Fibrosis Research: Methods and Protocols

392 Pages·2005·4.17 MB·English

by  David A. Zisman, Michael P. Keane, John A. Belperio, Robert M. Strieter (auth.), John Varga MD, David A. Brenner MD, Sem H. Phan MD, PhD (eds.)

Fibrosis Research
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M E T H O D S I N M O L E C U L A R M E D I C I N E™
Fibrosis Research
Methods and Protocols
Edited by
John Varga,
MD
Feinberg School of Medicine,
Northwestern University, Chicago, IL
David A. Brenner,
MD
College of Physicians and Surgeons, Columbia University,
New York, NY
Sem H. Phan, ,
MD PhD
University of Michigan Medical School, Ann Arbor, MI
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Cover Illustration: From Fig. 4 in Chapter 10, "Morphological Methods for Assessment of Fibrosis," by
Rakesh K. Kumar and Fig. 1 in Chapter 12, "Approaches to Evaluation of Fibrogenic Pathways in Surgical
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Fibrosis research : methods and protocols / edited by John Varga, David A. Brenner, Sem H. Phan.
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1. Fibrosis. 2. fibrosis–Research–Methodology.
[DNLM: 1. Fibrosis. 2. Cultured Cells. 3. Extracellular
Matrix–metabolism. 4. Models, Animal. QZ 150 F4436 2005]
I. Varga, John.
II. Brenner, David, 1953-
III. Phan, Sem Hin, 1949- IV. Series.
RB150.F52F54 2005
616.7'42–dc22
2005006567
Preface
Fibrosis or scar, defined pathologically as inappropriate repair by connec-
tive tissue, is increasingly recognized as an important feature of many chronic
diseases (Table 1), and as such, represents an enormous health burden. The
United States government estimates that 45% of deaths in the United States
can be attributed to fibrosing disorders. Fibrosis can affect virtually every
tissue and organ system. Traditionally, fibrosis has been viewed as the
irreversible, end-stage sequel to a multitude of diverse disease processes (Table
2). Excessive scarring following physical, thermal, metabolic, ischemic,
infectious, inflammatory, or immunological injury can occur in any part of the
body, and can cause destruction of the affected structures. Fibrotic tissue is
characterized by a loss of normal architecture, paucity of stromal cells, and
replacement of blood vessels and other essential parenchymal structures by
dense, homogeneous, and increasingly stable extracellular matrix. The scar is
composed primarily of type I collagen, but types III and IV collagens,
proteoglycans, fibronectin, glycoproteins, and matricellular proteins are also
prominent. The process leads to progressive distortion of tissue architecture
with consequent dysfunction and ultimate failure of fibrotic organs. Many of
the key morphological features of fibrosis are common to scarring affecting
the lungs, the liver, the kidneys, the heart, or the skin.
Fibrosis is the “dark side” of normal tissue repair. Following injury, a com-
plex and tightly orchestrated repertoire of cellular responses is called into play,
and normally the wound is rapidly and efficiently repaired. This process is
spatially and temporally self-limited. In contrast, under some conditions repair
is excessive, resulting in pathological scar formation. The pathogenesis of
fibrosis remains poorly understood. Fibrosis involves a “fibrogenic cascade”
integrating multiple molecular pathways and cellular targets. Historically, the
link between inflammation and fibrosis has been emphasized, providing the
rationale for anti-inflammatory or immunosuppressive therapies for fibrosis. It
has become increasingly evident that these types of interventions are generally
ineffective. The lack of effective treatments, and the high mortality and
increasing morbidity attributed to chronic fibrotic diseases, has stimulated an
explosion of research into the cellular, molecular, and genetic basis of fibrosis.
