Masaki Fujimoto

Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy

Abstract Transforming growth factor-β (TGF-β) has been implicated in the development of diabetic glomerulopathy. In order to evaluate a role of Smad3, one of the major signaling molecules downstream of TGF-β, in the pathogenesis of diabetic glomerulopathy, Smad3-null mice were made diabetic with streptozotocin injection and analyzed 4 weeks after induction of diabetes. Electron microscopy revealed that the thickness of glomerular basement membrane (GBM) in wild-type diabetic mice was significantly higher than that in non-diabetic mice, whereas no appreciable GBM thickening was found in Smad3-null diabetic mice. Urinary albumin excretion was dramatically increased in wild-type diabetic mice, whereas Smad3-null diabetic mice did not show any overt albuminuria. Northern blotting revealed that mRNA levels of fibronectin and α3 chain of type IV collagen (α3Col4) in renal cortex of wild-type diabetic mice were approximately twice as much as those of non-diabetic mice, whereas their mRNA levels were not increased in Smad3-null diabetic mice. Real-time polymerase chain reaction (PCR) also confirmed diabetes-induced upregulation of fibronectin and α3Col4 in glomeruli of wild-type mice. Glomerular expression of TGF-β1, as assessed by real-time PCR, was enhanced to a similar degree in wild-type and smad3-null diabetic mice, indicating that the observed differences between wild-type and Smad3-null mice are not attributable to difference in the expression of TGF-β1. These data clearly demonstrate a critical role of Smad3 in the early phase of diabetic glomerulopathy. This may be due at least partly to the present findings that diabetes-induced upregulation of fibronectin and α3Col4 is dependent on Smad3 function.

High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells

Objective—Osteopontin is upregulated in the diabetic vascular wall and in vascular smooth muscle cells cultured under high glucose concentration. In the present study, we analyzed the mechanism of high glucose-induced upregulation of osteopontin in cultured rat aortic smooth muscle cells. Methods and Results—We found that an inhibitor of Rho-associated protein kinase, Y-27632, suppressed osteopontin mRNA expression under high glucose concentration. Transfection of cells with a constitutive active Rho mutant, pSR&agr;-myc-RhoDA, enhanced osteopontin mRNA expression. Furthermore, incubation of cells under high glucose concentration activated Rho, indicating that Rho/Rho kinase pathway mediates high-glucose–stimulated osteopontin expression. Treatment of cells with an inhibitor of protein kinase C, GF109203X, and azaserine, an inhibitor of the hexosamine pathway, suppressed high glucose-induced Rho activation. Glucosamine treatment was shown to activate Rho. Treatment of cells with an inhibitor of MEK1, PD98059, suppressed osteopontin mRNA expression under high glucose concentration. Incubation of cells under high glucose concentration activated ERK. Finally, transfection of cells with pSR&agr;-myc-RhoDA also activated ERK. Conclusions—In conclusion, our present findings support a notion that Rho/Rho kinase pathway functions downstream of protein kinase C and the hexosamine pathways and upstream of ERK in mediating high-glucose–induced upregulation of osteopontin expression.

Targeted Disruption of TGF-β–Smad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascular Injury

The role of transforming growth factor (TGF)-&bgr; and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-&bgr;, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-&bgr; caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-&bgr; in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-&bgr;. TGF-&bgr; increased transcript level of &agr;2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-&bgr; were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

CCN3 Inhibits Neointimal Hyperplasia Through Modulation of Smooth Muscle Cell Growth and Migration

Objective—CCN3 belongs to the CCN family, which constitutes multifunctional secreted proteins that act as matrix cellular regulators. We investigated the pathophysiological roles of CCN3 in the vessels. Methods and Results—We examined the effects of CCN3 on the proliferation and migration of rat vascular smooth muscle cells (VSMC). CCN3 knockout mice were created, and vascular phenotypes and neointimal hyperplasia induced by photochemically induced thrombosis were investigated. CCN3 suppressed the VSMC proliferation induced by fetal bovine serum. The neutralizing antibody for transforming growth factor-&bgr; did not affect the growth inhibitory effect of CCN3. Moreover, CCN3 enhanced the mRNA expression of cyclin-dependent kinase inhibitors, p21 and p15. Gamma secretase inhibitor, an inhibitor of Notch signaling, partially inhibited the enhanced expression of p21 induced by CCN3. CCN3 also inhibited the VSMC migration. Finally, the histopathologic evaluation of the arteries 21 days after the endothelial injury revealed a 6-fold enhancement of neointimal thickening in the null mice compared with the wild-type mice. Conclusion—CCN3 suppresses neointimal thickening through the inhibition of VSMC migration and proliferation. Our findings indicate the involvement of CCN3 in vascular homeostasis, especially on injury, and the potential usefulness of this molecule in the modulation of atherosclerotic vascular disease.

The roles of transforming growth factor-β and Smad3 signaling in adipocyte differentiation and obesity.

We aimed at elucidating the roles of transforming growth factor (TGF)-β and Smad3 signaling in adipocyte differentiation (adipogenesis) and in the pathogenesis of obesity. TGF-β/Smad3 signaling in white adipose tissue (WAT) was determined in genetically obese (ob/ob) mice. The effect of TGF-β on adipogenesis was evaluated in mouse embryonic fibroblasts (MEF) isolated both from WT controls and Smad3 KO mice by Oil red-O staining and gene expression analysis. Phenotypic analyses of high-fat diet (HFD)-induced obesity in Smad3 KO mice compared to WT controls were performed. TGF-β/Smad3 signaling was elevated in WAT from ob/ob mice compared to the controls. TGF-β significantly inhibited adipogenesis in MEF, but the inhibitory effects of TGF-β on adipogenesis were partially abolished in MEF from Smad3 KO mice. TGF-β inhibited adipogenesis independent from the Wnt and β-catenin pathway. Smad3 KO mice were protected against HFD-induced insulin resistance. The size of adipocytes from Smad3 KO mice on the HFD was significantly smaller compared to the controls. In conclusion, the TGF-β/Smad3 signaling pathway plays key roles not only in adipogenesis but also in development of insulin resistance.

