Branislava Dobutovic

Regulation of Inducible Nitric Oxide Synthase (iNOS) and its Potential Role in Insulin Resistance, Diabetes and Heart Failure.

Nitric oxide synthases (NOS) are the enzymes responsible for nitric oxide (NO) generation. NO is a reactive oxygen species as well as a reactive nitrogen species. It is a free radical which mediates several biological effects. It is clear that the generation and actions of NO under physiological and pathophysiological conditions are regulated and extend to almost every cell type and function within the circulation. In mammals 3 distinct isoforms of NOS have been identified: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The important isoform in the regulation of insulin resistance (IR) is iNOS. Understanding the molecular mechanisms regulating the iNOS pathway in normal and hyperglycemic conditions would help to explain some of vascular abnormalities observed in type 2 diabetes mellitus (T2DM). Previous studies have reported increased myocardial iNOS activity and expression in heart failure (HF). This review considers the recent animal studies which focus on the understanding of regulation of iNOS activity/expression and the role of iNOS agonists as potential therapeutic agents in treatment of IR, T2DM and HF.

Human cytomegalovirus infection and atherothrombosis.

Vascular endothelium, as a key regulator of hemostasis, mediates vascular dilatation, prevents platelet adhesion, and inhibits thrombin generation. Endothelial dysfunction caused by acute or chronic inflammation, such as in atherosclerosis, creates a proinflammatory environment which supports leukocyte transmigration toward inflammatory sites, and at the same time promotes coagulation, thrombin generation, and fibrin deposition in an attempt to close the wound. Life-long persistent infection with human cytomegalovirus (HCMV) has been associated with atherosclerosis. In vivo studies have revealed that HCMV infection of the vessel wall affects various cells including monocytes/macrophages, smooth muscle cells (SMCs) and endothelial cells (ECs). HCMV-infected SMCs within vascular lesions display enhanced proliferation and impaired apoptosis, which contribute to intima-media thickening, plaque formation and restenosis. Monocytes play a central role in the process of viral dissemination, whereas ECs may represent a viral reservoir, maintaining persistent infection in HCMV-infected atherosclerotic patients following the primary infection. Persistent infection leads to dysfunction of ECs and activates proinflammatory signaling involving nuclear factor κB, specificity protein 1, and phosphatidylinositol 3-kinase, as well as expression of platelet-derived growth factor receptor. Activation of these pathways promotes enhanced proliferation and migration of monocytes and SMCs into the intima of the vascular wall as well as lipid accumulation and expansion of the atherosclerotic lesion. Moreover, HCMV infection induces enhanced expression of endothelial adhesion molecules and modifies the proteolytic balance in monocytes and macrophages. As a consequence, infected endothelium recruits naive monocytes from the blood stream, and the concomitant interaction between infected ECs and monocytes enables virus transfer to migrating monocytes. Endothelial damage promotes thrombin generation linking inflammation and coagulation. HCMV, in turn, enhances the thrombin generation. The virus carries on its surface the molecular machinery necessary to initiate thrombin generation, and in addition, may interact with the prothrombinase protein complex thereby facilitating thrombin generation. Thus, infection of endothelium may significantly increase the production of thrombin. This might not only contribute to thrombosis in patients with atherosclerosis, but might also induce thrombin-dependent proinflammatory cell activation. This review summarizes the existing evidence on the role of HCMV in vascular inflammation.

Thrombin and vascular inflammation

Vascular endothelium is a key regulator of homeostasis. In physiological conditions it mediates vascular dilatation, prevents platelet adhesion, and inhibits thrombin generation. However, endothelial dysfunction caused by physical injury of the vascular wall, for example during balloon angioplasty, acute or chronic inflammation, such as in atherothrombosis, creates a proinflammatory environment which supports leukocyte transmigration toward inflammatory sites. At the same time, the dysfunction promotes thrombin generation, fibrin deposition, and coagulation. The serine protease thrombin plays a pivotal role in the coagulation cascade. However, thrombin is not only the key effector of coagulation cascade; it also plays a significant role in inflammatory diseases. It shows an array of effects on endothelial cells, vascular smooth muscle cells, monocytes, and platelets, all of which participate in the vascular pathophysiology such as atherothrombosis. Therefore, thrombin can be considered as an important modulatory molecule of vascular homeostasis. This review summarizes the existing evidence on the role of thrombin in vascular inflammation.

