Rosa Maisto

Protection from Endotoxic Uveitis by Intravitreal Resolvin D1: Involvement of Lymphocytes, miRNAs, Ubiquitin-Proteasome, and M1/M2 Macrophages

This study investigated the protective effects of intravitreal Resolvin D1 (RvD1) against LPS-induced rat endotoxic uveitis (EIU). RvD1 was administered into the right eye at a single injection of 5 μL volume containing 10–100–1000 ng/kg RvD1 1 h post-LPS injection (200 μg, Salmonella minnesota) into thefootpad of Sprague-Dawley rats. 24 h later, the eye was enucleated and examined for clinical, biochemical, and immunohistochemical evaluations. RvD1 significantly and dose-dependently decreased the clinical score attributed to EIU, starting from the dose of 10 ng/kg and further decreased by 100 and 1000 ng/kg. These effects were accompanied by changes in four important determinants of the immune-inflammatory response within the eye: (i) the B and T lymphocytes, (ii) the miRNAs pattern, (iii) the ubiquitin-proteasome system (UPS), and (iv) the M1/M2 macrophage phenotype. LPS+RvD1 treated rats showed reduced presence of B and T lymphocytes and upregulation of miR-200c-3p, miR 203a-3p, miR 29b-3p, and miR 21-5p into the eye compared to the LPS alone. This was paralleled by decreases of the ubiquitin, 20S and 26S proteasome subunits, reduced presence of macrophage M1, and increased presence of macrophage M2 in the ocular tissues. Accordingly, the levels of the cytokine TNF-α, the chemokines MIP1-α and NF-κB were reduced.

Activation of Melanocortin Receptors MC1 and MC5 Attenuates Retinal Damage in Experimental Diabetic Retinopathy

We hypothesize that melanocortin receptors (MC) could activate tissue protective circuit in a model of streptozotocin- (STZ-) induced diabetic retinopathy (DR) in mice. At 12–16 weeks after diabetes induction, fluorescein angiography (FAG) revealed an approximate incidence of 80% microvascular changes, typical of DR, in the animals, without signs of vascular leakage. Occludin progressively decreased in the retina of mice developing retinopathy. qPCR of murine retina revealed expression of two MC receptors, Mc1r and Mc5r. The intravitreal injection (5 μL) of the selective MC1 small molecule agonist BMS-470539 (33 μmol) and the MC5 peptidomimetic agonist PG-901 (7.32 nM) elicited significant protection with regular course and caliber of retinal vessels, as quantified at weeks 12 and 16 after diabetes induction. Mouse retina homogenate settings indicated an augmented release of IL-1α, IL-1β, IL-6, MIP-1α, MIP-2α, MIP-3α, and VEGF from diabetic compared to nondiabetic mice. Application of PG20N or AGRP and MC5 and MC1 antagonist, respectively, augmented the release of cytokines, while the agonists BMS-470539 and PG-901 almost restored normal pattern of these mediators back to nondiabetic values. Similar changes were quantified with respect to Ki-67 staining. Finally, application of MC3-MC4 agonist/antagonists resulted to be inactive with respect to all parameters under assessment.

Daily Oxygen/O3 Treatment Reduces Muscular Fatigue and Improves Cardiac Performance in Rats Subjected to Prolonged High Intensity Physical Exercise

Rats receiving daily intraperitoneal administration of O2 and running on a treadmill covered an average distance of 482.8 ± 21.8 m/week as calculated during 5-week observation. This distance was increased in rats receiving daily intraperitoneal administration of an oxygen/O3 mixture at a dose of 100; 150; and 300 μg/kg with the maximum increase being +34.5% at 300 μg/kg and still present after stopping the administration of oxygen/O3. Oxygen/O3 decreased the mean arterial blood pressure (−13%), the heart rate (−6%), the gastrocnemius and cardiac hypertrophy, and fibrosis and reduced by 49% the left ventricular mass and relative wall thickness measurements. Systolic and diastolic functions were improved in exercised oxygen/O3 rats compared to O2 rats. Oxygen/O3 treatment led to higher MPI index starting from the dose of 150 μg/kg (p < 0.05) and more effective (+14%) at a dose of 300 μg/kg oxygen/O3. Oxygen/O3 dose-dependently increased the expression of the antioxidant enzymes Mn-SOD and GPx1 and of eNOS compared to the exercised O2 rats. The same doses resulted in decrease of LDH levels, CPK, TnI, and nitrotyrosine concentration in the heart and gastrocnemius tissues, arguing a beneficial effect of the ozone molecule against the fatigue induced by a prolonged high intensity exercise.

