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Fibrosis is a hallmark of systemic sclerosis

  1. Top of page
  2. Fibrosis is a hallmark of systemic sclerosis
  3. The origins of myofibroblasts: endothelial–mesenchymal transition
  4. What induces EndoMT?
  5. New insights into EndoMT: c-Abl, protein kinase Cδ, and small-molecule kinase inhibitors
  6. Is EndoMT important in SSc pathophysiology?
  7. REFERENCES

Over the last decade, considerable attention has been paid to the origin of the myofibroblast, the mesenchymal cell type most responsible for the excessive matrix production and deposition in tissue and vessel walls found in fibrotic disorders and fibroproliferative vasculopathies, respectively (1). In this context, the myofibroblast has increasingly been regarded as a crucial effector cell in the pathogenesis of systemic sclerosis (SSc; scleroderma), a multifaceted connective tissue disorder characterized by both skin and internal organ fibrosis and a severe proliferative vasculopathy affecting small and medium-sized arterioles and capillaries (2–4).

In SSc, organ dysfunction and failure are ultimately caused by overproduction and accumulation, in affected tissue, of extracellular matrix components, such as types I and III collagen and fibronectin (2, 3). Moreover, the concomitant fibroproliferative vasculopathy, characterized by subendothelial intimal fibrosis, may lead to severe vascular complications, including digital ulceration and gangrene, pulmonary arterial hypertension, and scleroderma renal crisis. These events heavily jeopardize patients' quality of life and are associated with very poor prognosis (4).

Despite numerous recent advances in the understanding of the molecular regulation of genes encoding collagens and other extracellular matrix proteins, the complex mechanisms responsible for the heterogeneous pathogenesis of SSc remain unknown (5). As a consequence, no targeted and disease-modifying therapies are available to control, arrest, or reverse disease progression and fibrosis (6). It is well known that fibroblasts are dysregulated, that they transform into myofibroblasts, and that they produce an excessive amount of collagen and other extracellular matrix components in SSc (7). For this reason, the pivotal goal of a research agenda today is the identification of the origin of tissue and subendothelial myofibroblasts, together with the intracellular transduction pathways involved in the transcriptional activation of the genes required for the recruitment and transdifferentiation of myofibroblasts from their putative cellular precursors.

The origins of myofibroblasts: endothelial–mesenchymal transition

  1. Top of page
  2. Fibrosis is a hallmark of systemic sclerosis
  3. The origins of myofibroblasts: endothelial–mesenchymal transition
  4. What induces EndoMT?
  5. New insights into EndoMT: c-Abl, protein kinase Cδ, and small-molecule kinase inhibitors
  6. Is EndoMT important in SSc pathophysiology?
  7. REFERENCES

Myofibroblasts are atypical fibroblasts expressing α-smooth muscle actin (α-SMA) and exhibiting an uncontrolled activated phenotype. They are in an intermediate state between a fibroblast and a smooth muscle cell phenotype, combining the structural features of both cell types (1). Traditionally, myofibroblasts within the fibrotic scar were considered to be derived from proliferating tissue resident fibroblasts (1). Besides resident fibroblasts, vessel wall pericytes, and adventitial fibroblasts, bone marrow–derived circulating fibroblast precursors, also known as fibrocytes, and epithelial cells via epithelial–mesenchymal transition have also been found to potentially contribute to the accumulation of myofibroblasts in fibrotic conditions (1, 7, 8).

Recently, it has been reported with increasing frequency that vascular endothelial cells (ECs) have an ability to acquire matrix-producing myofibroblastic features, providing proof of principle for the process of endothelial–mesenchymal transition (EndoMT) (9–11). EndoMT is a process of nonmalignant cellular transdifferentiation by which ECs disaggregate, lose polarity, and undergo a change in their typical shape, becoming elongated and acquiring the ability to migrate into the surrounding tissue. It is a phenotypic conversion characterized by the loss of vascular EC markers such as CD31, von Willebrand factor, and VE-cadherin, and the emergence of mesenchymal cell markers such as α-SMA, vimentin, type I procollagen, and S100A4/fibroblast-specific protein 1 (9–12).

