MD2 Inhibits Choroidal Neovascularization via Antagonizing TLR4/MD2 Mediated Signaling Pathway

Abstract Purpose To explore the pathological mechanism of Toll-like receptor 4 (TLR4) mediating neovascular age-related macular degeneration (nAMD) and the potential role of the TLR4 coreceptor myeloid differentiation protein 2 (MD2). Methods In the study, we inhibited MD2 with the chalcone derivative L2H17 and we utilized a laser-induced choroidal neovascularization (CNV) mouse model and Tert-butyl hydroperoxide (TBHP)-challenged rhesus choroid-retinal endothelial (RF/6A) cells to assess the effect of MD2 blockade on CNV. Results Inhibiting MD2 with L2H17 reduced angiogenesis in CNV mice, and significantly protected against retinal dysfunction. In retina and choroid/retinal pigment epithelium (RPE) tissues, L2H17 reduced phospho-ERK, phospho-P65 but not phospho-P38, phospho-JNK, and reduced the transcriptional levels of IL-6, TNF-α, ICAM-1 but not VCAM-1. L2H17 could protect RF/6A against TBHP-induced inflammation, oxidative stress, and apoptosis, via inhibiting the TLR4/MD2 signaling pathway and the following downstream mitogen-activated protein kinase (MAPK) and nuclear transcription factor-κB (NF-κB) activation. Conclusions Inhibiting MD2 with L2H17 significantly reduced CNV, suppressed inflammation, and oxidative stress by antagonizing TLR4/MD2 pathway in an MD2-dependent manner. MD2 may be a potential therapeutic target and L2H17 may offer an alternative treatment strategy for nAMD.


Introduction
Age-related macular degeneration (AMD), with visual symptoms of blurred, distorted, or no vision over time, is a leading cause of vision loss among the elderly. 1 It is a consensus that AMD is a multi-factor disease strongly related to smoking, oxidative stress, inflammation, obesity, and cardiovascular diseases. Among them, oxidative stress and inflammatory response have been proved to play an essential role in the occurrence and development of AMD. 2 Advanced AMD includes two forms that are dry AMD and neovascular AMD (nAMD) associated with vision loss. 3 Choroidal neovascularization (CNV) is the growth of abnormal blood vessels causing impaired vision and is responsible for 90% of acute vision loss caused by AMD. [4][5][6] So far, the pathological mechanisms of nAMD are still not completely clear.
Recent studies have reported that Toll-like receptor 4 (TLR4) is a well-characterized pattern recognition receptor associated with nAMD. 7 Myeloid differentiation 2 (MD2) is the co-receptor of TLR4 and is required for TLR4 activation.
MD2 directly recognizes lipopolysaccharide (LPS) and forms TLR4/MD2 complex with LPS, leading to the activation of the TLR4. 8 In addition to exogenous ligands, TLR4 can also recognize non-microbial endogenous pathogen-associated molecular patterns via an MD2-dependent manner. For instance, Rocha et al. recently found that saturated fatty acids can be identified by the CD14-TLR4-MD2 complex and trigger inflammatory pathways. 9 Mateja et al. and our team have also reported that TLR4/MD2 complex plays a vital role in leading to oxidative stress and chronic inflammation. [10][11][12][13] These findings suggest that MD2 may be an attractive therapeutic target for nAMD treatment.
In the study, we inhibited MD2 with chalcone derivative L2H17. 14 Furthermore, we investigated the effects of MD2 in mediating nAMD and explored the pathological mechanism. The study showed that inhibitor targeting MD2 reduced angiogenesis in vivo and alleviated the oxidative stress and inflammatory response. MD2 inhibition may bring hope for attractive treatment strategies of nAMD.

