Time course of histopathology of bleomycin-induced pulmonary fibrosis using an intratracheal sprayer in mice.

Idiopathic pulmonary fibrosis (IPF) is a poor prognosis disease that affects approximately 5 million people worldwide, and the detailed mechanisms underlying the pathogenesis of IPF remain unclear. Bleomycin-induced pulmonary fibrosis has been widely used as a representative animal model of IPF that induces fibrosis in lung tissue. The lungs of rodent consist of five lobes and each bronchus enters each lobe of the lung at a different bifurcation angle, path length, and diameter. The method of administration of bleomycin is considered as important thing to establish appropriate animal models. We conducted a time-dependent histopathological study to examine how pulmonary fibrosis develops in each lung lobe when bleomycin was intratracheally sprayed in ICR mice. And we then explored the suitable points for evaluation of anti-fibrotic agents in this model. As a result, we found that homogeneous fibrosis was induced in the 5 lobes of the lungs following initial inflammation. The expression of transforming growth factor (TGF)-ß1 and phospho-Smad2 (pSmad2) was observed from Day 1, and their positivity increased until Day 21. In conclusion, we have observed a detailed time course of histological changes in bleomycin-induced pulmonary fibrosis in ICR mice using the aerosolization technique. We found that our protocol can induce a highly homogeneous lesion in the lung and that the most suitable time point to assess anti-fibrotic agents is 14 days after treatment in this model.

Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity.

AIMS: Calcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored. METHODS AND RESULTS: An aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype. CONCLUSION: Sortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Discovery of novel spiro[chromane-2,4'-piperidine] derivatives as potent and orally bioavailable G-protein-coupled receptor 119 agonists.

Herein, we describe the discovery, synthesis, and evaluation of a novel series of spiro[chromane-2,4'-piperidine] derivatives as G-protein-coupled receptor 119 agonists. Their initial design exploited the conformational restriction in the linker-to-tail moiety, which was a key concept in this study, to give lead compound 11 (EC50 = 369 nM, Emax = 82%). An extensive structure-activity relationship study resulted in the identification of the optimized drug candidate (R)-29 (EC50 = 54 nM, Emax = 181%). The defining structural features of the series were a terminal benzyl-type bulky substituent and a methylene linker between the sulfonyl and phenyl groups, both of which were in the head moiety as well as the spiro-type scaffold in the linker-to-tail moiety. An in vivo oral glucose-tolerance test using C57BL/6N mice showed that (R)-29 reduced glucose excursion at a dose of 3 mg/kg in a dose-dependent manner.

Transcriptional control of intestinal cholesterol absorption, adipose energy expenditure and lipid handling by Sortilin.

The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Here we show that in the low-density lipoprotein receptor-deficient (Ldlr-/-) atherosclerosis model, Sortilin deficiency (Sort1-/-) in female mice suppresses Niemann-Pick type C1-Like 1 (Npc1l1) mRNA levels, reduces body and white adipose tissue weight, and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor. Female Ldlr-/-Sort1-/- mice on a high-fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 and Adiponectin, an adipokine that when reduced is associated with obesity and cardiovascular disease-related factors. Additionally, Sort1 deficiency suppressed cholesterol absorption in both female mice ex vivo intestinal tissue and human colon Caco-2 cells in a similar manner to treatment with the NPC1L1 inhibitor ezetimibe. Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.

Mechanism of lipid-lowering action of the dipeptidyl peptidase-4 inhibitor, anagliptin, in low-density lipoprotein receptor-deficient mice.

