Fenofibrate reduces cardiac remodeling by mitochondrial dynamics preservation in a renovascular model of cardiac hypertrophy.

Fenofibrate, a PPAR-α agonist clinically used to lower serum lipid levels, reduces cardiac remodeling and improves cardiac function. However, its mechanism of action is not completely elucidated. In this study we examined the effect of fenofibrate on mitochondria in a rat model of renovascular hypertension, focusing on mediators controlling mitochondrial dynamics and autophagy. Rats with two-kidney one-clip (2K1C) hypertension were treated with fenofibrate 150 mg/kg/day (2K1C-FFB) or vehicle (2K1C-VEH) for 8 weeks. Systolic blood pressure and cardiac functional were in-vivo assessed, while cardiomyocyte size and protein expression of mediators of cardiac hypertrophy and mitochondrial dynamics were ex-vivo examined by histological and Western blot analyses. Fenofibrate treatment counteracted the development of hypertension and the increase of left ventricular mass, relative wall thickness and cross-sectional area of cardiomyocytes. Furthermore, fenofibrate re-balanced the expression Mfn2, Drp1 and Parkin, regulators of fusion, fission, mitophagy respectively. Regarding autophagy, the LC3-II/LC3-I ratio was increased in 2K1C-VEH and 2K1C-FFB, whereas the autophagy was increased only in 2K1C-FFB. In cultured H9C2 cardiomyoblasts, fenofibrate reversed the Ang II-induced mRNA up-regulation of hypertrophy markers Nppa and Myh7, accumulation of reactive oxygen species and depolarization of the mitochondrial membrane exerting protection mediated by up-regulation of the Uncoupling protein 2. Our results indicate that fenofibrate acts directly on cardiomyocytes and counteracts the pressure overload-induced cardiac maladaptive remodeling. This study reveals a so far hidden mechanism involving mitochondrial dynamics in the beneficial effects of fenofibrate, support its repurposing for the treatment of cardiac hypertrophy and provide new potential targets for its pharmacological function.

Surgical Margins in Canine Cutaneous Soft-Tissue Sarcomas: A Dichotomous Classification System Does Not Accurately Predict the Risk of Local Recurrence.

Adjuvant treatments are recommended in dogs with incompletely excised cutaneous soft-tissue sarcoma (STS) to reduce the risk of local recurrence (LR), although guidelines are lacking on how to manage clean but close margins (CbCM). This retrospective study investigates the impact of CbCM on LR of canine STS. Ninety-eight surgically excised canine STS at first presentation were included. Tissue samples were routinely trimmed and analyzed. Cumulative incidence of LR was estimated for each category of margins (tumor-free, infiltrated, CbCM), and included CbCM in the tumor-free and infiltrated category, respectively. The prognostic impact on LR was then adjusted for relevant prognostic factors. Cumulative incidence of LR at three years differed significantly between the three categories (p = 0.016), and was estimated to be 42% with infiltrated margins, 23% with CbCM, 7% with tumor-free margins. Both when CbCM were grouped with infiltrated margins (p = 0.033; HR = 5.05), and when CbCM were grouped with tumor-free margins (p = 0.011; HR = 3.13), a significant difference between groups was found. STS excised with infiltrated margins had the greatest risk of LR. The rate of LR with CbCm was greater than recurrence rate of tumor-free margins. The category CbCM may be considered as a separate prognostic category.

The German Shorthair Pointer Dog Breed (Canis lupus familiaris): Genomic Inbreeding and Variability.

The German Shorthaired Pointer (GSHP) is a breed worldwide known for its hunting versatility. Dogs of this breed are appreciated as valuable companions, effective trackers, field trailers and obedience athletes. The aim of the present work is to describe the genomic architecture of the GSHP breed and to analyze inbreeding levels under a genomic and a genealogic perspective. A total of 34 samples were collected (24 Italian, 10 USA), and the genomic and pedigree coefficients of inbreeding have been calculated. A total of 3183 runs of homozygosity (ROH) across all 34 dogs have been identified. The minimum and maximum number of Single Nucleotide Polymorphisms (SNPs) defining all ROH are 40 and 3060. The mean number of ROH for the sample was 93.6. ROH were found on all chromosomes. A total of 854 SNPs (TOP_SNPs) defined 11 ROH island regions (TOP_ROH), in which some gene already associated with behavioral and morphological canine traits was annotated. The proportion of averaged observed homozygotes estimated on total number of SNPs was 0.70. The genomic inbreeding coefficient based on ROH was 0.17. The mean inbreeding based on genealogical information resulted 0.023. The results describe a low inbred population with quite a good level of genetic variability.

