Chi F. Hung

Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis.

RATIONALE The origin of cells that make pathologic fibrillar collagen matrix in lung disease has been controversial. Recent studies suggest mesenchymal cells may contribute directly to fibrosis. OBJECTIVES To characterize discrete populations of mesenchymal cells in the normal mouse lung and to map their fate after bleomycin-induced lung injury. METHODS We mapped the fate of Foxd1-expressing embryonic progenitors and their progeny during lung development, adult homeostasis, and after fibrosing injury in Foxd1-Cre; Rs26-tdTomato-R mice. We studied collagen-I(α)1-producing cells in normal and diseased lungs using Coll-GFP(Tg) mice. MEASUREMENTS AND MAIN RESULTS Foxd1-expressing embryonic progenitors enter lung buds before 13.5 days post-conception, expand, and form an extensive lineage of mesenchymal cells that have characteristics of pericytes. A collagen-I(α)1-expressing mesenchymal population of distinct lineage is also found in adult lung, with features of a resident fibroblast. In contrast to resident fibroblasts, Foxd1 progenitor-derived pericytes are enriched in transcripts for innate immunity, vascular development, WNT signaling pathway, and cell migration. Foxd1 progenitor-derived pericytes expand after bleomycin lung injury, and activate expression of collagen-I(α)1 and the myofibroblast marker αSMA in fibrotic foci. In addition, our studies suggest a distinct lineage of collagen-I(α)1-expressing resident fibroblasts that also expands after lung injury is a second major source of myofibroblasts. CONCLUSIONS We conclude that the lung contains an extensive population of Foxd1 progenitor-derived pericytes that are an important lung myofibroblast precursor population.

Role of IGF-1 pathway in lung fibroblast activation

BackgroundIGF-1 is elevated in pulmonary fibrosis and acute lung injury, where fibroblast activation is a prominent feature. We previously demonstrated that blockade of IGF pathway in murine model of lung fibrosis improved outcome and decreased fibrosis. We now expand that study to examine effects of IGF pathway on lung fibroblast behaviors that could contribute to fibrosis.MethodsWe first examined mice that express αSMA promoter upstream of GFP reporter treated with A12, a blocking antibody to IGF-1 receptor, after bleomycin induced lung injury. We then examined the effect of IGF-1 alone, or in combination with the pro-fibrotic cytokine TGFβ on expression of markers of myofibroblast activation in vitro, including αSMA, collagen α1, type 1, collagen α1, type III, and TGFβ expression.ResultsAfter bleomycin injury, we found decreased number of αSMA-GFP + cells in A12 treated mice, validated by αSMA immunofluorescent staining. We found that IGF-1, alone or in combination with TGF-β, did not affect αSMA RNA expression, promoter activity, or protein levels when fibroblasts were cultured on stiff substrate. IGF-1 stimulated Col1a1 and Col3a1 expression on stiff substrate. In contrast, IGF-1 treatment on soft substrate resulted in upregulation of αSMA gene and protein expression, as well as Col1a1 and Col3a1 transcripts. In conclusion, IGF-1 stimulates differentiation of fibroblasts into a myofibroblast phenotype in a soft matrix environment and has a modest effect on αSMA stress fiber organization in mouse lung fibroblasts.

Impact of long‐term treatment with neurotoxic dideoxynucleoside antiretrovirals: implications for clinical care in resource‐limited settings

A minority of HIV‐infected patients taking an antiretroviral (ARV) regimen containing dideoxynucleosides (d‐drugs) such as stavudine (d4T) and didanosine (DDI) experiences dose‐limiting neuropathic pain and paraesthesias, usually within weeks of starting these drugs. Because d‐drugs are among the few affordable options available in developing countries, continuing d‐drug therapy would be a desirable strategy for many HIV‐infected individuals. Therefore, we evaluated the safety of continuing d‐drug therapy.

