Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilagederived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis in vivo and in vitro, as well as of tumor metastasis in vivo.An accumulating body of scientific evidence now shows that the process of new capillary formation, or angiogenesis, is an essential component of a number of serious pathologies including solid tumor growth and metastasis, diabetic retinopathy, rheumatoid arthritis, and many others (1). Given the potential therapeutic benefit that an angiogenesis inhibitor could have in the treatment of these diseases, much of the research attention in this field is now focused on the discovery of suppressors of neovascularization.A wide variety of experimental strategies have been employed in the course of angiostatic drug discovery. Our approach, and that of other groups, has focused on the study of avascular tissues such as cartilage as enriched sources of inhibitors of angiogenesis (2-8). We have now purified a protein from cartilage that is a potent antiangiogenic molecule and have identified it as being troponin I (TnI). MATERIALS AND METHODSPurification of TnI from Cartilage. Tn I was purified from veal scapulae by using a modification of a protocol previously described (7). Briefly, veal scapulae were vacuum-frozen immediately after slaughter and stored at Ϫ20°C until used. Cartilage was scraped first with a periosteal elevator (Arista, New York) and then with a scalpel blade (No. 10, Bard-Parker) until clean of all muscle and connective tissue. Cartilage slices were extracted in 2 M NaCl, precipitated with HCl and ammonium sulfate (25-20%), and fractionated by using a series of chromatography steps: gel filtration on A-1.5 M Sepharose (Bio-Rad) in the presence of 4 M guanidine⅐HCl, ion exchange on a Bio-Rex 70 (Bio-Rad) cation exchange column, gel filtration on a Sephadex G-75 (superfine) (Pharmacia) column, reversed-phase HPLC on a Hi-Pore 304 column (Bio-Rad), and gel filtration on a Progel-TSK G3000SWXL column (30 cm ϫ 7.8 mm) (Supelco). Fractions obtained from each column step were tested for their ability to inhibit capillary endothelial cell (EC) proliferation as described be...

Oxidative stress and cytosolic Ca(2+) overload have important roles on apoptosis in dorsal root ganglion (DRG) neurons after spinal cord injury (SCI). Hypericum perforatum (HP) has an antioxidant property in the DRGs due to its ability to modulate NADPH oxidase and protein kinase C pathways. We aimed to investigate the protective property of HP on oxidative stress, apoptosis, and Ca(2+) entry through transient receptor potential melastatin 2 (TRPM2) and transient receptor potential vanilloid 1 (TRPV1) channels in SCI-induced DRG neurons of rats. Rats were divided into four groups as control, HP, SCI, and SCI + HP. The HP groups received 30 mg/kg HP for three concessive days after SCI induction. The SCI-induced TRPM2 and TRPV1 currents and cytosolic free Ca(2+) concentration were reduced by HP. The SCI-induced decrease in glutathione peroxidase and cell viability values were ameliorated by HP treatment, and the SCI-induced increase in apoptosis, caspase 3, caspase 9, cytosolic reactive oxygen species (ROS) production, and mitochondrial membrane depolarization values in DRG of SCI group were overcome by HP treatment. In conclusion, we observed a protective role of HP on SCI-induced oxidative stress, apoptosis, and Ca(2+) entry through TRPM2 and TRPV1 in the DRG neurons. Our findings may be relevant to the etiology and treatment of SCI by HP. Graphical Abstract Possible molecular pathways of involvement of Hypericum perforatum (HP) on apoptosis, oxidative stress, and calcium accumulation through TRPM2 and TRPV1 channels in DRG neurons of SCI-induced rats. The TRPM2 channel is activated by ADP-ribose and oxidative stress through activation of ADP-ribose pyrophosphate although it was inhibited by N-(p-amylcinnamoyl) anthranilic acid (ACA) and 2-aminoethyl diphenylborinate (2APB). The TRPV1 channel is activated by oxidative stress and capsaicin and it is blocked by capsazepine. Injury in the DRG can result in augmented ROS release, leading to Ca(2+) uptake through TRPM2 and TRPV1 channels. Mitochondria were reported to accumulate Ca(2+), provided intracellular Ca(2+) rises, thereby leading to depolarization of mitochondrial membranes and release of apoptosis-inducing factors such as caspase 3 and caspase 9. HP via regulation of NADPH oxidase and PKC inhibits TRPM2 and TRPV1 channels. The molecular pathway may be a cause of SCI-induced pain and neuronal death, and the subject should be urgently investigated.