Anti-VEGF reduces inflammatory features in macular edema secondary to retinal vein occlusion

INTRODUCTION

Retinal vein occlusion (RVO) is the second most common retinal vascular disease after diabetic retinopathy,resulting in visual impairment. The incidence of RVO is about 0.5%-1.8% in the general population

. The complications due to RVO include macular edema (RVO-ME), retinal neovascularization with secondary vitreous hemorrhage,neovascular glaucoma,

., which largely impaired the vision of patients. The pathogenesis of RVO-ME is multifactorial.The occluded and damaged blood vessels as well as retinal ischemia can result in local hypoxia with the increased hypoxia inducible factor-1 alpha (HIF-1α), resulting in elevated secretion of vascular endothelial growth factor(VEGF), which could cause vascular hyperpermeability and neovascularization

. Anti-VEGF treatment has been shown to be beneficial to patients with RVO-ME and becomes the first-line therapy in the treatment of RVO-ME

. Besides VEGF, other factors including inflammatory cells and cytokines were also associated with the pathogenesis of RVO-ME.

During the clinical practice, we noticed that the RVOME patients with HRF benefit from anti-VEGF injections,demonstrating the improved visual acuity (VA), reduced central macular thickness (CMT) and HRF number, as well as the decreased non-perfusion area (NPA). Besides the direct anti-VEGF effect, we hypothesized that anti-VEGF reagents might exert anti-inflammatory effect in patients with RVOME. To address this question, we retrospectively reviewed 28 eyes from 28 treatment-naïve patients, who underwent three consecutive intravitreal injections of anti-VEGF reagents.The VA, CMT, the HRF number, and NPA size before and after intravitreal injections were quantified and compared.

Increasing evidence suggested that hyperreflective foci(HRF) in retina were identified as the active inflammatory cells, especially microglia and macrophages, by using optical coherence tomography (OCT) or optical coherence tomography angiography (OCTA), indicating the inflammatory conditions in retina for patient with RVO-ME. HRF were first mentioned by Coscas

in patients with age-related macular degeneration (AMD) with spectral-domain OCT. Subsequently,HRF have been involved in many retinal diseases, such as RVO, diabetic retinopathy, choroideremia, and other retinal degenerative diseases

. Although its pathogenesis is still debated, HRF likely characterizes a progressive nature of an inflammatory retinal microenvironment.

The intravitreal injection was conducted at the temporal limbus through the eyeball’s pars plana under aseptic conditions in the operating room. Twenty-eight patients received three consecutive intravitreal injections of ranibizumab at the concentration of 0.5 mg/ 0.05 mL (Novartis Pharma Stein AG, Switzerland,

=18) or conbercept at the concentration of 0.5 mg/ 0.05 mL(Chengdu Kang Hong Biotech Co., Ltd., Sichuan Province,China,

=10) with a 30-gauge needle. Each injection interval allowed a variation of 1wk. The participants were treated with three monthly intravitreal injections until the macular edema(ME) was resolved.

1.1 临床资料 收集2011年1—11月我院门诊血清TPPA、RPR均阳性需要治疗的梅毒患者824例，其中男446例，女378例。男性平均年龄45.8岁，女性平均年龄37.9岁。

SUBJECTS AND METHODS

This study was approved by the Clinical Research Ethical Committee of Shanghai General Hospital affiliated to Shanghai Jiao Tong University (Permits No.2020KY205-2) and adhered to the principles of the Declaration of Helsinki. Informed consents were signed by all the participants.

The present study was a retrospective cohort study,including 28 treatment-naïve patients, aged 64.2±2.1 years old. The patients were comprised of 14 males (50%) and 14 females (50%). The RVO included 14 BRVO and 14 CRVO.This retrospective study was conducted in the Department of Ophthalmology, Shanghai General Hospital affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai, China between August 26, 2019, and July 30,2020. Participants who received intravitreal injections of anti-VEGF drugs for three months were included in the study. The eyes with any co-existing ocular diseases, including diabetic retinopathy, hypertensive retinopathy, AMD, or uveitis,

.,were excluded.

