Vascular Endothelial Growth Factor Isoforms and Their Receptors Are Expressed in Human Osteoarthritic Cartilage
2003; Elsevier BV; Volume: 162; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63808-4
ISSN1525-2191
AutoresHiroyuki Enomoto, Isao Inoki, Koichiro Komiya, Takayuki Shiomi, Eiji Ikeda, Ken-ichi Obata, Hideo Matsumoto, Yoshiaki Toyama, Yasunori Okada,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoTo assess the possible involvement of vascular endothelial growth factor (VEGF) in the pathology of osteoarthritic (OA) cartilage, we examined the expression of VEGF isoforms and their receptors in the articular cartilage, and the effects of VEGF on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in OA chondrocytes. Reverse transcriptase-polymerase chain reaction analyses demonstrated that mRNAs for three VEGF isoforms (VEGF121, VEGF165, and VEGF189) are detectable in all of the OA and normal (NOR) cartilage samples. However, the mRNA expression of their receptors (VEGFR-1 = Flt-1, VEGFR-2 = KDR and neuropilin-1) was recognized only in the OA samples. The protein expression of VEGFR-1 and VEGFR-2 in OA chondrocytes was also demonstrated by immunohistochemistry of the OA cartilage tissue and cultured OA chondrocytes. In situ hybridization and immunohistochemistry indicated that VEGF is expressed in the chondrocytes in the superficial and transitional zones of OA cartilage. A linear correlation was obtained between VEGF immunoreactivity and Mankin scores in the cartilage (r = 0.906, P < 0.001). The production levels of VEGF determined by enzyme-linked immunosorbent assay were significantly 3.3-fold higher in OA than in NOR samples (P < 0.001). Among MMP-1, -2, -3, -7, -8, -9, and -13, TIMP-1 and -2 measured by their sandwich enzyme immunoassay systems, the production of MMP-1 and MMP-3 but not TIMP-1 or TIMP-2 was significantly enhanced by the treatment of cultured OA chondrocytes with VEGF (P < 0.05), whereas no such effect was obtained with cultured NOR chondrocytes. These results demonstrate that VEGF and its receptors are expressed in OA cartilage, and suggest the possibility that VEGF is implicated for the destruction of OA articular cartilage through the increased production of MMPs. To assess the possible involvement of vascular endothelial growth factor (VEGF) in the pathology of osteoarthritic (OA) cartilage, we examined the expression of VEGF isoforms and their receptors in the articular cartilage, and the effects of VEGF on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in OA chondrocytes. Reverse transcriptase-polymerase chain reaction analyses demonstrated that mRNAs for three VEGF isoforms (VEGF121, VEGF165, and VEGF189) are detectable in all of the OA and normal (NOR) cartilage samples. However, the mRNA expression of their receptors (VEGFR-1 = Flt-1, VEGFR-2 = KDR and neuropilin-1) was recognized only in the OA samples. The protein expression of VEGFR-1 and VEGFR-2 in OA chondrocytes was also demonstrated by immunohistochemistry of the OA cartilage tissue and cultured OA chondrocytes. In situ hybridization and immunohistochemistry indicated that VEGF is expressed in the chondrocytes in the superficial and transitional zones of OA cartilage. A linear correlation was obtained between VEGF immunoreactivity and Mankin scores in the cartilage (r = 0.906, P < 0.001). The production levels of VEGF determined by enzyme-linked immunosorbent assay were significantly 3.3-fold higher in OA than in NOR samples (P < 0.001). Among MMP-1, -2, -3, -7, -8, -9, and -13, TIMP-1 and -2 measured by their sandwich enzyme immunoassay systems, the production of MMP-1 and MMP-3 but not TIMP-1 or TIMP-2 was significantly enhanced by the treatment of cultured OA chondrocytes with VEGF (P < 0.05), whereas no such effect was obtained with cultured NOR chondrocytes. These results demonstrate that VEGF and its receptors are expressed in OA cartilage, and suggest the possibility that VEGF is implicated for the destruction of OA articular cartilage through the increased production of MMPs. Cartilage is composed of highly differentiated chondrocytes and extracellular matrix (ECM), and essentially an avascular