IKK2 inhibition attenuates laser-induced choroidal neovascularization

Abstract

Choroidal neovascularization (CNV) is aberrant angiogenesis associated with exudative age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Inflammation has been suggested as a risk factor for AMD. The IKK2/NF-κB pathway plays a key role in the inflammatory response through regulation of the transcription of cytokines, chemokines, growth factors and angiogenic factors. We investigated the functional role of IKK2 in development of the laser-induced CNV using either Ikk2 conditional knockout mice or an IKK2 inhibitor. The retinal neuronal tissue and RPE deletion of IKK2 was generated by breeding Ikk2(-/flox) mice with Nestin-Cre mice. Deletion of Ikk2 in the retina caused no obvious defect in retinal development or function, but resulted in a significant reduction in laser-induced CNV. In addition, intravitreal or retrobulbar injection of an IKK2 specific chemical inhibitor, TPCA-1, also showed similar inhibition of CNV. Furthermore, in vitro inhibition of IKK2 in ARPE-19 cells significantly reduced heat shock-induced expression of NFKBIA, IL1B, CCL2, VEGFA, PDGFA, HIF1A, and MMP-2, suggesting that IKK2 may regulate multiple molecular pathways involved in laser-induced CNV. The in vivo laser-induced expression of VEGFA, and HIF1A in RPE and choroidal tissue was also blocked by TPCA-1 treatment. Thus, IKK2/NF-κB signaling appears responsible for production of pro-inflammatory and pro-angiogenic factors in laser-induced CNV, suggesting that this intracellular pathway may serve as an important therapeutic target for aberrant angiogenesis in exudative AMD.

Figure 1. Schematic diagram shows targeting strategy by inhibiting IKK2/NF-κB pathway to prevent choroidal neovascularization (CNV) in AMD.

Chronic inflammation and oxidative stress caused by pathologic changes, tissue damage or local components activation in subretinal space induce expression and secretion of cytokines, growth factors and extracellular matrix molecules by RPE, which in turn initiate CNV. NF-κB is a key transcription factor necessary for inflammatory and stress responses. Inhibition of NF-κB activation by IKK2 inhibitor is postulated to prevent chronic inflammation- and oxidative stress-induced CNV formation.

Figure 2. IKK2 is deleted in the RPE and retinal neurons in Ikk2^−/flox/Nestin-Cre⁺ mice.

(A) Nestin-promoter-driven Cre mediated specific deletion of the LoxP flanked genomic sequence was tested on mT/mG/Nestin-Cre⁺ mice (a) and performed on Ikk2^−/flox/Nestin-Cre⁺ mice (b). The mT/mG (membrane-Tomato/membrane-Green) mouse strain contains a Cre-reporter transgene knocked into the Rosa26 locus. When bred with the nestin-Cre mice, the Cre recombinase mediated the genomic deletion of the LoxP (solid triangles)-flanked tdTomato (red) STOP cassette and in turn led to expression of the downstream membrane-targeted enhanced green fluorescent protein (GFP). A similar nestin-Cre mediated homologous recombination to obtain a neuronal and RPE deletion of Ikk2 in retina was designed by breeding the Ikk2^−/flox mouse carrying one Ikk2 mutant allele and one Ikk2-flox allele with the mouse line expressing Cre recombinase under control of Nestin promoter (Nestin-Cre). The hematoxylin- and eosin-stained wild type mouse eye section on left show anatomy of retina, choroid, and sclera. (B, C) The RPE and neuronal retinal cell deletion of the LoxP-flanked tdTomato STOP cassette mediated by Nestin-Cre was confirmed by the red-to-green fluorescent color conversion on the retinas of the mT/mG/Nestin-Cre⁺ cre reporter mice, whereas the adjacent choroid tissue did not show such red-to-green change, as shown at lower (B) and higher (C) magnification images. Red cells represent cre-negative cells, while green cells are cre-positive cells. (D) Reverse transcription PCR showed that Ikk2 mRNA was eliminated in both retina and brain, but not in the control tissue, i.e., the skin of the Ikk2^−/flox/Nestin-Cre⁺ mice. (E) Western clot analysis showed that the IKK2 protein was absent in the retina and brain, but not in the skin of the Ikk2^−/flox/Nestin-Cre⁺ mice. Size bar = 20 μm in B and C.

Figure 3. IKK2 is not required for retina development and function.

(A, B) The paraffin-embedded retinal sections from the wild type control (WT) (A) and Ikk2^−/flox/Nestin-Cre⁺ (Mut) (B) mice at age of 2 month old were stained by hematoxylin and eosin (H&E) method. (C) The quantitative comparison of the cell layers in the outer nuclear layer (ONL) and inner nuclear layer (INL) between WT (n = 3 mice) and Mut (n = 3 mice) mice at 2 month old. (D) Visual acuity was tested on wild type (WT) control and Ikk2^−/flox/Nestin-Cre⁺ (Mut) mice at the age of 2 and 5 month old by OKR. Error bars indicate standard deviation.

