Collagenase IV Research Articles

Collagenase IV and clusterin-modified polycaprolactone-polyethylene glycol nanoparticles for penetrating dense tumor tissues.

Purpose: Novel collagenase IV (ColIV) and clusterin (CLU)-modified polycaprolactone-polyethylene glycol (PCL-PEG) nanoparticles that load doxorubicin (DOX) were designed and fully evaluated in vitro and in vivo.Methods: PCL-PEG-ColIV was synthesized by linking PCL-PEG and ColIV through a carbodiimide method. DOX-loaded nanoparticles (DOX-PCL-PEG-ColIV) were self-assembly prepared, followed by noncovalently adsorbing CLU on the DOX-PCL-PEG-ColIV surface to obtain DOX-PCL-PEG-ColIV /CLU nanoparticles, which can penetrate through the tumor extracellular matrix (ECM) and inhibit phagocytosis by macrophage. The physicochemical properties of nanoparticles were characterized. The cellular uptake and antiphagocytosis ability of nanoparticles in MCF-7 tumor cells and RAW264.7 cells were investigated. The penetration ability of nanoparticles was individually evaluated in the two-dimensional (2D) and three-dimensional (3D) ECM models. The tissue distribution and antitumor effect of nanoparticles were evaluated in MCF-7 cell-bearing nude mice.Results: Compared with DOX-PCL-PEG-COOH nanoparticles, DOX-PCL-PEG-ColIV/CLU nanoparticles could effectively overcome the phagocytosis by RAW264.7 and showed excellent cellular uptake in MCF-7 cells. In addition, they showed remarkable penetration ability through the 2D and 3D ECM models. DOX-PCL-PEG-ColIV/CLU nanoparticles significantly reduced the drug distribution in the liver and spleen and enhanced the drug accumulation in tumor tissue compared with DOX-PCL-PEG-COOH or DOX-PCL-PEG-ColIV nanoparticles. DOX-PCL-PEG-ColIV/CLU nanoparticles showed remarkable antitumor effect but did not cause severe pathological damages in the main tissues, including the heart, liver, spleen, lung, and kidney.Conclusion: Novel ColIV and CLU-modified PCL-PEG nanoparticles showed excellent cellular uptake, ECM penetration, antiphagocytosis, and antitumor effects both in vitro and in vivo.

Intravenous administration of human umbilical cord blood-mononuclear cells dose-dependently relieve neurologic deficits in rat intracerebral hemorrhage model

Human umbilical cord blood (HUCB) is now considered as a valuable source for stem cell-based therapies. Previous studies showed that intravascular injection of the HUCB significantly improves neurological functional recovery in a model of intracerebral hemorrhage (ICH). To extend these findings, we examined the behavioral recovery and injured volume in the presence of increasing doses of human umbilical cord blood derived mononuclear cells (HUC-MCs) after intracerebral hemorrhage in rats. The experimental ICH was induced by intrastriatal administration of bacterial collagenase IV in adult rats. One day after the surgery, the rats were randomly divided into 4 groups to receive intravenously either BrdU positive human UC-MCs (4 × 10(6), 8 × 10(6) and 16 × 10(6) cells in 1 ml saline, n=10, respectively) as treated groups or the same amount of saline as lesion group (n=10). There was also one group (control n=10) that received only the vehicle solution of collagenase. The animals were evaluated for 14 days with modified limb placing and corner turn tests. The transplanted human UC-MCs were also detected by immunohistochemistry with labeling of BrdU. Two weeks after infusion, there was a significant recovery in the behavioral performance when 4 × 10(6) or more UC-MCs were delivered (P<0.05-0.001). Injured volume measurements disclosed an inverse relationship between UC-MCs dose and damage reaching significance at the higher UC-MCs doses. Moreover, human UC-MCs were localized by immunohistochemistry only in the injured area. Intravenously transplanted UC-MCs can accelerate the neurological function recovery of ICH rat and diminish the striatum lesion size by demonstrating a dose relationship between them.

