Squamous metaplasia (SM) is common in smokers and is associated with airway obstruction in chronic obstructive pulmonary disease (COPD). A major mechanism of airway obstruction in COPD is thickening of the small airway walls. We asked whether SM actively contributes to airway wall thickening through alteration of epithelial-mesenchymal interactions in COPD. Using immunohistochemical staining, airway morphometry, and fibroblast culture of lung samples from COPD patients; genome-wide analysis of an in vitro model of SM; and in vitro modeling of human airway epithelial-mesenchymal interactions, we provide evidence that SM, through the increased secretion of IL-1β, induces a fibrotic response in adjacent airway fibroblasts. We identify a pivotal role for integrin-mediated TGF-β activation in amplifying SM and driving IL-1β-dependent profibrotic mesenchymal responses. Finally, we show that SM correlates with increased severity of COPD and that fibroblast expression of the integrin α v β 8 , which is the major mediator of airway fibroblast TGF-β activation, correlated with disease severity and small airway wall thickening in COPD. Our findings have identified TGF-β as a potential therapeutic target for COPD.

The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models -conditional deletion in mouse fibroblasts of the TGF-β-activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts -we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -β (TGF-β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-β family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.Large T antigen (TAg), an oncogene encoded by the small DNA tumor virus simian virus 40 (SV40), is a powerful tool for elucidating the mechanisms of growth regulation and control of cell proliferation. Expression of this viral protein is sufficient to transform multiple primary cell types (reviewed in references 19 and 47), and when expressed ectopically in transgenic mice, it causes neoplasia in numerous tissues (reviewed in reference 47).Transformation induced by T antigen is accomplished by targeting cellular components. For example, the amino-terminal region of T antigen (Fig. 1) inactivates the retinoblastoma (pRb) family of tumor suppressors via an LXCXE motif (reviewed in reference 37) that mediates binding to pRb proteins and a J domain (56) that interacts with hsc70 and participates in pRb inactivation. T antigen alleviates the growth-suppressive functions of the Rb family by a J-domain-dependent mechanism (52,(54)(55)(56)69) and in the case of murine polyomavirus by both J-domain-dependent and -independent mechanisms (50). The carboxy-terminal region of T antigen binds, stabilizes, and inactivates the tumor suppressor p53 (12, 31, 42) (Fig. 1). Some reports argue that the elimination of pRb and p53 tumor suppressor functions is the only T antigen activity that contributes to transformation (23). However, other reports suggest that T antigen targets additional cellular proteins and that these interactions contribute to transformation as well (1,16,31,46,62).Studies with transgenic mice have revealed that T antigen is able to induce neoplasia in numerous cell types (reviewed in reference 47). A major complication to the interpretation of the neoplastic phenotype is that the changes induced depend on the cell type...

Brain arteriovenous malformations (BAVMs) are a rare but potentially devastating hemorrhagic disease. Transforming growth factor-␤ signaling is required for proper vessel development, and defective transforming growth factor-␤ superfamily signaling has been implicated in BAVM pathogenesis. We hypothesized that expression of the transforming growth factor-␤ activating integrin, ␣v␤8, is reduced in BAVMs and that decreased ␤8 expression leads to defective neoangiogenesis. We determined that ␤8 protein expression in perivascular astrocytes was reduced in human BAVM lesional tissue compared with controls and that the angiogenic response to focal vascular endothelial growth factor stimulation in adult mouse brains with local Cre-mediated deletion of itgb8 and smad4 led to vascular dysplasia in newly formed blood vessels. In addition, common genetic variants in ITGB8 were associated with BAVM susceptibility, and ITGB8 genotypes associated with increased risk of BAVMs correlated with decreased ␤8 immunostaining in BAVM tissue. These three lines of evidence from human studies and a mouse model suggest that reduced expression of integrin ␤8 may be involved in the pathogenesis of sporadic BAVMs. (Am J Pathol