In vitro assessment of clevidipine using the profilin1 hypertensive mouse model.

Hypertension represents a major risk factor for cardiovascular events, associating with vascular hypertrophy and dysfunction in resistance vessels. Clevidipine is a novel antihypertensive drug working as a selective calcium channel antagonist with an ultra-short half-life that lowers arterial blood pressure by reducing systemic arterial resistance. The aim was to assess the effect of clevidipine on the hypertrophic vessels of profilin1 hypertensive transgenic mice compared to sodium nitroprusside (SNP) and labetalol using wire myograph techniques. The effects of clevidipine, SNP and labetalol on the hypertrophic vessels were studied on mesenteric arterial function from 8 profilin1 hypertrophic mice and eight non-transgenic controls. Our results showed a significant difference between the effects of the three drugs on the hypertrophic mesenteric arteries of transgenic profilin1 mice compared to the non-transgenic controls. The half maximal effective concentration (EC50) of clevidipine, SNP and labetalol in profilin1 mice (1.90 ± 0.05, 0.97 ± 0.07, 2.80 ± 0.05 nM, respectively) were significantly higher than the EC50 in non-transgenic controls (0.91 ± 0.06, 0.32 ± 0.06, 0.80 ± 0.09 nM, respectively). Moreover, the increase in the EC50 for clevidipine (2-fold) to produce the same effect on both normal and hypertrophic arteries was less than that of SNP (3-fold) and labetalol (3.5-fold). Therefore, we concluded clevidipine exhibited the lowest dose shift to relax the hypertrophic vessels compared to SNP and labetalol in the profilin1 hypertrophic animal mouse model.

Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases: important role of Rac/NADPH oxidase.

'Oxidative stress' is a term defining states of elevated reactive oxygen species (ROS) levels. Normally, ROS control several physiological processes, such as host defence, biosynthesis of hormones, fertilization and cellular signalling. However, oxidative stress has been involved in different pathologies, including metabolic syndrome and numerous cardiovascular diseases. A major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology is the NADPH oxidase (NOX) family of enzymes. NOX is a multi-component enzyme complex that consists of membrane-bound cytochrome b-558, which is a heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP-binding protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1 has been reported to be a key regulator of oxidative stress through its co-regulatory effects on both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a brief outline about the important role of the Rac1-NOX axis in the pathophysiology of both metabolic syndrome and cardiovascular disease.

Myocardial Rac1 exhibits partial involvement in thyroxin-induced cardiomyocyte hypertrophy and its inhibition is not sufficient to improve cardiac dysfunction or contractile abnormalities in mouse papillary muscles.

: Development of cardiac hypertrophy after thyroxin (T4) treatment is well recognized. Recently, we observed that T4-induced cardiac hypertrophy is associated with increased cardiac Rac1 expression and activity. Whether this Rac1 increase has a role in inducing this cardiac phenotype is, however, still unknown. Here, we showed that T4 treatment (500 µg/kg/d) for 2 weeks resulted in increased myocardial Rac1 activity with subsequent hypertension, cardiac hypertrophy, and left ventricular systolic dysfunction in vivo. Isolated right ventricular papillary muscles of T4-treated mice maintained their peak isometric active developed tension but exhibited significant decreases in their corresponding time to peak and in relaxation times. Positive inotropic responses to increasing pacing rate and ß-adrenergic stimulation were also depressed in these muscles. Pravastatin (10 mg/kg/d), a Rac1 inhibitor, significantly decreased myocardial Rac1 activity, hypertension, and cardiomyocyte size in T4-treated mice but could not attenuate gross heart weight or functional cardiac changes in these mice. Our data showed that T4 could activate different signaling pathways with distinct cardiovascular outcomes. We also provide the first mechanistic evidence for the partial involvement of Rac1 activation in T4-induced cardiomyocyte hypertrophy and reveal a putative role for Rac1 in the development of T4-induced hypertension.

Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury.

The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O(2)(·-)). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia-reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O(2)(·-) generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared with WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-h reperfusion) caused significantly larger MI in TG mice compared with WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91(phox), O(2)(·-), and P(21)-activated kinase. Thus these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.

Rac-induced left ventricular dilation in thyroxin-treated ZmRacD transgenic mice: role of cardiomyocyte apoptosis and myocardial fibrosis.

