Eleonora Santini

Acute retinal ganglion cell injury caused by intraocular pressure spikes is mediated by endogenous extracellular ATP

Elevated intraocular pressure may lead to retinal ganglion cell injury and consequent visual deficits. Chronic intraocular pressure increase is a major risk factor for glaucoma, a leading blinding disease, and permanent visual deficits can also occur following acute pressure increments due to trauma, acute glaucoma or refractive surgery. How pressure affects retinal neurons is not firmly established. Mechanical damage at the optic nerve head, reduced blood supply, inflammation and cytotoxic factors have all been called into play. Reasoning that the analysis of retinal neurons soon after pressure elevation would provide useful cues, we imaged individual ganglion cells in isolated rat retinas before and after short hydrostatic pressure increments. We found that slowly rising pressure to peaks observed in trauma, acute glaucoma or refractive surgery (50–90 mmHg) did not damage ganglion cells, whereas a rapid 1 min pulse to 50 mmHg injured 30% of these cells within 1 h. The severity of damage and the number of affected cells increased with stronger or repeated insults. Degrading extracellular ATP or blocking the P2X receptors for ATP prevented acute pressure‐induced damage in ganglion cells. Similar effects were observed in vivo. A short intraocular pressure transient increased extracellular ATP levels in the eye fluids and damaged ganglion cells within 1 h. Reducing extracellular ATP in the eye prevented damage to ganglion cells and accelerated recovery of their response to light. These data show that rapid pressure transients induce acute ganglion cell injury and unveil the causal role of extracellular ATP elevation in such injury.

Effect of short-term folic acid supplementation on insulin sensitivity and inflammatory markers in overweight subjects

Objective:Inflammation plays a pivotal role in the atherosclerotic process, and some chemokines seem to be crucial in the pathogenesis of vascular damage. High-serum homocysteine, recently recognized as an independent risk factor for vascular disease might increase cytokine and chemokine levels, thus amplifying endothelial damage; moreover, it might worse insulin resistance, thus further contributing to enhance cardiovascular risk. The effect of folic acid supplementation in improving in vivo endothelial function is still debated. In this study, we investigated the effect of folic acid supplementation on insulin sensitivity and peripheral markers of inflammation in overweight healthy subjects.Design:The study was performed as an unmasked randomized placebo-controlled trial of 12 weeks duration.Subjects:Sixty healthy volunteers with normal glucose tolerance and BMI between 25 and 29 kg/m2 were enrolled.Measurements:Biochemical parameters and plasma concentrations of homocysteine and of some inflammatory molecules were measured at baseline and at the end of the study, together with an estimation of insulin sensitivity.Results:Subjects receiving folic acid supplementation showed a decrement of homocysteine and an amelioration of insulin sensitivity; this treatment was also associated with a significant drop in the circulating concentration of monocyte chemoattractant protein-1, interleukin-8 and C-reactive protein, in the absence of any significant variation of BMI or fat mass.Conclusions:In healthy overweight subjects a short-term folic acid supplementation reduces the circulating level of some inflammatory mediators independently of weight change, thus suggesting a potential therapeutic role for folic acid in the protection from atherogenesis and cardiovascular diseases.

Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue

AIMS We assessed the impact of vascular and perivascular tumour necrosis factor-alpha (TNF-α) on the endothelin (ET)-1/nitric oxide (NO) system and the molecular pathways involved in small arteries from visceral fat of obese patients (Obese) and Controls. METHODS AND RESULTS Isolated small arteries from 16 Obese and 14 Controls were evaluated on a pressurized micromyograph. Endogenous ET-1 activity was assessed by the ETA blocker BQ-123. TNF-α and NO were tested by anti-TNF-α infliximab (IFX) and N(ω)-nitro-l-arginine methylester (L-NAME). Gene and protein expression of TNF-α, ET-1, ETA, and ETB receptors were determined by RT-PCR and IHC on arterial wall and in isolated adipocytes. Obese showed a blunted L-NAME-induced vasoconstriction, which was potentiated by IFX, and an increased relaxation to BQ-123, unaffected by L-NAME but attenuated by IFX. Perivascular adipose tissue (PVAT) removal reversed these effects. Obese showed intravascular superoxide excess, which was decreased by apocynin (NAD(P)H oxidase inhibitor), L-NAME, and BQ-123 incubations, and abolished by IFX. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular/perivascular TNF-α and TNF-α receptor expression were also detected. The arterial expression and phosphorylation of c-Jun N-terminal kinase (JNK) were higher in Obese vs. Controls, and downregulated by IFX. CONCLUSIONS In small arteries of Obese, PVAT-derived TNF-α excess, and an increased vascular expression of ET-1 and ETA receptor, contribute to the ET-1/NO system imbalance, by impairing tonic NO release. Reactive oxygen species excess, via NAD(P)H oxidase activation, induces the endothelial nitric oxide synthase uncoupling, which in turn generates superoxide and impairs NO production. The up-regulated JNK pathway represents a crucial molecular signalling involved in this process.

