Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.     

Regulation of hepatic fibrosis and extracellular matrix genes by the th response: new insight into the role of tissue inhibitors of matrix metalloproteinases.

Hepatic fibrosis is the hallmark of Schistosoma mansoni infection and often results in portal hypertension and bleeding from esophageal varices. The fibrotic process is highly dependent on type 2 cytokines, yet their role in the regulation of extracellular matrix remodeling genes remains largely unknown. Here, we examined the expression of matrix metalloproteases (MMP) -2, -3, -9, -12, and -13 and their inhibitors, tissue inhibitor of metalloproteases (TIMP) -1, -2, and -3, in the livers of infected mice and correlated their expression profiles with fibrosis and type 2 cytokine production. Expression of MMP-2, -3, -9, -12, and -13 and of TIMP-1 and -2 mRNA rapidly increased at the onset of egg laying in infected mice, while TIMP-3 was unchanged. Because TIMP are presumed to be important regulators of the extracellular matrix, and their expression correlated with the development of fibrosis, we studied their role in fibrogenesis by infecting TIMP-1- and TIMP-2-deficient mice. Strikingly, our data revealed no role for TIMP-1 or -2 in the fibrotic pathology induced by S. mansoni eggs. Because of these findings, we infected IL-10/IFN-gamma-deficient mice that develop an exaggerated fibrotic response to determine whether changes in type 2 cytokine dominance influence the pattern of MMP and TIMP expression. Fibrosis and type 2 cytokine production correlated with increased MMP-2/MMP-9 vs TIMP-1/TIMP-2 expression. These data, in addition to our knockout studies, demonstrate that TIMP-1/TIMP-2 play no essential role in fibrogenesis in schistosomiasis. Indeed, our findings suggest that inhibiting profibrotic cytokines or specific MMP may be a more effective strategy to ameliorate fibrotic pathology.     

Matrix metalloproteinases, tissue inhibitors of metalloproteinases, aminoterminal propeptide of procollagen type III, and hyaluronan in sera and tissue of patients with capsular contracture after augmentation with Trilucent breast implants

In various fibrotic diseases, matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), play an important role. In our study, serum concentrations of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were determined by enzyme-linked immunosorbent assay in 17 female patients with Baker grade II (n =9), III (n =7), and IV (n =1) capsular contracture after bilateral cosmetic mamma augmentation with Trilucent implants (AEI, Inc., Caversham, United Kingdom). Samples of capsular tissue for standard histology and immunohistochemistry were obtained from all patients. Sera from 20 female patients who had plastic surgery for reduction mammaplasty were used as the control group. The aminoterminal propeptide of procollagen type III (PIIINP) and hyaluronan were analyzed as markers for fibrogenesis in both groups, too. Statistical analysis was performed using the Mann-Whitney test and Spearman rank correlation. Patients with capsular contracture presented significantly higher concentrations of TIMP-1 and TIMP-2 in their sera than did the control group (p < 0.05), which correlated with Baker grade (r = 0.7 versus r = 0.65; p < 0.05). The concentration of MMP-2 was significantly higher in the sera of patients with capsule fibrosis, whereas there were no significant differences in MMP-1, MMP-9, and PIIINP serum concentrations. Patients with capsule fibrosis had a significantly lower MMP-to-TIMP ratio (1.1 +/- 0.4, p <0.05) than the control group (1.5 +/- 0.4), which correlated with the Baker classification (r =0.7; p <0.05). The hyaluronan serum concentration of patients with capsular contracture was significantly higher (p < 0.05) and correlated with the Baker grade (r = 0.73; p < 0.05), whereas PIIINP showed no difference. In the histologic evaluation, there was a chronic inflammatory reaction in the capsules around the breast implants and refracting material within the substance. Immunohistochemically, TIMP-1 and TIMP-2 showed an intensive accumulation, and MMP-2 showed a local reaction. PIIINP could be detected, too, whereas there was no staining for MMP-1 and MMP-9.The elevated systemic MMP-2 concentration and the local positive staining in the tissue might be due to the chronic inflammatory reaction. Nevertheless, the balance between MMPs and their natural inhibitors is disturbed in patients with capsule contracture. The elevated systemic concentration of TIMPs might be a pathway in the pathogenesis of severe fibrosis after breast augmentation with alloplastic material. Hyaluronan might be a useful marker for early prediction of capsule fibrosis, whereas PIIINP is not useful as a predictor.     

Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis

Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. In the acute phases of liver injury and as liver fibrosis progresses, there is increased expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Among the changes described, striking features include increased expression of gelatinase A (MMP-2) and membrane type 1-MMP (MT(1)-MMP; MMP-14) as well as TIMP-1 and TIMP-2. These molecules and other family members are involved in regulating degradation of both normal and fibrotic liver matrix. This article outlines recent progress in this field and discusses the mechanisms by which MMPs and TIMPs may contribute to the progression and regression of liver fibrosis. Recently described properties of MMPs and TIMPs of relevance to the pathogenesis of liver fibrosis are outlined. The proposal that regression of liver fibrosis is mediated by decreased expression of TIMPs and involves degradation of fibrillar collagens by a combination of MT(1)-MMP and gelatinase A, in addition to interstitial collagenase, is explored.     

Cardiorenal fibrosis and dysfunction in aging: Imbalance in mediators and regulators of collagen

Cardiorenal fibrosis is a biological process that increases with age and contributes to dysfunction of the heart and kidney. While numerous circulating and tissue hormones, cytokines and enzymes have been identified in the development of cardiorenal fibrosis, several reports have suggested that the anti-fibrotic natriuretic peptide system (NPS), pro-fibrotic renin–angiotensin–aldosterone system (RAAS), transforming growth factor-beta 1 (TGF-β1), matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are fundamental regulators and mediators of this process. However, the simultaneous assessment of these components in the development of age-mediated cardiorenal fibrotic remodeling is not completely understood. Thus, we assessed cardiorenal structure and function, the circulating NPS and RAAS and the cardiorenal tissue gene expression of collagen (Col) I, Col III, TGF-β1, MMP-9 and TIMP-1 in 2 and 20 month old Fischer rats. Our studies determined that aging was characterized by an increase in cardiorenal fibrosis that was accompanied with cardiorenal dysfunction. These alterations were associated with lower circulating atrial and C-type natriuretic peptides and higher angiotensin II and aldosterone levels in the aged rats. Moreover, we observed a decrease in Col I and III and an increase in TIMP- mRNA expressions in the aged heart and kidney, while TGF-β1 expression increased and MMP-9 decreased only in the aged kidney. We conclude that the age-mediated alterations in these fibrotic regulator and mediator profiles favors collagen accumulation due to an imbalance between the NPS and RAAS as well as a decline in the degradative pathway, thus suggesting a therapeutic opportunity to target these components.     

Plasma transforming growth factor beta1, metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 in acute viral hepatitis type B

AIM: Antiproliferative, pro-apoptotic and immunosuppressive activity effects suggest crucial role of transforming growth factor (TGF)-beta1, metalloproteinase (MMP)-1 and its tissue inhibitor (TIMP)-1 in the pathogenesis of acute liver injury that in some patients precede development of chronic liver diseases and fibrogenesis. The aim of this study was to evaluate effect of acute HBV infection on plasma TGF-beta1, MMP-1 and TIMP-1 levels. METHODS: TGF-beta1, MMP-1 and TIMP-1 plasma concentrations were measured with an enzyme immunoassay in 39 patients with acute viral hepatitis type B. Baseline measurement was performed within the first week of jaundice and then weekly up to the fourth week of the disease. Results were compared to baseline and normal values and to liver function tests. RESULTS: Plasma concentrations of TGF-beta1, TIMP-1 and MMP-1 were significantly elevated in the first week of acute viral B hepatitis in comparison to normal. Analysis of individual values demonstrated significant positive correlation between plasma concentrations of TGF-beta1 and TIMP-1. There was no correlation between MMP-1 and TGF-beta1 or TIMP-1. Significant correlation was demonstrated between both TGF-beta1 and ALT or AST as well as between TIMP-1 and ALT, AST or bilirubin. Elevated baseline levels of both TGF-beta1 and TIMP-1 decreased gradually in consecutive weeks of the disease. TGF-beta1 but not TIMP-1 plasma concentrations were significantly lower in 3rd and 4th week than baseline values. MMP-1 concentration remained on baseline level in the 2nd week of the disease. However in the 3rd week its values increased suddenly but the significant difference in comparison to baseline was observed only in 4th week. CONCLUSIONS: These results indicate important role of TGF-beta1, TIMP-1 and MMP-1 in acute viral hepatitis, that seems to be connected first of all with hepatocytes damage. Their role in extracellular matrix metabolism during acute liver injury needs further evaluation.     

