Serum levels of anti-heat shock protein 27 antibodies in patients with chronic liver disease.

Heat shock protein 27 (HSP27), an intracellular molecular chaperone, is involved in the pathogenesis of cancer by promoting both tumor cell proliferation and resistance to therapy. HSP27 is also present in the circulation and circulating HSP27 (sHSP27) can elicit an autoimmune response with production of antibodies. Levels of sHSP27 are enhanced in patients with hepatocellular carcinoma (HCC); it is, however, unknown whether changes in HSP27 antibody levels occur in patients with HCC and can be exploited as a circulating biomarker of HCC. Our aim was to assess the potential association between newly diagnosed HCC and serum anti-HSP27 antibody levels. In this cross-sectional study, anti-HSP27 antibody levels were measured in serum samples from 71 HCC patients, 80 subjects with chronic liver disease, and 38 control subjects by immunoenzymatic assay. Anti-HSP27 antibody levels did not differ significantly among groups. However, in patients with chronic active hepatitis/cirrhosis, anti-HSP27 levels were significantly higher in subjects with a positive history of alcoholism (p = 0.03). Our data do not support the hypothesis that anti-HSP27 antibody levels may help identify patients with HCC among subjects with chronic liver disease. However, our finding that alcohol-related liver disease is associated with higher anti-HSP27 levels is novel and deserves further investigations.

Retinal heat shock protein 25 in early experimental diabetes.

Diabetic retinopathy is the leading cause of blindness in adults, and oxidative stress has been pathogenically associated with retinal neurodegeneration. Cellular stresses induce expression of heat shock proteins (HSPs) and this results in cytoprotection. Our aim was to assess retinal expression of HSP25 in early experimental diabetes. Mice were rendered diabetic by streptozotocin injection. Ten weeks after diabetes onset retinal HSP25 expression were studied by real-time PCR, immunoblotting and immunohistochemistry (IHC). Expression of nitrotyrosine and Cu/Zn superoxide dismutase (SOD), was assessed by IHC and apoptosis by TUNEL. Retinal HSP25 mRNA and protein expression was significantly increased in diabetic as compared to non-diabetic animals and localised predominantly within the retinal ganglion cells (RGC) layer. This was paralleled overexpression of nitrotyrosine and SOD and enhanced apoptosis. In early experimental diabetes, HSP25 is overexpressed in the RGC layer in parallel with markers of oxidative stress and apoptosis.

Stretch reduces nephrin expression via an angiotensin II-AT(1)-dependent mechanism in human podocytes: effect of rosiglitazone.

Increased glomerular permeability to proteins is a characteristic feature of diabetic nephropathy (DN). The slit diaphragm is the major restriction site to protein filtration, and the loss of nephrin, a key component of the slit diaphragm, has been demonstrated in both human and experimental DN. Both systemic and glomerular hypertension are believed to be important in the pathogenesis of DN. Human immortalized podocytes were subjected to repeated stretch-relaxation cycles by mechanical deformation with the use of a stress unit (10% elongation, 60 cycles/min) in the presence or absence of candesartan (1 microM), PD-123319 (1 microM), and rosiglitazone (0.1 microM). Nephrin mRNA and protein expression were assessed using quantitative real-time PCR, immunoblotting, and immunofluorescence, and the protein expression of AT(1) receptor and angiotensin II secretion were evaluated. Exposure to stretch induced a significant approximately 50% decrease in both nephrin mRNA and protein expression. This effect was mediated by an angiotensin II-AT(1) mechanism. Indeed, podocyte stretching induced both angiotensin II secretion and AT(1) receptor overexpression, podocyte exposure to angiotensin II reduced nephrin protein expression, and both the AT-1 receptor antagonist candesartan and a specific anti-angiotensin II antibody completely abolished stretch-induced nephrin downregulation. Similar to candesartan, the peroxisome proliferator-activated receptor (PPAR)-gamma agonist, rosiglitazone, also inhibited stretch-induced nephrin downregulation, suggesting interference with stretch-induced activation of the angiotensin II-AT(1) receptor system. Accordingly, rosiglitazone did not alter stretch-induced angiotensin II secretion, but it prevented AT(1) upregulation in response to stretch. These results suggest a role for hemodynamic stress in loss of nephrin expression and allude to a role of PPAR-gamma agonists in the prevention of this loss.

Effect of the monocyte chemoattractant protein-1/CC chemokine receptor 2 system on nephrin expression in streptozotocin-treated mice and human cultured podocytes.

OBJECTIVE: Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes. RESEARCH DESIGN AND METHODS: Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting. RESULTS: In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin. CONCLUSIONS: These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.

Anti-heat shock protein 27 antibody levels and diabetes complications in the EURODIAB study.

