Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.     

Amelioration of Hyperglycemia with a Sodium-Glucose Cotransporter 2 Inhibitor Prevents Macrophage-Driven Atherosclerosis through Macrophage Foam Cell Formation Suppression in Type 1 and Type 2 Diabetic Mice

Direct associations between hyperglycemia and atherosclerosis remain unclear. We investigated the association between the amelioration of glycemia by sodium-glucose cotransporter 2 inhibitors (SGLT2is) and macrophage-driven atherosclerosis in diabetic mice. We administered dapagliflozin or ipragliflozin (1.0 mg/kg/day) for 4-weeks to apolipoprotein E-null (Apoe ^−/−) mice, streptozotocin-induced diabetic Apoe ^−/− mice, and diabetic db/db mice. We then determined aortic atherosclerosis, oxidized low-density lipoprotein (LDL)-induced foam cell formation, and related gene expression in exudate peritoneal macrophages. Dapagliflozin substantially decreased glycated hemoglobin (HbA1c) and glucose tolerance without affecting body weight, blood pressure, plasma insulin, and lipids in diabetic Apoe ^−/− mice. Aortic atherosclerotic lesions, atheromatous plaque size, and macrophage infiltration in the aortic root increased in diabetic Apoe ^−/− mice; dapagliflozin attenuated these changes by 33%, 27%, and 20%, respectively. Atherosclerotic lesions or foam cell formation highly correlated with HbA1c. Dapagliflozin did not affect atherosclerosis or plasma parameters in non-diabetic Apoe ^−/− mice. In db/db mice, foam cell formation increased by 4-fold compared with C57/BL6 mice, whereas ipragliflozin decreased it by 31%. Foam cell formation exhibited a strong correlation with HbA1c. Gene expression of lectin-like ox-LDL receptor-1 and acyl-coenzyme A:cholesterol acyltransferase 1 was upregulated, whereas that of ATP-binding cassette transporter A1 was downregulated in the peritoneal macrophages of both types of diabetic mice. SGLT2i normalized these gene expressions. Our study is the first to demonstrate that SGLT2i exerts anti-atherogenic effects by pure glucose lowering independent of insulin action in diabetic mice through suppressing macrophage foam cell formation, suggesting that foam cell formation is highly sensitive to glycemia ex vivo.     

Vasoprotective Effects of Urocortin 1 against Atherosclerosis In Vitro and In Vivo

Aim

Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis.

Methods

We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe ^−/−) mice.

Results

Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe ^−/− mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions.

Conclusions

This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.     

Glucose-Induced Glucagon-Like Peptide 1 Secretion Is Deficient in Patients with Non-Alcoholic Fatty Liver Disease

Background & Aims

The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gastrointestinal peptide hormones regulating postprandial insulin release from pancreatic β-cells. GLP-1 agonism is a treatment strategy in Type 2 diabetes and is evaluated in Non-alcoholic fatty liver disease (NAFLD). However, the role of incretins in its pathophysiology is insufficiently understood. Studies in mice suggest improvement of hepatic steatosis by GLP-1 agonism. We determined the secretion of incretins after oral glucose administration in non-diabetic NAFLD patients.

Methods

N = 52 patients (n = 16 NAFLD and n = 36 Non-alcoholic steatohepatitis (NASH) patients) and n = 50 matched healthy controls were included. Standardized oral glucose tolerance test was performed. Glucose, insulin, glucagon, GLP-1 and GIP plasma levels were measured sequentially for 120 minutes after glucose administration.

Results

Glucose induced GLP-1 secretion was significantly decreased in patients compared to controls (p<0.001). In contrast, GIP secretion was unchanged. There was no difference in GLP-1 and GIP secretion between NAFLD and NASH subgroups. All patients were insulin resistant, however HOMA2-IR was highest in the NASH subgroup. Fasting and glucose-induced insulin secretion was higher in NAFLD and NASH compared to controls, while the glucose lowering effect was diminished. Concomitantly, fasting glucagon secretion was significantly elevated in NAFLD and NASH.

Conclusions

Glucose-induced GLP-1 secretion is deficient in patients with NAFLD and NASH. GIP secretion is contrarily preserved. Insulin resistance, with hyperinsulinemia and hyperglucagonemia, is present in all patients, and is more severe in NASH compared to NAFLD. These pathophysiologic findings endorse the current evaluation of GLP-1 agonism for the treatment of NAFLD.     

