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• W1989387146 abstract "In contrast to factors that promote mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, expression of the glomerular CCN3 (nephroblastoma overexpressed gene [NOV]) gene is reduced before the proliferative phase and increased in glomeruli and serum when mesangial cell proliferation subsides. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 systemically was overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than threefold for up to 56 days. At day 5 after disease induction, CCN3-transfected rats showed an increase in glomerular endothelial area and in mRNA levels of the pro-angiogenic factors vascular endothelial growth factor and PDGF-C. At day 7, CCN3 overexpression decreased mesangial cell proliferation, including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis, and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphologic changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGF-receptor–β, and fibronectin, and increased PDGF-receptor–α and PDGF-C mRNA). These data identify a dual role for CCN3 in experimental glomerulonephritis with pro-angiogenic and antimesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes. In contrast to factors that promote mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, expression of the glomerular CCN3 (nephroblastoma overexpressed gene [NOV]) gene is reduced before the proliferative phase and increased in glomeruli and serum when mesangial cell proliferation subsides. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 systemically was overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than threefold for up to 56 days. At day 5 after disease induction, CCN3-transfected rats showed an increase in glomerular endothelial area and in mRNA levels of the pro-angiogenic factors vascular endothelial growth factor and PDGF-C. At day 7, CCN3 overexpression decreased mesangial cell proliferation, including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis, and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphologic changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGF-receptor–β, and fibronectin, and increased PDGF-receptor–α and PDGF-C mRNA). These data identify a dual role for CCN3 in experimental glomerulonephritis with pro-angiogenic and antimesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes. Diabetic nephropathy and glomerulonephritides, in particular the most common type IgA nephropathy, account for >50% of end-stage renal disease cases in most Western countries. Both diabetic nephropathy and the majority of progressive glomerulonephritides are characterized histologically by glomerular mesangial cell proliferation and/or matrix accumulation.1Klahr S. Schreiner G. Ichikawa I. The progression of renal disease.N Engl J Med. 1988; 318: 1657-1666Crossref PubMed Scopus (730) Google Scholar, 2Striker L.J. Peten E.P. Elliot S.J. Doi T. Striker G.E. Mesangial cell turnover: effect of heparin and peptide growth factors.Lab Invest. 1991; 64: 446-456PubMed Google Scholar Ample evidence links the PDGF system to these processes and the roles of PDGF-B and PDGF-D chains in mediating mesangioproliferative changes are particularly well established.3Floege J. Johnson R.J. Multiple roles for platelet-derived growth factor in renal disease.Miner Electrolyte Metab. 1995; 21: 271-282PubMed Google Scholar, 4Johnson R.J. Floege J. Couser W.G. Alpers C.E. Role of platelet-derived growth factor in glomerular disease.J Am Soc Nephrol. 1993; 4: 119-128Crossref PubMed Google Scholar, 5Floege J. Eitner F. Alpers C.E. A new look at platelet-derived growth factor in renal disease.J Am Soc Nephrol. 2008; 19: 12-23Crossref PubMed Scopus (239) Google Scholar Inhibition of PDGF-BB or PDGF-DD in mesangioproliferative nephritis can prevent renal failure, glomerulosclerosis, and tubulointerstitial fibrosis.6Floege J. Ostendorf T. Janssen U. Burg M. Radeke H.H. Vargeese C. Gill S.C. Green L.S. Janjic N. Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers.Am J Pathol. 1999; 154: 169-179Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 7Ostendorf T. Kunter U. Grone H.J. Bahlmann F. Kawachi H. Shimizu F. Koch K.M. Janjic N. Floege J. Specific antagonism of PDGF prevents renal scarring in experimental glomerulonephritis.J Am Soc Nephrol. 2001; 12: 909-918Crossref PubMed Google Scholar, 8Ostendorf T. van Roeyen C.R. Peterson J.D. Kunter U. Eitner F. Hamad A.J. Chan G. Jia X.C. Macaluso J. Gazit-Bornstein G. Keyt B.A. Lichenstein H.S. LaRochelle W.J. Floege J. A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.J Am Soc Nephrol. 2003; 14: 2237-2247Crossref PubMed Scopus (82) Google Scholar, 9Ostendorf T. Rong S. Boor P. Wiedemann S. Kunter U. Haubold U. van Roeyen C.R. Eitner F. Kawachi H. Starling G. Alvarez E. Smithson G. Floege J. Antagonism of PDGF-D by human antibody CR002 prevents renal scarring in experimental glomerulonephritis.J Am Soc Nephrol. 2006; 17: 1054-1062Crossref PubMed Scopus (57) Google Scholar, 10Boor P. Konieczny A. Villa L. Kunter U. van Roeyen C.R. LaRochelle W.J. Smithson G. Arrol S. Ostendorf T. Floege J. PDGF-D inhibition by CR002 ameliorates tubulointerstitial fibrosis following experimental glomerulonephritis.Nephrol Dial Transplant. 2007; 22: 1323-1331Crossref PubMed Scopus (53) Google ScholarAlthough there is abundant information on promitogenic and profibrotic mediators in the glomerulus, little is known about endogenous factors that might counteract and terminate these processes. In a cDNA array, we recently detected a prominent down-regulation of CCN3 (also known as nephroblastoma overexpressed gene, NOV) in PDGF-B– or PDGF-D–stimulated mesangial cells.11van Roeyen C.R. Eitner F. Scholl T. Boor P. Kunter U. Planque N. Grone H.J. Bleau A.M. Perbal B. Ostendorf T. Floege J. CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.Kidney Int. 2008; 73: 86-94Crossref PubMed Scopus (38) Google Scholar We subsequently identified CCN3 as an endogenous inhibitor of mesangial cell growth in vitro and a modulator of PDGF-induced mitogenesis in mesangioproliferative disease.11van Roeyen C.R. Eitner F. Scholl T. Boor P. Kunter U. Planque N. Grone H.J. Bleau A.M. Perbal B. Ostendorf T. Floege J. CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.Kidney Int. 2008; 73: 86-94Crossref PubMed Scopus (38) Google Scholar CCN3 belongs to the CCN protein family (cysteine-rich, angiogenic inducer 61 [Cyr61], connective tissue growth factor [CTGF], NOV), all of which are involved in the regulation of cell proliferation, migration, attachment, and differentiation.12Perbal B. NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.Mol Pathol. 2001; 54: 57-79Crossref PubMed Scopus (308) Google Scholar In the developing human kidney, CCN3 mRNA and/or protein is expressed in the mesonephric and paramesonephric ducts as well as in the stromal and other tubular cells.12Perbal B. NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.Mol Pathol. 2001; 54: 57-79Crossref PubMed Scopus (308) Google Scholar, 13Kocialkowski S. Yeger H. Kingdom J. Perbal B. Schofield P.N. Expression of the human NOV gene in first trimester fetal tissues.Anat Embryol (Berl). 2001; 203: 417-427Crossref PubMed Scopus (50) Google Scholar In the adult rat kidney, CCN3 localized to arterial smooth muscle cells, the medullary interstitium, and podocytes.11van Roeyen C.R. Eitner F. Scholl T. Boor P. Kunter U. Planque N. Grone H.J. Bleau A.M. Perbal B. Ostendorf T. Floege J. CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.Kidney Int. 2008; 73: 86-94Crossref PubMed Scopus (38) Google Scholar, 13Kocialkowski S. Yeger H. Kingdom J. Perbal B. Schofield P.N. Expression of the human NOV gene in first trimester fetal tissues.Anat Embryol (Berl). 