RICERCA ITALIANA     

TISSUTAL METABOLISM AND GENIC EXPRESSION: NEWS PERSPECTIVES IN SURGERY

Università degli Studi dell'Insubria Varese-Como

Abstract

Sperimental Phase:
-General Surgery
Foresee the use of intraparenchimal microdialisys to identify and quantify directly the entity of cellular damage into the interstitial
hepatic space.The implement of a sperimental study in the swine concerning the effects of hepatic preservation with predetermined ischemic damage levels. The evaluation of relation between ischemic damage, tolerance induction and survival. Foresee to set a sperimental model of colo-rectal surgery in the mouse, to verify the potential measurement of studying parameters (piruvate, lactate, glucose, glycerol) in peritoneal fluid withdrawed by microdialisys.
Clinical phase:
-Vascular surgery
The aim of our study will be to assess the extracellular metabolism of patients affected by critical limb ischemia, who underwent surgical revascularization or endovascular revascularization.
-Vascular Biology
In this context, the main aim of the present study will be to investigate the metabolism of leukocytes and endothelial cells (with particular regard to parameters directly related to the regulation of the inflammatory process and of ATH, and to the role of Ang II) in subjects candidate to surgery for peripheral revascularization. <<<

Principal Investigator

Renzo DIONIGI Università degli Studi INSUBRIA Varese-Como

Research Objectives

The aim of this work is the study of the cellular methabolism different districts and organs involved by surgical procedure.

Sperimental Phase
As we previously said, hepatic regeneration after liver resection is a well known event. Hepatocites, usually in G0 phase, are
stimulated to move into G1 phase and to start proliferating; what and why this phenomenon happen it is still unclear.
It is very likely that specific growth factrs such as EGF, PGF etc, produced locally or carried by the portal system, may stimulatehepatic cells to proliferate and to compensate the resected parenchyma. In order to measure the amount of liver regeneration several techniques have been proposed (see ahead); among them immunohystochemical mesurament of Ki67 cell's expression seem to be aneasy and precise methods. Microdialysis seems to be an optimal technique to collect interstitial fluids of the studied organ, and to measure different molecules such ad ODFR, markers of ischemia and cell damage as well ad growth factors.
The aims of the study are to evaluate the ODFR, markers of cell damages and growth factors production during liver ischemia and reperfusion with different type of ilar clamping, liver regeneration, using intra-parenchymal microdyalisis in rats
Aim of the Study
The above research have the following aims:
1) To continuously monitor and measure the intra-parenchymal production ODFR, markers of cell membrane destruction and
growth factors during ilar clamping performed with different techniques
2) To continuously monitor and measure the intra-parenchymal production ODFR, markers of cell membrane destruction and
growth factors during hepatic reperfusion after ilar clamping
3) To continuously monitor and measure the intra-parenchymal production ODFR, markers of cell membrane destruction and
growth factors during during hepatic regeneration
Liver transplantation and ischemic injury. Effect on tolerance induction To say, an organ already damaged by a bad preservation, will fail soon for toxicity of the immunosuppressants and recurrent rejection episodes. Thus, there is a need for special methods to investigate the metabolism of transplanted tissues through the transplant operation and later on during the attempts to induce tolerance. These techniques show a promising improvement in the accuracy of our understanding of tissue metabolism after transplantation, which could in turn foster the efficacy of treatment.
The unit will develop the proposed program, which will be stratified as per following key activities:
Implementation of an experimental study on liver preservation and postoperative recovery under predetermined ischemic damage
Evaluation of liver function under high dose, short term CyA administration and chimerism amplification by spleen cells
Study of correlation between ischemic damage, tolerance induction and graft survival.

Intraperitoneal Microdialysis: A new method to monitor patients after colo-rectal surgery.
The first phase of the study will verify the possibility for performing bowel resection in rats. Feasibility of intraperitoneal
microdialysis measuring of markers of ischemic damages ( LACTATE; PYRUVATE; GLYCEROL ect) using microdialysis technique.

