Rossella Bianchi

Structural Alterations in Subcutaneous Small Arteries of Normotensive and Hypertensive Patients With Non–Insulin-Dependent Diabetes Mellitus

Background — It is not presently known whether non–insulin-dependent diabetes mellitus (NIDDM) is associated with the presence of structural alterations in small arteries or whether the combination of hypertension and NIDDM may have an additive effect on endothelial dysfunction. Therefore, we investigated subcutaneous small arteries in 12 normotensive subjects (NT group), 18 patients with essential hypertension (EH group), 13 patients with NIDDM, and 11 patients with NIDDM and EH (NIDDM+EH group). Methods and Results — Subcutaneous small arteries were evaluated by a micromyographic technique. The internal diameter, the media-to-lumen ratio, remodeling and growth indices, and the collagen-to-elastin ratio were calculated. Concentration-response curves to acetylcholine, bradykinin, the endothelium-independent vasodilator sodium nitroprusside, and endothelin-1 were performed. The media-to-lumen ratio was higher in the EH, NIDDM, and NIDDM+EH groups compared with the NT group. EH patients showed the presence of eutrophic remodeling, whereas NIDDM and NIDDM+EH patients showed 40% to 46% cell growth. The collagen-to-elastin ratio was significantly increased in the EH and NIDDM+EH groups compared with the NT group. The vasodilatation to acetylcholine and bradykinin was similarly reduced in EH, NIDDM, and NIDDM+EH groups compared with the NT group. The contractile responses to endothelin-1 were similarly reduced in EH, NIDDM, and NIDDM+EH patients. Conclusions — Our data suggest that the effects of NIDDM and EH on small artery morphology are quantitatively similar but qualitatively different and that the presence of hypertension in diabetic patients has little additive effect on small artery morphology and none on endothelial dysfunction.

Effect of Treatment With Candesartan or Enalapril on Subcutaneous Small Artery Structure in Hypertensive Patients With Noninsulin-Dependent Diabetes Mellitus

Structural alterations of subcutaneous small resistance arteries are associated with a worse clinical prognosis in hypertension and noninsulin-dependent diabetes mellitus (NIDDM). However, no data are presently available about the effects of antihypertensive therapy on vascular structure in hypertensive patients with NIDDM. Therefore, we have investigated the effect of an angiotensin-converting enzyme inhibitor, enalapril, and a highly selective angiotensin receptor blocker, candesartan cilexetil, on indices of subcutaneous small resistance artery structure in 15 patients with mild hypertension and NIDDM. Eight patients were treated with candesartan (8 to 16 mg per day) and 7 with enalapril (10 to 20 mg per day) for 1 year. Each patient underwent a biopsy of the subcutaneous fat from the gluteal region at baseline and after 1 year of treatment. Small arteries were dissected and mounted on a micromyograph and the media-to-internal lumen ratio was evaluated; moreover, endothelium-dependent vasodilation to acetylcholine was assessed. A similar blood pressure-lowering effect and a similar reduction of the media-to-lumen ratio of small arteries was observed with the 2 drugs. Vascular collagen content was reduced and metalloproteinase-9 was increased by candesartan, but not by enalapril. Changes of circulating indices of collagen turnover and circulating matrix metalloproteinase paralleled those of vascular collagen. The 2 drugs equally improved endothelial function. In conclusion, antihypertensive treatment with drugs that inhibit the renin-angiotensin-aldosterone system activity is able to correct, at least in part, alterations in small resistance artery structure in hypertensive patients with NIDDM. Candesartan may be more effective than enalapril in reducing collagen content in the vasculature.

Expression of Fos immunoreactivity in the rat supraspinal regions following noxious visceral stimulation.

We used immunohistochemical detection of the Fos protein to study the neuronal activation in the brain of methoxyfluorane-anesthetized rats after noxious deep somatic or visceral stimulation. The anesthesia was effective in triggering gene induction in many brain regions. Nevertheless, Fos appeared de novo in several brain nuclei following noxious stimulation in anesthetized animals. This could be of clinical relevance, as it suggests that the gas anesthetic does not suppress noxious stimulus-evoked reactivity in brain neurons. Two types of visceronociceptive stimuli were used to compare the effects of a diffuse visceral inflammation (peritoneal inflammation) with those of a more restricted inflammation (urinary bladder inflammation). In the same supraspinal areas, there were very few immunostained neurons in unstimulated controls, whereas Fos-positive cells were slightly more numerous in anesthetized controls and significantly more numerous after noxious stimulation. The peritoneal inflammation induced more Fos-labeled neurons than the restricted visceral stimulation. Labeled cells were found in these cases mainly in the ventrolateral medulla, parabrachial complex, dorsal raphe nucleus, periaqueductal gray, several hypothalamic and thalamic nuclei, amygdaloid complex, and cortex. Altogether these findings indicated that somatic and visceral inputs generally activate the same neuronal groups. However, a separation between the activation of somatic and visceral pathways was found in some brain nuclei, such as the parabrachial complex, hypothalamic, and thalamic nuclei.