Vascular and parenchymal lesions along with enhanced neurogenesis characterize the brain of asymptomatic stroke-prone spontaneous hypertensive rats.

BACKGROUND AND OBJECTIVES: Spontaneously hypertensive stroke-prone rats (SHRSPs) develop hypertension, cerebrovascular abnormalities and a stroke phenotype in association with higher levels of proteinuria. Here, we focus on cerebral abnormalities preceding lesions detectable by MRI. METHODS: Longitudinal assessment of brain histology was performed in salt-loaded male SHRSPs (n = 26) and Wistar-Kyoto (WKY) normotensive control animals (n = 27). Groups of rats were sacrificed at different time points: Time 0, before the salt diet administration; Time 1, when proteinuria achieved 40 mg/day; Time 2, when proteinuria exceeded 100 mg/day. RESULTS: At Time 0, no brain lesions were observed. At Time 1, changes of the cortical penetrating arteries, vasogenic oedema, lacunae and focal cell loss appeared in SHRSPs and worsened at Time 2, although no lesions were yet detected by MRI. Staining for proliferation markers revealed a significant boost of cellular mitosis in the subventricular zone (SVZ) of SHRSPs. Moreover, we observed higher immunopositivity for nestin, glial fibrillary acidic protein and doublecortin (markers for neural stem cells, astrocytes and immature neurons, respectively). At Time 2, apoptotic caspase-3 as well as 4-hydroxynonenal-positive neurons were associated to decreased nestin and doublecortin staining. High expression levels of glial fibrillary acidic protein were maintained in the SVZ. No comparative alterations and SVZ activation were recorded in WKYs. CONCLUSION: Appearance of vascular changes in SHRSPs, before any MRI-detectable brain lesion, is coupled to active neural proliferation in the SVZ. With disease progression, only newborn astrocytes can survive, likely because of the neurotoxicity triggered by brain oedema and oxidative stress.

Immunohistochemical expression of oncological proliferation markers in the hearts of rats during normal pregnancy.

AIM: Pregnancy is characterized by left ventricular hypertrophy that is potentially accounted for by cardiomyocyte proliferation, although no such evidence is currently available. This study investigates if the left ventricular mass (LVM) increase during pregnancy implies cell hyperplasia. MATERIALS & METHODS: In nonpregnant and late-pregnant rats, cardiac function and LVM were evaluated by MRI, and cardiomyocyte dimensions and proliferations were assessed quantitatively by morphometric analysis and immunohistochemistry using oncological markers (Ki67 and MCM2). RESULTS: In late-pregnant rats, LVM and cardiomyocyte area were greater. No mitotic figures were found nor was there any significant difference between groups in Ki67 expression. MCM2 expression was related to LVM. CONCLUSION: During pregnancy, rat cardiomyocytes undergo hypertrophy but not hyperplasia; the expression of MCM2, related to LVM, suggests it could be a marker of protein synthesis. The application of oncological markers to physiological contexts may provide insight into their role within the cell cycle.

Vagal stimulation, through its nicotinic action, limits infarct size and the inflammatory response to myocardial ischemia and reperfusion.

