In vitro efficacy of a sterically stabilized immunoliposomes targeted to membrane type 1 matrix metalloproteinase (MT1-MMP).

The poor selective cytotoxicity of anticancer drugs lead to dose-limiting adverse effects which compromise the clinical outcome. Solid tumors recruit new blood vessels to support their growth, and epitopes that are uniquely expressed on tumor cells and tumor endothelial cells (ECs) can function as targets for immunoliposomal anticancer drugs. Membrane type 1 matrix metalloproteinase (MT1-MMP), an important protein related to tumor growth and angiogenesis, is expressed on malignant tumor cells and is activated ECs. Selective delivery could be achieved by targeting MT1-MMP, as well as other angiogenic ECs. In this regard, an anti-MT1-MMP Fab' antibody was used to prepare a MT1-MMP targeted sterically stabilized immunoliposomes (SIL[anti-MT1-MMP(Fab')]). The binding and intracellular distribution of SIL[anti-MT1-MMP(Fab')] and a non-targeted sterically stabilized liposomes (SL) were examined using human fibrosarcoma HT-1080 cells. SIL[anti-MT1-MMP(Fab')] was taken up by the cells in a lipid concentration, temperature, and time dependent manner, ultimately accumulating in the lysosomes. The cytotoxicity of doxorubicin (DXR)-containing SIL[anti-MT1-MMP(Fab')] (DXR-SIL[anti-MT1-MMP(Fab')]) was significantly higher than that of DXR-containing SL. The cellular internalization of SIL[anti-MT1-MMP(Fab')] was inhibited by endocytosis inhibitors, suggesting that their internalization was mediated via clathrin- or caveolae-dependent endocytosis. Furthermore, the efficient binding of SIL[anti-MT1-MMP(Fab')] was observed on human umbilical vein endothelial cells (HUVEC). Based on these results, it would be expected that DXR-SIL[anti-MT1-MMP(Fab')] may achieve direct tumor cell kill and indirect tumor cell kill via the destruction of the tumor endothelium in vivo. This strategy may have the potential for overcoming some major limitations in conventional chemotherapy in vivo.

Serum levels of matrix metalloproteinase 3 (stromelysin 1) for monitoring synovitis in rheumatoid arthritis.

CONTEXT: Matrix metalloproteinase 3 (MMP-3) is expressed in synovial tissues and involved in cartilage destruction in rheumatoid arthritis and osteoarthritis. OBJECTIVE: To study whether measurement of MMP-3 serum concentrations is useful to monitor the activity of rheumatoid synovitis. DESIGN: Levels of MMP-3 in serum and synovial tissue samples obtained from 29 rheumatoid arthritis patients and 20 osteoarthritis patients were measured by the 1-step sandwich enzyme immunoassay system. RESULTS: Levels of MMP-3 in the serum and synovial samples were significantly higher in rheumatoid arthritis than in osteoarthritis (P < .001), and the levels correlated directly with each other (r = 0.712, P < .001; N = 49). Immunohistochemistry demonstrated almost exclusive localization of MMP-3 to the lining cells in rheumatoid synovium. The immunoreactivity correlated directly with the scores of synovial inflammatory cell infiltration (r = 0.606, P < .001; n = 29) and the MMP-3 levels in the synovial tissues (r = 0.564, P = .001; n = 29) and those in the serum samples (r = 0.529, P = .003; n = 29) in rheumatoid arthritis. Levels of MMP-3 in rheumatoid serum samples dropped to low values at 1 and 2 weeks after total knee arthroplasty, while the levels of C-reactive protein increased at 1 week and the erythrocyte sedimentation rate and counts of white blood cells and platelets were unchanged at 1 and 2 weeks postoperative. CONCLUSIONS: Our results demonstrate that MMP-3 levels in the serum of rheumatoid arthritis patients correlate with the levels produced by the synovial lining cells and suggest that the activity of rheumatoid synovitis can be monitored by measuring serum levels of MMP-3.

Development of an ELISA for esRAGE and its application to type 1 diabetic patients.

We recently identified a naturally occurring soluble form of RAGE (the receptor for advanced glycation endproducts, receptor for AGE) in cultured human vascular cells, and named it endogenous secretory RAGE (esRAGE). esRAGE is generated by alternative RNA splicing and is able to capture AGE, and exerts protection against AGE-induced endothelial cell injury. In the present study, the presence of esRAGE in human circulation was demonstrated for the first time, and a highly sensitive and specific sandwich ELISA system for esRAGE was developed to see whether esRAGE could be related to an individual resistance to the development of diabetic vascular complications. Sera from 47 type 1 diabetic subjects without clinical nephropathy (urinary albumin excretion <300mg/g creatinine) and 55 healthy controls were analyzed by the ELISA. Circulating esRAGE concentrations in diabetic patients with simple and proliferative retinopathy (0.09+/-0.02ng/mL, n=16 and 0.08+/-0.02ng/mL, n=8, respectively) were significantly lower than in those without retinopathy (0.13+/-0.06ng/mL, n=23). The results indicate that esRAGE can be a useful biomarker to indicate individual variations in susceptibility to diabetic retinopathy.

Monoclonal antibodies with defined recognition sequences in the stem region of CD44: detection of differential glycosylation of CD44 between tumor and stromal cells in tissue.

