Macrophage programming is regulated by a cooperative interaction between fatty acid binding protein 5 and peroxisome proliferator-activated receptor γ.

Resolution of inflammation is an active process that is tightly regulated to achieve repair and tissue homeostasis. In the absence of resolution, persistent inflammation underlies the pathogenesis of chronic lung disease such as chronic obstructive pulmonary disease (COPD) with recurrent exacerbations. Over the course of inflammation, macrophage programming transitions from pro-inflammatory to pro-resolving, which is in part regulated by the nuclear receptor Peroxisome Proliferator-Activated Receptor γ (PPARγ). Our previous work demonstrated an association between Fatty Acid Binding Protein 5 (FABP5) expression and PPARγ activity in peripheral blood mononuclear cells of healthy and COPD patients. However, a role for FABP5 in macrophage programming has not been examined. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FABP5 is necessary for PPARγ activation. In turn, PPARγ acts directly to increase FABP5 expression in primary human alveolar macrophages. We further illustrate that lack of FABP5 expression promotes a pro-inflammatory macrophage programming with increased secretion of pro-inflammatory cytokines and increased chromatin accessibility for pro-inflammatory transcription factors (e.g., NF-κB and MAPK). And finally, real-time cell metabolic analysis using the Seahorse technology shows an inhibition of oxidative phosphorylation in FABP5-deficient macrophages. Taken together, our data indicate that FABP5 and PPARγ reciprocally regulate each other's expression and function, consistent with a novel positive feedback loop between the two factors that mediates macrophage pro-resolving programming. Our studies highlight the importance of defining targets and regulatory mechanisms that control the resolution of inflammation and may serve to inform novel interventional strategies directed towards COPD.

Cigarette smoke-induced airspace disease in mice develops independently of HIF-1α signaling in leukocytes.

The pathogenesis of chronic obstructive pulmonary disease (COPD), a prevalent disease primarily caused by cigarette smoke exposure, is incompletely elucidated. Studies in humans and mice have suggested that hypoxia-inducible factor-1α (HIF-1α) may play a role. Reduced lung levels of HIF-1α are associated with decreased vascular density, whereas increased leukocyte HIF-1α may be responsible for increased inflammation. To elucidate the specific role of leukocyte HIF-1α in COPD, we exposed transgenic mice with conditional deletion or overexpression of HIF-1α in leukocytes to cigarette smoke for 7 mo. Outcomes included pulmonary physiology, aerated lung volumes via microcomputed tomography, lung morphometry and histology, and cardiopulmonary hemodynamics. On aggregate, cigarette smoke increased the aerated lung volume, quasi-static lung compliance, inspiratory capacity of all strains while reducing the total alveolar septal volume. Independent of smoke exposure, mice with leukocyte-specific HIF-1α overexpression had increased quasi-static compliance, inspiratory capacity, and alveolar septal volume compared with mice with leukocyte-specific HIF-1α deletion. However, the overall development of cigarette smoke-induced lung disease did not vary relative to control mice for either of the conditional strains. This suggests that the development of murine cigarette smoke-induced airspace disease occurs independently of leukocyte HIF-1α signaling.

Balanced Wnt/Dickkopf-1 signaling by mesenchymal vascular progenitor cells in the microvascular niche maintains distal lung structure and function.

The well-described Wnt inhibitor Dickkopf-1 (DKK1) plays a role in angiogenesis as well as in regulation of growth factor signaling cascades in pulmonary remodeling associated with chronic lung diseases (CLDs) including emphysema and fibrosis. However, the specific mechanisms by which DKK1 influences mesenchymal vascular progenitor cells (MVPCs), microvascular endothelial cells (MVECs), and smooth muscle cells (SMCs) within the microvascular niche have not been elucidated. In this study, we show that knockdown of DKK1 in Abcg2pos lung mouse adult tissue resident MVPCs alters lung stiffness, parenchymal collagen deposition, microvessel muscularization and density as well as loss of tissue structure in response to hypoxia exposure. To complement the in vivo mouse modeling, we also identified cell- or disease-specific responses to DKK1, in primary lung chronic obstructive pulmonary disease (COPD) MVPCs, COPD MVECs, and SMCs, supporting a paradoxical disease-specific response of cells to well-characterized factors. Cell responses to DKK1 were dose dependent and correlated with varying expressions of the DKK1 receptor, CKAP4. These data demonstrate that DKK1 expression is necessary to maintain the microvascular niche whereas its effects are context specific. They also highlight DKK1 as a regulatory candidate to understand the role of Wnt and DKK1 signaling between cells of the microvascular niche during tissue homeostasis and during the development of chronic lung diseases.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Targeting c-Src Reverses Accelerated GPX-1 mRNA Decay in Chronic Obstructive Pulmonary Disease Airway Epithelial Cells.

