Fibroblast Biology Research Articles

THE CARDIAC FIBROBLAST: Therapeutic Target in Myocardial Remodeling and Failure

Cardiac fibroblasts play a central role in the maintenance of extracellular matrix in the normal heart and as mediators of inflammatory and fibrotic myocardial remodeling in the injured and failing heart. In this review, we evaluate the cardiac fibroblast as a therapeutic target in heart disease. Unique features of cardiac fibroblast cell biology are discussed in relation to normal and pathophysiological cardiac function. The contribution of cardiac fibrosis as an independent risk factor in the outcome of heart failure is considered. Candidate drug therapies that derive benefit from actions on cardiac fibroblasts are summarized, including inhibitors of angiotensin-aldosterone systems, endothelin receptor antagonists, statins, anticytokine therapies, matrix metalloproteinase inhibitors, and novel antifibrotic/anti-inflammatory agents. These findings point the way to future challenges in cardiac fibroblast biology and pharmacotherapy.

Altered fibroblast function following myocardial infarction

Adequate wound healing and scar formation is an essential response to myocardial infarction (MI), and fibroblasts are primary cellular components regulating the process. How fibroblast functions are altered post-MI and to what extent these abnormalities persist in vitro is not well understood. Accordingly, we isolated myocardial fibroblasts from MI and non-MI (remote) regions at 7 days post-MI ( n = 35) and from the free wall and septum of unoperated control C57BL/6 mice ( n = 14). Proliferation was increased 182 ± 28% in MI, but not in remote, fibroblasts compared with unoperated controls ( P = 0.01). Migration decreased 61 ± 8%, adhesion to laminin decreased 79 ± 8%, adhesion to collagen IV increased 196 ± 27%, and collagen synthesis increased 169 ± 24% in fibroblasts isolated from the MI region (all P < 0.05). Migration, adhesion, and collagen synthesis changes were similar in remote fibroblasts, and the phenotypic differences were maintained through passage four. Transforming growth factor β1 (TGFβ1) is a bioactive molecule that has been shown to affect fibroblast function. Stimulation of unoperated control fibroblasts with 10 ng/ml TGFβ 1 increased proliferation 137 ± 7% ( P = 0.03 vs. unstimulated), increased adhesion to collagen IV 149 ± 6% ( P < 0.01), and increased collagen I levels 187 ± 10% ( P = 0.01). TGFβ1 may, therefore, explain some of the changes in post-MI fibroblast phenotype. These data demonstrate for the first time region specific alterations in post-MI fibroblast biology that are maintained in vitro. Additionally, our model provides a novel in vitro template for examining the cellular mechanisms of wound healing and scar formation post-MI.

The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology.

Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, is a cysteine-rich proadhesive matricellular protein that plays an essential role in the formation of blood vessels, bone, and connective tissue. As expression of this protein is potently induced by transforming growth factor-beta (TGFbeta), it has been hypothesized that CTGF mediates several of the downstream actions of TGFbeta. In particular, CTGF is profibrotic, as CTGF is overexpressed in fibrotic disease and synergizes with TGFbeta to promote sustained fibrosis in vivo. Over the last several years, key data regarding the developmental role and structure and function relationship of CTGF have emerged. In addition, increased information concerning the mechanisms underlying the control of CTGF expression in normal and fibrotic cells and the signal transduction pathways through which CTGF acts on cells has been uncovered. This review summarizes the current state of knowledge regarding CTGF biology.

Perspective of cytokine regulation for periodontal treatment: fibroblast biology.

Efforts to understand the pathogenesis of periodontal diseases have been underway for decades. Studies of immunological aspects in addition to the structural components of gingival fibroblasts showed that the fibroblasts actively participate in immune and inflammatory events in periodontal diseases. Future strategies for the prevention and treatment of periodontal diseases should biologically regulate fibroblast activities. These cells are surrounded by monocyte-derived proinflammatory cytokines such as interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and lymphocyte-derived interleukin-6 (IL-6) in inflamed gingival tissue. Recent anti-cytokine therapy for inflammatory diseases including rheumatoid arthritis aimed to inhibit the binding of cytokines to targeted cells such as fibroblasts and condrocytes. IL-1beta and TNF-alpha are thought to be therapeutic targets because these cytokines are essential for the initiation of inflammatory immune reactions and are produced for prolonged periods in inflammatory diseases. IL-6 is also a target, because it is abundantly present in inflammatory lesions and activates fibroblasts in the presence of soluble IL-6 receptor. In addition, these cytokines accelerate gingival fibroblasts to produce collagenolytic enzymes, resulting in tissue destruction. Soluble receptors for IL-1beta and TNF-alpha are suggested to be candidates for therapeutic molecules, but soluble receptor for IL-6 is suggested to be a factor-stimulating fibroblast. This paper will review the utilization of soluble receptors specific to inflammatory cytokines which potentially stimulate fibroblasts to regulate biological events involved in the pathogenesis of periodontal diseases.

