R. Christopher Benyon

Scar-Associated Macrophages Are a Major Source of Hepatic Matrix Metalloproteinase-13 and Facilitate the Resolution of Murine Hepatic Fibrosis

Both the identity and source of the rodent collagenase(s) that mediates matrix remodeling in liver fibrosis remain elusive. We have recently demonstrated an unequivocal role for scar-associated macrophages (SAMs) in the spontaneous resolution of liver fibrosis and sought to determine whether SAMs are the source of matrix metalloproteinase (MMP) 13 (collagenase 3), considered to be the primary interstitial collagenase in rodents. In this study, we demonstrate an association between MMP13 expression and the presence of SAMs in the regression of experimental liver fibrosis. mmp13 gene expression was restricted to regions of fibrosis that were rich in SAMs. Both MMP13 mRNA and protein colocalized to large phagocytes within and directly apposed to hepatic scars. Using the CD11b-DTR-transgenic mouse to deplete SAMs in a model of chronic CCl4 injury, we found that SAM depletion resulted in a 5-fold reduction in mmp13 message (p = 0.005). Furthermore, resolution of CCl4-induced fibrosis was retarded in MMP13-deficient mice. Thus, SAMs selectively, during resolution of fibrosis induce and use the major collagenase MMP13 to mediate the resorption of interstitial matrix and successfully remodel the fibrotic liver.

Human mast cell .heterogeneity: Histamine release from mast cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE-dependent and nonimmunologic stimuli

We have compared the ability of anti-IgE, calcium ionophore A23187, substance P, compound 48/80, poly-L-lysine, and morphine to release histamine from mast cells of human skin, lung, adenoids, tonsils, and colon. Use of a single collagenase/hyaluronidase dispersion technique for all tissues has allowed comparisons of reactivity to be made that are free from methodological variations. Mast cells from all tissues examined secreted histamine in response to anti-IgE and calcium ionophore A23187. However, only skin mast cells were responsive to substance P, compound 48/80, poly-L-lysine, and morphine. Activation of human skin mast cells by these nonimmunologic stimuli clearly distinguishes them from the mast cells of human lung, adenoids, tonsils, and colon and is indicative of functional heterogeneity within the human mast cells population. We propose that the presence of functional receptor sites for neuropeptides and basic compounds on skin mast cells that are not present in mast cell populations from mucosal or lymphoid sources reflects a specialized role for these cells in vascular homeostasis.

Characterization of neuropeptide-induced histamine release from human dispersed skin mast cells.

1 Human skin mast cells, unlike other human mast cells so far studied, released histamine in a concentration‐related manner in response to substance P, vasoactive intestinal peptide (VIP) and somatostatin (1 μM to 30 μM). In contrast, eledoisin, physalaemin, neurokinin A, neurokinin B, calcitonin gene‐related peptide (CGRP), neurotensin, bradykinin and Lys‐bradykinin induced negligible histamine release. 2 The low histamine releasing activity of physalaemin, eledoisin, neurokinin A and neurokinin B relative to substance P suggests that the human skin mast cell activation site is distinct from the tachykinin NK‐1, NK‐2 or NK‐3 receptors described in smooth muscle. 3 The relative potencies of substance P and its fragments SP2–11, SP3–11, SP4–11 and SP1–4 in releasing histamine from human skin mast cells suggests that both the basic N‐terminal amino acids and the lipophilic C‐terminal portion of substance P are essential for activity. 4 Peptide‐induced histamine release, like that induced by compound 48/80, morphine and poly‐L‐lysine, is rapid, reaching completion in 10–20 s, is largely independent of extracellular calcium but requires intact glycolysis and oxidative phosphorylation. 5 The substance P analogue, [D‐Pro4, D‐Trp7,9,10] SP4–11 (SPA), not only reduced substance P‐induced histamine release in a concentration‐related manner but also inhibited that induced by VIP, somatostatin, compound 48/80, poly‐L‐lysine and morphine but not anti‐IgE. 6 The similar characteristics of histamine release induced by substance P, VIP, somatostatin, compound 48/80, poly‐L‐lysine and morphine suggest that they share a common pathway of activation‐secretion coupling distinct from that of IgE‐dependent activation. Furthermore, the ability of human skin mast cells to respond to basic non‐immunological stimuli including neuropeptides may reflect a specialised function for these cells.

Type I Collagen Promotes the Malignant Phenotype of Pancreatic Ductal Adenocarcinoma

Purpose: The purpose of this study was to determine the role of functional interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) in the formation of the desmoplastic reaction (DR) in pancreatic cancer and to characterize the effect of type I collagen (the predominant component of the DR) on pancreatic cancer cell phenotype. Experimental Design: PSCs and type I collagen were identified in sections of pancreatic cancer using immunohistochemistry, and their anatomic relationship was studied. Interactions among pancreatic cancer cell lines (MIA PaCa-2, Panc-1, and AsPC-1), primary cultures of human PSCs, and type I collagen were investigated in a series of tissue culture models. Results: In vivo, the DR causes gross distortion of normal pancreas, bringing cancer cells into close contact with numerous PSCs and abundant type I collagen. In tissue culture models of pancreatic cancer, conditioned media from each cell line increased PSC [3H]thymidine incorporation up to 6.3-fold that of controls, and AsPC-1 cells also increased PSC collagen synthesis 1.3-fold. Type I collagen was observed to increase long-term survival of pancreatic cancer cells treated with 5-fluorouracil, by up to 62% in clonogenic assays. This was because type I collagen increased the proliferation of cancer cells ([3H]thymidine incorporation was up to 2.8-fold that of cells cultured on tissue culture plastic) and reduced apoptosis of AsPC-1 cells in response to 5-fluorouracil (by regulating mcl-1). Conclusions: These experiments elucidate a mechanism by which the DR in pancreatic cancer may form and, via the collagen within it, promote the malignant phenotype of pancreatic cancer cells, suggesting significant detriment to the host.