2001; 203: 417-427Crossref PubMed Scopus (50) Google ScholarGiven the inhibition of mesangial cell proliferation by CCN3 in vitro and the regulated CCN3 expression during anti-Thy1.1 nephritis, a rat model of mesangioproliferative glomerulonephritis, we asked whether CCN3 could be manipulated therapeutically. For this, we overexpressed CCN3 in skeletal muscle by electroporation and studied the effects of systemic CCN3 increases on the acute and chronic course of mesangioproliferative anti-Thy1.1 glomerulonephritis.Materials and MethodsAnimal ExperimentsAll studies were approved by the local Institutional Review Board. Male Wistar rats weighing 180 g (Charles River, Sülzfeld, Germany) were used for this study. Animals were held in rooms with constant temperature and humidity, with 12:12-hour light cycles, and had ad libitum access to drinking water (ozone-treated and acidified) and standard rat chow.Anti-Thy1.1 mesangial proliferative glomerulonephritis was induced in all rats by intravenous injection of 1 mg/kg anti-Thy1.1 monoclonal antibody (clone OX-7; European Collection of Animal Cell Cultures, Salisbury, England). To study the kinetics of CCN3 serum concentrations during acute anti-Thy1.1 nephritis, 54 rats were sacrificed and serum samples were obtained at 4 hours and at 1, 2, 4, 7, 9, 14, 21, and 28 days after disease induction (n = 6 each). Healthy rats injected with PBS served as controls (n = 6).To study the effects of CCN3 overexpression on the course of anti-Thy1.1 nephritis, disease was induced in 28 rats. Muscle electroporation was performed 16 hours after disease induction as described.14Molnar M.J. Gilbert R. Lu Y. Liu A.B. Guo A. Larochelle N. Orlopp K. Lochmuller H. Petrof B.J. Nalbantoglu J. Karpati G. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles.Mol Ther. 2004; 10: 447-455Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 15Muramatsu T. Arakawa S. Fukazawa K. Fujiwara Y. Yoshida T. Sasaki R. Masuda S. Park H.M. In vivo gene electroporation in skeletal muscle with special reference to the duration of gene expression.Int J Mol Med. 2001; 7: 37-42PubMed Google Scholar In brief, under short inhalation anesthesia with isoflurane (Abbott, Wiesbaden, Germany) the left hind limb was shaved and the gastrocnemius muscle was injected with 150 U/animal of hyaluronidase type IV (Sigma-Aldrich, Deisenhofen, Germany). Two hours later, the rats were shortly anesthetized (isoflurane) and 500 μg expression vector encoding either human CCN3 cDNA (n = 14) or LacZ (n = 14) driven by the cytomegalovirus-promotor were injected into the primed gastrocnemius muscle. Five minutes later, the muscle was exposed to transcutaneous electroporation stimulation using 8 low-voltage pulses of 175 V and 20 ms at 1-Hz frequency using the Electro Square Porator ECM 830 (BTX; Genetronics, San Diego, CA) with circular paddle electrodes. The rats were sacrificed at day 5 (n = 6 each) or day 7 (n = 8 each) after disease induction.Progressive glomerulonephritis was induced in 32 rats by uninephrectomy followed by intravenous injection of 1 mg/kg anti-Thy1.1 antibody at day 0. Muscle electroporation was performed 16 hours after disease induction (n = 16 each) as described earlier. Renal function was controlled by measurement of proteinuria and serum creatinine in urine and serum samples, respectively, taken at days 6 and 56. During the experiment, 9 rats (6 in the LacZ group and 3 in the CCN3 group) died as a result of the anesthesia.In an additional experiment, 28 healthy Wistar rats were electroporated as described earlier with expression vectors encoding for CCN3 (n = 14) or LacZ (n = 14) and the rats were sacrificed at day 5 (n = 6 each) or day 7 (n = 8 each).One day before sacrifice, systolic blood pressure was measured by tail cuff plethysmography, using a programmed sphygmomanometer (Softron Co., Tokyo, Japan). In addition, 5-bromo-2′-deoxyuridine (BrdU, 100 mg/kg) was injected intraperitoneally in all rats. Subsequently, all rats were placed in metabolic cages for 24-hour stool-free urine collections.After sacrifice of the various groups, renal tissue for histology, immunohistochemistry, and RNA analyses were harvested and processed immediately. From the remaining cortical tissue, glomeruli were isolated by differential sieving as described.8Ostendorf T. van Roeyen C.R. Peterson J.D. Kunter U. Eitner F. Hamad A.J. Chan G. Jia X.C. Macaluso J. Gazit-Bornstein G. Keyt B.A. Lichenstein H.S. LaRochelle W.J. Floege J. A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.J Am Soc Nephrol. 