A therosclerosis is characterized with plaque formation in large and medium-sized blood vessels. The stiffened and narrowed blood vessels limit blood circulation and increase plaque thrombogenicity, which threatens the functionality of vital organs such as the heart and brain. [1][2][3] The development of atherosclerosis is a chronic pathological process. Vascular remodeling and inflammation, endothelial dysfunction, smooth muscle cell (SMC) proliferation and migration, and accumulation of cholesterol-rich lipoproteins in blood vessel walls are early events of atherogenesis, resulting in the recruitment of circulating monocytes, their adhesion to endothelium via adhesion molecules, and their differentiation into macrophages. 4 The subendothelial accumulation of cholesterol-laden macrophages is morphologically recognized as foam cells. In humans, these fatty streaks can progress to more advanced lesions characterized by a lipid-rich necrotic core and a fibrous cap consisting of SMCs and collagen.Lesion rupture can result from the decreased viability of SMCs that is necessary for collagen production and for the structural integrity of the fibrous cap following the release of matrix metalloproteinases from apoptotic macrophages. 4-8 Editorial see p 2472 Clinical Perspective on p 2534Hydrogen sulfide (H 2 S), a member of the gasotransmitter family, plays a number of important physiological roles within the body, including protection against cardiovascular disease.9-11 Cystathionine γ-lyase (CSE) endogenously produces H 2 S in the cardiovascular system, 12,13 and the deficiency of CSE in mice leads to decreased endogenous H 2 S level, age-dependent increase in blood pressure, impaired endothelium-dependent vasorelaxation, and accumulation of homocysteine in the blood.14 Administration of NaHS (a H 2 S donor) protects rat aortic SMCs from the cytotoxicity caused Background-Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H 2 S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H 2 S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H 2 S in atherosclerosis development. Methods and Results-Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N-acetylcysteine or ezetimibe, inhibited the acceler...

Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B ␤-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.The extracellular matrix (ECM) 1 provides mechanical support to cells and regulates signals reaching the cell that govern cell localization, differentiation, proliferation, and apoptosis. Components of the ECM, particularly the glycosaminoglycans (GAGs), are able to sequester bioactive molecules such as growth factors (1), proteases (2), and inhibitors. Turnover of the ECM is a highly regulated process necessary for movement of cells and for release of growth factors. Matrix metalloproteases (MMPs) are believed to be key participants in this remodeling; there are at least 20 MMPs, all able to digest various ECM components (3, 4).The MMPs, in turn, are regulated by tissue inhibitors of metalloproteinases or TIMPs. The major function of the TIMPs is to inhibit MMPs; any imbalance in which the activities of MMPs outweigh the TIMP levels will favor tissue destruction and pathological processes (5, 6). The TIMPs also possess growth stimulatory and regulatory activities (7,8). The four members of the TIMP family all have similar secondary structures of six loops stabilized by six highly conserved disulfide bonds. The TIMPs all bind tightly, albeit with widely varying affinity, to the various MMPs. The x-ray structure (9) shows that the N-terminal cysteine chelates the active site zinc. TIMPs have N-and C-terminal domains, each with three loops. The N-terminal domain of TIMP-1 folds readily and displays full inhibitory activity (10).TIMP-3 has several features that distinguish it from the other TIMPs. First, it is the only TIMP to bind tightly to the ECM: it was first observed as a transformation-sensitive protein bound to the ECM of chick embryo fibroblasts (11) and extractable with SDS or guanidine. This protein was subsequently shown ...

Purpose This study aimed to detect cell-surface vimentin (CSV) on the surface of epithelial-mesenchymal transitioned (EMT) circulating tumor cells (CTCs) from blood of patients with epithelial cancers. Experimental Design In this study, 101 patients undergoing post-surgery adjuvant chemotherapy for metastatic colon cancer were recruited. EMT CTCs were detected from blood of patients using 84-1 monoclonal antibody against CSV as a marker. EMT CTCs isolated were characterized further using EMT-specific markers, fluorescent in situ hybridization and single cell mutation analysis. Results Using 84-1 antibody, we detected CSV exclusively on EMT CTCs from a variety of tumor types but not in the surrounding normal cells in the blood. The antibody exhibited very high specificity and sensitivity towards different epithelial cancer cells. With this antibody, we detected and enumerated EMT CTCs from patients. From our observations, we defined a cutoff of < five or ≥ five EMT CTCs as optimal threshold with respect to therapeutic response using ROC curves. Using this defined threshold, the presence of ≥ five EMT CTCs was associated with progressive disease, while patients with less than five EMT CTCs showed therapeutic response. Conclusion Taken together, number of EMT CTCs detected correlated with the therapeutic outcome of the disease. These results establish cell-surface vimentin as a universal marker for EMT CTCs from a wide variety of tumor types and thus provide the foundation for emerging CTC detection technologies and for studying the molecular regulation of these EMT CTCs.

Although circulating tumor cells (CTCs) have potential as diagnostic biomarkers for cancer, determining their prognostic role in cancer patients undergoing treatment is a challenge. We evaluated the prognostic value of programmed death-ligand 1 (PD-L1) expression in CTCs in colorectal and prostate cancer patients undergoing treatment. Peripheral blood samples were collected from 62 metastatic colorectal cancer patients and 30 metastatic prostate cancer patients. CTCs were isolated from the samples using magnetic separation with the cell-surface vimentin(CSV)-specific 84-1 monoclonal antibody that detects epithelial-mesenchymal transitioned (EMT) CTCs. CTCs were enumerated and analyzed for PD-L1 expression using confocal microscopy. PD-L1 expression was detectable in CTCs and was localized in the membrane and/or cytoplasm and nucleus. CTC detection alone was not associated with poor progression-free or overall survival in colorectal cancer or prostate cancer patients, but nuclear PD-L1 (nPD-L1) expression in these patients was significantly associated with short survival durations. These results demonstrated that nPD-L1 has potential as a clinically relevant prognostic biomarker for colorectal and prostate cancer. Our data thus suggested that use of CTC-based models of cancer for risk assessment can improve the standard cancer staging criteria and supported the incorporation of nPD-L1 expression detection in CTCs detection in such models.

Background Detection, isolation and enumeration of circulating tumor cells (CTCs) from cancer patients has become an important modality in clinical management of patients with breast cancer. Although CellSearch, an EpCAM based method that is used to isolate epithelial CTCs has gained immense importance, its inability to detect mesenchymal CTCs from breast cancer patients raises concerns for its utility as a clinical management tool. Methods To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTC from sarcoma tumors. Here in this study, we tested the sensitivity and specificity of detecting CTC from blood collected at a random time during therapy from each of the 58 patients with metastatic breast cancer utilizing 84-1 (mAb against CSV to detect epithelial mesenchymal transitioned CTC) and CellSearch methods. Also we tested the possibility of improving the sensitivity and specificity of detection using additional parameters including nuclear EpCAM localization and epithelial mesenchymal ratios. Results CTC counts using CSV were significant in differentiating treatment responding (stable) and treatment non-responding (progression) populations in comparison to the CellSearch method. The results also indicated that a summation of CTCs detected from both methods with a threshold of 8 CTCs/7.5mL increased the specificity of CTC detection substantially in comparison with other tested combinations as determined by ROC curves. Conclusions Collectively, utilizing a summation of CellSearch and CSV methods provide new insights into using CTC enumeration to assess therapeutic response and thus provides a new approach to personalized medicine in breast cancer patients.