tissue. However, during endochondral ossification in a developing bone, neovascularization from subchondral bone takes place in the growth plate of cartilage, resulting in resorption of cartilage ECM and replacement by bone matrix.1Schenk RK Spiro D Wiener J Cartilage resorption in the tibial epiphyseal plate of growing rats.J Cell Biol. 1967; 34: 275-291Crossref PubMed Scopus (196) Google Scholar In pathological conditions such as rheumatoid arthritis (RA) and osteoarthritis (OA), damaged articular cartilage is frequently covered with and invaded by the granulation tissue with high vascularity, ie, pannus tissue. These findings observed in the pathophysiological conditions suggest the involvement of angiogenic factors in the process. In fact, a number of angiogenic molecules including basic fibroblast growth factor,2Baron J Klein KO Yanovski JA Novosad JA Bacher JD Bolander ME Cutler Jr, GB Induction of growth plate cartilage ossification by basic fibroblast growth factor.Endocrinology. 1994; 135: 2790-2793Crossref PubMed Scopus (81) Google Scholar vascular endothelial growth factor (VEGF),3Gerber HP Vu TH Ryan AM Kowalski J Werb Z Ferrara N VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.Nat Med. 1999; 5: 623-628Crossref PubMed Scopus (1752) Google Scholar and transforming growth factor-β4Jingushi S Scully SP Joyce ME Sugioka Y Bolander ME Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.J Orthop Res. 1995; 13: 761-768Crossref PubMed Scopus (53) Google Scholar are present in growth plate cartilage. Among them, VEGF, which is produced from hypertrophic chondrocytes, is considered to be a coordinator of ECM remodeling, angiogenesis, and bone formation in the growth plate.3Gerber HP Vu TH Ryan AM Kowalski J Werb Z Ferrara N VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.Nat Med. 1999; 5: 623-628Crossref PubMed Scopus (1752) Google Scholar VEGF is also known to be expressed in the cells of RA synovial tissue and the expression is related to angiogenesis in the synovium.5Ikeda M Hosoda Y Hirose S Okada Y Ikeda E Expression of vascular endothelial growth factor isoforms and their receptors Flt-1, KDR, and neuropilin-1 in synovial tissues of rheumatoid arthritis.J Pathol. 2000; 191: 426-433Crossref PubMed Scopus (113) Google Scholar However, limited information is so far available for the VEGF expression in the articular cartilage under the pathophysiological conditions. VEGF has a strong angiogenic activity with specific mitogenic and chemotactic actions on endothelial cells.6Keck PJ Hauser SD Krivi G Sanzo K Warren T Feder J Connolly DT Vascular permeability factor, an endothelial cell mitogen related to PDGF.Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1893) Google Scholar, 7Leung DW Cachianes G Kuang WJ Goeddel DV Ferrara N Vascular endothelial growth factor is a secreted angiogenic mitogen.Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4581) Google Scholar Alternative splicing of VEGF mRNA generates the five different isoforms with 121, 145, 165, 189, and 206 amino acid residues, which are named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206, respectively. VEGF was originally recognized in a tumor-conditioned medium,8Senger DR Galli SJ Dvorak AM Perruzzi CA Harvey VS Dvorak HF Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science. 1983; 219: 983-985Crossref PubMed Scopus (3495) Google Scholar but recent studies have demonstrated that it is expressed by various types of cells including vascular smooth muscle cells, monocytes, mesangial cells, and megakaryocytes,9Mohle R Green D Moore MA Nachman RL Rafii S Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.Proc Natl Acad Sci USA. 1997; 94: 663-668Crossref PubMed Scopus (642) Google Scholar in some of which the expression is constitutive. Thus, it is conceivable that the biological function of VEGF is dictated mainly by the expression of its receptors on the cells in various tissues. There are two types of well-known receptors of VEGF, ie, fms-like tyrosine kinase, Flt-1 (VEGFR-1)10de Vries C Escobedo JA Ueno H Houck K Ferrara N Williams LT The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor.Science. 