Cardiovascular & Haematological Disorders - Drug Targets - Volume 9, Issue 2, 2009

In an aging population vascular disorders well exemplified by the chronic limb ischemia, chronic heart failure, cerebral ischemia and age-related macular degeneration represent a serious medical and socio-economical problem. While there is always a not easily identifiable first pathogenic noxa, all of these diseases are characterized by ischemia, chronic inflammation and tissue degeneration. Orthodox medicine has provided several optimal drugs targeting various pathological situations but, even with their concomitant applications, it is not possible to reduce the chronic oxidative stress. Here it is proposed to associate the approach of ozonated autohemotherapy as a modifier of the biological response capable to block the pathological progress.

Cardiac performance after myocardial infarction is compromised by ventricular remodeling, which represents a major cause of late infarct-related chronic heart failure and death. In recent years, the scientists' interest has focused on the hypothesis that the administration of bone marrow progenitors, following myocardial infarction, could ameliorate left ventricular remodeling by continuing to differentiate along the haematopoietic lineage. This approach has been developed minding to the consolidated use of transfusions to restore lost or depleted blood components and, therefore, as an enriched dose of various progenitors, generally autologous, injected peripherally or directly in the infarcted area. Since the safety of this therapy was not yet established, for ethical reasons pioneering researchers involved in these studies used animal models as surrogate of the human biologic system. Herein this hypothesis of therapy resulted in an increased use of living animals and in the reappraisal of models of myocardial damage with limited discussion on the theoretical basis of animal models applied to cell-based therapies. Recently, the European Union and its commission for surveillance of laboratory animals advanced a new proposal to restrict the use of living animals. This review will focus on the history of models utilization in biomedicine, with particular attention to animal models, and delineate an operative comparison between the two best known models of myocardial injury, namely coronary ligation and cryodamage, in the perspective of adult stem cell research applied to cardiovascular regenerative medicine.

Glycolysis is one of the principle pathways of ATP generation in cells and is present in all cell tissues; in erythrocytes, glycolysis is the only pathway for ATP synthesis since mature red cells lack the internal structures necessary to produce the energy vital for life. Red cell deficiencies have been detected in all erythrocyte glycolytic pathways, although their frequencies differ owing to diverse causes, such as the affected enzyme and severity of clinical manifestations. The number of enzyme deficiencies known is endless. The most frequent glycolysis abnormality is pyruvate kinase deficiency, since around 500 cases are known, the first of which was reported in 1961. However, only approximately 200 cases were due to mutations. In contrast, only one case of phosphoglycerate mutase BB type mutation, described in 2003, has been detected. Most mutations are located in the coding sequences of genes, while others, missense, deletions, insertions, splice defects, premature stop codons and promoter mutations, are also frequent. Understanding of the crystal structure of enzymes permits molecular modelling studies which, in turn, reveal how mutations can affect enzyme structure and function.

Objective: To investigate the expressions of eNOS3 and Ve-cadherin at the first week of reperfused acute myocardial infarction (AMI). Methods:16 of mini-swines (20 to 30 Kg) were randomly assigned to the sham-operated group and the AMI group. Pathologic myocardial tissue was collected at 7-day of LAD reperfusion and assessed by immunofluorescence and laser co-focus microscope scan, in-situ hybridization, real-time quantitative polymerase chain reaction and western blot. Results: At 7-day of reperfusion, the eNOS3 mRNA and protein expressions in the infarcted and marginal areas were lower than those in the normal area and sham-operated area (all P<0.05), similarly the infarcted and marginal areas had lower Ve-cadherin mRNA expression than the normal area and the sham-operated area (all P<0.05), and Ve-cadherin protein expression was lower in the infarcted area compared with the marginal area, the normal area and the sham-operated area (all P<0.05). Conclusion: Lowexpressions of eNOS3 and Ve-cadherin in the salvaged sub-healthy microvascular endothelium of infarcted and marginal areas suggest that endothelial system is impaired at 7-day of reperfused acute myocardial infarction.

The process of atherothrombosis is known to involve endothelial pathology (first drug target), plaque formation (second drug target) and thrombosis (third drug target). However it has recently been postulated that, even before endothelial pathology occurs, the very first step in the process of atherothrombosis is dysfunction of the arterial glycocalyx that lies between the endothelial cells and the blood [1]. So there are really four drug targets, and perhaps the arterial glycocalyx will become the most important for future early prevention of people at risk. We will review the data available on the relationship of glycocalyx dysfunction to risk factors for atherothrombosis and indicate the areas of research that are required to elucidate this important new subject. Up to the present time, hyperglycaemia and oxidised LDL have been identified as causing glycocalyx dysfunction, and we will seek publications on drugs that modify these effects. Attempts are being made to explore the possibility of drug-induced reversal of hyperglycaemia-induced glycocalyx dysfunction. Progress is, however, dependent on grants being made available for work with the essential large animal (pig) experimental model for testing glycocalyx function. Such grants have hitherto not been sufficiently forthcoming, and this needs to be brought urgently to the attention of the pharmaceutical industry.