Cell lines and reagents
The rhesus choroid-retinal endothelial (RF/6A) cells were a gift from Pro. ShengZhou Wu (Wenzhou Medical University, Zhejiang, China). The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM)/F12 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Invitrogen, USA), 100 U/mL penicillin, and 100 lg/mL streptomycin (Invitrogen, USA) in a humidified environment at 37 C with 5% CO 2 . The fresh medium was changed every 2-3 days. Anti-P38, anti-p-P38, anti-ERK, anti-p-ERK, anti-JNK, anti-p-JNK, and anti-b-actin were purchased from Cell Signaling Technology (Danvers, MA). Cell proliferation assay (MTS, CellTiter 96 AQueous One Solution) was purchased from Promega (Beijing, China). Chalcone analog L2H17 (C 16 H 14 O 4 , 270kD) was synthesized and structurally identified in our laboratory. 15 And through our previous research, it has been proved to bind directly to MD2. Before the compounds were used in biological experiments, they were recrystallized from CHCl3/EtOH. Highperformance liquid chromatography (HPLC) was used to determine the purity (>99%). L2H17 was dissolved in dimethyl sulfoxide (DMSO) for in vitro experiments and in 0.5% sodium carboxymethylcellulose (CMC-Na) solution in vivo experiments.

Animals
Adult male C57BL/6j mice (6-8 weeks old) were purchased from SLRC Laboratory Animal (Shanghai, China) and were maintained and bred at the Animal Center of Wenzhou Medical University. All experimental procedures were performed in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (No. wydw2021-0638).
Drug administration and laser-induced mouse CNV model C57BL/6j mice were randomly divided into 4 groups: normal control (without laser photocoagulation), vehicle treatment, and L2H17-treated low and high dose (10, 50 mg/kg, respectively) groups. The normal control, vehicle treatment and L2H17-treated mice were administered with 0.9% saline, 0.5% sodium CMC-Na solution, and L2H17 (10, 50 mg/kg, respectively) by gavage once daily beginning 3 days prior (day 3) to CNV induction (day 0) through day þ7 after CNV induction.
Micron IV system (Phoenix Research Laboratories, Pleasanton, CA, USA) was used to make CNV model. The mice were anesthetized by intraperitoneal injection with 0.5% pentobarbital (10 mL/kg). Pupils were dilated with tropicamide phenylephrine eye drops (0.5%, 50 lL). The rupture of Brush's membrane-choroid induced by laser photocoagulation (659 nm laser, 50 lm spot size, 240mW, 50 ms duration) was performed in each mouse. Four laser spots were made in the 3, 6, 9, and 12 o'clock positions between retinal vessels. The model is successfully induced when the burns produce a bubble.

Scotopic electroretinogram(ERG)recordings
At 7 days after CNV induction, an electroretinographic recording was conducted with corneal electrodes (Roland Consult, Brandenburg, Germany). The mice were darkadapted for 12 h. After anesthesia and pupil dilation, ERG patterns were successively recorded under the standard recommendation of International Society for Clinical Electrophysiology of Vision (ISCEV) guidelines. The grounding electrode was placed under the mice, and the recording electrodes were placed into the visor subcutaneously. Rod response was recorded at the stimulus intensities of 0.01 cd/m 2 , and the stimulus intensities for the standard combined response was 3.0 cd/m 2 . An average of 5 recordings was taken. Following exposure to steady background luminance of 30 cd/m 2 for 10 mins, cone response, flicker response, and oscillatory potentials were also recorded. The incubation and amplitudes of a-and b-waves were measured at the peaks and valleys of the records relative to the baseline before the stimulus.

Fluorescein fundus angiography
At 7 days after CNV induction, the mice were given mydriasis and anaesthesia firstly, and then were injected with 0.2 mL of 1% FLUORESCITE V R to display the fundus vessels. The angiographic images were obtained with Micron IV system (Phoenix Research Laboratories, Pleasanton, CA, USA). The pixel intensity of leakage areas was quantified with Image J TM software (NIH, Bethesda, MD).

Choroidal flat mount
At 7 days after CNV induction, the entire ocular globes were enucleated and fixed in 4% paraformaldehyde (PFA) for 1 h immediately. The RPE-choroid-sclera complex eyecups were washed 3 times with PBS at a 5-minute interval. A choroidal flat mount was made. Then Alexa Fluor 594-conjugated isolectin GS-IB4 (1:100) was used to stain the newly formed blood vessel. The images were viewed by using a fluorescence microscopy system (LEICA, Germany).