Peroxisome Proliferator-Activated Receptors and Atherosclerosis

The peroxisome proliferator-activated receptors (PPARs) represent the family of 3 nuclear receptor isoforms-PPARα, -γ, and -δ/β, which are encoded by different genes. As lipid sensors, they are primarily involved in regulation of lipid metabolism and subsequently in inflammation and atherosclerosis. Atherosclerosis considers accumulation of the cells and extracellular matrix in the vessel wall leading to the formation of atherosclerotic plaque, atherothrombosis, and other vascular complications. Besides existence of natural ligands for PPARs, their more potent synthetic ligands are fibrates and thiazolidindiones. Future investigations should now focus on the mechanisms of PPARs activation, which might present new approaches involved in the antiatherosclerotic effects revealed in this review. In addition, in this review we are presenting latest data from recent performed clinical studies which have focus on novel approach to PPARs agonists as potential therapeutic agents in the treatment of complex disease such as atherosclerosis.

Insulin, Thrombin, ERK1/2 Kinase and Vascular Smooth Muscle Cells Proliferation

Vascular smooth muscle cells (VSMC) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMC allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMC proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs). Here we overview the work on ERKs 1 to 2, emphasising when possible their biological activities in VSMC proliferation. It is clear from numerous studies including our own, that ERK1/ERK2 pathway has an important role in VSMC proliferation induced by insulin (INS) and thrombin. Despite the physiological and pathophysiological importance of INS and thrombin, possible signal transduction pathways involved in INS and thrombin regulation of VSMCs proliferation remains poorly understood. Thus, this review examines recent findings in signaling mechanisms involved in INS and thrombin- triggered VSMCs proliferation with particular emphasis on ERK1/2 signaling pathways. Future investigations should now focus on the mechanisms of MAPK activation which might therefore represent a new mechanism involved in the antiproliferative effect revealed in this review.

Nitric Oxide and its Role in Cardiovascular Diseases

Nitric oxide synthases (NOS) are the enzymes responsible for nitric oxide (NO) generation. NO is a free radical which reacts with various molecules to cause multiple biological effects. It is clear that the generation and actions of NO under physiological and pathophysiological conditions are exquisitely regulated and extend to almost every cell type and function within the circulation. While the molecule mediates many physiological functions, an excessive presence of NO is toxic to cells. The enzyme NOS, constitutively or inductively, catalyses the production of NO in several biological systems. NO is derived not only from NOS isoforms but also from NOS-independent sources. In mammals, to date, three distinct NOS isoforms have been identified: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important physiological processes. This review focuses on recent advances in the understanding of the interactions between NOS enzymes and pathophysiology of cardiovascular diseases (CVD) and the role of NO agonists as potential therapeutic agents in treatment of CVD.

Effects of ghrelin on protein expression of antioxidative enzymes and iNOS in the rat liver.