OBJECTIVE: To assess whether serum anti-heat shock protein 27 (HSP27) antibody levels are associated with micro- and macrovascular complications of type 1 diabetes. RESEARCH DESIGN AND METHODS: Anti-HSP27 IgG antibody levels were measured in 531 type 1 diabetic subjects recruited as part of the cross-sectional analysis of the EURODIAB Prospective Complications Study. Case subjects (n = 363) were defined as individuals with one or more diabetes complications and control subjects (n = 168) as individuals with no evidence of any diabetes complication. RESULTS: Anti-HSP27 levels were comparable in case and control subjects (19.6 arbitrary units/ml [11.3-32.7] vs. 20.4 arbitrary units/ml [11.7-35.3], geometric mean [interquartile range]), and there was no correlation between HSP27 and anti-HSP27 levels (r = 0.01, P = 0.81). In logistic regression analysis, anti-HSP27 was not associated with the presence of complications, even after adjustment for main risk factors. CONCLUSIONS: Anti-HSP27 antibody levels are not a marker of vascular complications in type 1 diabetes.

Phytosterol-enriched yogurt increases LDL affinity and reduces CD36 expression in polygenic hypercholesterolemia.

Dietary enrichment with phytosterols (plant sterols similar to cholesterol) is able to reduce plasma cholesterol levels due to reduced intestinal absorption. The aim of this study was to investigate the effect of phytosterol-enriched yogurt consumption on the major serum lipid parameters, low density lipoprotein (LDL) receptor activity, LDL-receptor affinity, and CD36 expression in hypercholesterolemic subjects. Fifteen patients affected by polygenic hypercholesterolemia were evaluated in a single-blind randomized crossover study after a 4 weeks treatment with a phytosterol-enriched yogurt containing 1.6 g esterefied phytosterols (equivalent to 1.0 g free phytosterol). Lipid parameters were compared with a phytosterol-free placebo-controlled diet. The effect of the two treatments on each variable, measured as percentage change, was compared by paired samples t test and covariance analysis. The treatment induced a modest but significant decrease in LDL-cholesterol levels (4.3%, P = 0.03) and a significant increase in high density lipoprotein (HDL) 3-cholesterol (17.1%, P = 0.01). Phytosterol consumption had no effect on LDL-receptor activity whereas patient LDL-receptor affinity significantly increased (9.7%, P = 0.01) and CD36 expression showed a marked significant decrease (18.2%, P = 0.01) in the phytosterol-enriched yoghurt patients. Our data show that the oral administration of a phytosterol-enriched yogurt has modest but significant effects on commonly measured lipid parameters. The improvement of LDL-receptor affinity and the reduction in CD36 expression may reflect an important antiatherogenic effect.

Serum heat shock protein 27 and diabetes complications in the EURODIAB prospective complications study: a novel circulating marker for diabetic neuropathy.

OBJECTIVE: Heat shock protein 27 (HSP27) is a member of the small heat shock protein family of proteins. HSP27 expression is enhanced in target tissues of diabetic microvascular complications, and changes in circulating serum HSP27 levels (sHSP27) have been reported in patients with macrovascular disease. We investigated whether sHSP27 levels were associated with micro- and macrovascular complications in type 1 diabetic patients. RESEARCH DESIGN AND METHODS: A cross-sectional, nested, case-control study from the EURODIAB Prospective Complications Study of 531 type 1 diabetic patients was performed. Case subjects (n = 363) were defined as those with one or more complications of diabetes; control subjects (n = 168) were defined as those with no evidence of any complication. We measured sHSP27 levels and investigated their associations with diabetes complications. RESULTS: Mean sHSP27 levels were significantly higher in case subjects with distal symmetrical polyneuropathy (DSP) than in control subjects, even after adjustment for age and albumin excretion rate (AER) (785.9 vs. 574.7 pg/ml, P = 0.03). In logistic regression analysis, sHSP27 levels in the upper quartile were associated with a twofold increased odds ratio (OR) of DSP, independently of conventional risk factors, markers of inflammation, and AER (OR 2.41 [95% CI 1.11-5.24]). CONCLUSIONS: In this large cohort of type 1 diabetic subjects, we found an independent association between sHSP27 and DSP. This suggests that sHSP27 levels may be a novel marker for diabetic neuropathy.

The monocyte chemoattractant protein-1/cognate CC chemokine receptor 2 system affects cell motility in cultured human podocytes.