Immunohistochemistry
The eyeballs were removed from the euthanized mice and fixed in 4% paraformaldehyde for 24 h. Then, the eyes were embedded in paraffin. The tissue was cut into 5 mM thick sections. After deparaffinization and rehydration, 3% H 2 O 2 was used to inhibit endogenous peroxidase activity. These slides were then incubated with anti-p-P65 antibody (1:50, Cell Signaling Technology, Danvers, MA) overnight at 4 C. Slides were then washed 3 times with PBS, and incubated with secondary antibody (1:100, Cell Signaling Technology, Danvers, MA) for 1.5 h at room temperature. Finally, the immunoreactivity was visualized by DAB and hematoxylin was used to counterstain the slides. And the slices were scanned with a Nikon microscope equipped with a digital camera.

Western blot analysis
RF/6A cells were pretreated with L2H17 (2.5, 5, or 10 mM), vehicle control (DMSO) for 2 h, followed by incubation with TBHP (100 mM) for 1 h. At the end of the treatment, RF/6A cells were washed with PBS and suspended in RIPA lysis buffer with 1 mM PMSF (Beyotime Institute of Biotechnology, Jiangsu, China). Under a dissecting microscope, the retina and choroid/RPE tissue were lifted off the enucleated eyes. The tissue was washed with PBS and suspended in RIPA lysis buffer with 1 mM PMSF. Debris was removed by centrifugation at 17850 r/min for 15 mins at 4 C. The protein concentration was determined by the bicinchoninic acid (BCA, Beyotime Institute of Biotechnology, Jiangsu, China). Then 20-40 lg of protein per well was loaded and separated on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and was transferred to polyvinylidene fluoride (PVDF) membranes (Amersham, Chalfont, UK). The membranes were blocked with skim milk for 1 h at room temperature and then incubated with the following primary antibodies: anti-JNK, anti-p-JNK, anti-P38, anti-p-P38, anti-ERK, anti-p-ERK, anti-P65, antip-P65, anti-b-actin at 4 C overnight. After washing three times with TBST, membranes were incubated with secondary antibodies coupled to HRP (Beyotime Institute of Biotechnology, Jiangsu, China) for 1 h at room temperature and examined with iBright FL1000 Imaging System (Invitrogen, USA). The density analysis was performed using Image J.

MTS cell viability assay
RF/6A cells were seeded at a density of 1 Â 10 4 /well in 96well microplates and incubated for 24 h. The cells were pretreated with L2H17 (2.5, 5, or 10 lM), or vehicle control (DMSO) for 2 h, followed by incubation with TBHP (100 mM) for 22 h. MTS reagent (20 mL) was added to each well and incubated at 37 C for 1 to 2 h. The absorbance values were read at 490 nm with a 96-well plate reader (Multiskan SkyHigh, Thermo Scientific, USA). Each group had three replicates, and the cell viability was calculated by the percentage of the TBHP untreated control.

Cell apoptosis analysis
RF/6A cells (2 Â 10 5 cells/well) were seeded in 6-well plates and allowed to adhere overnight, and then the cells were pretreated with L2H17 (2.5, 5, and 10 lM) or vehicle (DMSO) for 2 h, followed by incubation with TBHP (100 mM) for 22 h. The cells were harvested and stained with Annexin V for 10 mins and PI for 5 mins. Flow-cytometric analysis was performed using Cytoflex LX (Beckman Coulter, USA). The experiments were repeated for 4 times, and the result was shown as histogram data.

Detection of reactive oxygen species
The ROS Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China) was used to verify the generation of intracellular ROS. RF/6A cells were seeded at 1 Â 10 4 cells/well in 96-well plates and allowed to adhere overnight. The cells were pretreated for 2 h at 37 C with L2H17 (2.5, 5, and 10 lM) or vehicle (DMSO), followed by incubation with TBHP (100 mM) for 22 h. Three experiments were performed in replicates of 4 for each treatment group. The medium was removed, followed by incubation with DCFH-DA for 20 mins at 37 C. Fluorescence was visualized on a LEICA digital inverted fluorescence microscope (488-nm excitation and 525-nm emission).