Vagal activity has protective effects in ischemic heart disease. We tested whether vagal stimulation (VS) could modulate the inflammatory reaction, a major determinant of cardiac injury after ischemia/reperfusion. Four groups of male rats underwent myocardial ischemia (30 minutes) and reperfusion (24 hours). One group underwent VS (40 minutes), 1 VS plus atrial pacing (VS + Pacing), and 1 VS plus nicotinic inhibition by mecamylamine (VS + MEC). After 24 hours, the area at risk, infarct size, inflammation parameters, and apoptosis were quantified. Infarct size was reduced in all VS-treated rats (controls, 53 ± 18%; VS, 6.5 ± 3%; VS + Pacing, 23 ± 6%; VS + MEC, 33 ± 9%; P < 0.005 vs. controls). The infarct size in the VS + MEC group was larger than that in VS-treated animals, despite similar heart rate, suggesting partial loss of protection. The number of macrophages, neutrophils, and apoptotic cells in the area at risk and the plasma cytokines levels were significantly reduced in all VS-treated animals. In conclusion, VS decreases infarct size and inflammatory markers during ischemia/reperfusion independent of the heart rate. The anti-inflammatory and antiapoptotic properties of the nicotinic pathway are the primary underlying mechanism. The vagally mediated modulation of inflammatory responses may prove valuable in the clinical management of acute coronary syndromes and of heart failure.

Beneficial effects of treatment with transglutaminase inhibitor cystamine on the severity of inflammation in a rat model of inflammatory bowel disease.

Inflammatory bowel disease (IBD) represents a socially and clinically relevant disorder, characterized by intestinal chronic inflammation. Cystamine (CysN) is a multipotent molecule with healthy effects and, moreover, it is an inhibitor of transglutaminases (TGs), including the TG type 2 (TG2), an enzyme with pleiotropic functions, involved in different pathways of inflammation and central in the pathogenesis of some human disorders as the IBD. Our aim was to evaluate the effect of CysN in an IBD rat model. A total of 30 rats were divided into 4 groups: controls without treatment (CTR; n=7); receiving the 2,4,6-trinitrobenzene sulfonic acid enema (TNBS group; n=8); treated with TNBS enema plus oral CysN (TNBS-CysN group; n=8); treated with CysN (CysN group; n=7). After killing, bowel inflammation was evaluated applying specific scores. TG activity, TG2 and isopeptide bond immunohistochemical expression, and tumor necrosis factor-α (TNF-α) were evaluated in the colonic tissue, such as interleukin-6 (IL-6) serological levels (ELISA). TG2 was also evaluated on the luminal side of the colon by immunoautoradiography. Colonic samples from IBD patients were compared with animal results. TNBS-CysN group developed a less severe colitis compared with the TNBS group (macroscopic score 0.43±0.78 vs 3.28±0.95, microscopic score 6.62±12.01 vs 19.25±6.04, P<0.05, respectively) associated with a decrease of TG activity, TG2 and isopeptide bond immunohistochemical expression, TNF-α and IL-6 levels. No statistically significant differences were found between CysN and CTR groups. The colonic immunolocalization of TG2 was comparable in humans affected by IBD and TNBS-administered animals. This is the first demonstration that treatment with a CysN has an anti-inflammatory effect, reducing severity of colitis in a rat model. CysN could be tested as a possible treatment or co-treatment in IBD therapeutic trials.

Multiple neurogenic and neurorescue effects of human mesenchymal stem cell after transplantation in an experimental model of Parkinson's disease.

Stimulation of endogenous repair in neurodegenerative diseases, such as Parkinson's disease (PD), appears to be a novel and promising therapeutic application of stem cells (SCs). In fact SCs could propel local microenvironmental signals to sustain active endeavors for damaged neurons substitution, normally failing in non-supportive pathological surroundings. In this study, we demonstrated that two different doses of naïve human adult mesenchymal stem cells (hMSCs), implanted in the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA), positively survived 23 days after transplantation. Their fate was directly influenced by the surrounding host environment while grafted hMSCs, dose dependently, regionally sustained the survival of striatal/nigral dopaminergic terminals and enhanced neurogenesis in the Subventricular Zone (SVZ). The number of proliferative cells (Ki67/Proliferating Cell Nuclear Antigen +) as well as neuroblasts migration significantly augmented in the lesioned striatum of transplanted animals compared to controls. No SVZ astrogenesis was detected in all experimental conditions, irrespectively of graft presence. Activation of endogenous stem cell compartments and rescue of dopaminergic neurons, supported by the persistent release of specific cytokine by MSCs in vivo, appeared in principle able to contrast the neurodegenerative processes induced by the 6-OHDA lesion. Our results suggest that reciprocal influences between grafted cells and endogenous neural precursors could be important for the observed neurorescue effect on several brain regions. Altogether, our data provide remarkable cues regarding the potential of hMSCs in promoting endogenous reparative mechanisms that may prove applicable and beneficial for PD treatment.

