Giuseppe Busca

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

Abstract 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.

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 Parkinsons 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.

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 &bgr;-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 &bgr;-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 &bgr;-blockade (4.11±1.23%, P<0.05 versus both SBb and BSx). &bgr;-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 &agr;-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 &agr;-adrenergic but not &bgr;-adrenergic receptors.

ANGIOTENSIN II INCREASES TISSUE‐SPECIFIC INHIBITOR OF METALLOPROTEINASE‐2 EXPRESSION IN RAT AORTIC SMOOTH MUSCLE CELLS IN VIVO: EVIDENCE OF A PRESSURE‐INDEPENDENT EFFECT

1 Angiotensin (Ang) II plays a major role in vascular remodelling. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in the tissue remodelling processes. The aim of the present study was to investigate whether AngII modulates TIMP‐2 expression in rat aortic smooth muscle cells in vivo. 2 Angiotensin II (200 ng/kg per min, s.c.) or AngII + losartan (10 mg/kg per day, s.c.) or normal saline was administered continuously by osmotic minipumps to Sprague‐Dawley rats for 1 week. In addition, the effect of endogenous AngII on TIMP‐2 expression was evaluated in renovascular hypertensive rats (two kidney, one clip (2K1C) and one kidney, one clip (1K1C) models). Control rats (sham 2K1C and sham 1K1C rats) underwent sham‐clipping of the left renal artery. At the end of the treatment, plasma renin activity was measured by radioimmunoassay, aortic TIMP‐2 mRNA expression was evaluated by real‐time polymerase chain reaction and/or northern blotting and protein expression was evaluated by immunohistochemistry. Systolic blood pressure (SBP) was measured twice a week by the tail‐cuff method. 3 Exogenous AngII administration produced the expected increase in SBP (P = 0.02) compared with the control saline‐treated group. The increase in SBP was abolished in AngII + losartan‐treated rats. Administration of AngII caused a significant increase in TIMP‐2 expression (P = 0.01) in rat aortic smooth muscle cells that was abolished in AngII + losartan‐treated rats. In renovascular hypertensive rats, SBP was higher (P < 0.0001) in 2K1C and 1K1C rats compared with the corresponding sham‐operated rats. Plasma renin activity was higher (P < 0.01) in 2K1C rats compared with the other groups. The expression of TIMP‐2 was significantly (P < 0.05) increased only in 2K1C rats. 4 Our in vivo data demonstrate that exogenous and endogenous AngII increases TIMP‐2 expression in rat aortic smooth muscle cells. This effect is not dependent on the AngII‐induced increase in blood pressure and is mediated by angiotensin AT1 receptors.

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 β-blockade, whereas interventions interfering with α-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), β-adrenoceptor blockade (propranolol [Pro], 40 mg/kg per day PO), or α-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, α-adrenoceptors are importantly involved in the pathogenesis of cardiovascular deterioration and disease progression.

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.

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

BackgroundBone 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.MethodsWe 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)1β, IL-6, tumor necrosis factor (TNF)α 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.ResultsConcerning 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 ; TNFα inversely correlated with LV fractional shortening.ConclusionAfter 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.

Role of the renal nerves in the control of renin synthesis during different sodium intakes in the rat.

Objective The aim of this study was to evaluate the role of the renal nerves in the regulation of renin synthesis in normotensive rats at different sodium balance. Methods Forty-eight male Sprague–Dawley rats were divided in six experimental groups, combining three diets at different NaCl content (normal 0.4%, low 0.04% or high 4.0%), and the surgical, bilateral renal denervation or the sham procedure. After 7 days of dietary treatment, all rats were sacrificed and plasma renin activity (PRA) was measured. Renin messenger RNA (mRNA) levels in the renal cortex were determined by semiquantitative polymerase chain reaction. Results PRA was higher in animals fed the low sodium diet compared with those at standard diet, while it was lower in animals fed the 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. Renin gene expression significantly increased in rats fed with the low sodium diet compared with the standard diet group, and significantly decreased in rats fed the high sodium diet. Renal denervation significantly reduced renin mRNA levels in rats receiving the low sodium diet, but did not produce any significant change in normal or high-sodium groups. Conclusion The activation of renin gene expression during sodium depletion in rats is dependent on the presence of the renal nerves, while the suppression of renin gene expression during a sodium load seems to be due to the macula densa mechanism alone.

Effects of adenosine receptor agonists on renal function in anaesthetized rats

Objectives To investigate the effects of the interaction between adenosine receptors and renal nerves on urinary sodium excretion and glomerular filtration rate. Methods and design The effects on water and sodium excretion and glomerular filtration rate of A1 [2-chloro-N6-cyclopentyl-adenosine (CCPA)] and A2 [2-hesinyl-5′-N-ethyl-carboxamido-adenosine (2HE-NECA)] adenosine agonists were studied in anaesthetized rats with one kidney surgically denervated. Arterial blood pressure, heart rate and rate of urine flow from each kidney were continuously recorded; inulin clearance was used as an index of glomerular filtration rate. The experiments were performed with three groups of rats, into which, after a control period of 20 min, CCPA, 2HE-NECA or vehicle was infused for two subsequent 20 min periods. Result During infusion of CCPA, the slight decrease in arterial pressure was associated with a transient decrease in glomerular filtration rate and marked long-lasting decreases in heart rate, water and sodium excretion and fractional sodium excretion. The response of the innervated kidney was similar to the response of the denervated kidney. Infusion of 2HE-NECA caused decreases in arterial pressure, glomerular filtration rate and excretion of water and sodium associated with an increase in heart rate. The reduction of water and sodium excretion from the innervated kidney was larger than that from the denervated kidney. Conclusions Activation both of A1 and of A2 receptor causes a reduction in urinary water and sodium excretion. The renal response to activation of A2 receptors is enhanced by the presence of renal nerves, whereas the response to activation of A1 receptors is not influenced by renal nerves.

Effects of adenosine-receptor agonists on renin release in anaesthetized rats

Objective To investigate the effects of the interaction between adenosine receptors and renal nerves on renin release. Materials and methods The effects on renin secretion of A1 (2-chloro-N 6-cyclopentil-adenosine) and A2 (2-hexynil-5‘-N-ethyl-carboxamido-adenosine) adenosine-receptor agonists were studied in two groups of anaesthetized rats, each with one kidney surgically denervated. Arterial blood pressure and the renal blood flow of innervated and denervated kidneys were continuously recorded. Cannulae were inserted into both renal veins through femoral veins. After 1 h of rest, A1 and A2 agonists were intravenously infused for 30 min in the two groups of rats. Plasma renin activity was measured by radioimmunoassay in blood samples drawn simultaneously from both renal veins and the femoral artery before and after the drug infusion. Results Infusions of A1 and A2 agonists produced comparable hypotensive effects. During A1 agonist administration, the heart rate decreased significantly, but it did not change after A2 agonist treatment. Renal blood flow was reduced by administration of the A1 agonist in both kidneys, while A2 agonist administration significantly reduced the renal blood flow of the innervated kidney only. The veno-arterial difference in plasma renin activity decreased after the A1 agonist infusion in both kidneys, but after the A2 agonist infusion it increased significantly in the innervated kidney only. Conclusions Renal nerves do not influence the inhibition of renin release mediated by A1 adenosine receptors. In vivo, A2-receptor agonist administration can stimulate renin release only in the presence of intact renal nerves.