The pathways inducing the critical transition from compensated hypertrophy to cardiac dilation and failure remain poorly understood. The goal of our study is to determine the role of Rac-induced signaling in this transition process. Our previous results showed that Thyroxin (T4) treatment resulted in increased myocardial Rac expression in wild-type mice and a higher level of expression in Zea maize RacD (ZmRacD) transgenic mice. Our current results showed that T4 treatment induced physiologic cardiac hypertrophy in wild-type mice, as demonstrated by echocardiography and histopathology analyses. This was associated with significant increases in myocardial Rac-GTP, superoxide and ERK1/2 activities. Conversely, echocardiography and histopathology analyses showed that T4 treatment induced dilated cardiomyopathy along with compensatory cardiac hypertrophy in ZmRacD mice. These were linked with further increases in myocardial Rac-GTP, superoxide and ERK1/2 activities. Additionally, there were significant increases in caspase-8 expression and caspase-3 activity. However, there was a significant decrease in p38-MAPK activity. Interestingly, inhibition of myocardial Rac-GTP activity and superoxide generation with pravastatin and carvedilol, respectively, attenuated all functional, structural, and molecular changes associated with the T4-induced cardiomyopathy in ZmRacD mice except the compensatory cardiac hypertrophy. Taken together, T4-induced ZmRacD is a novel mouse model of dilated cardiomyopathy that shares many characteristics with the human disease phenotype. To our knowledge, this is the first study to show graded Rac-mediated O(2)·(-) results in cardiac phenotype shift in-vivo. Moreover, Rac-mediated O(2)·(-) generation, cardiomyocyte apoptosis, and myocardial fibrosis seem to play a pivotal role in the transition from cardiac hypertrophy to cardiac dilation and failure. Targeting Rac signaling could represent valuable therapeutic strategy not only in saving the failing myocardium but also to prevent this transition process.

Vascular remodeling-associated hypertension leads to left ventricular hypertrophy and contractile dysfunction in profilin-1 transgenic mice.

Hypertension is a major health problem and a main risk factor for cardiovascular diseases. We have shown that overexpression of profilin-1 in blood vessels of transgenic mice generates mechanical tone and led to vascular remodeling/hypertension. However, little is known whether cardiac contractile performance in these mice is compromised. We investigated the in vivo contractile function and in vitro contractile performance using isolated papillary muscles from both right ventricle and left ventricle of profilin-1 mice at older age. Our results showed mild left ventricular hypertrophy and moderate systolic dysfunction in profilin-1 mice as evident by increased heart/body weight ratio and echocardiography analysis. Under near physiological conditions, right ventricle papillary muscles of profilin-1 mice maintained their peak isometric active developed tension, and the rate of force development over the entire frequency range of 4-14 Hz. Positive inotropic responses to increasing Ca and ß-adrenergic stimulation were also maintained. Conversely, left ventricular papillary muscles of profilin-1 mice exhibited depressed peak isometric, peak isometric active developed tension and rate of force development, and depressed positive inotropic responses to increasing Ca and ß-adrenergic stimulation. We here provide functional evidence that a significant contractile dysfunction in profilin-1 mice exists. Targeting vascular profilin-1 signaling could represent a promising therapeutic approach in hypertensive patients.

Cardiovascular antioxidant therapy: a review of supplements, pharmacotherapies, and mechanisms.

Oxidant stress plays an important role in the pathogenesis of atherosclerosis. In the late 1980s, biological studies demonstrated that oxygen-free radicals oxidize low-density lipoprotein-cholesterol, resulting in the creation of foam cells and inciting the cascade of biological events that ultimately result in the formation of atherosclerosis. In vitro studies showed the ability of antioxidant vitamins to scavenge free radicals and block the oxidation of low-density lipoprotein. This data was supported in vivo by early observational studies suggesting the benefit of antioxidants, particularly vitamin E, in the prevention of coronary artery disease. On the basis of these studies, the use of antioxidant supplements by the general population increased substantially and became a multibillion dollar industry. Despite strong biological evidence and promising observational data, more rigorous scientific evaluation did not support a causational relationship between vitamin supplements and lowering coronary artery disease risk. Several prospective, double-blind, placebo-controlled trials showed no benefit and possibly harmful effects. Therapies such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and statins, which are known to have benefit in preventing and treating atherosclerosis by reducing blood pressure and cholesterol, also have a "pleiotropic" effect in reducing the formation of reactive oxygen species (ROS). Advances in molecular biology and the study of ROS led to a better understanding of the mechanisms that govern their production and role in atherogenesis. This progress identified unforeseen pathways by which these drugs favorably alter the balance in ROS production, and have raised possibilities for future targeted therapies in the prevention of atherosclerosis.