Interplay between Intravitreal RvD1 and Local Endogenous Sirtuin-1 in the Protection from Endotoxin-Induced Uveitis in Rats

Rat endotoxin-induced uveitis (EIU) is a well-established model of human uveitis. In this model, intravitreal injection of resolvin D1 (RvD1, 10–100–1000 ng/kg) 1 hour after subcutaneous treatment of Sprague-Dawley rats with lipopolysaccharide (LPS, 200 μg/rat) significantly prevented the development of uveitis into the eye. RvD1 dose-dependently increased the expression of sirtuin-1 (SIRT1) within the eye, while it decreased the expression of acetyl-p53 and acetyl-FOXO1. These effects were accompanied by local downregulation of some microRNAs related to the expression and activity of SIRT1. These were miR-195-5p, miR-200a-3p, miR-34a-5p, and miR-145-5p. An increase of manganese superoxide dismutase and decrease of caspase 3 were evident after RvD1 treatment. In another set of experiments, the protective effects of RvD1 (1000 ng/kg) were partly abolished by the pretreatment of the rats with EX527 (10 mg/kg/day, i.p.), a specific inhibitor of SIRT1 activity, for 7 days prior to the induction of EIU in rats. Similarly, the effects of RvD1 (1000 ng/kg) on the SIRT1 protein expression were abolished by Boc2, N-t-butoxycarbonyl-PLPLP, a specific formyl-peptide receptor type 2/lipoxin A receptor antagonist. Therefore, an interplay of the SIRT1 activity on the RvD1 mediated resolution of EIU is argued.

Involvement of Proteasome and Macrophages M2 in the Protection Afforded by Telmisartan against the Acute Myocardial Infarction in Zucker Diabetic Fatty Rats with Metabolic Syndrome

This study investigated the involvement of proteasome and macrophages M2 in the protection afforded by telmisartan against the acute myocardial infarction in Zucker diabetic fatty (ZDF) rats with metabolic syndrome. ZDF rats were treated for three weeks with telmisartan at doses of 7 and 12 mg/kg/day. After treatment, rats were subjected to a 25 min occlusion of the left descending coronary artery followed by 2 h reperfusion (I/R). At the end of the I/R period, biochemical, immunohistochemical, and echocardiographic evaluations were done. Telmisartan treatment (7 mg/kg and 12 mg/kg) reduced the myocardial infarct size, the expression of proteasome subunits 20S and 26S, and the protein ubiquitin within the heart. The compound has led to an increased M2 macrophage phenotype within the cardiac specimens and a modification of the cardiac cytokine and chemokine profile. This was functionally translated in improved cardiac performance as evidenced by echography after 2 h reperfusion. 7 mg/kg/day telmisartan was sufficient to improve the left ventricular ejection fraction LVEF of the rat heart recorded after I/R (e.g., vehicle 38 ± 2.2%; telmisartan 54 ± 2.7%) and was sufficient to improve the diastolic function and the myocardial performance index up to values of 0.6 ± 0.01 measured after I/R.

Inhibition of Ocular Aldose Reductase by a New Benzofuroxane Derivative Ameliorates Rat Endotoxic Uveitis

The study investigated the effects of the aldose reductase (AR) inhibitor benzofuroxane derivative 5(6)-(benzo[d]thiazol-2-ylmethoxy) benzofuroxane (herein referred to as BF-5m) on the biochemical and tissue alterations induced by endotoxic uveitis in rats. BF-5m has been administered directly into the vitreous, in order to assess the expression and levels of (i) inflammatory markers such as the ocular ubiquitin-proteasome system, NF-κB, TNF-α, and MCP-1; (ii) prooxidant and antioxidant markers such as nitrotyrosine, manganese superoxide dismutase (MnSOD), and glutathione peroxidase (GPX); (iii) apoptotic/antiapoptotic factors caspases and Bcl-xl; (iv) markers of endothelial progenitor cells (EPCs) recruitment such as CD34 and CD117. 5 μL of BF-5m (0.01; 0.05; and 0.1 μM) into the right eye decreased in a dose-dependent manner the LPS-induced inflammation of the eye, reporting a clinical score 1. It reduced the ocular levels of ubiquitin, 20S and 26S proteasome subunits, NF-κB subunits, TNF-α, MCP-1, and nitrotyrosine. BF-5m ameliorated LPS-induced decrease in levels of MnSOD and GPX. Antiapoptotic effects were seen from BF-5m by monitoring the expression of Bcl-xl, an antiapoptotic protein. Similarly, BF-5m increased recruitment of the EPCs within the eye, as evidenced by CD34 and CD117 antibodies.

Melanocortin receptor agonists MCR1-5 protect photoreceptors from high-glucose damage and restore antioxidant enzymes in primary retinal cell culture

Retinal photoreceptors are particularly vulnerable to local high‐glucose concentrations. Oxidative stress is a risk factor for diabetic retinopathy development. Melanocortin receptors represent a family of G‐protein‐coupled receptors classified in five subtypes and are expressed in retina. Our previous data indicate that subtypes 1 and 5 receptor agonists exert a protective role on experimental diabetic retinopathy. This study focuses on their role in primary retinal cell cultures in high‐glucose concentrations. After eye enucleation from wild‐type male C57BL/6 mice, retinal cells were isolated, plated in high‐glucose concentration and treated with melanocortin receptors 1 and 5 agonists and antagonists. Immunocytochemical and biochemical analysis showed that treatment with melanocortin receptors 1 and 5 agonists reduced anti‐inflammatory cytokines and chemokines and enhanced manganese superoxide dismutase and glutathione peroxidase levels, preserving photoreceptor integrity. According with these evidences, we propose a major role of melanocortin receptors 1 and 5 on primary retinal cell response against high glucose or oxidative insults.