In crescentic glomerulonephritis (GN), monocyte chemoattractant protein-1 (MCP-1) is overexpressed within the glomeruli, and MCP-1 blockade has renoprotective effects. Adult podocytes are in a quiescent state, but acquisition of a migratory/proliferative phenotype has been described in crescentic GN and implicated in crescent formation. The cognate CC chemokine receptor 2 (CCR2), the MCP-1 receptor, is expressed by other cell types besides monocytes and has been implicated in both cell proliferation and migration. We investigated whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in cultured podocytes. MCP-1 binding to CCR2 enhanced podocyte chemotaxis/haptotaxis in a concentration-dependent manner and had a modest effect on cell proliferation. Closure of a wounded podocyte monolayer was delayed by CCR2 blockade, and CCR2 was overexpressed at the wound edge, suggesting a role for CCR2 in driving podocyte migration. Immunohistochemical analysis of kidney biopsies from patients with crescentic GN demonstrated CCR2 expression in both podocytes and cellular crescents, confirming the clinical relevance of our in vitro findings. In conclusion, the MCP-1/CCR2 system is functionally active in podocytes and may be implicated in the migratory events triggered by podocyte injury in crescentic GN and other glomerular diseases.

Partial characterisation of the human GFAT promoter: effect of single nucleotide polymorphisms on promoter function.

The 5'-flanking region of the human glutamine:fructose-6-phosphate amidotransferase (GFAT) gene was characterised as a functional active promoter and the GFAT gene contained multiple transcription start sites. A novel single nucleotide polymorphism identified at position -1412 (G to C) had a functional effect on promoter activity and EMSA revealed specific binding of nuclear proteins to this region.

Mechanical stretch induces monocyte chemoattractant activity via an NF-kappaB-dependent monocyte chemoattractant protein-1-mediated pathway in human mesangial cells: inhibition by rosiglitazone.

Hemodynamic abnormalities are important in the pathogenesis of the glomerular damage in diabetes. Glomerular macrophage infiltration driven by the chemokine monocyte chemoattractant protein-1 (MCP-1) is an early event in diabetic nephropathy. The thiazolidinedione rosiglitazone ameliorates albumin excretion rate in diabetic patients with microalbuminuria and has anti-inflammatory properties, raising the possibility of a relationship between its renoprotective and anti-inflammatory activity. Investigated was whether mesangial cell stretching, mimicking in vitro glomerular capillary hypertension, enhances MCP-1 expression and monocyte chemoattractant activity. The effect of the combination of stretch with high glucose on MCP-1 production was studied and, finally, the effect of rosiglitazone on these processes was assessed. Stretching of human mesangial cells significantly enhanced their monocyte chemoattractant activity. This effect was mediated by MCP-1 as it was paralleled by a significant rise in both MCP-1 mRNA and protein levels and was completely abolished by MCP-1 blockade. Combined exposure to both stretch and high glucose further increased MCP-1 production. Stretch activated the IkappaB-NF-kappaB pathway, and NF-kappaB inhibition, with the use of the specific inhibitor SN50, completely abolished stretch-induced MCP-1, indicating that stretch-induced MCP-1 was NF-kappaB dependent. The addition of rosiglitazone significantly diminished stretch-induced NF-kappaB activation, MCP-1 production, and monocyte chemotaxis. In conclusion, stretching of mesangial cells stimulates their monocyte chemoattractant activity via an NF-kappaB-mediated, MCP-1-dependent pathway, and this effect is prevented by rosiglitazone.

GLUT-1 overexpression: Link between hemodynamic and metabolic factors in glomerular injury?

Mesangial matrix deposition is the hallmark of hypertensive and diabetic glomerulopathy. At similar levels of systemic hypertension, Dahl salt-sensitive but not spontaneously hypertensive rats (SHR) develop glomerular hypertension, which is accompanied by upregulation of transforming growth factor beta1 (TGF-beta1), mesangial matrix expansion, and sclerosis. GLUT-1 is ubiquitously expressed and is the predominant glucose transporter in mesangial cells. In mesangial cells in vitro, GLUT-1 overexpression increases basal glucose transport, resulting in excess fibronectin and collagen production. TGF-beta1 has been shown to upregulate GLUT-1 expression. We demonstrated that in hypertensive Dahl salt-sensitive (S) rats fed 4% NaCl (systolic blood pressure [SBP]: 236+/-9 mm Hg), but not in similarly hypertensive SHR (SBP: 230+/-10 mm Hg) or their normotensive counterparts (Dahl S fed 0.5% NaCl, SBP: 145+/-5 mm Hg; and Wistar-Kyoto, SBP: 137+/-3 mm Hg), there was an 80% upregulation of glomerular GLUT-1 protein expression (P< or =0.03). This was accompanied by a 2.7-fold upregulation of TGF-beta1 protein expression in glomeruli of DSH compared with DSN rats (P=0.02). TGF-beta1 expression was not upregulated and did not differ in the glomeruli of Wistar-Kyoto and SHR rats. As an in vitro surrogate of the in vivo hemodynamic stress imposed by glomerular hypertension, we used mechanical stretching of human and rat mesangial cells. We found that after 33 hours of stretching, mesangial cells overexpressed GLUT-1 (40%) and showed an increase in basal glucose transport of similar magnitude (both P< or =0.01), which could be blocked with an anti TGF-beta1-neutralizing antibody. These studies suggest a novel link between hemodynamic and metabolic factors that may cooperate in inducing progressive glomerular injury in conditions characterized by glomerular hypertension.

