Malay Mandal

Regulation of matrix metalloproteinases: an overview.

Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell-matrix composition. MMP genes show a highly conserved modular structure. Ample evidence exists on the role of MMPs in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis, cardiovascular diseases, pulmonary diseases and cancer. The expression patterns of MMPs have interesting implications for the use of MMP inhibitors as therapeutic agents. Insights might be gained as to the preference for a general MMP inhibitor as opposed to an inhibitor designed to be specific for certain MMP family members as it relates to a defined disease state, and may give clues to potential side effects. The signalling pathways that lead to induction of expression of MMPs are still incompletely understood, but certain patterns are beginning to emerge. Regarding inhibition of MMP expression at the level of kinase pathways, it is possible that selective chemical inhibitors for distinct signalling pathways (e.g. MAPK, PKC) will hopefully, soon be available for initial clinical trials. Overexpression of selective dual specificity MAPK phosphatases have been shown to prevent MMP promoter activation which could also be used as a novel strategy to prevent activation of AP-1 and ETS transcription factors and MMP promoters in vivo. Interactions between members of different transcription factors provide fine-tuning of the transcriptional regulation of MMP promoter activity. MMPs play a crucial role in tumor invasion. Although the expression of MMPs in malignancies has been studied widely, the specific role of distinct MMPs in the progression of cancer may be more complex than has been assumed. For example, it has recently been shown that MMP-3, MMP-7, MMP-9 and MMP-12 can generate angiostatin from plasminogen, indicating that their expression in peritumoral area may in fact serve to limit angiogenesis and thereby inhibit tumor growth and invasion. The recent view about the role of stromal cells in the progression of cancer cell growth and metastasis is particularly interesting, and additional studies about the regulation of MMP gene expression and activity in malignancies are needed to understand the role and regulation of MMPs in tumor cell invasion.

Clinical implications of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are important for normal development, wound healing, and a wide variety of pathological processes, including the spread of metastatic cancer cells, arthritic destruction of joints, atherosclerosis, pulmonary fibrosis, emphysema and neuroinflammation. In the central nervous system (CNS), MMPs have been shown to degrade components of the basal lamina, leading to disruption of the blood brain barrier and to contribute to the neuroinflammatory responses in many neurological diseases. Inhibition of MMPs have been shown to prevent progression of these diseases. Currently, certain MMP inhibitors have entered into clinical trials. A goal to the future should be to design selective synthetic inhibitors of MMPs that have minimum side effects. MMP inhibitors are designed in such a way that these can not only bind at the active site of the proteinases but also to have the characteristics to bind to other sites of MMPs which might be a promising route for therapy. To name a few: catechins, a component isolated from green tea; and Novastal, derived from extracts of shark cartilage are currently in clinical trials for the treatment of MMP-mediated diseases.

Protective role of magnesium in cardiovascular diseases: A review

A considerable number of experimental, epidemiological and clinical studies are now available which point to an important role of Mg2+ in the etiology of cardiovascular pathology. In human subjects, hypomagnesemia is often associated with an imbalance of electrolytes such as Na+, K+ and Ca2+. Abnormal dietary deficiency of Mg2+ as well as abnormalities in Mg2+ metabolism play important roles in different types of heart diseases such as ischemic heart disease, congestive heart failure, sudden cardiac death, atheroscelerosis, a number of cardiac arrhythmias and ventricular complications in diabetes mellitus. Mg2+ deficiency results in progressive vasoconstriction of the coronary vessels leading to a marked reduction in oxygen and nutrient delivery to the cardiac myocytes.Numerous experimental and clinical data have suggested that Mg2+ deficiency can induce elevation of intracellular Ca2+ concentrations, formation of oxygen radicals, proinflammatory agents and growth factors and changes in membrane permeability and transport processes in cardiac cells. The opposing effects of Mg2+ and Ca2+ on myocardial contractility may be due to the competition between Mg2+ and Ca2+ for the same binding sites on key myocardial contractile proteins such as troponin C, myosin and actin.Stimulants, for example, catecholamines can evoke marked Mg2+ efflux which appears to be associated with a concomitant increase in the force of contraction of the heart. It has been suggested that Mg2+ efflux may be linked to the Ca2+ signalling pathway. Depletion of Mg2+ by alcohol in cardiac cells causes an increase in intracellular Ca2+, leading to coronary artery vasospasm, arrhythmias, ischemic damage and cardiac failure. Hypomagnesemia is commonly associated with hypokalemia and occurs in patients with hypertension or myocardial infarction as well as in chronic alcoholism.The inability of the senescent myocardium to respond to ischemic stress could be due to several reasons. Mg2+ supplemented K+ cardioplegia modulates Ca2+ accumulation and is directly involved in the mechanisms leading to enhanced post ischemic functional recovery in the aged myocardium following ischemia. While many of these mechanisms remain controversial and in some cases speculative, the beneficial effects related to consequences of Mg2+ supplementation are apparent. Further research are needed for the incorporation of these findings toward the development of novel myocardial protective role of Mg2+ to reduce morbidity and mortality of patients suffering from a variety of cardiac diseases.