Mapping and Congenic Dissection of Genetic Loci Contributing to Hyperglycemia and Dyslipidemia in Mice

Background

Patients with dyslipidemia have an increased risk of developing type 2 diabetes, and diabetic patients often have dyslipidemia. Potential genetic connections of fasting plasma glucose with plasma lipid profile were evaluated using hyperlipidemic mice.

Methods

225 male F₂ mice were generated from BALB/cJ (BALB) and SM/J(SM) Apoe-deficient (Apoe^−/−) mice and fed a Western diet for 5 weeks. Fasting plasma glucose and lipid levels of F₂ mice were measured before and after 5 weeks of Western diet and quantitative trait locus (QTL) analysis was performed using data collected from these two time points. 144 SNP(single nucleotide polymorphism) markers across the entire genome were typed.

Results

One major QTL (logarithm of odds ratio (LOD): 6.46) peaked at 12.7 cM on chromosome 9,Bglu16, and 3 suggestive QTLs on chromosomes 15, 18 and X were identified for fasting glucose, and over 10 loci identified for lipid traits. Bglu16 was adjacent to a major QTL, Hdlq17, for high-density lipoprotein (HDL) cholesterol (LOD: 6.31, peak: 19.1 cM). A congenic strain with a donor chromosomal region harboring Bglu16 and Hdlq17 on the Apoe^−/− background showed elevations in plasma glucose and HDL levels. Fasting glucose levels were significantly correlated with non-HDL cholesterol and triglyceride levels, especially on the Western diet, but only marginally correlated with HDL levels in F₂ mice.

Conclusions

We have demonstrated a correlative relationship between fasting glucose and plasma lipids in a segregating F₂ population under hyperlipidemic conditions, and this correlation is partially due to genetic linkage between the two disorders.     

GLP-1 Receptor Activation Inhibits VLDL Production and Reverses Hepatic Steatosis by Decreasing Hepatic Lipogenesis in High-Fat-Fed APOE*3-Leiden Mice

Objective

In addition to improve glucose intolerance, recent studies suggest that glucagon-like peptide-1 (GLP-1) receptor agonism also decreases triglyceride (TG) levels. The aim of this study was to evaluate the effect of GLP-1 receptor agonism on very-low-density lipoprotein (VLDL)-TG production and liver TG metabolism.

Experimental Approach

The GLP-1 peptide analogues CNTO3649 and exendin-4 were continuously administered subcutaneously to high fat diet-fed APOE*3-Leiden transgenic mice. After 4 weeks, hepatic VLDL production, lipid content, and expression profiles of selected genes involved in lipid metabolism were determined.

Results

CNTO3649 and exendin-4 reduced fasting plasma glucose (up to −30% and −28% respectively) and insulin (−43% and −65% respectively). In addition, these agents reduced VLDL-TG production (−36% and −54% respectively) and VLDL-apoB production (−36% and −43% respectively), indicating reduced production of VLDL particles rather than reduced lipidation of apoB. Moreover, they markedly decreased hepatic content of TG (−39% and −55% respectively), cholesterol (−30% and −55% respectively), and phospholipids (−23% and −36% respectively), accompanied by down-regulation of expression of genes involved in hepatic lipogenesis (Srebp-1c, Fasn, Dgat1) and apoB synthesis (Apob).

Conclusion

GLP-1 receptor agonism reduces VLDL production and hepatic steatosis in addition to an improvement of glycemic control. These data suggest that GLP-receptor agonists could reduce hepatic steatosis and ameliorate dyslipidemia in patients with type 2 diabetes mellitus.     

A GIP Receptor Agonist Exhibits β-Cell Anti-Apoptotic Actions in Rat Models of Diabetes Resulting in Improved β-Cell Function and Glycemic Control

Aims

The gastrointestinal hormone GIP promotes pancreatic islet function and exerts pro-survival actions on cultured β-cells. However, GIP also promotes lipogenesis, thus potentially restricting its therapeutic use. The current studies evaluated the effects of a truncated GIP analog, D-Ala²-GIP_1–30 (D-GIP_1–30), on glucose homeostasis and β-cell mass in rat models of diabetes.