Impact of disrupting adenosine A3 receptors (A3⁻/⁻ AR) on colonic motility or progression of colitis in the mouse.

BACKGROUND: Pharmacological studies suggest that adenosine A3AR influences motility and colitis. Functional A3⁻/⁻AR knockout mice were used to prove whether A3AR activation is involved in modulating either motility or colitis. METHODS: A3AR was probed by polymerase chain reaction (PCR) genotyping, Western blot, and immunochemistry. Motility was assessed in vivo by artificial bead-expulsion, stool-frequency, and FITC-dextran transit. Colitis was induced with dextran sodium sulfate (DSS) in A3⁻/⁻AR or wildtype (WT) age- and sex-matched controls. Progression of colitis was evaluated by histopathology, changes in myeloperoxidase (MPO), colon length, CD4(+) -cells, weight-loss, diarrhea, and the guaiac test. RESULTS: Goat anti-hu-A3 antiserum identified a 66 kDa immunogenic band in colon. A3AR-immunoreactivity is expressed in SYN(+) -nerve varicosities, s-100(+) -glia, and crypt cells, but not 5-HT(+) (EC), CD4(+) (T), tryptase(+) (MC), or muscle cells. A3AR immunoreactivity in myenteric ganglia of distal colon >> proximal colon by a ratio of 2:1. Intestinal transit and bead expulsion were accelerated in A3⁻/⁻AR mice compared to WT; stool retention was lower by 40%-60% and stool frequency by 67%. DSS downregulated A3AR in epithelia. DSS histopathology scores indicated less mucosal damage in AA3⁻/⁻AR mice than WT. A3⁻/⁻AR phenotype protected against DSS-induced weight loss, neutrophil (MPO), or CD4(+) -T cell infiltration, colon shortening, change in splenic weight, diarrhea, or occult-fecal blood. CONCLUSIONS: Functional disruption of A3AR in A3⁻/⁻AR mice alters intestinal motility. We postulate that ongoing release of adenosine and activation of presynaptic-inhibitory A3AR can slow down transit and inhibit the defecation reflex. A3AR may be involved in gliotransmission. In separate studies, A3⁻/⁻AR protects against DSS colitis, consistent with a novel hypothesis that A3AR activation contributes to development of colitis.

Cardiac remodeling caused by transgenic overexpression of a corn Rac gene.

Rac1-GTPase activation plays a key role in the development and progression of cardiac remodeling. Therefore, we engineered a transgenic mouse model by overexpressing cDNA of a constitutively active form of Zea maize Rac gene (ZmRacD) specifically in the hearts of FVB/N mice. Echocardiography and MRI analyses showed cardiac hypertrophy in old transgenic mice, as evidenced by increased left ventricular (LV) mass and LV mass-to-body weight ratio, which are associated with relative ventricular chamber dilation and systolic dysfunction. LV hypertrophy in the hearts of old transgenic mice was further confirmed by an increased heart weight-to-body weight ratio and histopathology analysis. The cardiac remodeling in old transgenic mice was coupled with increased myocardial Rac-GTPase activity (372%) and ROS production (462%). There were also increases in α(1)-integrin (224%) and ß(1)-integrin (240%) expression. This led to the activation of hypertrophic signaling pathways, e.g., ERK1/2 (295%) and JNK (223%). Pravastatin treatment led to inhibition of Rac-GTPase activity and integrin signaling. Interestingly, activation of ZmRacD expression with thyroxin led to cardiac dilation and systolic dysfunction in adult transgenic mice within 2 wk. In conclusion, this is the first study to show the conservation of Rho/Rac proteins between plant and animal kingdoms in vivo. Additionally, ZmRacD is a novel transgenic model that gradually develops a cardiac phenotype with aging. Furthermore, the shift from cardiac hypertrophy to dilated hearts via thyroxin treatment will provide us with an excellent system to study the temporal changes in cardiac signaling from adaptive to maladaptive hypertrophy and heart failure.

