Department Of Biopathology And Therapy Of Inflammation

PREVIOUS PROJECTS

In collaboration with former research staff: Cristina Lupu, Florea Lupu, Aurelian Radu, Geo Serban, Maria Calb
Surface alteration of blood platelets in diabetes mellitus (C Lupu, M Calb, 1988, Atherosclerosis; C Lupu et al., 1992, Platelets; J Mol Cell Cardiol 1993;1994, Platelets).
Structure and function of valvular en dothelial cells in normal and pathological conditions (I Manduteanu et al., 1988, J Mol Cell Cardiol).
Interaction of valvular endothelial cells with blood cells (I Manduteanu et al., 1992, J Submicrosc Cytol Pathol; C Lupu et al., 1993, Platelets; I Manduteanu et al., 1999, Endothelium)
The use of liposomes as drug delivery vehicles (M Voinea et al., 2002, Vascular Pharmacol; M. Voinea et al., 2002, J Cell Mol Med; M. Voinea et al., 2004, Eur J Pharmacol; M. Voinea et al., 2005, Pharm Res; M. Voinea Calin et al., 2009, Cell Tiss Res)
Mechanisms involved in the effects of anti-inflammatory drugs on activated endothelial cells (I Manduteanu et al. 2002, Pharmacology; Eur J Pharmacol 2003, Manduteanu I et al. 2007, Pharmacology; E Dragomir et al. 2004, J Diab Complications).
Modulation of MCP-1 and fractalkine expression by high glucose conditions in vascular cells: effects of anti-inflammatory drugs (E Dragomir et al. 2006, Vascular Pharmacol; E Dragomir et al., 2008 Thromb Haemost).

CURRENT PROJECTS

Molecular links between chronic inflammation and accelerated atherosclerosis: role of resistin and chemokines (fractalkine and CXCL16); new avenues for targeted therapy

Recent evidence supports a central role of inflammatory processes in all phases of atherosclerosis, but the underlying molecular mechanisms are incompletely understood. We hypothesize that resistin (a newly discovered cytokine with an unclear function in humans), fractalkine and CXCL16 (chemokines and cell adhesion molecules) may constitute markers of cardiovascular risk and may contribute to the pathogenesis of accelerated atherosclerosis.
The goal of the project is to study the role of newly described cytokines and chemokines as relevant links between inflammation and accelerated atherosclerosis and find new targets of clinical relevance.
Our main objectives are: (1) to study the effects of resistin and high glucose conditions (as inducers of accelerated atherosclerosis) on the modulation of the expression and function of fractalkine and CXCL16 and on their receptors in cells with key roles in the initiation and progression of atheroma formation. (2) to establish a correlation between the level of plasma cytokines/chemokines and the evolution from inflammation to overt atherosclerosis; (3) to develop a targeted therapy using liposomes as carriers for delivery of inhibitors of chemokine receptor interactions.
We reported that resistin up-regulates fractalkine expression in human endothelial cells and that resistin and TNF-α have no additive effects on fractalkine up-regulation or on the involved signaling molecules (I Manduteanu et al., 2009).
We have also demonstrated that resistin and high glucose concentrations have similar effects on monocyte adhesion to human endothelial cells and indicate resistin, fractalkine and P-sel as important molecular targets for designing drugs to prevent or reduce inflammation-associated atherosclerosis and/or diabetes (I Manduteanu et al., 2010, Biochem Biophys Res Commun).

Ongoing experiments are focused on:

The effect of vascular smooth muscle cells - monocytes interactions on inflammatory molecules and reactive oxygen species production: role of fractalkine – CX3CR1 axis in the process

The prolonged intimal retention of monocytes-macrophages is a central pathogenic process of atherogenesis. Increasing evidence suggests that the adhesive interactions between migrated monocytes and vascular smooth muscle cells may have important contributions in this process. However, the cellular, molecular and signal transduction mechanisms are not clearly understood.
Fractalkine is a chemokine and an adhesion molecule expressed by a range of cell types including endothelial cells, smooth muscle cells (fig. b) and monocytes. In atherosclerotic lesions, fractalkine (CX3CL1) and its receptor (CX3CR1) expressed by smooth muscle cells (SMC) and monocytes/macrophages (fig. a), mediate the heterotypic anchorage and chemotaxis of these cells.
Our specific goal is to search whether, during the close interaction of monocytes with SMC, the binding of fractalkine to its receptor modulates the expression of pro-atherogenic molecules and oxidative stress.
Our recent data indicate that the cross-talk between SMC and monocytes augments the infl ammatory response in both cell types as revealed by the increased expression of TNFα, IL-1β, IL-6, CX3CR1 and MMP-2 and -9. Up-regulation of TNFα (fig. 2A), MMP-9 (fig. 2B) and CX3CR1 (fig. 2C) is further increased upon interaction of SMC with activated monocytes and is dependent on fractalkine/CXRCR1 pair.
The AP-1 transcription factor is activated by fractalkine-receptor binding during the monocyte-SMC co-culture and trigger the gene and protein expression of TNFα, MMP-9 and CX3CR1. These data imply that the fractalkine/CX3RCR1 axis may represent a therapeutic target to impede the infl ammatory process associated with atherosclerosis (E Butoi Dragomir et al. 2011, BBA).
Moreover, preliminary results indicate that the cross-talk between smooth muscle cells and LPS-activated monocytes induces the reactive oxygen species in both cell types by up-regulating the NOX1 expression in SMC and Nox2 expression in monocytes. The signaling mechanism and the transcription factors responsible for oxidative stress induction and Nox subunities increases will be also investigated.