Recent research progress has led to new and evolving concepts regarding
the mechanisms that drive the process of fibrosis. Novel insights include the
identification and characterization of an increasingly large panel of molecules
v
vi Preface
Table 1
Selected Diseases Where Fibrosis Is a Major Cause of Morbidity or Mortality
Organ-specific
Lung fibrosis (idiopathic and drug-induced pulmonary fibrosis, asthma,
sarcoidosis, COPD)
Liver fibrosis (alcoholic cirrhosis, posthepatitis C cirrhosis, schistosomiasis,
primary biliary cirrhosis, sclerosing cholangitis)
Kidney fibrosis (diabetic nephropathy, lupus glomerulossclerosis)
Heart fibrosis (post-MI scar)
Vascular fibrosis (postangioplasty arterial restenosis, atherosclerosis
Skin fibrosis (burn scar, hypertrophic scar, keloid, nephrogenic fibrosing
dermatopathy)
Eye fibrosis (retro-orbital fibrosis, postcataract surgery, proliferative
vitreoretinopathy)
Bone marrow (idiopathic and drug-induced myelofibrosis)
Other (Peyronie’s disease, thyroid, Dupuytren’s contracture, dermatomyositis)
Systemic
Systemic sclerosis
Chronic graft vs host disease
Table 2
Association of Fibrosis With Types of Injury: Representative Examples
Burn Skin and soft tissue scarring and contraction
Physical Hypertrophic scar, keloid, postangioplasty vascular restenosis
Radiation Pulmonary fibrosis; skin fibrosis; myelofibrosis
Surgery Adhesions of visceral organs
Metabolic Alcoholic cirrhosis, diabetic nephropathy, dialysis-related
fibrosing dermatopathy
Ischemic Post-MI cardiac fibrosis, post-stroke brain scarring
Autoimmune Systemic sclerosis, myositis, lupus nephritis, chronic graft
vs host disease
Inflammatory Retroperitoneal fibrosis, asthma, sarcoidosis, atherosclerosis,
primary biliary cirrhosis
Infectious Hepatitic C-associated cirrhosis, schistosomal cirrhosis, HIV-
associated lymphoid fibrosis
Malignant Myelofibrosis
Preface vii
that mediate abnormal deposition of extracellular matrix, as well as the cellular
constituents that produce and/or react to these molecules. Significant unan-
swered issues involve basic cellular and molecular mechanisms related to cell
proliferation, motility, differentiation, and gene expression. For instance,
recent discoveries on the heterogeneity of fibroblast populations in fibrotic
lesions have relevance to understanding cellular differentiation and program of
gene expression that determines cell phenotype. The demonstration of epithe-
lial–mesenchymal transdifferentiation as a key element in the pathogenesis of
renal fibrosis illustrates the need for further research into basic mechanisms to
advance the field. The importance of the myofibroblastic phenotype requires
improvement in current understanding of how activation and/or trans-
differentiation is regulated in the context of progressive fibrosis. Elucidation
of mechanisms involved in the regulation of extracellular matrix production
and degradation is critical for understanding the basis for the abnormal deposition
of connective tissue elements that is the hallmark of fibrosis. The advent of
powerful techniques for global assessment of gene expression in cells and
tissues should prove an impetus for rapid progress in this area. At the tissue
and organismal level, improvement in methods for evaluating the extent,
severity, and activity of fibrosis is essential for more precise and less invasive
diagnostic and prognostic assessment, as well as for monitoring of therapeutic
efficacy.
It is now widely appreciated that the diverse fibroproliferative diseases, be
they immunological, metabolic, inflammatory, genetic, or iatrogenic in nature,
all have important pathogenetic features in common. That is to say, despite
their differing anatomic distribution and clinical manifestations, etiology and
natural history, the pathogenesis of these disorders appears to be related. Thus,
regardless of whether fibrosis involves the lung parenchyma or airways, the
renal glomerulus or interstitium, the parenchyma or biliary tree of the liver, the
gastrointestinal tract, the heart, the dermis or tendons, or the eye, in each case
secretion and activation of profibrotic cytokines, particularly transforming growth
factor (TGF)-β, expansion and activation of mesenchymal cell populations, and
extracellular matrix synthesis and organization similarly result in progressive
destruction of normal tissue (Table 3).
Fibrosis research is an emerging field. The challenges facing investigators
engaged in fibrosis research are many: to describe and annotate the full reper-
toire of fibrotic mediators and effector cells involved in the fibrotic response,
and characterize their deregulation in pathological fibrosis; to identify genes
and their variants linked to fibrosis, and define their role in pathogenesis; to
recognize shared pathogenetic themes that are common to distinct forms of
fibrosis; and ultimately, to translate these insights into the development of specific
viii Preface
Table 3
Underlying Pathophysiologic Processes Common to Different Fibrotic Diseases
Genetic susceptibility
Inflammation
T-cell activation, Th2 polarization; macrophage activation; eosinophil and mast
cell chemotaxis; cytokine and chemokine production
Vascular injury, endothelial adhesion molecule upregulation
Mesenchymal cell activation
Increased synthesis of extracellular matrix
Secretion and activation of TGF-β, other fibrotic cytokines, autocrine
stimulation and amplification
Migration
Cell differentiation
Myofibroblast transdifferentiation
Epithelial–mesenchymal transition
Bone marrow-derived mesenchymal progenitor cell influx and differentiation
Scar formation and persistence; tissue destruction
Connective tissue contraction
Blood vessel obliteration, defective vasculogenesis, tissue hypoxia
Matrix stabilization, collagen crosslinking
Defective matrix remodeling and degradation
and sensitive diagnostic tools and safe and effective therapies to slow, prevent,
and reverse organ fibrosis. For this undertaking to succeed, interactions be-
tween laboratory researchers and clinical investigators and clinicians from
many different specialities is a must, since fibrosis impacts all fields of Medi-
cine. The 22 chapters of Fibrosis Research: Methods and Protocols highlight
the interdisciplinary nature of fibrosis research, and emphasize the broad
applicability of these experimental methodologies to all areas of fibrosis
research.