Complement activation in heart diseases: Role of oxidants

Increasing evidence demonstrated that atherosclerosis is an immunologically mediated disease. Myocardial ischemia/reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Three major components are recognized as the major contributing factors in reperfusion injury. These are: (1) molecular oxygen; (2) cellular blood elements (especially the neutrophils); and (3) components of the activated complement system. The latter two often act in concert. Endothelial and leukocyte responses are involved in tissue injury, orchestrated primarily by the complement cascade. Anaphylatoxins and assembly of the membrane attack complex contribute directly and indirectly to further tissue damage. Tissue damage mediated by neutrophils can be initiated by complement fragments, notably C5a, which are potent stimulators of neutrophil superoxide production and adherence to coronary artery endothelium. The complement cascade, particularly the alternative pathway, is activated during myocardial ischemia/reperfusion. Complement fragments such as the anaphylatoxins C3a and C5a, are produced both locally and systematically, and the membrane attack complex is deposited on cell membranes and subsequent release of mediators such as histamine and platelet activating factor (PAF), thereby causing an increase in vascular permeability with concomitant manifestation of cellular edema. Complement increases the expression of CD18 on the neutrophils and increases P-selectin expression on the surface of the endothelium. Mitochondria may be a source of molecules that activate complements during ischemia/reperfusion injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. Tissue salvage can be achieved by depletion of complement components, thus making evident a contributory role for the complement cascade in ischemia/reperfusion injury. The complexities of the complement cascade provide numerous sites as potential targets for therapeutic interventions designed to modulate the complement response to injury. The latter is exemplified by the ability of soluble form of complement receptor 1 (sCR1) to decrease infarct size in in vitro models of ischemia/reperfusion injury. The mechanism(s) that initiates complement activation is not clearly known, although loss of CD59 (protectin) from cells compromised by ischemia/reperfusion may contribute to direct damage of the coronary vascular bed by the terminal complement complex. Therapeutic approaches to ischemia/reperfusion injury in general, and especially those involving complements, are at the very beginning and their potential benefits have still to be adequately evaluated. It may be noted that complement activation has both positive and negative effects and, therefore, might be modulated rather than abruptly blunted.

Structure and evolutionary aspects of matrix metalloproteinases: A brief overview

The matrix metalloproteinases (MMPs) are zinc dependent endopeptidases known for their ability to cleave one or several extracellular matrix (ECM) constituents, as well as non-matrix proteins. They comprise a large family of proteinases that share common structural and functional elements and are products of different genes. All members of this family contain a signal peptide, a propeptide and a catalytic domain. The catalytic domain contains two zinc ions and at least one calcium ion coordinated to various residues. All MMPs, with the exception matrilysin, have a hemopexin/vitronectin-like domain that is connected to the catalytic domain by a hinge or linker region. The hemopexin-like domain influences tissue inhibitor of metalloproteinases (TIMP) binding, the binding of certain substrates, membrane activation, and some proteolytic activities. It has been proposed that the origin of MMPs could be traced to before the emergence of vertebrates from invertebrates. It appears conceivable that the domain assemblies occurred at an early stage of the diversification of different MMPs and that they progressed through the evolutionary process independent of one another, and perhaps parallel to each other.

Hypothalamic-pituitary-thyroid axis status of humans during development of ageing process.

We have investigated hypothalamic-pituitary-thyroid function in four groups of healthy elderly male humans. Group A (n=18, age range 20-45 years) served as healthy younger controls, group B (n=10, age range 50-60 years), group C (n=15, age range 60-70 years) and group D (n=16, age range 70-85 years) are the subjects of this study. Groups C and D showed significantly lower T3 and thyroid-stimulating hormone (TSH), and higher T4 levels with respect to controls. Evidence for TSH circadian modulation was found in group A (control) and group B subjects. The TRH-stimulated TSH peak was reduced among all elderly subjects with respect to controls and appeared to be pronounced with the ageing process. The maximal prolactin response was also inhibited with increasing age. Our study suggest that a resetting of the pituitary threshold for the TSH feed-back suppression along with complex alterations in peripheral thyroid hormone concentrations may, in turn, develop in older people and that appeared to manifest prominently among the oldest population. Additionally, the TSH nocturnal response appeared to be impaired with increasing age indicating an alteration of hypothalamic function.