Clinical Phase
The aim of our study will be to assess the extracellular metabolism of patients affected by critical limb ischemia, who underwent surgical revascularization or endovascular revascularization.In this context, the main aim of the present study will be to investigate the metabolism of leukocytes and endothelial cells (with particular regard to parameters directly related to the regulation of the inflammatory process and of ATH, and to the role of Ang II) in subjects candidate to surgery for peripheral revascularization. <<<

Timescale

24 months

National and international background

Principles of Microdialysis
Microdialysis is a way to examine the interstitial space of intact tissue. The microdialysis unit is a semipermeable membrane which is perfused with a neutral solution using a high performance pump. The microdialysis catheter is placed in the interstitial space of the tissue to be examined. Small molecules can pass in and out of the membrane during perfusion. The ingoing and outgoing microdialysate can be compared. The net changes reflect what is happening in the interstitial space. Microdialysis catheters, designed for use in man, are now available from CMA/Microdialysis. The catheters can easily be inserted in human fat and muscle tissues and the metabolism in these tissues can be investigated.
Studies of metabolite levels in peripheral tissues: Microdialysis can be used to continuously monitor changes in the concentration of various metabolises in adipose tissue and muscle. In particular glucose, lactate and pyruvate (reflecting carbohydrate metabolism) and glycerol (reflecting lipid breakdown through lipolysis) have been investigated. It is also possible to study other small molecules which easily move through the membrane such as adenosine, urea and amino acids. It is possible to determine the true interstitial concentration of various metabolises with microdialysis. One way is to perfuse the tissue at a high speed (1 -5 ml/min) with increasing concentrations of the metabolite to be determined. The differences in the concentrations of the metabolite in the ingoing dialysis solvent versus the outgoing dialysis solvent are determined. From these differences the true concentration of the metabolite in the tissue can be calculated by a simple formula. Another way is to perfuse the tissue at a very low speed (0.3ml/min) with a long dialysis membrane (30 mm). Then, the recovery (i.e.uptake of molecules from the interstitial space) is 100%, thus giving the true tissue concentration directly.
Determination of the tissue flow: Changes in the concentration of a metabolite in the interstitial space is determined by three major factors. Those are local production, local uptake or breakdown by the cells and removal by the tissue flow. In metabolic experiments it is therefore essential to get information about tissue flow. A technique has recently been worked out to indirectly measure the tissue flow with microdialysis. The flow marker is added to the ingoing microdialysis solvent. This marker is usually ethanol. The ratio of outgoing versus ingoing ethanol is determined. This ratio decreases when more ethanol is removed from the microdialysis device by the tissue indicating an increase in blood flow. An increase in the ratio means that less ethanol is removed from the microdialysis system reflecting a decrease in the tissue flow. The ethanol technique is so far only semiquantitative, i.e., it can be used
to determine if blood flow increases or decreases but not at which rates these changes are occurring. However, various
mathematical models are under development which might in the future be used to determine the true rate of tissue flow with the ethanol technique. When the ethanol technique is combined with measurements of metabolites (which can be made simultaneously in the same microdialysis probe) it is possible to get a rather good idea about changes in the production or uptake of a certain metabolite by the tissue. Using the ethanol technique the effects of cathecolamines and insulin on interstitial flow in muscle and fat has been investigated in some detail.
Regulation of metabolism: Metabolically active agents can be added to the ingoing microdialysate. During microdialysis the drugs leave the probe and act on the surroundig tissue influencing the tissue flow and the production and/or uptake of metabolites by the cells in the tissue. These metabolise changes in the tissue will be reflected by corresponding changes of the concentration of metabolites in the outgoing microdialysate. It is also possible to determine the metabolically active agent in the ingoing versus outgoing dialysate and thereby get an idea about the concentration of this agent in the tissue surrounding the probe. Since microdialysis only takes place in a very small part of the total tissue it is possible to add a drug at a very high concentration (i.e., millimolar) to the microdialysis solvent causing a high local concentration of the drug, without getting any generalised effect of it on the organism. With microdialysis it has been possible to perform detailed pharmacological investigations of the adrenergic and insulin systems in human adipose tissue and skeletal muscle. Using this "pharmacological" approach the adrenergic receptors involved in the regulation of lipolysis and lactate metabolism as well as the mechanisms for how insulin acts on lipolysis and on carbohydrate metabolism and interstitial flow have been explored in some detail in human adipose tissue and muscle.
