Giovanna Castoldi

MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin II-dependent hypertension.

MicroRNAs play an important role in myocardial diseases. MiR‐133a regulates cardiac hypertrophy, while miR‐29b is involved in cardiac fibrosis. The aim of this study was to evaluate whether miR‐133a and miR‐29b play a role in myocardial fibrosis caused by Angiotensin II (Ang II)‐dependent hypertension. Sprague–Dawley rats were treated for 4 weeks with Ang II (200 ng/kg/min) or Ang II + irbesartan (50 mg/kg/day in drinking water), or saline by osmotic minipumps. At the end of the experimental period, cardiac miR‐133a and miR‐29b expression was measured by real‐time PCR, and myocardial fibrosis was evaluated by morphometric analysis. A computer‐based prediction algorithm led to the identification of collagen 1a1 (Col1A1) as a putative target of miR‐133a. A reporter plasmid bearing the 3′‐untranslated regions (UTRs) of Col1A1 mRNA was constructed and luciferase assay was performed. MiR‐133a suppressed the activity of luciferase when the reporter gene was linked to a 3′‐UTR segment of Col1A1 (P < 0.01). Mutation of miR‐133a binding sites in the 3′‐UTR of Col1A1 mRNA abolished miR‐133a‐mediated repression of reporter gene activity, showing that Col1A1 is a real target of miR‐133a. In vivo, Ang II caused an increase in systolic blood pressure (P < 0.0001, tail cuff) and myocardial fibrosis in presence of a decrease in miR‐133a (P < 0.01) and miR‐29b (P < 0.01), and an increase in Col1A1 expression (P < 0.01). These effects were abolished by Ang II administration + irbesartan. These data demonstrate a relationship between miR‐133a and Col1A1, suggesting that myocardial fibrosis occurring in Ang II‐dependent hypertension is regulated by the down‐regulation of miR‐133a and miR‐29b through the modulation of Col1A1 expression. J. Cell. Physiol. 227: 850–856, 2012.

NF‐κB mediated miR‐26a regulation in cardiac fibrosis

Micro‐RNAs (miRNAs) are a class of small non‐coding RNAs, recently emerged as a post‐transcriptional regulator having a key role in various cardiac pathologies. Among them, cardiac fibrosis that occurs as a result from an imbalance of extracellular matrix proteins turnover and is a highly debilitating process that eventually lead to organ dysfunction. An emerging theme on is that miRNAs participate in feedback loop with transcription factors that regulate their transcription. NF‐κB, a key transcription factor regulator controls a series of gene program in various cardiac diseases through positive and negative feedback mechanism. But, NF‐κB mediated miRNA regulation in cardiac fibrosis remains obscure. Bioinformatics analysis revealed that miR‐26a has targets collagen I and CTGF and possesses putative NF‐κB binding element in its promoter region. Here, we show that inhibition of NF‐κB in cardiac fibroblast restores miR‐26a expression, attenuating collagen I, and CTGF gene expression in the presence of Ang II, conferring a feedback regulatory mechanism in cardiac fibrosis. The target genes for miR‐26a were confirmed using 3′‐UTR luciferase reporter assays for collagen I and CTGF genes. Using NF‐κB reporter assays, we determine that miR‐26a overexpression inhibits NF‐κB activity. Finally, we show that miR‐26a expression is restored along with the attenuation of collagen I and CTGF genes in cardiac specific IkBa triple mutant transgenic mice (preventing NF‐κB activation) subjected to 4 weeks transverse aortic banding (TAC), compared to wild type (WT) mice. The data indicate a potential role of miR‐26a in cardiac fibrosis and, offer novel therapeutic intervention. J. Cell. Physiol. 228: 1433–1442, 2013.

Prevention of diabetic nephropathy by compound 21, selective agonist of angiotensin type 2 receptors, in Zucker diabetic fatty rats

