Department Of Pathophysiology And Pharmacology

PREVIOUS PROJECTS

Biochemical mapping of endothelial cell surface: evidence for differentiated microdomains (Ghinea N. and Simionescu N., J. Cell Biol. 1985; Ghinea N. et al., J Submicrosc Cytol Pathol, 1987; Leabu M. et al., J Submicrosc Cytol Pathol, 1987)
Detection of Albumin Binding Proteins (Ghinea N. et al., J Cell Biol, 1988; Popov D., J Mol Cell Cardiol, 1992)
Interaction of AGE-albumin with normal and diabetic capillary endothelium (Schmidt A.M. et al., Proc Natl Acad Sci USA, 1994; Popov D. and Simionescu M. Arch Physiol Biochem, 2006; Simionescu M. et al., Cell Tiss Res, 2009)
Pathomorphological changes of micro- and macrovasculature in diabetes (Popov D. et al., Acta Diabetol, 1996, 1997; Simionescu M. et al., Am J Pathol, 1996; Popov D. and Simionescu M., Eur Respir J, 1997; Mompeo B. et al., J Submicrosc Cytol Pathol, 1998; Costache G. et al., J Submicrosc Cytol Pathol, 2000; Popov D. et al., Cell Tiss Res, 2002; popov D. et al., Diabetologia 2003)
Vascular reactivity of resistance arteries in hyperlipemia associated with hyperglycaemia (Georgescu A. and Popov D., J Am Aging Assoc, 2000; Georgescu A. et al., Fundam Clin Pharmacol, 2001, 2003; Georgescu A. et al., Vasc Pharmacol, 2003, 2006)
Mechanisms involved in nebivolol effects on renal artery in diabetes associated with hypertension (Georgescu A. et al., Eur J Pharmacol, 2005, 2008; Georgescu A. et al., Pharmacology 2007, 2008)
Polymorphisms of the leptin gene (Constantin A. et al., Biochem Biophys Res Commun 2009)
Effect of high glucose concentration/diabetes mellitus on human blood platelets (Alexandru N. et al., Platelets 2007; Alexandru N. et al., J Cell Mol Med, 2008; Alexandru N. et al., Clin Chem Lab Med, 2008)
Contribution of circulating microparticles to human peripheral venular dysfunction: focus on chronic venous insufficiency (Georgescu A. et al., J Thromb Haemostasis, 2009)

CURRENT PROJECTS

Identification of molecular events that accompany vascular wall dysfunction in Metabolic Syndrome - related disorders

Goal: Based on the previous data on the remarkable plasticity of vascular smooth muscle cells (SMC) in diabetes mellitus, we are now engaged in deciphering the signalling pathways beyond this phenotypic change. The outcome of the project is to identify the dysfunctional molecules potentially tailored for therapeutic applications, for the benefit of diabetic patients who are at increased cardiovascular risk.
Objectives: 1. evaluation of protein expression of phosphorylated/activated molecules within the insulin cascade, engaged in the control of signalling response amplitude;
2. assessment of protein tyrosine Phosphatase 1B (PTP1B) expression, an enzyme involved in dephosphorylation of insulin receptor substrates, in endoplasmic reticulum stress control, and in regulation of the rate and duration of signalling response.
Results: We have reported that long-term high glucose concentration, acute oxidative stress, and insulin stimulation in vitro imbalance the expression of activated kinases Akt and ERK1/2 and of dephosphorylating PTP1B in the insulin signalling pathway (Popov D. et al., Biochem Biophys Res Commun, 2009). These results have been further confirmed in vivo, in the aorta of streptozotocin-injected hamster (Nemecz M. et al., Ann Rom Soc Cell Biol, 2010). The current research is directed towards substantiation of oxidant/antioxidant imbalance induced by high glucose concentration and insulin in human aortic SMC.

Vascular cells – adipocyte cross-talk within the white adipose tissue.

Objectives: 1. to assess the structure, function, and biochemistry of human subcutaneous fat arterioles in obesity and in obesity associated with Type 2 diabetes;
2. to decipher the effect of human proinflammatory adipokines on endothelial cells (ECs).
Results: 1. Structurally, obesity activates ECs and enhances the accumulation of collagen within the vascular wall, compared to the subcutaneous arterioles of lean subjects. Functionally, obesity increases the sensitivity of adrenergic response, diminishes eNOS protein expression, NO production, endothelium-dependent and insulin-induced vasodilatation (Fig. 1). Biochemically, obesity augments the levels of IRS-1, IRS-2, PI3K and Akt within the insulin cascade. The changes are significantly augmented in obesity associated with Type 2 diabetes, and are likely to have adverse consequences on vascular wall–adipocyte interplay (Georgescu A. et al. Clinical Science, 2011).
2. Human ECs were exposed in vitro to the conditioned medium (CM) of confluent or differentiated preadipocytes (Fig. 2). The level and activity of stress activated kinases and inflammatory mediated signalling molecules are currently examined (Constantin A, work in progress).