Development of a chromogenic assay for blood-plasma collagenase IV and its initial application

Objective To establish a chromogenic assay for blood-plasma collagenase Ⅳ in order to evaluation the reference range of collagenase Ⅳ in the plasma of healthy individuals.Methods The assay was based on measurement of terminal amino group with succinylated gelatin as substrates and TNBS as chromogenic reagent.The optical density of each reaction was determined at 405 nm using a Sunrise microplate reader.Chromatographic and detection conditions were optimized and performance of the methed was evaluated by recovery experiments and precision experiments.It was compared with ELISA.The levels of collagenase Ⅳ in 112 health persons'plasma were determined and the data were analyzed by SPSS statistical software.Results The whole determing time was within 1.5 h,the linear range of this method was 1.5-10.0 mg/L,and the minimum detection limit was 0.965 mg/L.They were well correlated with ELISA results (R~2=0.999 7,P＜0.01).The within-run CV was less than 3.16%and between-run CV was less than 9.81%.The 95%confidence interval of collagenase Ⅳ in healthy plasma was 33.38-49.80 mg/L.Conclusion This chromogenic assay for blood-plasma collagenase Ⅳ can be used for measurement of collagenase Ⅳ in blood-plasma and the reference range of collagenase Ⅳ in healthy plasma was established. Key words: Collagenases; Trinitrobenzen sulfonic acid; Staining and labeling

Retrieval of Porcine Ovarian Follicles by Different Methods

A series of studies has been conducted to establish a base infrastructure for an ovarian follicle culture system in the porcine and this study was designed to develop an effective retrieval protocol of preantral follicles. Five different methods using collagenase type I (A) or IV (B, Cl, C2 and C3), which employed different treatment durations and/or conditions, were employed and sliced ovarian tissue of prepubertal gilts was provided for the retrieval. A significant increase in total number of follicles retrieved was detected when collagenase IV (methods B or C) was used. In total, more ovarian follicles were retrieved by method B undertaking agitation and method C2 without the agitation than method Cl and C3, while the number of preantral follicles collected was the largest in method B. Neither incubation in 5% CO 2 in air atmosphere instead of the agitation nor increased duration of enzymatic treatment up to 120 minutes improved the efficiency of follicle retrieval. There were no differences in the number of follicles retrieved from intact ovaries and from used ovaries for oocyte collection. These results demonstrate the collagenase IV treatment with agitation is effective for retrieving porcine preantral follicles from the ovaries.

Transforming growth factor β increases collagenase IV in hepatic lipocytes

Transforming growth factor β increases collagenase IV in hepatic lipocytes

Basement membrane and the SIKVAV laminin-derived peptide promote tumor growth and metastases

Laminin, the major glycoprotein component of basement membrane, promotes the malignant phenotype. Cells which are adherent to laminin are more malignant than the non-adherent cells and in certain tumor cells, the number of laminin receptors is positively correlated with malignancy. Laminin also increases collagenase IV activity, an enzyme demonstrated to be critical for tumor spread. A site on laminin, containing the amino acid sequence SIKVAV, has been identified which when injected intravenously with B16F10 melanoma cells, causes an increase in the number of colonies on the surface of the lungs. This peptide does not affect tumor cell arrest in the vasculature or the immune system. It does promote angiogenesis in various in vitro and in vivo models, thereby facilitating tumor cell survival. When a complex mixture of laminin-enriched basement membrane components (Matrigel) is coinjected with tumor cells subcutaneously, tumor incidence and growth increases. Various tumor cell lines and primary isolates, which previously could not form tumors in mice, can be induced to grow rapidly in the presence of Matrigel. Slowly growing tumors or arrested tumors can also be induced to grow more quickly with additional injections of Matrigel. When an SIKVAV-containing synthetic peptide is coinjected with B16F10 tumor cells and Matrigel subcutaneously in mice, larger tumors are formed than that observed with either Matrigel or cells alone. Such studies define the role of laminin in tumor growth and spread and generate new models for studying therapeutic agents. Of particular interest is the ability to grow primary isolates which generally do not grow in mice.