1992; 255: 989-991Crossref PubMed Scopus (1903) Google Scholar and kinase insert domain-containing receptor, KDR (VEGFR-2).11Terman BI Carrion ME Kovacs E Rasmussen BA Eddy RL Shows TB Identification of a new endothelial cell growth factor receptor tyrosine kinase.Oncogene. 1991; 6: 1677-1683PubMed Google Scholar, 12Terman BI Dougher-Vermazen M Carrion ME Dimitrov D Armellino DC Gospodarowicz D Bohlen P Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.Biochem Biophys Res Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1435) Google Scholar Neuropilin-1 (NRP-1) is an isoform-specific co-receptor of VEGFR-2, and enhances the bioactivity of VEGF165 by increasing the binding affinity of the molecule to VEGFR-2.13Soker S Takashima S Miao HQ Neufeld G Klagsbrun M Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2117) Google Scholar Because the primary function of VEGF was considered to be at angiogenesis, previous studies on the receptors have focused on endothelial cells, and demonstrated the expression in the vascular endothelial cells in the pathological tissues with angiogenesis.14Neufeld G Cohen T Gengrinovitch S Poltorak Z Vascular endothelial growth factor (VEGF) and its receptors.EMBO J. 1999; 13: 9-22Google Scholar Interestingly, binding of VEGF to its receptors on the endothelial cells stimulates not only their proliferation and chemotaxis but also production of ECM-degrading metalloproteinases, ie, matrix metalloproteinases (MMPs).15Lamoreaux WJ Fitzgerald ME Reiner A Hasty KA Charles ST Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro.Microvasc Res. 1998; 55: 29-42Crossref PubMed Scopus (253) Google Scholar, 16Unemori EN Ferrara N Bauer EA Amento EP Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells.J Cell Physiol. 1992; 153: 557-562Crossref PubMed Scopus (468) Google Scholar, 17Zucker S Mirza H Conner CE Lorenz AF Drews MH Bahou WF Jesty J Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation.Int J Cancer. 1998; 75: 780-786Crossref PubMed Scopus (264) Google Scholar These MMPs are thought to play a key role in the degradation of basement membrane of blood vessels and their surrounding ECM, facilitating endothelial cell migration.18Nelson AR Fingleton B Rothenberg ML Matrisian LM Matrix metalloproteinases: biologic activity and clinical implications.J Clin Oncol. 2000; 18: 1135-1149Crossref PubMed Google Scholar However, little is known about the expression of VEGF receptors or biological activity of VEGF in articular cartilage. In the present study, we examined the expression of VEGF isoforms and their receptors in OA and normal (NOR) articular cartilage, and the effect of VEGF on the production of MMPs and their common inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] in cultured OA and NOR chondrocytes. Our results demonstrate that VEGF and its receptors are expressed in OA cartilage and VEGF stimulates OA chondrocytes to produce MMP-1 and MMP-3 without affecting the levels of TIMPs. Nonosteophytic cartilage samples were obtained at arthroplasty from 3 hip joints and 46 knee joints (49 samples) with OA (72 ± 8 years old, mean age ± SD) diagnosed according to the American Rheumatism Association Criteria.19Altman R Asch E Bloch D Bole G Borenstein D Brandt K Christy W Cooke TD Greenwald R Hochberg M Howell D Kaplan D Koopman W Longley III, S Mankin H McShane DJ Medsger TJ Meenan R Mikkelsen W Moskowitz R Murphy W Rothschild B Segal M Sokoloff L Wolfe F Development of criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of the knee.Arthritis Rheum. 1986; 29: 1039-1049Crossref PubMed Scopus (5545) Google Scholar NOR control cartilage samples without macroscopic changes were taken from 17 hip joints (17 samples) with femoral neck fracture (79 ± 9 years old). All of the samples were cut into slices (∼3-mm thick), fixed with periodate-lysine-paraformaldehyde or 4% paraformaldehyde fixatives for ∼24 hours at 4°C, and embedded in paraffin wax after decalcification with 0.5 mol/L of ethylenediaminetetraacetic acid, pH 7.4. Periodate-lysine-paraformaldehyde-fixed paraffin sections (4 μm thick) were stained with hematoxylin and eosin or toluidine blue, and subjected to histological/histochemical