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. 2001 Oct;159(4):1521-30.
doi: 10.1016/S0002-9440(10)62537-0.

Fractalkine: A Novel Angiogenic Chemokine in Rheumatoid Arthritis

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Free PMC article

Fractalkine: A Novel Angiogenic Chemokine in Rheumatoid Arthritis

M V Volin et al. Am J Pathol. .
Free PMC article

Abstract

Angiogenesis is an important aspect of the vasculoproliferation found in the rheumatoid arthritic (RA) pannus. We have previously implicated members of the CXC chemokine family as potent angiogenic mediators in RA. We investigated the possibility that the sole member of the CX(3)C chemokine family, fractalkine (fkn), induces angiogenesis and that fkn might mediate angiogenesis in RA. Recombinant human fkn significantly induced migration of human dermal microvascular endothelial cells (HMVECs), a facet of the angiogenic response, in the pmol/L range in a concentration-dependent manner (P < 0.05). Fkn also induced the formation of significantly more endothelial tubes on Matrigel than did a negative control (P < 0.05). Fkn significantly induced 2.3-fold more blood vessel growth than control in the in vivo Matrigel plug assays (P < 0.05). We identified HMVEC expression of the fkn receptor, CX(3)CR1. Next, we determined if RA synovial fluid (SF)-induced angiogenesis was fkn-dependent. SFs from six RA patients immunodepleted of soluble fkn induced 56% less migration of HMVECs than did sham-depleted RA SFs (P < 0.05). In vivo, immunodepletion of fkn from six RA SFs significantly inhibited their angiogenic activity in Matrigel plug assays (P < 0.05). Immunodepletion of fkn from five RA synovial tissue homogenates inhibited their ability to induce angiogenesis in in vivo Matrigel plug assays (P < 0.05). These results establish a new function for fkn as an angiogenic mediator and suggest that it may mediate angiogenesis in RA.

Figures

Figure 1.
Figure 1.
Fkn induces HMVEC migration. Results represent the mean number of cells/well ± SE of one representative assay of four. *, P < 0.05, significantly different from PBS control.
Figure 2.
Figure 2.
Anti-fkn inhibits fkn-induced, but not bFGF-induced HMVEC migration. A: Anti-fkn inhibited fkn-induced HMVEC migration. B: Anti-fkn did not inhibit bFGF-induced HMVEC migration. Results represent the mean number of cells/well ± SE of one of three similar assays. *, P < 0.05, significantly different from goat IgG control.
Figure 3.
Figure 3.
Fkn does not induce HMVEC proliferation. Results represent the mean absorbance of quadruplicate wells ± SE of one representative assay of four assays. No values were significantly different (P < 0.05) from media alone.
Figure 4.
Figure 4.
Fkn induces EC tube formation in vitro. A: Representative assay showing fractalkine-induced HMVEC tube formation and PBS control (original magnification, ×22). Individual tubes are shown in fractalkine-treated well (arrows) and non-tube-forming ECs are identified in a PBS-treated well (arrowheads). B: Fkn and PMA both induce HMVEC tube formation relative to their negative controls. C: Anti-fkn inhibits fkn-induced, but not PMA-induced HMVEC tube formation. Values represent the mean number of HMVEC tube branches/well ± SE for three or four assays. *, P < 0.05, significantly different from vehicle control.
Figure 5.
Figure 5.
HMVECs express CX3CR1. A: Agarose gel showing 320-bp CX3CR1 reverse transcriptase-PCR products from HMVECs and THP-1 cells. B: Western blot showing 50-kd CX3CR1 band in both HMVECs and THP-1 cells. MW, molecular weight markers.
Figure 6.
Figure 6.
Fkn induces angiogenesis in Matrigel plugs in vivo. A: Representative assay showing Masson trichrome staining of blood vessels in Matrigel plugs. Fkn-induced blood vessel formation compared to PBS control (original magnification, ×66). Blood vessels are shown in fractalkine-treated well (arrows). B: Values represent the concentration of hemoglobin (g/dl)/Matrigel plug weight (mg) ± SE for 18 assays. C: Values represent the concentration of hemoglobin (g/dl)/Matrigel plug weight (mg) as a percentage of the positive control aFGF-induced hemoglobin (g/dl)/Matrigel plug weight (mg) for between seven to nine assays. *, P < 0.05 significantly different from vehicle control.
Figure 7.
Figure 7.
RA SF angiogenic activity is decreased by immunodepletion of fkn in vivo. SFs from six RA patients were pooled, immunodepleted of fkn, and assayed for their angiogenic ability in Matrigel plugs implanted in mice. Results represent the mean concentration of hemoglobin (g/dl)/Matrigel plug weight (mg) ± SE. *, P < 0.05, significantly different from IgG control.
Figure 8.
Figure 8.
RA ST angiogenic activity is decreased by immunodepletion of fkn in vivo. ST homogenates prepared from five RA patients were pooled, immunodepleted of fkn, and assayed for their angiogenic ability in Matrigel plugs implanted in mice. A: Representative assay showing Masson trichrome staining of blood vessels in Matrigel plugs. Lack of RA ST-induced blood vessel formation after immunodepletion of fkn with anti-fkn compared to IgG control (original magnification, ×66). Blood vessels are indicated by arrows. B: Results represent the mean concentration of hemoglobin (g/dl)/Matrigel plug weight (mg) ± SE. *, P < 0.05, significantly different from IgG control.

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