Potential advantages of cell administration on the inflammatory response compared to standard ACE inhibitor treatment in experimental myocardial infarction.

BACKGROUND: Bone Marrow (BM) progenitor cells can target the site of myocardial injury, contributing to tissue repair by neovascolarization and/or by a possible direct paracrine effect on the inflammatory cascade. Angiotensin Converting Enzyme inhibitors (ACE-I) are effective in reducing mortality and preventing left ventricular (LV) function deterioration after myocardial infarction. METHODS: We investigated the short term effects of BM mononuclear cells (BMMNCs) therapy on the pro-inflammatory cytokines (pro-CKs) and on LV remodelling and compared these effects over a standard ACE-I therapy in a rat model of myocardial cryodamage. Forty two adult inbread Fisher-F344 rats were randomized into three groups: untreated (UT; n = 12), pharmacological therapy (ACE-I; n = 14, receiving quinapril), and cellular therapy (BMMNCs; n = 16, receiving BMMNCs infusion). Rats underwent to a standard echocardiogram in the acute setting and 14 days after the damage, before the sacrifice. Pro-CKs analysis (interleukin (IL)1beta, IL-6, tumor necrosis factor (TNF)alpha was performed (multiplex proteome arrays) on blood samples obtained by direct aorta puncture before the sacrifice; a control group of 6 rats was considered as reference. RESULTS: Concerning the extension of the infarcted area as well as the LV dimensions, no differences were observed among the animal groups; treated rats had lower left atrial diameters and higher indexes of LV function. Pro-Cks were increased in infarcted-UT rats if compared with controls, and significantly reduced by BMMNCs and ACE-I ; TNFalpha inversely correlated with LV fractional shortening. CONCLUSION: After myocardial infarction, both BMMNCs and ACE-I reduce the pattern of pro-Ck response, probably contributing to prevent the deterioration of LV function observed in UT rats.

Survival benefits of different antiadrenergic interventions in pressure overload left ventricular hypertrophy/failure.

We observed previously that in rats with aortic banding (Bd), development of left ventricular (LV) hypertrophy is opposed by beta-blockade, whereas interventions interfering with alpha-adrenoceptor function also inhibit interstitial fibrosis. To assess whether these differential structural effects do translate into different effects on LV function and on heart failure mortality, Bd or sham Bd 8-week-old rats were randomized to vehicle treatment (Vh), chemical sympathectomy ([Sx] 6-hydroxydopamine, 150 mg/kg IP twice a week), beta-adrenoceptor blockade (propranolol [Pro], 40 mg/kg per day PO), or alpha-adrenoceptor blockade (doxazosin [Dox], 5 mg/kg per day PO). After monitoring survival for 10 weeks, the survivors were anesthetized to undergo echocardiography and intraarterial blood pressure measurement. Bd-Vh rats showed increased LV and lung weights, as well as LV dilation, depressed endocardial and midwall fractional shortening and a restrictive transmitral diastolic flow velocity pattern. Compared with Bd-Vh rats, all of the actively treated Bd rats showed less LV hypertrophy, LV dilation, and lung congestion but no less depression of midwall fractional shortening. In contrast, Sx and Dox but not Pro treatment were also associated with lesser degrees of diastolic dysfunction and, even more importantly, with a striking increase in survival (sham banded rats, 100%; Bd-Vh, 40%; Bd-Pro, 51%; Bd-Sx, 83%; and Bd-Dox, 82%). Although Pro, Sx, and Dox provide similar midterm protection from development of LV hypertrophy and dysfunction and from circulatory congestion, only Sx and Dox favorably affected mortality. These findings indicate that in the aortic banding rat model, alpha-adrenoceptors are importantly involved in the pathogenesis of cardiovascular deterioration and disease progression.

