Ma Jie, Mehta Manisha, Lam Godfrey, Cyr Desireé, Ng Tat Fong, Hirose Tatsuo, Tawansy Khaled A, Taylor Andrew W, Lashkari Kameran
Schepens Eye Research Institute, Mass. Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA.
Department of Pathology, Boston University School of Medicine, Boston, MA.
Mol Vis. 2014 Jun 21;20:881-93. eCollection 2014.
The clinical phenotype of advanced stage retinopathy of prematurity (ROP, stages 4 and 5) cannot be replicated in an animal model. To dissect the molecular events that can lead up to advanced ROP, we examined subretinal fluid (SRF) and surgically dissected retrolental membranes from patients with advanced ROP to evaluate its influences on cell proliferation, angiogenic properties, and macrophage polarity.
We compared our findings to SRF collected from patients with uncomplicated rhegmatogenous retinal detachment (RD) without proliferative vitreoretinopathy and surgically dissected epiretinal membrane from eyes with macular pucker. All subretinal fluid samples were equalized for protein. The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells. Findings were compared with SRF collected from participants with uncomplicated rhegmatogenous RD without proliferative vitreoretinopathy. The ability of SRF to induce nitric oxide production was measured in vitro using murine J774A.1 macrophages. Cytokine profiles of SRF from ROP and RD eyes were measured using a multienzyme-linked immunosorbent assay (ELISA). Fluorescent immunohistochemistry of retrolental membranes from ROP was performed to detect the presence of leukocytes and the composition of tissue macrophages using markers for M1 and M2 differentiation.
The cytokine composition in SRF revealed that in ROP, not only were several proangiogenic factors were preferentially elevated but also the profile of proinflammatory factors was also increased compared to the RD eyes. SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects. SRF from eyes with ROP but not RD robustly induced nitric oxide production in macrophages. Furthermore, fluorescent immunostaining revealed a preponderance of M1 over M2 macrophages in retrolental fibrous membranes from ROP eyes. The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP. In contrast, SRF from RD eyes exhibits a suppressive environment for endothelial cell proliferation and angiogenesis.
Our investigation demonstrates that the microenvironment in advanced ROP eyes is proangiogenic and proinflammatory. These findings suggest that management of advanced ROP should not be limited to the surgical removal of the fibrovascular membranes and antiangiogenic therapy but also directed to anti-inflammatory therapy and to promote M2 activation over M1 activity.
早产视网膜病变（ROP，4期和5期）的临床表型无法在动物模型中复制。为了剖析可导致晚期ROP的分子事件，我们检查了晚期ROP患者的视网膜下液（SRF）并手术切除晶状体后膜，以评估其对细胞增殖、血管生成特性和巨噬细胞极性的影响。
我们将研究结果与从无增殖性玻璃体视网膜病变的单纯孔源性视网膜脱离（RD）患者收集的SRF以及黄斑皱襞患者手术切除的视网膜前膜进行比较。所有视网膜下液样本的蛋白质含量均一化。使用纤维蛋白凝块试验中毛细血管形成的组合来测量ROP眼SRF的血管生成潜力，并用人视网膜微血管内皮细胞的DNA合成来测试其增殖作用。将结果与从无增殖性玻璃体视网膜病变的单纯孔源性RD参与者收集的SRF进行比较。使用小鼠J774A.1巨噬细胞在体外测量SRF诱导一氧化氮产生的能力。使用多酶联免疫吸附测定（ELISA）测量ROP和RD眼SRF的细胞因子谱。对ROP的晶状体后膜进行荧光免疫组织化学检测，以使用M1和M2分化标志物检测白细胞的存在和组织巨噬细胞的组成。
SRF中的细胞因子组成显示，与RD眼相比，在ROP中，不仅几种促血管生成因子优先升高，而且促炎因子谱也增加。ROP眼的SRF支持细胞增殖和内皮索形成，而RD眼的SRF具有抑制作用。ROP眼而非RD眼的SRF在巨噬细胞中强烈诱导一氧化氮产生。此外，荧光免疫染色显示ROP眼晶状体后纤维膜中M1巨噬细胞多于M2巨噬细胞。ROP中SRF的细胞因子谱和生物学特性促进促血管生成环境，这支持与晚期ROP相关的纤维膜的维持和增殖。相比之下，RD眼的SRF对内皮细胞增殖和血管生成表现出抑制环境。
我们的研究表明，晚期ROP眼的微环境是促血管生成和促炎的。这些发现表明，晚期ROP的治疗不应仅限于手术切除纤维血管膜和抗血管生成治疗，还应针对抗炎治疗并促进M2激活而非M1激活。