Gene expression analysis
RNA was isolated from the retina and choroid/RPE tissue and RF/6A cells using TRIZOL (Thermo Fisher). Reverse transcription and quantitative PCR were performed using the two-step M-MLV platinum SYBR Green qPCR Supermix-UDG kit (Thermo Fisher). RT-qPCR analysis was performed using Eppendorf realplex4 instrument according to the manufacturer's instructions (Eppendorf, Hamburg, Germany). Primers were provided with Tsingke (Beijing, China). The primer sequences are listed in Supplementary  Table S1. The relative expression level of each gene was estimated and normalized to the amount of b-actin.

Statistical analysis
The results were presented as mean ± SEM and were analyzed using GraphPad Pro 6.0 (GraphPad, San Diego, CA). The student's t-test was used to analyze the differences between the two groups. One-Way ANOVA was used to analyze the differences among more than two groups. p < 0.05 was considered statistically significant.

MD2 specific inhibitor L2H17 reduced angiogenesis and protected retinal function
Our previous studies synthesized a group of chalcone derivatives. Among them, L2H17 was identified as an effective MD2 inhibitor. 15 As shown in Figure 1, L2H17 (10 or 50 mg/kg/day) could significantly reduce the neovascular area compared to vehicle treatment (Figure 1(A, B)). At the 7 days after laser injury, the effect of L2H17 on laserinduced leakage was also examined with fundus fluorescein angiography. Treatment with L2H17 (50 mg/kg/day) significantly reduced the leakage compared to vehicle treatment. But there is no significant difference between the low dose treatment group (10 mg/kg/day) and the vehicle-treated group (Figure 1(C, D)).
Next, we explored whether L2H17 could protect against laser-induced retinal dysfunction using electroretinography (ERG). The data indicated laser injury caused a significant reduction in the amplitudes of the ERG a-and b-waves in CNV mice. However, the reduction was significantly reversed in L2H17-treated mice (Figure 1(E-G)). Based on the data above, we concluded that MD2 inhibition effectively reduced laser injury and improved retinal function in vivo.

MD2 inhibition suppressed inflammatory injury in CNV mice
We investigated whether L2H17 could reverse the laserinduced inflammatory injury in CNV mice. As expected, L2H17 treatment reduced the transcriptional levels of TNFa, IL-6, and ICAM-1 in the retina and choroid/RPE tissue (Figure 2(A-C)). Unexpectedly, the transcriptional level of VCAM-1 was not different among vehicle-treated and L2H17-treated groups (Figure 2(D)). We next detected NF-jB activation, represented by the levels of p-P65, using immunohistochemistry. We found that L2H17 treatment significantly reduced p-P65 expression (Figure 2(E, F)) when compared to the vehicle-treated group. So, we believed that L2H17 treatment could effectively suppress inflammatory injury in CNV mice.

MD2 inhibition reduced activation of TLR4-mediated signaling pathway in CNV mice
To investigate the underlying mechanism of pharmacological effect of L2H17 on CNV, we examined if the TLR4 signaling pathway was involved in the process. Thus, we explored the activation of MAPK signaling pathways, including JNK, P38, and ERK, which are the downstream signaling proteins of TLR4/MD2 pathway. Laser photocoagulation increased phosphorylation of JNK, P38, and ERK in CNV mice when compared to the normal control group (Figure 3(A-D)). L2H17 administration significantly reduced the phosphorylation of ERK (p < 0.05) (Figure 3(D)). However, L2H17 did not prevent the phosphorylation of JNK and P38 (p > 0.05) ( Figure  3(B, C)). The results still revealed that TLR4-medicated signaling could be inhibited by L2H17.

