Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice

Emerging evidence suggests that transforming growth factor-beta (TGF-beta) is an important mediator of diabetic nephropathy. We showed previously that short-term treatment with a neutralizing monoclonal anti-TGF-beta antibody (alphaT) in streptozotocin-diabetic mice prevents early changes of renal hypertrophy and increased matrix mRNA. To establish that overactivity of the renal TGF-beta system mediates the functional and structural changes of the more advanced stages of nephropathy, we tested whether chronic administration of alphaT prevents renal insufficiency and glomerulosclerosis in the db/db mouse, a model of type 2 diabetes that develops overt nephropathy. Diabetic db/db mice and nondiabetic db/m littermates were treated intraperitoneally with alphaT or control IgG, 300 microgram three times per week for 8 wk. Treatment with alphaT, but not with IgG, significantly decreased the plasma TGF-beta1 concentration without decreasing the plasma glucose concentration. The IgG-treated db/db mice developed albuminuria, renal insufficiency, and glomerular mesangial matrix expansion associated with increased renal mRNAs encoding alpha1(IV) collagen and fibronectin. On the other hand, treatment with alphaT completely prevented the increase in plasma creatinine concentration, the decrease in urinary creatinine clearance, and the expansion of mesangial matrix in db/db mice. The increase in renal matrix mRNAs was substantially attenuated, but the excretion of urinary albumin factored for creatinine clearance was not significantly affected by alphaT treatment. We conclude that chronic inhibition of the biologic actions of TGF-beta with a neutralizing monoclonal antibody in db/db mice prevents the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes.     

Decreased matrix degradation in diabetic nephropathy: effects of ACE inhibition on the expression and activities of matrix metalloproteinases

Aims/hypothesis: Extracellular matrix accumulation is thought to be involved in the pathogenesis of diabetic nephropathy. Increased matrix synthesis has been well documented but the effects of diabetes on degradative pathways, particularly in the in vivo setting, have not been fully explored. Furthermore, the effect of renoprotective therapies on matrix accumulation through these pathways has not been examined. We investigated the degradative pathway of type IV collagen and the effects of ACE inhibition in experimental diabetic nephropathy. Methods: Diabetes was induced in 16 rats by administrating streptozocin; 8 of the diabetic rats were allocated at random to receive the ACE inhibitor perindopril (2 mg/l) in their drinking water and 8 age and weight matched rats served as controls. Gene expression of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) was measured by RT-PCR and type IV collagen content by immunohistochemistry. MMP activities were determined by degradation of a radiolabelled substrate and by zymography. Results: Six months of diabetes was associated with a decrease in mRNA and enzymatic activity of MMP-9 (21 % and 51 % respectively, p < 0.05 vs control) and a 51 % increase in TIMP-1 mRNA (p < 0.05 vs control). By contrast, MMP-2 mRNA was increased but its activity decreased (43 % and 43 % respectively, p < 0.05 vs control). Total degradative capacity of kidney tissue from diabetic rats was also lower (Control: 48 ± 7 %, Diabetic: 33 ± 6 %, p < 0.05). Activation of latent MMPs with amino-phenylmercuric acetate increased matrix degradation by two-fold. However the relative decrease associated with experimental diabetes still remained. All diabetes-associated changes in MMP and TIMP mRNA and activities were attenuated by perindopril treatment in association with reduced type IV collagen accumulation. Conclusions/interpretation: These results indicate that the impairment of matrix degradation contributes to matrix accumulation in diabetic nephropathy and that the beneficial effects of ACE inhibition could in part be mediated by modulation of changes in matrix degradative pathways. [Diabetologia (2002) 45: 268–275] Received: 13 June 2001 and in revised form: 14 September 2001     

Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli

Aims/hypothesis We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-β1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells. Methods THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/−THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA. Results In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF. Conclusions/interpretation Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target. N. A. Wahab and L. Schaefer made an equal contribution to the work reported in this paper     