Modulation of plasminogen activation and type IV collagenase activity by a synthetic peptide derived from the laminin A chain

Laminin is a large multidomain glycoprotein with diverse biological activities which include stimulation of neurite outgrowth, enhancement of tumor metastasis, and promotion of cell growth, adhesion, and differentiation. A 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A chain (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg- NH2) was identified which promotes metastasis and stimulates collagenase IV activity in the culture medium of B16 melanoma cells (Kanemoto et al., 1990). We report that this peptide, here designated LamA2091-2108, is also a potent stimulator of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, resulting in a 22-fold increase in the kcat/Km of the activation reaction. The activity of purified type I and type IV collagenase was inhibited by LamA2091-2108 with IC50 values of 3 and 43 microM, respectively. These data support an alternative mechanism for the appearance of collagenase activity in the culture media of melanoma cells, namely, that the peptide stimulates plasminogen activation, subsequently generating collagenase activity.

Identification of an amino acid sequence from the laminin A chain that stimulates metastasis and collagenase IV production.

Tumor cells attach, degrade, and migrate through basement membranes as they metastasize. Laminin, a major glycoprotein of basement membranes, promotes the metastatic activity of tumor cells by stimulating the attachment and migration of the cells and their secretion of collagenase IV. We have identified a synthetic peptide of 19 amino acids (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg) from the sequence of the A chain of laminin that increases experimental metastases of the lungs by murine melanoma cells. The peptide is active when injected either intravenously or intraperitoneally. The peptide increased collagenase IV activity, a key enzyme in the breakdown of basement membranes, to the same extent as laminin. This peptide represents an active site on laminin for promotion of the metastatic phenotype and generates a probe for studying the regulation of malignant activities.

Type IV collagenase activity of a primary HSV-2-induced hamster fibrosarcoma and its in vivo metastases and in vitro clones.

The expression of a basement membrane (BM) collagen-degrading metalloprotease (Type IV collagenase) was studied in a herpes simplex virus (HSV)-2 transformed hamster fibrosarcoma and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. The primary parent tumor was shown to release more or less Type IV collagenolytic activity compared with its sublines (derived from lung nodules that developed after resection of the primary tumor). Normal baby hamster kidney and hamster embryo fibroblasts did not secrete detectable amounts of BM collagenase, whereas normal hamster lung fibroblast secreted intermediate levels of Type IV collagenase activity. The collagenase IV activity of the parent tumor and its in vivo and in vitro derived sublines was assayed in vitro and compared with the ability of the cells lines to spontaneously metastasize in vivo. No correlation between the ability to secrete type IV collagenase and metastatic propensity was detected. Although all cell lines secreted type IV collagenase, the highest activity was recorded for a nonmetastatic variant.

Expression of Collagenase IV (Basement Membrane Collagenase) Activity in Murine Tumor Cell Hybrids That Differ in Metastatic Potential&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Expression of a basement membrane collagen-degrading metalloprotease activity (collagenase IV) was studied in a series of murine cell hybrids derived from fusions between highly metastatic cells (B16-F10RR) or moderately metastatic cells (UV-2237RR) and tumorigenic cells (K-1735 clone 16) or normal cells [peritoneal macrophages (PEC) or C3H mouse embryo fibroblasts (C3H-F)]. The collagenase IV activity of the parent cells and the hybrids was assayed in vitro and compared to the metastatic propensity of the same cells evaluated in both syngeneic (C57BL/6 X C3H/HeN)F1 mice and BALB/c nude mice. The level of collagenase IV activity secreted by the parent lines correlated with their metastatic capacity. The highly metastatic B16-F10RR line secreted the highest enzyme activity, whereas the tumorigenic but nonmetastatic K-1735 clone 16 and the normal parents PEC and C3H-F secreted the lowest enzyme activity. The enzyme activity was completely inhibited with EDTA. The hybrid derived from fusion of cells from two metastatic cell lines as well as hybrids derived from a metastatic and a nonmetastatic tumor cell line expressed higher levels of collagenase IV activity than either parent, and this expression was associated with a high ability to produce metastases in both nude and syngeneic mice. Fusion of metastatic cells with normal cells produced hybrid cells that exhibited suppression of both collagenase IV activity and metastatic capacity. Collagenase IV activity and metastatic propensity can, therefore, be altered by somatic cell hybridization; in the series of hybrids examined in these experiments the expression of type IV collagen-degrading metalloprotease activity and the metastatic ability were closely correlated, which suggests that collagenase IV activity and other properties required for metastasis are genetically linked.