• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jtoxpJSTP Web homeInformation for AuthorsSubmissionJSTP Journals and PublicationsJournal Description
J Toxicol Pathol. Jun 2011; 24(2): 137–142.
Published online Jun 30, 2011. doi:  10.1293/tox.24.137
PMCID: PMC3234605

Expression of Vascular Endothelial Growth Factor (VEGF) Associated with Histopathological Changes in Rodent Models of Osteoarthritis

Abstract

Vascular endothelial growth factor (VEGF) and its receptors have recently reported to be expressed in human osteoarthritis (OA), suggesting that VEGF could be implicated in the pathogenesis of this disease. In the present study, expression of VEGF in the articular cartilage was determined in three different OA models: medial meniscectomy and monoiodoacetate (MIA) injection in rats and age-associated spontaneous joint cartilage destruction in guinea pigs. VEGF was detected by immunohistochemical analysis in the regenerative and hypertrophic chondrocytes, perichondrium and osteophyte areas and chondrocyte clones. Stain intensity of VEGF immunoreactivity increased simultaneously with the degree of cartilage destruction and reparation. These results suggest that VEGF is a key factor in the articular cartilage in human OA and animal OA models.

Keywords: Osteoarthritis, Vascular endothelial growth factor (VEGF), Immunohistochemistry, Medial meniscectomy, Monoiodoacetate (MIA)

Osteoarthritis (OA) is a degenerative disease of joint cartilage that occurs in a large proportion of elderly people. In OA, cartilage matrix is lost gradually, which eventually devastates functional joints. Joint pain and movement limitation are the primary symptoms associated with this disease. Contributing to OA pathogenesis, cartilage damage induces disease-related factors, including proteolytic enzymes of the matrix metalloproteinase (MMPs) and aggrecanase families, cytokines, chemokines and growth factors1–7. However, causal genes, the molecular biological background and the signal pathways of this disease are largely unknown. There are a number of OA models, such as anterior surgical ligament transection (ACLT), medial meniscectomy1,7–9, collagenase injection10, extracellular matrix loss11,12, impact-induced trauma13, monoiodoacetate (MIA) injection14–16, the age-associated spontaneous OA-like model17,18 and STR/OrtCrlj mice19,20. It is essential that these OA models are investigated in creating new drugs for OA disease and for each different stage in drug development, such as the screening and preclinical stages. However, each of these animal models likely reflects only a subset of cases due to the heterogeneity of human OA. Moreover, though human OA and these models actually have some common pathological appearances, analyses of histopathological similarities between human OA and these animal models have just started. Recently, it was reported that VEGF and its receptors are expressed in human OA accompanying the progression of this disease, and this suggested the possibility that a mechanism via VEGF is implicated for destruction of OA articular cartilage21.

In the present study, we examined the expression of VEGF in the articular cartilage in OA-like models; rat medial meniscectomy, rat MIA injection and guinea pig age-associated spontaneous joint cartilage destruction. We then found that the immunoreactivity of VEGF in the cartilage commonly enhances with the degree of cartilage destruction and reparation in these models of rodents.

We used two OA-like models of the rat: medial meniscectomy and MIA injection. Moreover, an aged guinea pig model showing spontaneous joint cartilage destruction was also investigated. This is abbreviated here as the “SPOA model.” In the medial meniscectomy model, twelve male Fischer rats (F344/DuCrlCrlj, 12 weeks old) were used, and the surgery was carried out at the facilities of Charles River Laboratories Japan Inc. (Yokohama, Japan). At 1, 2 and 5 weeks after medial meniscectomy and sham operation (n = 3 per group) as a control, rats were killed under anesthesia by isoflurane inhalation, and the right knee joints were removed for pathological evaluation. In the MIA model, four male Lewis rats (8 weeks old) were used and treated with a single intra-articular injection of 0.3 mg MIA (Wako Pure Chemical Industries, Ltd., Osaka, Japan). At 1 week after MIA or saline injection (n = 2 per group) as a control, rats were killed under anesthesia, and the right knee joints were removed for pathological evaluation. In the SPOA model, eight Hartley guinea pigs purchased from Charles River Laboratories Japan Inc. were used at 16 months of age and 10 weeks age as control (n = 4 per group), and their joints were examined. All animal experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved in advance by the Committee of Animal Experiments in Research Laboratories of Mitsubishi Tanabe Pharma Corporation.

