Application of TPM1 in the treatment of wet age-related macular degeneration

Abstract

This invention discloses the application of TPM1 in the treatment of wet age-related macular degeneration (wAMD). The study found that TPM1 promotes angiogenesis, inflammation, and fibrosis in the eye. The anti-angiogenic effect of inhibiting TPM1 is similar to that of inhibiting VEGF-A, but the effect of inhibiting TPM1 in reducing inflammation and fibrosis is more significant, thus making it a potential target for the treatment of wAMD. Furthermore, single-cell sequencing revealed that TPM1 is specifically highly expressed in pericytes of CNV lesions in wAMD patients; functional experiments confirmed that knocking down TPM1 significantly inhibits pericyte proliferation and migration, suggesting that it participates in wAMD progression and unresponsiveness to anti-VEGF therapy by regulating pericyte function.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to application of TPM1 in treatment of wet age-related macular degeneration. BACKGROUND

[0002] According to clinical and pathological characteristics, age-related macular degeneration (AMD) can be divided into dry (atrophic) and wet (neovascular) subtypes. Among them, wet AMD (wAMD) accounts for only about 10%-15% of all AMD, but causes more than 80% of severe visual loss cases. The typical pathological manifestation of wAMD is that abnormal neovascularization of choroidal origin breaks through Bruch's membrane and forms choroidal neovascularization (CNV) under the retina. The structure of such neovascularization is fragile and has increased permeability, and is prone to leakage and hemorrhage, which directly leads to acute decline of central vision.

[0003] A large amount of evidence shows that vascular endothelial growth factor (VEGF) is a key molecular driver factor for CNV formation and leakage. Based on this, anti-angiogenic therapy (anti-VEGF) has become a first-line treatment strategy for wAMD, which can effectively inhibit neovascular activity and reduce leakage, thereby stabilizing or improving vision in most patients. However, about 10% of wAMD patients do not respond to initial anti-VEGF therapy; in long-term follow-up, about half of the patients still have significant visual decline within 5 years, which is often associated with CNV residual, atrophy progression and fibrotic scar formation. This suggests that anti-VEGF resistance / non-response has a complex pathological basis, and it is urgent to identify and analyze the key molecules and cellular mechanisms involved in this process to open up new treatment paths.

[0004] Existing studies believe that anti-VEGF non-response is driven by multiple factors, among which pericytes, as important supporting cells of capillary walls, are wrapped on the surface of endothelial cells and provide structural and survival signals, which can to some extent "coat" the new blood vessels and protect endothelial cells from anti-angiogenic therapy clearance. The support mediated by pericytes enables endothelial cells to maintain survival under relatively low VEGF conditions, thereby reducing the efficacy of anti-VEGF therapy.

[0005] By combining transcriptome and proteome analysis in mouse laser-induced CNV model, we found that TPM1 (tropomyosin-1) was significantly upregulated after CNV formation; TPM1 expression was significantly increased in mouse RPE / choroid complex at day 3 after CNV. Notably, TPM1 remained upregulated after anti-VEGF intervention, suggesting that it might be involved in CNV pathogenesis and treatment response regulation through mechanisms other than the VEGFA pathway.

[0006] Tropomyosin is a typical actin-binding protein that plays a structural and regulatory role in the contractile apparatus of muscle and non-muscle cells. During the maturation of pericytes to a contractile phenotype, smooth muscle-related contractile proteins, including actin, myosin, and tropomyosin, are highly expressed, conferring vascular wall tension regulation and stability.

[0007] Previous studies have reported that TPM1 in systemic circulation has immunomodulatory properties, can promote PKA phosphorylation and activate the FABP5 / NF-κB signaling pathway, and induce the expression of endogenous TPM1 in the aging retina. As a key blood-derived pro-aging factor, TPM1 can induce increased microglial cell activation in a mouse Alzheimer's disease model, leading to optic nerve remodeling and causing a decline in visual function in the aging retina, suggesting its potential as a target for anti-aging interventions. In addition, TPM1 can regulate LPS-mediated neuroinflammatory responses and neuronal death by promoting microglial cell activation. However, the role of TPM1 in angiogenesis and fibrosis has not been mentioned in the above reports.