At the initial examination, comprehensive ophthalmic examinations were performed for every patient, including OCTA, fundus photography, best-corrected visual acuity(BCVA), intraocular pressure and anterior segment evaluation using slit-lamp biomicroscopy. Follow-up examinations were conducted 1wk after each intravitreal injection.

由于种子千粒重低籽粒瘦弱，幼苗顶土能力差，为了保证全苗，在播前精细整地，足墒下种，适当浅播，深3厘米左右。

Retinal microvasculature was visualized by using the RTVue XR Avanti OCT system (Optovue, Inc., Fremont, CA, USA),and the quantification was carried out using the manufacturer’s AngioVue software. The scanning was centered on the fovea with an area of 6×6 mm

.

CMT measured with OCTA was calculated as the average retinal thickness in a 1-mm-diameter circular region centered at the fovea which was automatically analyzed by OCTA.

To observe the efficacy of two different anti-VEGF reagents,we sub-grouped the patients and analyzed the effect based on ranibizumab and conbercept injections. In Table 2, for ranibizumab treatment, the VA increased in RVO (1.4±0.1

0.6±0.1,

=18,

<0.05), BRVO (1.2±0.2

0.3±0.1,

=8,

<0.05), and CRVO (1.6±0.2

0.9±0.2,

=10,

<0.05);and for conbercept treatment, the VA was increased in RVO(1.7±0.2

1.0±0.1,

=10,

<0.05), BRVO (1.7±0.3

0.8±0.2,

=6,

<0.05), and CRVO (1.7±0.1

1.2±0.2,

=4,

<0.05).No significant difference has been found in term of VA improvement for each sub-group between ranibizumab and conbercept treatment.

The NPA was outlined manually in enface image of the superficial capillary plexus (SCP) with 6×6 mm

scanning area in OCTA and analyzed automatically with the OCTA auto-segmentation software. The SCP was segmented as 3 μm below the internal limiting membrane and 15 μm below the inner plexiform layer.

The data were analyzed by using the IBM SPSS Statistics 21 software. All values are presented as a number or mean±standard deviation. The VA was expressed as the logarithm of the minimum angle of resolution (logMAR).A paired

-test was employed to compare BCVA, the number of HRF, and NPA between the baseline and after 3 consecutive monthly anti-VEGF injections. A

-value less than 0.05 was determined as statistically significant difference.

RESULTS

CMT is a sensitive parameter to evaluate RVO-ME. In our study, the CMT reduced significantly after anti-VEGF injections (Table 1 and Figure 2),RVO (460±34.0

268.8±12.0 μm,

=28,

<0.05), BRVO(413±47

255±11 μm,

=14,

<0.05), and CRVO (512±47

283±22 μm,

=14,

<0.05).

目前，随着我国社会主义发展进程的不断推进，以电网改造工作为主的建设内容逐渐被列为我国重要的议事日程当中。且国家电网在智能电网战略部署的督导作用下，形成重点加强区域电网建设、提高中低压配电网智能化运行水平的改造体系。另外，在先进技术的协调作用下，我国终端远程通讯技术基本上得到进一步优化，利于提升农网系统的自动化建设水平。由此可以看出，实行电网改造工作对于我国电网建设进程而言，具有多么重要的意义[1]。

BCVA improved significantly from baseline to the final follow-up, and the mean change of BCVA was -0.8±0.1 for RVO group (Table 1). Figure 1 demonstrated the changes of VA before and after the treatment. After three consecutive injections of anti-VEGF reagents, the VA significantly increased in all three groups, RVO (1.5±0.1

0.8±0.1,

=28,

<0.05), BRVO (1.4±0.2

0.6±0.1,

=14,

<0.05), and CRVO (1.6±0.1

1.0±0.2,

=14,

<0.05).