Effects of the New Aldose Reductase Inhibitor Benzofuroxane Derivative BF-5m on High Glucose Induced Prolongation of Cardiac QT Interval and Increase of Coronary Perfusion Pressure

This study investigated the effects of the new aldose reductase inhibitor benzofuroxane derivative 5(6)-(benzo[d]thiazol-2-ylmethoxy)benzofuroxane (BF-5m) on the prolongation of cardiac QT interval and increase of coronary perfusion pressure (CPP) in isolated, high glucose (33.3 mM D-glucose) perfused rat hearts. BF-5m was dissolved in the Krebs solution at a final concentration of 0.01 μM, 0.05 μM, and 0.1 μM. 33.3 mM D-glucose caused a prolongation of the QT interval and increase of CPP up to values of 190 ± 12 ms and 110 ± 8 mmHg with respect to the values of hearts perfused with standard Krebs solution (11.1 mM D-glucose). The QT prolongation was reduced by 10%, 32%, and 41%, respectively, for the concentration of BF-5m 0.01 μM, 0.05 μM, and 0.1 μM. Similarly, the CPP was reduced by 20% for BF-5m 0.05 μM and by 32% for BF-5m 0.1 μM. BF-5m also increased the expression levels of sirtuin 1, MnSOD, eNOS, and FOXO-1, into the heart. The beneficial actions of BF-5m were partly abolished by the pretreatment of the rats with the inhibitor of the sirtuin 1 activity EX527 (10 mg/kg/day/7 days i.p.) prior to perfusion of the hearts with high glucose + BF-5m (0.1 μM). Therefore, BF-5m supplies cardioprotection from the high glucose induced QT prolongation and increase of CPP.

The melanocortin MC5R as a new target for treatment of high glucose-induced hypertrophy of the cardiac H9c2 cells

The study explored the anti-hypertrophic effect of the melanocortin MC5R stimulation in H9c2 cardiac myocytes exposed to high glucose. This has been done by using α-MSH and selective MC5R agonists and assessing the expression of GLUT4 and GLUT1 transporters, miR-133 and urotensin receptor levels as a marker of cardiac hypertrophy. The study shows for the first time an up-regulation of MC5R expression levels in H9c2 cardiomyocytes exposed to high glucose medium (33 mM D-glucose) for 48 h, compared to cells grown in normal glucose medium (5.5 mM D-glucose). Moreover, H9c2 cells exposed to high glucose showed a significant reduction in cell viability (-40%), a significant increase in total protein per cell number (+109%), and an increase of the urotensin receptor expression levels as an evidence of cells hypertrophy. The pharmacological stimulation of MC5R with α-MSH (90 pM)of the high glucose exposed H9c2 cells increased the cell survival (+50,8%) and reduced the total protein per cell number (-28,2%) with respect to high glucose alone, confirming a reduction of the hypertrophic state as per cell area measurement. Similarly, PG-901 (selective agonist, 10-10 M) significantly increased cell viability (+61,0 %) and reduced total protein per cell number (-40,2%), compared to cells exposed to high glucose alone. Interestingly, the MC5R agonist reduced the GLUT1/GLUT4 glucose transporters ratio on the cell membranes exhibited by the hypertrophic H9c2 cells and increased the intracellular PI3K activity, mediated by a decrease of the levels of the miRNA miR-133a. The beneficial effects of MC5R agonism on the cardiac hypertrophy caused by high glucose was also observed also by echocardiographic evaluations of rats made diabetics with streptozotocin (65 mg/kg i.p.). Therefore, the melanocortin MC5R could be a new target for the treatment of high glucose-induced hypertrophy of the cardiac H9c2 cells.

Intraperitoneal Administration of Oxygen/Ozone to Rats Reduces the Pancreatic Damage Induced by Streptozotocin

Background: The rat model of streptozotocin (STZ)-induced pancreatic damage was used to examine whether a systemic oxygen/ozone mixture could be beneficial for the pancreas by reducing the machinery of the local detrimental mediators released by STZ. Results: The results showed that oxygen/ozone administration (150 µg/Kg i.p.) for ten days in STZ rats increased the endogenous glutathione-s-transferase (GST) enzyme and nuclear factor-erythroid 2-related factor 2 (Nrf2) into the pancreatic tissue, together with reduction of 4-hydroxynonenal (4-HNE) and PARP-1 compared to STZ rats receiving O2 only. Interestingly, these changes resulted in higher levels of serum insulin and leptin, and pancreatic glucagon immunostaining. Consequently, glucose metabolism improved as evidenced by the monitoring of glycemia throughout. Conclusions: This study provides evidence that systemic administration of oxygen/ozone reduces the machinery of detrimental mediators released by STZ into the pancreas with less local damage and better functionality.