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Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque's fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis, consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress, and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis showed that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1's effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.

Objective: IGF-1 (insulin-like growth factor 1) exerts pleiotropic effects including promotion of cellular growth, differentiation, survival, and anabolism. We have shown that systemic IGF-1 administration reduced atherosclerosis in Apoe -/ - (apolipoprotein E deficient) mice, and this effect was associated with a reduction in lesional macrophages and a decreased number of foam cells in the plaque. Almost all cell types secrete IGF-1, but the effect of macrophage-derived IGF-1 on the pathogenesis of atherosclerosis is poorly understood. We hypothesized that macrophage-derived IGF-1 will reduce atherosclerosis. Approach and Results: We created macrophage-specific IGF-1 overexpressing mice on an Apoe - / - background. Macrophage-specific IGF-1 overexpression reduced plaque macrophages, foam cells, and atherosclerotic burden and promoted features of stable atherosclerotic plaque. Macrophage-specific IGF1 mice had a reduction in monocyte infiltration into plaque, decreased expression of CXCL12 (CXC chemokine ligand 12), and upregulation of ABCA1 (ATP-binding cassette transporter 1), a cholesterol efflux regulator, in atherosclerotic plaque and in peritoneal macrophages. IGF-1 prevented oxidized lipid-induced CXCL12 upregulation and foam cell formation in cultured THP-1 macrophages and increased lipid efflux. We also found an increase in cholesterol efflux in macrophage-specific IGF1-derived peritoneal macrophages., Conclusions: Macrophage IGF-1 overexpression reduced atherosclerotic burden and increased features of plaque stability, likely via a reduction in CXCL12-mediated monocyte recruitment and an increase in ABCA1-dependent macrophage lipid efflux.

Insulin-like growth factor-1 (IGF-1) decreases atherosclerosis in apolipoprotein E ( Apoe )-deficient mice when administered systemically. However, mechanisms for its atheroprotective effect are not fully understood. We generated endothelium-specific IGF-1 receptor (IGF1R)-deficient mice on an Apoe -deficient background to assess effects of IGF-1 on the endothelium in the context of hyperlipidemia-induced atherosclerosis. Endothelial deficiency of IGF1R promoted atherosclerotic burden, when animals were fed on a high-fat diet for 12 wk or normal chow for 12 mo. Under the normal chow feeding condition, the vascular relaxation response to acetylcholine was increased in the endothelial IGF1R-deficient aorta; however, feeding of a high-fat diet substantially attenuated the relaxation response, and there was no difference between endothelial IGF1R-deficient and control mice. The endothelium and its intercellular junctions provide a barrier function to the vasculature. In human aortic endothelial cells, IGF-1 upregulated occludin, claudin 5, VE-cadherin, JAM-A, and CD31 expression levels, and vice versa, specific IGF1R inhibitor, picropodophyllin, an IGF1R-neutralizing antibody (αIR3), or siRNA to IGF1R abolished the IGF-1 effects on junction and adherens proteins, suggesting that IGF-1 promoted endothelial barrier function. Moreover, endothelial transwell permeability assays indicated that inhibition of IGF-1 signaling elevated solute permeability through the monolayer of human aortic endothelial cells. In summary, endothelial IGF1R deficiency increases atherosclerosis, and IGF-1 positively regulates tight junction protein and adherens junction protein levels and endothelial barrier function. Our findings suggest that the elevation of the endothelial junction protein level is, at least in part, the mechanism for antiatherogenic effects of IGF-1. NEW & NOTEWORTHY Endothelial insulin-like growth factor-1 (IGF-1) receptor deficiency significantly elevated atherosclerotic burden in apolipoprotein E-deficient mice, mediated at least in part by downregulation of intercellular junction proteins and, thus, elevated endothelial permeability. This study revealed a novel role for IGF-1 in supporting endothelial barrier function. These findings suggest that IGF-1's ability to promote endothelial barrier function may offer a novel therapeutic strategy for vascular diseases such as atherosclerosis.