Sympathectomy or doxazosin, but not propranolol, blunt myocardial interstitial fibrosis in pressure-overload hypertrophy.

The adaptive changes that develop in the pressure-overloaded left ventricular (LV) myocardium include cardiomyocyte hypertrophy and interstitial fibrosis. Although the former is known to depend to a sizeable extent on sympathetic (over)activity, little information exists whether the same applies to the latter, ie, whether excess catecholamine exposure contributes to the imbalance between collagen deposition by fibroblasts and degradation by matrix metalloproteases (MMPs), eventually leading to LV collagen accumulation. Sprague-Dawley rats were subjected to abdominal aortic banding (B) or sham operation (S) and treated with beta-blockade (Bb, oral propranolol, 40 mg/kg per day), chemical sympathectomy (Sx, 6-hydroxydopamine, 150 mg/kg intraperitoneal twice per week) or vehicle (Vh). Ten weeks later, systolic blood pressure, LV weight, collagen abundance (computer-aided histology), zymographic matrix metalloproteinase (MMP)-2 activity and its specific tissue inhibitor concentration (TIMP-2) were measured. Both sympathectomy and beta-blockade failed to attenuate the banding-induced blood pressure elevation but significantly attenuated the attendant LV hypertrophy. As expected, pressure-overload hypertrophy was associated with interstitial fibrosis (collagen: 4.37+/-1.23% BVh versus 1.23+/-0.44% SVh, P<0.05), which was abolished by sympathectomy (2.55+/-1.31%, P=not significant versus SSx) but left unchanged by beta-blockade (4.11+/-1.23%, P<0.05 versus both SBb and BSx). beta-blockade, but not sympathectomy, was also associated with an increased TIMP-2/MMP-2 ratio (P<0.05), indicating reduced interstitial collagenolytic activity. In separate groups of banded and sham-operated rats, treatment with the alpha-receptor blocker doxazosin (10 mg/kg per day) displayed similar antifibrotic and biochemical effects as sympathectomy. Thus in the course of experimental pressure overload, the sympathetic nervous system plays a major pro-fibrotic role, which is mediated via alpha-adrenergic but not beta-adrenergic receptors.

Angiotensin II modulates frizzled-2 receptor expression in rat vascular smooth muscle cells.

Ang II (angiotensin II) has multiple effects on vascular smooth muscle cells through the modulation of different classes of genes. Using the mRNA differential-display method to investigate gene expression in rat aortic smooth muscle cells in culture in response to 3 h of Ang II stimulation, we observed that Ang II down-regulated the expression of a member of the family of transmembrane receptors for Wnt proteins that was identified as Fzd2 [Fzd (frizzled)-2 receptor]. Fzds are a class of highly conserved genes playing a fundamental role in the developmental processes. In vitro, time course experiments demonstrated that Ang II induced a significant increase (P<0.05) in Fzd2 expression after 30 min, whereas it caused a significant decrease (P<0.05) in Fzd2 expression at 3 h. A similar rapid up-regulation after Ang II stimulation for 30 min was evident for TGFbeta1 (transforming growth factor beta1; P<0.05). To investigate whether Ang II also modulated Fzd2 expression in vivo, exogenous Ang II was administered to Sprague-Dawley rats (200 ng.kg(-1) of body weight.min(-1); subcutaneously) for 1 and 4 weeks. Control rats received normal saline. After treatment, systolic blood pressure was significantly higher (P<0.01), whereas plasma renin activity was suppressed (P<0.01) in Ang II- compared with the saline-treated rats. Ang II administration for 1 week did not modify Fzd2 expression in aorta of Ang II-treated rats, whereas Ang II administration for 4 weeks increased Fzd2 mRNA expression (P<0.05) in the tunica media of the aorta, resulting in a positive immunostaining for fibronectin at this time point. In conclusion, our data demonstrate that Ang II modulates Fzd2 expression in aortic smooth muscle cells both in vitro and in vivo.

The translocation of marrow MNCs after experimental myocardial cryoinjury is proportional to the infarcted area.