The book is divided into four parts. Part 1 features a brief introduction to the
problem of fibrosis, with clinical overviews of pulmonary and renal fibrosis as
examples illustrative of the many fibrosing disorders listed in Table 1, and a
bird’s-eye view of TGF-β as a paradigm profibrotic mediator. Part 2 focuses
on widely used research methodologies utilizing cultured cells to model various
aspects of the fibrotic response in vitro. The isolation, characterization, and
propagation of mesenchymal cells are described, highlighting similarities and
differences between methods that are appropriate for different types of
Preface ix
fibroblasts. Chapters 7 to 10 discuss approaches for studying collagen gene
regulation and TGF-βproduction. Part 3 focuses on experimental methodologies
utilizing animal models to study the pathogenesis of fibrosis, discussing the
advantages and limitations of each. Part 4 covers the evolving genetic
approaches to identifying “fibrosis genes” or allelic polymorphisms in human
populations, microarray studies for describing global patterns of gene expression
associated with fibrosis, and proteomic approaches to the same.
As editors of this new and innovative book, Fibrosis Research: Methods
and Protocols, we hope that it will reach a broad audience, and prove to be
helpful to investigators studying any aspect of pathological fibrosis. It is our
further hope that the book will stimulate discussion and promote interdisciplinary
research collaborations, with the goal of understanding the daunting problem
of fibrosis.
We wish to acknowledge Francesco Ramirez, Sergio Jimenez, Humphrey
Gardner, William Schnaper, Partricia Greenwel, Steven Ackerman, and the
many other colleagues who have provided helpful suggestions and stimulating
discussion in preparation of this book.
John Varga, MD
David A. Brenner, MD
Sem H. Phan, MD,PhD
Contents
Preface ..............................................................................................................v
Contributors .................................................................................................. xiii
PART I. THE CLINICAL SCOPE OF FIBROSIS
1 Pulmonary Fibrosis
David A. Zisman, Michael P. Keane, John A. Belperio,
Robert M. Strieter, and Joseph P. Lynch, III....................................3
2 Renal Fibrosis
H. William Schnaper...........................................................................45
3 Transforming Growth Factor-β: A Key Mediator of Fibrosis
Alain Mauviel......................................................................................69
PART II. REGULATION OF EXTRACELLULAR MATRIX METABOLISM:
CULTURED CELLS
4 Isolation and Culture of Skin Fibroblasts
Laure Rittié and Gary J. Fisher...........................................................83
5 Isolation and Culture of Hepatic Stellate Cells
Ralf Weiskirchen and Axel M. Gressner.............................................99
6 Isolation and Phenotypic Characterization of Lung Fibroblasts
Carolyn J. Baglole, Sireesha Y. Reddy, Stephen J. Pollock,
Steven E. Feldon, Patricia J. Sime, Terry J. Smith,
and Richard P. Phipps...................................................................115
7 Methods for Measuring Type I Collagen Synthesis In Vitro
David C. Rishikof, Ping-Ping Kuang,
Mangalalaxmy Subramanian, and Ronald H. Goldstein..............129
8 Methods for Assessing the Molecular Mechanisms Controlling
Gene Regulation
Richard A. Rippe and Branko Stefanovic..........................................141
9 Methods for Measuring TGF-β Using Antibodies, Cells, and Mice
Vladimir Jurukovski, Branka Dabovic, Vesna Todorovic,
Yan Chen, and Daniel B. Rifkin....................................................161
PART III. STUDYING FIBROSIS USING ANIMAL MODELS
10 Morphological Methods for Assessment of Fibrosis
Rakesh K. Kumar...............................................................................179
xi