Summary: Microdialysis is now available for clinical and experimental in vivo studies of the metabolism at the cellular level in man; metabolites with a small molecular weight can be investigated easily. CMA/Mcrodialysis has developed microdialysis catheter, pumps and analytical chemistry instruments which are designed for human experiments (CMA 60, CMA 106, CMA 600). A number of different metabolites can be simultaneously measured using the same microdialysis catheter. The metabolite levels can be continuously monitored by microdialysis in skeletal muscle and adipose tissue; these tissues are easily available for human studies. By combining measurements of metabolites with measurements of the escape of ethanol from the microdialysis system it is possible to get an idea about local production and uptake of metabolises in the tissue, since ethanol is a tissue flow marker. Microdialysis can also be used in pharmacological experiments. It is possible to add metabolically active agents at very high concentrations locally to the tissue through the microdialysis system and then investigate the changes in tissue metabolite levels (and in tissue flow) which are induced by these agents. The microdialysis technique is easy to handle and safe for the subjects. It can be used to study children, elderly and critically ill patients without causing important discomfort.
ATHEROSCLEROSIS AND INFLAMMATION
Although critical factors in the complex process of atherogenesis (ATH) are represented by the deposition of cholesterol in the artery wall and by the oxidative modifications of lipoproteins (1-3), increasing evidence is pointing to a major role played by inflammatory mechanisms. In particular, the attention is focussed upon the imbalance of cytokine production (4;5). Endothelial cells (EC) obtained from human atheroma bind monocyte and T lymphocytes while normal EC do not, and VCAM-1 expression increases on EC overlying nascent atheroma (6). Inflammatory processes are also responsable of plaque instability and eventual rupture (7). In this regard, it has been shown that soluble adhesion molecules are involved in the development of atherosclerotic plaque, and their presence is documented not only in the plaque, but also in plasma obtained from patients. In particular, besides VCAM-1, P-selectin and ICAM-1 are strongly expressed on the endothelium overlying human atherosclerotic plaques (8).
During the initiation of ATH, mononuclear leukocytes are recruited into the vessel wall across an intact endothelium that expresses leukocyte adhesion molecules (9) and cell transmigration is governed by chemotactic factors produced in the subendothelial layer and/or by inflammatory cells. In this context, monocyte- and polymorphonuclear neutrophil (PMN)-derived interleukin (IL)-8 plays a pivotal role acting in an autocrine/paracrine fashion, boosting the systemic inflammatory response and locally recruiting inflammatory cells. Its expression is induced in monocytes and macrophages following the addition of oxidized LDL and cholesterol, respectively (10,11). Atheromatous tissue contains IL-8, most of which is derived from intimal macrophages (12). IL-8 is presumed to accelerate ATH by increasing the monocyte-endothelium adhesiveness (13), by its mitogenic and chemoattractant actions on smooth muscle cells (14), and by mediating angiogenesis in the atherosclerotic plaque (15). Besides IL-8, another cytokine of particular interest in ATH is TNF-alpha. TNF-alpha is a pleiotropic cytokine notably involved in T cell-mediated immunity (16) which contributes to the inflammatory process by inducing other mediators, such as plasminogen activator inhibitor type-1, which is considered a risk factor for myocardial infarction (17). An increased expression of TNF-alpha has been reported in human ATH (18), whereas HDL may protect endothelial cells from TNF-alpha-induced apoptosis (19).
Peripheral vascular disease is often characterized by the occurrence of vascular obstruction in various districts and the incidence of myocardial ischemia and stroke is very high in patients suffering from femoral obstruction (20). Femoral endarterectomy (EA) is a safe procedure with an acceptable perioperative morbidity. However, restenosis is common after the procedure. Cell-cell interactions and signaling mechanisms are complex in the inflammatory network (21). Among plaque-infiltrating lymphocytes, the Th1 subpopulation of CD4+ T cells dominates and these cells are the major productors of IL-2 and interferon (IFN)-gamma. There are differences in cytokines expression between plaques but the limited number of plaques available for analysis makes it difficult to characterize cytokine patterns. Th1 and Th2 pathways control each other by producing cytokines (mainly IFN-gamma and IL-10, respectively) that reciprocally inhibit the other pathway (22). The balance between pro-inflammatory and anti-inflammatory stimuli may be critical for the development and complications of ATH. Among the cytokines that are involved in plaque stabilization, a pivotal role is exerted by TGF-beta. For instance, in plaques obtained from patients undergoing carotid EA, TGF-beta1 mRNA was found to be increased in asymptomatic as compared with symptomatic patients (23).