BACKGROUND: The selective homing of peripherally injected marrow MNCs (MMNCs) has recently been demonstrated in a model of cryodamaged heart. However, the mechanism underlying this phenomenon is still unknown. In the hypothesis that this process is related to the necrotic area extension, the infarcted area was correlated with the number of homed MMNCs in a model of experimental cryodamaged heart. STUDY DESIGN AND METHODS: A total of 12 donor and 12 recipient inbred isogenic adult (4 weeks old) Fisher rats were used to mimic autologous transplantation. Myocardial damage was obtained in recipient rats by cryoinjury. MMNCs were purified, labeled with PKH26 (a red fluorescent cell dye), and infused 7 days after the injury through the femoral vein of recipient rats. One week after peripheral administration, the number of homed MMNCs was assessed and the infarct size was correlated with the number of cells present in the target. RESULTS: Labeled cells were found only in the injured myocardium of the treated animals (n = 6), where a mean of 12 +/- 3 PKH26+ cells per section examined were found; a significant correlation was found between the infarct size and the estimated number of cells (p = 0.008) CONCLUSION: These data indicate that the homing of MMNCs is related to the extent of the myocardial injury, suggesting that cellular therapy for regeneration of damaged myocardium should be individualized by taking into consideration the extension of the area to repair.

Left ventricular remodeling after experimental myocardial cryoinjury in rats.

The standard coronary ligation, the most studied model of experimental myocardial infarction in rats, is limited by high mortality and produces unpredictable areas of necrosis. To standardize the location and size of the infarct and to elucidate the mechanisms of myocardial remodeling and its progression to heart failure, we studied the functional, structural, and ultrastructural changes of myocardial infarction produced by experimental myocardial cryoinjury. The cryoinjury was successful in 24 (80%) of 30 male adult CD rats. A subepicardial infarct was documented on echocardiograms, with an average size of about 21%. Macroscopic examination reflected closely the stamp of the instrument used, without transition zones to viable myocardium. Histological examination, during the acute setting, revealed an extensive area of coagulation necrosis and hemorrhage in the subepicardium. An inflammatory infiltrate was evident since the 7th hour, whereas the reparative phase started within the first week, with proliferation of fibroblasts, endothelial cells, and myocytes. From the 7th day, deposition of collagen fibers was reported with a reparative scar completed at the 30th day. Ultrastructural study revealed vascular capillary damage and irreversible alterations of the myocytes in the acute setting and confirmed the histological findings of the later phases. The damage was associated with a progressive left ventricular (LV) remodeling, including thinning of the infarcted area, hypertrophy of the noninfarcted myocardium, and significant LV dilation. This process started from the 60th day and progressed over the subsequent 120 days period; at 180 days, a significant increase in LV filling pressure, indicative of heart failure, was found. In conclusion, myocardial cryodamage, although different in respect to ischemic damage, causes a standardized injury reproducing the cellular patterns of coagulation necrosis, early microvascular reperfusion, hemorrhage, inflammation, reparation, and scarring observed in myocardial infarction with a late evolution toward heart failure. This model is therefore suitable to study myocardial repair after injury.

Homing of peripherally injected bone marrow cells in rat after experimental myocardial injury.

BACKGROUND AND OBJECTIVES: Significant progress has been achieved during the past 10 years in cell transplantation and recent research has focused on the possibility of improving ventricular function after myocardial infarction. Most studies in the field of cardiac tissue repair are performed by direct intramyocardial injection of cells of different origin. Since this approach requires a surgical intervention, in this study we investigated the feasibility of non-invasive administration of bone marrow mononuclear cells (BMMNCs) by assessing the fate of peripherally injected, purified, labeled cells in cryodamaged hearts. DESIGN AND METHODS: Ten donor and ten recipient inbred isogenic adult (4 weeks old) Fisher rats were used as models to mimic autologous transplantation. Myocardial damage was obtained in recipient rats by placing a frozen metal probe on the anterior left ventricular wall for 15 seconds (freeze-thaw injury technique). BMMNCs were purified and labeled with a red fluorescent cell dye. Seven days after the injury about 15-25x10(6) cells were infused through the femoral vein of recipient rats. Seven days after the infusion, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. RESULTS: Labeled cells were found only in the injured area of the heart and not in the normal tissue, and a limited number of cells were identified in the spleen of all the animals. Most of the labeled cells in the infarcted area were Thy-1(+) and some were CD34(+). INTERPRETATION AND CONCLUSIONS: Our data suggest that peripherally injected BMMNCs can traffic through the circulation to the site of damage; we hypothesize that tissue injury leads to the priming of a cytokine cascade acting as chemoattractant for the infused cells.