伊贝沙坦对大鼠糖尿病模型肾脏肥大和肾小球毛细血管基底膜厚度的影响

目的 观察伊贝沙坦对大鼠糖尿病模型肾脏肥大和肾小球毛细血管基底膜厚度的影响。方法 将SD大鼠分为糖尿病肾病组(A组)、伊贝沙坦治疗组(B组)、健康对照组(C组),A和B组大鼠制成糖尿病模型,B组予以50mg／kg伊贝沙坦灌胃。观察第4、8、12周大鼠的血糖、体重、尿白蛋白、24h尿蛋白的改变及第12周时的肌酐清除率(Ccr)、肾重、肾脏肥大指数、肾组织总蛋白含量、肾小球面积和体积、肾小球毛细血管基底膜(GBM)厚度的改变。通过免疫组化观察肾结缔组织生长因子(CTGF)和转化生长因子(TGF)-βl的表达。结果 A组和B组大鼠血糖较C组明显升高且维持在一个较高水平(P＜0．01)。A组大鼠的体重较C组明显下降,B组有所增加(P＜0．05)。随时间的推移(第4、8、12周),A组大鼠尿蛋白、尿白蛋白逐渐增加,B组明显减少(P＜0．01)。A组大鼠Ccr较C组显著升高(P＜0．01),B组Ccr明显下降(P＜0．05)。至第12周时,A组大鼠肾脏重量、肾脏肥大指数、肾组织总蛋白含量、肾小球面积和体积均较C组明显增加(P＜0．01),B组均较A组降低(P＜0．01,P＜0．05)。免疫组化半定量分析显示,A组大鼠CTGF与TGF-βl的表达均高于C组(P＜0．01),B组明显低于A组(P＜0．01,P＜0．05)。A组大鼠GBM较C组明显增厚(P＜0．01),B组较A组明显变薄(P＜0．01）。肾小球CTGF、TGF—βl的表达与肾脏体积呈正相关(r＝0．83,r＝0．83;P＜0．05)。结论 早期应用伊贝沙坦可抑制糖尿病大鼠早期肾脏肥大和CTGF表达等。     

Caveolin-1 deficiency protects against mesangial matrix expansion in a mouse model of type 1 diabetic nephropathy

Aims/hypothesis

Glomerular matrix protein accumulation, mediated largely by resident mesangial cells (MCs), is central to the pathogenesis of diabetic nephropathy. We previously showed that caveolin (CAV)-1/caveolae mediate matrix upregulation by MCs in response to high glucose and TGFβ, two important pathogenic mediators of diabetic glomerular sclerosis. Here, we evaluated the in vivo role of CAV-1/caveolae in the development of diabetic nephropathy.

Methods

Diabetes was induced in Cav1-knockout (KO) mice and their wild-type (WT) counterparts by streptozotocin injection. After 10 months, kidneys were evaluated for the development of nephropathy, including glomerular sclerosis and upregulation of matrix proteins. Parallel experiments assessing glucose-induced matrix upregulation were carried out in MCs isolated from KO mice.

Results

KO diabetic mice developed hyperglycaemia and renal hypertrophy, but were protected from developing albuminuria and glomerular sclerosis compared with WT mice. KO mice were significantly protected from the upregulation of glomerular collagen I, fibronectin, connective tissue growth factor (CTGF) and TGFβ. In vitro, glucose induced collagen I A1 promoter activation and collagen I, fibronectin and CTGF protein upregulation in WT but not KO MCs. Re-expression of Cav1 in KO cells restored this response.

Conclusions/interpretation

Cav1 deletion rendered significant protection from glomerular matrix accumulation and albuminuria in a mouse model of type 1 diabetes. These studies provide a foundation for the development of renal-targeted interference with CAV-1/caveolae as a novel approach to the treatment of diabetic nephropathy.     

Thyroid hormone ameliorates diabetic nephropathy in a mouse model of type II diabetes

Conventional therapies for diabetic patients, such as strict glycemic control, do not completely stop the progression of diabetic nephropathy. Serum-free tri-iodothyronine (T?) levels were lower in patients with type II diabetes. The purpose of this study was to test a hypothesis that treatment with T? would improve diabetic nephropathy in db/db mice, a model of type II diabetes. Male db/db mice (16 weeks) were treated with T? for 4 weeks. Urinary excretions of albumin and blood glucose levels were measured. Kidneys were collected for histological examination and molecular assays of transforming growth factor-β1 (TGF-β1) expression and phosphatidylinositol 3-kinase (PI3K). T? attenuated albuminuria in db/db mice, suggesting an improved kidney function. T? significantly decreased accumulation of collagenous components in cortical interstitium (interstitial fibrosis) and expansion of mesangial matrix in glomeruli (glomerulosclerosis) and prevented the loss of glomeruli in db/db mice. Therefore, T? improved the renal structural damage seen in diabetic mice. Notably, diabetic nephropathy was accompanied by a significant decrease in PI3K activity and an increase in TGF-β1 expression in kidneys. T? restored renal PI3K activity, attenuated hyperglycemia, and decreased renal TGF-β1 expression in db/db mice. These effects of T? were abolished by simultaneous treatment with PI3K inhibitor (LY294002). These data suggest that T? prevented progressive kidney damage and remodeling in db/db mice by improving insulin signaling (e.g. PI3K activity).     