IGF-I induces vascular endothelial growth factor in human mesangial cells via a Src-dependent mechanism.

BACKGROUND: Both insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) have been implicated in the pathogenesis of early renal dysfunction in diabetes. We investigated whether IGF-I affects VEGF gene expression and protein secretion in human mesangial cells. Furthermore, we studied the intracellular signaling pathway involved and the interaction of IGF-I with mechanical stretch, a known VEGF inducer. METHODS: Human mesangial cells were exposed to IGF-I in the presence and in the absence of (1) anti-IGF-I type I receptor antibody (alpha IR3) (1 microg/mL), a monoclonal antibody blocking the IGF-I type I receptor; (2) wortmannin (600 nmol/L), a phosphatidylinositol 3-kinase (PI3K) inhibitor; (3) 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a specific Src inhibitor (10 micromol/L); and (4) cyclic stretch (approximately 10% elongation). RESULTS: IGF-I induced a dose-dependent increase in VEGF protein levels (10(-11) mol/L, 5%; 10(-10) mol/L, 14%; 10(-9) mol/L, 46%; 10(-8) mol/L, 66%; 10(-7) mol/L, 68%; P < 0.001). IGF-I-induced VEGF production rose by 6 hours with a peak at 12 hours, and declined by 24 hours (52%, 72%, and 34%, respectively; P < 0.01 at 12 hours). A corresponding 50% increase in VEGF mRNA levels was seen at 6 hours (P < 0.01). IGF-I-induced VEGF protein secretion was not affected by the addition of wortmannin (IGF-I, 76% vs. IGF-I + wortmannin, 79% increase over control; P = NS), but was abolished by alpha IR3 (IGF-I, 69% vs. IGF-I +alpha IR3, 0%; P < 0.001) and significantly reduced by PP2 (IGF-I, 50% vs. IGF-I + PP2, 14%; P < 0.01). Simultaneous exposure of human mesangial cells to both IGF-I and stretch failed to further increase VEGF production (IGF-I, 1.49 +/- 0.05; stretch, 1.76 +/- 0.05; and IGF-I + stretch, 1.83 +/- 0.11). CONCLUSION: IGF-I induces VEGF gene expression and protein secretion in human mesangial cells via a Src-dependent mechanism.

Vascular endothelial growth factor receptors in human mesangium in vitro and in glomerular disease.

Mesangial cell proliferation and growth factor over-expression are characteristic features of several glomerular diseases. Vascular endothelial growth factor (VEGF), a potent mitogen, is expressed in podocytes in the glomerulus, and VEGF receptors (flt-1, KDR, and neuropilin-1) are present on endothelial cells and other cell types. This study examined whether human mesangial cells (HMC) express VEGF receptors in vitro and ex vivo and evaluated the effect of VEGF on HMC proliferation. All receptor types were detected in HMC in vitro by immunofluorescence and Western blotting. VEGF(165) induced a dose-responsive increase in (3)H-thymidine incorporation (25 ng/ml VEGF(165) : 2.3-fold increase; 50 ng/ml : 3.8-fold; 100 ng/ml : 4. 8-fold; 200 ng/ml : 3.4-fold; P = 0.016) and in cell number (50 ng/ml VEGF(165) : 1.2-fold increase; 100 ng/ml : 1.6-fold; 200 ng/ml : 1.4-fold; P = 0.005), effects prevented by an anti-VEGF(165) polyclonal neutralizing antibody (100 microg/ml). The proliferative effect was confirmed by a tetrazolium dye-based assay (100 ng/ml VEGF(165) : 1.4-fold increase). In ex vivo experiments, VEGF receptors in biopsy material from normal and diseased kidneys were detected by immunohistochemistry. No mesangial flt-1 receptor staining was seen in normal renal cortical tissue samples, and only weak mesangial KDR staining was detected. In contrast, mesangial flt-1 and KDR receptor staining were both clearly seen in biopsy samples from proliferative renal diseases. In conclusion, flt-1, KDR, and neuropilin-1 are present on cultured HMC, and VEGF(165) induces HMC proliferation. In addition, the flt-1 and KDR receptors are expressed in the mesangium in mesangioproliferative disease.