Giulio Gabbiani

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.

The myofibroblast in wound healing and fibrocontractive diseases.

The demonstration that fibroblastic cells acquire contractile features during the healing of an open wound, thus modulating into myofibroblasts, has open a new perspective in the understanding of mechanisms leading to wound closure and fibrocontractive diseases. Myofibroblasts synthesize extracellular matrix components such as collagen types I and III and during normal wound healing disappear by apoptosis when epithelialization occurs. The transition from fibroblasts to myofibroblasts is influenced by mechanical stress, TGF‐β and cellular fibronectin (ED‐A splice variant). These factors also play important roles in the development of fibrocontractive changes, such as those observed in liver cirrhosis, renal fibrosis, and stroma reaction to epithelial tumours. Copyright

Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction

Au cours de la contraction du tissu de granulation, de nombreux fibroblastes acquièrent des caractéristiques ultrastructurelles qui les rendent semblables à des cellules musculaires lisses. Il est probable que ces éléments modifiés jouent un rôle dans le processus de contraction des plaies.

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.

Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.

The discovery of the myofibroblast has opened new perspectives for the comprehension of the biological mechanisms involved in wound healing and fibrotic diseases. In recent years, many advances have been made in understanding important aspects of myofibroblast basic biological characteristics. This review summarizes such advances in several fields, such as the following: i) force production by the myofibroblast and mechanisms of connective tissue remodeling; ii) factors controlling the expression of α-smooth muscle actin, the most used marker of myofibroblastic phenotype and, more important, involved in force generation by the myofibroblast; and iii) factors affecting genesis of the myofibroblast and its differentiation from precursor cells, in particular epigenetic factors, such as DNA methylation, microRNAs, and histone modification. We also review the origin and the specific features of the myofibroblast in diverse fibrotic lesions, such as systemic sclerosis; kidney, liver, and lung fibrosis; and the stromal reaction to certain epithelial tumors. Finally, we summarize the emerging strategies for influencing myofibroblast behavior in vitro and in vivo, with the ultimate goal of an effective therapeutic approach for myofibroblast-dependent diseases.

Contraction of granulation tissue in vitro: similarity to smooth muscle

Strips of granulation tissue from three different experimental models contract in vitro when treated with substances that induce contraction of smooth muscle. Because the fibroblasts in such tissues have some ultrastructural features typical of smooth muscle, our findings indicate that fibroblasts are able to modulate toward a cell type that is morphologically and functionally close to smooth muscle.

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.

Heterogeneity of myofibroblast phenotypic features: an example of fibroblastic cell plasticity

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of α-SM actin, the actin isoform present in SM cells and myoepithelial cells and particularly abundant in vascular SM cells. Myofibroblasts have been suggested to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. When contraction stops and the wound is fully epithelialized, myofibroblasts containing α-SM actin disappear, probably as a result of apoptosis, and the scar classically becomes less cellular and composed of typical fibroblasts with well-developed rough endoplasmic reticulum but with no more microfilaments. In contrast, α-SM actin expressing myofibroblasts persist in hypertrophic scars and in fibrotic lesions of many organs, including stroma reaction to epithelial tumours, where they are allegedly involved in retractile phenomena as well as in extracellular matrix accumulation. The mechanisms leading to the development of myofibroblastic features remain to be investigated. In vivo and in vitro investigations have shown that γ-interferon exerts an antifibrotic activity at least in part by decreasing α-SM actin expression whereas heparin increases the proportion of α-SM actin positive cells. Recently, we have observed that the subcutaneous administration of transforming growth factor-β1 to rats results in the formation of a granulation tissue in which α-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor, basic fibroblast growth factor and tumour necrosis factor-α, despite their profibrotic activity, do not induce α-SM actin in myofibroblasts. In conclusion, fibroblastic cells are relatively undifferentiated and can assume a particular phenotype according to the physiological needs and/or the microenvironmental stimuli. Further studies on fibroblast adaptation phenomena appear to be useful for the understanding of the mechanisms of development and regression of pathological processes such as wound healing and fibrocontractive diseases.

Arterial Smooth Muscle Cell Heterogeneity. Implications for Atherosclerosis and Restenosis Development

Abstract—During atheromatous plaque formation or restenosis after angioplasty, smooth muscle cells (SMCs) migrate from the media toward the intima, where they proliferate and undergo phenotypic changes. The mechanisms that regulate these phenomena and, in particular, the phenotypic modulation of intimal SMCs have been the subject of numerous studies and much debate during recent years. One view is that any SMCs present in the media could undergo phenotypic modulation. Alternatively, the seminal observation of Benditt and Benditt that human atheromatous plaques have the features of a monoclonal or an oligoclonal lesion has led to the hypothesis that a predisposed, medial SMC subpopulation could play a crucial role in the production of intimal thickening. The presence of a distinct SMC population in the arterial wall implies that under normal conditions, SMCs are phenotypically heterogeneous. The concept of SMC heterogeneity is gaining wider acceptance, as shown by the increasing number of publications on this subject. In this review, we discuss the in vitro studies that demonstrate the presence of distinct SMC subpopulations in arteries of various species, including humans. Their specific features and their regulation will be highlighted. Finally, the relevance of an atheroma-prone phenotype to intimal thickening formation will be discussed.

Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media.

Actin of smooth muscle cells of rat and human aortic media shows a predominance of the alpha-isoform. In experimental rat aortic intimal thickening, in human atheromatous plaque, and in cultured aortic smooth muscle cells, there is a typical switch in actin expression with a predominance of the beta-form and a noticeable amount of gamma-form. This pattern of actin expression represents a new reliable protein-chemical marker of experimental and human atheromatous smooth muscle cells.