Silvana Balzan

The opening of the ATP-sensitive mitochondrial potassium channel (mitok-ATP) is a common goal of cardioprotective strategies in the setting of acute and chronic myocardial disease. The biologically active thyroid hormone (TH), 3-5-3-triiodothyronine (T3), has been indicated as a potential activator of mitoK-ATP but the underlying mechanisms are still elusive. Here we describe a novel role of T3 in the transcriptional regulation of mitoK and mitoSur, the recently identified molecular constituents of the channel. To mimic human ischemic heart damage, we used a rat model of a low T3 state as the outcome of a myocardial ischemia/reperfusion event, and neonatal rat cardiomyocytes (NRCM) challenged with hypoxia or H₂O₂. Either in the in vivo or in vitro models, T3 administration to recover the physiological concentrations was able to restore the expression level of both the channel subunits, which were found to be downregulated under the stress conditions. Furthermore, the T3-mediated transcriptional activation of mitoK-ATP in the myocardium and NRCM was associated with the repression of the TH-inactivating enzyme, deiodinase 3 (Dio3), and an up-regulation of the T3-responsive miR-133a-3p. Mechanistically, the loss and gain of function experiments and reporter gene assays performed in NRCM, have revealed a new regulatory axis whereby the silencing of Dio3 under the control of miR-133a-3p drives the T3-dependent modulation of cardiac mitoK and mitoSur transcription.

Atherosclerosis is a systemic inflammation of the arteries characterized by atherosclerotic plaque due to the accumulation of lipids, inflammatory cells, apoptotic cells, calcium and extracellular matrix (ECM) proteins. Stable plaques present a chronic inflammatory infiltration, whereas vulnerable plaques present an ‘active’ inflammation involved in the thinning of the fibrous cap that predisposes to plaque rupture. Several complex biological cellular processes lead plaques to evolve from stable to vulnerable predisposing them to rupture and thrombosis. In this review, we analyze some emerging circulating biomarkers related to inflammation, ECM and lipid infiltration, angiogenesis, metalloproteinases and microRNA (miRNA), as possible diagnostic and prognostic indicators of plaque vulnerability.

Serum angiopoietin-2 level is elevated in several diseases suggesting its possible role as a mediator of angiogenesis and vascular network remodeling. Triiodothyronine and thyroxine have well documented effects on angiogenesis in vitro, but only few reports have studied angiopoietin-2 in thyroid-disease patients. The aim of the present study was to measure soluble angiopoietin-2 serum levels in a group of thyroid-disease patients with different levels of free triiodothyronine and thyroxine. Angiopoietin-2 were quantified by ELISA in sera of fifteen healthy volunteers and forty-two thyroid ambulatory patients: nine with hyperthyroidism, four in therapy for hyperthyroidism, seven with subclinal hyperthyroidism, twelve with hypothyroidism, five with thyroiditis and five in therapy for thyroiditis. Median angiopoietin-2 level was significantly elevated in hyperthyroid patients (p < 0.01) and it was significantly increased vs all the other groups (p < 0.0001). In hyperthyroid patients anti thyroid therapy seems to reduce angiopoietin-2 level. A significant positive correlation was observed between Log angiopoietin-2 levels and serum concentration of Log free triiodothyronine (r = 0.4, P < 0.001) and Log free thyroxine (r = 0.4, P < 0.001) respectively. In conclusion, increased levels of angiopoietin-2 are present in hyperthyroid patients, and seems to correlate with free triiodothyronine and free thyroxine levels but not with anti-thyroid antibodies. These findings suggest angiopoietin-2 as a mediator of angiogenesis and vascular network remodeling in this disease, but further studies will be needed to determine the role of this biomarker in the pathophysiology and progression of hyperthyroidism.

