Summary Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents were oxidized, cysteine-nitrosylated, and depleted of the channel stabilizing subunit calstabin1, compared to RyR1 from younger (3–6 months) adults. This RyR1 channel complex remodeling resulted in “leaky” channels with increased open probability leading to intracellular calcium leak in skeletal muscle. Similarly, six-month old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month old wild type mice. Treating aged mice with S107, stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca2+ release, muscle specific force and exercise capacity. Taken together these data indicate that leaky RyR1 contribute to age-related loss of muscle function.

Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two‐hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity‐binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 ± 11 nM and 125 ± 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type‐1 repeat (TSP‐1) domain of CTGF binds to the exon 7‐coded region of VEGF165 and that the COOH‐terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full‐length CTGF and the deletion mutant possessing the TSP‐1 domain inhibited VEGF165‐induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein‐to‐protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.

IntroductionMycobacterium tuberculosis infects one-third of the world's population (1) and is transmitted by the aerosol route. Although the mechanisms whereby M. tuberculosis evades the host immune response are increasingly well understood (2), those by which M. tuberculosis engages the immune response to drive tissue destruction and hence transmission are relatively poorly characterized (3). The events underlying this immunopathology are not well defined, in part because the mouse, one of the most useful models in which to study M. tuberculosis immunology, does not develop lung pathology similar to that of humans (4, 5). In humans, M. tuberculosis subverts the host immune response to drive proteolytic destruction of the extracellular matrix scaffold. The current paradigm of tuberculosis (TB) pathology proposes that caseation leads directly to cavitation (2, 4, 6). However, this model overlooks that fact that destruction of lung extracellular matrix must be driven by proteases. Fibrillar collagens provide the lung's tensile strength and are highly resistant to enzymatic degradation (7,8). Only collagenolytic MMPs can cleave these helical collagens at neutral pH (9).MMPs are a family of zinc-dependent proteases that can collectively degrade all components of the extracellular matrix (8). MMP activity is tightly regulated at the level of transcription and activation by proteolytic cleavage. MMPs are specifically inhibited by tissue inhibitor of metalloproteinases (TIMPs) (9). Excessive MMP activity is implicated in diverse pulmonary pathologies characterized by extracellular matrix destruction (8). However, despite the potentially key role of MMPs in lung matrix destruction in human TB, the central mechanisms resulting in tissue damage have not been defined.

Cellular functions within tissues are strictly regulated by the tissue microenvironment which comprises extracellular matrix and extracellular matrix-deposited factors such as growth factors, cytokines and chemokines. These molecules are metabolized by matrix metalloproteinases (MMP), a disintegrin and metalloproteinases (ADAM) and ADAM with thrombospondin motifs (ADAMTS), which are members of the metzincin superfamily. They function in various pathological conditions of both neoplastic and non-neoplastic diseases by digesting different substrates under the control of tissue inhibitors of metalloproteinases (TIMP) and reversion-inducing, cysteine-rich protein with Kazal motifs (RECK). In neoplastic diseases MMP play a central role in cancer cell invasion and metastases, and ADAM are also important to cancer cell proliferation and progression through the metabolism of growth factors and their receptors. Numerous papers have described the involvement of these metalloproteinases in non-neoplastic diseases in nearly every organ. In contrast to the numerous review articles on their roles in cancer cell proliferation and progression, there are very few articles discussing non-neoplastic diseases. This review therefore will focus on the properties of MMP, ADAM and ADAMTS and their implications for non-neoplastic diseases of the cardiovascular system, respiratory system, central nervous system, digestive system, renal system, wound healing and infection, and joints and muscular system.