The effect of selective antihypertensive drugs on the vascular remodeling-associated hypertension: insights from a profilin1 transgenic mouse model.

Hypertension represents a major risk factor for cardiovascular diseases. We have developed a novel transgenic mouse model by overexpressing the cDNA of human profilin1 in the blood vessels of transgenic mice, which led to vascular hypertrophy and hypertension. We assessed the effects of losartan, amlodipine, or atenolol on vascular hypertrophy-associated hypertension, by treating the profilin1 transgenic mice for 4 weeks. Our myograph results showed improvement in the contraction response toward phenylephrine and in the relaxation response toward acetylcholine and sodium nitrite in losartan- and amlodipine-treated profilin1 mice. Western blot analyses using mesenteric arteries of losartan- and amlodipine-treated profilin1 mice showed significant decreases in their signaling, respectively, as follows: the expression of α1 integrin (104% and 93%) and ß1 integrin (116% and 109%); p-ERK1/2 (149% and 130%) and p-JNK (171% and 137%); the phospho-myosin light chain 20 (117% and 150%); and the ROCKII expression (125% and 180%). Conversely, there were significant increases in the endothelial nitric oxide synthase expression (82% and 80%) and activation (p-endothelial nitric oxide synthase) (78% and 76%). On the other hand, atenolol-treated profilin1 mice showed no significant change in all measured parameters. In conclusion, the profilin1 gene may represent a new therapeutic target in the treatment of vascular hypertrophy-associated hypertension.

Vascular hypertrophy-associated hypertension of profilin1 transgenic mouse model leads to functional remodeling of peripheral arteries.

Increased mechanical stress/hypertension in the vessel wall triggers the hypertrophic signaling pathway, resulting in structural remodeling of vasculature. Vascular hypertrophy of resistance vessels leads to reduced compliance and elevation of blood pressure. We showed before that increased expression of profilin1 protein in the medial layer of the aorta induces stress fiber formation, triggering the hypertrophic signaling resulting in vascular hypertrophy and, ultimately, hypertension in older mice. Our hypothesis is that profilin1 induced vascular hypertrophy in resistance vessels, which led to elevation of blood pressure, both of which contributed to the modulation of vascular function. Our results showed significant increases in the expression of alpha(1)- and beta(1)-integrins (280 + or - 6.3 and 325 + or - 7.4%, respectively) and the activation of the Rho/Rho-associated kinase (ROCK) II pathway (260 and 350%, respectively, P < 0.05) in profilin1 mesenteric arteries. The activation of Rho/ROCK led to the inhibition of endothelial nitric oxide synthase expression (39 + or - 5.4%; P < 0.05) and phosphorylation (35 + or - 4.5%; P < 0.05) but also an increase in myosin light chain 20 phosphorylation (372%, P < 0.05). There were also increases in hypertrophic signaling pathways in the mesenteric arteries of profilin1 mice such as phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase (312.15 and 232.5%, respectively, P < 0.05). Functional analyses of mesenteric arteries toward the vasoactive drugs were assessed using wire-myograph and showed significant increases in the vascular responses of profilin1 mesenteric arteries toward phenylephrine, but significant decreases in response toward ROCK inhibitor Y-27632, ACh, sodium nitrite, and cytochalasin D. The changes in vascular responses in the mesenteric arteries of profilin1 mice are due to vascular hypertrophy and the elevation of blood pressure in the profilin1 transgenic mice.

Activation of adenosine low-affinity A3 receptors inhibits the enteric short interplexus neural circuit triggered by histamine.

We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl(-) secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (I(sc), Cl(-) secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N(3)-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC(50) for IB-MECA was 0.8 microM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex I(sc) responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that eADO acts at low-affinity A3 receptors in addition to high-affinity A1 receptors to suppress coordinated responses triggered by immune-histamine H2 receptor activation. The short interplexus circuit activated by histamine involves adenosine, acetylcholine, substance P, and serotonin. We postulate that A3 receptor modulation may occur in gut inflammatory diseases or allergic responses involving mast cell and histamine release.