Influence of C-peptide on early glomerular changes in diabetic mice

C‐peptide has been shown to ameliorate diabetes‐induced functional and structural renal changes in animal models as well as in patients with type 1 diabetes. This study aims to examine the molecular effects of C‐peptide on early glomerular changes in a mouse model of type 1 diabetes.

The reduced form of coenzyme Q10 improves glycemic control in patients with type 2 diabetes: An open label pilot study

Coenzyme Q10 (CoQ10) provides the energy for vital cellular functions and is known to act as an antioxidant. We conducted an open label study to examine the clinical effects of supplementation of the reduced form of CoQ10, ubiquinol, in addition to conventional glucose-lowering agents in patients with type 2 diabetes. Nine subjects (3 males and 6 females) with type 2 diabetes and receiving conventional medication were recruited. The subjects were assigned to receive an oral dose of 200 mg ubiquinol daily for 12 weeks. The effect of ubiquinol on blood pressure, lipid profile, glycemic control, oxidative stress, and inflammation were examined before and after ubiquinol supplementation. In addition, five healthy volunteers were also assigned to receive an oral dose of 200 mg ubiquinol daily for 4 weeks to examine the effects of ubiquinol on insulin secretion. In patients with diabetes, there were no differences with respect to blood pressure, lipid profile, oxidative stress marker, and inflammatory markers. However, there were significant improvements in glycosylated hemoglobin (53.0 ± 4.3 to 50.5 ± 3.7 mmol/mol, P = 0.01) (7.1 ± 0.4 to 6.8 ± 0.4%, P = 0.03). In healthy volunteers, the insulinogenic index (0.65 ± 0.29 to 1.23 ± 0.56, P = 0.02) and the ratio of proinsulin to insulin were significantly improved (3.4 ± 1.8 to 2.1 ± 0.6, P = 0.03). The results of our study are consistent with the suggestion that the supplementation of ubiquinol in subjects with type 2 diabetes, in addition to conventional antihyperglycemic medications, improves glycemic control by improving insulin secretion without any adverse effects

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy

Transforming growth factor-beta (TGF-beta) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-beta(1) signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-beta(1) in cultured mesangial cells. In addition, the expression of TGF-beta type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-beta(1). An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.

A low-grade increase of serum pancreatic exocrine enzyme levels by dipeptidyl peptidase-4 inhibitor in patients with type 2 diabetes

A potential adverse effect of dipeptidyl peptidase-4 inhibitors (DPP-4i) on the pancreas remains controversial. We evaluated the DPP-4i effects on pancreatic amylase and lipase activity in patients with type 2 diabetes. These enzymes were slightly but significantly increased, suggesting DPP-4i cause a low-grade inflammatory change in the exocrine pancreas.

Japanese diabetic patients with Werner syndrome exhibit high incidence of cancer

To the Editor Diabetes is associated with the occurrence and progression of malignant tumors [1, 2]. Werner syndrome (WS), an autosomal recessive disorder classified as a progeroid syndrome, occurs because of mutation of the WRN gene encoding a RecQ-type DNA helicase. Because WS patients often have diabetes and malignant tumors, we initiated a nation-wide epidemiological survey in Japan to clarify the current relationship between the prevalence of diabetes and malignant tumors in them. We sent 6921 survey sheets to hospitals with[200 beds. This survey confirmed 336 new WS patients; detailed clinical data were obtained for 163 patients with diabetes, malignant tumors, or both [3]. These patients were categorized as diabetic (n = 102) and non-diabetic (n = 61). The correlation between diabetes and epithelial tumors (cancers) or non-epithelial tumors was examined using the chi-square test. The proportions of patients grouped according to age were 1.2, 9.8, 23.3, 62.6, and 1.8 % for patients in their 20s, 30s, 40s, 50s, and 60s, respectively. The prevalence of cancers and non-epithelial tumors was 11.7 and 19.0 %, respectively. No significant difference was observed in the morbidity rates of non-epithelial tumors in diabetic or non-diabetic patients (21.6 vs. 21.3 %, p = 0.485). However, diabetic patients showed significantly higher cancer prevalence than non-diabetic patients (16.6 vs. 4.9 %, p = 0.013). The types of malignant tumors in these patients and their prevalence rates are shown in Table 1. The prevalence of non-epithelial tumors and cancers is similar in WS patients [4]; non-epithelial tumors are seldom observed in the general population. Insulin resistance is related to the high cancer prevalence in diabetic patients [5]. Diabetes in WS patients is usually caused by high insulin resistance [6], which may contribute to cancer development. WS patients usually die in their 40s [4]. However, in our study, more than 60 % WS patients were in their 50s, which confirmed that the average life expectancy of Japanese WS patients has increased by 5–10 years. Because cancer incidence has increased with age, aging may be a risk factor for cancer development, especially in diabetic WS patients. Thus, our results demonstrate that diabetic WS patients show high cancer prevalence. It is therefore important to monitor the development of not only non-epithelial tumors but also cancers in these patients, especially when WS is complicated with diabetes. Communicated by Renato Lauro.