ANG II increases TIMP-1 expression in rat aortic smooth muscle cells in vivo.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tissue remodeling processes. TIMP-1 is the main native inhibitor of MMPs and it contributes to the development of tissue fibrosis. It is known that ANG II plays a fundamental role in vascular remodeling. In this study, we investigated whether ANG II modulates TIMP-1 expression in rat aortic smooth muscle cells. In vitro, ANG II induces TIMP-1 mRNA expression in a dose-dependent manner. The maximal increase in TIMP-1 expression was present after 3 h of ANG II stimulation. The ANG II increase in TIMP-1 expression was mediated by the ANG type 1 receptors because it was blocked by losartan. The increase in TIMP-1 expression was present after the first ANG II treatment, whereas repeated treatments (3 and 5 times) did not modify TIMP-1 expression. In vivo, exogenous ANG II was administered to Sprague-Dawley rats (200 ng. kg(-1). min(-1) sc) for 6 and 25 days. Control rats received physiological saline. After treatment, systolic blood pressure was significantly higher (P < 0.01), whereas plasma renin activity was suppressed (P < 0.01), in ANG II-treated rats. ANG II increased TIMP-1 expression in the aorta of ANG II-treated rats both at the mRNA (P < 0.05) and protein levels as evaluated by Western blotting (P < 0.05) and/or immunohistochemistry. Neither histological modifications at the vascular wall nor differences in collagen content in the tunica media were present in both the ANG II- and saline-treated groups. Our data demonstrate that ANG II increases TIMP-1 expression in rat aortic smooth muscle cells. In vivo, both short- and long-term chronic ANG II treatments increase TIMP-1 expression in the rat aorta. TIMP-1 induction by ANG II in aortic smooth muscle cells occurs in the absence of histological changes at the vascular wall.

Neuronal nitric oxide synthase and renin stimulation by sodium deprivation are dependent on the renal nerves.

OBJECTIVE: The aim of the present study was to evaluate the role of the renal nerves in the regulation of neuronal nitric oxide synthase (nNOS) gene expression in normotensive rats on different sodium balance. METHODS: Thirty-six male Sprague-Dawley rats were divided into six experimental groups combining three diets with different NaCl content (normal, 0.4%; low, 0.04%; or high, 4.0%), and bilateral renal denervation or sham denervation. After 7 days of dietary treatment, all rats were sacrificed and plasma renin activity (PRA) measured. The nNOS and renin messenger RNA (mRNA) levels in the renal cortex were determined by semiquantitative polymerase chain reaction. RESULTS: PRA was higher in animals with low sodium diet compared with those with standard diet, while it was lower in animals with high sodium diet. Renal denervation decreased PRA in normal and low sodium groups, while it did not alter the PRA values in the high sodium group. The nNOS gene expression significantly increased in rats fed with the low sodium diet compared with the standard diet group, and it significantly decreased in rats with the high sodium diet. Renal denervation significantly reduced nNOS mRNA levels in rats receiving the low sodium diet, but did not significantly influence nNOS mRNA in normal and high sodium groups. Renin mRNA was influenced by diets and denervation in a parallel way to nNOS mRNA. CONCLUSION: The renal nerves mediate the increase of renin and nNOS mRNA during sodium restriction, while the suppression of nNOS and renin gene expression during a sodium load is independent of the presence of the renal nerves. The parallel changes in renin and nNOS mRNA during different sodium intakes suggest that nNOS can be part of the complex, and still largely unclarified, macula densa mechanism of renin regulation.