Differential release of histamine and eicosanoids from human skin mast cells activated by IgE‐dependent and non‐immunological stimuli

1 Cells were dispersed from human foreskin using a mixture of collagenase and hyaluronidase and separated into mast cell‐depleted (<1%) or enriched (>75%) preparations by density‐gradient centrifugation. 2 Challenge of gradient fractions with ∍‐chain‐specific anti‐human IgE stimulated the release of histamine, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The release of eicosanoids was significantly correlated with that of histamine, suggesting that they are derived from the mast cell population of the dispersate. In highly purified (76.2 ± 4.2%) mast cell preparations, maximum net release of histamine, PGD2 and LTC4 was 3432 ± 725, 84.9 ± 10.8 and 6.6 ± 1.2 pmol/106 nucleated cells. 3 The non‐immunological stimuli substance P, vasoactive intestinal peptide (VIP), somatostatin, compound 48/80, morphine and poly‐L‐lysine released similar amounts of histamine to anti‐IgE, but 12 to 21 fold less PGD2 and LTC4. 4 These studies suggest that IgE‐dependent and non‐immunological stimuli activate human skin mast cells by different secretory mechanisms, a hypothesis supported by our previous findings of differences in Ca2+ requirements and time‐course of histamine release. Activation by the non‐immunological mechanism may be of importance in vivo due to the close anatomical association between skin mast cells and dermal nerve‐terminals containing neuropeptides.

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.

Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells

BACKGROUND/AIMS Connective tissue growth factor is a recently described mitogenic protein implicated in a variety of fibrotic disorders. Connective tissue growth factor may be a downstream mediator of the pro-fibrotic and mitogenic actions of transforming growth factor-beta, promoting extracellular matrix deposition and fibrogenesis. As transforming growth factor-beta is considered important to the pathogenesis of hepatic fibrosis, we examined the possible contribution of connective tissue growth factor to this process. METHODS Connective tissue growth factor expression was examined in normal and fibrotic human and rat livers using RT-PCR and ribonuclease protection assays, and in primary cultures of rat hepatic stellate cells by Northern and Western blotting. RESULTS Ribonuclease protection assays demonstrated connective tissue growth factor mRNA was increased 3-5-fold in human fibrotic liver compared with normal. RT-PCR showed this mRNA was increased in carbon-tetrachloride-treated rat liver. Northern analysis showed connective tissue growth factor mRNA was increasingly expressed during progressive activation of cultured rat hepatic stellate cells. Western analysis confirmed that freshly isolated hepatic stellate cells secreted relatively little connective tissue growth factor compared with hepatic stellate cells activated in culture. Hepatic stellate cells stimulated with transforming growth factor-beta showed increased expression of connective tissue growth factor mRNA and protein. CONCLUSIONS Connective tissue growth factor mRNA is consistently upregulated in human liver cirrhosis of various aetiologies, supporting a role for this growth factor in hepatic fibrogenesis. Our studies suggest that hepatic stellate cells may be an important source of hepatic connective tissue growth factor in vivo, particularly following stimulation with transforming growth factor-beta.

Regulation of hepatic stellate cell proliferation and collagen synthesis by proteinase-activated receptors

BACKGROUND/AIMS Thrombin and MC tryptase, which are agonists for proteinase-activated receptors-1 and -2, respectively, are both increased in injured liver. We have examined if rat stellate cells express these receptors and if receptor agonists influence stellate cell activation. METHODS Expression of mRNA for proteinase activated receptors-1 and -2 were examined by RT-PCR and Northern blotting in lysates of cultured stellate cells and receptor protein examined by Western blotting. The effects of receptor agonists on cell proliferation and collagen synthesis were examined by 3H-thymidine and 3H-proline incorporation assays, respectively. RESULTS Rat stellate cells activated by culture on plastic showed a progressive increase in expression of proteinase-activated receptor-1 and -2 mRNA and proteinase-activated receptor-2 protein as they transformed to a myofibroblastic phenotype. Proteinase-activated receptor-1 agonists thrombin and the peptide SFFLRN, and proteinase-activated receptor-2 agonists tryptase and the peptide SLIGRL induced stellate cell proliferation and the rapid phosphorylation of 44 and 42 kDa mitogen-activated protein kinases. PD98059, an inhibitor of these kinases, inhibited this proliferative response. Both tryptase and SLIGRL increased collagen secretion by stellate cells. CONCLUSIONS This study indicates that the natural proteinase-activated receptor agonists thrombin and MC tryptase might sustain liver fibrosis by promoting stellate cell proliferation and collagen synthesis.

Expression of matrix metalloproteinase‐2 and ‐14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis

Abstract: Background/Aims: Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP‐2 and MMP‐14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis.

Elastin accumulation is regulated at the level of degradation by macrophage metalloelastase (MMP‐12) during experimental liver fibrosis

Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP‐12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl4) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP‐12 elastin degradation was defined mechanistically using CD11b‐DTR and MMP‐12 knockout mice. In a CCl4 model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP‐12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP‐12 was bound in greater quantities to its inhibitor TIMP‐1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP‐12. Exposure of CCl4 in MMP‐12−/− mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP‐12−/− mice compared with WT. Conclusion: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage‐derived MMP‐12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies. (HEPATOLOGY 2012;55:1965–1975)