Craig A. Henke

Role of mesenchymal cell death in lung remodeling after injury.

Repair after acute lung injury requires elimination of granulation tissue from the alveolar airspace. We hypothesized that during lung repair, signals capable of inducing the death of the two principal cellular elements of granulation tissue, fibroblasts and endothelial cells, would be present at the air-lung interface. Bronchoalveolar lavage fluid obtained from patients during lung repair induced both fibroblast and endothelial cell death, while fluid obtained at the time of injury or from patient controls did not. The mode of cell death for endothelial cells was apoptosis. Fibroblast death, while morphologically distinct from necrosis, also differed from typical apoptosis. Only proliferating cells were susceptible to the bioactivities in lavage fluid, which were trypsin sensitive and lipid insoluble. Histological examination of lung tissue from patients after lung injury revealed evidence of apoptotic cells within airspace granulation tissue. Our results suggest that cell death induced by peptide(s) present at the air-lung interface may participate in the remodeling process that accompanies tissue repair after injury.

Fibrotic extracellular matrix activates a profibrotic positive feedback loop

Pathological remodeling of the extracellular matrix (ECM) by fibroblasts leads to organ failure. Development of idiopathic pulmonary fibrosis (IPF) is characterized by a progressive fibrotic scarring in the lung that ultimately leads to asphyxiation; however, the cascade of events that promote IPF are not well defined. Here, we examined how the interplay between the ECM and fibroblasts affects both the transcriptome and translatome by culturing primary fibroblasts generated from IPF patient lung tissue or nonfibrotic lung tissue on decellularized lung ECM from either IPF or control patients. Surprisingly, the origin of the ECM had a greater impact on gene expression than did cell origin, and differences in translational control were more prominent than alterations in transcriptional regulation. Strikingly, genes that were translationally activated by IPF-derived ECM were enriched for those encoding ECM proteins detected in IPF tissue. We determined that genes encoding IPF-associated ECM proteins are targets for miR-29, which was downregulated in fibroblasts grown on IPF-derived ECM, and baseline expression of ECM targets could be restored by overexpression of miR-29. Our data support a model in which fibroblasts are activated to pathologically remodel the ECM in IPF via a positive feedback loop between fibroblasts and aberrant ECM. Interrupting this loop may be a strategy for IPF treatment.

Future Directions in Idiopathic Pulmonary Fibrosis Research. An NHLBI Workshop Report

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

Pathologic Caveolin-1 Regulation of PTEN in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disorder refractory to current pharmacological therapies. Fibroblasts isolated from IPF patients display pathological activation of PI3K/Akt caused by low PTEN phosphatase activity. This enables these cells to escape the negative proliferative properties of polymerized collagen. The mechanism underlying low PTEN activity in IPF fibroblasts is unclear, but our prior studies indicate that membrane-associated PTEN expression is decreased in these cells. Caveolin-1 is an integral membrane protein whose expression is decreased in IPF lung tissue, but how low caveolin-1 contributes to pathological fibrosis is incompletely understood. The objective of this study was to examine the hypothesis that caveolin-1 regulates PTEN function in IPF fibroblasts. Here we demonstrate that caveolin-1 expression is a determinant of membrane PTEN levels and show that PTEN interacts with caveolin-1 via its caveolin-1-binding sequence. We demonstrate that caveolin-1 expression is low in IPF fibroblasts and that this correlates with low membrane PTEN levels, whereas overexpression of caveolin-1 restores membrane PTEN levels, inhibits Akt phosphorylation, and suppresses proliferation. We demonstrate that caveolin-1 and PTEN expression are low in myofibroblasts within IPF fibroblastic foci. These data indicate that IPF fibroblasts display low caveolin-1 expression, which results in low membrane-associated PTEN expression. This creates a membrane microenvironment depleted of inhibitory phosphatase activity, facilitating the aberrant activation PI3K/Akt and pathological proliferation.

Acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44.

Fibrosis results when myofibroblasts invade the wound fibrin provisional matrix. Extracellular matrix receptors on the cell surface mediate cell adhesion, migration, and invasion. Recent work with transformed cells indicates that these cells use the cell surface matrix receptor CD44 for migration and invasion. In this study, we examine whether lung fibroblasts, isolated from patients dying with acute alveolar fibrosis, use CD44 to invade a fibrin matrix. Consistent with a role for CD44 in mediating fibroblast invasion and subsequent tissue fibrosis, immunohistochemical analysis of lung tissue from patients who died from acute alveolar fibrosis after lung injury reveals CD44-expressing mesenchymal cells throughout newly formed fibrotic tissue. PCR, Western, and immunoprecipitation analysis demonstrate that the 85-kD CD44 isoform is expressed by acute lung injury fibroblasts. Consistent with a role in mediating matrix adhesion and migration ultrastructurally, CD44 was found uniformly over the cell surface and was found densely labeling filopodia and lamellipodia, highly motile structures involved in cell migration. To determine if lung injury fibroblasts use CD44 to invade fibrin, a fibrin gel model of fibrosis was used. By blocking the function of CD44 with monoclonal antibodies, fibroblast invasion into a fibrin matrix was inhibited. To examine the mechanism by which CD44 mediates fibroblast invasion, the role of CD44 in fibroblast migration and adhesion was evaluated. Anti-CD44 antibody blocked fibroblast migration on the provisional matrix proteins fibronectin, fibrinogen, and hyaluronic acid. Additionally, fibroblast CD44 mediated adhesion to the provisional matrix proteins fibronectin, fibrin, and hyaluronic acid, but not to laminin, a component of the basement membrane. These findings support the hypothesis that fibroblast CD44 functions as an adhesion receptor for provisional matrix proteins and is capable of mediating fibroblast migration and invasion of the wound provisional matrix resulting in the formation of fibrotic tissue.