3.1 建立良好的护患关系 良好的护患关系是医护活动顺利开展的必要条件，患者的依从性常与护患关系有着密切的联系［6］。融洽的护患关系可产生良好的心理气氛和情绪反应，促使患者遵循治疗方案。如果护患间没有充分的交流，没有形成良好的信任关系，就难以促使患者建立良好的服药依从性。因此，护士应针对老年人的特点，并结合其知识层次和性格，进行有效的沟通交流，建立良好的护患关系。

The HRF number was manually counted in the whole retina within a 6-mm diameter centered on the fovea using a foveaspanning horizontal B-scan. HRF in OCTA was defined as a discrete and well-circumscribed dot-shaped lesion of equal or higher reflectivity than the retinal pigment epithelium (PRE)band. The maximal diameter of HRF was limited within the 20 to 50 μm range in order to exclude small counting noise signals(less than 20 μm) and prevent large hyperreflective clumps,such as hard exudates. Poor-quality images with a signal strength index less than 4/10 were excluded. The quantification of HRF was conducted independently by two experienced physicians.

The baseline clinical features of 28 eyes were shown in Table 1. The participants are comprised of 14 females (50%) and 14 males (50%). The mean age of patients was 64.2±2.1 years old, ranging from 50 to 78 years old, with 63.2±2.5 years old for BRVO and 64.8±3.4 years old for CRVO. The RVO included both BRVO (50%,

=14)and CRVO (50%,

=14). All participants underwent three consecutive monthly injections of ranibizumab (18 patients)or conbercept (10 patients). Eight patients with BRVO and 10 patients with CRVO were injected with ranibizumab; and 6 patients with BRVO and 4 patients with CRVO were injected with conbercept. The mean interval between baseline and final follow-up was 108.1±8.7 (range 56-240)d.

For ranibizumab treatment group, the CMT decreased in RVO(506±38

284±17 μm,

=18,

<0.05), and CRVO (578±46

290±29 μm,

=10,

<0.05; Table 2). As for conbercept treatment groups, the CMT decreased in RVO (399±61

242±12 μm,

=10,

<0.05; Table 2). No obvious difference for the reduction of CMT was observed for each sub-group between ranibizumab and conbercept treatment.

中国教师：在大的格局和总体布局当中，广州以校际间均衡、区域间均衡为目标，以好教育为统领，组合拳打得十分精彩，并得到了肯定。教育部《教育现代化进程监测评价指标体系研究》专题组发布的《全国15个副省级城市教育现代化监测评价与比较研究报告（2015）》显示，在4个现代化一级监测指标中，广州好几个综合排名位居前列，其中教育普及发展第二、教育条件保障第二、教育质量要素第四。那么，接下来在办好家门口的学校方面，有什么好的做法吗？

就拿最开始的喂饭方式来讲，正确的方式是把勺子平行地伸进嘴巴，放在宝宝的舌头上面，让他自己把嘴唇闭住把食物从勺子上抿下来。而事实中，很多喂养者都急于把食物喂进去，选择的方式是更有“效率”地把勺子与嘴巴呈30度角，从上往下地把食物塞进去，然后宝宝被动地合上嘴巴。

HRF was identified as active macrophages and/or microglia in retina on OCT or OCTA examination. In this study, HRF was shown to be distributed throughout the whole retina, especially in the inner retina. HRF number significantly reduced in RVO(76.5±4.8

47.8±4.3,

=28,

<0.05), BRVO (68.1±5.6

40.6±4.7,

=14,

<0.05), and CRVO (84.9±7.3

55±6.9,

=14,

<0.05), respectively, following anti-VEGF treatment(Table 1 and Figure 3).

For ranibizumab treatment, the number of HRF decreased in RVO (83.8±6.7

55.8±5.8,

=18,

<0.05), and CRVO(90.7±9.3

66.7±6.7,

=10,

<0.05; Table 2). As for conbercept treatment group, the number of HRF decreased in RVO (64.9±4.5

33.0±2.2,

=10,

<0.05; Table 2). No significant difference for HRF reduction was detected among three groups between ranibizumab and conbercept treatments.