grading according to Mankin and colleagues.20Mankin HJ Dorfman H Lippiello L Zarins A Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data.J Bone Joint Surg Am. 1971; 53: 523-537Crossref PubMed Scopus (1992) Google Scholar For the experimental use of the surgical samples, informed consent was obtained from the patients according to the hospital ethical guidelines. Total RNA was extracted directly from articular cartilage samples (13 OA and 5 NOR samples) by the acid guanidium-phenol-chloroform method using Isogen (Nippon Gene, Toyama, Japan) according to the manufacturer's protocol. By using a random oligonucleotide hexamer (Takara, Otsu, Japan), randomly primed cDNAs were prepared from 2 μg of total RNA by Superscript II reverse transcriptase (Life Technologies, Inc., Rockville, MD). A 1-μl aliquot of the reaction product was subjected to RT-PCR analysis on the expression of VEGF, VEGFR-1, VEGFR-2, NRP-1, and β-actin at 30 cycles. PCR was performed in a 50-μl reaction volume containing 800 nmol/L of each primer, 220 μmol/L of dNTPs, and 1 U of Ex TaqDNA polymerase (Takara, Otsu, Japan). The thermal cycle was 1 minute at 94°C, 1 minute at 64°C for VEGF and VEGFR-1, 63°C for VEGFR-2 and NRP-1, or 65°C for β-actin, and 1 minute at 72°C, followed by 3 minutes at 72°C for the final extension. The nucleotide sequences of the PCR primers were 5′-TGCCTTGCTGCTCTACCTCC-3′ (forward, on exon 1) and 5′-TCACCGCCTCGGCTTGTCAC-3′ (reverse, on exon 8) for VEGF; 5′-GATGTTGAGGAAGAGGAGGATT-3′ (forward) and 5′-AAGCTAGTTTCCTGGGGGTATA-3′ (reverse) for VEGFR-1; 5′-GATGTGGTTCTGAGTCCGTCT-3′ (forward) and 5′-CATGGCTCTGCTTCTCCTTTG-3′ (reverse) for VEGFR-2; 5′-CAACGATAAATGTGGCGATACT-3′ (forward) and 5′-TATACTGGGAAGAAGCTGTGAT-3′ (reverse) for NRP-1; 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ (forward) and 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′ (reverse) for β-actin. The above RT-PCR analysis enabled us to differentiate each isoform of VEGF by the difference in size of the amplified DNA fragments. The expected sizes of the amplified cDNA fragments of VEGF121, VEGF145, VEGF165, VEGF189, VEGF206, VEGFR-1, VEGFR-2, NRP-1, and β-actin were 0.41, 0.48, 0.54, 0.61, 0.66, 1.1, 0.56, 0.82, and 0.66 kb, respectively. An aliquot of the PCR product was electrophoresed in a 2% agarose gel, and stained with ethidium bromide. To confirm the specific amplification from the target mRNAs, the RT-PCR products were subcloned into the pBluescript KS vector (Stratagene, La Jolla, CA) and analyzed by sequencing with fluorescent T7 primer (Amersham Pharmacia Biotech, Buckinghamshire, UK) using a Thermo Sequenase fluorescent-labeled primer cycle sequencing kit (Amersham Pharmacia Biotech) and ALF DNA sequencer II (Amersham Pharmacia Biotech). Paraffin sections from the paraformaldehyde-fixed samples (seven OA and five NOR cartilage samples) were treated with proteinase K (5 μg/ml; Sigma Chemical Co., St. Louis, MO) in 10 mmol/L of Tris-HCl, pH 8.0, and 1 mmol/L of ethylenediaminetetraacetic acid at 37°C for 15 minutes, and postfixed in 4% paraformaldehyde at room temperature for 10 minutes. They were then rinsed in 0.1 mol/L of phosphate buffer and incubated in 0.2 mol/L of HCl at room temperature for 10 minutes. After washing in 0.1 mol/L of phosphate buffer, they were dehydrated in ethanol and air-dried. Single-stranded sense and anti-sense digoxigenin-labeled RNA probes were generated by in vitro transcription of the cDNA with T3 or T7 RNA polymerase using the DIG RNA labeling kit (Boehringer Mannheim, Mannheim, Germany) following the protocol from manufacturer. Template DNA was a 517-bp cDNA encoding human VEGF121, which was cloned in pBluescript KS vector. This cDNA clone was kindly provided by Dr. Herbert A. Weich (Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany). Hybridization with the digoxigenin-labeled RNA probes was performed at 50°C for 16 hours in 40 μl of buffer containing 50% formamide, 10 mmol/L Tris-HCl, pH 7.6, 0.2 μg/μl tRNA, 1× Denhardt's solution (0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrolidone), 10% dextran sulfate, 600 mmol/L NaCl, 0.25% sodium dodecyl sulfate, and 1 mmol/L ethylenediaminetetraacetic