Retinol-binding protein-4 in women with untreated essential hypertension

BACKGROUND Retinol-binding protein-4 (RBP4) is a novel adipokine able to modulate the action of insulin in several tissues. A variable degree of insulin resistance characterizes the vast majority of hypertensive (HYP) patients. The aim of this study was to evaluate the relationship between RBP4 and essential hypertension, exploring potential links between RBP4 and other adipokines with some proxies of early vascular damage in female naive HYP patients. METHODS Serum RBP4, leptin, adiponectin, and resistin levels were determined in 35 HYP and 35 normotensive lean women with normal glucose tolerance paired by age and body mass index (BMI) served as controls (CTL); carotid intima-media thickness (IMT) was also measured. RESULTS A striking difference was observed in RBP4 levels between HYP and CTL with significantly higher levels in the former than in the latter. No relationship was observed between glomerular filtration rate (GFR) and RBP4. Adiponectin levels were slightly but significantly lower in HYP than in CTL, whereas no differences were observed in resistin and leptin concentrations between the two groups of women. In the whole study group, a strong linear relationship was observed between IMT value and both RBP4 (rho = 0.321, P = 0.0076) and resistin (rho = 0.340, P = 0.0048); these two adipocytokines, together with cholesterol, were the only variables independently related to IMT (r(2) = 0.24; P = 0.004) by a stepwise analysis. CONCLUSIONS RBP4 levels are increased in naive HYP women and correlated with the degree of IMT suggesting a participation of this adipocytokine in the modulation of the atherosclerotic process exerted by the adipose tissue as endocrine organ.

The P2X7 receptor-inflammasome complex has a role in modulating the inflammatory response in primary Sjögren's syndrome.

Innate and adaptive immunity may contribute to gland dysfunction in patients with primary Sjögrens syndrome (pSS). The P2X7 receptor (P2X7R)–NLRP3 inflammasome complex modulates the release of the inflammatory cytokines IL‐1β and IL‐18. The presence of P2X7R in salivary glands suggests an interesting scenario for the initiation and amplification of the innate immune response in pSS. Therefore, the aim of this study was to assess the role of the P2X7R–NLRP3 inflammasome in pSS.

Effects of different LDL particles on inflammatory molecules in human mesangial cells.

Aims/hypothesisInflammation is a mechanism of glomerular damage in chronic glomerulopathies. LDL may increase the production of inflammatory cytokines in renal tissues. However, the relative role of native, oxidised and glycated LDL in promoting this process has been only partially elucidated.MethodsWe tested the inflammatory and proapoptotic effects of native, oxidised and glycated LDL in human mesangial cells (HMCs) by measuring levels of IL6, CD40 and macrophage migration inhibitory factor (MIF) genes, MIF protein, release of IL6, soluble CD40, fibronectin and laminin, early and late apoptosis, and extracellular regulated kinases (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation.ResultsIL6 and CD40 mRNA were dose-dependently upregulated by all three species; this was closely paralleled by their increased release. MIF mRNA was potently stimulated by modified LDL, as confirmed by immunostaining. Fibronectin and laminin release was stimulated by both oxidised and glycated, but not native, LDL. All LDL species induced some increase in late, but not early, apoptosis, and similarly activated JNK2/3 phosphorylation; in contrast, ERK1/2 phosphorylation was more strongly upregulated by oxidised than either native or glycated LDL.ConclusionsIn HMCs, the production and release of IL6 and CD40 is stimulated by both native and modified LDL, while MIF is more strongly stimulated by oxidised LDL. Regarding the pattern of mesangial expansion, fibronectin and laminin are upregulated by oxidised and glycated LDL. Apoptosis, if modest, is induced by all species. Intracellular signalling of native and modified LDL involves JNK2/3 and, perhaps more specifically, ERK1/2. Tight control of the lipid profile may be useful in preserving kidney function in patients with metabolic alterations.

The complex P2X7 receptor/inflammasome in perivascular fat tissue of heavy smokers

Smoking is a recognized cardiovascular risk factor. Perivascular visceral adipose tissue (PVAT) is a source of inflammatory molecules, thus contributing to atherosclerosis progression. The P2X7 receptor (P2X7R)‐inflammasome complex, crucial in determining IL‐1β and IL‐18 release, participates in this scenario. We evaluated whether smoking might affect the PVAT inflammatory phenotype and explored the putative role of the axis P2X7R‐inflammasome in this picture.