The anti-fibrotic effect of liver growth factor is associated with decreased intrahepatic levels of matrix metalloproteinases 2 and 9 and transforming growth factor beta 1 in bile duct-ligated rats

Liver growth factor (LGF), a mitogen for liver cells, behaves as an anti-fibrotic agent even in extrahepatic sites, but its mechanistic basis is unknown. We aimed to determine the intrahepatic expression pattern of key modulators of liver fibrosis in bile duct-ligated rats (BDL) after injection of LGF. BDL rats received either LGF (4.5 microg/ratXdose, two doses/week, at time 0 or 2 or 5w after operation, depending on the group (BDL+LGF groups, n=20) or saline (BDL+S groups, n=20). Groups were compared in terms of fibrosis (histomorphometry), liver function (aminopyrine breath test), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor beta 1 (TGF-beta1) and liver endoglin content (Western blotting), and serum tissue inhibitor of metalloproteinases 1 (TIMP-1) levels (ELISA). In BDL+LGF rats, the fibrotic index was significantly lower at 5w, p=0.006, and at 8w, p=0.04, than in BDL+S rats. Liver function values in BDL+LGF rats were higher than those obtained in BDL+S rats (80% at 5w and 79% at 8w, versus 38% and 29%, p<0.01, taking healthy controls as 100%). Notably, in BDL+LGF rats the intrahepatic expression levels of both MMPs were lower at 2w (MMP-2, p=0.03; MMP-9, p=0.05) and 5w (MMP-2, p=0.05, MMP-9, p=0.04). In addition, the hepatic TGF-beta1 level in BDL+LGF rats was lower at 2w (36%, p=0.008), 5w (50%) and 8wk (37%), whereas intrahepatic endoglin expression remained constant in all BDL rats studied. LGF ameliorates liver fibrosis and improves liver function in BDL rats. The LGF-induced anti-fibrotic effect is associated with a decreased hepatic level of MMP-2, MMP-9 and TGF-beta1 in fibrotic rats.     

TIMPs and MMPs expression in CSF from patients with TSP/HAM

The tropical spastic paraparesis or human T-cell lymphotropic virus associated myelopathy (TSP/HAM), has been related with an overexpression of matrix metalloproteinases (MMPs), especially MMP-9. Initial studies of reverse zymography with cerebrospinal fluid (CSF) from TSP/HAM patients, and controls showed the presence of TIMPs, endogenous MMP inhibitors. We determined in CSF the levels of TIMPs by immunoanalysis in 25 patients with TSP/HAM, and compared with two groups: controls and patients with acute and subacute inflammatory neurological diseases. We found that TIMP-2, TIMP-3 and TIMP-4 levels were significantly higher than in controls in both TSP/HAM and inflammatory patients, while TIMP-1 was increased only in the inflammatory group. Levels of MMP-3 and MMP-9 from the two groups of patients showed a significant upregulation in CSF. In the CSF of around the 70% of TSP-HAM and inflammatory patients the presence MMP-9 was detected by zymography, but not in controls. MMP-2 was only overexpressed in the acute inflammatory group. The active form of MMP-2 was observed in both groups of patients with a similar high frequency (60%). MMPs overexpressions are independent of the evolution time of the disease in TSP/HAM. The chronic overexpression of these extracelullar matrix proteins detected in CSF of TSP/HAM should be indirectly produced by secreted viral proteins being responsible for the progression of this disease, accounting for the observed differences with acute inflammatory patients. Our results support the existence of an imbalance between MMPs and their endogenous tissue inhibitors, which could be a pathogenic factor in the chronicity of TSP/HAM.     

Unbalanced collagenases/TIMP-1 expression and epithelial apoptosis in experimental lung fibrosis

In this study, we examined the sequential expression of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors as well as the presence of apoptosis in a model of pulmonary fibrosis induced in rats with paraquat and hyperoxia. Animals showing neither clinical nor morphological changes with this double aggression were classified as "resistant". Rats were killed at 1, 2, 3, and 6 wk, and lungs were used for collagen content, gene expression by real-time PCR, gelatinolytic activity by zymography, apoptosis by in situ DNA fragmentation, and protein localization by immunohistochemistry. Our results showed a significant decrease of collagenases MMP-8 and MMP-13, with an increase of TIMP-1 and transforming growth factor-beta. Immunoreactive TIMP-1 was increased in experimental rats and primarily localized in alveolar macrophages. Expression of gelatinases MMP-2 and MMP-9 mRNAs was not affected, but lung zymography revealed an increase in progelatinase B, progelatinase A, and its active form. Epithelial apoptosis was evident from the first week, whereas at later periods, interstitial cell apoptosis was also noticed. Resistant animals behave as controls. These findings suggest that an imbalance between collagenases and TIMPs, excessive gelatinolytic activity, and epithelial apoptosis participate in the fibrotic response in this experimental model.     