Objective: Laboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette-Guérin (BCG))., Methods: Seven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC 90 . The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently., Results: Our panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance., Conclusion: BCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective- IGF-1 (insulin-like growth factor 1) is a major autocrine/paracrine growth factor, which promotes cell proliferation, migration, and survival. We have shown previously that IGF-1 reduced atherosclerosis and promoted features of stable atherosclerotic plaque in Apoe -/ - mice-an animal model of atherosclerosis. The aim of this study was to assess effects of smooth muscle cell (SMC) IGF-1 signaling on the atherosclerotic plaque. Approach and Results- We generated Apoe -/- mice with IGF1R (IGF-1 receptor) deficiency in SMC and fibroblasts (SM22α [smooth muscle protein 22 α]-CreKI/IGF1R-flox mice). IGF1R was decreased in the aorta and adventitia of SM22α-CreKI/IGF1R-flox mice and also in aortic SMC, embryonic, skin, and lung fibroblasts isolated from SM22α-CreKI/IGF1R-flox mice. IGF1R deficiency downregulated collagen mRNA-binding protein LARP6 (La ribonucleoprotein domain family, member 6) and vascular collagen, and mice exhibited growth retardation. The high-fat diet-fed SM22α-CreKI/IGF1R-flox mice had increased atherosclerotic burden and inflammatory responses. α-SMA (α-smooth muscle actin)-positive plaque cells had reduced proliferation and elevated apoptosis. SMC/fibroblast-targeted decline in IGF-1 signaling decreased atherosclerotic plaque SMC, markedly depleted collagen, reduced plaque fibrous cap, and increased plaque necrotic cores. Aortic SMC isolated from SM22α-CreKI/IGF1R-flox mice had decreased cell proliferation, migration, increased sensitivity to apoptosis, and these effects were associated with disruption of IGF-1-induced Akt signaling. Conclusions- IGF-1 signaling in SMC and in fibroblast is a critical determinant of normal vascular wall development and atheroprotection.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal disease. Histone deacetylase 6 (HDAC6) alters function and fate of various proteins via deacetylation of lysine residues, and is implicated in TGF-β1-induced EMT (epithelial-mesenchymal transition). However, the role of HDAC6 in pulmonary fibrosis is unknown., Methods: HDAC6 expression in IPF and control lungs was assessed by quantitative real-time PCR (qRT-PCR) and immunoblots. Lung fibroblasts were treated with TGF-β1 ± HDAC6 inhibitors (Tubacin, Tubastatin, ACY1215, or MC1568), and fibrotic markers such as type I collagen were assessed using qRT-PCR and immunoblots. Mice were treated with bleomycin (oropharyngeal aspiration; single dose) ± Tubastatin (intraperitoneally injection; daily for 21 days), and lung collagen expression was gauged using immunoblots and trichrome staining. In a separate experiment, HDAC6 wild-type (WT) and knockout (KO) mice were administered bleomycin, and lungs were evaluated in the same manner., Results: HDAC6 expression was deregulated in IPF lungs. Among the HDAC6 inhibitors tested, only Tubastatin significantly repressed TGF-β1-induced expression of type-1 collagen in lung fibroblasts, and this finding was coupled with decreased Akt phosphorylation and increased Akt-PHLPP (PH domain and Leucine rich repeat Protein Phosphatase) association. Tubastatin repressed TGF-β1-induced S6K phosphorylation, HIF-1α expression, and VEGF expression. Tubastatin also repressed TGF-β1-induced inhibition of LC3B-II (a marker of autophagosome formation). In bleomycin-treated mouse lungs, HDAC6 expression was increased, and Tubastatin repressed type-1 collagen expression. However, in HDAC6 KO mice, bleomycin-induced type-1 collagen expression was not repressed compared to WT mice. Knockdown of HDAC6, as well as HDAC10, another potential Tubastatin target, did not inhibit TGF-β1-induced collagen expression in lung fibroblasts., Conclusions: HDAC6 expression is altered during lung fibrogenesis. Tubastatin represses TGF-β1-induced collagen expression, by diminishing Akt phosphorylation and regulating downstream targets such as HIF-1α-VEGF axis and autophagy. Tubastatin-treated WT mice are protected against bleomycin-induced fibrosis, but HDAC6 KO mice are not. Our data suggest that Tubastatin ameliorates pulmonary fibrosis, by targeting the TGFβ-PI3K-Akt pathway, likely via an HDAC6-independent mechanism.