Vascular hypertrophy and hypertension caused by transgenic overexpression of profilin 1.

We have overexpressed either the cDNA of human profilin 1 or expressed the mutant (88R/L) in the blood vessels of transgenic FVB/N mice. Reverse transcription-PCR indicated selective overexpression of profilin 1 and 88R/L in vascular smooth muscle cells. Polyproline binding showed increased profilin 1 and 88R/L proteins in transgenic mice compared with control (~30%, p < 0.05). Rhodamine-phalloidin staining revealed increase stress fiber formation in vascular smooth muscle cells of profilin 1 compared with 88R/L and control. Hematoxylin and eosin staining showed clear signs of vascular hypertrophy in the aorta of profilin 1 mice versus 88R/L and control. However, there were no differences between 88R/L and control mice. Western blotting confirmed the activation of the hypertrophic signaling cascades in aortas of profilin 1 mice. Phospho-ERK1/2 was significantly higher in profilin 1 than 88R/L and control (512.3 and 361.7%, respectively, p < 0.05). Profilin 1 mice had significant increases in phospho-JNK as compared with 88R/L and control (371.4 and 346%, respectively, p < 0.05). However, there were no differences between 88R/L and control mice in both kinases. There was a significant increase in ROCK II kinase in the aorta of profilin 1 mice compared with controls (>400%, p < 0.05). Tail cuff and circadian monitoring of blood pressure showed significant increases in systolic and mean arterial blood pressures of profilin 1 mice starting at age 6 months compared with controls (~25 mm Hg, p < 0.05). These results suggest that increased actin polymerization in blood vessels triggers activation of the hypertrophic signaling cascades and results in elevation of blood pressure at advanced age.

Cyclic AMP signaling contributes to neural plasticity and hyperexcitability in AH sensory neurons following intestinal Trichinella spiralis-induced inflammation.

Trichinella spiralis infection causes hyperexcitability in enteric after-hyperpolarising (AH) sensory neurons that is mimicked by neural, immune or inflammatory mediators known to stimulate adenylyl cyclase (AC)/cyclic 3',5'-adenosine monophosphate (cAMP) signaling. The hypothesis was tested that ongoing modulation and sustained amplification in the AC/cAMP/phosphorylated cAMP related element binding protrein (pCREB) signaling pathway contributes to hyperexcitability and neuronal plasticity in gut sensory neurons after nematode infection. Electrophysiological, immunological, molecular biological or immunochemical studies were done in T. spiralis-infected guinea-pigs (8000 larvae or saline) after acute-inflammation (7 days) or 35 days p.i., after intestinal clearance. Acute-inflammation caused AH-cell hyperexcitability and elevated mucosal and neural tissue levels of myeloperoxidase, mast cell tryptase, prostaglandin E2, leukotrine B4, lipid peroxidation, nitric oxide and gelatinase; lower level inflammation persisted 35 days p.i. Acute exposure to blockers of AC, histamine, cyclooxygenase or leukotriene pathways suppressed AH-cell hyperexcitability in a reversible manner. Basal cAMP responses or those evoked by forskolin (FSK), Ro-20-1724, histamine or substance P in isolated myenteric ganglia were augmented after T. spiralis infection; up-regulation also occurred in AC expression and AC-immunoreactivity in calbindin (AH) neurons. The cAMP-dependent slow excitatory synaptic transmission-like responses to histamine (mast cell mediator) or substance P (neurotransmitter) acting via G-protein coupled receptors (GPCR) in AH neurons were augmented by up to 2.5-fold after T. spiralis infection. FSK, histamine, substance P or T. spiralis acute infection caused a 5- to 30-fold increase in cAMP-dependent nuclear CREB phosphorylation in isolated ganglia or calbindin (AH) neurons. AC and CREB phosphorylation remained elevated 35 days p.i.. Ongoing immune activation, AC up-regulation, enhanced phosphodiesterase IV activity and facilitation of the GPCR-AC/cAMP/pCREB signaling pathway contributes to T. spiralis-induced neuronal plasticity and AH-cell hyperexcitability. This may be relevant in gut nematode infections and inflammatory bowel diseases, and is a potential therapeutic target.

Hypertension caused by transgenic overexpression of Rac1.