2003; 14: 2237-2247Crossref PubMed Scopus (82) Google Scholar Serum and urine biochemistries were assessed by an autoanalyzer (Vitros 250; Ortho Clinical Diagnostics, Neckargemuend, Germany).Renal Morphology and Immunohistochemical AnalysisTissues for light microscopy and immunoperoxidase staining were fixed in methyl Carnoy's solution and embedded in paraffin. Sections (4 μm) were stained with PAS reagent and counterstained with hematoxylin (PAS staining). Glomerulosclerosis and tubulointerstitial damage was graded on a scale as follows: 0, no glomeruli/cortical area affected, 1, >0% to ≤25% glomeruli/cortical area affected; 2, >25% to ≤50% glomeruli/cortical area affected; 3, >50% to ≤75% glomeruli/cortical area affected; and 4, >75% to ≤100% of glomeruli/cortical area affected.In the PAS-stained sections, the total number of mitoses in 50 consecutive glomerular cross-sections was evaluated. Immunohistochemical analyses were performed following a previously published protocol in a blinded manner.16Eitner F. Ostendorf T. Van Roeyen C. Kitahara M. Li X. Aase K. Grone H.J. Eriksson U. Floege J. Expression of a novel PDGF isoform PDGF-C, in normal and diseased rat kidney.J Am Soc Nephrol. 2002; 13: 910-917PubMed Google Scholar Renal tissues were stained using the following antibodies: rabbit polyclonal anti-rat fibronectin (AB 1954; Chemicon, Hampshire, United Kingdom), goat polyclonal anti-human collagen type IV (SouthernBiotec, Birmingham, AL), mouse monoclonal anti-human α-smooth muscle actin (clone 1A4; Dako, Hamburg, Germany), mouse monoclonal anti-rat CD68 (clone ED-1; Serotec, Oxford, United Kingdom), mouse monoclonal anti-human proliferating cell nuclear antigen (PCNA) (PC-10; Oncogene, San Diego, CA), mouse monoclonal anti-rat epithelial aminopeptidase P (clone JG12; BenderMedSystems, Vienna, Austria), mouse monoclonal anti-BrdU (clone Bu-1; Amersham, Freiburg, Germany), mouse monoclonal anti-human desmin (clone D33; Dako), and polyclonal anti-CCN3 antibody (K19M, described by Kyurkchiev et al17Kyurkchiev S. Yeger H. Bleau A.M. Perbal B. Potential cellular conformations of the CCN3(NOV) protein.Cell Commun Signal. 2004; 2: 9Crossref PubMed Scopus (37) Google Scholar). Negative controls for the immunohistochemical procedures consisted of substitution of the primary antibody with nonimmune IgG.For PCNA, BrdU, and CD68 staining, positively stained cells in 50 randomly selected glomeruli were counted. For the quantification of positively stained glomerular areas of JG12, α-smooth muscle actin (α-SMA), collagen type IV, and fibronectin, 50 glomeruli were evaluated by computer-based morphometry using the analysis v3.1 software (Soft imaging System GmbH, Münster, Germany) as described previously.10Boor P. Konieczny A. Villa L. Kunter U. van Roeyen C.R. LaRochelle W.J. Smithson G. Arrol S. Ostendorf T. Floege J. PDGF-D inhibition by CR002 ameliorates tubulointerstitial fibrosis following experimental glomerulonephritis.Nephrol Dial Transplant. 2007; 22: 1323-1331Crossref PubMed Scopus (53) Google Scholar In all analyses, the investigator was unaware of the origin of the slides.Real-Time Quantitative RT-PCRTotal RNA was isolated from isolated glomeruli using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantification of the RNA content and sample purity, cDNA synthesis, and real-time quantitative RT-PCR were assessed and performed, respectively, as described.8Ostendorf T. van Roeyen C.R. Peterson J.D. Kunter U. Eitner F. Hamad A.J. Chan G. Jia X.C. Macaluso J. Gazit-Bornstein G. Keyt B.A. Lichenstein H.S. LaRochelle W.J. Floege J. A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.J Am Soc Nephrol. 