Christine Chaponnier

Myofibroblasts and mechano-regulation of connective tissue remodelling

During the past 20 years, it has become generally accepted that the modulation of fibroblastic cells towards the myofibroblastic phenotype, with acquisition of specialized contractile features, is essential for connective-tissue remodelling during normal and pathological wound healing. Yet the myofibroblast still remains one of the most enigmatic of cells, not least owing to its transient appearance in association with connective-tissue injury and to the difficulties in establishing its role in the production of tissue contracture. It is clear that our understanding of the myofibroblast — its origins, functions and molecular regulation — will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally.

Tissue repair, contraction, and the myofibroblast

After the first description of the myofibroblast in granulation tissue of an open wound by means of electron microscopy, as an intermediate cell between the fibroblast and the smooth muscle cell, the myofibroblast has been identified both in normal tissues, particularly in locations where there is a necessity of mechanical force development, and in pathological tissues, in relation with hypertrophic scarring, fibromatoses and fibrocontractive diseases as well as in the stroma reaction to epithelial tumors. It is now accepted that fibroblast/myofibroblast transition begins with the appearance of the protomyofibroblast, whose stress fibers contain only β‐ and γ‐cytoplasmic actins and evolves, but not necessarily always, into the appearance of the differentiated myofibroblast, the most common variant of this cell, with stress fibers containing α‐smooth muscle actin. Myofibroblast differentiation is a complex process, regulated by at least a cytokine (the transforming growth factor‐β1), an extracellular matrix component (the ED‐A splice variant of cellular fibronectin), as well as the presence of mechanical tension. The myofibroblast is a key cell for the connective tissue remodeling that takes place during wound healing and fibrosis development. On this basis, the myofibroblast may represent a new important target for improving the evolution of such diseases as hypertrophic scars, and liver, kidney or pulmonary fibrosis.

Mechanical Tension Controls Granulation Tissue Contractile Activity and Myofibroblast Differentiation

We have examined the role of mechanical tension in myofibroblast differentiation using two in vivo rat models. In the first model, granulation tissue was subjected to an increase in mechanical tension by splinting a full-thickness wound with a plastic frame. Myofibroblast features, such as stress fiber formation, expression of ED-A fibronectin and alpha-smooth muscle actin (alpha-SMA) appeared earlier in splinted than in unsplinted wounds. Myofibroblast marker expression decreased in control wounds starting at 10 days after wounding as expected, but persisted in splinted wounds. In the second model, granuloma pouches were induced by subcutaneous croton oil injection; pouches were either left intact or released from tension by evacuation of the exudate at 14 days. The expression of myofibroblast markers was reduced after tension release in the following sequence: F-actin (2 days), alpha-SMA (3 days), and ED-A fibronectin (5 days); cell density was not affected. In both models, isometric contraction of tissue strips was measured after stimulation with smooth muscle agonists. Contractility correlated always with the level of alpha-SMA expression, being high when granulation tissue had been subjected to tension and low when it had been relaxed. Our results support the assumption that mechanical tension is crucial for myofibroblast modulation and for the maintenance of their contractile activity.

The NH2-terminal peptide of α–smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of α–smooth muscle actin (α-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of α-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of α–skeletal actin has no effect on myofibroblasts, whereas the NH2-terminal peptide of β–cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing α-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of α-SMA in wound contraction. The α-SMA–FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.

Evidence of an Intracellular Reservoir in the Nasal Mucosa of Patients with Recurrent Staphylococcus aureus Rhinosinusitis

Severe infections due to Staphylococcus aureus require prolonged therapy for cure, and relapse may occur even years after the first episode. Persistence of S. aureus may be explained, in part, by nasal carriage of S. aureus, which occurs in a large percentage of healthy humans and represents a major source of systemic infection. However, the persistence of internalized S. aureus within mucosal cells has not been evaluated in humans. Here, we provide the first in vivo evidence of intracellular reservoirs of S. aureus in humans, which were assessed in endonasal mucosa specimens from patients suffering from recurrent S. aureus rhinosinusitis due to unique, patient-specific bacterial clonotypes. Heavily infected foci of intracellular bacteria located in nasal epithelium, glandular, and myofibroblastic cells were revealed by inverted confocal laser scan fluorescence and electron microscopic examination of posttherapy intranasal biopsy specimens from symptom-free patients undergoing surgery on the sinuses. Intracellular residence may provide a sanctuary for pathogenic bacteria by protecting them from host defense mechanisms and antibiotic treatment during acute, recurrent S. aureus rhinosinusitis.

Medical aspects of the actin cytoskeleton

The actin cytoskeleton is affected in many disease states. The mechanisms by which altered structure or expression of actin or of actin-binding proteins cause specific defects are beginning to emerge. Notable recent findings concern the roles in tumor suppression of proteins that link actin to the cell membrane, the specific functions of actin isoforms in cells with a developed contractile apparatus, and the variety of complications caused by release of filamentous actin into extracellular fluids.

β- and γ-cytoplasmic actins display distinct distribution and functional diversity

Using newly generated monoclonal antibodies, we have compared the distribution of β- and γ-cytoplasmic actin in fibroblastic and epithelial cells, in which they play crucial roles during various key cellular processes. Whereas β-actin is preferentially localized in stress fibers, circular bundles and at cell-cell contacts, suggesting a role in cell attachment and contraction, γ-actin displays a more versatile organization, according to cell activities. In moving cells, γ-actin is mainly organized as a meshwork in cortical and lamellipodial structures, suggesting a role in cell motility; in stationary cells, γ-actin is also recruited into stress fibers. β-actin-depleted cells become highly spread, display broad protrusions and reduce their stress-fiber content; by contrast, γ-actin-depleted cells acquire a contractile phenotype with thick actin bundles and shrinked lamellar and lamellipodial structures. Moreover, β- and γ-actin depleted fibroblasts exhibit distinct changes in motility compared with their controls, suggesting a specific role for each isoform in cell locomotion. Our results reveal new aspects of β- and γ-actin organization that support their functional diversity.

Pathological situations characterized by altered actin isoform expression

Modulation of actin isoform expression is a well‐established feature of developmental phenomena. As one might expect, it is also characteristic of several pathological situations that are the subject of the present review. α‐Smooth muscle actin has proven to be a reliable marker for identifying (a) vascular smooth muscle cells during vascular development and vascular diseases, and (b) myofibroblasts during wound healing, fibrocontractive diseases, and stromal reaction to epithelial tumours. The hallmark of a differentiated myofibroblast relies on the acquisition of an organized contractile apparatus characterized by α‐smooth muscle actin‐expressing stress fibres. More and more data suggest that α‐smooth muscle actin plays a direct role in myofibroblast contractile activity through its N‐terminal domain AcEEED. Newly developed antibodies against α‐skeletal and α‐cardiac actins have allowed the detection of subpopulations of α‐skeletal positive cardiomyocytes in adult, hypertrophic, and failing heart. These antibodies have also permitted us to identify the differentiation degree of malignant cells in tumours such as rhabdomyosarcoma. Whether the differential expression of actin isoforms in human diseases is functionally relevant is not yet fully established, although studies on human actin mutations, actin null mice, and the N‐terminal end of α‐smooth muscle actin support this possibility. Copyright

Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung.

Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.

Intimal Smooth Muscle Cells of Porcine and Human Coronary Artery Express S100A4, a Marker of the Rhomboid Phenotype In Vitro

We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S- and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with &agr;-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S- and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein. In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes.