Neil J. Kelly

Development of a Mouse Model of Metabolic Syndrome, Pulmonary Hypertension, and Heart Failure with Preserved Ejection Fraction

&NA; Pulmonary hypertension (PH) associated with heart failure with preserved ejection fraction (PH‐HFpEF; World Health Organization Group II) secondary to left ventricular (LV) diastolic dysfunction is the most frequent cause of PH. It is an increasingly recognized clinical complication of the metabolic syndrome. To date, no effective treatment has been identified, and no genetically modifiable mouse model is available for advancing our understanding for PH‐HFpEF. To develop a mouse model of PH‐HFpEF, we exposed 36 mouse strains to 20 weeks of high‐fat diet (HFD), followed by systematic evaluation of right ventricular (RV) and LV pressure‐volume analysis. The HFD induces obesity, glucose intolerance, insulin resistance, hyperlipidemia, as well as PH, in susceptible strains. We observed that certain mouse strains, such as AKR/J, NON/shiLtJ, and WSB/EiJ, developed hemodynamic signs of PH‐HFpEF. Of the strains that develop PH‐HFpEF, we selected AKR/J for further model validation, as it is known to be prone to HFD‐induced metabolic syndrome and had low variability in hemodynamics. HFD‐treated AKR/J mice demonstrate reproducibly higher RV systolic pressure compared with mice fed with regular diet, along with increased LV end‐diastolic pressure, both RV and LV hypertrophy, glucose intolerance, and elevated HbA1c levels. Time course assessments showed that HFD significantly increased body weight, RV systolic pressure, LV end‐diastolic pressure, biventricular hypertrophy, and HbA1c throughout the treatment period. Moreover, we also identified and validated 129S1/SvlmJ as a resistant mouse strain to HFD‐induced PH‐HFpEF. These studies validate an HFD/AKR/J mouse model of PH‐HFpEF, which may offer a new avenue for testing potential mechanisms and treatments for this disease.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor

&NA; Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high‐fat diet (HFD) in some inbred mouse strains. We conducted a genome‐wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD‐induced PH. Mice from 36 inbred and wild‐derived strains were fed with regular diet or HFD for 20 weeks beginning at 6‐12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end‐systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest‐path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH‐related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high‐fat feeding. Network‐based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest‐path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high‐fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network‐based approach adds evidence for a role for Egfr in murine PH.

Variable Susceptibility to Cigarette Smoke–Induced Emphysema in 34 Inbred Strains of Mice Implicates Abi3bp in Emphysema Susceptibility

&NA; Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke‐induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta‐analysis of moderate‐to‐severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (&Dgr; CL 7.0 ± 2.2 &mgr;m) and CBA/J was the least susceptible (&Dgr; CL −0.3 ± 1.2 &mgr;m). By integrating mouse and human genome‐wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke‐induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.

Macrophage Elastase Suppresses White Adipose Tissue Expansion with Cigarette Smoking

Macrophage elastase (MMP12) is a key mediator of cigarette smoke (CS)-induced emphysema, yet its role in other smoking related pathologies remains unclear. The weight suppressing effects of smoking are a major hindrance to cessation efforts, and MMP12 is known to suppress the vascularization on which adipose tissue growth depends by catalyzing the formation of antiangiogenic peptides endostatin and angiostatin. The goal of this study was to determine the role of MMP12 in adipose tissue growth and smoking-related suppression of weight gain. Whole body weights and white adipose depots from wild-type and Mmp12-deficient mice were collected during early postnatal development and after chronic CS exposure. Adipose tissue specimens were analyzed for angiogenic and adipocytic markers and for content of the antiangiogenic peptides endostatin and angiostatin. Cultured 3T3-L1 adipocytes were treated with adipose tissue homogenate to examine its effects on vascular endothelial growth factor (VEGF) expression and secretion. MMP12 content and activity were increased in the adipose tissue of wild-type mice at 2 weeks of age, leading to elevated endostatin production, inhibition of VEGF secretion, and decreased adipose tissue vascularity. By 8 weeks of age, adipose MMP12 levels subsided, and the protein was no longer detectable. However, chronic CS exposure led to macrophage accumulation and restored adipose MMP12 activity, thereby suppressing adipose tissue mass and vascularity. Our results reveal a novel systemic role for MMP12 in postnatal adipose tissue expansion and smoking-associated weight loss by suppressing vascularity within the white adipose tissue depots.

Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain

&NA; Rationale: Macrophage elastase (matrix metalloproteinase [MMP]‐12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C‐terminal domain (CTD). Objectives: To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer. Methods: We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non‐small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and KrasLSL/G12D mouse models of lung cancer. Measurements and Main Results: We show that SR20 triggers tumor apoptosis by up‐regulation of gene expression of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL‐mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer. Conclusions: The MMP12 CTD initiates TRAIL‐mediated tumor cell death through its conserved SR20 peptide.

Extreme Trait Whole-Genome Sequencing Identifies PTPRO as a Novel Candidate Gene in Emphysema with Severe Airflow Obstruction

&NA; Rationale: Genetic association studies in chronic obstructive pulmonary disease have primarily tested for association with common variants, the results of which explain only a portion of disease heritability. Because rare variation is also likely to contribute to susceptibility, we used whole‐genome sequencing of subjects with clinically extreme phenotypes to identify genomic regions enriched for rare variation contributing to chronic obstructive pulmonary disease susceptibility. Objectives: To identify regions of rare genetic variation contributing to emphysema with severe airflow obstruction. Methods: We identified heavy smokers that were resistant (n = 65) or susceptible (n = 64) to emphysema with severe airflow obstruction in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We filtered whole‐genome sequencing results to include only rare variants and conducted single variant tests, region‐based tests across the genome, gene‐based tests, and exome‐wide tests. Measurements and Main Results: We identified several suggestive associations with emphysema with severe airflow obstruction, including a suggestive association of all rare variation in a region within the gene ZNF816 (19q13.41; P = 4.5 × 10−6), and a suggestive association of nonsynonymous coding rare variation in the gene PTPRO (P = 4.0 × 10−5). Association of rs61754411, a rare nonsynonymous variant in PTPRO, with emphysema and obstruction was demonstrated in all non‐Hispanic white individuals in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We found that cells containing this variant have decreased signaling in cellular pathways necessary for survival and proliferation. Conclusions: PTPRO is a novel candidate gene in emphysema with severe airflow obstruction, and rs61754411 is a previously unreported rare variant contributing to emphysema susceptibility. Other suggestive candidate genes, such as ZNF816, are of interest for future studies.

PPARγ in emphysema: blunts the damage and triggers repair?

Cigarette smoke is the most common cause of pulmonary emphysema, which results in an irreversible loss of lung structure and function. Th1 and Th17 immune responses have been implicated in emphysema pathogenesis; however, the drivers of emphysema-associated immune dysfunction are not fully understood. In this issue of the JCI, Shan and colleagues found that peroxisome proliferator-activated receptor γ (PPARγ) is downregulated in APCs isolated from the lungs of emphysematous chronic smokers and mice exposed to cigarette smoke. Furthermore, treatment with a PPARγ agonist prevented emphysema development and appeared to reduce emphysema-associated lung volume expansion in mice exposed to cigarette smoke. Further work will need to be done to evaluate the potential of PPARγ agonists to restore lung capacity in emphysematous patients.

Automated Measurement of Blood Vessels in Tissues from Microscopy Images

The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification. We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton‐based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method.

UNIT 12.44 Automated Measurement of Blood Vessels in Tissues from Microscopy Images

The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification. We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton‐based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method.

Automated Measurement of Blood Vessels in Tissues from Microscopy Images: Automated measurement of blood vessels

The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification. We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton‐based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method.