Renal connective tissue growth factor induction in experimental diabetes is prevented by aminoguanidine

The aim of this study was to determine whether aminoguanidine (AG), an inhibitor of advanced glycation, prevents expression of the profibrotic cytokine, connective tissue growth factor (CTGF), as well as accumulation of the previously reported CTGF-dependent matrix protein, fibronectin, in a model of experimental diabetic nephropathy. Diabetic animals were randomly allocated into groups receiving 32 wk of AG or vehicle. Diabetic rats showed increases in CTGF mRNA and protein expression as well as in advanced glycation end-product (AGE) and fibronectin immunostaining, compared with nondiabetic rats. In the diabetic kidney, the increase in CTGF gene and protein expression as well as expression of the extracellular matrix protein fibronectin were prevented by AG. To further explore the relationship between AGEs and mesangial CTGF and fibronectin production, cultured human mesangial cells were exposed in vitro to soluble AGE-BSA and carboxymethyl lysine-BSA, and this led to induction of both CTGF and fibronectin. On the basis of our in vitro findings in mesangial cells linking AGEs to CTGF expression, the known prosclerotic effects of CTGF, and the ability of AG to attenuate mesangial expansion, it is postulated that the antifibrotic effects of AG in this animal model may be partially mediated by CTGF.     

High glucose alters matrix metalloproteinase expression in two key vascular cells: potential impact on atherosclerosis in diabetes

Diabetes is a major risk factor for atherosclerosis. Hyperglycemia is an underlying contributing factor; however, the mechanisms that mediate the vascular complications are not yet fully understood. In the present study, we provide evidence that elevated glucose induces discordant matrix metalloproteinase (MMP) expression from two key vascular cells, endothelial cells and macrophages. Our results clearly indicate that high glucose (25 mM) induced endothelial cell expression and activity of the collagenase, MMP-1 and the gelatinase, MMP-2, whilst reducing expression of the stromelysin, MMP-3 (P<0.05). Similarly, our results show that high glucose (25 mM) induces expression and activity of MMP-9 from monocyte-derived macrophages (P<0.05). High glucose culture did not affect metalloproteinase inhibitor (TIMP-1) expression. Our results suggest for the first time that high glucose exposure induced discordant regulation of the MMP/TIMP system in vascular cells. The increased MMP-1, MMP-2 and MMP-9 activities induced by high glucose exposure could promote matrix degradation thereby accelerating atherogenesis and potentially reducing plaque stability in diabetes.     

金属蛋白酶-2和金属蛋白酶组织抑制物-1与糖尿病肾病关系的实验研究

目的　观察链脲佐菌素 (STZ)实验性糖尿病大鼠肾脏基质金属蛋白酶 2 (MMP 2 )及金属蛋白酶组织抑制物 1(TIMP 1)蛋白的表达及功能、形态学改变 ,探讨MMP 2、TIMP 1在糖尿病肾病(DN)发生机制中的意义。　方法　 2 0只雄性Wistar大鼠随机分为糖尿病组和正常对照组 ,分别于第1、2、4、6、8周测定尿白蛋白排泄率 (UAER) ,第 9周用Western印迹方法检测MMP 2、TIMP 1蛋白表达水平 ,电镜观察肾小球基底膜厚度 (GBMT)。　结果　糖尿病组较正常组TIMP 1蛋白表达明显增加 ,MMP 2蛋白表达显著降低 (P <0 .0 1)。 4、6、8周UAER显著增加 ,GBMT明显增厚 (P <0 .0 1)。电镜发现糖尿病组肾小球基底膜弥慢性增厚 ,局部有系膜细胞插入和双轨征。　结论　持续高血糖可使大鼠肾脏MMP 2下降 ,TIMP 1增加。导致肾小球基底膜增厚 ,UAER增加。MMP 2、TIMP 1失衡可能对糖尿病肾病功能和形态学改变有重要意义。     

