Beate Eckes

Monocyte Chemoattractant Protein-1 Enhances Gene Expression and Synthesis of Matrix Metalloproteinase-1 in Human Fibroblasts by an Autocrine IL-1α Loop

Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C chemokine superfamily, has recently been shown to be involved in the pathogenesis of tissue fibrosis. In vitro studies demonstrated that MCP-1 up-regulates type I collagen gene expression via endogenous production of TGF-β in rat lung fibroblasts. We here show that recombinant human MCP-1 affects gene expression of interstitial collagenase (matrix metalloproteinase-1 (MMP-1)) in primary human skin fibroblasts and a stable fibroblast cell line. MMP-1 mRNA was induced by MCP-1 (10 ng/ml) as early as 6 h and reached a maximal expression at 24 h. MCP-1 also caused an increase of MMP-2 mRNA expression in both types of fibroblasts at 48 h. Interestingly, tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA was also up-regulated by MCP-1, and TIMP-1 mRNA expression peaked at 48 h in both types of fibroblasts. Immunoblot analysis demonstrated increased levels of MMP-1 and TIMP-1 protein in the culture supernatants of primary fibroblasts stimulated with MCP-1. In addition, MCP-1 strongly induced IL-1α mRNA expression in dermal fibroblasts in parallel with the induction of MMP-1. Preincubation with IL-1 receptor antagonist almost completely abrogated the expression of MMP-1 mRNA, and partially inhibited MMP-1 synthesis induced by MCP-1. Transient transfection of primary skin fibroblasts with a MMP-1 promoter-reporter construct indicated a dose-dependent increase in promoter activity by MCP-1 stimulation. These data demonstrate that MCP-1 up-regulates MMP-1 mRNA expression and synthesis in human skin fibroblasts at a transcriptional level and provide evidence that this is mediated by an IL-1α autocrine loop.

Fibroblast-matrix interactions in wound healing and fibrosis

The regulation of matrix deposition is a key event in many physiological and pathological situations. It involves the activity of mediators in autocrine and paracrine fashions and the contact of cells with the surrounding extracellular matrix as well. The tightly regulated balance of both mechanisms guarantees rapid and adaptive cellular responses to meet changes in the biological requirements of the environment. Disturbances lead to wound healing defects or the development of fibrosis. The molecular mechanisms for these regulatory events are only partially understood, but involve the activity of integrins and a structural continuum of extracellular matrix-receptor-cytoskeleton-nucleus for transfer of information and the regulation of activated genes.

Pathogenesis of fibrosis: type 1 collagen and the skin.

Abstract This review on the pathogenesis of fibrosis emphasizes the similarities between tissue repair, a tightly regulated salutary biological response, and fibrosis, an unregulated pathological process. It focuses on the transcriptional regulation of type I collagen, the role of cytokines in fibroblast activation, integrins as examples of cell-matrix signaling pathways, and the heterogeneity of fibroblast populations as factors contributing to fibrosis. Tissue remodeling and the role of matrix metalloproteinases and metalloproteinase inhibitors are mentioned briefly. The capacity of extracellular matrix to modulate cellular function is a recurring theme.

Cell-matrix interactions in dermal repair and scarring

Regulation of cellular functions during dermal repair following injury is complex and critically dependent on the interaction of cells with the surrounding extracellular matrix (ECM). The ECM comprises various families of macromolecules that form the structural scaffold of the tissue, but also carry distinct biological activities. After injury to the skin, the defect is filled by a provisional matrix that is invaded by inflammatory cells, sprouting blood vessels and fibroblasts. In a later phase, the wound contracts, the tissue is replaced by mature connective tissue produced by activated fibroblasts, and a scar is formed. All cells involved communicate directly with the ECM by integrins and other matrix receptors. These transmit signals and induce adaptive responses to the environment by the embedded cells. The ECM or proteolytic fragments of individual ECM constituents exert defined biological activities influencing cell survival, differentiation of myofibroblasts, ECM synthesis and turnover, wound angiogenesis and scar remodeling. Extensive crosstalk exists between ECM and growth factors, and between growth factors and integrins. ECM-cell contact also enables direct transmission of mechanical tension, which then modulates many activities of all cellular players. Understanding this complex interplay is important to provide a basis for designing effective wound therapy and for strategic interference with mechanisms that have gone out of control in fibrotic conditions.