A considerable number of studies on hematological malignancies have recently demonstrated that the identification of rearrangements in immunoglobulin (Ig) and T-cell receptor (TCR) genes are important tools for diagnosis and follow-up of B- and T-cell disorders. The heterogeneity of these malignancies makes it difficult to carry out a precise assessment in all patients despite well-established morphological and immunophenotyping criteria. Clonal analysis of hematological malignancies is supported by the fact that all malignant cells have a common clonal origin with identically rearranged Ig and/or TCR genes. Identification of B- or T-cell clonality in polyclonal tissue such as the blood is indicative of a lymphoproliferative process. Germline gene segments of Ig heavy chain (IGH), Ig kappa (IGK), Ig lambda (IGL) and TCR are rearranged in each lymphocyte during B- and T-cell differentiation. A specific combination of gene segments and somatic mutations occurring during this process is responsible for the wide diversity of antigen-specific receptors and antibodies. Ig and TCR rearrangements are considered the “fingerprint” of each lymphocyte and therefore can be used as tumor- specific PCR targets for detection of residual malignant cells present after treatment. Determination of minimal residual disease (MRD) has a proven prognostic value and enables effective early interventional treatment. This is becoming routinely implemented in several treatment protocols and is increasingly used in guidelines for drug therapy and stem cell transplantation. In this review we focus on: (1) the process of gene rearrangements in B- and T-cells, (2) principles of polymerase chain reaction (PCR)-based assays and real-time PCR methods commonly used to detect and follow clonal Ig and TCR rearrangements, (3) multiplex primer sets recently designed by the BIOMED-2 concerted action group, and (4) application of these techniques in MRD detection.

Previous studies using different techniques have shown that adenoviral-mediated gene transfer to different tissues, including the kidney, is more efficient in neonatal mice. In this study, we report a simple technique that allows an efficient and long term expression of β-galactosidase (β-gal) in the heart of newborn mice. Newborn and adult C57BL6/J mice were subjected to a single retro-orbital venous plexus injection of recombinant adenoviral vectors (rAd) (2 x 10⁹ particles/ g body weight) carrying the lac Z gene. Seven days after the injection, positive β-gal staining was systematically observed in the heart, lung, intestine, liver, kidney and spleen of newborn mice. However, only the heart showed persistent expression of β-gal one year after the initial injection. In contrast, adult mice showed only significant but transient β- gal expression mainly in the liver. In summary, we have found that a single retro-orbital intravenous injection can be used to establish a long-term adenoviral-mediated gene transfer to cardiac cells of newborn mice.

Proteomics applications to study the molecular effects of drug administration (pharmacoproteomics) on left ventricular hypertrophy (LVH) and atherosclerosis are here reviewed. In most cases, an absence of complete normalization after treatment is revealed, in contrast to what is reported by classical approaches.

Since most thrombotic reactions occur on the vessel wall, interaction of anticoagulants with vascular components is critical. Heparin (H) is the primary drug for treatment and prevention of thrombosis. To improve H's efficacy and bioavailability, a covalent complex of H and its biological target, antithrombin (AT), was developed. While H has a short, variable intravenous half-life leading to unpredictable anticoagulation, clearance of covalent ATH complex is slower. H's variable anticoagulant effect arises from interactions with plasma and vessel wall proteins. ATH has increased bioavailability due to lower plasma protein and endothelial binding relative to H. Pharmacodynamic studies demonstrate that the AT moiety can regulate ATH binding to target tissues. For example, blood vessel binding is enhanced using ATH containing recombinant AT with oligomannose structures that can interact with endothelial mannose receptor lectins. Furthermore, recent work has shown that inhibition of factor VIIa/tissue factor complex by ATH is significantly faster than AT + H. Similarly, thrombin bound to endothelial thrombomodulin is inhibited more efficiently by ATH than by AT + H, which might improve regulation of thrombin generation. Overall, linking H to AT may prevent unwanted protein interactions and allow vessel wall sites to be targeted. This review examines ATH biodistribution.