Effects of high glucose and thiamine on the balance between matrix metalloproteinases and their tissue inhibitors in vascular cells

Pericyte survival in diabetic retinopathy depends also on interactions with extracellular matrix (ECM) proteins, which are degraded by matrix metalloproteinases (MMP). Elevated glucose influences ECM turnover, through expression of MMP and their tissue inhibitors, TIMP. We reported on reduced pericyte adhesion to high glucose-conditioned ECM and correction by thiamine. We aimed at verifying the effects of thiamine and benfotiamine on MMP-2, MMP-9 and TIMP expression and activity in human vascular cells with high glucose. In HRP, MMP-2 activity, though not expression, increased with high glucose and decreased with thiamine and benfotiamine; TIMP-1 expression increased with high glucose plus thiamine and benfotiamine; MMP-9 was not expressed. In EC, MMP-9 and MMP-2 expression and activity increased with high glucose, but thiamine and benfotiamine had no effects; TIMP-1 expression was unchanged. Neither glucose nor thiamine modified TIMP-2 and TIMP-3 expression. TIMP-1 concentrations did not change in either HRP or EC. High glucose imbalances MMP/TIMP regulation, leading to increased ECM turnover. Thiamine and benfotiamine correct the increase in MMP-2 activity due to high glucose in HRP, while increasing TIMP-1.     

Ferulic acid prevents pathological and functional abnormalities of the kidney in Otsuka Long-Evans Tokushima Fatty diabetic rats

We investigated the preventive effects of ferulic acid (FA) and alpha-tocopherol (AT) on the progression of diabetic nephropathy. Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats were used as type 2 diabetes and non-diabetes models, respectively. Two-thirds of the OLETF rats were fed 0.2% FA-containing or 0.5% AT-containing chow. Diabetic nephropathy was assessed based on urinary protein excretion and pathological changes which were scored based on the percentages of extracellular matrix area in the glomerular area. Furthermore, renal messenger RNA (mRNA) expression of intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-beta1) was quantified by real-time polymerase chain reaction. After 12 weeks of FA- or AT-supplementation, urinary protein in untreated-OLETF group was significantly higher than that in LETO group, thus FA-supplementation significantly decreased urinary protein excretion. Pathological scores in FA-supplemented group were significantly lower than those in untreated OLETF group. Supplementation with either FA or AT significantly prevented the elevation of TGF-beta1 mRNA expression caused by diabetes. Treatment with neither FA nor AT had a significant effect on COX-2 or ICAM-1 mRNA expressions. We have demonstrated the preventative effects of FA on diabetic nephropathy via suppression of TGF-beta1 upregulation, furthermore FA may be more potent than AT.     

Systemic delivery of TNF-related apoptosis-inducing ligand (TRAIL) elevates levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and prevents type 1 diabetes in nonobese diabetic mice

Recent studies have demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) is a modulator of the immune response. The relation between TRAIL and type 1 diabetes (T1D) as an autoimmune inflammatory disease in vivo is relatively unknown. To explore the potential role of TRAIL in the development of T1D, we examined its in vivo effects in nonobese diabetic (NOD) mice. NOD mice at 7 wk of age were iv injected with an adenovirus carrying either human TRAIL (Ad.hTRAIL) or β-galactosidase genes. Blood glucose was monitored weekly, and the expression of hTRAIL was evaluated in plasma and liver of mice. To investigate whether hTRAIL elicits its effect through the induction of tissue inhibitor of metalloproteinase-1 (TIMP-1), we examined the concentration of plasma TIMP-1 by ELISA and the inhibition of matrix metalloproteinase (MMP) by gelatin zymography. Here, we show that Ad.hTRAIL-transduced mice had significantly reduced blood glucose levels and markedly increased production of TIMP-1 compared with control β-galactosidase animals. Pancreatic tissue isolated from Ad.hTRAIL-treated NOD mice showed reduced MMP activities associated with significantly improved insulitis. In addition, TIMP-1 in vitro suppressed cytokine-induced apoptosis in insulin-producing INS-1 cells. These results indicate that T1D can be prevented by TRAIL overexpression through enhancement of TIMP-1 function. Elevated TIMP-1 production inhibits the activity of MMPs, which may contribute to suppress the transmigration of diabetogenic T cells into the pancreatic islets and protects pancreatic β-cells from cytokine-induced apoptosis. Therefore, TRAIL and TIMP-1 induction may be potential targets to prevent development of T1D.     