Lung pericyte-like cells are functional interstitial immune sentinel cells

Pericytes are perivascular PDGF receptor-β+ (PDGFRβ+) stromal cells required for vasculogenesis and maintenance of microvascular homeostasis in many organs. Because of their unique juxtaposition to microvascular endothelium, lung PDGFRβ+ cells are well situated to detect proinflammatory molecules released following epithelial injury and promote acute inflammatory responses. Thus we hypothesized that these cells represent an unrecognized immune surveillance or injury-sentinel interstitial cell. To evaluate this hypothesis, we isolated PDGFRβ+ cells from murine lung and demonstrated that they have characteristics consistent with a pericyte population (referred to as pericyte-like cells for simplicity hereafter). We showed that pericyte-like cells expressed functional Toll-like receptors and upregulated chemokine expression following exposure to bronchoalveolar lavage fluid (BALF) collected from mice with sterile lung injury. Interestingly, BALF from mice without lung injury also induced chemokine expression in pericyte-like cells, suggesting that pericyte-like cells are primed to sense epithelial injury (permeability changes). Following LPS-induced lung inflammation, increased numbers of pericyte-like cells expressed IL-6, chemokine (C-X-C motif) ligand-1, chemokine (C-C motif) ligand 2/ monocyte chemotactic protein-1, and ICAM-1 in vivo. Sterile lung injury in pericyte-ablated mice was associated with decreased inflammation compared with normal mice. In summary, we found that pericyte-like cells are immune responsive and express diverse chemokines in response to lung injury in vitro and in vivo. Furthermore, pericyte-like cell ablation attenuated inflammation in sterile lung injury, suggesting that these cells play an important functional role in mediating lung inflammatory responses. We propose a model in which pericyte-like cells function as interstitial immune sentinels, detecting proinflammatory molecules released following epithelial barrier damage and participating in recruitment of circulating leukocytes.

uPARAP Function in Cutaneous Wound Repair

Optimal skin wound healing relies on tight balance between collagen synthesis and degradation in new tissue formation and remodeling phases. The endocytic receptor uPARAP regulates collagen uptake and intracellular degradation. In this study we examined cutaneous wound repair response of uPARAP null (uPARAP-/-) mice. Full thickness wounds were created on dorsal surface of uPARAP-/- or their wildtype littermates. Wound healing evaluation was done by macroscopic observation, histology, gene transcription and biochemical analysis at specific intervals. We found that absence of uPARAP delayed re-epithelialization during wound closure, and altered stiffness of the scar tissue. Despite the absence of the uPARAP-mediated intracellular pathway for collagen degradation, there was no difference in total collagen content of the wounds in uPARAP-/- compared to wildtype mice. This suggests in the absence of uPARAP, a compensatory feedback mechanism functions to keep net collagen in balance.

Ablation of Pericyte-Like Cells in Lungs by Oropharyngeal Aspiration of Diphtheria Toxin

&NA; We demonstrated previously that FoxD1‐derived cells in the lung are enriched in pericyte‐like cells in mouse lung. These cells express the common pericyte markers and are located adjacent to endothelial cells. In this study, we demonstrate the feasibility of administering diphtheria toxin (DT) by oropharyngeal aspiration as an approach to ablating FoxD1‐derived cells. We crossed mice expressing Cre‐recombinase under the FoxD1 promoter to Rosa26‐loxP‐STOP‐loxP‐iDTR mice and generated a bitransgenic line (FoxD1‐Cre;Rs26‐iDTR) in which FoxD1‐derived cells heritably express simian or human diphtheria toxin receptor and are sensitive to DT. We delivered low‐dose (0.5 ng/g) and high‐dose (1ng/g × 2) to FoxD1‐Cre;Rs26‐iDTR mice and littermate control mice by oropharyngeal aspiration and evaluated ablation by flow cytometry and immunohistochemistry. FoxD1‐Cre mice showed a 40‐50% reduction in PDGFR&bgr;+ cells by flow cytometry at Days 2 and 7 after DT administration, with a return of PDGFR&bgr;+ cells at Day 28. Confocal microscopy revealed an observable reduction in pericyte markers. Bronchoalveolar lavage fluid analysis revealed no significant differences in total protein, bronchoalveolar lavage fluid red blood cell, or white blood cell counts at low dose. However, at high‐dose DT, there was a proinflammatory effect in the control mice and increased mortality associated with systemic toxicity in Cre+ mice. Low‐dose DT reduced lung PDGFR&bgr;+ stromal cells in the FoxD1‐Cre;iDTR transgenic model without a differential effect on lung inflammation in DT‐sensitive and DT‐insensitive animals. Low‐dose DT is a viable method for transient lineage‐specific stromal cell ablation in the lung that minimizes systemic toxicity.