Knee joints in the three models were fixed in 10% neutralized buffered formalin, decalcified with buffered EDTA (10% ethylenediaminetetraacetic acid, pH 7.4), transected in the frontal or sagittal plane and embedded in paraffin. The sections included the tibial plateau and femoral condyle and were stained with safranin O or toluidine blue and evaluated for cartilage damage and osteophyte formation. Immunohistochemistry was employed to assess expression and localization of VEGF according to the histopathological progress of damage/repair of the articular cartilage. Tissue sections were incubated overnight at 4 °C with anti-VEGF (A-20) rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) followed by horseradish per-oxidase-labeled goat antibody against rabbit IgG (Nichirei, Tokyo, Japan) and visualized with 3.3’-diaminobenzidine.

Initially, histopathological changes and VEGF immunohistochemistry were examined in the articular cartilage of the tibial medial plateau in the rat medial meniscectomy model. A knee joint from a sham-operated animal used as a control is depicted in Fig. 1, panels A and B. One week after medial meniscectomy, the articular cartilage showed fibrillation, loss and degeneration of chondrocytes (Fig. 1C). Two weeks after the surgery, a migrational response of the perichondrium and initial osteophyte formation were seen (Fig. 1E). Moreover, osteophyte development and reorganization of the regenerative articular cartilage were seen at 5 weeks after surgery (Fig. 1G). Expression of VEGF in the articular cartilage was not detected in normal rats and the animals studied one week after surgery (Fig. 1B, D). Two weeks after the surgery, VEGF was observed in the perichondrium and regenerative or hypertrophic chondrocytes (Fig. 1F). The stain intensity of VEGF became obvious as the chondrocytes in the osteophyte area increased at 5 weeks after surgery (Fig. 1H).

Fig. 1.
Histopathological changes of the articular cartilage of the tibial medial plateau in the frontal plane. The bars indicate 0.5 mm. A, C, E, G: Toluidine blue staining. B, D, F, H: Immunohistochemical staining of VEGF. A, B: Sham operation ...

Histopathological changes and VEGF immunohistochemistry were then examined in the articular cartilage in rats of the MIA injection model. A knee joint from a saline-injected animal as a control is depicted in Fig. 2, panels A and B. At 1 week after MIA injection, loss and degeneration of chondrocytes and a reduction of safranin O-positive proteoglycan staining were observed in the articular cartilage without osteophyte formation (Fig. 2C, 2E). Although expression of VEGF was not detected in the articular cartilage of normal rats (Fig. 2B), some regenerative or hypertrophic chondrocytes showed positive immunoreaction 1 week after MIA injection (Fig. 2D, 2F).

Fig. 2.
Histopathological changes of the articular cartilage of the femoral condyle after monoiodoacetate (MIA) injection in the frontal plane. The bars indicate 200 μm. A, C: Safranin O staining. B, D: Immunohistochemical staining ...

Finally, histopathological changes and VEGF immunohistochemistry were examined in the articular cartilage in the guinea pig SPOA model. Examples of young guinea pigs (10 weeks old) are shown in Fig. 3, panels A and B. In aged guinea pigs (16 months old), the articular cartilage became thin and included advanced tidemark, chondrocytes were degenerated and proteoglycan staining was reduced (Fig. 3C). Fibrillation and osteophyte formation were seen in the tibial medial plateau in the frontal plane (data not shown). Moreover, appearance of chondrocyte clones were seen in the destructive cartilage matrix (Fig. 3E). Although expression of VEGF was not detected in the articular cartilage of young guinea pigs (Fig. 3B), some regenerative or hypertrophic chondrocytes showed positive immunoreaction against VEGF in the articular cartilage of aged guinea pigs (Fig. 3D). Moreover, the chondrocyte clones showed a strongly positive reaction against VEGF in the superficial and middle zone (Fig. 3F).

Fig. 3.
Histopathological changes of the articular cartilage on age-associated spontaneous OA in guinea pigs. The bars indicate 200 μm. A, C, E: Safranin O staining. B, D, F: Immunohistochemical staining of VEGF. A, B: 10 weeks old. ...

OA is pathologically characterized by fibrillation and erosion in cartilage, by chondrocyte proliferation and osteophyte formation at the joint margins and by sclerosis of subchondral bone. Destruction of articular cartilage (chondrocytes) involving catabolism of matrix proteins of chondrocytes, such as type 2 collagen and aggrecan, is caused by proteolytic enzymes of the matrix metalloproteinase (MMPs) and aggrecanase families. It is also reputed that MMPs or MMP13 inhibitors protect cartilage degeneration2–4 and that mice deficient in a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and MMP9 are protected from cartilage damage in some models5,6. Furthermore, other factors such as transforming growth factor-beta (TGF-β), cytokines, chemokines and cathepsin have been advocated for involvement in OA pathogenesis, but the relationship between these factors and articular cartilage destruction is still unknown.

