Hussein D. Foda

Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) Binds to the Catalytic Domain of the Cell Surface Receptor, Membrane Type 1-Matrix Metalloproteinase 1 (MT1-MMP)

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2·MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs.

Matrix metalloproteinases in cancer invasion, metastasis and angiogenesis.

Matrix metalloproteinases (MMPs) are a family of proteinases that play an important role in cancer as well as in numerous other diseases. In this article, we summarize the current views on the role of MMPs in cancer with respect to invasion, metastasis and angiogenesis. A positive correlation between tumor progression and the expression of multiple MMP family members in tumor tissues has been demonstrated in numerous human and animal studies. It has been assumed that cancer cells are responsible for producing the MMPs in human tumors. However, recent evidence suggests that tumor cells have docking sites that bind stromal-cell-secreted MMPs. Furthermore, the role of MMPs produced by endothelial cells, especially MMP-2 and MT1-MMP, appear to be crucial for tumor angiogenesis, which is a requirement for cancer growth and dissemination.

Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs

Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP‐1, ‐2, and ‐3 and membrane type 1‐MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain‐induced proliferation and migration of HASM; and the strain‐induced increase in the release of MMPs was accompanied by an increase in tenascin‐C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

CXCR4 and CXCL12 (SDF-1) in Prostate Cancer Inhibitory Effects of Human Single Chain Fv Antibodies

Purpose: Metastasis is a major cause of morbidity in prostate cancer (PCa). Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs. Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis. In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities. Experimental Design: We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system. We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells. We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion. Results: Our results confirmed that PCa cell lines express cell-surface CXCR4. Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues. Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays. Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells. Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels. Conclusions: Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4. Recombinant scFv are an efficient mode of targeting tumor antigens. Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis.

Regulation of gelatinases in human airway smooth muscle cells: mechanism of progelatinase A activation.

Matrix metalloproteinases (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue destruction and remodeling, wound healing, and angiogenesis. The 72-kDa gelatinase A is the most widely distributed of all the MMPs, and along with the 92-kDa gelatinase B, both play an important role in the turnover of basement membrane. The role of MMPs in chronic airway inflammation and remodeling has received scant attention. In this study, we sought to examine the release and activation of gelatinases from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-α and phorbol 12-myristate 13-acetate (PMA) on this release and activation. The role of membrane type 1 MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating progelatinase A has been explored. We have demonstrated, using human airway smooth muscle cells in culture, that 1) ASM releases gelatinase A constitutively and when stimulated with PMA and tumor necrosis factor-α releases gelatinase B, and the release of gelatinase B is protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM cells with PMA or concanavalin A failed to activate progelatinase A despite these agents increasing cell expression of MT1-MMP; and 3) the inability of ASM cell membranes to activate progelatinase A is most likely secondary to the high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.Matrix metalloproteinases (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue destruction and remodeling, wound healing, and angiogenesis. The 72-kDa gelatinase A is the most widely distributed of all the MMPs, and along with the 92-kDa gelatinase B, both play an important role in the turnover of basement membrane. The role of MMPs in chronic airway inflammation and remodeling has received scant attention. In this study, we sought to examine the release and activation of gelatinases from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate (PMA) on this release and activation. The role of membrane type 1 MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating progelatinase A has been explored. We have demonstrated, using human airway smooth muscle cells in culture, that 1) ASM releases gelatinase A constitutively and when stimulated with PMA and tumor necrosis factor-alpha releases gelatinase B, and the release of gelatinase B is protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM cells with PMA or concanavalin A failed to activate progelatinase A despite these agents increasing cell expression of MT1-MMP; and 3) the inability of ASM cell membranes to activate progelatinase A is most likely secondary to the high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.

Pseudopulmonary embolism: Acute respiratory distress in the syndrome of heparin-induced thrombocytopenia

A 73-year-old man with myasthenia gravis was treated with daily plasmapheresis. During the course of treatment, the patient developed progressive thrombocytopenia and an episode of severe acute respiratory distress suggesting pulmonary embolism. The thrombocytopenia and respiratory impairment improved after discontinuation of heparin, and both recurred on heparin rechallenge. The presence of heparin-specific antibodies was confirmed by in vitro assay. The time frame of clinical events suggests a heparin-mediated mechanism for both the thrombocytopenia and respiratory compromise. We conclude that acute respiratory distress may be the presenting manifestation of the syndrome of heparin-associated thrombocytopenia in patients treated with dialysis or apheresis.

Pituitary adenylate cyclase-activating peptide (PACAP), a VIP-like peptide, has prolonged airway smooth muscle relaxant activity

Pituitary adenylate cyclase-activating peptide (PACAP), a widely distributed peptide belonging to the vasoactive intestinal peptide (VIP) family of peptides, stimulates the accumulation of cyclic adenosine monophosphate (cAMP) in many tissues, with greater potency and efficacy than VIP. We report that PACAP-38 was one-third as potent and 70% as efficacious as VIP in producing relaxation of isolated perifused guinea pig strips, although it was approximately twice as effective in stimulating cAMP accumulation. The PACAP-38-induced relaxation, however, was five to eight times as prolonged as that of VIP, and its cAMP stimulation was also more sustained. The prolonged action of PACAP-38 is probably due to its greater resistance to enzymatic degradation. The data suggest that airway relaxation is not solely dependent on the total content of cAMP in airways. PACAP-38 exhibits properties that may be useful in the management of airway constriction.

Attenuation of oxidant-induced lung injury by the synthetic matrix metalloproteinase inhibitor BB-3103.

Acute, diffuse lung injury often complicates sepsis, gastric acid aspiration, extensive trauma, and other conditions. The lung endothelial and epithelial cells are the early targets of this injury leading to increased pulmonary vascular permeability and pulmonary edema. Clinically, this condition is characterized by catastrophic respiratory failure, known as the a dult r espiratory d istress s yndrome (ARDS). Despite advances in our understanding of the pathogenesis of this type of lung injury and in the management of patients with this disorder, the outcome remains grave. There is an urgent need for an effective treatment. Recently matrix metalloproteinases (MMPs), especially gelatinases, have been implicated in the pathogenesis of acute lung injury. Gelatinase A and B and their activated forms are increased in the bronchoalveolar lavage fluid (BAL) of both animal models of acute lung injury and patients with ARDS. 1–4 In this study we sought to investigate the protective effect of an MMP inhibitor in an experimental model of acute lung injury caused by oxygen free radicals. We examined the MMP inhibitor BB-3103, a soluble, low-molecular-weight broad spectrum inhibitor obtained from British Biotechnology Ltd., Oxford, England.

Nitric oxide mediates oxidant tissue injury caused by paraquat and xanthine oxidase.

Abstract In both paraquat and X/XO models of lung injury, the injury, previously attributed to the generation of reactive oxygen species, was related to the induction of NO. synthesis, and was totally preventable by inhibition of this synthesis. The results support the view that the NO. radical itself is an essential intermediary in the pathogenesis of at least some forms of oxidant tissue damage. Another form of oxidant injury, caused by prolonged perfusion of the lung ex vivo, is not mediated by NO. however.