Enhanced expression of the cellular antioxidant glutathione peroxidase (GPX)-1 prevents cigarette smoke-induced lung inflammation and tissue destruction. Subjects with chronic obstructive pulmonary disease (COPD), however, have decreased airway GPX-1 levels, rendering them more susceptible to disease onset and progression. The mechanisms that downregulate GPX-1 in the airway epithelium in COPD remain unknown. To ascertain these factors, analyses were conducted using human airway epithelial cells isolated from healthy subjects and human subjects with COPD and lung tissue from control and cigarette smoke-exposed A/J mice. Tyrosine phosphorylation modifies GPX-1 expression and cigarette smoke activates the tyrosine kinase c-Src. Therefore, studies were conducted to evaluate the role of c-Src on GPX-1 levels in COPD. These studies identified accelerated GPX-1 mRNA decay in COPD airway epithelial cells. Targeting the tyrosine kinase c-Src with siRNA inhibited GPX-1 mRNA degradation and restored GPX-1 protein levels in human airway epithelial cells. In contrast, silencing the tyrosine kinase c-Abl, or the transcriptional activator Nrf2, had no effect on GPX-1 mRNA stability. The chemical inhibitors for c-Src (saracatinib and dasanitib) restored GPX-1 mRNA levels and GPX-1 activity in COPD airway cells in vitro. Similarly, saracatinib prevented the loss of lung Gpx-1 expression in response to chronic smoke exposure in vivo. Thus, this study establishes that the decreased GPX-1 expression that occurs in COPD lungs is at least partially due to accelerated mRNA decay. Furthermore, these findings show that targeting c-Src represents a potential therapeutic approach to augment GPX-1 responses and counter smoke-induced lung disease.

Cigarette smoke induction of S100A9 contributes to chronic obstructive pulmonary disease.

S100 calcium-binding protein A9 (S100A9) is elevated in plasma and bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), and aging enhances S100A9 expression in several tissues. Currently, the direct impact of S100A9-mediated signaling on lung function and within the aging lung is unknown. Here, we observed that elevated S100A9 levels in human BALF correlated with age. Elevated lung levels of S100A9 were higher in older mice compared with in young animals and coincided with pulmonary function changes. Both acute and chronic exposure to cigarette smoke enhanced S100A9 levels in age-matched mice. To examine the direct role of S100A9 on the development of COPD, S100a9-/- mice or mice administered paquinimod were exposed to chronic cigarette smoke. S100A9 depletion and inhibition attenuated the loss of lung function, pressure-volume loops, airway inflammation, lung compliance, and forced expiratory volume in 0.05 s/forced vital capacity, compared with age-matched wild-type or vehicle-administered animals. Loss of S100a9 signaling reduced cigarette smoke-induced airspace enlargement, alveolar remodeling, lung destruction, ERK and c-RAF phosphorylation, matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-9 (MMP-9), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and keratinocyte-derived chemokine (KC) release into the airways. Paquinimod administered to nonsmoked, aged animals reduced age-associated loss of lung function. Since fibroblasts play a major role in the production and maintenance of extracellular matrix in emphysema, primary lung fibroblasts were treated with the ERK inhibitor LY3214996 or the c-RAF inhibitor GW5074, resulting in less S100A9-induced MMP-3, MMP-9, MCP-1, IL-6, and IL-8. Silencing Toll-like receptor 4 (TLR4), receptor for advanced glycation endproducts (RAGE), or extracellular matrix metalloproteinase inducer (EMMPRIN) prevented S100A9-induced phosphorylation of ERK and c-RAF. Our data suggest that S100A9 signaling contributes to the progression of smoke-induced and age-related COPD.

Bone Marrow-Derived Proangiogenic Cells Mediate Pulmonary Arteriole Stiffening via Serotonin 2B Receptor Dependent Mechanism.