[0008] To date, there have been no reports on the specific function of TPM1 in the development of wAMD. Similarly, there is no direct evidence of its involvement in anti-VEGF treatment resistance. In addition, the cell-specific function of TPM1 in different vascular cell subpopulations (such as endothelial cells, pericytes, smooth muscle cells, etc.) still lacks systematic research. SUMMARY

[0009] The present invention first found that inhibition or silencing of TPM1 can inhibit ocular neovascularization, reduce ocular inflammatory response and fibrosis, which indicates that TPM1 can be used as a potential target for treating wet age-related macular degeneration. TPM1 Therefore, the first object of the present invention is to provide the use of a TPM1 inhibitor in the preparation of a medicament for treating wet age-related macular degeneration.

[0010] Preferably, the TPM1 inhibitor can inhibit ocular neovascularization, reduce ocular inflammatory response and fibrosis.

[0011]

[0012] ​Preferably, the TPM1 inhibitor is an agent that inhibits or silences TPM1 the expression of the gene.

[0013] Preferably, the agent that inhibits or silences TPM1 the expression of the gene comprises inhibiting or silencing the expression of the gene by means of RNA interference, gene knockout, chemical inhibition, miRNA-mediated silencing, transcription factor inhibition, or epigenetic modification. TPM1 the expression of the gene.

[0014] Preferably, the agent of RNA interference comprises siRNA, dsRNA, or shRNA against TPM1 the gene.

[0015] Preferably, the sequence of the shRNA against TPM1 the gene is as set forth in SEQ ID NO. 1-2; and the sequence of the siRNA against TPM1 the gene is as set forth in SEQ ID NO. 11.

[0016] Preferably, the medicament is an ophthalmic preparation.

[0017] Preferably, the ophthalmic preparation comprises eye drops, eye ointment, eye spray, eye gel, eye patch, intraocular injection, ophthalmic microspheres, ocular implant, periocular injection, and ophthalmic sustained-release preparation.

[0018] A second object of the present application is to provide a medicament for treating wet age-related macular degeneration, comprising a TPM1 inhibitor as an active ingredient.

[0019] Preferably, the medicament for treating wet age-related macular degeneration further comprises a pharmaceutically acceptable excipient or carrier.

[0020] The present application has found that TPM1 promotes angiogenesis, inflammation, and fibrosis in the eye. The anti-angiogenic effect of inhibiting TPM1 is similar to that of inhibiting VEGF-A, but in terms of reducing inflammation and fibrosis, the effect of inhibiting TPM1 is more significant, and thus can be used as a potential target for treating wAMD. In addition, single-cell sequencing has found that TPM1 is specifically highly expressed in pericytes in the CNV lesion of wAMD patients; functional experiments have confirmed that knocking down TPM1 significantly inhibits pericyte proliferation and migration, suggesting that it participates in the progression of wAMD and anti-VEGF therapy non-response by regulating pericyte function. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1Figure 6. The transcriptome sequencing procedure of laser-induced CNV model and the expression changes of TPM1 in choroid / RPE. (A) The schematic diagram of RNA sequencing after CNV modeling. (B) The expression of TPM1 in choroid / RPE complex of normal mice and mice after CNV modeling.

[0022] Figure 2 Figure 7. The construction of laser-induced CNV model and the detection of choroidal TPM1 expression at day 3 after modeling. (A) The schematic diagram of CNV model. (B-C) The detection of choroidal TPM1 expression at day 3 after CNV modeling by Western blot and qPCR, n=3. All data were presented as mean ± SD. *: p<0.05, ***: p<0.001.

[0023] Figure 3 Figure 8. The construction of laser-induced CNV model and the detection of choroidal TPM1 expression at day 7 after intravitreal injection of shVEGF-A. (A) The schematic diagram of CNV model. (B-C) The detection of choroidal TPM1 expression at day 7 after intravitreal injection of shVEGF-A in CNV model mice by Western blot and qPCR, n=4. All data were presented as mean ± SD. *: p<0.05, ***: p<0.001.