AIMS/INTRODUCTION: Dipeptidyl peptidase-4 inhibitors are used for treatment of patients with type 2 diabetes. In addition to glycemic control, these agents showed beneficial effects on lipid metabolism in clinical trials. However, the mechanism underlying the lipid-lowering effect of dipeptidyl peptidase-4 inhibitors remains unclear. Here, we investigated the lipid-lowering efficacy of anagliptin in a hyperlipidemic animal model, and examined the mechanism of action. MATERIALS AND METHODS: Male low-density lipoprotein receptor-deficient mice were administered 0.3% anagliptin in their diet. Plasma lipid levels were assayed and lipoprotein profile was analyzed using high-performance liquid chromatography. Hepatic gene expression was examined by deoxyribonucleic acid microarray and quantitative polymerase chain reaction analyses. Sterol regulatory element-binding protein transactivation assay was carried out in vitro. RESULTS: Anagliptin treatment significantly decreased the plasma total cholesterol (14% reduction, P < 0.01) and triglyceride levels (27% reduction, P < 0.01). Both low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol were also decreased significantly by anagliptin treatment. Sterol regulatory element-binding protein-2 messenger ribonucleic acid expression level was significantly decreased at night in anagliptin-treated mice (15% reduction, P < 0.05). Anagliptin significantly suppressed sterol regulatory element-binding protein activity in HepG2 cells (21% decrease, P < 0.001). CONCLUSIONS: The results presented here showed that the dipeptidyl peptidase-4 inhibitor, anagliptin, exhibited a lipid-lowering effect in a hyperlipidemic animal model, and suggested that the downregulation of hepatic lipid synthesis was involved in the effect. Anagliptin might have beneficial effects on lipid metabolism in addition to a glucose-lowering effect.

A single injection of gain-of-function mutant PCSK9 adeno-associated virus vector induces cardiovascular calcification in mice with no genetic modification.

BACKGROUND AND AIMS: Studying atherosclerotic calcification in vivo requires mouse models with genetic modifications. Previous studies showed that injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice promotes atherosclerosis. We aimed to study cardiovascular calcification induced by PCSK9 AAV in C57BL/6J mice. METHODS: 10 week-old C57BL/6J mice received a single injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9). Ldlr(-/-) mice served as positive controls. Mice consumed a high-fat, high-cholesterol diet for 15 or 20 weeks. Aortic calcification was assessed by fluorescence reflectance imaging (FRI) of a near-infrared calcium tracer. RESULTS: Serum levels of PCSK9 (0.14 µg/mL to 20 µg/mL, p < 0.01) and total cholesterol (82 mg/dL to 820 mg/dL, p < 0.01) increased within one week after injection and remained elevated for 20 weeks. Atherosclerotic lesion size was similar between PCSK9 AAV and Ldlr(-/-) mice. Aortic calcification was 0.01% ± 0.01 in PCSK9 AAV mice and 15.3% ± 6.1 in Ldlr(-/-) mice at 15 weeks (p < 0.01); by 20 weeks, the PCSK9 AAV mice aortic calcification grew to 12.4% ± 4.9. Tissue non-specific alkaline phosphatase activity was similar in PCSK9 AAV mice and Ldlr(-/-) mice at 15 and 20 weeks, respectively. As example of the utility of this model in testing modulators of calcification in vivo, PCSK9 AAV injection to sortilin-deficient mice demonstrated reduced aortic calcification by 46.3% (p < 0.05) compared to littermate controls. CONCLUSIONS: A single injection of gain-of-function PCSK9 AAV into C57BL/6J mice is a useful tool to study cardiovascular calcification in mice with no genetic manipulation.

Sortilin mediates vascular calcification via its recruitment into extracellular vesicles.

Vascular calcification is a common feature of major cardiovascular diseases. Extracellular vesicles participate in the formation of microcalcifications that are implicated in atherosclerotic plaque rupture; however, the mechanisms that regulate formation of calcifying extracellular vesicles remain obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin regulated the loading of the calcification protein tissue nonspecific alkaline phosphatase (TNAP) into extracellular vesicles, thereby conferring its calcification potential. Furthermore, SMC calcification required Rab11-dependent trafficking and FAM20C/casein kinase 2-dependent C-terminal phosphorylation of sortilin. In a murine model, Sort1-deficiency reduced arterial calcification but did not affect bone mineralization. Additionally, transfer of sortilin-deficient BM cells to irradiated atherosclerotic mice did not affect vascular calcification, indicating a primary role of SMC-derived sortilin. Together, the results of this study identify sortilin phosphorylation as a potential therapeutic target for ectopic calcification/microcalcification and may clarify the mechanism that underlies the genetic association between the SORT1 gene locus and coronary artery calcification.