Adipocytokine levels mark endothelial function in normotensive individuals

BackgroundEndothelial dysfunction is an independent risk factor for cardiovascular events. Inflammatory mediators released by the adipose tissue can lead to local insulin resistance and endothelial dysfunction. This study addressed the relationship of adipocytokines with endothelial function and blood pressure.MethodsIn 92 newly diagnosed, drug-naïve essential hypertensive patients (HT, mean age 49 yrs) without organ damage and 66 normotensive subjects (NT, mean age 47 yrs), by an automated system, we measured endothelium-dependent and -independent vasodilation as brachial artery flow-mediated dilation before and after administration of glyceryl-trinitrate. Retinol binding protein-4 (RBP4) and resistin levels were determined by ELISA and RIA, respectively. Oxidative stress was evaluated by measuring serum malondyaldehyde (MDA).ResultsFlow-mediated dilation was significantly (p = 0.03) lower in HT (5.3 ± 2.6%) than NT (6.1 ± 3.1%), while response to glyceryl-trinitrate (7.5 ± 3.7% vs 7.9 ± 3.4%) was similar. RBP4 (60.6 ± 25.1 vs 61.3 ± 25.9 μg/ml), resistin (18.8 ± 5.3 vs 19.9 ± 6.1 ng/ml) and MDA levels (2.39 ± 1.26 vs 2.08 ± 1.17 nmol/ml) were not different in HT and NT. RBP4 (r = −0.25; p = 0.04) and resistin levels (r = −0.29; p = 0.03) were related to flow-mediated dilation in NT, but not in HT (r = −0.03 and r = −0.10, respectively). In NT, multivariate analysis including RBP4 and confounders showed that only BMI or waist circumference remained related to flow- mediated dilation. In the multivariate model including resistin and confounders, BMI, age and resistin were significantly related to flow-mediated dilation, while only age significant correlated with this parameter when BMI was replaced by waist circumference.ConclusionsAdipocytokine levels may be independent predictors of endothelial dysfunction in the peripheral circulation of healthy subjects, providing a pathophysiological link between inflammation from adipose tissue and early vascular alterations.

Effects of endothelin-1 on fibroblasts from type 2 diabetic patients: Possible role in wound healing and tissue repair

Endothelin-1 (ET-1) promotes the contractile ability of fibroblasts, essential for wound closure and reconstitution of the dermis. Wound healing is impaired in type 2 diabetic patients (D). We compared the effect of ET-1 on proliferative transforming growth factor (TGFβ1) expression, fibronectin and laminin release), differentiative [α-smooth muscle actin (α-SMA) expression] and inflammatory [monocyte chemo-attractant protein (MCP-1) and interleukin-6 (IL-6) expression] responses in skin fibroblasts of healthy subjects (C) and D, testing the relative role of ETA and ETB receptors in mediating these responses. ET-1 did not influence TGFβ1, fibronectin or laminin production. α-SMA was more abundant and more stimulated in D, as well as MCP-1 and IL-6 expression and release. These effects were prevented by BMS-182874, selective antagonist of ETA, more abundant than ETB in both cell strains and whose expression rose more in D than C upon stimulation with ET-1. This peculiar pattern of responses to ET-1, presumably acquired during the chronic in vivo exposure to hyperglycemia along the natural history of the disease, may partially explain the increased susceptibility of D to chronic ulcerations.

Soluble Human Leukocyte Antigen-G Expression and Glucose Tolerance in Subjects with Different Degrees of Adiposity

CONTEXT Type 2 diabetes mellitus (T2DM) and obesity are characterized by a low-grade inflammation, which might be related to the development of insulin resistance. Human leukocyte antigen-G (HLA-G) shows antiinflammatory and tolerogenic properties, including the modulation of CD8+ T-cell cytotoxicity and regulation of CD4+ T-lymphocyte function. These functions are partially shared with IL-10, whose levels are reduced in insulin-resistant states. OBJECTIVE The aim was to explore the relationship between HLA-G and the metabolic and inflammatory pattern of obesity or T2DM. PATIENTS AND MAIN OUTCOME MEASURES Soluble HLA-G, IL-6, and IL-10 were measured and related with metabolic and biochemical parameters in 230 volunteers with normal glucose tolerance, impaired glucose tolerance, or T2DM by oral glucose tolerance test. RESULTS sHLA-G, detected in 144 subjects (sHLA-G positive), was more frequent in T2DM or impaired glucose tolerance subjects than in normal glucose tolerance (chi(2) =18.6; P < 0.0001), and its plasma levels increased progressively across the classes of glucose tolerance. sHLA-G-positive individuals had higher body mass index, systolic blood pressure, and cholesterol levels; a reduced degree of insulin sensitivity; and almost 2-fold higher levels of IL-6, a cytokine related to insulin sensitivity, whereas IL-10 was similar. In the sHLA-G-positive subgroup, by a multivariate regression model, sHLA-G was significantly related to 2-h glucose, the area under insulin curve, and IL-6 levels (multiple r(2) = 0.14; P < 0.001), independently of age, gender, and body mass index. CONCLUSIONS A frequent expression of sHLA-G, linked to a typical biomarker of insulin resistance like IL-6, seems to characterize subjects with an impaired glucose metabolism.