In pathological conditions, such as rheumatoid arthritis and OA, damaged articular cartilage is frequently covered with and invaded by granulation tissue with high vascularity, the so- called pannus tissue. These findings under pathophysiological conditions suggest the involvement of angiogenic factors in the process. Among these, VEGF, which is produced by hypertrophic chondrocytes, is considered to be a coordinator of extracellular matrix (ECM) remodeling, angiogenesis and bone formation in the growth plate22. In fact, expression of VEGF was reported in human OA patients21 and in the rabbit ACLT model23.

In the present study, we found that some regenerative chondrocytes, perichondria and clones showed immunoreaction against VEGF in the MIA model, the medial meniscectomy model and the SPOA model. We demonstrated that expression of VEGF in cartilage was common to three different OA models established by different stress mechanisms. Indeed, stain intensity of VEGF was gradually enhanced simultaneously with histopathological severity, which was evaluated based on such things as regeneration and hypertrophy of the chondrocytes and clones, migrational response of perichondrium, as well as osteophyte formation and development after articular cartilage destruction. Some authors have found that regenerative chondrocytes or clones increase with the severity of OA24. The present data suggest a possible action of VEGF on the articular chondrocytes in OA-like models of rodents. The primary function of VEGF was supposed to be that of angiogenesis. However, recent studies have demonstrated that the biological function of VEGF is dictated mainly by the expression of its receptors on the cells in various tissues besides blood vessels. Reportedly, at the cellular level, VEGF did not stimulate the proliferation of human OA chondrocytes21. However, VEGF stimulates OA chondrocytes to produce increased amounts of MMP-1 and MMP-3 without changing the production levels of TIMPs21. In animals models, in vivo, regenerative chondrocytes or clones subjected to different forms of stress produce VEGF, and VEGF may then stimulate OA chondrocytes to produce increased MMPs and other proteolytic enzymes for destruction of articular cartilage.

In conclusion, we demonstrated that VEGF is expressed in the articular cartilage of OA-like models of rodents just as previously reported in human OA patients. Our data also suggest that VEGF is likely to be causally involved in the destruction of articular cartilage and in its repair. However, the intrinsic functions of VEGF in the pathogenesis of OA remain to be elucidated; in particular the localization and specification of its subtypes and receptors need to be clarified.

Acknowledgments

We thank Mr. Mamoru Koyama and Ms. Yasuko Ogawa for their excellent technical assistance. We also thank Prof. Dr. Bernhard F. Becker (Munich University) for restyling our English.

