We demonstrate here differences in CNV development between Tnfrsf1b
−/− and Tnfrsf1a
−/− mice 2 weeks after laser-induced rupture of Bruch's membrane. The loss of receptor 1a in Tnfrsf1a
−/− mice led to exacerbation of CNV development compared with WT and Tnfrsf1b
−/− animals. In contrast, the loss of TNF-α receptor 1b reduced the inflammatory response and pathologic angiogenesis after laser treatment. Our data show that TNF-α receptors 1a and 1b play different roles in the pathogenesis of CNV formation after laser photocoagulation. Although this model does not exactly mimic all the aspects of CNV that occur in association with AMD, it shares two important features. First, the inflammatory invasion and the abnormalities in Bruch's membrane are similar to those in AMD. Second, the new vessels develop from the choroid, grow along the edges of the laser burn, and proliferate into the subretinal space.
16 Macrophage infiltration and increased expression of proinflammatory cytokines such TNF-α contribute to the CNV lesions after laser photocoagulation.
2–6,17–19 We demonstrated previously,
6 and also in this study, an increased expression of TNF-α in RPE/choroid after laser photocoagulation. We have also demonstrated that anti–TNF-α treatment (etanercept) reduces the size and the leakage of laser-induced CNV.
6 However, the exact role of TNF-α receptors 1a and 1b in the development of CNV is unknown. Tnfrsf1a is expressed ubiquitously, whereas Tnfrsf1b expression is tightly regulated and found predominantly on endothelial and hematopoietic cells.
20,21 Under physiological conditions, both TNFR knockout lines did not show any pathologic changes in blood vessel growth within the retina and choroid. However, 2 weeks after laser photocoagulation—the time of peak of CNV extension in rodents
22,23 —severe damage with large areas of fluorescein leakage and advanced histologic changes were observed in WT and Tnfrsf1a
−/− animals. We found a large number of cells immunoreactive for the macrophage/microglia marker F4/80 localized within the laser scars or surrounding the scars in Tnfrsf1a
−/− and WT mice. Moreover, an increase in the density of F4/80-positive cells was observed in Tnfrsf1a
−/− animals, and this correlated with a more severe CNV appearance in these mice compared with WT controls. In contrast, in Tnfrsf1b
−/− mice, CNV membranes had smaller lesions and decreased fluorescein leakage compared with WT and Tnfrsf1a
−/− mice. Reduced pathologic angiogenesis (2 weeks after laser treatment) was accompanied by decreased macrophage invasion after laser damage in these animals, suggesting that recruitment of inflammatory cells to the site of injury is critical in the development of CNV. It is unclear whether these cells were resident microglia cells or were recruited from peripheral blood monocytes. Depleting circulating macrophages with clodronic acid diminished the density of F4/80-positive cells in Tnfrsf1a
−/− and WT mice and reduced CNV formation compared with vehicle treated controls. However, in Tnfrsf1b
−/− mice, clodronic acid depletion did not change the macrophage invasion and CNV size, which appeared to be smaller than in other groups. These results suggest that signals through receptor 1b increase macrophage infiltration and recruitment of inflammatory cells to the site of injury. Because macrophages in CNV lesions are themselves a source of TNF-α,
4 it is possible that through an autocrine loop the inhibition of macrophage infiltration itself, because of the loss of receptor 1b in Tnfrsf1b
−/− animals, resulted in a lower level of TNF-α within the CNV lesions and thus further favored smaller lesion sizes. TNF-α regulates inflammatory cell activation and recruitment. This also favors the hypothesis that macrophage activation, TNF-α expression, and inflammatory cell recruitment may exert an autoregulatory and autocrine loop similar to the expression of VEGF by inflammatory cells. Furthermore, the apoptosis and subsequent clearance (efferocytosis) of inflammatory cells by macrophages are key mechanisms orchestrating successful resolution of inflammation. However, recently Michlewska et al.
24 demonstrated that TNF-α potentially inhibits efferocytosis of neutrophils by monocyte-derived macrophages and therefore exacerbates the process of inflammation. We found increased apoptotic activity in Tnfrsf1b
−/− mice compared with that in Tnfrsf1a
−/− and WT mice after laser photocoagulation. Lesions in Tnfrsf1b
−/− mice showed a higher number of TUNEL-positive cells localized in subretinal space. The higher number of apoptotic cells corresponded with reduced CNV lesions in these animals. This increased apoptotic activity in Tnfrsf1b
−/− mice after laser photocoagulation was in accordance with the findings of Goukassian et al.,
13 who demonstrated that ischemia-induced endothelial cell apoptosis was greater in the limbs of Tnfrsf1b
−/− mice than in those of WT and Tnfrsf1a
−/− mice after oxygen challenge. Furthermore, Luo et al.
12 demonstrated that Tnfrsf1a
−/− mice experienced enhanced, whereas Tnfrsf1b
−/− had reduced, ischemia-initiated angiogenesis and arteriogenesis compared with WT mice in a femoral artery ligation model, a commonly used in vivo arteriogenesis/angiogenesis model. The inhibited angiogenesis in Tnfrsf1b
−/− mice was associated with increased endothelial cell death and decreased endothelial cell migration mediated through Tnfrsf1a in these mice.
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