Figure 4. Induction of the laser-induced CNV is inhibited in the Ikk2^−/flox/Nestine-Cre⁺ mice.

The development of CNV after laser photocoagulation was quantified by scoring the fluorescence leakage and isolectin IB4 stain areas and measuring the thickness of choroidal neovascular membrane. (A–C) Fundus photographs and fluorescein angiogram (A–C) were taken from the wild type (WT), Ikk2^+/flox/Nestine-Cre⁺ (Ikk2^+/−), and Ikk2^−/flox/Nestine-Cre⁺ (Ikk2^−/−) mouse eyes that had been treated with laser injury for 7 days. The development of CNV was quantified by scoring the relative average size of fluorescence leakage in each animal (D) comparing to optic disk size. N = number of mice. (E–G) The images of Alexa Fluor 568 conjugated isolectin IB4 stained CNVs were taken from the indicated mouse eyes at 7 days post-laser injury. Arrows indicate the CNV identified by isolectin IB4 staining. (H) The summary of the mean CNV area from each genotypic animal was shown as relative to that induced in the WT eyes. N = number of mice. (I and J) Representative H&E stained eye sections show the neovascular membrane networks originating from the choroicapillaries passing through the broken Bruch's membrane. The white arrows with two arrowheads show the thickness of choroidal neovascular membrane. The scale bar represents 50 μm in length. (K) Quantification of the thickness of the choroidal neovascular membrane. Error bars indicate standard deviation. Two-tail T-test: * p<0.05, *** p<0.005. n indicates the number of laser-injured spots.

Figure 5. IKK2 chemical inhibitor TPCA-1 significantly reduces the laser-induced CNV formation.

(A) The size measurements on the laser induced vascular sprouting in the eyes injected with 20 μL TPCA-1 or control vehicle (PBS/20% DMSO). 20 μL of IKK2 chemical inhibitor TPCA-1 at indicated concentration was retrobulbarly injected immediately after the laser injury. CNVs were visualized by FITC-dextran staining 7 days after laser injury and quantified as area size relative to CNV area in the control mice injected with vehicle solution. (B) Visual function was measured by OKR tests performed on the laser-injured and TPCA-1 treated mice on day-6 and -21 post laser injury. The mice received a retrobulbar injection of 20 μL TPCA-1 at the concentrations indicated under the X-axis of the graph in one eye right after laser injury. “Untreated” represents the results from the eyes without any injection. “TPCA-1” represents the visual acuity from the eyes retrobulbarly injected with 20 μL TPCA-1. “c” represents the combined visual acuity test results from both TPCA-1 treated and untreated eyes. (C) The size of CNV area developed in the laser-induced and TPCA-1 treated eyes was normalized to the size of CNV area in the laser-induced WT eyes that had not been given any injection after laser injury. The TPCA-1 treatment was given once to the mice via either retrobulbar (20 μL 10 mM TPCA-1 or control vehicle) or intravitreal (2 μL 10 mM TPCA-1 or control vehicle) injection immediately after laser injury and, seven days later, the FITC-dextran stained images were taken and calculated. (D) The size of CNV area developed in the laser-induced and TPCA-1 treated eyes was normalized to the size of CNV area in the laser-induced, and control solvent vehicle injected eyes. The TPCA-1 treatment was given once to the mice via either retrobulbar (20 μL 10 mM TPCA-1 or control vehicle) or intravitreal (2 μL 10 mM TPCA-1 or control vehicle) injection at 3 days after laser injury. The images were taken and calculated at 7 days after laser injury. (E) Comparison of the relative sizes of CNV area in the laser-injured mice that had been treated with either retrobulbar injection of 20 μL of 10 mM TPCA-1 or intravitreal injection of 2 μL of 2 μg/μL mouse VEGF 164 affinity purified polyclonal antibody. They were normalized to the size of CNV area in the laser-injured mice that had been treated with control solvent vehicle and IgG isotype control antibody, respectively. Error bars indicate standard deviation. Two-tail T-test: * p<0.05; ** p<0.01, *** p<0.005. “n” indicates the number of mice used at each condition.

Figure 6. Retrobulbar administration of TPCA-1 causes no obvious toxicity to blood cells.

