Randy C. Mifflin

Myofibroblasts. I. Paracrine cells important in health and disease

Myofibroblasts are a unique group of smooth-muscle-like fibroblasts that have a similar appearance and function regardless of their tissue of residence. Through the secretion of inflammatory and anti-inflammatory cytokines, chemokines, growth factors, both lipid and gaseous inflammatory mediators, as well as extracellular matrix proteins and proteases, they play an important role in organogenesis and oncogenesis, inflammation, repair, and fibrosis in most organs and tissues. Platelet-derived growth factor (PDGF) and stem cell factor are two secreted proteins responsible for differentiating myofibroblasts from embryological stem cells. These and other growth factors cause proliferation of myofibroblasts, and myofibroblast secretion of extracellular matrix (ECM) molecules and various cytokines and growth factors causes mobility, proliferation, and differentiation of epithelial or parenchymal cells. Repeated cycles of injury and repair lead to organ or tissue fibrosis through secretion of ECM by the myofibroblasts. Transforming growth factor-β and the PDGF family of growth factors are the key factors in the fibrotic response. Because of their ubiquitous presence in all tissues, myofibroblasts play important roles in various organ diseases and perhaps in multisystem diseases as well.Myofibroblasts are a unique group of smooth-muscle-like fibroblasts that have a similar appearance and function regardless of their tissue of residence. Through the secretion of inflammatory and anti-inflammatory cytokines, chemokines, growth factors, both lipid and gaseous inflammatory mediators, as well as extracellular matrix proteins and proteases, they play an important role in organogenesis and oncogenesis, inflammation, repair, and fibrosis in most organs and tissues. Platelet-derived growth factor (PDGF) and stem cell factor are two secreted proteins responsible for differentiating myofibroblasts from embryological stem cells. These and other growth factors cause proliferation of myofibroblasts, and myofibroblast secretion of extracellular matrix (ECM) molecules and various cytokines and growth factors causes mobility, proliferation, and differentiation of epithelial or parenchymal cells. Repeated cycles of injury and repair lead to organ or tissue fibrosis through secretion of ECM by the myofibroblasts. Transforming growth factor-beta and the PDGF family of growth factors are the key factors in the fibrotic response. Because of their ubiquitous presence in all tissues, myofibroblasts play important roles in various organ diseases and perhaps in multisystem diseases as well.

Myofibroblasts. II. Intestinal subepithelial myofibroblasts

Intestinal subepithelial myofibroblasts (ISEMF) and the interstitial cells of Cajal are the two types of myofibroblasts identified in the intestine. Intestinal myofibroblasts are activated and proliferate in response to various growth factors, particularly the platelet-derived growth factor (PDGF) family, which includes PDGF-BB and stem cell factor (SCF), through expression of PDGF receptors and the SCF receptor c- kit. ISEMF have been shown to play important roles in the organogenesis of the intestine, and growth factors and cytokines secreted by these cells promote epithelial restitution and proliferation, i.e., wound repair. Their role in the fibrosis of Crohns disease and collagenous colitis is being investigated. Through cyclooxygenase (COX)-1 and COX-2 activation, ISEMF augment intestinal ion secretion in response to certain secretagogues. By forming a subepithelial barrier to Na+ diffusion, they create a hypertonic compartment that may account for the ability of the gut to transport fluid against an adverse osmotic gradient. Through the paracrine secretion of prostaglandins and growth factors (e.g., transforming growth factor-β), ISEMF may play a role in colonic tumorigenesis and metastasis. COX-2 in polyp ISEMF may be a target for nonsteroidal anti-inflammatory drugs (NSAIDs), which would account for the regression of the neoplasms in familial adenomatous polyposis and the preventive effect of NSAIDs in the development of sporadic colon neoplasms. More investigation is needed to clarify the functions of these pleiotropic cells.Intestinal subepithelial myofibroblasts (ISEMF) and the interstitial cells of Cajal are the two types of myofibroblasts identified in the intestine. Intestinal myofibroblasts are activated and proliferate in response to various growth factors, particularly the platelet-derived growth factor (PDGF) family, which includes PDGF-BB and stem cell factor (SCF), through expression of PDGF receptors and the SCF receptor c-kit. ISEMF have been shown to play important roles in the organogenesis of the intestine, and growth factors and cytokines secreted by these cells promote epithelial restitution and proliferation, i.e., wound repair. Their role in the fibrosis of Crohns disease and collagenous colitis is being investigated. Through cyclooxygenase (COX)-1 and COX-2 activation, ISEMF augment intestinal ion secretion in response to certain secretagogues. By forming a subepithelial barrier to Na(+) diffusion, they create a hypertonic compartment that may account for the ability of the gut to transport fluid against an adverse osmotic gradient. Through the paracrine secretion of prostaglandins and growth factors (e.g., transforming growth factor-beta), ISEMF may play a role in colonic tumorigenesis and metastasis. COX-2 in polyp ISEMF may be a target for nonsteroidal anti-inflammatory drugs (NSAIDs), which would account for the regression of the neoplasms in familial adenomatous polyposis and the preventive effect of NSAIDs in the development of sporadic colon neoplasms. More investigation is needed to clarify the functions of these pleiotropic cells.

Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

Mesenchymal Cells of the Intestinal Lamina Propria

The mesenchymal elements of the intestinal lamina propria reviewed here are the myofibroblasts, fibroblasts, mural cells (pericytes) of the vasculature, bone marrow-derived stromal stem cells, smooth muscle of the muscularis mucosae, and smooth muscle surrounding the lymphatic lacteals. These cells share similar marker molecules, origins, and coordinated biological functions previously ascribed solely to subepithelial myofibroblasts. We review the functional anatomy of intestinal mesenchymal cells and describe what is known about their origin in the embryo and their replacement in adults. As part of their putative role in intestinal mucosal morphogenesis, we consider the intestinal stem cell niche. Lastly, we review emerging information about myofibroblasts as nonprofessional immune cells that may be important as an alarm system for the gut and as a participant in peripheral immune tolerance.

Immunohistochemical study of myofibroblasts in normal colonic mucosa, hyperplastic polyps, and adenomatous colorectal polyps

CONTEXT Myofibroblasts are distinct cells with characteristics of both smooth muscle cells and fibroblasts. Through their ability to secrete cytokines, chemokines, prostaglandins, growth factors, and matrix components, they are thought to play critical roles in inflammation, growth, repair, and neoplasia. OBJECTIVE The goal of this study was to identify the distinct cell populations of the lamina propria of normal colon and colorectal polyps. DESIGN We studied the expression of alpha-smooth muscle actin (alphaSMA), smooth muscle myosin (SMM), desmin, vimentin, and c-kit by intestinal mesenchymal (stromal) cells in the normal colonic mucosa (n = 5), as well as in hyperplastic polyps (n = 5), sporadic colorectal adenomas (n = 47), and adenomas from patients with familial polyposis (n = 36). RESULTS In the normal colonic mucosa, the pericryptal stromal cells were alphaSMA+, SMM+, desmin-, and vimentin+, defining them as myofibroblasts. In contrast, cells of the muscularis mucosae were alphaSMA+, SMM+, desmin+, and vimentin-, defining them as smooth muscle cells. alpha-Smooth muscle actin also highlighted direct connections between the muscularis mucosae and the pericryptal myofibroblasts, and vimentin immunostaining showed a network of connections between the alphaSMA+ pericryptal myofibroblasts and the alphaSMA- fibroblasts in the interstitium. In all hyperplastic polyps and adenomatous polyps, the interstitial stromal cells (fibroblasts) now also express alphaSMA and form a syncytium of alphaSMA+ networklike connections throughout the lamina propria. Stromal cells of sporadic adenomas demonstrated the same immunohistochemical staining characteristics displayed by adenomas from patients with familial polyposis and by hyperplastic polyps. Conclusions.-These findings indicate that in normal colon, alphaSMA- fibroblasts are the predominant cell type in the lamina propria. However, the pericryptal (subepithelial) stromal cells are a distinct cell type (alphaSMA+ myofibroblast) that is immunophenotypically different from muscularis mucosae smooth muscle cells and are connected to the interstitial, nonpericryptal fibroblasts with which they exist as a network throughout the lamina propria of the normal colon. Furthermore, in both hyperplastic and neoplastic polyps, there are changes in nonpericryptal fibroblasts from vimentin+, alphaSMA-, and SMM- to vimentin+, alphaSMA+, and SMM+; thus, the interstitial fibroblasts are replaced by myofibroblasts. The factors that cause these changes and the origin of the myofibroblasts need to be determined to clarify the biology of colorectal tumorigenesis.