Materials and Methods

The insulinotropic and pro-survival potency of D-GIP_1–30 was evaluated in perfused pancreas preparations and cultured INS-1 β-cells, respectively, and receptor selectivity evaluated using wild type and GIP receptor knockout mice. Effects of D-GIP_1–30 on β-cell function and glucose homeostasis, in vivo, were determined using Lean Zucker rats, obese Vancouver diabetic fatty rats, streptozotocin treated rats, and obese Zucker diabetic fatty rats, with effects on β-cell mass determined in histological studies of pancreatic tissue. Lipogenic effects of D-GIP_1–30 were evaluated on cultured 3T3-L1 adipocytes.

Results

Acutely, D-GIP_1–30 improved glucose tolerance and insulin secretion. Chronic treatment with D-GIP_1–30 reduced levels of islet pro-apoptotic proteins in Vancouver diabetic fatty rats and preserved β-cell mass in streptozotocin treated rats and Zucker diabetic fatty rats, resulting in improved insulin responses and glycemic control in each animal model, with no change in body weight. In in vitro studies, D-GIP_1–30 exhibited equivalent potency to GIP_1–42 on β-cell function and survival, but greatly reduced action on lipoprotein lipase activity in 3T3-L1 adipocytes.

Conclusions

These findings demonstrate that truncated forms of GIP exhibit potent anti-diabetic actions, without pro-obesity effects, and that the C-terminus contributes to the lipogenic actions of GIP.     

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu,Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

Purpose

The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 ^−/−) mouse.

Methods

Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 ^−/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed.

Results

Corneal vital staining scores in the Sod1 ^−/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 ^−/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 ^−/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 ^−/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 ^−/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 ^−/− mice.

Conclusions

Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 ^−/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.     

Raised Soluble P-Selectin Moderately Accelerates Atherosclerotic Plaque Progression

Soluble P-selectin (sP-selectin), a biomarker of inflammatory related pathologies including cardiovascular and peripheral vascular diseases, also has pro-atherosclerotic effects including the ability to increase leukocyte recruitment and modulate thrombotic responses in vivo. The current study explores its role in progressing atherosclerotic plaque disease. Apoe ^−/− mice placed on a high fat diet (HFD) were given daily injections of recombinant dimeric murine P-selectin (22.5 µg/kg/day) for 8 or 16 weeks. Saline or sE-selectin injections were used as negative controls. In order to assess the role of sP-selectin on atherothrombosis an experimental plaque remodelling murine model, with sm22α-hDTR Apoe^−/− mice on a HFD in conjunction with delivery of diphtheria toxin to induce targeted vascular smooth muscle apoptosis, was used. These mice were similarly given daily injections of sP-selectin for 8 or 16 weeks. While plaque mass and aortic lipid content did not change with sP-selectin treatment in Apoe^−/− or SM22α-hDTR Apoe^−/− mice on HFD, increased plasma MCP-1 and a higher plaque CD45 content in Apoe ^−/− HFD mice was observed. As well, a significant shift towards a more unstable plaque phenotype in the SM22α-hDTR Apoe^−/− HFD mice, with increased macrophage accumulation and lower collagen content, leading to a lower plaque stability index, was observed. These results demonstrate that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.     

Osteoprotegerin Inhibits Aortic Valve Calcification and Preserves Valve Function in Hypercholesterolemic Mice

Background

There are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic Ldlr ^−/− Apob ^100/100 mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known.

Objectives

To determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic Ldlr ^−/− Apob ^100/100 mice.

Methods

Young Ldlr ^−/− Apob ^100/100 mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older Ldlr ^−/− Apob ^100/100 mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation.

Results

In Young Ldlr ^−/− Apob ^100/100 mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older Ldlr ^−/− Apob ^100/100 mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ∼80%, and attenuated aortic valve calcification by ∼ 70%. OPG also attenuated impairment of aortic valve function.

Conclusions

OPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone Ldlr ^−/− Apob ^100/100 mice.     

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

Background and Objectives

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.

Methods

GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET).

Results

GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.

Conclusions

GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.     

The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background

The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh^−/− mice to oxidative stress.

Methodology/Principal Findings

The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh^−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh^−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh^−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3⁺ regulatory T cells were observed only in Mutyh^−/− mice.

Conclusions

The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.     

Minor Contribution of Endogenous GLP-1 and GLP-2 to Postprandial Lipemia in Obese Men

Context

Glucose and lipids stimulate the gut-hormones glucagon-like peptide (GLP)-1, GLP-2 and glucose-dependent insulinotropic polypeptide (GIP) but the effect of these on human postprandial lipid metabolism is not fully clarified.

