Collagen microarchitecture mechanically controls myofibroblast differentiation.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation

A two-compartment mechanochemical model of the roles of\ud transforming growth factor β and tissue tension in dermal wound healing

The repair of dermal tissue is a complex process of interconnected phenomena, where cellular, chemical and mechanical aspects all play a role, both in an autocrine and in a paracrine fashion. Recent experimental results have shown that transforming growth factor−β (TGFβ) and tissue mechanics play roles in regulating cell proliferation, differentiation and the production of extracellular materials. We have developed a 1D mathematical model that considers the interaction between the cellular, chemical and mechanical phenomena, allowing the combination of TGFβ and tissue stress to inform the activation of fibroblasts to myofibroblasts. Additionally, our model incorporates the observed feature of residual stress by considering the changing zero-stress state in the formulation for effective strain. Using this model, we predict that the continued presence of TGFβ in dermal wounds will produce contractures due to the persistence of myofibroblasts; in contrast, early elimination of TGFβ significantly reduces the myofibroblast numbers resulting in an increase in wound size. Similar results were obtained by varying the rate at which fibroblasts differentiate to myofibroblasts and by changing the myofibroblast apoptotic rate. Taken together, the implication is that elevated levels of myofibroblasts is the key factor behind wounds healing with excessive contraction, suggesting that clinical strategies which aim to reduce the myofibroblast density may reduce the appearance of contractures

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor beta 1

Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1

A fibrocontractive mechanochemical model of dermal wound\ud closure incorporating realistic growth factor kinetics

Fibroblasts and their activated phenotype, myofibroblasts, are the primary cell types involved in the contraction associated with dermal wound healing. Recent experimental evidence indicates that the transformation from fibroblasts to myofibroblasts involves two distinct processes: the cells are stimulated to change phenotype by the combined actions of transforming growth factor β (TGFβ) and mechanical tension. This observation indicates a need for a detailed exploration of the effect of the strong interactions between the mechanical changes and growth factors in dermal wound healing. We review the experimental findings in detail and develop a model of dermal wound healing that incorporates these phenomena. Our model includes the interactions between TGFβ and collagenase, providing a more biologically realistic form for the growth factor kinetics than those included in previous mechanochemical descriptions. A comparison is made between the model predictions and experimental data on human dermal wound healing and all the essential features are well matched

Electrical coupling between ventricular myocytes and myofibroblasts in the infarcted mouse heart

Aims: Recent studies have demonstrated electrotonic coupling between scar tissue and the surrounding myocardium in cryoinjured hearts. However, the electrical dynamics occurring at the myocyte-nonmyocyte interface in the fibrotic heart remain undefined. Here, we sought to develop an assay to interrogate the nonmyocyte cell type contributing to heterocellular coupling and to characterize, on a cellular scale, its voltage response in the infarct border zone of living hearts. Methods and results: We used two-photon laser scanning microscopy in conjunction with a voltage-sensitive dye to record transmembrane voltage changes simultaneously from cardiomyocytes and adjoined nonmyocytes in Langendorff-perfused mouse hearts with healing myocardial infarction. Transgenic mice with cardiomyocyte-restricted expression of a green fluorescent reporter protein underwent permanent coronary artery ligation and their hearts were subjected to voltage imaging 7-10 days later. Reporter-negative cells, i.e. nonmyocytes, in the infarct border zone exhibited depolarizing transients at a 1:1 coupling ratio with action potentials recorded simultaneously from adjacent, reporter-positive ventricular myocytes. The electrotonic responses in the nonmyocytes exhibited slower rates of de- and repolarization compared to the action potential waveform of juxtaposed myocytes. Voltage imaging in infarcted hearts expressing a fluorescent reporter specifically in myofibroblasts revealed that the latter were electrically coupled to border zone myocytes. Their voltage transient properties were indistinguishable from those of nonmyocytes in hearts with cardiomyocyte-restricted reporter expression. The density of connexin43 expression at myofibroblast-cardiomyocyte junctions was ∼5% of that in the intercalated disc regions of paired ventricular myocytes in the remote, uninjured myocardium, whereas the ratio of connexin45 to connexin43 expression levels at heterocellular contacts was ∼1%. Conclusion: Myofibroblasts contribute to the population of electrically coupled nonmyocytes in the infarct border zone. The slower kinetics of myofibroblast voltage responses may reflect low electrical conductivity across heterocellular junctions, in accordance with the paucity of connexin expression at myofibroblast-cardiomyocyte contacts

Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation

Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer

A Role of Myocardin Related Transcription Factor-A (MRTF-A) in Scleroderma Related Fibrosis.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix

Endothelial to mesenchymal transition (EndoMT) in the pathogenesis of Systemic Sclerosis-associated pulmonary fibrosis and pulmonary arterial hypertension. Myth or reality?

Systemic Sclerosis (SSc) is a systemic autoimmune disease characterized by progressive fibrosis of skin and multiple internal organs and severe functional and structural microvascular alterations. SSc is considered to be the prototypic systemic fibrotic disorder. Despite currently available therapeutic approaches SSc has a high mortality rate owing to the development of SSc-associated interstitial lung disease (ILD) and pulmonary arterial hypertension (PAH), complications that have emerged as the most frequent causes of disability and mortality in SSc. The pathogenesis of the fibrotic process in SSc is complex and despite extensive investigation the exact mechanisms have remained elusive. Myofibroblasts are the cells ultimately responsible for tissue fibrosis and fibroproliferative vasculopathy in SSc. Tissue myofibroblasts in SSc originate from several sources including expansion of quiescent tissue fibroblasts and tissue accumulation of CD34+ fibrocytes. Besides these sources, myofibroblasts in SSc may result from the phenotypic conversion of endothelial cells into activated myofibroblasts, a process known as endothelial to mesenchymal transition (EndoMT). Recently, it has been postulated that EndoMT may play a role in the development of SSc-associated ILD and PAH. However, although several studies have described the occurrence of EndoMT in experimentally induced cardiac, renal, and pulmonary fibrosis and in several human disorders, the contribution of EndoMT to SSc-associated ILD and PAH has not been generally accepted. Here, the experimental evidence supporting the concept that EndoMT plays a role in the pathogenesis of SSc-associated ILD and PAH will be reviewed