Subepithelial Myofibroblasts are Novel Nonprofessional APCs in the Human Colonic Mucosa

The human gastrointestinal mucosa is exposed to a diverse normal microflora and dietary Ags and is a common site of entry for pathogens. The mucosal immune system must respond to these diverse signals with either the initiation of immunity or tolerance. APCs are important accessory cells that modulate T cell responses which initiate and maintain adaptive immunity. The ability of APCs to communicate with CD4+ T cells is largely dependent on the expression of class II MHC molecules by the APCs. Using immunohistochemistry, confocal microscopy, and flow cytometry, we demonstrate that α-smooth muscle actin+, CD90+ subepithelial myofibroblasts (stromal cells) constitutively express class II MHC molecules in normal colonic mucosa and that they are distinct from professional APCs such as macrophages and dendritic cells. Primary isolates of human colonic myofibroblasts (CMFs) cultured in vitro were able to stimulate allogeneic CD4+ T cell proliferation. This process was dependent on class II MHC and CD80/86 costimulatory molecule expression by the myofibroblasts. We also demonstrate that CMFs, engineered to express a specific DR4 allele, can process and present human serum albumin to a human serum albumin-specific and DR4 allele-restricted T cell hybridoma. These studies characterize a novel cell phenotype which, due to its strategic location and class II MHC expression, may be involved in capture of Ags that cross the epithelial barrier and present them to lamina propria CD4+ T cells. Thus, human CMFs may be important in regulating local immunity in the colon.

Intestinal myofibroblasts: targets for stem cell therapy

The subepithelial intestinal myofibroblast is an important cell orchestrating many diverse functions in the intestine and is involved in growth and repair, tumorigenesis, inflammation, and fibrosis. The myofibroblast is but one of several α-smooth muscle actin-positive (α-SMA(+)) mesenchymal cells present within the intestinal lamina propria, including vascular pericytes, bone marrow-derived stem cells (mesenchymal stem cells or hematopoietic stem cells), muscularis mucosae, and the lymphatic pericytes (colon) and organized smooth muscle (small intestine) associated with the lymphatic lacteals. These other mesenchymal cells perform many of the functions previously attributed to subepithelial myofibroblasts. This review discusses the definition of a myofibroblast and reconsiders whether the α-SMA(+) subepithelial cells in the intestine are myofibroblasts or other types of mesenchymal cells, i.e., pericytes. Current information about specific, or not so specific, molecular markers of lamina propria mesenchymal cells is reviewed, as well as the origins of intestinal myofibroblasts and pericytes in the intestinal lamina propria and their replenishment after injury. Current concepts and research on stem cell therapy for intestinal inflammation are summarized. Information about the stem cell origin of intestinal stromal cells may inform future stem cell therapies to treat human inflammatory bowel disease (IBD).

Subepithelial Myofibroblasts Express Cyclooxygenase-2 in Colorectal Tubular Adenomas

Purpose: Recent data support the hypothesis that the inducible isoform of cyclooxygenase (COX-2) plays a role in the early stages of colonic carcinogenesis and that nonsteroidal anti-inflammatory drugs (NSAIDs) retard the development of colon cancer by modulating COX-2. However, the cell types responsible for producing COX-2 in colorectal adenomas remain a subject of controversy. Experimental Design: COX-2 expression in normal colonic mucosa (n = 50), hyperplastic polyps (n = 43), sporadic adenomas (n = 67), and invasive colonic adenocarcinoma (n = 39) was studied in formalin-fixed and paraffin-embedded tissue sections from endoscopy biopsy and colonic resection specimens. Immunohistochemistry (avidin-biotin complex technique with double immunolabeling) was used to identify the phenotypes of COX-2-producing cells. Results: In colorectal adenomas, increased expression of COX-2 was detected and localized to α smooth muscle actin (∝SMA)-positive subepithelial stromal cells (myofibroblasts) in the periluminal region of the lamina propria in 63 (94%) of 67 cases. In contrast, in normal colonic mucosa and in hyperplastic polyps with intact epithelium, COX-2 expression was found only in macrophages and endothelial cells. In areas in which the surface epithelium was ulcerated in normal mucosa as well as hyperplastic or neoplastic polyps, COX-2 expression was increased in granulation tissue (and present in macrophages, endothelium, and myofibroblasts). In invasive carcinoma, COX-2 expression in myofibroblasts was limited to the adenomatous portion of the tumor and was detected in 62% of cases (n = 39). In addition, focal expression of COX-2 by malignant epithelial cells was observed in 23% of invasive adenocarcinoma. Conclusions: These results show that increased COX-2 expression in sporadic adenoma of the colon is common and is localized specifically to subepithelial intestinal myofibroblasts. These findings further support the hypothesis that myofibroblasts are important target cells for NSAID-mediated chemoprevention of colorectal cancer.