In vivo imaging of leukocyte recruitment to the atheroprone femoral artery reveals anti-inflammatory effects of rosuvastatin.

OBJECTIVE: To monitor the anti-inflammatory effect of rosuvastatin in leukocyte endothelial interactions in the atheroprone femoral artery in vivo. METHODS AND RESULTS: Male Apolipoprotein E null mice (ApoE-/- mice, 6 weeks old) were fed a high-fat diet (20% fat, 1.25% cholesterol) with or without the HMG CoA reductase inhibitor rosuvastatin (10 mg/kg/day) for 6 weeks. Significant leukocyte adhesion was observed in the femoral artery of ApoE-/- mice, but not of wild type mice, in the absence of rosuvastatin. Interestingly, no obvious plaque formation was observed in the artery at this time point. The number of adherent leukocytes was dramatically diminished in ApoE-/- mice treated with rosuvastatin. DHE-associated oxidative stress and the expression of gp91-phox, a component of NADPH oxidase, were induced in ApoE-/- mice and were abolished by rosuvastatin treatment. CONCLUSION: Our data documented leukocyte recruitment prior to lipid accumulation and subsequent inhibition by rosuvastatin. The underlying mechanism seemed to involve oxidative stress and an anti-inflammatory effect on the endothelium of atheroprone vessels.

Adipose inflammation initiates recruitment of leukocytes to mouse femoral artery: role of adipo-vascular axis in chronic inflammation.

BACKGROUND: Although inflammation within adipose tissues is known to play a role in metabolic syndrome, the causative connection between inflamed adipose tissue and atherosclerosis is not fully understood. In the present study, we examined the direct effects of adipose tissue on macro-vascular inflammation using intravital microscopic analysis of the femoral artery after adipose tissue transplantation. METHODS AND RESULTS: We obtained subcutaneous (SQ) and visceral (VIS) adipose tissues from C57BL/6 mice fed normal chow (NC) or a high fat diet (HF), then transplanted the tissues into the perivascular area of the femoral artery of recipient C57/BL6 mice. Quantitative intravital microscopic analysis revealed an increase in adherent leukocytes after adipose tissue transplantation, with VIS found to induce significantly more leukocyte accumulation as compared to SQ. Moreover, adipose tissues from HF fed mice showed significantly more adhesion to the femoral artery. Simultaneous flow cytometry demonstrated upregulation of CD11b on peripheral granulocyte and monocytes after adipose tissue transplantation. We also observed dominant expressions of the inflammatory cytokine IL-6, and chemokines MCP-1 and MIP-1ß in the stromal vascular fraction (SVF) of these adipose tissues as well as sera of recipient mice after transplantation. Finally, massive accumulations of pro-inflammatory and dendritic cells were detected in mice with VIS transplantation as compared to SQ, as well as in HF mice as compared to those fed NC. CONCLUSION: Our in vivo findings indicate that adipose tissue stimulates leukocyte accumulation in the femoral artery. The underlying mechanisms involve upregulation of CD11b in leukocytes, induction of cytokines and chemokines, and accumulation of pro-inflammatory cells in the SVF.

Combination of amlodipine and atorvastatin synergistically reduces leukocyte recruitment to mechanically injured mouse femoral artery.