The aim of this study was to evaluate the effect of compound 21 (C21), a selective AT2 receptor agonist, on diabetic nephropathy and the potential additive effect of C21, when associated with losartan treatment, on the development of albuminuria and renal fibrosis in Zucker diabetic fatty (ZDF) rats. The experiments lasted 15 wk (from 5 to 20 wk of age) and were performed in 40 ZDF rats and 12 control lean rats. ZDF rats were divided into 4 groups: 1) 9 rats were treated with losartan; 2) 10 rats were treated with C21; 3) 9 rats were treated with losartan plus C21; and 4) 12 rats were maintained without any treatment. ZDF rats showed an increase in blood glucose level, albuminuria, renal fibrosis, macrophage infiltration, and TNF-α expression and a reduction of glomerular nephrin expression compared with control lean rats. C21 treatment reduced renal glomerular, tubulointerstitial, and perivascular fibrosis, and macrophage infiltration and TNF-α expression in ZDF rats. C21 treatment caused a decrease in albuminuria in ZDF rats up to 11 wk of age. Losartan decreased macrophage infiltration, TNF-α expression, and renal glomerular and perivascular fibrosis, restored glomerular nephrin expression, but did not affect tubulointerstitial fibrosis. Losartan treatment caused a decrease in albuminuria in ZDF rats up to 15 wk of age. At the end of the protocol, only the combination of C21 plus losartan significantly reduced albuminuria in ZDF rats. These data demonstrate that C21 has beneficial effects on diabetic nephropathy, suggesting the combination of C21 and losartan as a novel pharmacological tool to slow the progression of nephropathy in type II diabetes.

Association between serum values of C-reactive protein and cytokine production in whole blood of patients with Type 2 diabetes

Diabetes mellitus accelerates atherosclerotic processes, and it is known that inflammation plays a key role in atherosclerosis. The aim of the present study was to evaluate in patients with Type 2 diabetes whether serum levels of CRP (C-reactive protein) are associated with cytokine production in whole blood. A total of 89 outpatients with Type 2 diabetes were enrolled, and blood pressure, body mass index, fasting blood glucose, glycated haemoglobin, cholesterol, triacylglycerols (triglycerides) and hs-CRP (high-sensitivity CRP) were measured. IL-6 (interleukin-6), IL-1beta (interleukin-1beta) and TNF-alpha (tumour necrosis factor-alpha) were measured before and after 24 h of incubation of whole blood with LPS (lipopolysaccharide) or saline. The basal values of IL-1beta, IL-6 and TNF-alpha were low and were not significantly related to hs-CRP levels. A univariate analysis showed that the level of IL-1beta and IL-6, obtained after 24 h of incubation of whole blood with LPS, increased significantly with increasing levels of hs-CRP and, after adjusting for potential confounders, IL-1beta still remained statistically significant. In our sample of patients with Type 2 diabetes, there was no association between serum hs-CRP levels and basal levels of IL-6, IL-1beta and TNF-alpha. Conversely, a significant association was observed between serum hs-CRP levels and IL-1beta and IL-6 production after 24 h of incubation of whole blood with LPS. In conclusion, our data suggest that patients with Type 2 diabetes and high hs-CRP levels may have an enhanced reactivity in response to specific stimuli that produce different interleukins, with possible implications in inflammatory atherosclerotic processes.

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.

Renal Antifibrotic Effect of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline in Diabetic Rats

Background and Aim: Diabetic nephropathy is the main cause of end-stage renal disease. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a physiological tetrapeptide hydrolyzed by the angiotensin-converting enzyme (ACE), has antifibrotic effects in the cardiovascular system and in the kidney in experimental models of hypertension, heart failure and renal disease. The aim of the study was to evaluate the effect of Ac-SDKP in diabetic nephropathy and the potential additive effect of Ac-SDKP, when compared to ACE inhibitors alone, on the development of renal fibrosis. Method: Diabetes was induced in 28 Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin. Control rats (n = 10) received only buffer solution. An ACE inhibitor (ramipril, 3 mg/kg/day) was administered to 11 diabetic rats. After 2 months, Ac-SDKP (1 mg/kg/day) was administered by osmotic minipumps for 8 weeks to 7 diabetic rats and to 6 diabetic rats treated with ramipril. Osmotic minipumps delivered saline solution in the corresponding sham-treated rats (diabetic rats, n = 10, and ramipril-treated diabetic rats, n = 5). Results: Diabetic rats showed a significant increase in blood glucose level, urinary albumin excretion and renal fibrosis, and a reduction of glomerular nephrin expression with respect to control rats. Ac-SDKP administration significantly reduced renal fibrosis in diabetic rats, without significantly reducing urinary albumin excretion. Ramipril treatment caused a significant decrease in albuminuria and renal fibrosis and restored glomerular nephrin expression. Administration of Ac-SDKP, in addition to ramipril, further reduced renal fibrosis with respect to ramipril alone, while it did not improve the antiproteinuric effect of ramipril. Conclusion: Ac-SDKP administration reduces renal fibrosis in diabetic nephropathy. Addition of Ac-SDKP to ACE inhibition therapy improves the reduction of renal fibrosis with respect to ACE inhibition alone, suggesting a beneficial effect of this pharmacological association in diabetic nephropathy.