2003; 14: 2237-2247Crossref PubMed Scopus (82) Google Scholar All quantitative data from the real-time RT-PCR were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control and calculated using the ΔΔCT-method. Primer sequences are listed in Table 1. GAPDH cDNA amplification was used as an internal standard.Table 1Primers for Quantitative Real-Time RT-PCRGeneForward primerReverse primerProbeSYBR Green (quantitative PCR core kit for SYBR green I) (Eurogentec, Seraing, Belgium)GAPDH5′-ACAAGATGGTGAAGGTCGGTG-3′5′-AGAAGGCAGCCCTGGTAACC-3′MCP-15′-CCAGATGCAGTTAATGCCCC-3′5′-TCTCCAGCCGACTCATTGG-3′RANTES5′-ACTCCCTGCTGCTTTGCCT-3′5′-GTGTAAAAATACTCCTTCACGTGGG-3′TaqMan probe (qPCR core kit) (Eurogentec, Seraing, Belgium)GAPDH5′-ACAAGATGGTGAAGGTCGGTG-3′5′-AGAAGGCAGCCCTGGTAACC-3′5′-CGGATTTGGCCGTATCGGACGC-3′PDGF-A5′-TTCTTGATCTGGCCCCCAT-3′5′-TTGACGCTGCTGGTGTTACAG-3′5′-CAGTGCAGCGCTTCACCTCCACA-3′PDGF-B5′-GCAAGACGCGTACAGAGGTG-3′5′-GAAGTTGGCATTGGTGCGA-3′5′-TCCAGATCTCGCGGAACCTCATCG-3′PDGF-C5′-CAGCAAGTTGCAGCTCTCCA-3′5′-GACAACTCTCTCATGCCGGG-3′5′-CGACAAGGAGCAGAACGGAGTGCAA-3′PDGF-D5′-ATCGGGACACTTTTGCGACT-3′5′-GTGCCTGTCACCCGAATGTT-3′5′-TTGCGCAATGCCAACCTCAGGAG-3′PDGFR-α5′-GCCACGAAAGAGGTCAAGGA-3′5′-GCCTGATCTGGACGAAGCC-3′5′-TGAAGACAGTCACCATTTCTGTTCACGAGAA-3′PDGFR-β5′-AATGACCACGGCGATGAGA-3′5′-TCTTCCAGTGTTTCCAGCAGC-3′5′-CATCAACGTTACTGTGATCGAAAATGGCTATG-3′Col1a15′-GAAGGCAACAGTCGATTCACC-3′5′-GACTGTCTTGCCCCAAGTTCC-3′5′-ACAGCACGCTTGTGGATGGCTGC-3′Fibronectin5′-AAACAGGTCTGGACTCCCCA-3′5′-CAGAATGCTCGGCGTGATG-3′5′-TCTTCTGATGTCACCGCCAACTCATTCA-3′CCN35′-CTACAGAGTGGAGCGCGTGTT-3′5′-GGAAGATTCCTGTTGGTGACCC-3′5′-AAGAGCTGTGGAATGGGCTTGTCCAC-3′VEGF5′-GTACCTCCACCATGCCAAGTG-3′5′-ATGGGCTTTCTGCTCCCCT-3′5′-CCCAGGCTGCACCCACGACAG-3′GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCP-1, monocyte chemotactic protein-1; RANTES, regulated upon activation, normally T-expressed and presumably secreted. Open table in a new tab Measurement of CCN3 Concentration in SerumTransgenic CCN3 serum concentrations were measured using the human NOV/CCN3 DuoSet enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. For Western blot analysis the serum samples were purified and analyzed with a rabbit polyclonal anti-CCN3 antibody as described.11van Roeyen C.R. Eitner F. Scholl T. Boor P. Kunter U. Planque N. Grone H.J. Bleau A.M. Perbal B. Ostendorf T. Floege J. CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.Kidney Int. 2008; 73: 86-94Crossref PubMed Scopus (38) Google ScholarIn Vitro ExperimentsConditionally immortalized human glomerular endothelial cells (CiGEnCs) were obtained and cultured as described in detail previously.18Satchell S.C. Tasman C.H. Singh A. Ni L. Geelen J. von Ruhland C.J. O'Hare M.J. Saleem M.A. van den Heuvel L.P. Mathieson P.W. Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.Kidney Int. 2006; 69: 1633-1640Crossref PubMed Scopus (166) Google Scholar, 19Boor P. van Roeyen C.R. Kunter U. Villa L. Bucher E. Hohenstein B. Hugo C.P. Eriksson U. Satchell S.C. Mathieson P.W. Eitner F. Floege J. Ostendorf T. PDGF-C mediates glomerular capillary repair.Am J Pathol. 2010; 177: 58-69Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Proliferation assays were performed in unstimulated cells or CiGEnCs stimulated with recombinant CCN3 (R&D Systems) in basal and full medium using a BrdU incorporation assay according to the manufacturer's instructions (Roche, Mannheim, Germany). The vascular endothelial growth factor (VEGF) aptamer was described previously20Ostendorf T. Kunter U. Eitner F. Loos A. Regele H. Kerjaschki D. Henninger D.D. Janjic N. Floege J. VEGF(165) mediates glomerular endothelial repair.J Clin Invest. 