Ulf Janssen

Novel Approach to Specific Growth Factor Inhibition in Vivo : Antagonism of Platelet-Derived Growth Factor in Glomerulonephritis by Aptamers

Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective.

Regular ArticlesNovel Approach to Specific Growth Factor Inhibition in Vivo: Antagonism of Platelet-Derived Growth Factor in Glomerulonephritis by Aptamers

Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective.

Glomerular Oxidative and Antioxidative Systems in Experimental Mesangioproliferative Glomerulonephritis

Increased mesangial cell proliferation is a hallmark of many glomerulopathies in humans. Whereas the pathogenic role of reactive oxygen species (ROS) in the development of experimental mesangioproliferative glomerulonephritis (GN) is well established, very little is known about the mechanisms leading to increased ROS concentrations in the glomerulus. This study therefore examined glomerular ROS and the activities of oxidative and antioxidative enzymes during the early course of mesangioproliferative anti-Thy 1.1 GN. Anti-Thy 1.1 GN was induced in male Wistar rats, and glomeruli were isolated 2, 24, and 120 h after disease induction to examine ROS levels as well as oxidative and antioxidative enzyme expression in comparison to non-nephritic controls. At all time points, increased glomerular ROS levels, particularly of hydrogen peroxide and superoxide anions, were observed. Activities of NADH-dependent and NADPH-dependent oxidative enzymes were also increased during the course of GN, whereas no increased activity of xanthine oxidase, another potential source of ROS, was detectable. Despite glomerular oxidative stress, no compensatory increase of antioxidative enzyme activities occurred. On the contrary, catalase, superoxide dismutase, and glutathione peroxidase activities even decreased during the course of disease. In tubulointerstitial samples, no increase in oxidative activity was observed in the course of disease, thus confirming that detected ROS were of glomerular origin. Our data document for the first time a pronounced dysregulation of pro-oxidative and antioxidative enzymes in mesangioproliferative GN, leading to a net increase in glomerular ROS levels. Detailed knowledge of such pathways may lead to new therapeutic approaches for GN in humans.

Differential Expression of Mcp-1 and Its Receptor CCR2 in Glucose Primed Human Mesangial Cells

Background: Glomerular mononuclear cell infiltration is associated with the development of a diffuse glomerulosclerosis in patients with diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the recruitment and accumulation of monocytes and lymphocytes within the glomerulus. In the present study, we examined whether the ambient glucose concentration alters the expression of MCP-1 and its receptor CCR2 in primary human mesangial cells (HMC). Methods: MCP-1 mRNA expression was assessed by Northern blot and CCR2 mRNA expression by RT-PCR analysis. MCP-1 protein production was determined by ELISA. Migration studies were performed to assess functional MCP-1 receptor expression. Results: Exposure of HMC to 30 mMD-glucose led to a 30% increase in MCP-1 mRNA expression as compared to 5 mMD-glucose and osmotic controls while there was no difference in MCP-1 protein production. Simultaneously, CCR2 mRNA expression was down-regulated in HMC exposed to 30 mMD-glucose. 5 mMD-glucose primed HMC showed a dose-dependent migration towards MCP-1 that was dose-dependently inhibited by pertussis toxin, the broad-spectrum chemokine antagonist vMIP-II as well as the CCR2 receptor antagonist (1–8del)MCP-1 – demonstrating functional activity of MCP-1 receptor expression in primary HMC. In accordance with the downregulatory effects of 30 mMD-glucose on CCR2 mRNA expression no migratory response towards MCP-1 was observed under these conditions. The additional proinflammatory stimulus TNFα increased MCP-1 protein production in 30 as compared to 5 mMD-glucose primed HMC (2,194 ± 568 vs. 1,422 ± 379 pg MCP-1/104 cells × ml in 30 vs. 5 mMD-glucose primed HMC +24 h TNFα 500 U/ml, p = 0.002). However, this was not associated with an increased MCP-1 mRNA transcription. The 30 mMD-glucose induced downregulation of CCR2 mRNA expression was prevented in the presence of TNFα. Conclusion: High ambient glucose does not affect mesangial MCP-1 release and decreases its CCR2 receptor expression. However, in the presence of an inflammatory stimulus these effects of high glucose are reversed and an autocrine pathway of MCP-1 develops in mesangial cells.

Extracellular Actin Impairs Glomerular Capillary Repair in Experimental Mesangioproliferative Glomerulonephritis

Exogenous administration of actin prevents tumour growth in mice by specifically antagonizing angiogenin, a potent inducer of neovascularization. To investigate whether the angiogenin/actin system is also of importance in renal disease, we examined the effect of actin during glomerular capillary repair in anti-Thy-1.1 mesangioproliferative glomerulonephritis. Male Wistar rats were injected intravenously with actin, a control protein, i.e. albumin, or vehicle alone at 8, 16, 24, 32, 40 and 48 h after disease induction. On day 8, actin-treated rats showed significantly more microaneurysms and persistent mesangiolysis as compared to both control groups. This was associated with increased proteinuria in actin-treated rats. Moreover, actin-treated rats showed increased counts of glomerular macrophages (+40%) and polymorphonuclear leukocytes (+100%) on day 3 as well as a decrease in glomerular endothelial area on days 3 and 8. However, no difference in early glomerular endothelial as well as non-endothelial cell proliferation was noted in actin-treated rats as compared to controls. Actin treatment had no apparent influence on mesangial cell activation (i.e. de novo expression of α-smooth muscle actin) or glomerular accumulation of fibronectin or type IV collagen. Additional in vitro studies demonstrated that extracellular actin inhibits the angiogenin but not VEGF165-induced proliferation of (glomerular) endothelial cells. Moreover, actin inhibited other, yet unidentified, serum-derived angiogenic factors. In conclusion, exogenous actin impairs glomerular capillary repair in experimental mesangioproliferative glomerulonephritis possibly due to interference with angiogenic factors such as angiogenin. Our combined in vivo and in vitro observations suggest that the release of intracellular actin during mesangiolysis is an endogenous pathway by which glomerular capillary damage is augmented.