THE RENIN-ANGIOTENSIN SYSTEM AS A MODULATOR OF ATHEROSCLEROSIS AND INFLAMMATION
ATH is characterized by complex phenomena among which the renin-angiotensin system (RAS) is now recognized as an important modulating factor (24-26). In particular, Ang II may result in modifications of lipid metabolism and and plaque instability. Ang II promotes lipid oxidation and uptake by macrophages, migration, proliferation and apoptosis of vascular smooth muscle cells (VSMC) and matrix deposition (27). Ang II exerts its effects through the binding and activation of multiple receptors (28), and contributes to the atherosclerotic process mainly through the activation of the AT1-R type which results in the classical vascular effects of Ang II, including vasoconstriction, cell growth and matrix synthesis (29-31). Ang II through a NF-kappaB dependent pathway, induces TNF-alpha release from adult heart (32). Moreover, Ang II increases the release of cell adhesion molecules, neutrophil infiltration and IL-8 release (33;34). Cardiac Ang II is increased in patients with unstable angina and plays a pivotal role in coronary microvessel inflammation, contributing to the increase of T lymphocytes, macrophages and to upregulation of mRNA of inducible nitrous oxide synthase (iNOs) and of different inflammatory cytokines (35). The involvement of the RAS in coronary and microvessel inflammation is supported by the evidence that treatment with ACE inhibitors or AT1-R antagonists markedly reduces the expression of proinflammatory cytokines and iNOs genes (36). In in vitro studies, Ang II upregulates both mRNA and protein expression of TGF-beta in cardiac fibroblastes, myofibroblast and myocytes (37-39).
ROLE OF THE ANTIINFLAMMATORY ACTIVITY OF STATINS IN ATHEROSCLEROSIS
Statins have pharmacological activities independent from their lipid-lowering action, which nevertheless are likely to play a role in their therapeutic effects in ATH. These effects can be reconduced to the ability to interfere with the inflammatory mechanisms contributing to ATH (40), such as down-regulation of adhesion molecule expression (41), inhibition of expression of the chemokines such as MCP-1 in activated leukocytes and ECs (42).
Statins are likely to exert relevant effects on both IL-8 and TNF-alpha production and activity, and contribute also to improving endothelial dysfunction (43). After myocardial infarction and heart failure, eNOS is dramatically decreased. Statins directly enhance endothelial nitric oxide synthase (eNOS) activity (44) and long-term treatment with statins increases the expression of eNOS (45) while in atherosclerotic vessels it is decreased (46).
Simvastatin was found to lower serum levels of the IL-6, IL-8 and MCP-1 after 6 weeks of treatment and to decrease the mRNA expression of proinflammatory cytokines in peripheral blood mononuclear cells from hypercholesterolemic patients (47). Recently it has been reported that people in the highest IL-8 quartile (measured in peripheral blood) had an increased risk of incident CAD in apparently healthy individuals compared with those in the lowest quartile (48). In a pilot study it has been demonstrated that IL-8 production by PMNs from high-risk patients was reduced after 30-day treatment with simvastatin and this reduction was already detectable at the 3th day of treatment (49).
The clinical relevance of "low-grade inflammation" has become increasingly convincing. In epidemiological studies, high-sensitivity C-reactive-protein (CRP-hs) levels (particularly when associated with an high LDL/HDL ratio) are related to the highest cardiovascular risk (50). In a CARE substudy comparing 391 patients who had underwent a vascular event with matched subjects, the subgroup of patients showing the greater relative risk was the one with "inflammation present" (CPR-hs, serum soluble amiloide) and, when treated with pravastatin benefitted from therapy (51). Moreover, the patients in the highest quartile when divided according to CRP and submitted to coronary angioplastry had the highest mortality if not treated with statins whereas, patients assuming a statin therapy showed a risk similar to the patients without "low grade inflammation" (52). Even more surprisingly, in the AF/TxCAPS (53) the risk reduction during lovastatin treatment was present in patients with "normal" LDL/HDL ratio and CRP-hs levels above the median, and it was of the same magnitude when compared with the reduction of risk in dyslipidemic patients who did not show high CRP.
Circumstantial evidence suggests that statins may interact with the RAS. Hypercholesterolemia induces AT1-R overexpression on ECs, circulating leukocytes and platelets (54) while in hypercholesterolemic patients AT1-R density was reduced by statins (25;26). Treatment with statin prevented the Ang II-induced ROS production in PMN of rats and the fMLP-induced respiratory burst in PMN from healthy donors (55). Simvastatin and atorvastatin decreased AT1-R density and the statin treatment reversed the elevated blood pressure response to Ang II. Moreover, fluvastatin enhances the inhibitory effect of valsartan on vascular neointimal formation, suggesting that a cross-talk may exist between AT1-Rs and statins in vascular smooth muscle cells (56). <<<