acid. After hybridization, the sections were washed in a buffer containing 50% formamide and 2× standard saline citrate at 50°C for 30 minutes, followed by digestion with ribonuclease A (10 μg/ml; Wako Pure Chemical Industries, Osaka, Japan) at 37°C for 30 minutes. After washing in 2× standard saline citrate at 50°C for 20 minutes and twice in 0.2× standard saline citrate at 50°C for 20 minutes, they were treated with 0.3% hydrogen peroxide and 0.1% sodium azide in distilled water for 30 minutes at room temperature to block endogenous peroxidase activity. After blocking nonspecific binding with 10% normal horse serum, they were incubated with mouse anti-digoxigenin antibody (1/750 dilution; Boehringer Mannheim) at room temperature for 90 minutes, then incubated with biotinylated horse antibodies against mouse immunoglobulin (IgG) (1/200 dilution; Vector Laboratories, Burlingame, CA) for 30 minutes, and finally reacted with an avidin-biotin-peroxidase complex solution (1/100 dilution; DAKO, Glostrup, Denmark) for 30 minutes. Color was developed with 0.2 mg/ml of 3,3′-diaminobenzidine tetrahydrochloride in 50 mmol/L Tris-HCl, pH 7.6, containing 0.003% hydrogen peroxide, and the sections were counterstained with hematoxylin. Sections from the periodate-lysine-paraformaldehyde-fixed samples (44 OA and 12 NOR cartilage samples) were treated with 0.3% H2O2 and 10% normal horse serum to block endogenous peroxidase and nonspecific binding, respectively. The sections were then treated with rabbit polyclonal antibodies against human VEGF (1/50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 90 minutes. After the reactions with goat antibodies against rabbit IgG conjugated to peroxidase labeled-dextran polymer (no dilution; En Vision+ Rabbit; DAKO) at room temperature for 30 minutes, the color was developed with 3,3′-diaminobenzidine tetrahydrochloride in 50 mmol/L of Tris-HCl, pH 7.6, containing 0.006% H2O2. Counterstaining was performed with hematoxylin. As for a control, sections were reacted by replacing the first antibodies with nonimmune rabbit IgG (DAKO) or with the anti-VEGF antibodies that were incubated with the blocking peptide for VEGF (Santa Cruz Biotechnology) at room temperature for 2 hours before the immunostaining. For immunohistochemistry of VEGFR-1 and VEGFR-2, paraffin sections of OA (five samples) and NOR cartilage (five samples) were reacted with goat polyclonal antibodies against VEGFR-1 (1/50 dilution; R&D Systems, Minneapolis, MN) or mouse monoclonal antibody against VEGFR-2 (1/50 dilution; Santa Cruz Biotechnology), and then treated with biotinylated rabbit IgG to goat IgG and horse IgG to mouse IgG (Vector Laboratories), respectively. As for a control, primary antibodies were replaced with nonimmune goat or mouse IgG (Santa Cruz Biotechnology). After the reactions, the sections were incubated with avidin-biotin-peroxidase complex (DAKO) and the color was developed with 3,3′-diaminobenzidine tetrahydrochloride as described above. Full-thickness cartilage slices (18 OA and 12 NOR samples) were cultured in serum-free Dulbecco's modified Eagle's medium: Nutrient Mixture F-12 (Life Technologies, Inc.) containing 0.2% lactalbumin hydrolysate for 3 days. After centrifugation at 1500 rpm for 5 minutes, the culture media were stored at −20°C until used for assay. The levels of VEGF (ng/ml) were measured using the enzyme-linked immunosorbent assay (ELISA) system for human VEGF, which recognizes VEGF121 and VEGF165 isoforms (R&D Systems). The values were expressed as ng/g dry tissue weight. Isoforms of VEGF were also analyzed by immunoblotting. The media (2 ml/lane) from OA and NOR cartilage slices (two samples each), which showed high and negligible levels of VEGF by ELISA, were first concentrated with 3.3% trichloroacetic acid and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% total acrylamide) under the reducing condition. The proteins separated in the gels were transferred onto nitrocellulose filters, and the filters were incubated for ∼12 hours at room temperature with rabbit polyclonal antibodies against human VEGF (1/100 dilution; Santa Cruz Biotechnology). They were reacted with