Proteomic Analysis of Aorta and Protective Effects of Grape Seed Procyanidin B2 in db/db Mice Reveal a Critical Role of Milk Fat Globule Epidermal Growth Factor-8 in Diabetic Arterial Damage

Background

Atherosclerosis is one of the major complications of type 2 diabetic patients (T2DM), leading to morbidity and mortality. Grape seed procyanidin B2 (GSPB2) has demonstrated protective effect against atherosclerosis, which is believed to be, at least in part, a result of its antioxidative effects. The aim of this study is to identify the target protein of GSPB2 responsible for the protective effect against atherosclerosis in patients with DM.

Methods and Results

GSPB2 (30 mg/kg body weight/day) were administrated to db/db mice for 10 weeks. Proteomics of the aorta extracts by iTRAQ analysis was obtained from db/db mice. The results showed that expression of 557 proteins were either up- or down-regulated in the aorta of diabetic mice. Among those proteins, 139 proteins were normalized by GSPB2 to the levels comparable to those in control mice. Among the proteins regulated by GSPB2, the milk fat globule epidermal growth factor-8 (MFG-E8) was found to be increased in serum level in T2DM patients; the serum level of MFG-E8 was positively correlated with carotid-femoral pulse wave velocity (CF-PWV). Inhibition of MFG-E8 by RNA interference significantly suppressed whereas exogenous recombinant MFG-E8 administration exacerbated atherogenesis the db/db mice. To gain more insights into the mechanism of action of MFG-E8, we investigated the effects of MFG-E8 on the signal pathway involving the extracellular signal-regulated kinase (ERK) and monocyte chemoattractant protein-1 (MCP-1). Treatment with recombinant MFG-E8 led to increased whereas inhibition of MFG-E8 to decreased expression of MCP-1 and phosphorylation of ERK1/2.

Conclusion

Our data suggests that MFG-E8 plays an important role in atherogenesis in diabetes through both ERK and MCP-1 signaling pathways. GSPB2, a well-studied antioxidant, significantly inhibited the arterial wall changes favoring atherogenesis in db/db mice by down-regulating MFG-E8 expression in aorta and its serum level. Measuring MFG-E8 serum level could be a useful clinical surrogate prognosticating atherogenesis in DM patients.     

Regulatory role of NADPH oxidase in glycated LDL-induced upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts and diabetic mice

Cardiovascular disease is the predominant cause of death in diabetic patients. Fibroblasts are one of the major types of cells in the heart or vascular wall. Increased levels of glycated low-density lipoprotein (glyLDL) were detected in diabetic patients. Previous studies in our group demonstrated that oxidized LDL increased the amounts of NADPH oxidase (NOX), plasminogen activator inhibitor-1 (PAI-1), and heat shock factor-1 (HSF1) in fibroblasts. This study examined the expression of NOX, PAI-1, and HSF1 in glyLDL-treated wild-type or HSF1-deficient mouse embryo fibroblasts (MEFs) and in leptin receptor-knockout (db/db) diabetic mice. Treatment with physiologically relevant levels of glyLDL increased superoxide and H₂O₂ release and the levels of NOX4 and p22phox (an essential component of multiple NOX complexes) in wild-type or HSF1-deficient MEFs. The levels of HSF1 and PAI-1 were increased by glyLDL in wild-type MEFs, but not in HSF1-deficient MEFs. Diphenyleneiodonium (a nonspecific NOX inhibitor) or small interfering RNA for p22phox prevented glyLDL-induced increases in the levels of NOX4, HSF1, or PAI-1 in MEFs. The amounts of NOX4, HSF1, and PAI-1 were elevated in hearts of db/db diabetic mice compared to wild-type mice. The results suggest that glyLDL increased the abundance of NOX4 or p22phox via an HSF1-independent pathway, but that of PAI-1 via an HSF1-dependent manner. NOX4 plays a crucial role in glyLDL-induced expression of HSF1 and PAI-1 in mouse fibroblasts. Increased expression of NOX4, HSF1, and PAI-1 was detected in cardiovascular tissue of diabetic mice.     