Group I metabotropic glutamate receptors modulate glutamate and γ-aminobutyric acid release in the periaqueductal grey of rats

In this study, we investigated the effects of group I metabotropic glutamate (mglu) receptor ligands on glutamate and gamma-aminobutyric acid (GABA) extracellular concentrations at the periaqueductal grey level by using in vivo microdialysis. An agonist of group I mglu receptors, (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG, 1 and 2 mM], as well as a selective agonist of mglu(5) receptors, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 2 and 4 mM), both increased dialysate glutamate and GABA concentrations. 7-(Hydroxyimino)cyclopropa-[b]-chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 1 mM), a selective mglu(1) receptor antagonist, and 2-methyl-6-(phenylethynyl)pyridine (MPEP, 0.5 mM), a selective mglu(5) receptor antagonist, perfused in combination with DHPG, antagonized the effect induced by DHPG on the extracellular glutamate and GABA concentrations. MPEP (0.5 mM), perfused in combination with CHPG, antagonized the increased glutamate and GABA extracellular levels induced by CHPG. MPEP (1 mM) decreased the extracellular concentrations of glutamate but did not modify the dialysate GABA concentrations. Moreover, as the intra-periaqueductal grey perfusion of (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [(RS)-CPP, 100 microM], a selective N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, did not change the extracellular concentrations of glutamate, this suggests that the MPEP-induced decrease in glutamate is not a consequence of NMDA receptor blockade. These data show that group I mglu receptors in the periaqueductal grey may modulate the release of glutamate and GABA in awake, freely moving rats. In particular, mglu(5), but not mglu(1), receptors seem to be functionally active on glutamate terminals.

Evidence for the involvement of SDF‐1 and CXCR4 in the disruption of endothelial cell‐branching morphogenesis and angiogenesis by TNF‐α and IFN‐γ

Vigorous inflammatory responses are associated with tissue damage, particularly when toxic levels of inflammatory cytokines are produced. Despite proangiogenic factors being present early at sites of inflammation, vascular repair occurs toward the end of the inflammatory response, suggesting modulation of the proangiogenic response. Endogenous inhibitors of angiogenesis induced during acute inflammation are poorly characterized. Here, we looked for endothelial cell‐derived modulators of angiogenesis that may account for delayed neovascularization during inflammation. Gene profiling of endothelial cells showed that the inflammatory cytokines tumor necrosis factor α (TNF‐α) and interferon‐γ (IFN‐γ) selectively promote expression of the antiangiogenic molecules, IFN‐inducible protein‐10, monokine induced by IFN‐γ, tryptophanyl‐tRNA synthetase, and tissue inhibitor of metalmetalloproteinase‐1, and inhibit expression of the proangiogenic molecules, platelet‐endothelial cell adhesion molecule‐1, vascular endothelial growth factor receptor‐2, stromal cell‐derived factor‐1 (SDF‐1), collagen type IV, endothelial cell growth factor‐1, and carcinoembryonic antigen‐related cell adhesion molecule‐1. Reduced endothelial cell expression of SDF‐1 protein by TNF‐α and IFN‐γ disrupts extracellular matrix‐dependent endothelial cell tube formation, an in vitro morphogenic process that recapitulates critical steps in angiogenesis. Replacement of SDF‐1 onto the endothelial cell surface reconstitutes this morphogenic process. In vivo, TNF‐α and IFN‐γ inhibit growth factor‐induced angiogenesis and SDF‐1 expression in endothelial cells. These results demonstrate that SDF‐1/CXC chemokine receptor‐4 constitutes a TNF‐α‐ and IFN‐γ‐regulated signaling system that plays a critical role in mediating angiogenesis inhibition by these inflammatory cytokines.

Dose-dependent Mercuric Chloride Tubular Injury in Rat Kidney

Mercuric chloride (HgCl 2 ) produces an acute renal failure in experimental animal models. Since the mechanism of tubular injury has not completely been clarified, this morpho-quantitative study was undertaken to better understand the effects of 2 sublethal doses (T1=1 mg/kg and T3.5=3.5 mg/kg) of HgCl 2 in rat proximal tubules. Morphometrical analysis was performed to quantify both cytoplasmic and nuclear changes found in treated in respect to saline-injected proximal tubules. In the controls, single-cell damage was occasional and nucleolar changes were absent. HgCl 2 induced progressively severe proximal tubule atrophy. In the T1 group, necrosis was limited to pars recta cells and nucleolar segregation was often partial. In the T3.5 group, atrophy was extensive in both convoluted and straight tracts, the nucleolus was completely segregated and coiled body-like inclusions were detached from it. Ultrastructural analysis confirmed dose-dependent changes within straight proximal tubules, i.e., necrosis, apoptosis, nucleolar segregation, swollen mitochondria, vacuolization, and disrupted brush border. In conclusion, in the rat kidney HgCl 2 induced dose-dependent alterations not only in the cytoplasm but also in the nucleus of proximal tubule cells. These findings will be useful for better understanding of the pathogenesis of mercury nephrotoxicity and its genotoxic effect.