Introduction We investigated the effects of ghrelin on protein expression of the liver antioxidant enzymes superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), nuclear factor κB (NFκB) and inducible nitric oxide synthase (iNOS). Furthermore, we aimed to investigate whether extracellular regulated protein kinase (ERK1/2) and protein kinase B (Akt) are involved in ghrelin-regulated liver antioxidant enzymes and iNOS protein expression. Material and methods Male Wistar rats were treated with ghrelin (0.3 nmol/5 µl) injected into the lateral cerebral ventricle every 24 h for 5 days, and 2 h after the last treatment the animals were sacrificed and the liver excised. The Western blot method was used to determine expression of antioxidant enzymes, iNOS, phosphorylation of Akt, ERK1/2 and nuclear factor κB (NFκB) subunits 50 and 65. Results There was significantly higher protein expression of CuZnSOD (p < 0.001), MnSOD (p < 0.001), CAT (p < 0.001), GPx, (p < 0.001), and GR (p < 0.01) in the liver isolated from ghrelin-treated animals compared with control animals. In contrast, ghrelin significantly (p < 0.01) reduced protein expression of iNOS. In addition, phosphorylation of NFκB subunits p65 and p50 was significantly (p < 0.001 for p65; p < 0.05 for p50) reduced by ghrelin when compared with controls. Phosphorylation of ERK1/2 and of Akt was significantly higher in ghrelin-treated than in control animals (p < 0.05 for ERK1/2; p < 0.01 for Akt). Conclusions The results show that activation of Akt and ERK1/2 is involved in ghrelin-mediated regulation of protein expression of antioxidant enzymes and iNOS in the rat liver.

Regulation of Endothelial Nitric Oxide Synthase in Pathophysiological Conditions

The nitric oxide (NO) cascade and endothelial NO synthase (eNOS) are best known for their role in endothelium-mediated relaxation of vascular smooth muscle (VSM). NO generated by eNOS has been established as a key regulatory signaling molecule in the vasculature. The activities of eNOS are controlled by intracellular calcium/calmodulin (CaM) and by binding of the molecular chaperone heat-shock protein 90 (Hsp90). A number of studies have demonstrated a close association between insulin resistance (IR) and NO bioactivity. Some recent studies demonstrate that insulin signaling is essential for normal cardiovascular (CV) function and lack of it such as IR result in CV dysfunction and disease. A key step in the initiation and progression of atherosclerosis is a reduction in the bioactivity of endothelial cell-derived NO. Multiple changes in endothelial function and eNOS activity accompany the onset and development of Type 2 diabetes mellitus (T2DM) and contribute to the development of cardiovascular disease (CVD). This review focuses on recent findings about regulation of eNOS in pathophysiological conditions such are: IR, T2DM and CVD.

Evaluation of the Possible Contribution of Antioxidants Administration in Metabolic Syndrome

The metabolic syndrome (MetS) is common, and its associated risk burdens of diabetes and cardiovascular disease (CVD) are a major public health problem. The hypothesis that main constituent parameters of the MetS share common pathophysiologic mechanisms provides a conceptual framework for the future research. Exercise and weight loss can prevent insulin resistance and reduce the risk of diseases associated with the MetS. Interrupting intracellular and extracellular reactive oxygen species (ROS) overproduction could also contribute to normalizing the activation of metabolic pathways leading to the onset of diabetes, endothelial dysfunction, and cardiovascular (CV) complications. On the other hand, it is difficult to counteract the development of CV complications by using conventional antioxidants. Indeed, interest has focused on strategies that enhance the removal of ROS using either antioxidants or drugs that enhance endogenous antioxidant defense. Although these strategies have been effective in laboratory experiments, several clinical trials have shown that they do not reduce CV events, and in some cases antioxidants have actually worsened the outcome. More research is needed in this field.

Involvement of the ADAM 12 in Thrombin-Induced Rat's VSMCs Proliferation

Cardiovascular disease is the largest single cause of mortality and its major underlying pathology is atherosclerosis. The proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of the various vascular diseases, including atherosclerosis and hypertension. Thrombin (Thr) is involved in the abnormal proliferation of VSMCs associated with atherosclerosis and hypertension. ADAMs (A Disintegrin And Metalloproteinase) are transmembrane metalloproteinases, belonging to the adamalysins group, that are distinct from matrix metalloproteinases (MMPs) in a way as they have an extracellular disintegrin domain and cytoplasmic domain that can associate with intracellular proteins. There is limited knowledge about the presence of ADAM metalloproteinase activity in Thr-induced VSMCs proliferation. Therefore, this review examines recent findings in signaling mechanisms employed by Thr in modulating the regulation of proliferation of VSMCs with particular emphasis on involvement of ADAM 12 which has been identified as an important mediator of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of Thr in vascular biology and vascular diseases.