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A disintegrin and metalloproteinases (ADAMs) are involved in various biological events including cell adhesion, cell fusion, membrane protein shedding, and proteolysis. In the present study, our reverse transcription-PCR analysis showed that among the 12 different ADAM species with a putative metalloproteinase motif, prototype membrane-anchored ADAM28m and secreted-type ADAM28s are selectively expressed in human breast carcinoma tissues. By real-time quantitative PCR, their expression levels were significantly higher in carcinomas than in nonneoplastic breast tissues. In situ hybridization, immunohistochemistry, and immunoblotting analyses indicated that ADAM28 is predominantly expressed in an active form by carcinoma cells within carcinoma tissues. A direct correlation was observed between mRNA expression levels and proliferative activity of the carcinoma cells. Treatment of ADAM28-expressing breast carcinoma cells (MDA-MB231) with insulin-like growth factor-I (IGF-I) increased cell proliferation, cleavage of IGF binding protein (IGFBP)-3, as well as IGF-I cell signaling; these processes were all significantly inhibited by treatment with ADAM inhibitor or anti-ADAM28 antibody. Down-regulation of ADAM28 expression in MDA-MB231 cells with small interfering RNA significantly reduced cell proliferation, IGFBP-3 cleavage, and growth of xenografts in mice. In addition, cleavage of IGFBP-3 in breast carcinoma tissues was correlated with ADAM28 expression levels and inhibited by treatment with ADAM inhibitor or anti-ADAM28 antibody. These results show that ADAM28 is overexpressed in an activated form in human breast carcinoma cells and suggest that ADAM28 is involved in cell proliferation through enhanced bioavailability of IGF-I released from the IGF-I/ IGFBP-3 complex by selective IGFBP-3 cleavage in human breast carcinomas.

ADAMs (a disintegrin and metalloproteinases) are multifunctional molecules involved in cell-cell fusion, cell adhesion, membrane protein shedding, and proteolysis. In the present study, we examined the mRNA expression of 13 different ADAM species with putative metalloproteinase activity in human astrocytic tumors, nonneoplastic brain tissues, and other intracranial tumors by reverse transcriptase-polymerase chain reaction, and found that prototype membrane-anchored ADAM12 (ADAM12m) is predominantly expressed in glioblastomas. Real-time quantitative polymerase chain reaction indicated that the expression level of ADAM12m is remarkably at least 5.7-fold higher in glioblastomas (n ‫؍‬ 16) than in nonneoplastic brain tissues (n ‫؍‬ 6), low grade (n ‫؍‬ 7) and anaplastic astrocytic tumors (n ‫؍‬ 9) (P < 0.05 for each group), and intracranial neurinomas (n ‫؍‬ 5) (P < 0.01). In situ hybridization showed that glioblastoma cells are responsible for the gene expression. ADAMs (a disintegrin and metalloproteinases) are a gene family of multidomain membrane-anchored proteins comprising of more than 30 members in various animal species (see http://www.people.virginia.edu/ϳjw7g/Tableof theADAMs.html) and are implicated in pathophysiological conditions, which include neuronal development, 1 cancer development and progression, 2,3 and inflammatory responses 4 through proteolysis, cell adhesion, cell fusion, and cell-matrix interaction. 5,6 They contain several distinct domains with structural homology to the reprolysin/adamalysin family of snake venom metalloproteinases.7 A typical ADAM protein includes an N-terminal signal peptide, and propeptide, metalloproteinase, disintegrin, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains. The metalloproteinase domains of several ADAMs have a catalytic site with the conventional zinc-dependent metalloproteinase sequence (HEXGHXXGXXHD), which is highly homologous to that of the matrix metalloproteinases (MMPs).

The avascularity of cardiac valves is abrogated in several valvular heart diseases (VHDs). This study investigated the molecular mechanisms underlying valvular avascularity and its correlation with VHD. Chondromodulin-I, an antiangiogenic factor isolated from cartilage, is abundantly expressed in cardiac valves. Gene targeting of chondromodulin-I resulted in enhanced Vegf-A expression, angiogenesis, lipid deposition and calcification in the cardiac valves of aged mice. Echocardiography showed aortic valve thickening, calcification and turbulent flow, indicative of early changes in aortic stenosis. Conditioned medium obtained from cultured valvular interstitial cells strongly inhibited tube formation and mobilization of endothelial cells and induced their apoptosis; these effects were partially inhibited by chondromodulin-I small interfering RNA. In human VHD, including cases associated with infective endocarditis, rheumatic heart disease and atherosclerosis, VEGF-A expression, neovascularization and calcification were observed in areas of chondromodulin-I downregulation. These findings provide evidence that chondromodulin-I has a pivotal role in maintaining valvular normal function by preventing angiogenesis that may lead to VHD.