Fibroblast biology in thyroid diseases

Several thyroid diseases are manifested by structural and functional alterations in connective tissue. Hypothyroidism in its more severe and sustained form results in generalized deposition of the glycosaminoglycan hyaluronan in many tissues. This condition is termed myxedema and is easily remedied

The role of oxidised regenerated cellulose/collagen in wound repair: effects in vitro on fibroblast biology and in vivo in a model of compromised healing

Irrespective of underlying chronic wound pathology, delayed wound healing is normally characterised by impaired new tissue formation at the site of injury. It is thought that this impairment reflects both a reduced capacity to synthesize new tissue and the antagonistic activities of high levels of proteinases within the chronic wound environment. Historically, wound dressings have largely been passive devices that offer the wound interim barrier function and establish a moist healing environment. A new generation of devices, designed to interact with the wound and promote new tissue formation, is currently being developed and tested. This study considers one such device, oxidised regenerated cellulose (ORC) /collagen, in terms of its ability to promote fibroblast migration and proliferation in vitro and to accelerate wound repair in the diabetic mouse, a model of delayed wound healing. ORC/collagen was found to promote both human dermal fibroblasts proliferation and cell migration. In vivo studies considered the closure and histological characteristics of diabetic wounds treated with ORC/collagen compared to those of wounds given standard treatment on both diabetic and non-diabetic mice. ORC/collagen was found to significantly accelerate diabetic wound closure and result in a measurable improvement in the histological appearance of wound tissues. As the diabetic mouse is a recognised model of impaired healing, which may share some characteristics of human chronic wounds, the results of this in vivo study, taken together with those relating the positive effects of ORC/collagen in vitro, may predict the beneficial use of this device in the clinical setting.

Molecular aspects of scleroderma

Scleroderma is a complex disease characterized by activation of the immune system, small-vessel vasculopathy, and fibrosis of the skin and other organs. This review is limited to the discussion of scleroderma fibroblast biology and the cellular and molecular mechanisms that contribute to the abnormal deposition of collagen. Selected aspects of abnormal extracellular matrix (ECM) regulation by scleroderma fibroblasts are critically reviewed. These include the role of transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) and their receptors in the fibrotic process in scleroderma and the overview of the transcription factors involved in regulation of the human alpha2 (I) collagen (COL1A2) gene.

Molecular aspects of scleroderma.

Scleroderma is a complex disease characterized by activation of the immune system, small-vessel vasculopathy, and fibrosis of the skin and other organs. This review is limited to the discussion of scleroderma fibroblast biology and the cellular and molecular mechanisms that contribute to the abnormal deposition of collagen. Selected aspects of abnormal extracellular matrix (ECM) regulation by scleroderma fibroblasts are critically reviewed. These include the role of transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) and their receptors in the fibrotic process in scleroderma and the overview of the transcription factors involved in regulation of the human alpha2 (I) collagen (COL1A2) gene.

Free-Ranging Fibroblasts

The fibroblast has been the prototype cell for studying a range of phenomena, including cell proliferation, morphology, adhesion, and movement. Yet, our current understanding of fibroblast biology is based predominantly on observing these cells in two-dimensional (2D) culture. Cukierman et al. (see the Perspective by Geiger) have designed a 3D matrix system to culture fibroblasts and report key biological differences compared with the traditional 2D culture system. A comprehensive description of cell-matrix adhesions reveals striking structural and morphological differences that may justify a reevaluation of current models. E. Cukierman, R. Pankov, D. R. Stevens, K. M. Yamada, Taking cell-matrix adhesions to the third dimension. Science 294 , 1708-1712 (2001). [Abstract] [Full Text] B. Geiger, Encounters in space. Science 294 , 1661-1663 (2001). [Full Text]

Free-Ranging Fibroblasts

The fibroblast has been the prototype cell for studying a range of phenomena, including cell proliferation, morphology, adhesion, and movement. Yet, our current understanding of fibroblast biology is based predominantly on observing these cells in two-dimensional (2D) culture. Cukierman et al. (see the Perspective by Geiger) have designed a 3D matrix system to culture fibroblasts and report key biological differences compared with the traditional 2D culture system. A comprehensive description of cell-matrix adhesions reveals striking structural and morphological differences that may justify a reevaluation of current models.E. Cukierman, R. Pankov, D. R. Stevens, K. M. Yamada, Taking cell-matrix adhesions to the third dimension. Science 294, 1708-1712 (2001). [Abstract] [Full Text]B. Geiger, Encounters in space. Science 294, 1661-1663 (2001). [Full Text]

Characterization of fibroblastic cell plasticity in the lamina propria of the rat uterine cervix at term.