Angiogenesis is important for recovery after tissue damage in myocardial ischemia/reperfusion and triiodothyronine (T3) has documented effects on angiogenesis. The angiopoietins 1/2 and tyrosine kinase receptor represent an essential system in angiogenesis controlling endothelial cell survival and vascular maturation. Recently, in a 3-day ischemia/reperfusion rat model, the infusion of a low dose of T3 was shown to improve the post ischemic recovery of cardiac function. Adopting this model, our study aimed to investigate the effects of T3 on the capillary index and the expression of angiogenic genes as the angiopoietins 1/2 and tyrosine kinase receptor system, in the thoracic aorta and in the left ventricle. In the thoracic aorta, T3 infusion significantly improved the angiogenic sprouting and angiopoietin 2 expression. Instead, Sham-T3 group did not show a significant increment of capillary density and angiopoietin 2 expression. In the area at risk of the left ventricle, T3 infusion did not increase capillary density but restored levels of angiopoietin 1 which were reduced in I/R group. Angiopoietin 1 levels were similar to Sham group and unchanged by T3 administration. In the remote zone, T3 induced a significant increment both of angiopoietin 1/2. In conclusion, T3 infusion induced a different response of angiopoietin 1/2 between the ventricle (the area at risk and the remote zone) and the thoracic aorta, probably reflecting the different action of angiopoietin 1/2 in cardiomyocytes and endothelial cells. Overall these data suggest a new aspect of T3-mediated cardioprotection through angiogenesis.

It is now widely accepted that the microcirculation plays a role in the complications of atherosclerosis, but the microcirculation response to atherosclerosis risk factors like diabetes, hypercholesterolemia and hypertension, is still unclear. Alterations in the endothelial production of IL6, NO and ET-1 are known to be correlate with these diseases. Simulating the presence of hyperglycemia, hypercholesterolemia and hypertension, this in vitro study investigated the effect of glucose, angiotensin II, and nLDL treatments on IL-6, ET-1 and NO in HMEC-1. The medium concentrations of IL6 and ET-1 were measured by ELISA assay, whereas NO by a colorimetric assay. The mRNA and protein expressions of IL-6, Pre-po-ET-1 and eNOS by extracted cells were also investigated by RT-PCR. NO concentration in the medium of HMEC-1 increased in a dose-dependent manner by glucose after 24 hours and by nLDL both at 6 and 24 h, with higher values at 6 hours. The eNOS mRNA expression at 6h induced by nLDL, showed a parallel trend to the medium NO. No increment dose dependent NO concentration was observed by angiotensin II.nLDL induced a dose-dependent increase of ET-1 medium levels, more accentuated in 6h respect to 24h. The expression of prepro-ET-1 showed a parallel dose-dependent increased after 6 hours. Both glucose and nLDL increased IL-6 levels in a dose-dependent manner at 6 and 24 h. In conclusion, glucose treatment on HMEC-1 cells exerted a mild stimulus on NO and IL-6 production. nLDL treatment showed a similar glucose stimulus on NOx, but it induced an intense pro-inflammatory activity and showed the ability to stimulate ET-1 synthesis.

Endothelial system acts as a large endocrine organ in the human body; however, little is still known about the regulative role of THs on endothelial cells. Aim of the present study was to investigate the expression of the TH deiodinases (D1, D2, and D3) and TH receptors (TRα1, TRα2, and TRβ1) in an endothelial microvascular cultured cell model (HMEC-1), after stimulation with triiodothyronine (T3, 10–100 nM), thyroxine (T4, 10–100 nM), and reverse T3 (rT3, 1–10 nM). DIO1 was significantly inhibited by T4 at 10 and 100 nM (p p p p p p p p p p

Lectin-like oxidized low-density lipoprotein (LOX-1) has been identified in endothelial cells as the main receptor of oxidized low-density lipoprotein (OxLDL). LOX-1 is upregulated in the presence of pathological conditions including atherosclerosis, hypertension, and diabetes because it acts as a mediator of "endothelial dysfunction". It promotes the generation of superoxide anion (O2(-)), the inhibition of nitric oxide (NO) production and the increment of endothelial adhesiveness to monocytes. Recently, it was reported that OxLDL, binding to LOX-1, determined a significant increase in the generation of reactive oxygen species (ROS), suggesting the involvement of signaling pathways such as mitogen-activated protein kinases (MAPKs). It is now generally accepted that ROS act indirectly on the modulation of LOX-1 expression because ROS oxidize native LDL. Moreover, LOX-1 activation per se may stimulate ROS generation. Accordingly, our findings showed that high levels of ROS can directly increase LOX-1 production in microvascular endothelial cells (HMEC-1). It has been reported that OxLDL, usually > 20 μg protein/ml, induced apoptosis in a variety of cell types. At low concentrations (< 5 μg protein/ml) OxLDL appears to be associated with cell proliferation and low levels of ROS-induced capillary tube formation in endothelial cells. Our data and those of the literature indicate the existence of a direct control of LOX-1 by ROS. Although ROS in large amounts clearly have detrimental effects on cell biology, small amounts of ROS could have a beneficial effect, suggesting its therapeutic potential for reducing ischemic tissue.