Attenuation of diabetes-induced cardiac inflammation and pathological remodeling by low-dose radiation

In the present study, novel preventive effects of repeated low-dose radiation exposure on diabetes-induced cardiac inflammation and cardiac damage were investigated. C57BL/6J mice were given multiple low doses of streptozotocin (STZ, 60 mg/kg × 6) to generate type 1 diabetes. A week after the last STZ injection, hyperglycemic mice were diagnosed and treated with and without whole-body low-dose radiation exposure (25 mGy X rays) once every 2 days for 2, 4, 8, 12 and 16 weeks. Diabetes caused significant increases in cardiac inflammation, shown by time-dependent increases in mRNA and protein expressions of interleukin 18 (IL-18), tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule 1 (ICAM-1), plasminogen activator inhibitor 1 (PAI-1), and monocyte chemoattractant protein 1 (MCP-1). Repeated exposure of control mice to low-dose radiation caused mild increases in these inflammatory factors, except for ICAM-1. Repeated exposure of diabetic mice to low-dose radiation significantly reduced diabetes-increased cardiac expression of IL-18, TNF-α, MCP-1 and PAI-1 at both the mRNA and protein levels. Furthermore, cardiac histopathological abnormalities, oxidative damage and fibrosis were significant in diabetic mice but to a lesser extent in diabetic mice with repeated low-dose radiation exposure. These results suggest that although low-dose radiation contributes to mild cardiac inflammation in control mice, it can significantly reduce diabetes-induced cardiac inflammation and associated pathological changes. Therefore, low-dose radiation may potentially become a novel approach to the prevention of diabetic cardiovascular complications.     

The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions

Previous studies have demonstrated the importance of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of diabetic nephropathy in terms of inflammation, but the direct role of the MCP-1/CCR2 system on podocyte apoptosis under diabetic conditions has never been explored. In vitro, mouse podocytes were exposed to a medium containing 30?mM glucose (HG) with or without CCR2 siRNA or CCR2 inhibitor (RS102895). Podocytes were also treated with MCP-1 or TGF-β1 with or without anti-TGF-β1 antibody, CCR2 siRNA, or CCR2 inhibitor. In vivo, 20?db/m and 20?db/db mice were divided into two groups, and ten mice from each group were treated with RS102895. Western blot and Hoechst 33342 or TUNEL staining were performed to identify apoptosis. HG-induced apoptosis and TGF-β1 levels were significantly abrogated by CCR2 inhibition. In addition, treatment with MCP-1 directly induced apoptosis via CCR2. Moreover, TGF-β1- and MCP-1-induced apoptosis were significantly ameliorated by the inhibition of CCR2 and anti-TGF-β1 antibody, respectively. Glomerular expression of cleaved caspase-3 and apoptotic cells within glomeruli were also significantly increased in db/db mice compared to db/m mice, and these increases were significantly attenuated in db/db?+?RS102895 mice. These results suggest that interactions between the MCP-1/CCR2 system and TGF-β1 may contribute to podocyte apoptosis under diabetic conditions.     

Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice

Atherosclerosis is a major complication of diabetes. Up to 16 weeks of age, the db/db mouse is insulin-resistant and hyperglycemic and is a good model of Type 2 diabetes. After approximately 16 weeks of age, the mice develop pancreatic beta cell failure that can progress to a Type 1 diabetes phenotype. We have previously shown that glucose increases production of endothelial 12/15 lipoxygenase (12/15LO) products in vitro. In young 10-week-old Type 2 diabetic db/db mice, we found significant elevations in levels of urinary 12/15LO products, 12S-hydroxyeicosatetraenoic acid (12S-HETE) and 13S-hydroxyoctadecaenoic acid (13S-HODE) in vivo compared with C57BLKS/J mice. Using isolated primary aortic endothelial cells (ECs) from db/db mice and WEHI78/24 mouse monocyte cells in static adhesion assays, we found increased WEHI monocyte adhesion to db/db ECs (14 +/- 2 monocytes/field for db/db ECs versus 4 +/- 1 monocytes/field for C57BLKS/J ECs, p < 0.002). Thus, ECs from db/db mice appear to be "pre-activated" to bind monocytes. Analysis of db/db ECs revealed a 2-fold elevation in 12/15LO protein compared with C57BLKS/J EC. To determine that 12/15LO products were responsible for the increased monocyte adhesion observed with db/db ECs, we inhibited expression of murine 12/15LO using either an adenovirus expressing a ribozyme to 12/15LO (AdRZ) or with the 12/15LO inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. Treatment of db/db ECs for 48 h with AdRZ or 4 h with 10 microm cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate significantly reduced monocyte adhesion to db/db endothelium (p < 0.009). Thus, inhibition of the murine 12/15LO in db/db mice significantly reduced monocyte/endothelial interactions. We also found that adhesion of monocytes to diabetic db/db ECs was mediated by interactions of alpha4beta1 integrin on monocytes with endothelial vascular cell adhesion molecule 1 and connecting segment 1 fibronectin and interactions of beta2 integrins with endothelial intercellular adhesion molecule 1. In summary, regulation of the 12/15LO pathway is important for mediating early vascular changes in diabetes. Modulation of the 12/15LO pathway in the vessel wall may provide therapeutic benefit for early vascular inflammatory events in diabetes.     

RhoA induces expression of inflammatory cytokine in adipocytes

Rho GTPase regulates actin cytoskeleton organization and assembly in many cell types, however, its significance in adipose tissue is not well characterized. Here, we demonstrate high RhoA activity in adipose tissues of C57BL/6J mice. To determine the effect of RhoA activation on 3T3-L1 cells, stable cell lines overexpressing G14VRhoA fused to destabilizing domain of FKBP12 (DD-G14VRhoA-L1) were generated. Treatment of DD-G14VRhoA-L1 cells with Shield1 following their differentiation into adipocytes, resulted in the appearance of thick cortical actin filaments, and increased the mRNA expression levels of plasminogen activator inhibitor type-1 (PAI-1) and monocyte chemoattractant protein-1 (MCP-1). The induction of PAI-1 and MCP-1 was inhibited by treatment with a Rho-associated kinase (ROCK) inhibitor, Y-27632. In 3T3-L1 adipocytes, tumor necrosis factor-α activated RhoA and increased mRNA expression of PAI-1 and MCP-1, and their treatment with Y-27632 partially inhibited these changes. The results indicate that RhoA-ROCK pathway induces inflammatory cytokine expression in adipocytes.     

Supplementation with 9-cis β-carotene-rich alga Dunaliella improves hyperglycemia and adipose tissue inflammation in diabetic mice

Several species of the alga Dunaliella contain high levels of β-carotene. Low dietary β-carotene intake is associated with type 2 diabetes. Dunaliella contains high levels of all-trans and 9-cis isomers of β-carotene and is the best known naturally occurring nutritional source of 9-cis β-carotene. Since vitamin A has been suggested to play a role in glucose and lipid metabolism, we aimed to study the effect of Dunaliella supplementation on diabetes in mice. Ten diabetic db/db mice were fed chow diet fortified with 8 % 9-cis-rich Dunaliella powder. Ten db/db and heterozygous db/+ mice each served as controls and were fed chow diet alone. The control db/db mice developed severe hyperglycemia with fasting glucose levels reaching 400 mg dL^?1. Dunaliella significantly inhibited the elevation of plasma glucose (p?=?0.007). The area under the curve of the glucose tolerance test was 24 % lower in Dunaliella-treated mice than in the control db/db mice. Triglyceride elevation was significantly lower in the Dunaliella group than in the db/db group (p?=?0.007). The mRNA levels of several pro-inflammatory genes in adipose tissue, including monocyte chemotactic protein-1, intercellular adhesion molecule, vascular adhesion molecule-1, receptor-associated protein factor 6, and p-selectin, were elevated in the db/db group as compared to the db/+, whereas their levels were significantly lower in the Dunaliella-treated group. These results suggest that 9-cis β-carotene-rich Dunaliella may inhibit diabetes in db/db mice by reducing inflammation in adipose tissue. This study also emphasizes the importance of β-carotene isomers in our diet.     