References

1. Hayami T, Pickarski M, Wesolowski GA, McLane J, Bone A, Destefano J, Rodan GA, Duong le T. The role of subchondral bone remodeling in osteoarthritis: reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model. Arthritis Rheum. 50: 1193–1206 2004. [PubMed]
2. Janusz MJ, Hookfin EB, Heitmeyer SA, Woessner JF, Freemont AJ, Hoyland JA, Brown KK, Hsieh LC, Almstead NG, De B, Natchus MG, Pikul S, Taiwo YO. Moderation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis Cartilage. 9: 751–760 2001. [PubMed]
3. Janusz MJ, Bendele AM, Brown KK, Taiwo YO, Hsieh L, Heitmeyer SA. Induction of osteoarthritis in the rat by surgical tear of the meniscus: Inhibition of joint damage by a matrix metalloproteinase inhibitor. Osteoarthritis Cartilage. 10: 785–791 2002. [PubMed]
4. Baragi VM, Becher G, Bendele AM, Biesinger R, Bluhm H, Boer J, Deng H, Dodd R, Essers M, Feuerstein T, Gallagher BM, Jr, Gege C, Hochgürtel M, Hofmann M, Jaworski A, Jin L, Kiely A, Korniski B, Kroth H, Nix D, Nolte B, Piecha D, Powers TS, Richter F, Schneider M, Steeneck C, Sucholeiki I, Taveras A, Timmermann A, Van Veldhuizen J, Weik J, Wu X, Xia B. A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: Evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models. Arthritis Rheum. 60: 2008–2018 2009. [PubMed]
5. Botter SM, Glasson SS, Hopkins B, Clockaerts S, Weinans H, van Leeuwen JP, van Osch GJ. ADAMTS5-/- mice have less subchondral bone changes after induction of osteoarthritis through surgical instability: implications for a link between cartilage and subchondral bone changes. Osteoarthritis Cartilage. 17: 636–645 2009. [PubMed]
6. Itoh T, Matsuda H, Tanioka M, Kuwabara K, Itohara S, Suzuki R. The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis. J Immunol. 169: 2643–2647 2002. [PubMed]
7. Appleton CT, Pitelka V, Henry J, Beier F. Global analysis of gene expression in early experimental osteoarthritis. Arthritis Rheum. 56: 1854–1868 2007. [PubMed]
8. Williams JM, Felten DL, Peterson RG, O’Connor BL. Effects of surgically induced instability on rat knee articular cartilage. J Anat. 134: 103–109 1982. [PMC free article] [PubMed]
9. Hayami T, Pickarski M, Zhuo Y, Wesolowski GA, Rodan GA, Duong le T. Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transaction and meniscectomized models of osteoarthritis. Bone. 38: 234–243 2006. [PubMed]
10. van der Kraan PM, Vitters EL, van Beuningen HM, van de Putte LB, van den Berg WB. Degenerative knee joint lesions in mice after a single intra-articular collagenase injection. A new model of osteoarthritis. J Exp Pathol. 71: 19–31 2006 [PMC free article] [PubMed]
11. Williams JM, Ongchi DR, Thonar EJ. Repair of articular cartilage injury following intra-articular chymopapain-induced matrix proteoglycan loss. J Orthop Res. 11: 705–716 1993. [PubMed]
12. Williams JM, Uebelhart D, Thonar EJ, Kocsis K, Modis L. Alteration and recovery of spatial orientation of collagen network of articular cartilage in adolescent rabbits following intra-articular chymopapain injection. Connect Tissue Res. 34: 105–117 1996. [PubMed]
13. Mazieres B, Blanckaert A, Thiechart M. Experimental post-contusive osteoarthritis of the knee: quantitative microscopic study of the patella and the femoral condyles. J Rheumatol. 14: 119–121 1987. [PubMed]
14. Kobayashi K, Imaizumi R, Sumichika H, Tanaka H, Goda M, Fukunari A, Komatsu H. Sodium iodoacetateinduced experimental osteoarthritis and associated pain model in rats. J Vet Med Sci. 65: 1195–1199 2003. [PubMed]
15. Janusz MJ, Little CB, King LE, Hookfin EB, Brown KK, Heitmeyer SA, Caterson B, Poole AR, Taiwo YO. Detection of aggrecanase- and MMP-generated catabolic neoepitopes in the iodoacetate model of cartilage degeneration. Osteoarthritis Cartilage. 12: 720–728 2004. [PubMed]
16. Vermeirsch H, Biermans R, Salmon PL, Meert TF. Evaluation of pain behavior and bone destruction in two arthritic models in guinea pig and rat. Pharmacol Biochem Behav. 87: 349–359 2007. [PubMed]
17. Bendele AM, Hulman JF. Spontaneous cartilage degeneration in guinea pigs. Arthritis Rheum. 31: 561–565 1988. [PubMed]
18. de Bri E, Lei W, Svensson O, Chowdhury M, Moak SA, Greenwald RA. Effect of an inhibitor of matrix metalloproteinases on spontaneous osteoarthritis in guinea pigs. Adv Dent Res. 12: 82–85 1998. [PubMed]
19. Walton M. Degenerative joint disease in the mouse knee; histological observations. J Pathol. 123: 109–122 1977. [PubMed]
20. Mason RM, Chambers MG, Flannelly J, Gaffen JD, Dudhia J, Bayliss MT. The STR/ort mouse and its use as a model of osteoarthritis. Osteoarthritis Cartilage. 9: 85–91 2001. [PubMed]
21. Enomoto H, Inoki I, Komiya K, Shiomi T, Ikeda E, Obata K, Matsumoto H, Toyama Y, Okada Y. Vascular endothelial growth factor isoforms and their receptors are expressed in human osteoarthritic cartilage. Am J Pathol. 162: 171–181 2003. [PMC free article] [PubMed]
22. Gerber 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. 5: 623–628 1999. [PubMed]
23. Hashimoto S, Creighton-Achermann L, Takahashi K, Amiel D, Coutts RD, Lots M. Development and regulation of osteophyte formation during experimental osteoarthritis. Osteoarthritis Cartilage. 10: 180–187 2002. [PubMed]
24. Kouri JB, Arguello C. Use of microscopical techniques in the study of human chondrocytes from osteoarthritic cartilage. Micro Res Tech. 40: 22–36 1998 [PubMed]

Articles from Journal of Toxicologic Pathology are provided here courtesy of The Japanese Society of Toxicologic Pathology
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...