Indian Hedgehog Regulates Intestinal Stem Cell Fate Through Epithelial−Mesenchymal Interactions During Development

BACKGROUND & AIMS Intestinal stem cells (ISCs) are regulated by the mesenchymal environment via physical interaction and diffusible factors. We examined the role of Indian hedgehog (Ihh) in mesenchymal organization and the mechanisms by which perturbations in epithelial-mesenchymal interactions affect ISC fate. METHODS We generated mice with intestinal epithelial-specific disruption of Ihh. Gross and microscopic anatomical changes were determined using histologic, immunohistochemical, and in situ hybridization analyses. Molecular mechanisms were elucidated by expression profiling and in vitro analyses. RESULTS Deletion of intestinal epithelial Ihh disrupted the intestinal mesenchymal architecture, demonstrated by loss of the muscularis mucosae, deterioration of the extracellular matrix, and reductions in numbers of crypt myofibroblasts. Concurrently, the epithelial compartment had increased Wnt signaling, disturbed crypt polarity and architecture, defective enterocyte differentiation, and increased and ectopic proliferation that was accompanied by increased numbers of ISCs. Mechanistic studies revealed that Hh inhibition deregulates bone morphogenetic protein signaling, increases matrix metalloproteinase levels, and disrupts extracellular matrix proteins, fostering a proliferative environment for ISCs and progenitor cells. CONCLUSIONS Ihh regulates ISC self-renewal and differentiation. Intestinal epithelial Ihh signals to the mesenchymal compartment to regulate formation and proliferation of mesenchymal cells, which in turn affect epithelial proliferation and differentiation. These findings provide a basis for analyses of the role of the muscularis mucosae in ISC regulation.

Roles of myofibroblasts in prostaglandin E2-stimulated intestinal epithelial proliferation and angiogenesis

Prostaglandins (PG) are produced throughout the gastrointestinal tract and are critical mediators for a complex array of physiologic and pathophysiologic processes in the intestine. Intestinal myofibroblasts, which express cyclooxygenase (COX) and generate PGE(2), play important roles in intestinal epithelial proliferation, differentiation, inflammation, and neoplasia through secreting growth factors and cytokines. Here, we show that PGE(2) activated human intestinal subepithelial myofibroblasts (18Co) through Gs protein-coupled E-prostanoid receptors and the cyclic AMP/protein kinase A pathway. 18Co cells and primary colonic myofibroblast isolates expressed a number of growth factors; several of them were dramatically regulated by PGE(2). An epidermal growth factor-like growth factor, amphiregulin (AR), which was not expressed by untreated cells, was strongly induced by PGE(2). Expression of vascular endothelial growth factor A (VEGFA) was rapidly increased by PGE(2) exposure. Hepatocyte growth factor (HGF) was elevated in PGE(2)-treated myofibroblasts at both mRNA and protein levels. Thus, PGE(2)-activated myofibroblasts promoted the proliferation and migration of intestinal epithelial cells, which were attenuated by neutralizing antibodies to AR and HGF, respectively. Moreover, in the presence of PGE(2), myofibroblasts strongly stimulated the migration and tubular formation of vascular endothelial cells. Neutralizing antibody to VEGFA inhibited the observed stimulation of migration. These results suggest that myofibroblast-generated growth factors are important mediators for PGE(2)-induced intestinal epithelial proliferation and angiogenesis, which play critical roles in intestinal homeostasis, inflammation, and neoplasia.