Lisofylline,a novel antiinflammatory compound,protects mesangial cells from hyperglycemia- and angiotensin II-mediated extracellular matrix deposition

Chronic elevated glucose levels and activation of the renal renin-angiotensin system have been implicated in the pathogenesis of diabetic nephropathy. We tested the ability of lisofylline (LSF), a novel antiinflammatory compound, to prevent extracellular matrix (ECM) accumulation and growth factor production by human mesangial cells (HMCs) cultured in chronic elevated glucose (HG) or angiotensin II (AngII). HMCs were cultured in normal glucose (NG) (5.5 mm) and in HG (25 mm) for 7 d or with 10-7 m AngII for 4 h with or without LSF. Levels of the ECM protein fibronectin and TGF-beta in media were shown to increase in HG compared with NG. LSF decreased HG-induced fibronectin and TGF-beta production to control levels. Increased expression of collagen type IV and laminin was observed in AngII-cultured HMCs. LSF protected HMCs from the AngII induction of these key matrix proteins. cAMP-responsive binding element phosphorylation was significantly higher in both HG and AngII-cultured HMCs. LSF reduced phosphorylation of both cAMP-responsive binding element and p38 MAPK compared with control. These data demonstrate that LSF protects HMCs from HG- and AngII-mediated ECM deposition by the reduction of matrix protein secretion possibly through regulation of TGF-beta production and modulation of the p38 MAPK pathway. These results suggest that LSF may provide therapeutic benefit for prevention or treatment of diabetic nephropathy.     

Connective tissue growth factor is up-regulated in the diabetic retina: amelioration by angiotensin-converting enzyme inhibition

Connective tissue growth factor (CTGF) has been postulated to have prosclerotic and angiogenic properties. The aim of this present study was to characterize retinal CTGF expression in the absence and presence of diabetes and in the context of treatment with the angiotensin-converting enzyme (ACE) inhibitor, perindopril. Retinas were obtained from control, diabetic, and diabetic plus perindopril-treated (3 mg/d) rats. CTGF gene expression was quantitated by RT-PCR and localized by in situ hybridization. CTGF protein expression was analyzed by Western blotting and localized by immunohistochemistry. Diabetes was associated with a greater than 2-fold increase in CTGF mRNA levels, which was attenuated by perindopril treatment. CTGF immunoreactivity was increased almost 2-fold in diabetes and was ameliorated by the ACE inhibitor perindopril. By in situ hybridization and immunohistochemistry, the major site of CTGF gene expression in the retina of diabetic rats was the ganglion cell layer. Based on the known in vivo effects of CTGF, it is postulated that this growth factor plays a pivotal role in mediating diabetes-associated retinal pathology. Furthermore, the protective effects of ACE inhibitors on retinal pathology may partly be mediated via effects on retinal CTGF expression.     

基质金属蛋白酶-9和金属蛋白酶组织抑制物-1在IgA肾病肾组织中的表达

目的 探讨基质金属蛋白酶－9（MMP－9）／金属蛋白酶组织抑制物－1（TIMP－1）系统在IgA肾病肾组织中的表达及其对IgA肾病的进展的影响。方法 采用免疫组织化学和原位杂交技术,分别在蛋白质和基因水平检测38例IgA肾病患者肾组织中的MMP－9和TIMP－1的变化。结果 MMP－9在正常肾脏肾小球的脏层上皮细胞和内皮细胞有少量表达,在肾小管上皮细胞和间质血管壁也有少量表达;在IgA肾病中,MMP－9在系膜增殖性肾小球和间质血管壁的表达均明显增多（P＜0．001）,而在硬化肾小球内的表达则明显减少,肾小管细胞的MMP－9表达无明显变化。TIMP－1在正常肾组织中不能检出,在IgA肾病患者具有系膜增殖性病变的肾小球中有微量表达,在增殖在很重但尚未完全硬化的肾小球内表达增多,在肾小管间质表达最为明显（P＜0．001）,其主要见于肾小管细胞、间质细胞和血管内皮细胞。肾组织中的TIMP－1表达与血清肌酐水平呈显著相关（P＜0．05）,与肾小管间质的纤维化和炎细胞浸润程度亦明显相关（P值均＜0．01）。肾小球中的MMP－9表达与尿蛋白无明显相关性,但与血清肌酐水平呈显著负相关（P＜0．05）。结论 MMP－9和TIMP－1的异常表达可能是影响IgA肾病进展的因素之一。     

The effects of high glucose and atorvastatin on endothelial cell matrix production.