Morphometric changes induced by amino acid supplementation in skeletal and cardiac muscles of old mice.

Aging is associated with progressive structural disorganization of muscular and cardiac fibers, decreasing functional capacity, and increased rates of disease and death. Aging is also characterized by disturbances in protein synthesis with impaired cellular organelle functions, particularly in the mitochondria. The availability of amino acids is a key factor for the overall metabolism of mammals and exogenous supplements of amino acid mixtures (AAm) could be a valid therapeutic strategy to improve quality of life, avoiding malnutrition and muscle wasting in the elderly. We investigated the morphoquantitative effects of long-term AAm supplementation on the mitochondria and sarcomeres (by electron microscope) and on collagen matrix deposition (by histologic techniques) in both skeletal and cardiac muscles of young and aged mice. Our data showed that old animals have fewer mitochondria and massive fibrosis in both muscles. Long-term AAm supplementation increased the number and volume of mitochondria and sarcomeres and decreased fibrosis in both skeletal muscle and hearts in old rats. These findings indicate that AAm restored muscular morphologic parameters and probably improved the mechanical performance of these organs.

Beneficial effects of melatonin in protecting against cyclosporine A‐induced cardiotoxicity are receptor mediated

Abstract:  Melatonin, the chief product secreted by pineal gland, is capable of reducing free radical damage by acting directly as a free radical scavenger, and indirectly, by stimulating of antioxidant enzymes. Cyclosporine A (CsA) is the most widely used immunosuppressive drug, but its therapeutic use has several side effects including, i.e. nephrotoxicity and cardiotoxicity. This study was designed to examine the beneficial effects of melatonin in preventing CsA‐induced cardiotoxicity. Additionally, we investigated the ability of melatonin to protect the rat heart via melatonin receptor. In one group of Wistar rats, melatonin (1 mg/kg/day i.p.) was administered concurrently with CsA (15 mg/kg/day s.c.) for 21 days. In another group of animals, melatonin was injected with CsA and luzindole, an antagonist of melatonin receptors. Oxidative stress in heart tissue homogenates was estimated using thiobarbituric acid reactive substances (TBARS), reduced glutathione levels and antioxidant enzyme activities including catalase and superoxide dismutase. CsA administration for 21 days produced elevated levels of TBARS, marked depletion of cardiac antioxidant enzymes and caused morphological alterations in myocardial fibers. Melatonin markedly reduced TBARS levels, increased the antioxidant enzyme levels and normalized altered cardiac morphology. The protective effects of melatonin were lost when the animals received the melatonin receptor antagonist. In conclusion our study shows that, (a) melatonin significantly reduces CsA cardiotoxicity, and (b) the reduction in CsA‐induced cardiotoxicity was mediated by the binding of melatonin to its membrane receptors.

Provinol Prevents CsA-induced Nephrotoxicity by Reducing Reactive Oxygen Species, iNOS, and NF-kB Expression

Cyclosporine A (CsA) use is associated with several side effects, the most important of which is nephrotoxicity that includes, as we previously showed, tubular injury and interstitial fibrosis. Recently, many researchers have been interested in minimizing these effects by pharmacological interventions. To do this, we tested whether the administration of a red wine polyphenol, Provinol (PV), prevents the development of CsA-induced nephrotoxicity. Rats were treated for 21 days and divided into four groups: control; group treated with PV (40 mg/kg/day by oral administration in tap water); group treated with CsA (15 mg/kg/day by subcutaneous injection); group treated with CsA plus PV. CsA produced a significant increase of systolic blood pressure; it did not affect urinary output, but caused a significant decrease in creatinine clearance. These side effects were associated with an increase in conjugated dienes, which are lipid peroxidation products, inducible NO-synthase (iNOS), and nuclear factor (NF)-kB, which are involved in antioxidant damage. However, PV prevented these negative effects through a protective mechanism that involved reduction of both oxidative stress and increased iNOS and NF-kB expression induced by CsA. These results provide a pharmacological basis for the beneficial effects of plant-derived polyphenols against CsA-induced renal damage associated with CsA.

Changes in Hsp90 expression determine the effects of cyclosporine A on the NO pathway in rat myocardium.

Cyclosporine A (CsA) is associated with the development of cardiovascular toxicity in transplant patients but can exert myocardial protection against ischemia/reperfusion damages. We examined in a rat model of chronic CsA administration whether subtle variations in the NO pathway could account for these opposite effects. CsA treatment rapidly led to an increase in myocardial Hsp90 expression promoting the recruitment of Akt and calcineurin, thereby promoting eNOS activation through Ser1177 phosphorylation and Thr495 dephosphorylation, respectively. This was associated with an increase in myocardial VEGF expression and led to anti‐apoptotic effects in isolated cardiac myocytes. Upon longer CsA exposure, cardiac toxicity developed, as documented by the infiltration of connective tissue and the increase in iNOS expression. These later effects were associated with a dramatic decrease in the abundance and scaffold function of Hsp90, thereby unraveling the key role of Hsp90 in governing CsA effects.