Objective

To explore the responses of GLP-1, GLP-2 and GIP after a fat-rich meal compared to the same responses after an oral glucose tolerance test (OGTT) and to investigate possible relationships between incretin response and triglyceride-rich lipoprotein (TRL) response to a fat-rich meal.

Design

Glucose, insulin, GLP-1, GLP-2 and GIP were measured after an OGTT and after a fat-rich meal in 65 healthy obese (BMI 26.5–40.2 kg/m²) male subjects. Triglycerides (TG), apoB48 and apoB100 in TG-rich lipoproteins (chylomicrons, VLDL₁ and VLDL₂) were measured after the fat-rich meal.

Main Outcome Measures

Postprandial responses (area under the curve, AUC) for glucose, insulin, GLP-1, GLP-2, GIP in plasma, and TG, apoB48 and apoB100 in plasma and TG-rich lipoproteins.

Results

The GLP-1, GLP-2 and GIP responses after the fat-rich meal and after the OGTT correlated strongly (r = 0.73, p<0.0001; r = 0.46, p<0.001 and r = 0.69, p<0.001, respectively). Glucose and insulin AUCs were lower, but the AUCs for GLP-1, GLP-2 and GIP were significantly higher after the fat-rich meal than after the OGTT. The peak value for all hormones appeared at 120 minutes after the fat-rich meal, compared to 30 minutes after the OGTT. After the fat-rich meal, the AUCs for GLP-1, GLP-2 and GIP correlated significantly with plasma TG- and apoB48 AUCs but the contribution was very modest.

Conclusions

In obese males, GLP-1, GLP-2 and GIP responses to a fat-rich meal are greater than following an OGTT. However, the most important explanatory variable for postprandial TG excursion was fasting triglycerides. The contribution of endogenous GLP-1, GLP-2 and GIP to explaining the variance in postprandial TG excursion was minor.     

Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury

Introduction

Ligament and meniscal damage can cause joint disease. Arthritic joints contain increased amounts of epidermal growth factor receptor (EGFR) protein, and polymorphisms in EGFR are associated with arthritis risk. The role of endogenous EGFR regulation during joint disease due to ligament and meniscal trauma is unknown. Mitogen-inducible gene 6 (MIG-6) can reduce EGFR phosphorylation and downstream signaling. We examined the effect of EGFR modulation by MIG-6 on joint disease development after ligament and meniscus injury.

Methods

Knee ligament transection and meniscus removal were performed surgically on mice homozygous for a global inactivating mutation in MIG-6 (Mig-6^−/−) and in wild-type (WT) animals.

Results

Two weeks after surgery, Mig-6^−/−mice had bone erosion as well as greater fibrous tissue area and serum RANKL concentration than WT mice. Four weeks after surgery, Mig-6^−/−mice had less cartilage and increased cell proliferation relative to contralateral control and WT knees. Increased apoptotic cells and growth outside the articulating region occurred in Mig-6^−/−mice. Tibia trabecular bone mineral density (BMD) and the number of trabeculae were lower in surgically treated knees relative to the respective control knees for both groups. BMD, as well as trabecular thickness and number, were lower in surgically treated knees from Mig-6^−/−mice relative to WT surgically treated knees. Phosphorylated EGFR staining in surgically treated knees decreased for WT mice and increased for Mig-6^−/−mice. Fewer inflammatory cells were present in the knees of WT mice.

Conclusion

Mig-6^−/−mice have rapid and increased joint damage after ligament and meniscal trauma. Mig-6 modification could lessen degenerative disease development after this type of injury.     

Mutation in KERA Identified by Linkage Analysis and Targeted Resequencing in a Pedigree with Premature Atherosclerosis

Aims

Genetic factors explain a proportion of the inter-individual variation in the risk for atherosclerotic events, but the genetic basis of atherosclerosis and atherothrombosis in families with Mendelian forms of premature atherosclerosis is incompletely understood. We set out to unravel the molecular pathology in a large kindred with an autosomal dominant inherited form of premature atherosclerosis.