BACKGROUND: Statins are known to enhance atherosclerotic plaque stability through influences on extracellular matrix homeostasis. Net matrix production reflects the relative balance of matrix production and degradation through enzymes such as matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of MMP (TIMPs). The effects of statins on endothelial cell production of these parameters following co-exposure with a proatherogenic stimulus such as high glucose are not known. METHODS: Human endothelial cells were exposed for 72 h to 5 mm (control) or 25 mm (high) glucose +/- atorvastatin (1 micromol/l). Extracellular matrix homeostasis was assessed by measuring matrix metalloproteinase (MMP)-2 secretion, tissue inhibitor of MMP (TIMP)-1 and -2 secretion and net collagen IV production. Results were expressed as percentage +/- SEM of control values. RESULTS: Exposure to high glucose increased cellular collagen IV expression to 190.1 +/- 11.7% (P < 0.0001) of control levels. No change in MMP-2 secretion (111.6 +/- 5.2%; P > 0.05) was observed but both TIMP-1 and TIMP-2 expression were increased to 136.3 +/- 6.4% and 144.0 +/- 27.5%, respectively (both P < 0.05). The presence of atorvastatin in high glucose conditions reduced collagen IV expression to 136.1 +/- 20.6%. This was paralleled by increased secretion of MMP-2 to 145.8 +/- 7.8% (P < 0.01), increased TIMP-2 expression to 208.0 +/- 21.3% (P < 0.005 compared with high glucose) but no change in TIMP-1 expression (155.1 +/- 14.6%) compared with high glucose alone. The presence of atorvastatin in control conditions did not affect levels of collagen IV expression (114.5 +/- 13.2%). CONCLUSIONS: Endothelial cell exposure to high glucose was associated with a MMP/TIMP profile that increased extracellular matrix production which was attenuated by concurrent exposure to atorvastatin. Consequently, a mechanism by which the atherosclerotic plaque regression that is observed in patients taking these drugs has been demonstrated.     

Glucose-induced TGF-beta1 and TGF-beta receptor-1 expression in vascular smooth muscle cells is mediated by protein kinase C-alpha

Sclerosis and increased matrix expression in diabetes are mediated by glucose-induced transforming growth factor (TGF)-beta1 expression. The intracellular effects of high glucose occur at least in part by way of protein kinase C (PKC). We previously described a role for PKC-alpha in glucose-induced permeability. We now investigated the hypothesis that glucose-induced expression of TGF-beta1 and its receptors (TGF-beta-R1 and -R2) are mediated by activation of this PKC isoform. TGF-beta1 and TGF-beta-R expressions were determined in vascular smooth muscle cells (VSMCs) by immunocytochemistry and Western blotting. PKC isoforms were assessed by confocal microscopy. PKC isoforms were inhibited with antisense oligodeoxynucleotides. PKC-alpha was upregulated by overexpression or microinjection. High glucose (20 mmol/L) increased VSMC TGF-beta1 and TGF-beta-R1 expression but not TGF-beta-R2 expression. PKC inhibitors and specific PKC-alpha downregulation by antisense treatment prevented this effect, whereas antisense treatment against PKC-beta, -epsilon, and -zeta had no influence. PKC-alpha overexpression increased TGF-beta1 and TGF-beta-R1 expression but not TGF-beta-R2 expression. PKC-alpha microinjection into individual VSMCs also increased TGF-beta1 and TGF-beta-R immunofluorescence. Last, VSMCs from PKC-alpha-deficient mice did not respond to high glucose compared with VSMCs from wild-type mice. We propose that high glucose-induced TGF-beta1 and TGF-beta-R1 expression is mediated by PKC-alpha. Our findings suggest an autocrine feedback mechanism and a possible role for PKC-alpha in diabetic vascular disease.