Alessandro Cavarape

Increased Circulating Intercellular Adhesion Molecule-1 Levels in Type II Diabetic Patients: The Possible Role of Metabolic Control and Oxidative Stress

Blood levels of the circulating form of the integrin intercellular adhesion molecule-1 (ICAM-1), malondialdehyde (MDA), and hemoglobin A1c (HbA1c) were studied at baseline and 3 months after improved metabolic control in 25 type II diabetic patients without signs of macroangiopathy, and were compared with those in 15 matched healthy normal controls. Circulating ICAM-1 and MDA levels were increased in diabetic patients, both at baseline and 3 months later. However, with improving metabolic control HbA1c, circulating ICAM-1, and MDA significantly decreased. A significant correlation between circulating ICAM-1, HbA1c, and MDA was found in diabetic patients at each time. Multiple regression analysis considering circulating ICAM-1 as the dependent variable and HbA1c and MDA as independent variables, showed a significant correlation between the three variable at each time. Similar correlations were found in control subjects. These data show increased levels of circulating ICAM-1 in type II diabetic patients, independent of the presence of macroangiopathy. Moreover, these results suggest that oxidative stress and metabolic control might participate in determining increased circulating ICAM-1 levels in both type II diabetic patients and normal subjects.

Rho-Kinase Inhibition Blunts Renal Vasoconstriction Induced by Distinct Signaling Pathways In Vivo

In addition to intracellular calcium, which activates myosin light chain (MLC) kinase, MLC phosphorylation and hence contraction is importantly regulated by MLC phosphatase (MLCP). Recent evidence suggests that distinct signaling cascades of vasoactive hormones interact with the Rho/Rho kinase (ROK) pathway, affecting the activity of MLCP. The present study measured the impact of ROK inhibition on vascular F-actin distribution and on vasoconstriction induced by activation/inhibition of distinct signaling pathways in vivo in the microcirculation of the split hydronephrotic rat kidney. Local application of the ROK inhibitors Y-27632 or HA-1077 induced marked dilation of pre- and postglomerular vessels. Activation of phospholipase C with the endothelin ET B agonist IRL 1620, inhibition of soluble guanylyl cyclase with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibition of adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine (CPA) reduced glomerular blood flow (GBF) by about 50% through vasoconstriction at different vascular levels. ROK inhibition with Y-27632 or HA-1077, but not protein kinase C inhibition with Ro 31-8220, blunted ET B-induced vasoconstriction. Furthermore, the reduction of GBF and of vascular diameters in response to ODQ or CPA were abolished by pretreatment with Y-27632. ROK inhibitors prevented constriction of preglomerular vessels and of efferent arterioles with equal effectiveness. Confocal microscopy demonstrated that Y-27632 did not change F-actin content and distribution in renal vessels. The results suggest that ROK inhibition might be considered as a potent treatment of renal vasoconstriction, because it interferes with constriction induced by distinct signaling pathways in renal vessels without affecting F-actin structure.

High-fructose diet decreases catalase mRNA levels in rat tissues

Insulin resistance and hyperinsulinemia have recently been identified as independent determinants of several risk factors for cardiovascular disease. The generation of reactive oxygen species (ROS) may play an important role as a final common mediator by which glucose and insulin resistance might contribute to development of cardiovascular disease and hypertension. The aim of the present study was to evaluate changes on mRNA expression of antioxidant enzymes [catalase, Cu-Zn superoxide dismutase (Cu-ZnSOD), MnSOD], blood pressure and metabolic parameters in insulin resistance that follow feeding normotensive Wistar rats a high-fructose-enriched diet. In our investigation 26 normal male Wistar rats were fed a highfructose diet for 2 weeks (no.=14) or normal chow to serve as a control group (no.=12). In vivo insulin resistance was verified in a subgroup of control and fructose-fed rats by the euglycemic hyperinsulinemic clamp technique at 2 different insulin infusion rates, 29 (submaximal stimulation) and 290 (maximal stimulation) pmol/kg/min respectively. The glucose infusion rate (GIR) was not significantly different in the two groups during the submaximal infusion of insulin (1.4±0.8 mmol/kg/min in fructosefed rats vs 1.6±0.7 mmol/kg/min in control rats, NS) while in fructose-fed rats it was significantly lower (-29.8%) than in control rats during maximal infusion of insulin (2.6±0.3 mmol/kg/min vs 3.7±0.3 mmol/kg/min, p<0.05). Fructose feeding markedly reduced the expression of catalase mRNA and Cu-ZnSOD mRNA in the liver, catalase mRNA in the heart (p<0.05). A tendency of fructose feeding to reduce the expression of antioxidant enzymes in skeletal muscle and adipose tissue was also observed (NS). Fructose feeding also increased plasma uric acid (119.9±30.4 vs 42.1±10 μmol/l, p<0.05) and systemic blood pressure (128±4 vs 109±5 mmHg, p<0.05) respect to control animals. No significant changes were observed in plasma levels of glycemia and tryglycerides. Our study suggests that in non-hyperglycemic, fructose-fed insulin-resistant rats the expression of catalase is inhibited in liver and heart. This condition might lead to higher susceptibility to oxidative stress in insulin resistance. However, an adaptive cellular response to maintain the effectiveness of intracellular signaling pathways mediated by insulinactivated hydrogen peroxide generating systems may also be hypothesized.

