Amanda Genis

Melatonin receptor‐mediated protection against myocardial ischaemia/reperfusion injury: role of its anti‐adrenergic actions

Abstract:  Melatonin has potent cardioprotective properties. These actions have been attributed to its free radical scavenging and anti‐oxidant actions, but may also be receptor mediated. Melatonin also exerts powerful anti‐adrenergic actions based on its effects on contractility of isolated papillary muscles. The aims of this study were to determine whether melatonin also has anti‐adrenergic effects on the isolated perfused rat heart, to determine the mechanism thereof and to establish whether these actions contribute to protection of the heart during ischaemia/reperfusion. The results showed that melatonin (50 μm) caused a significant reduction in both isoproterenol (10−7 m) and forskolin (10−6 m) induced cAMP production and that both these responses were melatonin receptor dependent, since the blocker, luzindole (5 × 10−6 m) abolished this effect. Nitric oxide (NO), as well as guanylyl cyclase are involved, as l‐NAME (50 μm), an NO synthase inhibitor and ODQ (20 μm), a guanylyl cyclase inhibitor, significantly counteracted the effects of melatonin. Protein kinase C (PKC), as indicated by the use of the inhibitor bisindolylmaleimide (50 μm), also play a role in melatonin’s anti‐adrenergic actions. These actions of melatonin are involved in its cardioprotection: simultaneous administration of l‐NAME or ODQ with melatonin, before and after 35 min regional ischaemia, completely abolished its cardioprotection. PKC, on the other hand, had no effect on the melatonin‐induced reduction in infarct size. Cardioprotection by melatonin was associated with a significant activation of PKB/Akt and attenuated activation of the pro‐apoptotic kinase, p38MAPK during early reperfusion. In summary, the results show that melatonin‐induced cardioprotection may be receptor dependent, and that its anti‐adrenergic actions, mediated by NOS and guanylyl cyclase activation, are important contributors.

Fenofibrate protects endothelial cells against the harmful effects of TNF-alpha

Introduction: Fenofibrate exerts pleiotropic effects on endothelial cells (ECs) by, amongst others, increasing nitric oxide (NO) production. We aimed to investigate fenofi brate’s putative beneficial actions in healthy or TNF-alpha-induced dysfunctional ECs. Methods: Fenofi brate-induced pro-vasodilatory responses were assessed in aortic rings (50 - 125μM; 30min) with and without L-NMMA (100μM). Rat cardiac microvascular ECs were treated with fenofibrate (30 and 50μM; 1h). In the pre-treatment experiments, fenofibrate (50μM) was administered one hour before TNFalpha treatment (20ng/ml; 24h). NO-production (DAF-2/DA or Griess assay), mitochondrial ROS-production (MitoSox™), cell viability (propidium iodide staining), and changes in the expression/phosphorylation of critical endothelial proteins were measured by Western blotting. Results: Fenofibrate increased NO-production ˜2-fold in healthy ECs (p<0.05 vs. vehicle). A ˜23% pro-vasodilatory response was induced in aortic rings, which was reversed by L-NMMA (p<0.05 vs. fenofibrate). Fenofibrate pretreatment ameliorated TNF-alpha-induced endothelial dysfunction by reversing the loss of NO, improving oxidative stress, restoring cell viability and preventing caspase-3 activation. Protective effects were underpinned by ˜47% and ˜49% up-regulation of activated eNOS and AMP-kinase, respectively (p<0.05 vs. TNFalpha). Conclusions: Fenofibrate protects TNF-alpha-induced dysfunctional ECs via up-regulated eNOS-NO, reduced oxidative stress and improved cell viability. These novel findings warrant further investigations to explore the potential use of fenofibrate as an anti-endothelial dysfunction therapeutic agent.

A histomorphometric study on the effects of antiretroviral therapy (ART) combined with a high-calorie diet (HCD) on aortic perivascular adipose tissue (PVAT)

Perivascular adipose tissue (PVAT), surrounding arteries is metabolically active. Obesity and antiretroviral therapy (ART) may cause pathophysiological conditions in the aortic wall and surrounding PVAT. The aim of the study was to determine the histological effects on the aortic wall, aortic PVAT adipocyte morphology and leptin staining intensity in obese rats treated with ART. Wistar rats (N=36) were divided into four groups; a lean control (C/ART-), ART control (C/ART+), high-calorie diet (HCD) untreated (HCD/ART-) and HCD and ART experimental (HCD/ART+). The aorta and surrounding PVAT were stained with haematoxylin and eosin (H&E) and anti-leptin antibodies for immunohistochemistry (IHC). The C/ART+ group had a thinner tunica media compared to the HCD/ART- group. The tunica adventitia was thicker in the ART groups (C/ART+ and HCD/ART+) compared to the lean control group. White adipocytes in the HCD/ART- group was larger in size compared to the other three groups. The high-calorie diet groups (HCD/ART- and HCD/ART+) had increased adipocyte sizes, for both brown and differentiating adipocytes, compared to the control groups (C/ART- and C/ART+). The unilocular and differentiating adipocytes in the C/ART+ group showed intense leptin staining. Unilocular and differentiating adipocytes in the HCD/ART- and HCD/ART+ groups showed weak to no leptin staining intensity. The present study indicated that ART and a HCD, separately and combined, altered both the tunica media and adventitia of the aortic wall, whereas the HCD alone caused adipocytes to increase in size. The leptin staining intensity suggested that ART alone may lead to increased leptin expression, whereas ART combined with a HCD may cause leptin deficiency. Changes seen with ART in a rat model suggest that aortic wall thickness and PVAT adipocyte morphology alterations should be considered by clinicians in obese individuals receiving ART.