETs to dihydroxy epoxyeicosatrienoic acids. Monocrotaline induced the proliferation of pulmonary endothelial and vascular smooth muscle cells in vivo as determined by 5-Bromo-2'-deoxy-Uridine incorporation, and this effect was significantly blunted in animals treated with sEH inhibitors. Proliferation of cultured pulmonary smooth muscle cell, however, was not affected by EETs or sEH inhibitors suggesting that the in-vivo effects are a consequence of a direct EET-mediated protection against the inflammation induced by monocrotaline. CONCLUSION: sEH inhibition reduces pulmonary vascular remodeling and the development of pulmonary hypertension in the monocrotaline model of primary pulmonary hypertension in rats.

Cecal ligation and incision: an acute onset model of severe sepsis in rats.

BACKGROUND: Sepsis is a leading cause of death among critically ill patients. Up to now, severe sepsis with acute onset in animals has been induced mainly through injection of single bacteria species or endotoxin and not through a surgical procedure, which might adequately mirror the situation in septic patients. We therefore aimed to establish a surgical model of severe sepsis in rodents fulfilling international sepsis criteria. MATERIALS AND METHODS: Twenty-eight anesthetized/ventilated Sprague Dawley rats underwent laparotomy and cecal mobilization. The cecum was either replaced into the abdomen (SHAM, n = 14) or the cecum and the mesenteric blood vessels were ligated, and the cecum was opened through a 1.5 cm blade incision (cecal ligation and incision, CLI, n = 14). RESULTS: Within 390 min, mortality was 0% (SHAM) and 50% (CLI), respectively. Compared with SHAM, CLI resulted in a 43% reduction of mean arterial blood pressure and in severe metabolic acidosis as measured by arterial base excess and pH. CLI led to a 15-fold increase in mononuclear cell population and to a 5-fold accumulation of nitrite in peritoneal lavage. Abdominal swabs from the Douglas cavity in CLI-animals showed gram-positive and gram-negative bacterial growth on agar compared with sterile swabs from SHAM-animals. In CLI-animals, plasma IL-1beta level was increased to 435 pg/mL (SHAM: 10 pg/mL) and plasma IL-6 level to 19718 pg/mL (SHAM: 832 pg/mL). CONCLUSIONS: CLI causes bacterial peritonitis with subsequent systemic inflammation and organ dysfunction. Thus, CLI mimics clinical sepsis and provides a surgical short term model of severe sepsis in rodents.

Protein kinase G I immunoreaction is colocalized with arginine-vasopressin immunoreaction in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) contains the primary mammalian circadian clock. This clock is entrained to environmental rhythms by external stimuli called zeitgebers. This entrainment is accomplished by the activation of specific, interacting signal transduction cascades. Since a cyclic guanosine monophosphate-dependent mechanism play a crucial functional role for light entrainment in the late night and for transmission of cholinergic stimuli, we examined the expression of protein kinase (PKG) in the rat SCN by means of qualitative and semi-quantitative immunocytochemistry. Immunoreaction (IR) for the isozyme protein kinase G I (PKG I) was found in the dorsomedial part of the SCN considered as an important relay in the output pathways of the clock. Within the SCN, PKG-I IR was colocalized with arginine-vasopressin-IR. The intensities of the PKG-I-IR did not vary between day and night.

Inhalation of the BK(Ca)-opener NS1619 attenuates right ventricular pressure and improves oxygenation in the rat monocrotaline model of pulmonary hypertension.

BACKGROUND: Right heart failure is a fatal consequence of chronic pulmonary hypertension (PH). The development of PH is characterized by increased proliferation of vascular cells, in particular pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells. In the course of PH, an escalated right ventricular (RV) afterload occurs, which leads to increased perioperative morbidity and mortality. BK(Ca) channels are ubiquitously expressed in vascular smooth muscle cells and their opening induces cell membrane hyperpolarization followed by vasodilation. Moreover, BK activation induces anti-proliferative effects in a multitude of cell types. On this basis, we hypothesized that treatment with the nebulized BK channel opener NS1619 might be a therapy option for pulmonary hypertension and tested this in rats. METHODS: (1) Rats received monocrotaline injection for PH induction. Twenty-four days later, rats were anesthetized and NS1619 or the solvent was administered by inhalation. Systemic hemodynamic parameters, RV hemodynamic parameters, and blood gas analyses were measured before as well as 30 and 120 minutes after inhalation. (2) Rat PASMCs were stimulated with PDGF-BB in the presence and absence of NS1619. AKT, ERK1 and ERK2 activation were investigated by western blot analyses, and relative cell number was determined 48 hours after stimulation. RESULTS: Inhalation of a 12 µM and 100 µM NS1619 solution significantly reduced RV pressure without affecting systemic arterial pressure. Blood gas analyses demonstrated significantly reduced carbon dioxide and improved oxygenation in NS1619-treated animals pointing towards a considerable pulmonary shunt-reducing effect. In PASMC's, NS1619 (100 µM) significantly attenuated PASMC proliferation by a pathway independent of AKT and ERK1/2 activation. CONCLUSION: NS1619 inhalation reduces RV pressure and improves oxygen supply and its application inhibits PASMC proliferation in vitro. Hence, BK opening might be a novel option for the treatment of pulmonary hypertension.