Reactive oxygen species, including superoxide, are important mediators of the pathophysiology of hypertension. In the vasculature, superoxide antagonizes nitric oxide (NO*), resulting in increased vascular tone. The GTP binding protein Rac regulates a wide variety of cellular functions, including the activation of NADPH oxidase, the major source of O2*-in the blood vessel wall. An hypothesis is that Rac1 may act as an important regulator of vascular O2*- production, contributing to the balance between O2*- and NO* and maintaining consequent homeostasis of blood pressure. To alter the activity of vascular NADPH oxidase, the authors developed a transgenic animal model that overexpresses the human cDNA of the constitutively active mutant of Rac1 (RacCA) in smooth muscle cells using the smooth muscle +/--actin promoter. The RacCA transgenic had excessive amounts of O2*- in the vessel wall that, which led to heightened production of peroxynitrite, as detected by increased protein nitration and reduced NO* levels. RacCA mice developed moderate hypertension, which was corrected by N-acetyl-L-cysteine (NAC). RacCA transgenic mice also developed left ventricular hypertrophy as a secondary effect of pressure overload. The data suggest that Rac1 is a critical regulator of the redox state of blood vessels and homeostasis of blood pressure.

ADOA3R as a therapeutic target in experimental colitis: proof by validated high-density oligonucleotide microarray analysis.

Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)--induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation (90% of genes tested (33 of 37 genes). We conclude that our validated high-density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine-based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

Smooth muscle cell expression of a constitutive active form of human Rac 1 accelerates cutaneous wound repair.

BACKGROUND: Hyperoxia has been shown to improve wound healing; however, the mechanism for such therapeutic effects of oxygen remains hypothetical. Rac 1 regulates a wide variety of cellular activities, including cell proliferation and migration, and also is a key regulator for the activity of the nicotinamide dinucleotide phosphate oxidase the enzyme complex responsible for the production of a large fraction of cellular superoxide. METHODS: We generated transgenic mice that express either the cDNA of a constitutively active mutant of human Rac 1 (V12 mutant or Rac CA) or the dominant negative isoform (V12 and N17 mutant or Rac DN) in the blood vessels using mouse vascular smooth muscle promoter for alpha-actin. We placed 2 wounds of 6 mm in diameter at the middorsal region of each mouse and allowed about 3 weeks for the wounds to heal. RESULTS: The size of the wounds in Rac CA transgenic mice was reduced relative to wild type mice; healing of Rac DN mice was slower than wild type and Rac CA ( P < .05). Blood vessel formation appeared faster in Rac CA mice, a finding associated with enhanced expression of some angiogenic growth factors. CONCLUSION: The current studies suggest that Rac 1 activation accelerates the wound healing process and is associated with more efficient angiogenesis at the wound site.

Dietary sodium regulates angiotensin AT1a and AT1b mRNA expression in mouse brain.

Previous results showed that angiotensin (Ang) AT1a and AT1b receptor mRNA are expressed in mouse hypothalamus (HYP), brainstem (BS) and anterior pituitary (PIT). To extend these findings, we developed a real-time polymerase chain reaction (PCR) method to differentiate and quantify Ang AT1a and AT1b mRNA in mouse brain. An experiment was conducted in male C57Bl/6J mice to determine the effects of low and high dietary salt (0.04 or 8% NaCl for 2 weeks) on mRNA expression. Physiological measurements showed that high salt increased water intake (15.1 +/- 0.6 ml/day), whereas low salt decreased water intake (3.2 +/- 0.1 ml/day). There were no significant changes in body weight, hematocrit or plasma osmolality. Real-time PCR was effective in distinguishing AT1a and AT1b receptor mRNA. The PCR efficiencies for AT1a, AT1b and 18S ribosome were tested to be identical, making it possible to quantify mRNA levels. There were differences in angiotensin receptor expression, related to diet and brain region. In hypothalamus, both the high salt and low salt diet decreased AT1a expression (to 63 +/- 4% and 62 +/- 1%), although there were no changes in AT1b. In brainstem, there was a marked increase in AT1a (to 365 +/- 60%) and AT1b (to 372 +/- 23%) after high salt, although there was only a marked decrease for AT1b (to 23 +/- 5%) after low salt. In anterior pituitary, both high salt and low salt diet increased AT1a expression (to 152 +/- 8% and 123 +/- 9%), although there were no changes in AT1b. Results document that both AT1 receptor subtypes are present in mouse hypothalamus, brainstem and anterior pituitary, and that there is differential regulation of expression in response to changes in dietary salt.