NPA reflected the non-perfusion of retinal capillaries due to transient occlusion of leukocyte in retinal blood vessels or permanent dropout of retinal capillaries forming acellular capillaries. To see whether or not anti-VEGF reagent could reduce NPA, we compared the NPA before and after three consecutive injections. As shown in Table 1, there was slightly increase of NPA for CRVO (10.4±1.4 mm

) than BRVO (8.9±1.0 mm

), but no significant difference, in the defined macular region (6×6 mm

) at baseline. After anti-VEGF treatment, we observed gradual reduction of NPA after three consecutive injections in patients with RVO, 8.9±1.0 mm

(baseline), 8.5±1.0 mm

(after 1

injection), 8.1±1.0 mm

(after 2

injection), and 7.4±1.0 mm

(after 3

injection), although no significant difference was observed (Figure 4).

To see the effect of different anti-VEGF reagents on NPA, we analyzed and compared their effect on the changes of NPA.The data showed that, in ranibizumab and conbercept treatment group, NPA decreased in RVO, BRVO, and CRVO (Table 2)but with no significant difference. No obvious change was shown in NPA reduction between ranibizumab and conbercept treatments.

DISCUSSION

With an estimated 16 million patients worldwide, RVO become one of the most common retinal vascular diseases in adults

. In our study, the VA improved and the CMT decreased significantly in patients with RVO-ME after anti-VEGF treatments. The HRF also decreased significantly,accompanied with progressive reduction of NPA after three consecutive anti-VEGF treatments. This study indicated that retinal inflammation might play a contributory role in the pathogenesis of RVO-ME.

The pathogenesis of RVO-ME is complicated, in which ischemia and hypoxia plays essential roles in the formation of RVO-ME. Ischemia and hypoxia could stabilize HIF-1α and leads to elevated secretion of VEGF and other down-stream targets. In human, VEGF family contains five members,including VEGF-A (usually named as VEGF), VEGF-B,VEGF-C, VEGF-D, as well as placental growth factor (PGF).Both VEGF and PGF play important roles in the formation of ME through inducing the breakdown of blood-retinal barrier (BRB). Besides, the inflammation is considered as a key player in RVO. Previous studies reported that several inflammatory factors, other than VEGF and PGF, contributed to the pathogenesis of RVO-ME, including angiotensin II,interleukin (IL)-1β, IL-6, IL-8, basic fibroblast growth factor(bFGF), monocyte chemoattractant protein 1 (MCP-1), and PGF,

. Through binding VEGF receptors (VEGFR) on endothelial cells, VEGF and PGF induced the up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells, enhancing the leukocyte adhesion to the vessel wall,thus leading to leukostasis, retinal non-perfusion and BRB breakdown. Chronic inflammation will result in acellular capillaries, aggregating the retinal hypoxia. VEGF and PGF also facilitate the proliferation and activation of microglia and macrophage through VEGFR. The HRF on OCT or OCTA mainly refers to the inflammatory cells in retina, such as microglia and macrophages. These inflammatory cells,including leukocytes, will release pro-inflammatory factors,causing BRB breakdown, ME, neuronal damage, and visual deterioration

.

We hypothesized that anti-VEGF reagents, by antagonizing VEGF and/or PGF, blocked the activation of VEGFR both on endothelial cells and inflammatory cells (microglia,macrophage, and leukocyte,

.), thus down-regulated adhesion molecules of endothelial cell and deactivated the inflammatory cells as well as the inflammatory factor release. In this way, the leukostasis was alleviated and NPA was improved,and the HRF also decreased by anti-VEGF treatment, indicating an anti-inflammatory effect of anti-VEGF reagents.

Although no statistically significant difference was found,we observed the amelioration of NPA in patients with RVO after anti-VEGF treatment (Figure 4). The improved NPA might be due to transient adhesion of leukocyte to endothelial cells