[0024] Figure 4 Figure 9. The immunofluorescence imaging and quantitative analysis of CNV model choroidal neovascularization (IB4), fibroblasts (a-SMA) and macrophages / microglia (Iba1). (A-B) The IB4 (green), a-SMA (red) and Iba1 (red) immunofluorescence images of choroid in CNV model. Scale bar: 100 μm. The quantitative results of IB4 are shown in (C), the quantitative results of Iba1 are shown in (D), and the quantitative results of a-SMA are shown in (E), n=4. All data were presented as mean ± SD. *: p<0.05, ***: p<0.001.

[0025] Figure 5 Figure 10. The functional changes of HBVP after knocking down TPM1 in primary human brain perivascular cells (HBVP). (A-B) Single-cell RNA sequencing analysis of choroidal tissue of wAMD patients. (C) CCK-8 quantitative analysis of HBVP 72 h after knocking down TPM1 in HBVP, n=3. (D-E) HBVP scratch and healing rate quantitative analysis at 0 h, 24 h and 48 h after knocking down TPM1 in HBVP, n=3. Scale bar: 200 μm. All data were presented as mean ± SD. *: p<0.05, ***: p<0.001. DETAILED DESCRIPTION

[0026] The following examples are further illustrations of the application and are not intended to limit the same.

[0027] The experimental methods used in the following examples are routine methods unless otherwise specified, and the reagents and materials used are commercially available unless otherwise specified.

[0028] Example 1

[0029] I. Materials

[0030] 1) Plasmids

[0031] shRNAs targeting TPM1 and VEGF-A were designed using pLKO.1 as the vector through the sigma website.

[0032] The designed sequences were synthesized by Tsingke Biotechnology Company and connected with pLKO.1 vector using T4 DNA ligase. All plasmids were transformed using DH5a and extracted using a plasmid extraction kit (TIANGEN, DP117TA).

[0033] Table 1 shRNA sequence information

[0034] .

[0035] 2) Proteins and antibodies, primers

[0036] Table 2 Proteins and antibodies

[0037] .

[0038] Table 3 Primers

[0039] .

[0040] Table 4 siRNA oligo sequence

[0041] .

[0042] II. Methods

[0043] 1. Laser-induced CNV mouse model

[0044] 1) Four laser photocoagulation spots were irradiated in the area 1.5-2 mm away from the disc diameter of the adult mouse's eye (spot size 75 μm, power 90 mw, duration 75 ms). Laser photocoagulation was performed at 12, 3, 6 and 9 positions, avoiding large retinal blood vessels;

[0045] 2) After laser photocoagulation, the vitreous cavity was injected with shTPMl, shVEGF-A or shNC plasmid-PEI complex (1 pg^L) ;

[0046] 3) On day 7, the eyes were collected. The eyeballs were fixed in 4% PFA for 2 h, and the cornea, lens, lens and vitreous body were removed, and the choroid-RPE complex was separated. The following experiments were performed separately: the choroid-RPE complex was washed with PBS and softened in PBS containing 5% donkey serum and 0.5% Triton X-100 for 1 hour, then incubated with primary antibody Alexa Fluor 488 (1:500) conjugated with alloantigen protein GS-IB4, a-SMA and Iba-1 (1:100) at 4°C overnight. The next day, after washing with PBS, the corresponding species of secondary antibody was incubated at room temperature for 2 h; the choroid-RPE complex was washed with PBS and placed flat with the RPE facing up; images were taken using a fluorescence microscope, and CNV regions were analyzed using image analysis software program.

[0047] 2. RNA isolation, cDNA synthesis and quantitative real-time PCR (qRT-PCR)

[0048] Mouse choroidal tissues were collected, total RNA was isolated from cells using TRIzol reagent (TIANGEN) and synthesized cDNA using FastKing RT kit with DNase (TIANGEN), qRT-PCR was performed using SYBR Green (ROCHE) and ABI QuantStudio 6 Flex device (Life Technologies). The results were normalized to GAPDH transcripts. The relative fold change in gene expression was calculated using the delta-delta Ct method.

