Additional file 2 of Comparison of the regressive effects of aflibercept and brolucizumab on pigment epithelial detachment

Additional file 2: Supplementary figure 2. Mean changes from baseline in the maximum height (MH) of pigment epithelial detachment (PED) measured in the intravitreal aflibercept (IVA) and intravitreal brolucizumab (IVBr) groups in cases with ≥ 300 μm of PED before and at 1, 2, and 3 months after the first treatment

Additional file 1 of Comparison of the regressive effects of aflibercept and brolucizumab on pigment epithelial detachment

Additional file 1: Supplemantary figure 1. Changes in central macular thickness (CMT), central choroidal thickness (CCT), and best corrected visual acuity (BCVA) at the initial visit and 3 months after the first treatment

AMPK-Activated Protein Kinase Suppresses Ccr2 Expression by Inhibiting the NF-κB Pathway in RAW264.7 Macrophages

<div><p>C-C chemokine receptor 2 (Ccr2) is a key pro-inflammatory marker of classic (M1) macrophage activation. Although Ccr2 is known to be expressed both constitutively and inductively, the full regulatory mechanism of its expression remains unclear. AMP-activated protein kinase (AMPK) is not only a master regulator of energy homeostasis but also a central regulator of inflammation. In this study, we sought to assess AMPK’s role in regulating RAW264.7 macrophage Ccr2 protein levels in resting (M0) or LPS-induced M1 states. In both M0 and M1 RAW264.7 macrophages, knockdown of the AMPKα1 subunit by siRNA led to increased Ccr2 levels whereas pharmacologic (A769662) activation of AMPK, attenuated LPS-induced increases in Ccr2 expression in an AMPK dependent fashion. The increases in Ccr2 levels by AMPK downregulation were partially reversed by NF-κB inhibition whereas TNF-a inhibition had minimal effects. Our results indicate that AMPK is a negative regulator of Ccr2 expression in RAW264.7 macrophages, and that the mechanism of action of AMPK inhibition of Ccr2 is mediated, in part, through the NF-κB pathway.</p></div

Immunoelectron microscopy for SLURP-1 in the photoreceptors.

<p>A. Immunogold particles (SLURP-1) are mainly observed in the photoreceptor outer segments (OS) but not the inner segments (IS). B, Arrowheads and arrows indicate metal particles associated with the cell membranes and intracellular sites, respectively. C. The summary graph of SLURP-1 distribution in the OS and IS. The percentage of total particles examined was plotted in 20 nm bins from the cell membranes of the OS or IS. 124 and 111 particles were measured in the OS and IS, respectively. The OS exhibit a peak distribution of SLURP-1 just inside the cell membrane. Scale bars: 2 µm (A), 1 µm (B).</p

Characterization of Primary Antibodies.

<p>SLURP-1, secreted mammalian lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1; OPN1SW, a marker for S-opsin; ChAT, choline acetyltransferase; CHT1, high affinity choline transporter.</p

AMPKα1 negatively regulates Ccr2 expression in the M0 and the LPS-stimulated M1 macrophages.

<p>A: Reduced AMPKα1 protein levels in macrophages treated with AMPKα1 siRNA RAW264.7 were confirmed by Western blotting of whole cell lysates. β-actin was probed as an internal control. B: Whole cell lysates of RAW264.7 macrophages treated with either control or AMPKα1 siRNA were examined by Western blotting to confirm compensation by AMPKα2. Tissue lysates prepared from mouse retina were used as a positive control for expression of AMPKα2 protein. β-actin was probed as an internal control. C: Ccr2 expression on RAW264.7 macrophages was analyzed by flow cytometry. RAW264.7 macrophages were stimulated with 10–1000 ng/ml of LPS for 12 h. D: Flow cytometry analysis of Ccr2 expression on RAW264.7 macrophages treated with either control or AMPKα1 siRNA. RAW264.7 macrophages were stimulated with 100 ng/ml of LPS for 12 h to induce the M1 state. n = 3. ***, <i>p <</i> 0.001.</p

AMPKα1 reduction increases Ccr2 expression in the LPS-stimulated M1 macrophages through NF-κB signaling.

<p>A, B, C and D: RAW264.7 macrophages were pretreated with IKK inhibitor (BMS345541, 0.5–2 μM) and NF-κB inhibitors (LY303511, 1–3 μM; SM7368, 4 μM) for 2 h, followed by co-treatment with 100 ng/ml of LPS and different concentration of each inhibitor for 12 h. Ccr2 expression was analyzed by flow cytometry. n = 3. ***, <i>p <</i> 0.001.</p

Expression of SLURP-1 in the retina under dark adaptation.

<p>Samples were collected around midnight. SLURP-1 expression in the retina was not affected by dark adaptation. A. Confocal image showing SLURP-1 (green) and rhodopsin (red) immunoreactivity in mouse retina. SLURP-1 is localized in the photoreceptor outer segments (OS) and co-localizes with rhodopsin. B. Confocal image showing SLURP-1 (green) and S-opsin (red) immunoreactivity in mouse retina. SLURP-1 is localized in the photoreceptor OS and co-localizes with S-opsin. DNA was counterstained with TO-PRO-3 (blue). Scale bars: 20 µm (A, B); ONL, outer nuclear layer; INL, inner nuclear layer; RPE, retinal pigment epithelium; IS, inner segments. C. Semi quantitative RT-PCR bands of <i>SLURP-1</i> or <i>GAPDH</i> during the day (light) or night (dark). D. Real-time RT-PCR results of <i>SLURP-1</i> expression normalized against <i>GAPDH</i> expression during the day (light) or night (dark).</p

Pharmacological activation of AMPK counter-regulates Ccr2 expression in the LPS-stimulated M1 macrophages.

<p>A: RAW264.7 macrophages treated with either control or AMPKα1 siRNA were additionally treated with 25–100 μM of the AMPK activator, A769662. The phosphorylation of AMPKα (p-AMPKα) after A769662 treatment was examined by Western blotting. β-actin was probed as an internal control. B: RAW264.7 macrophages were pretreated with 25–100 μM A769662 for 2 h, followed by co-treatment with 100 ng/ml of LPS and each different concentration of A769662 for 12 h. Dimethyl sulfoxide (DMSO) was used as a control. Ccr2 expression was analyzed by flow cytometry. C: RAW264.7 macrophages treated with either control or AMPKα1 siRNA were pretreated with 50 μM A769662 for 2 h, followed by co-treatment with 100 ng/ml of LPS and 50 μM A769662 for 12 h. DMSO was used as a control. Ccr2 expression was analyzed by flow cytometry. n = 3. *, <i>p <</i> 0.05; ***, <i>p <</i> 0.001.</p

ChAT and CHT1 immunoreactivity in photoreceptor outer segments.

<p>A. ChAT immunoreactivity (green) is present in the inner plexiform layer (IPL) as two stratified bands, the amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), and photoreceptor outer segments (OS). B. CHT1 immunoreactivity (green) is also present in the IPL as two stratified bands, the amacrine cells in the INL and GCL, and photoreceptor OS. DNA was counterstained with TO-PRO-3 (blue). Scale bar: 50 µm; ONL, outer nuclear layer.</p