New risk factors for atherosclerosis in hypertension: focus on the prothrombotic state and lipoprotein(a).

Although adequate control of blood pressure is of basic importance in cardiovascular prevention in hypertensive patients, correction of additional risk factors is an integral part of their management. In addition to classical risk factors, epidemiological research has identified a number of other conditions that might significantly contribute to cardiovascular risk in the general population and might achieve specific relevance in patients with high blood pressure. In fact, more than 20% of patients with premature cardiovascular events do not have any of the traditional risk factors and, although effective intervention on blood pressure and additional risk factors has significantly reduced cardiovascular morbidity and mortality, the contribution to stroke, coronary artery disease and renal failure is still unacceptably high. Evaluation of new risk factors may further expand our capacity to predict atherothrombotic events when these factors are included along with the traditional ones in the assessment of global cardiovascular risk in hypertensive patients. Because it could be anticipated that the role of these novel factors will become increasingly evident in the future, researchers with an interest in hypertension and physicians dealing with problems related to cardiovascular prevention should give them appropriate consideration. This review summarizes the basic biology and clinical evidence of two emerging risk factors that are reciprocally related and contribute to the development and progression of organ damage in hypertension: the prothrombotic state and lipoprotein(a).

ANG II- and TxA2-induced mesenteric vasoconstriction in rats is mediated by separate cell signaling pathways

Studies in vitro have demonstrated that vasoconstrictor agents increase intracellular Ca(2+) and activate protein kinase C (PKC) to elevate vascular tone. The aim of the present study was to determine the importance of these signaling pathways for angiotensin II (ANG II) and thromboxane A(2) (TxA(2)) in regulating mesenteric blood flow (MBF) in vivo. In anesthetized rats increasing doses of ANG II or the TxA(2) agonist U-46619 were administered into the superior mesenteric artery to reduce MBF. Intra-arterial infusion of inhibitors served to examine the contribution of different pathways: 8-(diethylamino)octyl 3,4,5-trimethoxybenoate hydrochloride (TMB-8) to inhibit intracellular Ca(2+) release, nifedipine to block transmembrane Ca(2+) influx through the L-type Ca(2+) channel, and staurosporine to inhibit PKC. Each of the inhibitors attenuated ANG II-induced reductions in MBF, and all dose-response curves were shifted to the right to an approximately threefold higher ANG II dose. Combinations of the inhibitors revealed that their effects were additive; together they abolished the vasoconstrictor action of ANG II completely. In contrast, the dose-response curve for U-46619 was not affected by any of the inhibitors infused either separately or together. The results demonstrate that a rise in intracellular Ca(2+) and activation of PKC are major mediators of the vasoconstrictor effect of ANG II in mesenteric circulation, but they play a subordinate role, if any, for the effects of TxA(2). Because TxA(2) plays a major role only under pathological conditions, the uncontrolled vasoconstriction appears to be associated with the recruitment of novel signal transduction pathways.Studies in vitro have demonstrated that vasoconstrictor agents increase intracellular Ca2+ and activate protein kinase C (PKC) to elevate vascular tone. The aim of the present study was to determine the importance of these signaling pathways for angiotensin II (ANG II) and thromboxane A2(TxA2) in regulating mesenteric blood flow (MBF) in vivo. In anesthetized rats increasing doses of ANG II or the TxA2 agonist U-46619 were administered into the superior mesenteric artery to reduce MBF. Intra-arterial infusion of inhibitors served to examine the contribution of different pathways: 8-(diethylamino)octyl 3,4,5-trimethoxybenoate hydrochloride (TMB-8) to inhibit intracellular Ca2+ release, nifedipine to block transmembrane Ca2+ influx through the L-type Ca2+ channel, and staurosporine to inhibit PKC. Each of the inhibitors attenuated ANG II-induced reductions in MBF, and all dose-response curves were shifted to the right to an approximately threefold higher ANG II dose. Combinations of the inhibitors revealed that their effects were additive; together they abolished the vasoconstrictor action of ANG II completely. In contrast, the dose-response curve for U-46619 was not affected by any of the inhibitors infused either separately or together. The results demonstrate that a rise in intracellular Ca2+ and activation of PKC are major mediators of the vasoconstrictor effect of ANG II in mesenteric circulation, but they play a subordinate role, if any, for the effects of TxA2. Because TxA2 plays a major role only under pathological conditions, the uncontrolled vasoconstriction appears to be associated with the recruitment of novel signal transduction pathways.