Different organs contain fibroblasts with specific features and functions, indicating the complexity of fibroblast biology. In the rat cervical stroma, fibroblasts are preferentially located in the fibrous ring that surrounds the mucous layer. The purpose of this study was to investigate the morphological features and immunophenotype of fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats. Expression of the cytoskeletal proteins desmin, vimentin, and alpha-smooth muscle actin (alpha-SMA) were studied by immunohistochemistry. The optical density of immunohistochemical staining was quantified by image analysis. The ultrastructural features of fibroblastic cells were observed under transmission electron microscopy. Cervical fibroblastic cells always expressed vimentin and desmin but never alpha-SMA. During the first half of pregnancy (Day 5 [D5] to D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found from D15 to D22, with maximal expression at term (D23). Immunohistochemical expression for vimentin was constant throughout pregnancy and showed no differences with cycling and postpartum uterine cervices. Stromal cells from cycling and early pregnant rats displayed ultrastructural features characteristic of typical fibroblasts. In contrast, at the end of pregnancy, fibroblasts differentiated and showed increased secretory characteristics, reaching the ultrastructural features of a myofibroblast. Based on the differential expression of desmin and the electron microscopic observations, the foregoing results showed a modulation of the fibroblastic phenotype in the uterine cervix during pregnancy. To our knowledge, this is the first report that addresses the presence of myofibroblasts derived from resident fibroblasts in the fibrous ring of the rat uterine cervix. Fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.

Fibroblast biology: Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis

There is growing evidence that activated synovial fibroblasts, as part of a complex cellular network, play an important role in the pathogenesis of rheumatoid arthritis. In recent years, significant progress has been made in elucidating the specific features of these fibroblasts. It has been understood that although macrophage and lymphocyte secreted factors contribute to their activation, rheumatoid arthritis synovial fibroblasts (RA-SFs) do not merely respond to stimulation by pro-inflammatory cytokines, but show a complex pattern of molecular changes also maintained in the absence of external stimulation. This pattern of activation is characterized by alterations in the expression of regulatory genes and signaling cascades, as well as changes in pathways leading to apoptosis. These together result in the upregulation of adhesion molecules that mediate the attachment of RA-SFs to the extracellular matrix and in the overexpression of matrix degrading enzymes that mediate the progressive destruction of the joints. In addition, activated RA-SFs exert specific effects on other cell types such as macrophages and lymphocytes. While the initiating step in the activation of RA-SFs remains elusive, several key pathways of RA-SF activation have been identified. However, there is so far no single, specific marker for this phenotype of RA-SF. It appears that activated RA-SFs are characterized by a set of specific properties which together lead to their aggressive behavior.

Fibroblast biology: Effector signals released by the synovial fibroblast in arthritis

There is mounting evidence indicating that the synovial fibroblast is a direct effector of tissue injury and matrix remodeling in inflammatory synovitis. Through the elaboration of effector signals including cytokines and chemokines, mesenchymal cells stimulate or suppress inflammation via autocrine and paracrine mechanisms. Synovial fibroblasts are the principal cells mediating joint destruction through secretion of metalloproteinases, and recent evidence suggests that they may also promote bone resorption by stimulating osteoclastogenesis. Moreover, they may play an integral role in the initial phases of synovitis by releasing chemokines that recruit leukocytes to the joint, and cytokines that trigger angiogenesis. Studies focusing on synoviocyte-leukocyte interactions mediated via the cytokine network and the role of cell-cell contact in driving synoviocyte activation will help define the complex interplay that leads to the initiation and perpetuation of synovial inflammation.

Fibroblast biology: Development and differentiation of synovial fibroblasts in arthritis

Synovial fibroblasts occur as two phenotypes - intimal and subintimal. The specialised intimal phenotype includes expression of uridine diphosphoglucose dehydrogenase (UDPGD), vascular cell adhesion molecule-1 (VCAM-1) and complement decay-accelerating factor (DAF). These gene products contribute to specialised functions relating to tissue movement and leucocyte traffic.