1999; 104: 913-923Crossref PubMed Scopus (277) Google Scholar and used at a concentration of 1 μg/mL.Western Blot AnalysisGlomeruli were isolated by differential sieving, lysed in Triton X-100 lysis buffer, and treated with ultrasound. The soluble cell lysates were separated and blotted as described21van Roeyen C.R. Ostendorf T. Denecke B. Bokemeyer D. Behrmann I. Strutz F. Lichenstein H.S. LaRochelle W.J. Pena C.E. Chaudhuri A. Floege J. Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.Kidney Int. 2006; 69: 1393-1402Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and probed with polyclonal antibodies against PDGF-receptor–β (PDGFR-β) (Santa Cruz Biotechnology, Santa Cruz, CA), pSMAD1/5/8 (Cell Signaling Technology, Danvers, MA), phosphorylated Sma- and Mad-related protein, homolog of Drosophila (pSmad2) (Cell Signaling Technology), Smad2 (Cell Signaling Technology), CCN2 (Santa Cruz Biotechnology), β-actin (Sigma, Taufkirchen, Germany), and GAPDH (Santa Cruz Biotechnology). The primary antibodies (diluted 1:1000) were detected using horseradish peroxidase–conjugated antibodies visualized by the Amersham ECL system after intensive washing of the membrane.Statistical AnalysisAll values were expressed as means ± SD. To analyze the data, the two-tailed Mann-Whitney U-test or one-way analysis of variance with Scheffé post hoc analysis were used where appropriate. For comparison of serum CCN3 levels during the course of anti-Thy1.1 with healthy animals, one-way analysis of variance with the Dunnett post hoc test was used. For comparison of serum CCN3 levels with parameters of renal damage, the Spearman correlation coefficients (rs) were calculated. Statistical significance was defined as a P value < 0.05.ResultsMuscle Electroporation Results in Systemic Overexpression of CCN3We first assessed temporal changes of serum levels of endogenous CCN3 in rats with acute anti-Thy1.1 nephritis. This revealed a significant increase from day 4 to 14 with a maximum on day 7 after disease induction (Figure 1).Based on the earlier-described time information and the natural course of acute anti-Thy1.1 nephritis, we performed a muscle electroporation with a cytomegalovirus promoter-driven vector overexpressing human CCN3 or LacZ as a control at 16 hours after disease induction. This not only avoids interference of the CCN3 overexpression with the disease induction phase but also should serve to amplify the peak of the endogenous CCN3 overproduction on day 7 after disease induction. After muscle electroporation both LacZ activity and the transgenic CCN3 protein were detectable in skeletal muscle on days 5 and 7 after disease induction (Figure 2A). In parallel, serum levels of CCN3 increased 2.8-fold on day 5 and >8.4-fold on day 7 (Figure 2B) as measured with the anti-human–CCN3 ELISA, which detects both the endogenous rat as well as the transgenic human CCN3 proteins. In addition to the full-length CCN3 protein, a truncated CCN3 fragment could be detected by Western blot analysis of sera (Figure 2C). A similar truncated CCN3 fragment has been described in different biological fluids.12Perbal B. NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.Mol Pathol. 2001; 54: 57-79Crossref PubMed Scopus (308) Google Scholar, 22Lazar N. Manara C. Navarro S. Bleau A.M. Llombart-Bosch A. Scotlandi K. Planque N. Perbal B. Domain-specific CCN3 antibodies as unique tools for structural and functional studies.J Cell Commun Signal. 2007; 1: 91-102Crossref PubMed Scopus (24) Google ScholarFigure 2Overexpression of CCN3 in muscle and in the circulation. A: Transgenic protein expression could be detected in the transfected muscle of nephritic rats at days 5 and 7 after disease induction by immunohistochemical staining of CCN3 or by X-Gal staining. B: The secreted transgenic CCN3 protein (full length and truncated) could be detected by Western blot analysis in serum of healthy rats transfected with the CCN3 expression vector. Arrows indicate the full-length and truncated CCN3 protein detected in serum of CCN3-overexpressing rats. C: By using ELISA, serum samples of nephric and healthy rats showed increased CCN3 concentrations in comparison with