CD11b/ICAM-1 interaction, and anti-VEGF treatment disrupted the interaction between leukocyte and endothelial cells and re-opened the occluded capillaries.Thus we found the gradual improvement of NPA in patients with RVO after anti-VEGF therapy. But for some patients,the initial NPA cannot be identified easily due to massive,intensive hemorrhage obscured the direct observation of non-perfusion in retina. For some NPA caused by capillary dropout, it can be extrapolated that this non-perfusion cannot be alleviated due to the permanent capillary obliteration because anti-VEGF treatment cannot re-vascularize the NPA in a timely manner. So, the patients with RVO are suggested to initiate anti-VEGF treatment as soon as possible to decrease and eliminate the non-perfusion caused by early transient occlusion by leukocytes, and thus avoid permanent capillary dropout (acellular capillaries).There are still some shortcomings in the current research.First, the sample size of this clinical study was comparatively small, that may affect the comparisons. Second, the study was a short-term observation, which needs a long-term followup. Last, OCTA requires consistent cooperation from the patients, and small vibration might make it difficult to perform the comparison among different groups. Therefore, longterm evaluation of the efficacy of anti-VEGF reagents and large sample multi-center studies are needed in the treatment of RVO-ME. Besides OCTA, other multimodal ophthalmic imagings are required for evaluation of RVO-ME before and after the treatment.

李渔(1611－1680)，浙江金华府兰溪人，初名仙侣，后改名渔，字谪凡，号笠翁。他是明末清初时期著名的文学家、戏剧家、戏剧理论家、美学家，一生中创作了大量的文学作品，包括戏曲、小说、诗词、曲赋等，其中成就最高的是戏曲，他的作品将拟话本小说与戏曲艺术巧妙地融合为一个整体，创作出《笠翁传奇十种》《无声戏》《十二楼》等优秀戏曲作品。

怀揣着对乡村教育挚爱的坚定信念，她在泥泞的小路上往返于各村校之间。70多岁的老母亲患上了严重的白内障，需要做眼球摘除手术，为了工作，她无法陪护在母亲的病床前。孩子高考前，她亦没有时间陪伴。

In summary, retinal inflammation plays a critical role in RVO-ME. As detected with OCTA, besides macular edema,HRF and NPA were also observed. In the pathogenesis of RVO-ME, the retina was stressed under ischemia and hypoxia,which stabilized HIF-1α and increased the production of its down-stream targets, including VEGF, PGF, and VEGFR.Through binding VEGFR, VEGF/PGF enhanced the expression of adhesion molecules on endothelial cells, such as ICAM-1 and VCAM-1, which promoted leukostasis, leading to increased NPA

and aggregating the retinal hypoxia.VEGF/PGF also promoted the activation of inflammatory cells

activating VEGFR on above cells, which were observed as increased number of HRF on OCTA. The activated inflammatory cells, such as macrophage and microglia,increased production of the inflammatory factors, such as IL-1β and IL-6, and MCP-1, further aggravating BRB breakdown and macular edema. The increasing NPA and activation of inflammation constitute a vicious cycle in the pathogenesis of RVO-ME. Anti-VEGF treatments, by antagonizing VEGF and/or PGF, breakdown the vicious cycle and ameliorate the inflammation and retinal hypoxia, as proposed in Figure 5.However, the specific mechanisms for anti-VEGF therapy in the reduction of HRF and NPA warrant further investigation to fully elucidate the anti-inflammatory effect of anti-VEGF in RVO-ME treatment.

ACKNOWLEDGEMENTS

Supported by the National Natural Science Foundation of China (No.81970811; No.81970810;No.82171062); Domestic Science and Technology Cooperation Project of Shanghai Municipal Science and Technology Commission (No.21015800700).

None;

None;

None;

None;

None;

None;

None;

None;

None;

None;

None;

None;

None.

1 Klein R. The 15-year cumulative incidence of retinal vein occlusion.

2008;126(4):513.

2 Klein BEK, Meuer SM, Knudtson MD, Klein R. The relationship of optic disk cupping to retinal vein occlusion: the beaver dam eye study.

2006;141(5):859-862.

3 Pierru A, Girmens JF, Héron E, Paques M. Retinal vein occlusions.

2017;40(8):696-705.

4 Jonas JB, Monés J, Glacet-Bernard A, Coscas G. Retinal vein occlusions.

2017;58:139-167.

5 Miyagi S, Nishinaka A, Yamamoto T, Otsu W, Nakamura S, Shimazawa M, Kitaoka T, Hara H. Establishment of a pigmented murine model abundant with characteristics of retinal vein occlusion.

2021;204:108441.