Thrombospondin 2 regulates cell proliferation induced by Rac1 redox-dependent signaling.

Thrombospondin 2 (TSP2) is a matricellular protein controlling the apoptosis-proliferation balance in endothelial cells. Little is known about its transcriptional regulation compared with that of TSP1. We found that overexpression of a constitutively active mutant of Rac (Rac(V12)) specifically increases TSP2 mRNA levels without affecting TSP1 in human aortic endothelial cells (HAEC). Moreover, TSP2 induction by Rac(V12) is dependent upon reactive oxygen species (ROS) production, as gp91ds-tat peptide, an inhibitor of NADPH oxidase, and the flavoprotein inhibitor diphenylene iodinium (DPI) block TSP2 synthesis. Furthermore, we found that increasing Rac(V12) expression results in a biphasic proliferative curve, with proliferation initially increasing as Rac(V12) expression increases and then returning to levels less than that of control cells at higher expression. This growth inhibition is mediated by TSP2, as either DPI treatment, which blocks TSP2 synthesis, or pan-TSP blocking antibodies restore the proliferative ability of HAEC with high expression. Mechanistically, we show that the effect of TSP2 on cell proliferation is independent of the antiangiogenic TSP2 Hep1 sequence, which is capable of altering actin cytoskeletal reorganization but not proliferation in our experimental conditions. Finally, we show in vivo that Rac-induced TSP2 expression is observed in the aorta of transgenic mice selectively expressing Rac(V12) in smooth muscle cells. These results identify Rac-induced ROS as a new pathway involved in the regulation of TSP2 expression.

Rabbit chronic ileitis leads to up-regulation of adenosine A1/A3 gene products, oxidative stress, and immune modulation.

A rabbit model of chronic ileitis has helped decipher the mechanism of alteration of multiple electrolyte and nutrient malabsorptions in inflammatory bowel disease (IBD). This study examined alterations in the adenosine A1/A3 receptor, oxidant, antioxidant, and immune-inflammatory pathways in chronic ileitis. Chronic ileal inflammation was induced 13-15 days after infection with 10,000 Eimeria magna oocytes. Quantitative analysis in 16 rabbits was done for oxidants, antioxidants, A1 and A3 transcripts, transport, injury, and inflammatory mediators. Inflamed gut had villus blunting, crypt hyperplasia and fusion, and immune cell infiltration. Alkaline phosphatase and Na-glucose co-transport were reduced by 78% (P=0.001) and 89% (P=0.001), respectively. Real-time fluorescence monitoring (TaqMan)-polymerase chain reaction revealed a transcriptional up-regulation of 1.34-fold for A1 and 5.40-fold for A3 receptors in inflamed gut. Lipid peroxidation increased in the mucosa (78%, P=0.012), longitudinal muscle-myenteric plexus (118%, P=0.042), and plasma (104%, P=0.001). Mucosal antioxidants were altered by inflammation: reductions occurred in superoxide dismutase (32%, P=0.001) and catalase (43%, P=0.001), whereas increases occurred in glutathione (75%, P=0.0271) and glutathione reductase (86%, P=0.0007). Oxidant enzyme activities were elevated by 21% for xanthine oxidase (P=0.004), 172% for chloramine (P=0.022), 47% for gelatinase (P=0.041), and 190% for myeloperoxidase (P=0.002). Mast cell tryptase increased by 79% (P=0.006). Increases occurred in the plasma concentration of leukotriene B(4) (13-fold, P=0.003), thromboxane B(2) (61-fold, P=0.018), and tumor necrosis factor-alpha (9-fold, P=0.002). In conclusion, chronic ileitis and tissue injury are associated with discrete alterations in complex multi-level oxidant, antioxidant, and immune inflammatory components. The rabbit ileitis model is a suitable model to gain further insight into chronic inflammation and IBD. We hypothesize that adenosine A3 and A1 receptors may provide a novel target for therapy in chronic ileitis and perhaps IBD.