Methods and Results

Parametric linkage analysis was performed in a pedigree comprising 4 generations, of which a total of 11 members suffered from premature vascular events. A parametric LOD-score of 3.31 was observed for a 4.4 Mb interval on chromosome 12. Upon sequencing, a non-synonymous variant in KERA (c.920C>G; p.Ser307Cys) was identified. The variant was absent from nearly 28,000 individuals, including 2,571 patients with premature atherosclerosis. KERA, a proteoglycan protein, was expressed in lipid-rich areas of human atherosclerotic lesions, but not in healthy arterial specimens. Moreover, KERA expression in plaques was significantly associated with plaque size in a carotid-collar Apoe^−/− mice (r² = 0.69; p<0.0001).

Conclusion

A rare variant in KERA was identified in a large kindred with premature atherosclerosis. The identification of KERA in atherosclerotic plaque specimen in humans and mice lends support to its potential role in atherosclerosis.     

Period2 Deficiency Blunts Hypoxia-Induced Mobilization and Function of Endothelial Progenitor Cells

Background

In the clinic, variations in circadian rhythm are evident in patients with cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. In this study, we focused on the role of the circadian gene period2 (per2) in mobilization and function of endothelial progenitor cells (EPCs) in vitro and in vivo after myocardial infarction (MI) in mice.

Methods and Results

MI was produced by surgical ligation of the left anterior descending coronary artery in mice with and without per2 deficiency. Trans-thoracic echocardiography was used to evaluate cardiac function in mice. Per2^−/− mice with MI showed decreased cardiac function and increased infarct size. The number of CD34+ cells and capillary density were decreased in the myocardium of per2^−/− mice on immunohistochemistry. Flow cytometry revealed decreased number of circulating EPCs in per2^−/− mice after MI. In vitro, per2^−/− EPCs showed decreased migration and tube formation capacity under hypoxia. Western blot analysis revealed inhibited activation of extracellular signal-regulated kinase and Akt signaling in the bone marrow of per2^−/− mice and inhibited PI3K/Akt expression in per2^−/− EPCs under hypoxia.

Conclusions

Per2 modulates EPC mobilization and function after MI, which is important to recovery after MI in mice.     

Reduced VLDL clearance in Apoe−/−Npc1−/− mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe^−/−Npc1^−/− mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe^−/−Npc1^−/− liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe^−/−Npc1^−/− liver was unexpected. However, several other LXR target genes also increased in Apoe^−/−Npc1^−/− liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe^−/− mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe^−/−Npc1^−/− mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.     

Selective Deletion of the A1 Adenosine Receptor Abolishes Heart-Rate Slowing Effects of Intravascular Adenosine In Vivo

Objective

Intravenous adenosine induces temporary bradycardia. This is due to the activation of extracellular adenosine receptors (ARs). While adenosine can signal through any of four ARs (A1AR, A2AAR, A2BAR, A3AR), previous ex vivo studies implicated the A1AR in the heart-rate slowing effects. Here, we used comparative genetic in vivo studies to address the contribution of individual ARs to the heart-rate slowing effects of intravascular adenosine.

Methods and Results

We studied gene-targeted mice for individual ARs to define their in vivo contribution to the heart-rate slowing effects of adenosine. Anesthetized mice were treated with a bolus of intravascular adenosine, followed by measurements of heart-rate and blood pressure via a carotid artery catheter. These studies demonstrated dose-dependent slowing of the heart rate with adenosine treatment in wild-type, A2AAR^−/−, A2BAR^−/−, or A3AR^−/− mice. In contrast, adenosine-dependent slowing of the heart-rate was completely abolished in A1AR^−/− mice. Moreover, pre-treatment with a specific A1AR antagonist (DPCPX) attenuated the heart-rate slowing effects of adenosine in wild-type, A2AAR^−/−, or A2BAR^−/− mice, but did not alter hemodynamic responses of A1AR^−/− mice.

Conclusions

The present studies combine pharmacological and genetic in vivo evidence for a selective role of the A1AR in slowing the heart rate during adenosine bolus injection.     

MCP/CCR2 Signaling Is Essential for Recruitment of Mesenchymal Progenitor Cells during the Early Phase of Fracture Healing

Objective

The purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models.

Methods

The expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1^−/− and C-C chemokine receptor 2 (CCR2)^−/− mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1^−/− mice and were evaluated by histological staining and micro-CT.

Results

MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1^−/− and CCR2^−/− mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1^−/− mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1^−/− mouse. Conversely, transplantation of MCP-1^−/− mouse-derived grafts into host WT mice markedly decreased new bone formation.

Conclusions

MCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.