AGE-DEPENDENT CHANGE IN ARACHIDONIC ACID METABOLIC CAPACITY IN RAT ALVEOLAR MACROPHAGES

Arachidonic acid (AA) metabolism was assessed in cultured alveolar macrophages (AM) obtained from newborn (10 days old) and adult (2 months and 4 months old) rats. The AMs were stimulated with the calcium ionophore, A23187 (10μM). The released radiolabelled AA metabolites were measured by thin layer chromatography. The results showed that among different aged rats, the synthesis of 5‐1ipoxygenase (5‐LO) metabolites, LTB4, LTC4, LTD4 and 5‐HETE were increased with age inspite of similar levels of [14C]AA release. In response to A23187, 5‐LO metabolic capacity of 2 and 4 months old adult rat AMs were increased 21‐fold and 34‐fold, respectively, compared with 10 days old rat AMs. As the metabolic capacity increased, the release of prostaglandins and thromboxane B2 tended to decrease markedly. Newborn rats (10 days old) AM, at the initial developmental stage, did not produce a noticeable amount of 5‐LO metabolites which, conceivably, contribute to high susceptibility of neonatal lung to infection.

Role of Ca2+-dependent metalloprotease-2 in stimulating Ca2+ ATPase activity under peroxynitrite treatment in bovine pulmonary artery smooth muscle membrane.

We have determined effect of the oxidant peroxynitrite (ONOO ‐) on Ca 2+ ‐dependent matrix metalloprotease‐2 (MMP‐2) activity and the role of the protease on Ca 2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO ‐‐triggered conditions. The smooth muscle plasma membrane possesses a 72‐kDa protease activity in a gelatin‐containing zymogram. The 72‐kDa protease activity has been found to be inhibited by tissue inhibitor of metalloprotease‐2 (TIMP‐2), indicating that the protease is the matrix metalloprotease‐2 (MMP‐2). Treatment of the membrane suspension with ONOO ‐ caused stimulation of the MMP‐2 activity (as evidenced by 14 C‐gelatin degradation) and also increased Ca 2+ ATPase activity. The ONOO ‐‐triggered protease activity and the Ca 2+ ATPase activity were found to be inhibited by the antioxidants: vitamin E, thiourea, and mannitol. Pretreatment with catalase and superoxide dismutase did not significantly alter ONOO ‐‐stimulated MMP‐2 activity and Ca 2+ ATPase activity, indicating that peroxide and superoxide are not present in appreciable amount in ONOO ‐. Under both basal and ONOO ‐ triggered conditions, the MMP‐2 activity and the Ca 2+ ATPase activity were also inhibited by EGTA, 1:10‐phenanthroline, and TIMP‐2. However, the ONOO ‐‐stimulated MMP‐2 activity and the Ca 2+ ATPase activity were found to be insensitive to phenylmethylsulfonylfluoride, Bowman‐Birk inhibitor, chymostatin, leupeptin, antipain, N ‐ethylmaleimide, and pepstatin. These results suggest that ONOO ‐ caused stimulation of MMP‐2 activity and that the increased MMP‐2 activity subsequently played a pivotal role in stimulating Ca 2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane.

Identification, purification and partial characterization of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in bovine pulmonary artery smooth muscle.

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle. This inhibitor was purified by a series of anion-exchange, gel filtration and affinity chromatographic procedure. The purified inhibitor showed an apparent molecular mass of 30 kDa in SDS-PAGE. Amino terminal sequence analysis for the first 22 amino acids of the purified inhibitor was also found to be identical to bovine TIMP-1. This glycosylated inhibitor was found to be active against matrix metallorpoteinase-9 (MMP-9, gelatinase B), the ambient matrix metalloproteinase in the pulmonary smooth muscle. The purified TIMP-1 was also found to be sensitive to pure rabbit and human fibroblast collagenase and type IV collagenase. In contrast, it had minimum inhibitory activity against bacterial collagenase. It was also found to be inactive against the serine proteases trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation.

Role of membrane-associated CA2+ dependent matrix metalloprotease-2 in the oxidant activation of Ca2+ATPase by tertiary butylhydroperoxide

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca2++ATPase activity. The smooth muscle plasma membrane possesses a Ca2++ dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA and the tissue inhibitor of metalloprotease-2 (TIMP-2). Since 72 kDa is the molecular mass of MMP-2 and since in our present study the 72 kDa protease in the gelatin containing zymogram is inhibited by matrix metalloprotease inhibitors, EGTA and TIMP-2, it may be suggested that the 72 kDa protease is the MMP-2. In addition to the increasing Ca2++ATPase activity, t-buOOH also enhances the activity of the membrane associated Ca2++ dependent protease that degrades 14C-gelatin. The oxidant triggered protease activity and the Ca2++ATPase activity were found to be prevented by the antioxidant vitamin E, and also by the Ca2++ dependent matrix metalloprotease inhibitors: EGTA and TIMP-2. Adding MMP-2 to the smooth muscle plasma membrane suspension caused an increase in Ca2++ATPase activity and pretreatment with TIMP-2 prevents the increase in Ca2++ATPase activity. Combined treatment of the smooth muscle plasma membrane with low doses of MMP-2 and t-buOOH augments further the Ca2++ATPase activity caused by the respective doses of either t-buOOH or MMP-2. Pretreatment with TIMP-2 prevents the increase in Ca2++ATPase activity elicited by the low doses of MMP-2 and/or t-buOOH.