Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals.

Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.     

Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa

Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present. The HUVE and BAE cells were either 125I-surface labeled or metabolically labeled. Triton X-100 lysates of labeled cells were immunoprecipitated with polyclonal antibodies prepared against purified human platelet GP IIb-IIIa complex. Two membrane proteins were detected on both HUVE (Mr = 130,000 and 110,000) and BAE (Mr = 135,000 and 105,000) cells, which were similar to human platelet GP IIb (Mr = 125,000) and GP IIIa (Mr = 108,000). The two membrane proteins from HUVE cells and the two from BAE cells cosedimented in sucrose gradients, indicating that they exist as a complex. Unlike the human platelet GP IIb-IIIa complex, the HUVE and BAE membrane protein complexes were not dissociated by chelation of Ca2+. Platelet GP IIb and GP IIIa and the related membrane proteins on both HUVE and BAE cells showed similar changes in electrophoretic mobility upon disulfide reduction. These data demonstrate that human and bovine endothelial cells synthesize membrane proteins that have properties similar to the platelet membrane GP IIb-IIIa complex.     

Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells

Cultured endothelial cells secrete a platelet-derived growth factor-like molecule (PDGFc). We examined the effects of purified human alpha-thrombin on the production of PDGFc in cultures of human umbilical vein endothelial cells (HUVE) using a specific radioreceptor assay for PDGF. Addition of physiologically relevant concentrations of alpha-thrombin (0.1 to 10 U/ml) induced a time- and dose-dependent increase in the release of PDGFc into the culture medium. Significant stimulation of PDGFc release was observed as early as 1.5 h after addition of alpha-thrombin (10 U/ml) with a 4.9 +/- 1.1 fold increase at 24 h (mean +/- SEM of nine experiments, P less than 0.01). alpha-Thrombin treatment of HUVE did not affect cell viability as assessed by trypan blue dye exclusion. The receptor binding of PDGFc secreted by HUVE in response to alpha-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. alpha-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (less than 0.3 ng per 10(6) cells) compared to levels of PDGFc released into supernatant medium in response to alpha-thrombin (greater than 5.0 ng per 10(6) cells after a 24-h incubation with 10 U/ml alpha-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with alpha-thrombin failed to release PDGFc. Over a 3-h time course, however, alpha-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that alpha-thrombin induces secretion of PDGFc from HUVE by a nonlytic mechanism requiring the serine esterase activity of the enzyme. Although this effect does not initially require de novo protein synthesis, it does require cell-mediated conversion of PDGFc from an inactive to an active form.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10

Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

Dermal nevus cells from congenital nevi cannot penetrate the dermis in skin reconstructs

Congenital nevi are composed of pigment cells bearing common features with melanocytes but showing altered differentiation which leads to nesting and dermal involvement. Using a dead de-epidermized dermis seeded with a combination of keratinocytes and various sources of pigment cells (normal melanocytes, dermal nevus cells from congenital nevi, Bowes melanoma cells), we have studied the formation of nests and the dermal migration of pigment cells together with their secretion profiles of matrix metalloproteinases (MMP). Dermal fibroblasts were also used as control cells in epidermal reconstructs. Besides their morphologic features, the absence of pigment donation to keratinocytes was the major characteristic of dermal nevus cells. A positive correlation was established between the increasing percentage of seeded nevus cells and the patchy pigmentation of reconstructs, as well as the clustering of cells in junctional nests. However, the presence of nevus cells in the dermis of reconstructs was never detected, whereas melanoma cells and dermal fibroblasts could invade the dermis during the time span of the experiments. MMP9 was never expressed in congenital dermal nevus cells but pro-MMP2 was constitutively expressed by all strains of congenital nevus cells and dermal fibroblasts. Melanocytes produced comparable amounts of both pro-MMP2 and pro-MMP9, and Bowes melanoma cells secreted a marginal level of pro-MMP2. In view of their three-dimensional behaviour and secretion of MMPs, we propose that dermal congenital nevus cells correspond to an intermediate status of differentiation between normal melanocytes and melanoma cells. Activation of MMPs by a cofactor or the activation of another signalling pathway seems necessary to induce the dermal passage of nevus cells.     

HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion.     

Endogenous digitalis-like factor as a stimulator of endothelin secretion from endothelial cells

Effects of human urine-derived endogenous digitalis-like factor (EDLF) and ouabain on endothelin (ET) secretion were examined in cultured endothelial cells. ET was secreted in a linear fashion over 5 hours from bovine pulmonary artery endothelium into serum-free medium. EDLF stimulated ET secretion in a dose-dependent manner. In contrast, ouabain did not affect ET secretion at the concentration of 10(-9)-10(-5) M. These results indicate that human urine-derived EDLF is distinct from plant-derived ouabain and act as a stimulator of ET secretion by endothelial cells.     

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10^?9 M TPA and was maximal at 10^?8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10^?7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.     

Paracellular transport of insulin-like growth factor-I (IGF-I) across human umbilical vein endothelial cell monolayers

Insulin-like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF-IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF-I and IGF-II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio-receptor assays using both IGF-I and IGF-II and (ii) affinity label cross-linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP-2, -3, and -4 are secreted into serum-free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF-I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of ¹²⁵I-IGF-I and [³H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of ¹²⁵I-IGF-I across HUVE cell monolayers was not significantly different to that of [³H]inulin, a paracellular probe. Moreover, ¹²⁵I-IGF-I transport was not inhibited by either excess unlabelled IGF-I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit ¹²⁵I-IGF-I binding to HUVE cell monolayers. Our findings show that the movement of free IGF-I across HUVE cell monolayers occurs via a paracellular route and not by a receptor-mediated, transcellular pathway. J. Cell. Physiol. 170:290–298, 1997. © 1997 Wiley-Liss, Inc.     

Nitric oxide and endothelin secretion by brain microvessel endothelial cells: Regulation by cyclic nucleotides

Endothelin (ET)-1 was originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells. It possesses a wide range of biological activities within the cardiovascular system and in other organs, including the brain. Also secreted by endothelial cells, nitric oxide (NO), has recently been identified as a relaxing factor, as well as a pleiotropic mediator, second messenger, immune defence molecule, and neurotransmitter. Most of the data concerning the secretion of these two agents in vitro has been collected from studies on macrovascular endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we have analyzed the ability of brain microvessel endothelial cells in vitro to release ET-1 and NO, which, at the level of the blood-brain barrier, have perivascular astrocytes as potential targets. The present study was performed with immortalized rat brain microvessel endothelial cells, which display in culture a non transformed phenotype. Our data demonstrate that: (1) these cells release NO when induced by IFNγ and TNFα, (2) they constitutively secrete ET-1, and (3) cAMP potentiates the cytokine-induced NO release and exerts a biphasic regulation on ET-1 secretion: micromolar concentrations of 8-Br-cAMP inhibit and higher doses stimulate ET-1 secretion. This stimulation is blocked by EGTA and the calmodulin antagonist W7, but not by protein kinase C inhibitors, suggesting the involvement of the calmodulin branch of the calcium messenger system. These results suggest that cerebral microvessel endothelial cells may participate in vivo to the regulation of glial activity in the brain through the release of NO and ET-1. © 1993 Wiley-Liss, Inc.     

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2 fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9–mCherry fusion protein was detected 6 h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9–mCherry secretion, demonstrating the utility of this method in a biological experiment.     

Three-dimensional collagen matrices induce delayed but sustained activation of gelatinase A in human endothelial cells via MT1-MMP

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, plays an important role during angiogenesis. It is constitutively expressed by human endothelial cells as a latent enzyme and requires activation. Thrombin is the only described physiological inducer of gelatinase A in human endothelial cells. In this study, we investigated the mechanisms of gelatinase A activation by another physiological inducer, collagen. Endothelial cells were cultured on various ECM components for 24 h and the conditioned media were assessed for gelatinase A activity using gelatin zymography. The results demonstrated that type I collagen matrix specifically activates gelatinase A after 24 h in human umbilical vein and 48 h in neonatal foreskin endothelial cells. In contrast, thrombin activated gelatinase A after only 2 h. Activation by collagen was sustained over long periods of time in culture (96 h). Unlike thrombin-induced activation, collagen required active membrane type 1-MMP (MT1-MMP) on the endothelial cell surface to activate gelatinase A. In addition, collagen-induced activation of gelatinase A was inhibited by antibodies to the integrin receptor, alpha(2)beta(1), but not alpha(3)beta(1). Our findings, that collagen can provide long-term activation of gelatinase A are likely to be relevant to endothelial cell invasion during angiogenesis.     

uPA and MMP‐2 were involved in self‐assembled network formation in a two dimensional co‐culture model of bone marrow stromal cells and endothelial cells

Two dimensional (2D) co‐cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self‐assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), matrix metalloproteinase‐2 (MMP‐2) as well tissue inhibitors of matrix metalloproteinase‐2 (TIMP‐2) were investigated in this 2D co‐culture model. We found that the expression of uPA, MMP‐2 in the co‐cultured cells was significantly higher than those in mono‐cultured cells. In opposite, PAI‐1, expressed only by HUVECs is not regulated in the co‐culture. Inhibition assays confirm that uPA played a critical role in the formation of self‐assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP‐2 prevented the formation of self‐assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP‐2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP‐2 activity, and may contribute to endothelial cell migration and the formation of this self‐assembled network observed in these 2D co‐cultured cells. J. Cell. Biochem. 114: 650–657, 2013. © 2012 Wiley Periodicals, Inc.     

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.     

Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is constitutively released from human hepatocytes

Circulating NAMPT (PBEF/visfatin) has pleiotropic functions and is secreted from adipocytes. Since it is doubtful whether serum levels can be explained by secretion from adipocytes alone, we asked whether hepatocytes are also able to liberate NAMPT. Using HepG2 cells and primary rat and human hepatocytes, release of NAMPT into the cell culture supernatant was found to occur constitutively in a time-dependent manner. In primary human hepatocytes, secretion within 24 h was far higher than the cellular content, but was neither influenced by inhibitors of secretion nor by glucose, insulin or TNFα. As determined by size exclusion chromatography, HepG2 lysates and supernatants primarily contained the dimeric form of NAMPT which exhibited similar in vitro specific enzymatic activity. In primary human hepatocytes, secreted NAMPT was less active. Our results demonstrate that human hepatocytes are a potential source of circulating NAMPT.     

The activities of MMP‐9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts

The current study was designed to investigate whether the activities of TGC (total gelatinase and collagenase) as well as MMP‐9 (matrix metalloproteinase‐9, gelatinase B) secreted by the cultured fibroblasts and myoblasts were influenced by the specific extracellular substrates and by cyclic mechanical strain. Fibroblasts (Rat 2) and myoblasts (C2C12) were cultured with either fibronectin, laminin or collagen type I for 24 h and applied with or without a biaxial deformation at 1 Hz using the Flexcell FX‐4000 system. MMP‐9 activity was increased in fibroblasts when the cells were in contact with fibronectin and laminin, while in myoblasts, enhanced activity of the secreted enzyme was only observed when collagen was present. TGC activity expressed from myoblasts was increased in cells growing on all three types of extracellular proteins in response to the mechanical stimulation, but in fibroblasts, such an increase was only observed in cells grown on the laminin coating. In summary, our data demonstrate that the activities of MMP‐9 synthesized by fibroblasts tend to be regulated by the specific extracellular protein the cells are in contact with, whereas the gelatinolytic actions of proteases produced by myoblasts are more responsive to the mechanical deformation.