biotinylated goat IgG to rabbit IgG and color was developed with 3,3′-diaminobenzidine tetrahydrochloride as described above. As for a control, culture media (2 ml/lane) of OA chondrocytes (three samples) were subjected to immunoblotting for VEGF according to the above-mentioned method. Chondrocytes were isolated from OA (seven samples) and NOR cartilage (five samples) by digestion of the minced cartilage with 0.4% (w/v) Actinase E (Kaken, Tokyo, Japan) for 1 hour at 37°C and then with 0.025% (w/v) bacterial collagenase type I (Worthington Biochemical Corp., Freehold, NJ) for ∼16 hours at 37°C. Isolated cells were plated on 24-well culture flasks at a density of 5 × 105 cells/well in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 24 hours. The culture media were replaced with serum-free Dulbecco's modified Eagle's medium containing 0.2% lactalbumin hydrolysate, and then treated with recombinant VEGF165 (0, 10, or 50 ng/ml) (R&D Systems) for 3 days. After the treatment, the media were harvested and centrifuged at 3000 rpm for 5 minutes. The supernatants were stored at −20°C until used for assays of MMPs and TIMPs. To show the expression of VEGFR-1 and VEGFR-2 and exclude the possibility of the endothelial cell contamination in the isolated OA and NOR chondrocytes, the cells were cultured on Lab-Tek chamber slides (Nalge-Nunc International, Tokyo, Japan) and subjected to immunohistochemistry for the receptors and endothelial cell markers (CD31 and von Willebrand factor). They were reacted with antibodies against VEGFR-1 (1/50 dilution; R&D Systems) and VEGFR-2 (1/50 dilution; Santa Cruz Biotechnology), mouse monoclonal antibody against CD31 (1/200 dilution; DAKO), rabbit polyclonal antibodies against von Willebrand factor (1/200 dilution; DAKO), or nonimmune goat, mouse and rabbit IgG (DAKO). As for a control, human umbilical vein endothelial cells in culture were also subjected to the immunostaining with these antibodies. A chondrocytic phenotype of the cultured OA and NOR cells was confirmed by the positive immunostaining of aggrecan and type II collagen by using the mouse monoclonal antibody against human aggrecan (1/10 dilution; Abcam Limited, Cambridge, UK) and rabbit polyclonal antibodies against human type II collagen (1/20 dilution; Monosan, Am Uden, Netherlands) (data not shown). Concentrations (ng/ml) of MMP-1, -2, -3, -7, -8, -9, and -13 and TIMP-1 and -2 secreted into the culture media of OA and NOR chondrocytes were determined according to the corresponding sandwich enzyme immunoassay systems as we have described previously.21Nakamura H Ueno H Yamashita K Shimada T Yamamoto E Noguchi M Fujimoto N Sato H Seiki M Okada Y Enhanced production and activation of progelatinase A mediated by membrane-type 1 matrix metalloproteinase in human papillary thyroid carcinomas.Cancer Res. 1999; 59: 467-473PubMed Google Scholar The sandwich enzyme immunoassay systems for MMP-1, -3, -8, and -13 measure both precursor and active forms of the MMPs, but those for MMP-2, -7, and -9 detect only their latent forms (proMMPs). The EIA for TIMP-1 determines the whole amount of TIMP-1 including free TIMP-1 and the complexed forms with active MMPs and proMMP-9. However, the sandwich enzyme immunoassay for TIMP-2 detects free TIMP-2 and TIMP-2 complexed with active MMPs, but not the complex with proMMP-2. To study activation of proMMP-2 and proMMP-9, the culture media of OA chondrocytes were subjected to gelatin zymography according to our method.22Nakada M Nakamura H Ikeda E Fujimoto N Yamashita J Sato H Seiki M Okada Y Expression and tissue localization of membrane-type 1, 2, and 3 matrix metalloproteinases in human astrocytic tumors.Am J Pathol. 