EndoMT is a phenomenon that is reported to occur during embryonic cardiovascular development and also is reported to play an important role during several pathologic conditions, including cardiac, pulmonary, and renal fibrosis, carcinoma-associated interstitial fibrosis, and vascular pathologies (9, 11–16). Indeed, recent evidence has shown that a substantial proportion of myofibroblasts arise from ECs via EndoMT in experimental renal fibrosis and bleomycin-induced pulmonary fibrosis in mice (13, 15). A possible role of EndoMT in the neointimal thickening observed in transplant atherosclerosis and restenosis has also been suggested (11). In addition, it has recently been demonstrated that transdifferentiation of pulmonary arteriolar ECs into mesenchymal-like cells occurs in the hypoxia-induced vascular remodeling process of chronic pulmonary hypertension (11). A number of in vitro studies have demonstrated that ECs from a variety of vascular beds retain the ability to transition into mesenchymal-like cells under a variety of culture conditions (11). Findings in experimental wound repair have suggested that EndoMT may also take place during the formation of granulation tissue (11). Others have observed that microvascular ECs differentiate into mesenchymal cells in response to chronic inflammatory stimuli (10).

What induces EndoMT?

  1. Top of page
  2. Fibrosis is a hallmark of systemic sclerosis
  3. The origins of myofibroblasts: endothelial–mesenchymal transition
  4. What induces EndoMT?
  5. New insights into EndoMT: c-Abl, protein kinase Cδ, and small-molecule kinase inhibitors
  6. Is EndoMT important in SSc pathophysiology?
  7. REFERENCES

Among the many growth factors and cytokines shown to regulate EndoMT in the embryonic heart and in tissue fibrosis, the potent profibrotic transforming growth factor β (TGFβ) has been highlighted as a key player (11, 12). In fact, it has been shown to act as a potent inducer of EndoMT both in vitro and in vivo (11, 12). To date, cultured ECs of many different origins have been demonstrated to undergo EndoMT when exposed to TGFβ (9, 16–18).

Studies aimed at unraveling the molecular mechanisms behind TGFβ-induced EndoMT have shown that the transcriptional repressor Snail-1 is crucial for TGFβ-induced mesenchymal transdifferentiation of embryonic stem cell–derived ECs (19). Snail-1 is a zinc-finger transcription factor that also plays an important role in the process of epithelial–mesenchymal transition, causing potent inhibition of E-cadherin gene transcription in cultured epithelial cells (20). Kokudo et al found that TGFβ stimulation induced the expression of Snail-1 in ECs, and small interfering RNA (siRNA) knockdown of Snail-1 blocked TGFβ-induced EndoMT (19). Moreover, conditional overexpression of Snail-1 induced EndoMT even in the presence of a TGFβ type I kinase inhibitor, suggesting that Snail-1 acts downstream of TGFβ signals during EndoMT (19). However, the exact intracellular signaling pathways linking TGFβ stimulation to the up-regulation of Snail-1 and the initiation of expression of myofibroblastic markers, such as α-SMA, in ECs remained to be elucidated.

Indeed, TGFβ activation and signaling cascades are extremely complex and involve multiple intracellular molecules and pathways (21). Bioactive TGFβ in a dimeric form binds to a constitutively active serine/threonine transmembrane kinase known as TGFβ receptor type II (TGFβRII). The classic pathway of TGFβ signal transduction into the cell nucleus involves the ligand-bound TGFβRII, which recruits and then transphosphorylates TGFβRI. Signaling from phosphorylated TGFβRI to the nucleus then may occur through the Smad family of proteins or through different non-Smad pathways, such as the activation of the nonreceptor protein tyrosine kinase c-Abl (21, 22).