[0049] 3. Western blot experiment

[0050] 1) Preparation of liquid:

[0051] ① 10x Running Buffer: Add 144 g glycine, 10 g SDS powder, 30.3 g Tris powder in order, ddH2O as solvent, make up to 1 L, prepare as a storage solution. Dilute to 1x Running Buffer to prepare working solution for use;

[0052] ② 5x SDS Loading Buffer: Add SDS powder 4 g, bromophenol blue 20 mg, DTT 3.085 g, Tris-HCL (1M pH 6.8) 10 mL, glycerol 20 mL in order, ddH2O as solvent, make up to 40 mL;

[0053] ③ 10x Transfer Buffer: Add 30.3 g Tris powder, 144 g glycine in sequence, ddH2O as solvent, make up to 1 L, prepare as a storage solution. Dilute to 1x Transfer Buffer according to the formula of 10x Transfer Buffer 100 mL + methanol 200 mL + ddH2O 700 mL to prepare as a transfer working solution for use;

[0054] 2) Preparation of separation gel and concentration gel:

[0055] ① Preparation of 10% separation gel (10 mL):

[0056] Table 5 Preparation of 10% separation gel

[0057] .

[0058] ② Preparation of 5% concentration gel (5 mL):

[0059] Table 6 Preparation of 5% concentration gel

[0060] .

[0061] 3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution to the tank, and check for leakage. Remove the comb, and add the marker and sample to the gel hole in sequence. Add the electrophoresis solution to the electrophoresis tank, and set the program to 80 V for constant voltage electrophoresis for 30 min, and then to 120 V for constant voltage electrophoresis for 1 h;

[0062] 4) Membrane transfer: First, activate the PVDF membrane with methanol, and then remove the gel in step 3). Assemble the membrane transfer clamp in the order of "sandwich" structure, and make sure there are no air bubbles in the assembly. Assemble the membrane transfer device, add the transfer working solution, and set the program to 250 mA constant current for 2 h, and then transfer the membrane on ice;

[0063] 5) Blocking: After the membrane transfer is completed, remove the membrane, first add 1x TBST and place it on a shaker at room temperature for 5 min, discard, and then add 5% skim milk as blocking solution and place it on a shaker at room temperature for blocking. The blocking time is 1 h;

[0064] 6) Primary antibody incubation: Discard the blocking solution, wash with 1x TBST at room temperature on a shaker until the blocking solution is clean, discard the liquid, and then add the primary antibody prepared with 5% BSA as the antibody diluent, and incubate overnight at 4°C in a refrigerator on a shaker;

[0065] 7) Membrane washing: wash the membrane with 1 x TBST for 3 times, 10 min each time;

[0066] 8) Secondary antibody incubation: discard the solution, add the secondary antibody prepared with 5% skim milk at a ratio of 1:5000, incubate at room temperature on a shaker for 1 h;

[0067] 9) Repeat step 7);

[0068] 10) Exposure and development: prepare the developing solution (A solution: B solution = 1:1, prepare fresh each time), place the membrane on the plate, and add the developing solution to the membrane, shake it evenly, expose and develop it on the machine, and save the results.

[0069] 4. CCK-8 cell proliferation experiment

[0070] HBVP was seeded in a 96-well plate at a density of 5 x 10 3 cells / well, cultured overnight, and the next day, TPM1 and control siRNA knockdown experiments were performed. After 72 h, the culture medium was replaced with 100 μL of fresh PM culture medium, and CCK8 detection reagent was added at a volume of 10 μL / well, shaken, and the cells were returned to 37°C for 1 h. Then, the wavelength at 450 nm was measured using a microplate reader. The ratio of the experimental group to the control group was calculated as the statistical result.

[0071] 5. Scratch experiment

[0072] HBVP was seeded in a 6-well plate at a density of 1.5 x 10 5 cells / well, and the next day, when the confluence reached about 85%, siTPM1 and si-Control transfection experiments were performed. After 24 h, the culture medium was replaced with serum-free PM culture medium, and mitomycin C (1 µM) was added for 1 h. A 200 μL gun tip was used to make a scratch in the center of the plate, the culture medium was discarded, and fresh serum-free PM culture medium was added. The scratch state at 0 h was recorded by taking a photo, and the scratch state was recorded again after 24 h and 48 h. The percentage of scratch closure in the experimental group relative to the control group was calculated using ImageJ software (v1.49).

