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. 2000 Aug;59(8):607–614. doi: 10.1136/ard.59.8.607

Immunohistochemical localisation of protein tyrosine kinase receptors Tie-1 and Tie-2 in synovial tissue of rheumatoid arthritis: correlation with angiogenesis and synovial proliferation

T Uchida 1, M Nakashima 1, Y Hirota 1, Y Miyazaki 1, T Tsukazaki 1, H Shindo 1
PMCID: PMC1753215  PMID: 10913057

Abstract

OBJECTIVE—To investigate the involvement of Tie-1 and Tie-2, receptor tyrosine kinases required for angiogenesis, in synovial proliferation and angiogenesis of rheumatoid arthritis (RA).
METHODS—Synovial tissues from 10 patients with RA and three control subjects were analysed by double immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR).
RESULTS—Expression of Tie-1 and Tie-2 was seen in all synovia, but predominantly in papillary projected portions. In synovial lining cells, Tie-2 was expressed mainly in the basal layer and frequently colocalised with vimentin and proliferating cell nuclear antigen (PCNA), whereas Tie-1 was also expressed in the superficial layer. In stromal cells, Tie-2 immunoreactivity was restricted to vimentin positive fibroblast—but not macrophage derived cells, whereas Tie-1 expression was not dependent on the phenotype. Tie receptors were also highly expressed in the endothelium and surrounding pericytes of capillaries scattered over the papillary proliferated synovium without notable difference in the expression of the two receptors. Furthermore, Tie positive vessels often overexpressed PCNA. In normal synovia, expression of Tie receptors was restricted to the capillary endothelium. RT-PCR confirmed the expression of Tie-1 and Tie-2 in RA synovial tissues and also in the cultured synoviocytes.
CONCLUSION—The results suggest the possible involvement of overexpressed Tie-1 and Tie-2 in synovial lining and stromal cells in the pathophysiology of RA synovitis, probably through distinct mechanisms. Furthermore, expression of Tie receptors in actively growing vasculature may reflect the direct involvement of these receptors in angiogenesis and subsequent vascularisation.



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Figure 1  .

Figure 1  

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Expression of Tie-1 and Tie-2 in rheumatoid arthritis (RA) (A) and control (B) synovia. Three serial sections from patient No 4 were used for haematoxylin and eosin (HE) staining (left panel), immunostaining for Tie-1 (middle panel), and Tie-2 (right panel). Expression of Tie-1 and Tie-2 was widely distributed in RA synovium, such as lining cells and stromal components, including capillaries. The distribution of Tie-1 immunoreactivity was more widespread than that of Tie-2. Note the restricted expression of Tie-2 to the basal layer of stratified lining cells. Although the number of Tie-2 positive cells was somewhat less than for Tie-1, the intensity of staining was stronger than Tie-1 in both lining and stromal cells except for capillaries (insets). In control patient No 11, only the capillary components (arrow) were positive for Tie-2. Bars = 100 µm.

Figure 2  .

Figure 2  

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Double immunohistochemistry of synovial lining cells (A-D) and stromal cells (E-H). (A) Immunostaining for Tie-1. Tie-1 expression was seen throughout stratified synovial lining cells. (B) Double staining for Tie-2 (red) and CD68 (blue). Tie-2 immunoreactivity was predominantly localised in the basal layer of synovial lining cells, whereas CD68 immunoreactivity was restricted to the superficial layer. (C) Double staining with Tie-2 (red) and vimentin (blue). Colocalisation of Tie-2 and vimentin expression was obvious in the basal layer of the synovium (arrowheads) on the serial section shown in (B). (D) Double staining of Tie-2 (red) and proliferating cell nuclear antigen (PCNA) (blue). Similarly to section C, colocalisation of Tie-2 and PCNA was restricted to the basal layer (arrowheads). (E, F) Double staining of Tie-1 (red) and CD68 (blue)/vimentin (blue) in stromal cells. Tie-1 expression was frequently colocalised with both CD68 (E) and vimentin (F). (G, H) Double staining of Tie-2 (red) and CD68 (blue)/vimentin (blue) in stromal cells. In contrast with Tie-1, the distribution of Tie-2 expression was completely distinct from that of CD68 (G). On the other hand, Tie-2 expression was frequently concordant with vimentin positive cells (H) (arrowheads). A-D = patient No 4, E-H = patient No 8. Bars in each panel = 50 µm.

Figure 3  .

Figure 3  

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Tie-2 expression in the synovial vascular component. (A) Double immunohistochemistry for Tie-2 (red) and α smooth muscle actin (αSMA) (blue). Tie-2 was co-stained with αSMA, suggesting Tie-2 expression in pericytes surrounding capillaries. (B) Double staining with Tie-2 (red) and CD34 (blue) antibodies. Colocalisation of Tie-2 with CD34 was found only in capillaries with small lumen (red arrowheads) but not in tiny vessels lacking luminal structures (black arrowheads), suggesting the absence of Tie-2 expression at the initial stage of angiogenesis. (C) Tie-2 expression in capillary endothelium. Expression of Tie-2 and CD34 in a section from patient No 6 was shown in the three photomicrographs as follows. Left panel: Tie-2 immunoreactivity before the addition of CD34 antibody; middle panel: double staining for Tie-2 and CD34; right panel: CD34 expression after removal of Tie-2 staining by immersing in ethanol. (D) Double staining for Tie-2 (red) and proliferating cell nuclear antigen (PNCA) (blue). (E) Single staining for CD34 in a section adjacent to (D). PCNA was frequently overexpressed in Tie-2 positive endothelial and pericytic cells as indicated by black arrowheads. Inset in (D) is a representative capillary showing both immunoreactivities. Note several CD34 positive cells in (E) are negative for Tie-2 and PCNA in (D) (red arrowheads). Asterisks in (D) and (E) represent large vessels which did not react with either Tie-2 or PCNA or CD34. Bars in each panel = 50 µm.

Figure 4  .

Figure 4  

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Reverse transcriptase polymerase chain reaction (RT-PCR) of Tie-1 and Tie-2. (A) Tie-1 and Tie-2 in synovial tissues. Total RNA was extracted from each synovium and cDNA was synthesised from 1 µg RNA. PCR was performed with specific primers corresponding to each cDNA. PCR cycles to amplify Tie-1, Tie-2, and G3PDH were 35, 35, and 25 cycles, respectively. Amplified PCR products were then electrophoresed onto 1.5% agarose gel. To rule out possible genomic DNA contamination, control experiments were also performed without RT using the same reactions, and showed no amplification (data not shown). (B) Tie-1 and Tie-2 in cultured synoviocytes. Cultured synoviocytes obtained from two independent patients with RA (cs-1 and cs-2) and subcultured five times were subjected to RT-PCR with the same conditions as described above.

Selected References

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