New insights into EndoMT: c-Abl, protein kinase Cδ, and small-molecule kinase inhibitors

  1. Top of page
  2. Fibrosis is a hallmark of systemic sclerosis
  3. The origins of myofibroblasts: endothelial–mesenchymal transition
  4. What induces EndoMT?
  5. New insights into EndoMT: c-Abl, protein kinase Cδ, and small-molecule kinase inhibitors
  6. Is EndoMT important in SSc pathophysiology?
  7. REFERENCES

In a report in this issue of Arthritis & Rheumatism, Li and Jimenez provide evidence that the tyrosine kinase c-Abl and protein kinase Cδ (PKCδ) are crucial for TGFβ induction of EndoMT in vitro (23). Indeed, they show that imatinib mesylate and rottlerin or similar small-molecule kinase inhibitors may be effective therapeutic agents for the fibroproliferative vasculopathy of SSc and other fibrotic disorders in which EndoMT plays a pathogenetic role. Using fully differentiated primary mouse pulmonary ECs, the authors confirmed the occurrence of EndoMT following TGFβ exposure, as demonstrated by the initiation of expression and assembly of α-SMA in typical intracellular stress fibers, expression of type I collagen, and loss of endothelial VE-cadherin. Indeed, findings in other studies indicate that loss of endothelial cell–cell contacts (including loss of VE-cadherin expression) is a necessary and early step in the progression of EndoMT that precedes changes in EC morphology and the subsequent expression of α-SMA with the acquisition of a migratory phenotype (18).

Moreover, Li and Jimenez demonstrate that TGFβ-mediated EndoMT is associated with strong up-regulation of the expression of Snail-1, and that this transcriptional repressor is directly involved in TGFβ-induced α-SMA expression. In fact, transfection of Snail-1 siRNA caused a significant inhibition of TGFβ-induced α-SMA expression in pulmonary ECs (23).

Finally, they provide evidence that TGFβ induces the process of EndoMT via activation of the c-Abl and PKCδ kinases, since highly specific inhibition of their kinase activity with imatinib mesylate and rottlerin, respectively, or by knockdown of their transcripts with siRNA abrogated TGFβ-induced α-SMA and Snail-1 expression at both the messenger RNA and protein levels (23). In particular, imatinib mesylate and rottlerin appear to abrogate TGFβ-induced EndoMT through the inhibition of c-Abl– and PKCδ-mediated glycogen synthase kinase 3β (GSK3β) phosphorylation at residue Ser9, respectively, with subsequent phosphorylation and proteasomal degradation of Snail-1. In fact, GSK3β phosphorylation at residue Ser9 is known to block GSK3β activity, resulting in stabilization, transport, and accumulation of Snail-1 in the nucleus, where it may exert its transcriptional regulatory activity with consequent induction of mesenchymal cell markers (e.g., α-SMA and type I collagen) and repression of EC-specific markers (e.g., VE-cadherin) (19, 20, 23). The net result is the transition to a myofibroblastic phenotype, as summarized in Figure 7 of the report by Li and Jimenez (23).

The study by Li and Jimenez, elegantly elucidating the molecular basis of EndoMT induced by TGFβ, may suggest a new target pathway that could have potential therapeutic implications for SSc and fibroproliferative vasculopathies. In SSc, targeting of the TGFβ-dependent EndoMT process via small-molecule kinase inhibitors, such as imatinib mesylate and rottlerin, might represent a promising treatment for both tissue fibrosis and vasculopathy, the two major aspects of the disease. Indeed, EndoMT might substantially contribute not only to pathologic vascular remodeling in SSc, but also to dermal and internal organ fibrosis, as has recently been observed in experimental models of kidney and lung fibrosis (13, 15). Previous studies have shown that blocking of c-Abl with imatinib mesylate could prevent, or induce regression of, dermal fibrosis in different mouse models of SSc and could attenuate bleomycin-induced pulmonary fibrosis in mice (24–26). However, whether these effects would depend on the abrogation or reversal of the EndoMT process is unknown. In addition, PKCδ has been reported to contribute to the regulation of collagen gene expression in SSc fibroblasts (27).

A possible limitation of the study by Li and Jimenez is that their in vitro system might not reflect the in vivo reality. As they note, further studies using suitable preclinical animal models for the proliferative vasculopathy of SSc are now needed to fully support the data and the potential therapeutic role of c-Abl and PKCδ inhibitors through inhibition of EndoMT. In the future, randomized controlled clinical trials will disclose whether EndoMT blockade with small-molecule kinase inhibitors can really represent a new, efficient targeted treatment for SSc.