Background: We have previously shown that systemic infusion of insulin-like growth factor-1 (IGF-1) exerts anti-inflammatory and antioxidant effects and reduces atherosclerotic burden in apolipoprotein E (Apoe)-deficient mice. Monocytes/macrophages express high levels of IGF-1 receptor (IGF1R) and play a pivotal role in atherogenesis, but the potential effects of IGF-1 on their function are unknown., Methods and Results: To determine mechanisms whereby IGF-1 reduces atherosclerosis and to explore the potential involvement of monocytes/macrophages, we created monocyte/macrophage-specific IGF1R knockout (MΦ-IGF1R-KO) mice on an Apoe(-/-) background. We assessed atherosclerotic burden, plaque features of stability, and monocyte recruitment to atherosclerotic lesions. Phenotypic changes of IGF1R-deficient macrophages were investigated in culture. MΦ-IGF1R-KO significantly increased atherosclerotic lesion formation, as assessed by Oil Red O staining of en face aortas and aortic root cross-sections, and changed plaque composition to a less stable phenotype, characterized by increased macrophage and decreased α-smooth muscle actin-positive cell population, fibrous cap thinning, and decreased collagen content. Brachiocephalic artery lesions of MΦ-IGF1R-KO mice had histological features implying plaque vulnerability. Macrophages isolated from MΦ-IGF1R-KO mice showed enhanced proinflammatory responses on stimulation by interferon-γ and oxidized low-density lipoprotein and elevated antioxidant gene expression levels. Moreover, IGF1R-deficient macrophages had decreased expression of ABCA1 and ABCG1 and reduced lipid efflux., Conclusions: Our data indicate that macrophage IGF1R signaling suppresses macrophage and foam cell accumulation in lesions and reduces plaque vulnerability, providing a novel mechanism whereby IGF-1 exerts antiatherogenic effects., (© 2016 American Heart Association, Inc.)

Both cigarette smoke (CS) and asbestos cause lung inflammation and lung cancer, and at high asbestos exposure levels, populations exposed to both of these carcinogens display a synergistic increase in the development of lung cancer. The mechanisms through which these two toxic agents interact to promote lung tumorigenesis are poorly understood. Here, we begin to dissect the inflammatory signals induced by asbestos in combination with CS using a rodent inhalation model and in vitro cell culture. Wild-type C57BL/6 mice were exposed to room air as a control, CS, and/or asbestos (4 days per week to CS and 1 day per week to asbestos for 5 weeks). Bronchoalveolar lavage (BAL) fluid was collected following exposure and analyzed for inflammatory mediators. Asbestos-exposed mice displayed an increased innate immune response consistent with NLRP3 inflammasome activation. Compared to mice exposed only to asbestos, animals coexposed to CS + asbestos displayed attenuated levels of innate immune mediators and altered inflammatory cell recruitment. Histopathological changes in CS + asbestos-exposed mice correlated with attenuated fibroproliferative lesion development relative to their counterparts exposed only to asbestos. In vitro experiments using a human monocyte cell line (THP-1 cells) supported the in vivo results in that coexposure to cigarette smoke extract repressed NLRP3 inflammasome markers in cells treated with asbestos. These observations indicate that CS represses central components of the innate immune response to inhaled asbestos., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Aging constitutes a significant risk factor for fibrosis, and idiopathic pulmonary fibrosis (IPF) is characteristically associated with advancing age. We propose that age-dependent defects in the quality of protein and cellular organelle catabolism may be causally related to pulmonary fibrosis. Our research found that autophagy diminished with corresponding elevated levels of oxidized proteins and lipofuscin in response to lung injury in old mice and middle-aged mice compared to younger animals. More importantly, older mice expose to lung injury are characterized by deficient autophagic response and reduced selective targeting of mitochondria for autophagy (mitophagy). Fibroblast to myofibroblast differentiation (FMD) is an important feature of pulmonary fibrosis in which the profibrotic cytokine TGFβ1 plays a pivotal role. Promotion of autophagy is necessary and sufficient to maintain normal lung fibroblasts' fate. On the contrary, FMD mediated by TGFβ1 is characterized by reduced autophagy flux, altered mitophagy, and defects in mitochondrial function. In accord with these findings, PINK1 expression appeared to be reduced in fibrotic lung tissue from bleomycin and a TGFβ1-adenoviral model of lung fibrosis. PINK1 expression is also reduced in the aging murine lung and biopsies from IPF patients compared to controls. Furthermore, deficient PINK1 promotes a profibrotic environment. Collectively, this study indicates that an age-related decline in autophagy and mitophagy responses to lung injury may contribute to the promotion and/or perpetuation of pulmonary fibrosis. We propose that promotion of autophagy and mitochondrial quality control may offer an intervention against age-related fibrotic diseases., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)