CD44 is an enigmatic cell adhesion molecule acting as a major receptor for hyaluronan and playing roles in many biological and pathological processes such as lymphocyte homing, T-cell activation, wound healing, angiogenesis, and metastatic spread of tumor cells. However, the complexity of the molecule, with its alternatively spliced variants, extensive glycosylation, and processing by different proteases, has hampered detailed analysis. In this study, we prepared four monoclonal antibodies (285-2F12, 284-43F1, 268-1F5, and 294-6F2) and one polyclonal antibody (C6) that recognize defined sequences in the stem region of CD44H. Interestingly, two of the monoclonal antibodies, 268-1F5 and 294-6F2, failed to recognize the CD44 expressed in five of the seven human tumor cell lines examined by Western blotting. Treatment of the samples with a combination of neuraminidase and O-glycosidase as well as the expression of mutants with site-directed mutations at possible modification sites rendered the CD44 reactive to the antibodies. Thus, the reactivity of the antibodies is sensitive to O-glycosylation presumably near the recognition sites. Glycosylation of CD44 that affects reactivity to the antibodies was found to be regulated differentially between tumor and stromal cells in two breast and three oral carcinoma tissues. Antibody 268-1F5 reacted to the tumor cells, but not to the cells in the surrounding stroma. On the other hand, the reactivity of 294-6F2 to the cells was opposite between the two tumor types. Thus, these sets of antibodies are useful to detect and analyze the as-yet-unknown roles of site-specific glycosylation of CD44, particularly in tumors.

Production and activation of matrix metalloproteinase-2 in proliferative diabetic retinopathy.

PURPOSE: To investigate the matrix metalloproteinase (MMP) species and their activation associated with the pathogenesis of proliferative diabetic retinopathy (PDR). METHODS: Sandwich enzyme immunoassays were used to measure concentrations of MMP-1, -2, -3, -7, -8, -9, and -13 in vitreous samples from patients with PDR and nondiabetic vitreoretinal diseases. To evaluate activation ratios of the zymogen of MMP-2 (proMMP-2) and -9 (proMMP-9) in the vitreous samples and fibrovascular tissues, gelatin zymography was performed. Production and tissue localization of MMP-2, membrane type 1-MMP (MT1-MMP), tissue inhibitor of metalloproteinases (TIMP)-2, and MMP-9 in the fibrovascular tissues were examined by immunohistochemistry. mRNA expression of MT1-MMP in the tissues was determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Among the seven different MMPs examined in the vitreous samples, only the levels of MMP-2 and -9 were significantly higher in the PDR samples than in the control. However, activation ratios of proMMP-2 (10.6% +/- 11.8%) and proMMP-9 (2.5% +/- 5.1%) in PDR vitreous samples were low and not significantly different from those of the control. In contrast, high activation ratios of proMMP-2 (54.3% +/- 13.6%) and notable activation of proMMP-9 (19.5% +/- 7.8%) were observed in the fibrovascular tissues. Immunohistochemical study demonstrated the localization of MMP-2 and -9 in the endothelial cells and glial cells of the fibrovascular tissues. MMP-2 was colocalized with MT1-MMP and TIMP-2, which are an activator and an activation-enhancing factor, respectively, for proMMP-2. RT-PCR analysis indicated the gene expression of MT1-MMP in the tissues. CONCLUSIONS: These data demonstrate that proMMP-2 is efficiently activated in the fibrovascular tissues of PDR, probably through interaction with MT1-MMP and TIMP-2, and suggest the possibility that the activity of MMP-2 and MT1-MMP is involved in the formation of the fibrovascular tissues.

Vascular endothelial growth factor isoforms and their receptors are expressed in human osteoarthritic cartilage.

To assess the possible involvement of vascular endothelial growth factor (VEGF) in the pathology of osteoarthritic (OA) cartilage, we examined the expression of VEGF isoforms and their receptors in the articular cartilage, and the effects of VEGF on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in OA chondrocytes. Reverse transcriptase-polymerase chain reaction analyses demonstrated that mRNAs for three VEGF isoforms (VEGF(121), VEGF(165), and VEGF(189)) are detectable in all of the OA and normal (NOR) cartilage samples. However, the mRNA expression of their receptors (VEGFR-1 = Flt-1, VEGFR-2 = KDR and neuropilin-1) was recognized only in the OA samples. The protein expression of VEGFR-1 and VEGFR-2 in OA chondrocytes was also demonstrated by immunohistochemistry of the OA cartilage tissue and cultured OA chondrocytes. In situ hybridization and immunohistochemistry indicated that VEGF is expressed in the chondrocytes in the superficial and transitional zones of OA cartilage. A linear correlation was obtained between VEGF immunoreactivity and Mankin scores in the cartilage (r = 0.906, P < 0.001). The production levels of VEGF determined by enzyme-linked immunosorbent assay were significantly 3.3-fold higher in OA than in NOR samples (P < 0.001). Among MMP-1, -2, -3, -7, -8, -9, and -13, TIMP-1 and -2 measured by their sandwich enzyme immunoassay systems, the production of MMP-1 and MMP-3 but not TIMP-1 or TIMP-2 was significantly enhanced by the treatment of cultured OA chondrocytes with VEGF (P < 0.05), whereas no such effect was obtained with cultured NOR chondrocytes. These results demonstrate that VEGF and its receptors are expressed in OA cartilage, and suggest the possibility that VEGF is implicated for the destruction of OA articular cartilage through the increased production of MMPs.