A new experimental model for hypertension: the NG-nitro-L-arginine methyl ester (L-NAME) treated hamster

Objective: to decipher the molecular mechanisms beyond left ventricular remodelling.
Results: Chronic inhibition of NO production (for ~6 months) by L-NAME generates systemic hypertension in Golden Syrian hamsters. We show for the first time that cardiac remodelling in hypertensive hamsters implies complex structural alterations involving both the myocytes and the extracellular matrix (ECM): myofibrils disorganization, changes in gap junctions patterns, and excessive ECM deposition. Together, these changes are likely to affect the conduction properties of myocardium (Fig. 3) (Costache G., Dumitrescu M.).

Testing drugs aimed to alleviate Metabolic Syndrome-related vascular dysfunction employing the hypertensive - hypercholesterolemic hamster, an original experimental model that mimics human pathology

Objectives: 1. to substantiate the molecules beyond the contractility reducing effect of enoxaparin in aging and aging associated with diabetes; this property is independent on the anticoagulant effect of the drug (Georgescu A. et al., 2003); 2. to decipher the molecular events beyond the antihypertensive effect of irbesartan (angiotensin II type 1 receptor antagonist) in experimental hypertension associated with hypercholesterolemia.
Results: 1. To the beneficial effect of enoxaparin on vascular reactivity of resistance arteries contributes the MAP kinase pathway, via reduction of c-fos gene and of transcription factor AP-1 protein expression. This may be relevant for the improvement of circulation at the level of the microvasculature of aged persons and old–diabetic patients (Georgescu A. et al. Blood Coagul Fibrinolysis, 2011).
2. Experiments performed so far demonstrate that association of the high salt diet with a hypercholesterolemic diet significantly increases plasma cholesterol and triglyceride concentration, as well as blood pressure (Fig. 4); irbersartan administration (10 mg kg. b.w./day) corrects these disturbances.
3. Experimental hypertension + hypercholesterolemia produce major changes in blood platelets morphology, signalling mechanisms, and oxidative stress. These changes were significantly diminished by irbesartan administration, which functions as an antioxidant. This novel finding supplements the antihypertensive effect of irbesartan (Alexandru N. et al., Journal of Thrombosis and Haemostasis, 2011). Platelets activation is associated with a plethora of endogenous signalling mechanisms that conduct to induction of a robust oxidative/nitrative stress (Fig. 5) (Alexandru N. et al., Trends in Cardiovascular Medicine, 2010).

Circulating microparticles and endothelial progenitor cells (EPCs) in experimental hypertension associated with hypercholesterolemia (HH)

Objectives: employing HH model we aim to 1. Evaluate the modifications taking place in plasma biochemistry and blood pressure; 2. Assess the microparticles (MPs) and EPCs levels; 3. Examine the arteriolar wall structure, function, and signalling molecules and 4. Investigate the effects of irbesartan administration.

Results: Hypertension associated with hypercholesterolemia induces (i) a significant increase in plasma cholesterol and triglyceride and diastolic arterial blood pressure and heart rate, (iii) a marked elevation of MPs and a significant decrease in EPCs; the MPs to EPCs ratio may be considered as a marker of vascular dysfunction, (iv) structural modifications of the arterial wall correlated with altered protein expression of MMP2, MMP9, MMP12, TIMP1, TIMP2 and collagen type I and III, (v) a considerably altered reactivity of the arterial wall and (vi) an inflammatory process characterized by augmented expression of P-Selectin, E-Selectin, vonWillebrand factor, tissue factor, IL-6,MCP-1 and RANTES.
Irbesartan administration has the potential to correct all the altered parameters in HH animals and to mobilize EPCs by NO, chemokines and adhesion molecule-dependent mechanisms (Georgescu et al., Journal of Thrombosis and Haemostasis, 2012).

New equipments acquired from the CARDIOPRO project

-PALM MicroBeam laser microdissection system(located in the Laser Microdissection new laboratory) enables microsurgical collection of morphologically defined cell populations from a tissue section or cultures; the selected cellular material can be subjected to RNA and DNA isolation and subsequent molecular analysis. We will use this apparatus for various applications such as observation of intracellular processes in living cell cultures, recordings of cell/cell interaction, motility and growth, detection of cellular changes induced by pharmacological agents and for ion measurements.
-The Multi Wire Myograph System - 620M (located in the Myography laboratory) allows the simultaneous isometric measurement of vascular reactivity of 4 small or large blood vessel fragments (internal diameter from 60 to 450μm). The system will be employed in our projects to measure vascular reactivity in pathological conditions (diabetes, atherosclerosis, obesity, hypertension) vs. normal, and to assess the effects of drugs and inhibitors on the contractile/vasorelaxant force of the vascular wall.
-The Stemi 2000-C Stereomicroscope ensures an accurate preparation of the blood vessels, their proper insertion into the myograph chamber, and the digital recording of samples image.
-PowerLab 4/30: an integrated system of hardware and software designed to record, display, and analyse experimental data.
-TECAN, Infinite M200 PRO, a multimode microplate reader of absorption and fluorescence.
-The Veriti® 96-Well Thermal Cycler provides a precise control over PCR optimization.