Role of integrin alpha8 in murine model of lung fibrosis

Background Integrin α8 (ITGA8) heterodimerizes with integrin β1 and is highly expressed in stromal cells of the lung. Platelet-derived growth factor receptor beta (PDGFRβ+) cells constitute a major population of contractile myofibroblasts in the lung following bleomycin-induced fibrosis. Integrin α8β1 is upregulated in fibrotic foci in bleomycin-induced lung injury. However, the functional role of ITGA8 in fibrogenesis has not been characterized. In this study, we examined whether genetic deletion of ITGA8 from PDGFRβ+ cells in the lung altered fibrosis. Methods Pdgfrb-Cre/+;Itga8flox/- or Pdgfrb-Cre/+;Itga8flox/flox (Cre+) and control mice (Cre-) were used for in vitro and in vivo studies. Primary cultures of PDGFRβ+ cells were exposed to TGFβ, followed by RNA isolation for qPCR. For in vivo studies, Cre+ and Cre- mice were characterized at baseline and after bleomycin-induced fibrosis. Results PDGFRβ-selected cells from Cre+ animals showed higher levels of Col1a1 expression after treatment with TGFβ. However, Cre- and Cre+ animals showed no significant difference in measures of acute lung injury or fibrosis following bleomycin challenge. Conclusion While ITGA8 deletion in lung PDGFRβ+ stromal cells showed evidence of greater Col1a1 mRNA expression after TGFβ treatment in vitro, no functional difference was detected in vivo.

Characterization of human PDGFRβ-positive pericytes from IPF and non-IPF lungs

Pericytes are key regulators of the microvasculature through their close interactions with the endothelium. However, pericytes play additional roles in tissue homeostasis and repair, in part by transitioning into myofibroblasts. Accumulation of myofibroblasts is a hallmark of fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). To understand the contribution and role of pericytes in human lung fibrosis, we isolated these cells from non-IPF control and IPF lung tissues based on expression of platelet-derived growth factor receptor-β (PDGFR-β), a common marker of pericytes. When cultured in a specialized growth medium, PDGFR-β+ cells retain the morphology and marker profile typical of pericytes. We found that IPF pericytes migrated more rapidly and invaded a basement membrane matrix more readily than control pericytes. Exposure of cells to transforming growth factor-β, a major fibrosis-inducing cytokine, increased expression of α-smooth muscle actin and extracellular matrix genes in both control and IPF pericytes. Given that pericytes are uniquely positioned in vivo to respond to danger signals of both systemic and tissue origin, we stimulated human lung pericytes with agonists having pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Both control and IPF lung pericytes increased expression of proinflammatory chemokines in response to specific PAMPs and DAMPs released from necrotic cells. Our results suggest that control and IPF lung pericytes are poised to react to tissue damage, as well as microbial and fibrotic stimuli. However, IPF pericytes are primed for migration and matrix invasion, features that may contribute to the function of these cells in lung fibrosis.

Transgenic Animal Models in Lung Research

The use of transgenic animals in pulmonary research has greatly evolved in the past decade as refinements in genetic techniques have enabled enhanced ability to manipulate gene expression in transgenic animals. An important milestone in the development of transgenic animals is the successful introduction of bacterial and eukaryotic recombinase and recombination sites into the mouse genome. This advancement has improved cell-type specificity in which genetic modification occurs and expanded the repertoire of techniques that direct temporal control of gene modification in transgenic animals. These advances have tremendous implications for lung research where the origin of cells implicated in lung pathogenesis and contributions to disease by specific cell types can now be explored.

Mouse Models of Acute Lung Injury

Acute respiratory distress syndrome or ARDS remains a devastating complication of critical illness, resulting in significant annual morbidity, mortality, and healthcare expenditures. Although much is known about the physiology of ARDS, many aspects of its pathogenesis remain incompletely understood, and no effective pharmacologic therapies have been identified to date. Because of this, research focused on ARDS and its preclinical animal model correlate, acute lung injury, remains a priority for scientists focused on lung diseases, critical illness, and trauma. Mouse model systems allow the use of genetic models and a wide range of reagents to pursue highly mechanistic studies into the cellular and molecular mechanisms of acute lung injury. However, the challenges of using mice to study acute lung injury include identifying appropriate, clinically relevant models and integrating cellular and molecular data with physiological measurements of lung injury. This chapter provides a brief review of the advantages and challenges of mouse models and reviews different models of acute lung injury. It also includes practical information on specific methods to help the new investigator develop mouse models of acute lung injury in his or her laboratory.