Expression of integrin β1 by fibroblasts is required for tissue repair in vivo

In tissue repair, fibroblasts migrate into the wound to produce and remodel extracellular matrix (ECM). Integrins are believed to be crucial for tissue repair, but their tissue-specific role in this process is poorly understood. Here, we show that mice containing a fibroblast-specific deletion of integrin β1 exhibit delayed cutaneous wound closure and less granulation tissue formation, including reduced production of new ECM and reduced expression of α-smooth muscle actin (α-SMA). Integrin-β1-deficient fibroblasts showed reduced expression of type I collagen and connective tissue growth factor, and failed to differentiate into myofibroblasts as a result of reduced α-SMA stress fiber formation. Loss of integrin β1 in adult fibroblasts reduced their ability to adhere to, to spread on and to contract ECM. Within stressed collagen matrices, integrin-β1-deficient fibroblasts showed reduced activation of latent TGFβ. Addition of active TGFβ alleviated the phenotype of integrin-β1-deficient mice. Thus integrin β1 is essential for normal wound healing, where it acts, at least in part, through a TGFβ-dependent mechanism in vivo.

Anti–Glycoprotein VI Treatment Severely Compromises Hemostasis in Mice With Reduced α2β1 Levels or Concomitant Aspirin Therapy

Background—Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. Methods and Results—Mice lacking (−/−) or expressing half-levels (+/−) of the other major platelet collagen receptor, integrin &agr;2&bgr;1, were injected with the anti–GP VI antibody JAQ1 and analyzed on day 5. Anti–GP VI treatment resulted in a marked hemostatic defect in &agr;2−/− or &agr;2+/− mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti–GP VI–treated wild-type but not &agr;2−/− or &agr;2+/− platelets to collagen. This process required the simultaneous activation of the Gq and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti–GP VI–treated or GP VI/Fc receptor &ggr;-chain–deficient but not control mice. Conclusions—Anti–GP VI therapy may result in defective hemostasis in patients with reduced &agr;2&bgr;1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti–GP VI–based therapeutics in the prevention of cardiovascular disease.

Downregulation of collagen synthesis in fibroblasts within three-dimensional collagen lattices involves transcriptional and posttranscriptional mechanisms

Culturing human fibroblasts in a three‐dimensional collagen matrix leads to a reduction of collagen I by more than 90%, both on the level of mRNA steady‐state as well as protein. In order to differentiate changes in de novo transcription and posttranscriptional control, nuclear run on assays and pulse/chase experiments determining mRNA stability were used. Our results indicate that de novo transcription of the COL1A1 gene and proα1(I)collagen mRNA half‐life are both decreased by 50% in fibroblasts grown in three‐dimensional collagen lattices as compared to monolayer cultures. The extracellular matrix therefore elicits signals which are transduced from the cell surface to the inside of fibroblasts resulting in a specific reprogramming of transcriptional as well as posttranscriptional processes.

Signalling and regulation of collagen I synthesis by ET-1 and TGF-β1

Endothelin‐1 (ET‐1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC). ET‐1 signals are transduced by two receptor subtypes, the ETA‐ and ETB‐receptors which activate different Gα proteins. Here, we investigated the expression of both ET‐receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA‐receptor is the major ET‐receptor subtype expressed. To determine further signalling intermediates, we inhibited Gαi and three phospholipases. Pharmacologic inhibition of Gαi, phosphatidylcholine‐phospholipase C (PC‐PLC) and phospholipase D (PLD), but not of phospholipase Cβ, abolished the increase in collagen I by ET‐1. Inhibition of all phospholipases revealed similar effects on TGF‐β1 induced collagen I synthesis, demonstrating involvement of PC‐PLC and PLD in the signalling pathways elicited by ET‐1 and TGF‐β1. ET‐1 and TGF‐β1 each stimulated collagen I production and in an additive manner. ET‐1 further induced connective tissue growth factor (CTGF), as did TGF‐β1, however, to lower levels. While rapid and sustained CTGF induction was seen following TGF‐β1 treatment, ET‐1 increased CTGF in a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET‐1 treatment. Coincidentally at 5 days of permanent ET‐1 stimulation, a switch in ET‐receptor subtype expression to the ETB‐receptor was observed. We conclude that the signalling pathways induced by ET‐1 and TGF‐β1 leading to augmented collagen I production by fibroblasts converge on a similar signalling pathway. Thereby, long‐time stimulation by ET‐1 resulted in a changed ET‐receptor subtype ratio and in a biphasic CTGF induction.

Expression of TGF-β1, -β2 and -β3 in localized and systemic scleroderma

Abstract Scleroderma is a generalized or localized disorder which leads to fibrosis of the affected organs. TGF-β has been implicated as a causal agent in its pathogenesis. In mammals, TGF-β comprises a family of three members, β1, β2 and β3. Since cutaneous wound healing is thought to result either in formation of a scar or in scar-free tissue regeneration, depending on the relative amounts of the β3 isoform, the expression of all three isoforms was studied in skin biopsies of patients with either localized or systemic scleroderma. mRNA for all three isoforms was detected in inflammatory skin areas of both disease forms, but never in sclerotic or healthy skin. Immunohistochemical analysis confirmed expression of β1 and β2 proteins in inflammatory skin of patients, whereas β3 protein appeared to be present in the subepidermal area and also found throughout the dermis of patients and healthy dermis as well.

Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.