L-type calcium channel inhibitor diltiazem prevents aneurysm formation by blood pressure-independent anti-inflammatory effects.

Formation of abdominal aortic aneurysms is a progressive inflammatory process that involves infiltration and differentiation of monocytes in the vessel wall, proliferation and migration of smooth muscle cells, and eventually the degradation of the internal elastic lamina, which leads to outward vascular remodeling and distension of the vessel. Because calcium channel blockers exert multiple beneficial effects on the vascular system, we investigated the effect of the benzothiazepine-type calcium channel blocker diltiazem on aneurysm formation in a mouse model. Angiotensin II infusion induced massive suprarenal aortic aneurysm formation in male apolipoprotein E-deficient mice that was blocked by cotreatment with diltiazem even if the blood pressure was controlled by coinfusion of phenylephrine. Diltiazem prevented the angiotensin II-mediated induction of proinflammatory cytokines after 7 days of angiotensin II treatment in the aortic arch attributable to a reduction in the amount of locally infiltrating macrophages. To identify the underlying mechanism, vascular segments and cultured vascular cells as well as monocytes were studied. Diltiazem failed to reduce the angiotensin II-induced expression of proinflammatory chemokines and cytokines in isolated mouse thoracic aortic segments in organ culture. Furthermore, diltiazem did not affect the recruitment of proinflammatory Ly6C(+) monocytes in vivo pointing toward an effect of the compound on gene expression in monocytes/macrophages. Indeed, diltiazem prevented the interleukin-6-induced mRNA expression of interleukin-1ß and the monocyte chemoattractant protein CCL12 in peritoneal macrophages and RAW264.7 cells independent of the intracellular calcium concentration. Thus, diltiazem limits aortic aneurysm formation in mice by a blood pressure-independent anti-inflammatory effect on monocytic cells.

Pharmacological inhibition of the soluble epoxide hydrolase-from mouse to man.

Epoxyeicosatrienoic acids (EETs) build a family consisting of four arachidonic acid derived regioisomers that are generated by P450 epoxygenases. In the past years, growing interest in influencing EET level arose since EETs possess numerous beneficial effects in the cardiovascular system, for example, vasodilation, anti-inflammation and elicit renal and myocardial protection. Because EETs are primarily metabolized by the soluble epoxide hydrolase (sEH) and potent inhibitors of this enzyme are currently available, pharmacological sEH inhibition seems to be a feasible approach to elevate EET level in vivo. Hence, first clinical trials on sEH inhibition in man have begun. This review focuses on sEH inhibition as a novel pharmacological cardiovascular protective strategy with special regard to in vivo investigations.

Effects of intravenous and inhaled levosimendan in severe rodent sepsis.

PURPOSE: We aimed at comparing the effects of intravenous (i.v.) and inhaled (inh.) levosimendan (LEVO) on survival, inflammatory cytokines and the apoptotic mediator caspase-3 in a rat model of severe sepsis induced by cecal ligation and incision (CLI). METHODS: Twenty-eight anesthetized/ventilated male Sprague-Dawley rats (body weight 528 +/- 20 g) underwent laparotomy. Cecal mobilisation served as control (SHAM, n = 7). In all other groups, severe sepsis was induced by CLI. No further intervention occurred in the CLI-group (n = 7). 180 min after CLI, 24 microg/kg i.v. LEVO was administered in the CLI + LEVO-IV-group (n = 7), and 24 microg/kg inh. LEVO was administered via jet nebulizer in the CLI + LEVO-INH-group (n = 7). RESULTS: CLI induced arterial hypotension, with i.v. and inh. LEVO attenuating blood pressure decrease over 390 min [CLI 34(31/50), CLI + LEVO-IV 82(69/131)*, CLI + LEVO-INH 78(62/85)* mmHg; median(25/75% quartile), *P < 0.05]. CLI induced metabolic acidosis. I.v. and inh. LEVO avoided arterial pH [CLI 7.18(7.16/7.2), CLI + LEVO-IV 7.27(7.24/7.31)*, CLI + LEVO-INH 7.26(7.24/7.28)*] and base excess deterioration [CLI -19(-21.8/-17.9), CLI + LEVO-IV -13(-14.8/-12)*, CLI + LEVO-INH -12.7(-14/-12.2)* mmol/l]. Overall mortality in the CLI-group was 57% compared to 0%* in both LEVO-treated groups after 390 min. LEVO administration significantly attenuated the increase in proinflammatory interleukin (IL)-1beta [CLI 896(739/911), CLI + LEVO-IV 302(230/385)*, CLI + LEVO-INH 346(271/548) pg/ml] and IL-6 [CLI 35651(31413/35816), CLI + LEVO-IV 21156(18397/28026), CLI + LEVO-INH 13674(10105/24843) pg/ml] in the plasma and reduced cleaved caspase-3 expression in the spleen. CONCLUSIONS: In a rat model of severe sepsis induced by CLI, i.v. and inh. LEVO equally attenuated arterial hypotension, metabolic acidosis and prolonged survival. Moreover, i.v. and inh. LEVO inhibited proinflammatory mediator release and reduced splenic caspase-3 expression.