SOCS3 is an endogenous inhibitor of pathologic angiogenesis

Abstract

Inflammatory cytokines and growth factors drive angiogenesis independently; however, their integrated role in pathologic and physiologic angiogenesis is not fully understood. Suppressor of cytokine signaling-3 (SOCS3) is an inducible negative feedback regulator of inflammation and growth factor signaling. In the present study, we show that SOCS3 curbs pathologic angiogenesis. Using a Cre/Lox system, we deleted SOCS3 in vessels and studied developmental and pathologic angiogenesis in murine models of oxygen-induced retinopathy and cancer. Conditional loss of SOCS3 leads to increased pathologic neovascularization, resulting in pronounced retinopathy and increased tumor size. In contrast, physiologic vascularization is not regulated by SOCS3. In vitro, SOCS3 knockdown increases proliferation and sprouting of endothelial cells costimulated with IGF-1 and TNFα via reduced feedback inhibition of the STAT3 and mTOR pathways. These results identify SOCS3 as a pivotal endogenous feedback inhibitor of pathologic angiogenesis and a potential therapeutic target acting at the converging crossroads of growth factor- and cytokine-induced vessel growth.

Figure 1

SOCS3 attenuates pathologic neovascularization. (A) Schematic representation of SOCS3 functioning as a negative feedback modulator of angiogenic activation. (B-C) Socs3 expression in wild-type mice exposed to OIR compared with normoxia both at the mRNA and protein level (n = 3-4). (D) Socs3 expression is significantly up-regulated in pathologic neovessels obtained by laser microdissection of retinal layers (outlined on the right; n = 4). GCL indicates ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. (E) Retinal vasculature of Cre reporter mice Rosa mT^flox/mG. This strain universally expresses a red-fluorescent protein (mT) that is removed in tissues with Cre recombinase expression, revealing instead a green fluorescent protein (mGFP). In control mice, the retinal vasculature is red (top), whereas in Tie2-Cre mice, the retinal vasculature is green (bottom), demonstrating that Tie2-Cre recombinase is expressed in retinal vessels (n = 3). (F) Conditional Tie2-Socs3^ko mice have more pathologic neovascularization in OIR compared with Socs3^flox/flox control mice. The area of vaso-obliteration is unchanged (n = 13-34). (G,I) Increased growth of LLC (G; n = 5-7) and B16F10 melanoma tumors (I; n = 5-9) in Tie2-Socs3^ko compared with Socs3^flox/flox mice. (H,J) Increased tumor vessel density of LLC (H; n = 5-7) and B16F10 melanoma tumors (J; n = 5-9) in Tie2-Socs3^ko compared with Socs3^flox/flox mice. Scale bar in panel E indicates 200 μm; panel F, 1 mm; panels G and I, 1 cm; and in panels H and J, 100 μm. *P < .05, **P < .01, P < .001.

Figure 2

SOCS3 regulates vascular sprouting and endothelial activation. (A) Schematic representation of the signaling pathways regulated by SOCS3. (B) Increased sprouting of TNFα/IGF-1–stimulated aortic rings from Tie2-Socs3^ko compared with Socs3^flox/flox mice (n = 4-6). (C) Increased proliferation of human ECs treated with SOCS3 siRNA compared with controls (n = 9-10). (D) Expression of phospho-mTOR (P-mTOR) and P-STAT3 (green) in neovascular tufts (NV, red) of the OIR retina of Tie2-Socs3^ko (not shown) and Socs3^flox/flox control mice (n = 4). GCL indicates ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. (E) Increased mTOR and STAT3 phosphorylation, but not ERK, in IGF-1–stimulated ECs treated with Socs3 siRNA compared with controls (n = 3-5). (F) Prestimulation with TNFα (15 minutes) before IGF-1 further pronounces the increase of P-mTOR and P-STAT3 in IGF-1–stimulated ECs treated with Socs3 siRNA compared with controls (n = 3-6). Scale bar in panel B indicates 1 mm; and panel D, 50 μm. NS indicates not significant, *P < .05, **P < .01.