Radiofrequency-based carotid wall tracking: a comparison between two different systems.

Objectives: A direct measurement of carotid stiffness implies an accurate assessment of changes in carotid diameter and pressure during cardiac cycle. Radiofrequency-based wall-tracking systems (WTS) are capable to track arterial wall movement with adequate spatial and temporal resolution, and to provide carotid pressure estimate from calibrated distension waveforms. The aim of the present study was to compare the values of carotid distension and beta-stiffness index acquired in the same population by two commercially available WTS, in order to determine whether their measures can be pooled in clinical studies. In addition, a local carotid pulse pressure (PP) obtained from calibrated distension waveforms was compared with that obtained from pressure waveforms. Methods: In 105 patients, right common carotid artery (CCA) systo-diastolic excursions were assessed during the same session and by the same operator both by WTS implemented in Esaote system (QAS, MyLab) and in Aloka system (E-track; Alpha 10). In 78 patients, carotid PP was also estimated by applanation tonometry. Results: Despite comparable blood pressure and heart rate values during the two acquisitions, CCA distension was significantly lower (363 ± 162 vs. 458 ± 176 &mgr;m, P < 0.0001) and beta-stiffness index (11.9 ± 5.5 vs. 9.4 ± 3.8, P < 0.0001) higher with Esaote as compared to Aloka. PP obtained from calibrated pressure and distension waveforms was comparable (42.6 ± 11.4 vs. 43.0 ± 10.7 mmHg, P = 0.51). Conclusions: The values of carotid distension and stiffness obtained by two different WTS are not interchangeable and cannot be merged into a common database. Calibrated distension curves may provide an acceptable estimate of local carotid pressure.

Oxidative stress biomarkers and chromogranin A in uremic patients: effects of dialytic treatment.

OBJECTIVE To evaluate oxidative stress in uremia and dialysis and chromogranin A, a stress hormone that could be related to oxidative processes. METHODS Plasma oxidative stress biomarkers (-SH, 8-OHdG, and ox-LDL) and chromogranin A were measured in 89 outpatients (21 uremic patients, 17 in peritoneal dialysis, and 51 in haemodialysis), and in 18 subjects with normal renal function. RESULTS -SH groups were significantly reduced in heamodialysis, peritoneal, and uremic patients as compared with the control group (p=0.01), while 8-OHdG was increased (p<0.01). No differences were observed for ox-LDL. Chromogranin A was increased in uremic, peritoneal and haemodialysis patients (p<0.01), showing a positive correlation to 8-OHdG (p<0.01). CONCLUSION Oxidative stress biomarkers and chromogranin A levels differ between control subjects when compared to both uremic and dialysis patients. No differences were observed between uremic and dialysis patients, suggesting that uremia is the major source of the increase in oxidative stress and CgA levels in patients with end stage renal disease.

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.

Different regulation of miR‐29a‐3p in glomeruli and tubules in an experimental model of angiotensin II‐dependent hypertension: potential role in renal fibrosis

The aim of this study was to evaluate the role of the angiotensin II (Ang II) induced‐differential miRNA expression in renal glomerular and tubulo‐interstitial fibrosis in an experimental model of Ang II‐dependent hypertension. To clarify this issue, Sprague Dawley rats were treated with Ang II (200 ng/kg per minute, n = 15) or physiological saline (n = 14) for 4 weeks. Systolic blood pressure and albuminuria were measured every 2 weeks. At the end of the experimental period, renal glomerular and tubulo‐interstitial fibrosis was evaluated by histomorphometric analysis, after Sirius‐Red and Massons trichrome staining. Ang II increased systolic blood pressure (P < 0.0001), albuminuria (P < 0.01) and both glomerular and tubulo‐interstitial fibrosis (P < 0.01). Using laser capture microdissection and miRNA microarray analysis this study showed that miR‐29a‐3p was down‐regulated in renal tubules and up‐regulated in glomeruli. Real‐time polymerase chain reaction (PCR) experiments confirmed in Ang II‐treated rats a down‐regulation of miR‐29a‐3p in tubules (P < 0.01), while no significant changes were observed in glomeruli. Matrix metalloproteinase‐2 (MMP‐2) was identified as putative miR‐29a‐3p target (by TargetScan, miRanda, Tarbase software) and functionally confirmed by luciferase activity assay. These data demonstrate that the effects of Ang II on miR‐29a‐3p expression in renal tubules is different from the one exerted in the glomeruli and that miR‐29a‐3p targets MMP‐2. These results suggest that the development of renal fibrosis at glomerular and tubulo‐interstitial level depends on different molecular mechanisms.