6 Lin J, Gibbons A, Smiddy WE. Cost-utility of anti-vascular endothelial growth factor treatment for macular edema secondary to central retinal vein occlusion.

2021;5(7):656-663.

7 Light JG, Tian J, Wenick AS. Outcomes in retinal vein occlusions presenting with poor visual acuity treated with anti-vascular endothelial growth factor therapy.

2021;5(9):888-900.

8 Chhablani J, Stewart M, Paulose R, Gallego-Pinazo R, Dolz-Marco R.Clinical characteristics and treatment outcomes of recurrent central retinal vein occlusions.

2018;33(2):191-197.

9 Coscas G, De Benedetto U, Coscas F, Li Calzi CI, Vismara S, Roudot-Thoraval F, Bandello F, Souied E. Hyperreflective dots: a new spectral-domain optical coherence tomography entity for followup and prognosis in exudative age-related macular degeneration.

2013;229(1):32-37.

10 Ogino K, Murakami T, Tsujikawa A, Miyamoto K, Sakamoto A, Ota M, Yoshimura N. Characteristics of optical coherence tomographic hyperreflective foci in retinal vein occlusion.

2012;32(1):77-85.

11 Vujosevic S, Torresin T, Bini S, Convento E, Pilotto E, Parrozzani R,Midena E. Imaging retinal inflammatory biomarkers after intravitreal steroid and anti-VEGF treatment in diabetic macular oedema.

2017;95(5):464-471.

12 Romano F, Arrigo A, MacLaren RE, Charbel Issa P, Birtel J, Bandello F, Battaglia Parodi M. Hyperreflective foci as a pathogenetic biomarker in choroideremia.

2020;40(8):1634-1640.

13 Karlstetter M, Scholz R, Rutar M, Wong WT, Provis JM, Langmann T.Retinal microglia: just bystander or target for therapy?

2015;45:30-57.

14 Rogers S, McIntosh RL, Cheung N, Lim L, Wang JJ, Mitchell P,Kowalski JW, Nguyen H, Wong TY,. The prevalence of retinal vein occlusion: pooled data from population studies from the United States,Europe, Asia, and Australia.

2010;117(2):313-319.e1.

15 Hirabayashi K, Tanaka M, Imai A,

. Development of a novel model of central retinal vascular occlusion and the therapeutic potential of the adrenomedullin-receptor activity-modifying protein 2 system.

2019;189(2):449-466.

16 Daruich A, Matet A, Moulin A,

. Mechanisms of macular edema: beyond the surface.

2018;63:20-68.

17 Alizadeh E, Mammadzada P, André H. The different facades of retinal and choroidal endothelial cells in response to hypoxia.

2018;19(12):E3846.

18 Campochiaro PA. Seeing the light: new insights into the molecular pathogenesis of retinal diseases.

2007;213(2):348-354.

19 De Bats F, Gambrelle J, Feldman A, Mauget-Faysse M, Germain-Pastene M, Denis P. Role of intolerance to hypoxia in the occurrence of anterior bilateral ischaemic optic neuropathy at high altitude.

2010;33(10):724-727.

20 van Bergen T, Etienne I, Cunningham F, Moons L, Schlingemann RO,Feyen JHM, Stitt AW. The role of placental growth factor (PlGF) and its receptor system in retinal vascular diseases.

2019;69:116-136.

21 Klein R, Klein BE, Moss SE, Meuer SM. The epidemiology of retinal vein occlusion: the beaver dam eye study.

2000;98:133-141; discussion 141-143.

22 Rodrik-Outmezguine VS, Okaniwa M, Yao Z,

. Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor.

2016;534(7606):272-276.

23 de Smet MD. Insights into the physiopathology of inflammatory macular edema.

2017;58:168-177.

24 Noma H, Mimura T, Yasuda K, Shimura M. Role of soluble vascular endothelial growth factor receptor signaling and other factors or cytokines in central retinal vein occlusion with macular edema.

2015;56(2):1122-1128.

25 Jung SH, Kim KA, Sohn SW, Yang SJ. Association of aqueous humor cytokines with the development of retinal ischemia and recurrent macular edema in retinal vein occlusion.

2014;55(4):2290-2296.