Effects of BQ-123 on systemic and renal hemodynamic responses to endothelin-1 in the rat split hydronephrotic kidney

Objective To assess the site of action of endothelin-1 in vessels of different sizes in the kidney in vivo and investigate the function of endothelin A (ETA) receptors in mediating renal and systemic vasoconstriction. Design The luminal diameters of different vessels were measured and glomerular blood flow in cortical glomeruli was determined by intravital videomicroscopy in the split hydronephrotic kidney of anesthetized female Wistar rats. Methods The rats were infused with endothelin-1 (40 pmol/kg per min) with or without pretreatment with the selective ETA-receptor antagonist BQ-123 (0.5 mg/kg). Aortic clamping was used to control renal blood pressure during the endothelin-1 infusion. Results Exogenous endothelin-1 induced a significant rise (30 ± 3%) in mean arterial pressure and a marked, long-lasting fall in glomerular blood flow (53 ± 3%) related to reduction of the inner diameter of arcuate (−30%), interlobular arteries (−33%) and afferent arterioles (−17%). Aortic clamping to normalize renal blood pressure did not attenuate the vasoconstriction and reduction in glomerular blood flow. Pretreatment with BQ-123 significantly reduced both the endothelin-1-induced rise in mean arterial pressure (12 ± 1%) and the fall in glomerular blood flow (−23 ± 11%). BQ-123 blunted the response to endothelin-1 in arcuate (−12%), interlobular (−11%) and afferent vessels (−5%). Acetylcholine and nitroprusside completely reversed the vasoconstriction in BQ-123-pretreated animals. Conclusions BQ-123 largely prevented the hemodynamic effects of exogenously administered endothelin-1. Our direct in-vivo techniques showed that ETA receptors are, at least in part, involved in endothelin-1-mediated vasoconstriction in the rat kidney, and support the hypothesis that ETA receptors may help to control arterial pressure in anesthetized rats.

Endotoxin priming and liver damage by experimental duodenal obstruction in the rat.

To verify whether endotoxin (LPS) might act as a priming cofactor of liver injury caused by obstructing the duodenum, four groups of male Wistar rats were studied. The first two groups comprised rats in which a closed duodenal loop (CDL) was created: CDL, n= 6 and CDL + LPS, n= 7; the next two groups comprised sham‐operated animals: Sham n= 6 and Sham + LPS, n= 6. LPS, 400 μg/kg bodyweight, was administered i.p. to the rats belonging to groups CDL + LPS and Sham + LPS, 24 h before laparotomy. Twenty‐four hours after laparotomy the animals were killed. Damage to bile ducts, extent and grading of coagulative and lytic spotty necrosis in liver tissue were evaluated morphologically. Coagulative necrosis was severe in four of seven rats of the group CDL + LPS, mild in six of six rats of group CDL, and absent in four of six and five of six rats of groups Sham and Sham + LPS (χ 2 32.8, P= 0.0001). The animals of group CDL + LPS had more frequently diffuse lytic spotty necrosis than the animals in the three other groups (χ 2 9.57 P< 0.01). The results of our study indicate that, in rodents subjected to a closed duodenal loop, priming with LPS exacerbates liver injury due to cholate stasis.

Endotoxin priming exacerbates acute reflux pancreatitis in the rat.

