The Expression Of VEGF And It Receptors In Development And Remodeling In Rat Mandibular Condylar Cartilage

VEGF, also known as vascular permeability factor, has been associated with angiogenesis in numerous physiological and pathological states. It is a multifunctional cytokine that contributes to angiogenesis by both direct and indirect mechanisms. On one hand, it stimulates the endothelial cells to proliferate, migrate and alter their pattern of gene expression. On the other hand, it renders these same microvascular endothelial cells hyperpermeable, so that they releae fibrinogen into the extravascular space. Extravascular fibrin serves as a provisional matrix that favours and supports the growth of new blood vessels.For a long time, VEGF was considered an endothelial-specific factor. Only in the last few years has attention been focused on its synthesis and the presence of its receptors in non- endothelial tissues. As far as chondrogenic lineage, the expression of VEGF has been observed in the cartilaginousvertebral discs of mice and in growth-plate cartilage. Moreover, there is consistent evidence for the involvement of VEGF in pathological neovascularization of joints, with increased amounts of the favtor being in cartilage, synovial tissues and fluids from osteoarthritis and rheumatoid arthritis.VEGF has been demonstrated in mature hypertrophic chondrocytes in vitro systems and in developing human bone. VEGF, synthesized by the growth plate hypertrophic chondrocytes, is responsible for hypertrophic cartilage invision by metaphyseal blood vessels. Furthermore, VEGF has an autocrine activity also in proliferation and differentiation of chondrocytes and in growth plate morphogenesis and remoldeling.However, little is known about the expression of VEGF and it receptors (Flt-1 and Flk-1) in rat mandibular condylar cartilage or biological activity of VEGF in development and remodeling in rat condylar cartilage. Our purposes now were to (1) identify the the expression of VEGF isoforms and their receptors in rat condylar cartilage by Immunohistochemistry and Western-blot and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis; (2) compare the expression of VEGF and its receptors (Flt-1 and Flk-1) in the various stages of natural growth and remoldeling of condylar cartilage in the rat model with gradually induced occlusal disorders.Part I:The protein and mRNA expression of VEGF and its receptors (Flt-1 and Flk-1) were seen in rat condylar cartilage. VEGF immunoreactivity was primarily evident in the proliferative cell layer. In addition, the upper part of the hypertrophic cell layer showed strong immunoreactivity that gradually descreased as cells became closer to the osteogenic front. The lowest part of this layer, which encompassed calcified cartilage, had weak immunoreactivity. Neither fibrous nor prechondroblastic cell layer demonstrated evident immunoreactivity against VEGF. No immunoreactivity for VEGF receptors(Flk-1 and Flt-1) antibody was demonstrated in the fibrous or prechondroblastic cell layer of the mandibular condylar cartilage, except for weak immunoreactivity in some of the deepest prechondroblasts. Immunoreactivity became more strongly evident in the chondroblastic zone, where a well-defined reaction product was seen. Although, immunoreactivity was strongly defined in this area, isolated areas with little or sparse immunoreactivity were seen. Immunoreactivity extended to the upper part of the hypertrophic zone, where the signal intensity descreased. VEGF receptors (Flk-1 and Flt-1) immunoreactivity decreased in deeper hypertrophic cells and no immunoreactivity was seen in the deepest cells of the hypertrophic zone.In western blot, VEGF which was isolated from rat mandibular condylar condrocytes was a dimeric glycoprotein with 42 KDa of molecular mass and a monomeric glycoprotein with 21 KDa of molecular mas. The western blot analysis of rat mandibular condylar cartilage also indicated that the immunoreactive species had a molecular mass of approximately 180 KDa for Flt-1 and 200 KDa for Flk-1.To see what VEGF isoforms were expressed in the developing condylar cartilage, we carried out RT-PCR. The primers, corresponding to sequences in exons 2 and 8 of the VEGF gene, were expected to amplify multiple VEGF isoforms produced by alternative splicing. Two isoforms (VEGF120 and VEGF164) were observed in an agarose gel at the position of 368 and 500 bp, respectively, as expected. Flt-1 and Flk-1 and GAPDH were seen in an agarose gel at the position of 363 and 433 and 420 bp respectively. Part II:Fourteen rats were randomly divided into four groups: 1. the first group was control group. 2. In the second group rats, incisors were ground short once a week. 3. In the third group rats, the first lower molar on the left was pulled by the orthodontic spring to make it inclined medially to result in uncoordinated cusp-fossa occlusal contacts. 4. In the fourth group rats, on onehand incisors were ground short, on the other hand the first lower molar on the left was pulled by the orthodontic spring to make it inclined medially. We examined the histological features of the condylar cartilage by haematoxyline-eosine staining and the expression of VEGF and its receptors (Flk-1 and Fit-1) in these four group rats by immunohistochemistry. The results are as followed:1. HistologyIn all groups, the histological alterations in temporomandibular joints showed mainly changes in the thickness of condylar cartilage on the whole and that of different region of condylar cartilage. In the control group rats, the anterior region was the thinnest portion and the posterior region was the thinnest portion of condylar cartilage, and the thickness of middle region was between that of the anterior region and that of the posterior region. In the second group rats, the thickness of anterior and middle region of condylar cartilage increased, but that of posterior region decreased when compared to control group. In the third group rats, the thickness of whole condylar cartilag decreased significantly when compared to control group, there was a more obvious decrease on the right than on the left. In the fourth group rats, the thickness of whole condylar cartilag decreased only on the right, interestingly, the anterior and middle region of condylar cartilage became thicker than ever, and the posterior region of condylar cartilage became thinner when compared to control group.2. ImmunohistochemistryThe expression of VEGF and its receptors (Flk-1 and Flt-1) correlated with the change of thickness of distinct region of condylar cartilage. In the control group, the staining of VEGF and its receptors (Flk-1 and Flt-1) was predominant in the hypertrophic cell layer of the posterior region of the condylar cartilage. In the second group, VEGF and its receptors (Flk-1 and Flt-1) were abundantly expressed in the middle region of the condylar...