Molecules and mechanisms involved in cytokine and chemokine-dependent vascular inflammation as targets for novel nanotherapeutic strategies

Our goals are to search for the mechanisms by which fractalkine and resistin modulate the inflammation in endothelial cells, smooth muscle cells and monocytes/macrophages, to determine if they contribute to SMC-Mac cross-talk, to get insights in the signalling mechanisms and gene regulation involved and to develop new targeted nanotechnology-based therapeutic strategies to reduce vascular wall inflammation (Fig. 3).
This hypothesis (based on our preliminary data) will be tested by the objectives of the project:
Objective 1: To explore the role of subendothelial resistin on monocytes transmigration.
Objective 2: To analyze the resistin and fractalkine-induced monocytes and SMC phenotype modulation and the molecular mechanisms involved in the macrophage-SMC cross-talk.
Objective 3: To develop new targeted therapeutic strategies to reduce vascular wall inflammation based on nanotechnology.
Recently we have uncovered a novel pro-inflammatory mechanism of action of resistin in human endothelial cells: up-regulation of SOCS3 expression through STAT3 activation. The data indicate SOCS3 as a possible target to modulate the inflammatory processes in vascular cells (M Pirvulescu et al, 2012 BBRC).

Development of strategies for targeted therapies for cardiovascular diseases based on nanoparticles

Goal: Monocyte adhesion to/and migration through endothelium play a major role to the development of atherosclerosis. Therefore, our aim is to achieve targeted drug delivery using nanoparticles (liposomes, lipid and polymeric nanoparticles) that specifically are directed at either: 1) the vascular endothelium or 2) the monocyte-endothelium interactions and/or 3) monocyte transmigration
Objectives:
1: Designing immunoliposomes by coupling antibodies to specific endothelial cell adhesion molecules to the liposome surface.
Using specific immunoliposomes as nanocarriers for inhibition of signalling pathways we plan to develop a drug delivery strategy for selective targeting dysfunctional endothelial cells.
2. Targeting monocytes/macrophages to reduce the inflammatory process within the atherosclerotic plaque.
Considering the multiple roles of monocyte/macrophages during all stages of atherosclerosis we have previously examined whether systemic depletion of monocytes/ macrophages had a beneficial or adverse effect on the lesions development. We have reported that the depletion of monocytes/macrophages using clodronate-encapsulated liposomes in hyperlipemic hamsters is a two ways sword: it has a beneficial effect by decreasing the expression of IL-1β andMMP-2 and MMP-9 activities and an adverse effect by inducing a significant increase in the lipid and collagen content and expansion of valvular lesions (Calin M. et al 2009).
Ongoing experiments are designed to gain more insights into the consequences of local induction of apoptosis of monocytes/macrophages on the plaque development.
3: Nanoparticles designed to target chemokine-related inflammatory processes in vascular diseases.
Since chemokines are known to be critically involved in the recruitment of inflammatory cells into atherosclerotic plaque, we plan to develop a new therapeutic approach intended to achieve the local delivery of chemokine receptor antagonists mediated by targeted sensitive liposomes and prevent the accumulation of pro-inflammatory cells in the vascular wall.
4. Development and characterization of polymeric nanoparticles and lipid nanoemulsions as delivery systems for anti-inflammatory agents
Different types of nanoparticles will be designed, prepared by various methods and characterized for size, zeta potential, structure (electron microscopy), cytotoxicity, therapeutic agents entrapment efficiency and in vitro release. Nanoparticles able to efficiently entrap and deliver hydrophobic agents (such as polyphenols and signalling pathways inhibitors) to endothelium will be optimized. Moreover, efficient delivery systems for siRNA cellular transport aimed to knock down the expression of certain cytokines or chemokines will be developed and tested in vitro and in vivo.

New CARDIOPRO Equipments

- Flow chamber for live-cell microscopy environmental control (FCS2 chamber from Bioptechs, USA): allows observation of leucocytes adhesion in controlled flow conditions to cells plated onto a coverslip and placed in temperature controlled, optical chamber with a microscope Olympus IX81.
- Inverted Microscope Olympus IX81 with independent motorized unit (z axis), objectives from 4x- 60x, fluorescence filters for excitation in UV, blue, green and provided with a powerful software CellSense Dimensions.