Biology of fibroblasts

During recent years, fibroblasts have been recognized as a heterogenous cell type with a remarkable degree of differentiation and important functional properties in health and in disease. Our better understanding of the biology of fibroblasts has elucidated some of the mechanisms responsible for fibrosis of the kidney and other organs. In chronic inflammation, activation of differentiating fibroblasts and/or the selection of highly proliferative subsets results in a specialized phenotype of fibroblast, often called the myofibroblast, which is the pivotal cell in fibrogenesis and the subsequent destruction of the parenchyma. In addition, perivascular and epithelial cells may be recruited to transdifferentiate into fibroblast-like cells to joint in this process. So far, many aspects of cell regulation in fibrogenesis have been described, with most of the known regulatives being cytokines. However, further research is imperative in this interesting area to eventually overcome the fatal course of chronic fibrosis, which leaves the patient and the physician without any hope for sufficient treatment.

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture.

In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.

Human thyroid fibroblasts exhibit a distinctive phenotype in culture: characteristic ganglioside profile and functional CD40 expression.

Fibroblasts from different regions of the human body exhibit substantial phenotypic diversity, some of which relates to the capacity for cross-talk with cells of the immune system. We examine, for the first time, thyroid fibroblast biology in culture. Thyroid explants were placed in culture, and fibroblasts were outgrown and serially passaged. These fibroblasts take on a morphology in culture resembling cells from other anatomic regions. When treated with PGE2, they assume a stellate morphology similar to that of prostanoid-treated orbital fibroblasts. The ganglioside profile exhibited by these cells is distinct from that observed previously in orbital and dermal fibroblasts. They uniformly express Thy-1, a surface glycoprotein. Messenger RNA encoding CD40, a surface receptor found on bone marrow-derived cells, and CD40 protein were expressed constitutively at low levels. Interferon-gamma (500 U/ml) treatment for 48-72 h resulted in high levels of surface HLA-DR and CD40 display. When CD40 is engaged with CD40 ligand (CD40L), nuclear factor-kappaB binding activity is up-regulated as is interleukin (IL)-6 and IL-8 expression. IL-1beta treatment up-regulates the expression of IL-1alpha, IL-1beta, and PGE2. These observations suggest that thyroid fibroblasts possess the molecular machinery necessary for cross-talk with immunocompetent cells such as lymphocytes and mast cells through the CD40/CD40L complex, as well as through classic cytokine networks, and to participate potentially in the inflammatory response of the thyroid gland.

Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.

SYSTEMIC SCLEROSIS

The uniform failure of trials using potent nonselective immunosuppressive and cytotoxic agents to halt the progression of SSc indicate an urgent need for alternative types of therapeutics in future trials. The improved outcome among SSc patients in the past 20 years primarily has been secondary to the widespread use of ACE inhibitors, calcium channel blockers, and improved pharmacologic agents to manage gastrointestinal symptoms. It remains unknown whether early treatment with D-penicillamine or aggressive treatment of interstitial lung disease will slow progression of cutaneous or pulmonary fibrosis, respectively. Recent advances and accelerated research into the mechanisms of cellular activation, growth, and regulation hold great potential for understanding the pathogenesis of SSc. Hopefully the next decade will yield new information regarding fibroblast biology, endothelial cell injury, and human immune regulation so that specific biologic response agents may selectively halt disease progression. Progress probably will result only from well-designed multicenter trials of patients in the early stages of illness involving quantifiable outcome measures of disease.

Approach to the Patient with Diffuse Lung Disease

When evaluating diffuse lung infiltrates, the clinician should place special emphasis on the acuity of symptoms, nonpulmonary complaints and findings, environmental exposures, and risk factors for immunosuppressive diseases. Certain radiographic features, such as the distribution of opacities, hilar adenopathy, Kerley-B lines or pneumothorax, or pulmonary function tests demonstrating air flow limitation also narrow the differential diagnosis. One can direct the subsequent workup based on the narrowed differential diagnosis, the pace of disease, the activity of the ongoing inflammatory-immune process, and the age, overall medical condition, and wishes of the patient. Unless a specific diagnosis (for example, hypersensitivity pneumonitis, the treatment of which is withdrawal of the offending agent) can be made, therapy of noninfectious diffuse lung disease is quite unsatisfactory. Immunosuppressive therapy is indicated to arrest the active inflammatory process with the hope that objective signs of improvement will occur after a 3- to 12-month course. Important areas of basic research in pulmonary fibrosis include cell-cell and cell-matrix interactions in the lung interstitium and delineation of fibroblast biology and cytokine-mediated lung connective tissue pathology. More successful therapies will probably evolve from better understanding of the molecular and cellular biology of the lung fibrogenic process.