MD2 inhibition suppressed TBHP-induced production of reactive oxygen species in RF/6A cells
Oxidative stress has been reported to be involved in the pathological progress of CNV. 16,17 Thus, we examined whether L2H17 could reverse TBHP-induced generation of intracellular ROS in RF/6A cells. As visualized with fluorescence microscopy and the indicator DCFH-DA, RF/6A cells challenged with 100 lM TBHP exhibited an increase in intracellular fluorescence compared with non-treated controls, while this increase of ROS was markedly suppressed by L2H17 pretreatment dose-dependently (Figure 4(A, B)). Figure S2, TBHP was toxic to RF/6A cells with a IC50 of 100 lM. For subsequent experiments, TBHP, at the dose of 100 lM, was used to evaluate the protection effect of L2H17. As shown in Figure 5(A), TBHP-induced viability reduction was prevented by L2H17 (2.5, 5, or 10 mM). The results suggested that L2H17 pretreatment protected RF/6A cells from TBHP-induced cell death.

As shown in Supplementary
TBHP causes oxidative stress-induced apoptosis in cells. 18,19 In our RF/6A cells experimental model, flow cytometry data revealed that L2H17 protected against TBHP-induced apoptosis. Exposure of RF/6A cells to L2H17 (5, 10 mM) significantly decreased late apoptotic cells ( Figure 5(B, C)). Our findings revealed that MD2 inhibition could suppress TBHPinduced cell death by reducing apoptosis in RF/6A cells.
MD2 inhibition prevented TBHP-induced activation of the TLR4 signaling pathway in RF/6A cells Since TLR4 has been implicated in AMD, we evaluated whether the protective effects of L2H17 could be attributed to inhibiting the activation of the TLR4 signaling pathway   in RF/6A cells. To test the downstream signaling proteins, we detected the activation of MAPK signaling pathway (JNK, P38, ERK) and nuclear factor-jB (NF-jB). Results indicated that TBHP stimulation increased JNK, P38 and ERK phosphorylation ( Figure 5(D-F)). Pretreatment with L2H17 resulted in a significant decrease in phosphorylation of P38 and ERK ( Figure 5(E, F)). However, L2H17 did not prevent TBHP-induced increase of JNK phosphorylation ( Figure 5(D)). TBHP also activated the NF-jB pathway as assessed through nuclear P65 protein level. As shown in Figure 6(A, B) P65 phosphorylation was prevented by L2H17. The results partially demonstrate the potential molecular mechanism underlying the protective effect of L2H17 on nAMD through its prevention of TLR4 and its downstream signaling.