[0073] III. Results

[0074] 1. In order to explore new targets in the process of angiogenesis, we constructed a laser-induced mouse choroidal neovascularization (CNV) model for RNA sequencing. Sequencing analysis found that TPM1 was up-regulated after CNV formation (P < 0.05) (Fig. 1A-B). Figure 1

[0075] ​2. To validate the sequencing results above, we reconstructed a laser-induced choroidal angiogenesis (CNV) model to detect TPM1 expression. Figure 2 (A) Western blot and qPCR showed that TPM1 expression in the mouse choroid / RPE complex was significantly higher than at baseline on day 3 after CNV ( Figure 2 (BC in the middle).

[0076] 3. In addition, shVEGF-A was injected intravitreally in a mouse CNV model. Figure 3 (A) 7 days later, TPM1 still showed an upregulation trend in the choroid / RPE complex tissue of the CNV model after shVEGF-A treatment. Figure 3 (BC in the image). This suggests that TPM1 is induced during CNV formation, and conventional anti-VEGF therapy did not inhibit this induction; on the contrary, it may have further stimulated TPM1 expression.

[0077] 4. To further investigate the potential role of TPM1 in angiogenesis, we constructed knockdown plasmids of TPM1 and VEGF-A. We then injected shTPM1 or shVEGF-A into the vitreous cavity of a mouse CNV model, respectively, and simultaneously stained the choroidal CNV, labeling new blood vessels with IB4, macrophages / microglia with Iba1, and fibroblasts with α-SMA. Figure 4 The results showed that, compared with the control group, the IB4 positive area in the shTPM1 group was reduced by approximately 30%, and the IB4 positive area in the shVEGF-A group was reduced by approximately 35.2%, indicating that both TPM1 and VEGF-A inhibition could suppress CNV angiogenesis. Specifically, the Iba1 and α-SMA signal intensities in the shVEGF-A group were reduced by approximately 7.9% and 6.1% respectively compared with the control group, while the Iba1 signal in the shTPM1 group was reduced by approximately 51% and the α-SMA signal intensity was reduced by approximately 51.1% compared with the control group, showing significant differences. Figure 4 (CE in the text). In summary, these data indicate that knocking down TPM1 in the mouse CNV model not only inhibits angiogenesis itself, but also more effectively reduces the occurrence and development of inflammation and fibrosis.

[0078] 5. To investigate the specific cell types in which TPM1 plays a role in wAMD, we analyzed a single-cell RNA sequencing dataset of choroidal tissue from existing wAMD patients (GSE135922). We found that TPM1 was specifically highly expressed in a pericyte subset in CNV lesions, suggesting that TPM1 may primarily function in pericytes. Figure 5A-B). Therefore, we performed functional experiments to verify the role of TPM1 in human brain vascular pericytes (HBVPs) after knocking down TPM1 in HBVPs. The CCK-8 experiment results showed that TPM1 knockdown significantly inhibited the proliferation ability of HBVPs compared with the control group (Fig. 1C); the scratch test further confirmed that TPM1 knockdown led to a significant decrease in the migration ability of HBVPs (Fig. 1D-E); the above results showed that TPM1 played an important role in maintaining the proliferation and migration of pericytes, suggesting that it might affect the function of pericytes in the pathological process of wAMD, and thus participate in the formation of the mechanism of anti-VEGF therapy non-response. Figure 5 Figure 5

[0079] The above is only a preferred embodiment of the present application, and it should be pointed out that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined by the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.​​

Claims

1. Use of a TPM1 inhibitor for the manufacture of a medicament for the treatment of wet age-related macular degeneration, characterized in that, The TPM1 inhibitor is an shRNA against TPM1 the gene whose sequence is shown as SEQ ID NO. 1-2.

2. Use according to claim 1, characterized in that, The TPM1 inhibitor can inhibit ocular neovascularization, reduce the degree of inflammation and fibrosis in the eye.

3. Use according to claim 1 or 2, characterized in that, The drug is an ophthalmic preparation.

4. Use according to claim 3, characterized in that, The ophthalmic preparation includes eye drops, eye sprays, eye gels, eye patches, intraocular injections, eye microspheres, eye implants, and periocular injections.

Images