Recent insights into diabetic renal injury from the db/db mouse model of type 2 diabetic nephropathy

The db/db mouse is the most widely used animal model of type 2 diabetic nephropathy. Recent studies have utilized genetic backcrossing with transgenic mouse strains to create novel db/db strains that either lack or overexpress specific genes. These novel strains [ICAM-1-/-, CCL2-/-, MKK3-/-, osteopontin-/-, plasminogen activator inhibitor-1 (PAI-1)-/-, endothelial nitric oxide synthase-/-, SOD-Tg, rCAT-Tg] have provided valuable insights into the molecular mechanisms which promote diabetic renal injury. In addition, surgical removal of one kidney has been shown to accelerate injury in the remaining kidney of diabetic db/db mice. A number of novel therapeutic agents have also been tested in db/db mice, including inhibitors of inflammation (chemokine receptor antagonists, anti-CCL2 RNA aptamer, anti-c-fms antibody); oxidative stress (oxykine, biliverdin); the renin-angiotensin-aldosterone system (aliskiren, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, eplerenone); advanced glycation end products (AGE; pyridoxamine, alagebrium, soluble AGE receptor); angiogenesis (NM-3, anti-CXCL12 RNA aptamer, soluble Flt-1); lipid accumulation (statins, farnesoid X receptor agonists, Omacor); intracellular signaling pathways (PKC-β or JNK inhibitors); and fibrosis [transforming growth factor (TGF)-β antibody, TGF-βR kinase inhibitor, soluble betaglycan, SMP-534, CTGF-antisense oligonucleotide, mutant PAI-1, pirfenidone], which have identified potential therapeutic targets for clinical translation. This review summarizes the advances in knowledge gained from studies in genetically modified db/db mice and treatment of db/db mice with novel therapeutic agents.     

Effect of telmisartan on the expression of adiponectin receptors and nicotinamide adenine dinucleotide phosphate oxidase in the heart and aorta in type 2 diabetic rats

ABSTRACT: BACKGROUND: Diabetic cardiovascular disease is associated with decreased adiponectin and increased oxidative stress. This study investigated the effect of telmisartan on the expression of adiponectin receptor 2 (adipoR2) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in the heart and the expression of adiponectin receptor 1 (adipoR1) in aorta in type 2 diabetic rats. METHODS: Type 2 diabetes was induced by high-fat and high-sugar diet and intraperitoneal injection of a low dose of streptozotocin (STZ). Heart function, adipoR2, p22phox, NOX4, glucose transporter 4(GLUT4), monocyte chemoattractant protein-1(MCP-1) and connective tissue growth factor CTGFin the heart, and adipoR1, MCP-1 and nuclear factor kappa B (NF-kappaB) in aorta were analyzed in controls and diabetic rats treated with or without telmisartan (5mg/kg/d) by gavage for 12 weeks. RESULTS: Heart function, plasma and myocardial adiponectin levels, the expression of myocardial adipoR2 and GLUT4 were significantly decreased in diabetic rats (P<0.05). The expression of myocardial p22phox, NOX4, MCP-1, and CTGF was significantly increased in diabetic rats (P<0.05). The expression of adipoR1 was decreased and the expression of MCP-1 and NF-kappaB was increased in the abdominal aorta in diabetic rats (P<0.05). Telmisartan treatment significantly attenuated these changes in diabetic rats (P<0.05). CONCLUSIONS: Our results suggest that telmisartan upregulates the expression of myocardial adiponectin, its receptor 2 and GLUT4. Simultaneously, it downregulates the expression of myocardial p22phox, NOX4, MCP-1, and CTGF, contributing so to the improvement of heart function in diabetic rats. Telmisartan also induces a protective role on the vascular system by upregulating the expression of adipoR1 and downregulating the expression of MCP-1 and NF-kappaB in the abdominal aorta in diabetic rats. KEYWORDS: Telmisartan; Adiponectin receptor; NADPH oxidase; Type 2 diabetic; Cardiac; Aorta.     