Discussion
Currently, anti-VEGF is the primary treatment for nAMD. However, some patients develop atrophy of the retina and other complications after long-term anti-VEGF treatment, resulting in a persistent decrease in visual acuity. 20,21 So, exploring alternative treatment strategies for nAMD is urgently needed. The current pathological mechanism of nAMD has not been fully understood. Previous studies have revealed that oxidative injury and inflammation were the leading causes of AMD. 11  can inhibit the RhoA/ROCK signaling pathway converts microglia to M1 type to target inflammation and inhibit the development of CNV. 24 As such, anti-oxidative stress and anti-inflammatory therapy are expected to be new treatments for CNV (Figure 7). TLR4 and MD2 have been found to be highly expressed in the retina. 25 Several studies reported that TLR4 could initiate inflammatory response in an MD2-dependent manner. TLR4 activation leads to inflammatory cascade and retinal damage. MD2 is a co-receptor of TLR4 and plays a pivotal role in formation of MD2/TLR4/LPS complex. The complex activates TLR4 and mediates inflammatory response. 8 Recently, a few studies have demonstrated that endogenous ligands bind to TLR4/MD2, activating the TLR4 signaling pathway. [26][27][28] This data suggests that MD2 also plays a crucial role in the sterile inflammatory response mediated by TLR4. The activation of the TLR4 signaling pathway increased the phosphorylation of a series of downstream kinases, such as MAPKs. Finally, it activated the NF-jB, causing the expression of a series of inflammatory cytokines, such as IL-6, IL-8,TNF-a, and chemokines. 10,11,29,30 Li et al. demonstrated that Ab could activate the TLR4-MyD88-NF-jB signaling pathway in RPE cells, indicating that TLR4related pathways may be potential AMD therapeutic targets. 7 As an important protein in TLR4 signaling pathway, the role of MD2 in the development of CNV has not been reported. Our research is the first to address the role of MD2 in CNV. In this study, We showed that MD2 specific inhibitor, L2H17, could reduce angiogenesis and protect against retinal function in CVN model. Since we have confirmed at the molecular and cellular levels that L2H17 could directly binds to MD2, to verify whether the effect of L2H17 is related to its inhibition of TLR4 and its downstream inflammation-related pathways, RT-qPCR, immunohistochemistry, and Western blot were used to confirm the levels of inflammatory factors such as IL-6, TNF-a, ICAM, VCAM, and the expression levels of NF-jB and MAPKrelated proteins. The results showed that inhibition of MD2 could reduce the levels of various inflammatory factors to varying degrees, and inhibit the activities of MAPK and NF-jB. We concluded that L2H17 treatment might mitigate CNV via blocking TLR4/MD2-mediated pathways and MD2 may serve as a potential therapeutic target for the treatment of nAMD.
TBHP can induce oxidative stress and inflammatory response in cells, and oxidative damage and inflammatory response of RPE cells are the central link and important pathological factor in the development of AMD. In our previous studies, we have confirmed that MD2 inhibition can alleviate the oxidative-inflammatory response induced by TBHP in ARPE-19 cells, and the role of MD2 is TLR4dependent. 14 On this basis, we used RF/6A to study the molecular mechanism of chalcone derivative L2H17 inhibiting choroidal neovascularization, and further study the mechanism of MD2/TLR4 signaling pathway in the occurrence and development of CNV. In cultured RF/6A cells, we revealed that TBHP increased ROS levels, leading to apoptosis. ROS induction required MD2 since pharmacological inhibition of MD2 reduced ROS levels. The result suggested that L2H17 at the used doses may have anti-oxidative effects. However, the mechanism by which this antioxidant effect of L2H17 is exerted still needs further exploration. In our previous studies, L2H17 was reported to significantly reduce inflammation in retinal I/R injury and diabetic retinopathy by targeting MD2 protein. 14,31 Consistent with these results, this study showed that L2H17 down-regulated TNFa, IL-6, ICAM-1and VCAM-1 in vivo and in vitro. And IL-6 has been proved to be involved in angiogenesis. 32,33 And we extrapolate that L2H17 may inhibit CNV by blocking TLR4/MD2 mediated pathways and through its anti-inflammation and anti-oxidative effects.
Our study has some limitations. The laser-induced CNV model only simulates the state of AMD disease. It still needs a long process to develop from CNV to nAMD. The occurrence of CNV does not necessarily mean that nAMD has occurred. Therefore, the effectiveness of L2H17 on nAMD needs to be further explored in AMD patients. In this research, we used wild-type mice only, and the crucial role of MD2 in CNV needs to be further tested in the MD2 knockout mice. What's more, in order to reduce the impact of sex hormones on the results, we used only male mice, but the large number of women with AMD should not be overlooked, the mechanism by which sex differences in disease pathogenesis need to be studied further. And further research is needed in this study on the antioxidant effect of MD2 inhibition.
Overall, the study provides evidence that MD2 may serve as a potential therapeutic target, and compound L2H17 may offer an alternative treatment strategy for nAMD. Proposed mechanism for TLR4/MD2 signaling and the inhibiting effects of L2H17 on the progression of nAMD. Oxidative stress or laser damage activates TLR4 through MD2 in choroid-retinal cells. TLR4 activation is associated with MyD88 interaction. The MyD88 pathway activates NF-jB and MAPK to induce the expression of proinflammatory cytokines. Meanwhile, the level of ROS has also increased to a certain extent. Oxidative stress and unregulated inflammatory responses cause cell apoptosis. L2H17 reduces cell death caused by oxidative stress and inflammation by disrupting the formation of MD2-TLR4 complex.