control treated rats. Data are means ± SD relative to the mean of control transfected rats per each time point. *P < 0.05 versus LacZ control rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CCN3 Overexpression Augments Early Glomerular Capillary Repair on Day 5 of Acute Anti-Thy1.1 NephritisNephritic rats overexpressing LacZ or CCN3 in muscle remained normotensive and showed albuminuria and a minor decrease in renal function on day 5 with no significant differences between the two groups (data not shown).The most prominent finding in CCN3-overexpressing nephritic rats on day 5 as compared with the LacZ group was a significantly increased glomerular endothelial cell area, as assessed by immunostaining with the JG12 antibody (Figure 3A). In parallel, a nonsignificant trend toward an increased number of proliferating PCNA/JG12 double-positive endothelial cells was observed in the CCN3 group (Figure 3A). CCN3 overexpression also led to glomerular mRNA overexpression of the two pro-angiogenic factors VEGF and PDGF-C (Figure 3B).Figure 3Systemic overexpression of CCN3 induces angiogenesis. A: The overexpressed CCN3 protein induces an increased glomerular endothelial, JG12-positive area, and a trend toward increased glomerular endothelial cell proliferation as identified by double-immunostaining for PCNA (blue) and JG12 (brown). Data are means ± SD of n = 6 rats, each. B: The increased glomerular endothelial area is accompanied by glomerular mRNA overexpression of the pro-angiogenic factors VEGF and PDGF-C, as measured by real-time RT-PCR (n = 6 rats, each). A reduced mRNA expression of CCN3 and PDGFR-β could be observed by real-time RT-PCR in glomeruli of CCN3 in comparison with LacZ-overexpressing rats. Data are means ± SD relative to the mean of control transfected rats (n = 6, each). *P < 0.05 versus LacZ control rats. C: The percentage of CCN3-positive glomerular area is not changed by CCN3 overexpression as identified by immunostaining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)At the transcriptional level, the overexpressed CCN3 down-regulated glomerular CCN3 mRNA expression, whereas the CCN3-positive glomerular area was not altered (Figure 3, B and C). Glomerular matrix protein expression of fibronectin, and types I and IV collagen, was unchanged at the mRNA and/or protein level in CCN3 compared with LacZ-transfected nephritic rats (data not shown).In nephritic rats, on day 5, CCN3 versus LacZ overexpression did not affect overall glomerular cell proliferation (ie, the number of glomerular mitoses or PCNA-positive cells), mesangial cell proliferation (ie, PCNA/α-SMA double-positive cells), mesangial cell activation (as assessed by their de novo expression of α-SMA), or the number of infiltrating glomerular ED-1–positive macrophages (data not shown). However, there was significantly reduced expression of glomerular PDGFR-β chain mRNA in the CCN3 group (Figure 3B).Recombinant CCN3 Protein Affects the Proliferation of Glomerular Endothelial Cells in VitroCiGEnCs were stimulated with recombinant CCN3 protein under permissive (33°C, undifferentiated endothelial cells) and nonpermissive (37°C, differentiated glomerular endothelial cells) conditions in vitro. Under permissive conditions, incubation with CCN3 did not lead to a significantly increased cell proliferation when cells were growing in basal medium, but it potently induced proliferation of cells growing in full medium (Figure 4A). Next, we analyzed the proliferation of CiGEnCs stimulated with conditioned media from CiGEnCs and human mesangial cells that had been stimulated with CCN3. Conditioned medium from endothelial but not mesangial cells induced a significant increase in proliferating differentiated CiGEnCs (Figure 4B), suggesting that CCN3 induces an autocrine release of pro-angiogenic mediators in CiGEnCs. By using a VEGF-specific aptamer20Ostendorf T. Kunter U. Eitne" @default.
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