Progressive pulmonary fibrosis is mediated by TGF-beta isoform 1 but not TGF-beta3

Tissue repair is a well-orchestrated biological process involving numerous soluble mediators, and an imbalance between these factors may result in impaired repair and fibrosis. Transforming growth factor (TGF)-beta is a key profibrotic element in this process and it is thought that its three isoforms act in a similar way. Here, we report that TGF-beta3 administered to rat lungs using transient overexpression initiates profibrotic effects similar to those elicited by TGF-beta1, but causes less severe and progressive changes. The data suggest that TGF-beta3 does not lead to inhibition of matrix degradation in the same way as TGF-beta1, resulting in non-fibrotic tissue repair. Further, TGF-beta3 is able to downregulate TGF-beta1-induced gene expression, suggesting a regulatory role of TGF-beta3. TGF-beta3 overexpression results in an upregulation of Smad proteins similar to TGF-beta1, but is less efficient in inducing the ALK 5 and TGF-beta type II receptor (TbetaRII). We provide evidence that this difference may contribute to the progressive nature of TGF-beta1-induced fibrotic response, in contrast to the limited fibrosis observed following TGF-beta3 overexpression. TGF-beta3 is important in "normal wound healing", but is outbalanced by TGF-beta1 in "fibrotic wound healing" in the lung.     

Time course of matrix metalloproteases and tissue inhibitors in bleomycin-induced pulmonary fibrosis

To investigate simultaneously localization and relative activity of MMPs during extracellular matrix (ECM) remodeling in bleomycin-induced pulmonary fibrosis in rat, we analyzed the time course of the expression, activity and/or concentration of gelatinases MMP-2 and MMP-9, collagenase MMP-1, matrylisin MMP-7, TIMP-1 and TIMP-2, both in alveolar space (cellular and extracellular compartments) and in lung tissue. MMP and TIMP expression was detected (immunohistochemistry) in lung tissue. MMP activity (zymography) and TIMP concentration (ELISA) were evaluated in lung tissue homogenate (LTH), BAL supernatant (BALs) and BAL cell pellet (BALp) 3, 7, 14, and 28 days after bleomycin intratracheal instillation. Immunohistochemistry showed an extensive MMP and TIMP expression from day 7 in a wide range of structural and inflammatory cells in treated rats. MMP-2 was present mainly in epithelia, MMP-9 in inflammatory cells. MMP-2 and MMP-9 activity was increased respectively in BAL fluid and BAL cells, with a peak at day 7. TIMP-1 and TIMP-2 concentration (ELISA) enhancement was delayed at day 14. In conclusion gelatinases and their inhibitors are significantly activated during bleomycin-induced pulmonary fibrosis. Marked changes in gelatinases activity are observed early in the alveolar compartment, with a prevailing extracellular activity of MMP-2 and a predominant intracellular distribution of MMP-9, while enzyme activity changes in lung parenchyma were less evident. In the repairing phase the reduction of gelatinases activity is synchronous with a peak of alveolar concentration of their inhibitors.     

Basement membrane protein and matrix metalloproteinase deregulation in engineered human oral mucosa following infection with Candida albicans.

A variety of morphological changes in the basement membrane (BM) are known to occur in inflammatory diseases. Modifications of the BM can be associated with significant changes in protein content. Candida albicans (C. albicans) is normally a commensal organism and is a member of the natural flora of a large number of healthy individuals. However, under certain conditions, C. albicans can invade host tissues, causing inflammation and tissue damage. The aim of this study was to investigate the effect of C. albicans on the expression and production of structural (laminin-5 and type IV collagen) and inflammatory [matrix metalloproteinases (MMPs) and their inhibitors] proteins by human oral epithelial cells. Using engineered normal human oral mucosa infected with 10(5) C. albicans/cm2 for different periods of time, we were able to demonstrate that this yeast promotes significant laminin-5 and type IV collagen gene activation and protein secretion. These effects were accompanied by MMP-2 and MMP-9 gene activation. Interestingly, only the levels of active MMP-9 rose. The increase in MMP levels was paralleled by a decrease in the secretion of type 2 matrix metalloproteinase tissue inhibitors (TIMP-2). Our results demonstrated that C. albicans has a significant effect on tissue structure through BM protein and MMP modulation. This might help C. albicans overcome the mechanical and biological defenses of the tissue and allow it to disseminate, causing severe infections. If C. albicans uses MMPs (mainly MMP-9) to disseminate, inhibition of this protease could be of interest in treating a variety of inflammatory disorders, including oral candidiasis.     