Another point for consideration is that Li and Jimenez report a preventive effect of c-Abl and PKCδ inhibitors on TGFβ-induced EndoMT. Therefore, future studies should investigate whether imatinib mesylate and rottlerin are also able to revert fully established EndoMT in vitro and in vivo. To date, several observations suggest that the process of epithelial–mesenchymal transition, which shares similar molecular mechanisms with EndoMT, may be reversible (11). However, much less is known regarding the reversibility of EndoMT (11).

In their experiments, Li and Jimenez used primary mouse pulmonary ECs. Another interesting question to be addressed is whether ECs from different sources, such as the dermis, may show a peculiar propensity to undergo EndoMT when challenged with TGFβ, and whether the same or similar intracellular signaling pathways are involved. This may be relevant to SSc, in which proliferative vasculopathy affects multiple vascular beds and may manifest as digital ulceration and gangrene, pulmonary arterial hypertension, and scleroderma renal crisis (4).

In addition to TGFβ, it will be of major importance to investigate whether other pathways known to be activated in SSc may participate in the EndoMT process. In this regard, it has been shown that the Wnt intercellular signaling pathway and Notch signaling may be involved in EndoMT (11). Furthermore, EC-derived endothelin 1 has been shown to promote experimental cardiac fibrosis through stimulation of EndoMT (28). EndoMT may also be induced by chronic stimulation of proinflammatory cytokines, hemodynamic stress, mechanical injury, and tissue hypoxia (11), all of which are mechanisms involved in the pathogenesis of SSc. In fact, it has been demonstrated that chronic nitric oxide synthase inhibition may activate EndoMT, both in vitro and in vivo (11). A recent study provided evidence that abnormal fibrillin 1 expression and chronic oxidative stress mediate EndoMT in the tight skin mouse model (29).

Is EndoMT important in SSc pathophysiology?

  1. Top of page
  2. Fibrosis is a hallmark of systemic sclerosis
  3. The origins of myofibroblasts: endothelial–mesenchymal transition
  4. What induces EndoMT?
  5. New insights into EndoMT: c-Abl, protein kinase Cδ, and small-molecule kinase inhibitors
  6. Is EndoMT important in SSc pathophysiology?
  7. REFERENCES

Finally, we propose that, depending on the type of vasculature involved, the process of EndoMT may have multiple and different roles in the pathophysiology of SSc. Indeed, EndoMT may affect different vascular beds. In arterioles, it may preferentially lead to subendothelial accumulation of myofibroblasts and fibrotic tissue with subsequent neointimal thickening, leading to a fibroproliferative vasculopathy. At the capillary level, it may contribute to perivascular and interstitial fibrosis through the generation of myofibroblasts that can migrate into the surrounding tissue, as demonstrated in various experimental models (12). Similar to what has been hypothesized for cardiac fibrosis (12), we speculate that the importance of EndoMT for SSc may extend beyond the mere increase in the number of profibrotic myofibroblasts. EndoMT may favor loss of microvascular ECs and thus contribute to capillary rarefaction, leading to chronic tissue ischemia (12). This in turn could further up-regulate the expression of TGFβ and other profibrotic cytokines, promote the influx of inflammatory cells such as macrophages and T cells, and thereby exacerbate the fibrotic process.

According to this view, EndoMT may take center stage as a critical profibrotic switch and become a potential target for antifibrotic therapies. Considering the evidence that EndoMT may participate in different pathogenetic mechanisms of SSc, there is realistic hope that targeting this process will bring about a meaningful advance in the treatment of this disorder.

REFERENCES

  1. Top of page
  2. Fibrosis is a hallmark of systemic sclerosis
  3. The origins of myofibroblasts: endothelial–mesenchymal transition
  4. What induces EndoMT?
  5. New insights into EndoMT: c-Abl, protein kinase Cδ, and small-molecule kinase inhibitors
  6. Is EndoMT important in SSc pathophysiology?
  7. REFERENCES