Recent studies have demonstrated a potential synergistic effect of the combination of amlodipine with atorvastatin to reduce acute inflammation. The intraluminal wire injury of the mouse femoral artery induced significant leukocyte recruitment to the injured area and oxidative stress within 24 h. Administration of low-dose amlodipine (0.5 mg kg(-1) per day) or atorvastatin (1 mg kg(-1) per day) alone for 7 days failed to modulate leukocyte adhesion, whereas their co-administration for 7 days significantly inhibited leukocyte adhesion. Moreover, flow cytometric analysis showed that injury-induced oxidative stress and CD11b expression in three leukocyte fractions were elevated after injury and then reduced after the co-administration. Next, adoptive transfer of mononuclear cells (MNCs) was performed and MNCs were harvested from mice after wire injury exhibited adhesion to the recipient injured artery. Furthermore, the co-administration of low-dose atorvastatin and amlodipine to MNCs or the vasculature reduced the recruitment of MNCs to the injured artery. Our findings indicate that amlodipine and atorvastatin synergistically inhibit vascular inflammation. The underlying mechanisms of their effect involve, at least in part, stabilizing oxidative stress at the point of injury, suggesting the clinical efficacy of this drug combination for the treatment of vascular diseases.

Oxidative stress in mononuclear cells plays a dominant role in their adhesion to mouse femoral artery after injury.

Leukocyte recruitment plays a pivotal role during inflammation after vascular injury. The importance of oxidative stress in vascular injury and its modulation by angiotensin II receptor blockers (olmesartan) have been demonstrated. We examined the contribution of leukocyte-associated oxidative stress in acute-phase leukocyte recruitment and its modulation by olmesartan. Male mice were treated with olmesartan (5 mg/kg per day) or vehicle for 7 days before the transluminal wire injury of the femoral artery. Intravital microscopy of the artery revealed that the mechanical injury increased adherent leukocytes at both 24 hours and 7 days after the injury, which was significantly reduced by olmesartan treatment. Dihydroethidium-associated fluorescence intensity observed in vehicle-treated mice was significantly diminished under olmesartan treatment. Apocynin, a nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, showed a similar inhibitory effect on the leukocyte adhesion. Adoptive transfer of mononuclear cells, harvested from mice after wire injury, but not from those without wire injury, exhibited adhesion to the recipient injured artery. Furthermore, olmesartan treatment of mononuclear cells, but not of injured vasculature, reduced their recruitment to the injured artery. These data indicate that leukocyte recruitment to the mechanically injured artery is mediated by oxidative stress in leukocytes but not in vasculatures. Treatment with olmesartan blocked leukocyte recruitment by antagonizing mononuclear cells-associated oxidative stress.

Real-time imaging of mechanically injured femoral artery in mice reveals a biphasic pattern of leukocyte accumulation.

Wire injury of an artery has been recognized as a standard model of vascular inflammation and atherosclerosis; however, the mechanism of leukocyte recruitment has not been studied in this model. In this study, we documented the recruitment of leukocytes to the murine femoral artery after a wire injury. A transluminal mechanical injury was generated by insertion of a wire into the femoral artery of male C57BL/6J mice. The mice were anesthetized and ventilated after tracheotomy and protected from hypothermia by a warming lamp. Body temperature and blood pH did not significantly change during the experiment. The interaction between rhodamine 6G-labeled leukocytes and the injured femoral artery was monitored using an epifluorescent microscope, and the images were evaluated using a computer-assisted image analysis program. In the absence of injury, virtually no leukocyte adhesion was observed. In contrast, the number of adherent leukocytes increased 4 and 24 h after injury and declined 72 h after injury. The rolling flux of leukocytes increased 4 h after injury and remained high up to 7 days, but it was faster 72 h after injury. We identified another peak of leukocyte adhesion 7 days after injury. Injection of anti-P-selectin antibody significantly reduced leukocyte adhesion at the early and later phases. In conclusion, we have established a novel experimental system for direct observation of leukocyte recruitment to the injured femoral artery. Our system revealed a previously undetected, unique profile of leukocyte recruitment during vascular injury.