Effect of heparin and related glycosaminoglycan on PDGF-induced lung fibroblast proliferation, chemotactic response and matrix metalloproteinases activity

Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation are the key events in various biological and pathological processes in pulmonary fibrosis. In addition, biopsy specimens from the lungs of patients with pulmonary fibrosis show increased numbers of mast cells which have metachromatic granules containing heparin, histamine and proteases. Little is known about how these products influence pulmonary fibrosis. In the present study, we investigated the effect of heparin and related glycosaminoglycans on PDGF-induced lung fibroblast proliferation and chemotactic response in vitro. In addition, we examined the effect of heparin on both the induction of matrix metalloproteinases (MMPs) and MMPs activity in lung fibroblasts in vitro. Heparin, de-N-sulphated heparin but not heparan sulphate inhibited PDGF-induced lung fibroblast proliferation. In contrast, only heparin inhibited PDGF-stimulated human lung fibroblast chemotaxis. Negatively charged poly-L-glutamic acid had no effect on either fibroblast proliferation or chemotaxis. Thus the negative charge alone cannot account for the ant-proliferative and anti-chemotactic effects of heparin. Furthermore, heparin and heparan sulphate also had no inhibitory effect on induction of MMPS, including MMP-1 (interstitial collagenase), MMP-2 (gelatinase A) and MMP-9 (gelatinase B). Only heparin inhibited both MMP-1 and MMP-2/MMP-9 activity. Additionally, tissue inhibitor of metalloproteinase type 1 (TIMP-1) and type 2 (TIMP-2) inhibited PDGF-stimulated human lung fibroblast chemotaxis. The ability of heparin to inhibit fibroblast chemotaxis may account for the inhibitory effect of heparin on MMP activity. The above results suggested that heparin and related glycosaminoglycans differentially regulate PDGF-induced lung fibroblast proliferation, chemotaxis and MMPs activity and further that these effects may have a key role in extracellular matrix remodeling in inflammatory lung disease.     

TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.     

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.     

Tumor metastasis in an orthotopic murine model of head and neck cancer: possible role of TGF-beta 1 secreted by the tumor cells

In an orthotopic murine model of head and neck cancer, combined subcutaneous and intratumoral vaccination with recombinant vaccinia virus expressing interleukin-2 (rvv-IL-2) induced significant tumor regression early on therapy. However, its efficacy was restricted by recurrent tumor growth and loco-regional metastases. In this study, we explored the mechanism of tumor metastasis. We compared the levels of expression of a number of molecules involved in tumor metastasis, which included transforming growth factor-beta1 (TGF-beta1), E-cadherin, matrix metalloproteinases (MMPs): MT1-MMP, MMP-2, MMP-9, their tissue inhibitors (TIMPs): TIMP-1/TIMP-2, and pro-angiogenic factors CD31, VEGF-R2, and iNOS between primary and metastatic tumors by real-time RT-PCR and immunohistochemistry. We detected spontaneous lymph node and tongue metastasis. Metastasis was delayed in rvv-IL-2 treated mice. Cultured tumor cells expressed negligible amount of TGF-beta1. Untreated or metastatic tumors, on the other hand, expressed high levels of TGF-beta1 and secreted TGF-beta1 in the sera of tumor-bearing mice. Levels of TGF-beta1 in the sera suddenly jumped at the time when tumor metastasis started. In the metastatic tumors, levels of MT1-MMP, MMP-2, and MMP-9 were significantly elevated (P < 0.001), while levels of TIMP-1/TIMP-2 and E-cadherin were decreased (P < 0.001) compared to control or primary tumors. Levels of CD31, VEGF-R2, and iNOS were also significantly elevated in the metastatic lesions (P < 0.001). The concurrence of high levels of TGF-beta1 in the sera, expression of proteins involved in metastasis and initiation of metastasis suggested possible role of TGF-beta1 in on setting the metastatic cascade in this model.