Acetylshikonin from Zicao ameliorates renal dysfunction and fibrosis in diabetic mice by inhibiting TGF-β1/Smad pathway

Diabetic nephropathy (DN) is the major cause of end-stage renal disease in diabetic patients. Zicao, a well-known Chinese traditional medicine, has attracted much attention due to its beneficial effects in various medical fields. In this study, we attempted to investigate the effects and mechanisms of action of acetylshikonin, the main ingredient of Zicao, on renal dysfunction in DN. Our results showed that administration with acetylshikonin not only decreased blood urea nitrogen, urine creatinine and the mean kidney-to-body weight ratio in streptozotocin-induced diabetic mice, but also restored the loss of body weight, whereas the blood glucose was not changed. Masson’s trichrome staining showed that acetylshikonin treatment resulted in a marked decrease in kidney fibrosis from diabetic mice. The increased expression of fibrosis proteins, such as plasminogen activator inhibitor type 1 (PAI-1), connective tissue growth factor, and collagen III and IV, were reduced after acetylshikonin administration. In addition, the expressions of interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, intercellular adhesion molecule 1 and infiltration of macrophages in kidney tissues were decreased in acetylshikonin-treated diabetic mice. Acetylshikonin led to a reduction of transforming growth factor-β1 (TGF-β1) expression and Smad-2/3 phosphorylation, as accompanied by increased Smad7 expression. Furthermore, in vitro treatment with acetylshikonin markedly attenuated TGF-β1-induced the PAI-1, collagen III and IV, and Smad-2/3 phosphorylation in HK2 immortalized human proximal tubule epithelial cells. Acetylshikonin also prevented epithelial-to-mesenchymal transition induced by TGF-β1. Collectively, our study provides evidences that acetylshikonin attenuates renal fibrosis though inhibiting TGF-β1/Smad signaling pathway, suggesting that acetylshikonin may be a novel therapeutic agent for the treatment of DN.     

Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice

The hypothesis of this study was that cells of the monocyte/macrophage lineage (Mo/Mp) exhibit an impaired transition from pro-inflammatory to pro-healing phenotypes in wounds of diabetic mice, which contributes to deficient healing. Mo/Mp isolated from excisional wounds in non-diabetic db/+ mice exhibited a pro-inflammatory phenotype on day 5 post-injury, with high level expression of the pro-inflammatory molecules interleukin-1β, matrix metalloprotease-9 and inducible nitric oxide synthase. Wound Mo/Mp exhibited a less inflammatory phenotype on day 10 post-injury, with decreased expression of the pro-inflammatory molecules and increased expression of the alternative activation markers CD206 and CD36. In contrast, in db/db mice, the pro-inflammatory phenotype persisted through day 10 post-injury and was associated with reduced expression of insulin-like growth factor-1, transforming growth factor-β1 and vascular endothelial growth factor. Reduced levels of these growth factors in wounds of db/db mice may have contributed to impaired wound closure, reduced granulation tissue formation, angiogenesis and collagen deposition. The persistent pro-inflammatory wound Mo/Mp phenotype in db/db mice may have resulted from elevated levels of pro-inflammatory interleukin-1β and interferon-γ and reduced levels of anti-inflammatory interleukin-10 in the wound environment. Our findings are consistent with the hypothesis that dysregulation of Mo/Mp phenotypes contributes to impaired healing of diabetic wounds.     

Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy

Monocyte chemoattractant protein (MCP-1) is an important mediator for macrophage recruitment in atherosclerosis and various glomerulonephritis. However, the role of MCP-1 and its receptor CCR2 in the progression of diabetic nephropathy remains unknown. Using a type 1 diabetic nephropathy model that shows noticeable glomerulosclerosis, we examined the role of MCP-1/CCR2 by propagermanium (Pro; CCR2 antagonist) treatment, and confirmed it by transfection of plasmids carrying the 7ND (a mutant of MCP-1) gene. We measured the mesangial matrix expansion, type IV collagen (Col4), transforming growth factor (TGF)-beta1 positive area, and macrophage infiltration in glomeruli after 12 weeks. Mesangial matrix expansion and macrophage infiltration were increased in diabetic mice and inhibited by Pro or 7ND-treatment. Increased glomerular expression of Col4 and TGF-beta1 in diabetic mice was also ameliorated. Thus blocking the MCP-1/CCR2 pathway ameliorated glomerulosclerosis, indicating that the MCP-1/CCR2 pathway plays a crucial role in the progression of diabetic nephropathy.     

烟酰胺核糖抑制db/db小鼠糖尿病心肌病的作用及机制研究

目的:探讨烟酰胺核糖(NR)对2型糖尿病小鼠心肌病的治疗作用及其机制。方法:2型糖尿病模型db/db鼠和及其严格对照小鼠db/+小鼠,将小鼠分为Con (db/+)组,DM (db/db)组,DM+NR组。采用超声测小鼠心脏功能,western-blot及免疫组化测SIRT1表达含量,DHE染色、MDA含量和MnSOD活性检测反映氧化应激水平。结果:与对照组相比,db/db小鼠心脏功能显著下降(LVEF:42.3±7.2vs 73.7±10.2, P0.01;LVFS:22.1±4.2vs 42.7±6.9, P0.01),SIRT1表达量显著下调(P0.01)。NR喂养提高SIRT1表达量(P0.01),并有效改善db/db小鼠心脏功能(LVEF:53.1±8.1vs 42.3±7.2, P0.01;LVFS:33.4±6.9vs 22.1±4.2, P0.01)。同时,NR喂养显著降低了db/db小鼠心肌组织的凋亡水平和氧化应激水平(P0.05)。结论:NR有效改善了db/db小鼠的心功能障碍,降低了db/db小鼠的心肌凋亡水平和氧化应激水平,这些作用的发挥可能与NR增加SIRT1的表达量有关。     

Urokinase-Type Plasminogen Activator-Induced Monocyte Adhesion Is Modulated by Kininogen, Kallikrein, Factor XII, and Plasminogen

Urokinase-type plasminogen activator (u-PA) was found to induce monocyte adhesion through a u-PA receptor (u-PAR)-mediated cAMP-dependent signal transduction pathway (J. Biol. Chem.270, 30282–30285, 1995). In the present study, the effects of kininogen, kallikrein, factor XII, and plasminogen on u-PA-induced monocyte adhesion were examined since these proteins are abundant in plasma and closely related to u-PA in fibrinolysis and inflammation. Monocyte adhesion to a standard plastic surface by u-PA was shown to be inhibited by the activated, two-chain forms of kininogen (HKa) and kallikrein. The latter occurred only at higher, though physiological, concentrations and was dependent on its catalytic activity. Monocyte adhesion was promoted by factor XII and plasminogen via a noncatalytic mechanism. The findings indicated that u-PA-induced monocyte adhesion was downregulated by HKa and kallikrein and upregulated by factor XII and plasminogen at physiological concentrations. Therefore, these contact system proteins may be important modulators of u-PA-induced monocyte adhesion, a process which is involved in many pathophysiological events.