(A–F) Flow cytometric analyses of blood cell population. Single cell suspension was prepared from spleen, thymus and bone marrow of the mice at 7 days after laser-injury followed immediately by a single retrobulbar injection of 56 μg TPCA-1 in 20 μL vehicle solution (PBS/20% DMSO) or control vehicle only in 20 μL. The representative plots are shown and numerical values represent the average (±SD, n>5). Statistics were performed using ProStat 5.5 program, and showing no significant difference in all the groups. P≤0.05 is considered as significant. (A, B) The CD4⁺ and CD8a⁺ T cells from the TCRβ-gated populations in spleen (A) and thymus (B) were calculated and plotted. (C, D) Spleen (C) and bone marrow (D) cells were isolated from TPCA-1 treated (+) and untreated (−) mice and stained with anti-B220, anti-IgM, and anti-IgD antibodies. Data in (C) show the percentile of mature (IgD^highIgM^low) and immature (IgD^lowIgM^high) B cells in the B220-gated splenocytes, and data in (D) show the percentages of mature recirculating (R2, IgD^highIgM^int), transitional immature (R3, IgM^highIgD^int), immature (R4, IgM^highIgD⁻) and precursor (R5, IgM⁻IgD⁻) B cell populations in the B220-gated bone marrow cells. (E) Spleen cells were isolated from TPCA-1 treated (+) and untreated (−) mice and stained with anti-CD11b, anti-Ly6C and anti-Ly6G antibodies. The numerical values are shown as the average of neutrophil (Ly6G⁺/Ly6C^int), monocyte (Ly6G⁻/Ly6C⁺) and mature macrophage (Ly6G⁻/Ly6C⁻) from the CD11b gate in the spleens. (F) Spleen cells were stained with 7AAD to detect dead cells. The numerical values are shown as the average of the CD11b⁺ 7AAD⁺ populations in splenic lymphoid cells. (G) Hematoxylin and eosin (H&E) histological staining was performed to evaluate toxicity to liver and spleen at 7 days after treatment. The scale bar represents 50 μm in length. (H) Relative cell viability of HEK293T cells (left panel) and ARPE-19 cells (right panel) was shown at 12 hours after being cultured with 50 μM TPCA-1 or 10 ng/mL TNFα or combination of both. The cells were pretreated with 50 μM TPCA-1 for 30 minutes prior to addition of 10 ng/mL TNFα for combination treatment. Cell viability was measured using MTT assay. Data are represented as the mean±SD from three independent experiments. Asterisk indicates Two-tail T-test: *** p<0.005.

Figure 7. TPCA-1 inhibits NF-κB signaling pathway and the thermal injury-induced angiogenesis-associated factors.

(A) TPCA-1 inhibits the TNFα-induced κB-Luciferase activity in a dosage dependent manner. 293T cells containing κB-Luciferase reporter were pretreated with or without TPCA1 for 30 minutes, then stimulated with 10 ng/mL TNFα to induce κB-Luciferase expression in the presence of the indicated amount of TPCA-1 for 5 hours, and followed by cell lysis and luciferase assay. (B) TPCA-1 inhibits TNFα-induced IκBα degradation in a dosage dependent manner. ARPE-19 cells were pretreated with TPCA-1 at indicated concentration for 30 minutes prior to addition of 10 ng/mL of TNFα for 15 and 30 minutes in the presence of the same amount of TPCA-1. Protein lysates were analyzed for the levels of IκBα and tubulin (as a loading control) by Western blotting. (C) TPCA-1 treatment inhibits the thermal injury-induced p65 phosphorylation at serine 536 amino acid residue. The ARPE-19 cells at 80% confluence in the complete DMEM/F-20 medium were pretreated with 5 μM TPCA-1 for 30 minutes and followed by heat shock for 5 seconds with addition of 10 mL HEPES-buffered saline preheated at 55°C; and then the heating medium was immediately replaced with normal complete DMEM/F-20 medium containing 5 μM TPCA-1 and further cultured at 37°C for 10, 30, 60, and 120 minutes before the protein lysates were collected. Western blotting was used to detect the total NF-κB p65 protein and the activated form of NF-κB p65 phosphorylated at Serine 536. (D and E) TPCA-1 treatment reduces thermal injury-induced and NF-κB dependent transcription of signaling protein, cytokine and chemokine, such as IκBα, IL1B and CCL2 (D), as well as of those associated with angiogenesis, such as VEGF-A, PDGFA, HIF-1A, and MMP-2 (E) in ARPE-19 cells. Cells were treated similarly as described in C. Four hours after the thermal treatment, RNAs were isolated for qPCR analysis. Relative mRNA levels were normalized to β-actin first, and then the fold increase or decrease by heat shock. Cells were treated with heat shock at the presence or absence of TPCA-1 or treated with TPCA-1 without heat shock were compared to control cells without heat shock and TPCA treatment. Error bars indicate standard deviation. Two-tail T-test: * p<0.05, n = 3. The red * indicates the comparison between the heat shocked cells and control cells. The purple * indicates the comparison between the heat shocked cells at the presence or absence of TPCA-1. (F) Relative mRNA level of Vegfa, Hif1a, and Hmox1 was shown in the RPE/Choroid/Sclera tissues collected from laser injured eyes treated with or without TPCA-1. Mice received the retrobulbar injection of 56 μg TPCA-1 or vehicle solution and the RNA samples were prepared from RPE/Choroid/Sclera tissues at day 1 and day 2 post injury. Each RNA sample was pooled from 4 eyes in the same condition and the control sample is from the eyes without laser injury. Red and green * show the significant difference between the indicated sample and control sample without laser injury. Purple and light blue *** show the significant difference between the laser injured samples at the presence or absence of TPCA-1. Error bars indicate standard deviation. Two-tail T-test: * p<0.05, *** p<0.005, n = 3.