Acute lung injury. Pathogenesis of intraalveolar fibrosis.

In patients dying with acute lung injury, interstitial mesenchymal cells migrate into the airspace where they replicate and deposit connective tissue. We therefore hypothesized that peptides capable of promoting mesenchymal cell migration and replication would be present in the alveolar airspace. To examine this hypothesis, patients with severe acute diffuse lung injury (n = 26) underwent bronchoalveolar lavage. Acutely ill patients without lung injury served as controls (n = 12). Recovered effluent was examined for mesenchymal cell growth-promoting and migration-promoting activity. Lavage cell supernates from both patients and controls were devoid of bioactivity. However, substantial growth-promoting and migration-promoting activity was present in lavage fluid from nearly every patient, whereas little or none was present in fluid from controls. Characterization of the bioactivity indicated a significant proportion consisted of three peptides related to PDGF: (a) a 14-kD peptide that shared with PDGF several biophysical, biochemical, receptor-binding, and antigenic properties; (b) a 29-kD peptide that appeared identical to PDGF of platelet origin; and (c) a 38-kD peptide that was biophysically and antigenically similar to PDGF. These data indicate that peptide moieties are present in the airspace of patients after acute lung injury that can signal mesenchymal cell migration and replication.

Fibrotic myofibroblasts manifest genome-wide derangements of translational control.

Background As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast–the primary effector of the fibroproliferative response. We have recently shown that aberrant beta 1 integrin signaling in fibrotic fibroblasts results in defective PTEN function, unrestrained Akt signaling and subsequent activation of the translation initiation machinery. How this pathological integrin signaling alters the gene expression pathway has not been elucidated. Results Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord with this, we found systems-level indications for TGF-β -driven EMT as one source of IPF myofibroblasts. Conclusions These findings establish the power of systems level genome-wide analysis to provide mechanistic insights into fibrotic disorders such as IPF. Our data point to derangements of translational control downstream of aberrant beta 1 integrin signaling as a fundamental component of IPF pathobiology and indicates that TGF-β -driven EMT is one source for IPF myofibroblasts.

Pathological alteration of FoxO3a activity promotes idiopathic pulmonary fibrosis fibroblast proliferation on type i collagen matrix.

Idiopathic pulmonary fibrosis (IPF) is a prevalent, progressive, and incurable fibroproliferative lung disease. The phenotype of IPF fibroblasts is characterized by their ability to elude the proliferation-suppressive properties of polymerized type I collagen. The mechanism underlying this pathological response is incompletely understood but involves aberrant activation of the phosphatidylinositol 3-kinase-Akt signaling pathway owing to inappropriately low phosphatase and tensin homolog phosphatase activity. Akt can phosphorylate and inactivate the forkhead box O3a (FoxO3a) transcriptional factor, which, when transcriptionally active, increases the expression of the CDK inhibitor p27 and promotes cell cycle arrest. Herein, we demonstrate that IPF fibroblasts display high levels of inactive FoxO3a compared with nonfibrotic control fibroblasts because of their high Akt activity. We found that p27 levels are decreased in IPF compared with control fibroblasts cultured on polymerized collagen. Furthermore, overexpression of FoxO3a in IPF fibroblasts increases p27 levels and suppresses the ability of IPF fibroblasts to proliferate on polymerized collagen. In contrast, the expression of dominant-negative FoxO3a augmented control fibroblast proliferation. IHC examination of fibroblastic foci in IPF lung tissue demonstrates the presence of inactive FoxO3a in cells within fibroblastic foci. These data indicate that the ability of IPF fibroblasts to circumvent the proliferation-suppressive properties of polymerized collagen involves inactivation of FoxO3a by high Akt activity, resulting in down-regulation of p27.

Telomerase and telomere length in pulmonary fibrosis.

In addition to its expression in stem cells and many cancers, telomerase activity is transiently induced in murine bleomycin (BLM)-induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease, we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). The results showed that telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples, some of which were obtained from lung cancer resections. Less than 4% of the human IPF lung fibroblast samples exhibited shortened telomeres, whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in late-generation telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblasts was associated with the binding of acetylated histone H3K9 to the TERT promoter region. These findings indicate that significant telomerase induction was evident in fibroblasts from fibrotic murine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length.

Identification and partial characterization of angiogenesis bioactivity in the lower respiratory tract after acute lung injury.

Survival after acute lung injury (ALI) depends on prompt alveolar repair, a process frequently subverted by the development of granulation tissue within the alveolar airspace. Immunohistochemical examination of the intraalveolar granulation tissue confirmed that capillaries as well as myofibroblasts were the principal cellular constituents. We therefore hypothesized that angiogenesis factors would be present on the air-lung interface after ALI. To evaluate this hypothesis, bronchoalveolar lavage fluid from patients with ALI (n = 25) and patient controls (n = 8) was examined for angiogenesis bioactivity by its ability of induce endothelial cell migration. While lavage fluid from controls had no bioactivity, lavage fluid from 72% of patients with ALI promoted endothelial cell migration. Heparin affinity, ion exchange, and gel filtration chromatography resolved the bioactivity into at least two moieties. One appeared identical to the well characterized endothelial cell growth factor, basic fibroblast growth factor. The other was a 150-kD non-heparin binding protein that mediated endothelial cell migration and attachment in vitro, and the growth of new vessels in vivo. These data are consistent with the hypothesis that the growth of capillaries into the alveolar airspace results from angiogenesis factors present on the alveolar surface of the lung after ALI.