1999; 154: 417-428Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar After sodium dodecyl sulfate-polyacrylamide gel electrophoresis using gelatin-containing gels, the gels were washed in 2.5% Triton X-100 to remove sodium dodecyl sulfate, incubated for 22 hours at 37°C in 50 mmol/L Tris-HCl, pH 7.4, containing 0.15 mol/L NaCl, 10 mmol/L CaCl2, and 0.02% NaN3, and then stained with 0.1% Coomassie Brilliant Blue R250. Activation ratios were estimated by computer-assisted densitometric scanning. The proliferation of chondrocytes was assayed by the cell proliferation ELISA system (Amersham Pharmacia Biotech) according to the manufacture's protocol. In brief, chondrocytes were plated on 96-well culture flasks at a density of 2 × 105 cells/well in Dulbecco's modified Eagle's medium containing 0.2% lactalbumin hydrolysate for 24 hours. The culture media were replaced with the same media and treated with recombinant VEGF165 (0, 10, 50, and 100 ng/ml) (R&D Systems) for 48 hours. After adding 5-bromo-2′-deoxyuridine (BrdU) to give a final concentration of 10 μmol/L, the cells were incubated for additional 2 hours at 37°C. Incorporation of BrdU to the chondrocytes was detected using peroxidase-labeled anti-BrdU antibody by the immunoperoxidase method and the optical density was determined in a microplate reader (Bio-Rad Laboratories, Hercules, CA). Mann-Whitney U-test was used to compare the data of the OA and NOR samples. Simple linear regression or Spearman's rank correlation was used for analysis of relationship between different parameters recorded in this study, and one-way repeated-measures analysis of variance was used for comparison between more than three parameters. The expression of VEGF isoforms and their receptors was examined by RT-PCR using total RNA extracted directly from OA and NOR cartilage. As shown in Figure 1, three isoforms of VEGF121, VEGF165, and VEGF189 were expressed in all of the samples from both OA and NOR cases, whereas VEGF145 and VEGF206 were not detected in the samples. On the other hand, mRNA expression of their receptors was recognized only in OA cartilage: VEGFR-1 (92%, 12 of 13 cases), VEGFR-2 (69%, 9 of 13 cases), and NRP-1 (100%, 13 of 13 cases). Interestingly, the samples expressing VEGFR-2 were also positive for both VEGFR-1 and NRP-1, the latter of which is a co-receptor of VEGFR-2 (Figure 1). The specific amplification from the target mRNAs was confirmed by sequencing the amplified DNA products (data not shown). Cells expressing VEGF mRNA were identified by in situ hybridization. Chondrocytes in the superficial and transitional zones of the OA cartilage were labeled with the anti-sense RNA probe, and some clustered chondrocytes were also positive (Figure 2C). The signal in the chondrocytes of the NOR cartilage was negligible (Figure 2A). The sense probe gave only a background signal in the chondrocytes of OA (Figure 2D) and NOR cartilage (Figure 2B). The 44 cartilage specimens from nonosteophytic areas of OA cartilage (44 cases) had the typical OA changes such as surface irregularities, fibrillation, and fissuring. Mankin scores of the samples ranged from 2 to 12 (5.9 ± 2.4, mean ± SD; n = 44). The 12 control samples from NOR articular cartilage (12 cases) had little or no microscopic changes with Mankin scores 0 or 1 (0.6 ± 0.5, n = 12). Immunohistochemistry demonstrated that VEGF localizes to the OA chondrocytes mainly in the superficial and transitional zones in 95% of the samples (42 of 44 samples) (Figure 3, B and C). The chondrocytes located in the radial zone were stained, when the cartilage had deep fissures reaching the zone. Clustered chondrocytes close to the fissures were frequently labeled (Figure 3C). When the percentage of the immunostained chondrocytes to the whole cells was calculated, ∼25% of the total chondrocytes on average (28.6 ± 23.0%) were immunostained positively in the OA samples. VEGF staining was also found in 50% of the NOR cartilage samples (6 of 12 samples), but only a few chondrocytes in the superficial zone (2.5 ± 2.6%) were immunostained (Figure 3A). The percentage of the VEGF-positive chondrocytes was significantly higher in the OA than in the NOR cartilage (P < 0.001). The specificity of the VEGF immunostaining was assured, because the staining was abolished with the antibody absorbed with the neutralization peptide (Figure 3D) or no staining was observed with nonimmune rabbit IgG (data not shown). A linear correlation was found between the percentage of immunostained chondrocytes and Mankin score (r = 0.906, n = 56, P < 0.001) (Figure 4).Figure 4Correlation of VEGF i
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