RATIONALE: Pulmonary arterial hypertension is a deadly disease of the pulmonary vasculature for which no disease-modifying therapies exist. Small-vessel stiffening and remodeling are fundamental pathological features of pulmonary arterial hypertension that occur early and drive further endovascular cell dysfunction. Bone marrow (BM)-derived proangiogenic cells (PACs), a specialized heterogeneous subpopulation of myeloid lineage cells, are thought to play an important role in pathogenesis. OBJECTIVE: To determine whether BM-derived PACs directly contributed to experimental pulmonary hypertension (PH) by promoting small-vessel stiffening through 5-HT2B (serotonin 2B receptor)-mediated signaling. METHODS AND RESULTS: We performed BM transplants using transgenic donor animals expressing diphtheria toxin secondary to activation of an endothelial-specific tamoxifen-inducible Cre and induced experimental PH using hypoxia with SU5416 to enhance endovascular injury and ablated BM-derived PACs, after which we measured right ventricular systolic pressures in a closed-chest procedure. BM-derived PAC lineage tracing was accomplished by transplanting BM from transgenic donor animals with fluorescently labeled hematopoietic cells and treating mice with a 5-HT2B antagonist. BM-derived PAC ablation both prevented and reversed experimental PH with SU5416-enhanced endovascular injury, reducing the number of muscularized pulmonary arterioles and normalizing arteriole stiffness as measured by atomic force microscopy. Similarly, treatment with a pharmacological antagonist of 5-HT2B also prevented experimental PH, reducing the number and stiffness of muscularized pulmonary arterioles. PACs accelerated pulmonary microvascular endothelial cell injury response in vitro, and the presence of BM-derived PACs significantly correlated with stiffer pulmonary arterioles in pulmonary arterial hypertension patients and mice with experimental PH. RNA sequencing of BM-derived PACs showed that 5-HT2B antagonism significantly altered biologic pathways regulating cell proliferation, locomotion and migration, and cytokine production and response to cytokine stimulus. CONCLUSIONS: Together, our findings illustrate that BM-derived PACs directly contribute to experimental PH with SU5416-enhanced endovascular injury by mediating small-vessel stiffening and remodeling in a 5-HT2B signaling-dependent manner.

Inactivation of Tsc2 in Abcg2 lineage-derived cells drives the appearance of polycystic lesions and fibrosis in the adult kidney.

Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36-37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.

De novo generation of adipocytes from circulating progenitor cells in mouse and human adipose tissue.

White adipocytes in adults are typically derived from tissue resident mesenchymal progenitors. The recent identification of de novo production of adipocytes from bone marrow progenitor-derived cells in mice challenges this paradigm and indicates an alternative lineage specification that adipocytes exist. We hypothesized that alternative lineage specification of white adipocytes is also present in human adipose tissue. Bone marrow from transgenic mice in which luciferase expression is governed by the adipocyte-restricted adiponectin gene promoter was adoptively transferred to wild-type recipient mice. Light emission was quantitated in recipients by in vivo imaging and direct enzyme assay. Adipocytes were also obtained from human recipients of hematopoietic stem cell transplantation. DNA was isolated, and microsatellite polymorphisms were exploited to quantify donor/recipient chimerism. Luciferase emission was detected from major fat depots of transplanted mice. No light emission was observed from intestines, liver, or lungs. Up to 35% of adipocytes in humans were generated from donor marrow cells in the absence of cell fusion. Nontransplanted mice and stromal-vascular fraction samples were used as negative and positive controls for the mouse and human experiments, respectively. This study provides evidence for a nontissue resident origin of an adipocyte subpopulation in both mice and humans.

Critical Genomic Networks and Vasoreactive Variants in Idiopathic Pulmonary Arterial Hypertension.

RATIONALE: Idiopathic pulmonary arterial hypertension (IPAH) is usually without an identified genetic cause, despite clinical and molecular similarity to bone morphogenetic protein receptor type 2 mutation-associated heritable pulmonary arterial hypertension (PAH). There is phenotypic heterogeneity in IPAH, with a minority of patients showing long-term improvement with calcium channel-blocker therapy. OBJECTIVES: We sought to identify gene variants (GVs) underlying IPAH and determine whether GVs differ in vasodilator-responsive IPAH (VR-PAH) versus vasodilator-nonresponsive IPAH (VN-PAH). METHODS: We performed whole-exome sequencing (WES) on 36 patients with IPAH: 17 with VR-PAH and 19 with VN-PAH. Wnt pathway differences were explored in human lung fibroblasts. MEASUREMENTS AND MAIN RESULTS: We identified 1,369 genes with 1,580 variants unique to IPAH. We used a gene ontology approach to analyze variants and identified overrepresentation of several pathways, including cytoskeletal function and ion binding. By mapping WES data to prior genome-wide association study data, Wnt pathway genes were highlighted. Using the connectivity map to define genetic differences between VR-PAH and VN-PAH, we found enrichment in vascular smooth muscle cell contraction pathways and greater genetic variation in VR-PAH versus VN-PAH. Using human lung fibroblasts, we found increased stimulated Wnt activity in IPAH versus controls. CONCLUSIONS: A pathway-based analysis of WES data in IPAH demonstrated multiple rare GVs that converge on key biological pathways, such as cytoskeletal function and Wnt signaling pathway. Vascular smooth muscle contraction-related genes were enriched in VR-PAH, suggesting a potentially different genetic predisposition for VR-PAH. This pathway-based approach may be applied to next-generation sequencing data in other diseases to uncover the contribution of unexpected or multiple GVs to a phenotype.