Recently, endotoxaemia has been reported as a prognostic marker in acute pancreatitis. However, the role of endotoxin in inducing or aggravating acute pancreatitis is not fully understood. We administered endotoxin 400 μg/kg i.p. to rats 24 h before performing either a closed duodenal loop (group B) or a sham operation (group D). Pancreatic damage and overall survival were compared with the results obtained in rats not exposed to endotoxin undergoing either closed duodenal loop (group A) or sham treatment (group C). In a first set of experiments, 24 h after laparotomy blood samples were collected and the animals were sacrificed; survival up to 8 days was estimated in a second set of experiments. Group B had higher lipase concentrations and more severe tissue damage than group C (P<0.05). A larger number of abscesses was observed in both group B and group D as compared to group C (P<0.05). Survival was significantly shorter in group B (P<0.0001). We conclude that priming with endotoxin worsens the extent of pancreatic damage induced by the closed duodenal loop procedure in the rat, possibly favouring selective homing of neutrophils to the site of inflammation, in similarity to what happens in the Shwartzman phenomenon.

Systemic and intratubular effects of cyclosporin-A and tacrolimus on the rat kidney

Cyclosporin-A and tacrolimus can cause hypertension and renal failure through endothelin receptors. The importance of tubular function was never investigated. The aim of this study was to compare the effects of intratubular injection of cyclosporin-A and tacrolimus with effects observed during systemic infusion. In 20 rats, either cyclosporin-A or tacrolimus was infused, 30 and 1 mg/kg i.v., respectively, in 30 min. Before and after administration, glomerular filtration rate, single nephron filtration rate, proximal and distal absolute reabsorption and percent reabsorption were measured by clearance and micropuncture techniques. In 22 other rats, single nephron filtration rate, absolute reabsorption, percent reabsorption, were measured at the last proximal and early distal tubules before and during intraluminal microinjection of either cyclosporin-A or tacrolimus. During cyclosporin-A and tacrolimus i.v. infusion, glomerular filtration rate fell from 536+/-43 to 448+/-37 microl/min (P<0.026) and from 408+/-33 to 284+/-81 microl/min (P<0. 02), single nephron filtration rate from 26.4+/-2.0 to 20.6+/-1.9 (P<0.002) and from 21.6+/-2.2 to 17.4+/-2.0 nl/min, respectively (P<0.02). The last proximal absolute reabsorption remained unchanged with cyclosporin-A (16.8+/-2.2 vs. 15.1+/-1.7 nl/min, P>0.444), but was slightly reduced by tacrolimus (14.4+/-1.7 vs. 11.3+/-1.7 nl/min, P<0.05). During microinjection, single nephron filtration rate was increased by cyclosporin-A (20+/-1 vs. 63+/-8 nl/min, P<0.0001), and tacrolimus (from 17+/-2 to 49+/-9 nl/min, P<0.0001), and so was reabsorption, independent of the sampling site. Cyclosporin-A and tacrolimus, indeed, raise single nephron filtration rate directly when injected intraluminally. Since this effect occurs in the direction opposite to that recorded during systemic infusion, it must be mediated through different pathways. The i.v. infusion of cyclosporin-A, but not tacrolimus, impairs glomerulo-tubular balance.

Pre-Procedural Statin Use Is Associated with Improved Long-Term Survival and Reduced Major Cardiovascular Events in Patients Undergoing Carotid Artery Stenting: A Retrospective Study

Carotid artery stenting (CAS) is a minimal invasive procedure used to resolve carotid occlusion that can be affected by peri-procedural complications. Statin use before CAS has shown to reduce peri-procedural risk and improve survival, though time-dependent cofactors that influence mortality has not been considered. The aim of this study was to evaluate long-term survival of patients who undergo CAS considering new occurred major adverse cardiovascular event (MACE) as time-dependent cofactor. In this study, 171 high cardiovascular risk patients (age 72 ± 8 years, 125 males) were enrolled after CAS procedure and were followed for a median of 8.4 years. Death occurred in 44% of patients with a mean time to death of 69 ± 39 months and MACE in 34% with a mean time of 35 ± 42 months. In patients who used or not statins at baseline, death occurred in 33% and 65%, respectively (p < 0.001). Survival analysis showed that statin use reduced risk of death (hazard ratio HR 0.36, 95% confidence interval CI 0.23–0.58, p < 0.0001). Including MACE as time-dependent variable did not change beneficial effects of statins. Additionally, statin use was associated with a protective effect on MACE (HR 0.48, 95% CI 0.27–0.85, p = 0.012); particularly, the prevalence of stroke was reduced by 59% (p = 0.018). In multivariate analysis, effects of statins were independent of demographic and anthropometric variables, prevalence of cardiovascular risk factors, renal function, antiplatelet use, and MACE occurrence. In conclusion, use of statins before CAS procedure is associated with increased long-term survival and reduced MACE occurrence. This evidence supports the hypothesis that statin use before CAS might be beneficial in high risk patients.