ADAM8 is a negative regulator of retinal neovascularization and of the growth of heterotopically injected tumor cells in mice - PubMed (original) (raw)

ADAM8 is a negative regulator of retinal neovascularization and of the growth of heterotopically injected tumor cells in mice

Victor H Guaiquil et al. J Mol Med (Berl). 2010 May.

Abstract

ADAM8 is a member of the "a disintegrin and metalloproteinase" (ADAM) family of membrane-anchored metalloproteinases. ADAM8-deficient mice have no evident spontaneous developmental or pathological defects, and little is currently known about the role of ADAM8 in disease. Here, we investigated the contribution of ADAM8 to pathological neovascularization in mice using an oxygen-induced retinopathy (OIR) model and heterotopical injection of tumor cells. We found an increase in retinal re-vascularization but fewer neovascular tufts in the OIR model and increased growth of heterotopically injected tumor cells in Adam8-/- mice compared with wild-type controls. These results suggest that ADAM8 functions to limit both of these processes in wild-type mice. In cell-based assays, overexpression of ADAM8 increased the ectodomain shedding of several co-expressed membrane proteins with roles in angiogenesis (CD31, Tie-2, Flk-1, Flt-1, EphrinB2, EphB4, VE-cadherin, KL-1, E-selectin, and neuregulin-1beta2). Thus, dysregulated expression of ADAM8 in endothelial cells in vivo could potentially increase the processing of these and other substrate proteins. Taken together, our findings suggest that inhibiting ADAM8 could be useful for promoting re-vascularization and thereby preventing formation of neovascular tufts in proliferative retinopathies. On the other hand, blocking ADAM8 could be detrimental in the context of rapidly growing tumors.

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Conflict of interest statement

Conflict of interest The authors reported no conflict of interest.

Figures

Fig. 1

Increased hypoxia-induced retinal neovascularization in _Adam8_−/− mice. _Adam8_−/− mice and wild-type controls were subjected to the oxygen-induced retinopathy (OIR) model (see Material and methods for details). a Analysis of whole-mount retinas prepared at P17 showed a smaller central avascular area in _Adam8_−/− mice compared with wild-type controls (representative examples are shown, scale bar=1 mm). b Quantification of the size of the central avascular area compared with the size of the retina corroborated that the central avascular area is smaller in _Adam8_−/− mice compared with controls (mean relative size of avascular area in _Adam8_−/− mice=10.5%±1.4% SEM, _n_=21; in wild-type controls=19.6%±1.3% SEM, _n_=23, Student’s t test, p<0.0001). c _Adam8_−/− and wild-type mice developed similar capillary regression at P12 of the OIR model, but starting at P15, re-vascularization of the retina was significantly increased in _Adam8_−/− mice compared with controls. d The average number of neovascular nuclei in tufts on the vitreous side of the internal limiting membrane (see Material and methods for details) was significantly reduced in the absence of ADAM8. Each point represents the average number of nuclei per retinal section of one animal. (_Adam8_−/− mice 4.0±1.1 SEM, _n_=18; wild-type mice 11.3±0.7 SEM, _n_=22, p<0.0001.) e The number of tufts that were clearly visible in retinal whole mounts at 4× magnification was decreased in _Adam8_−/− mice compared with wild-type controls (_Adam8_−/− mice 31.3±4.1 SEM, _n_=19; wild-type mice 56.8±9.1 SEM, _n_=21, p<0.0098). f Representative images of sections of a retina whole mount from an _Adam8_−/− mouse or wild-type control, stained with isolectin B4-FITC (arrows point to neovascular tufts, scale bar=100 μm)

Fig. 2

Expression of ADAM8 in retinas of mice subjected to oxygen-induced retinopathy. a–c Representative examples of a neovascular tuft from retinas of wild-type mice (top panels in a, b and all panels in c, or _Adam8_−/− mice (lower panels in a, b) stained with antibodies against ADAM8 (Alexa 488) in a, b, and c, or against the endothelial cell marker CD31 (Cy3 in a), the pericyte marker NG2 (Texas Red in b) or the macrophage marker F4/80 (PE in c). Merged images are shown in the _right_-hand panels in a–c, scale bar=20 μm. The expression of ADAM8 in neovascular tufts does not overlap with CD31, NG2, or F4/80, and no staining with the ADAM8 antibody was seen in neovascular tufts in _Adam8_−/− retinas. d Intracellular ADAM8 staining (Alexa 488) co-localizes with lectin-TRITC labeled capillaries in a wild-type retina. e Immunohistochemical analysis shows high expression of ADAMs 8, 9, and 15 in neovascular tufts in sections of retinas from wild-type mice subjected to the OIR model, but only ADAMs 9 and 15 are detected in neovascular tufts from retinas of _Adam8_−/− mice after OIR

Fig. 3

Increased growth of heterotopically injected B16F0 tumor cells in _Adam8_−/− mice. a Heterotopic injection of 106 B16F0 melanoma cells into the flanks of _Adam8_−/− mice yielded larger tumors compared with wild-type controls (data are from five separate experiments, mean tumor size in _Adam8_−/− mice 0.86 g±0.1 SEM, _n_=36; wild-type mice 0.43 g±0.06 SEM, _n_=35; Student’s t test p<0.001). b B16F0 heterotopic tumor growth measured with a caliper was accelerated in _Adam8_−/− mice compared with wild-type controls (at day 17 _Adam8_−/− mice volume 2.8 cm3±0.5 SEM, _n_=10; in wild-type mice 1.4 cm3± 0.4 SEM, _n_=10; Student’s t test p<0.044). c, d Staining of tumor sections with anti-CD31 did not reveal differences in the spacing or numbers of vessels per field between tumors from _Adam8_−/− mice and wild-type controls [representative example in c, quantitation of vessels per field by Prism4.0a software is shown in d (_Adam8_−/− mice vessels per field 34.1±3.1 SEM, _n_=14; in wild-type mice 31.0±3.3 SEM, _n_=17; Student’s t test p<0.25)]

Fig. 4

Ectodomain shedding of membrane receptors with roles in angiogenesis by ADAM8 in “gain of function” overexpression experiments. To identify potential substrates for ADAM8, several alkaline–phosphatase-tagged membrane proteins were overexpressed in Cos7 cells together with empty vector (−), wild-type mouse ADAM8 (8), or the catalytically inactive ADAM8E>Q mutant (8EQ; see Materials and methods for details). a Each graph shows the AP activity released into the supernatant as a percentage of the total AP activity in the cell lysate and supernatant and is representative of at least three separate experiments with two wells per experiment. Overexpressed ADAM8 can increase the ectodomain shedding of Tie-2, Flt-1, VE-cadherin, NRG-1β2, Flk-1, EphB4, KL-1, ephrinB2, CD31, and E-selectin, but does not affect the processing of VCAM-1 and ICAM-1, or NRG-1β1 and TRANCE (not shown). b Western blot analysis confirmed the overexpression of ADAM8 and ADAM8E>Q in Cos-7 cells

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ADAM8 is a negative regulator of retinal neovascularization and of the growth of heterotopically injected tumor cells in mice - PubMed (original) (raw)