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

Abstract

The invention discloses application of TPM1 in treatment of wet age-related macular degeneration. Research finds that TPM1 promotes angiogenesis, inflammation and fibrosis in eyes. The anti-angiogenesis effect of inhibiting TPM1 is similar to that of inhibiting VEGF-A, but the effect of inhibiting TPM1 is more remarkable in the aspect of reducing inflammation and fibrosis, so that the compound can be used as a potential target for treating wAMD. In addition, single cell sequencing finds that the TPM1 has high specific expression in CNV focus peripheral cells of a wAMD patient; functional experiments prove that the TPM1 is knocked down to significantly inhibit periodontocyte proliferation and migration, which prompts that the TPM1 participates in wAMD progress by regulating periodontocyte functions and does not respond to anti-VEGF treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of TPM1 in the treatment of wet age-related macular degeneration. Background Technology

[0002] Based on clinical and pathological characteristics, age-related macular degeneration (AMD) can be divided into two main subtypes: dry (atrophic) and wet (neovascular). While wet AMD (wAMD) accounts for only about 10%-15% of all AMD cases, it causes severe vision loss in over 80% of cases. The typical pathological manifestation of wAMD is the breaching of Bruch's membrane by abnormal neovascularization originating from the choroid, forming choroidal neovascularization (CNV) under the retina. These neovascularizations are fragile, highly permeable, and prone to leakage and hemorrhage, directly causing an acute decline in central vision.

[0003] Extensive evidence suggests that vascular endothelial growth factor (VEGF) is a key molecular driver of CNV formation and leakage. Based on this, anti-VEGF therapy has become a first-line treatment strategy for wAMD, effectively inhibiting neovascularization and reducing leakage, thereby stabilizing or improving vision in most patients. However, approximately 10% of wAMD patients do not respond to initial anti-VEGF therapy; in long-term follow-up, about half of these patients still experience significant vision loss within 5 years, often associated with CNV persistence, atrophy progression, and fibrotic scar formation. This suggests that anti-VEGF tolerance / non-responsiveness has a complex pathological basis, necessitating the identification and elucidation of key molecular and cellular mechanisms involved in this process to open up new therapeutic pathways.

[0004] Current research suggests that non-responsiveness to anti-VEGF is driven by multiple factors, among which pericytes, as important supporting cells of the capillary wall, coat the surface of endothelial cells and provide structural and survival signals. They can, to some extent, "armor" new blood vessels, protecting endothelial cells from clearance by anti-angiogenic therapy. Pericyll-mediated support allows endothelial cells to survive under relatively low VEGF conditions, thereby reducing the efficacy of anti-VEGF therapy.

[0005] In a mouse laser-induced CNV model, our combined transcriptomic and proteomic analysis revealed that tropomyosin-1 (TPM1) was significantly upregulated after CNV formation; TPM1 expression was significantly increased in the mouse RPE / choroid complex on day 3 after CNV. Notably, TPM1 remained upregulated even after anti-VEGF intervention, suggesting that it may participate in the regulation of CNV course and treatment response 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 both muscles and non-muscle cells. During the maturation of pericytes into a contractile phenotype, smooth muscle-related contractile proteins (including actin, myosin, and tropomyosin) are highly expressed, conferring regulation and stability of vascular wall tension.

[0007] Previous studies have reported that circulating TPM1 possesses immunomodulatory properties, inducing endogenous TPM1 expression in the aging retina by promoting PKA phosphorylation and activating the FABP5 / NF-κB signaling pathway. As a key blood-derived pro-aging factor, TPM1 can induce increased microglial activation in a mouse Alzheimer's disease model, thereby triggering optic nerve remodeling and leading to age-related visual function decline, suggesting its potential as an anti-aging intervention target. Furthermore, TPM1 can also regulate LPS-mediated neuroinflammatory responses and neuronal death by promoting microglial activation. However, the aforementioned reports have not mentioned the effects of TPM1 on angiogenesis and fibrosis.

[0008] To date, no studies have reported on the specific function of TPM1 in the development and progression of wAMD. Similarly, no direct evidence has been reported regarding its involvement in the process of developing tolerance to anti-VEGF therapy. Furthermore, systematic research on the cell-specific functions of TPM1 in different vascular cell subsets (such as endothelial cells, pericytes, and smooth muscle cells) remains lacking. Summary of the Invention

[0009] This invention is the first to discover inhibition or silencing. TPM1 The gene can inhibit ocular neovascularization, reduce ocular inflammation and fibrosis, suggesting that TPM1 may be a potential target for the treatment of wet age-related macular degeneration.

[0010] Therefore, the first objective of this invention is to provide the use of TPM1 inhibitors in the preparation of medicaments for treating wet age-related macular degeneration.

[0011] Preferably, the TPM1 inhibitor can inhibit ocular neovascularization, reduce ocular inflammation, and decrease the degree of fibrosis.

[0012] Preferably, the TPM1 inhibitor is an inhibitor or a silencer. TPM1 Gene reagents.

[0013] Preferably, the suppression or silencing TPM1 Gene-related agents include methods such as RNA interference, gene knockout, chemical repression, miRNA-mediated silencing, transcription factor repression, or epigenetic modification to suppress or silence genes. TPM1 Gene reagents.

[0014] Preferably, the RNA interference reagent includes those targeting... TPM1 The gene's siRNA, dsRNA, or shRNA.

[0015] Preferably, the target TPM1 The shRNA sequence of the gene is shown in SEQ ID NO. 1-2; the target TPM1 The sequence of the gene's siRNA is shown in SEQ ID NO.11.

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

[0017] Preferably, the ophthalmic preparations include eye drops, eye ointments, eye sprays, ophthalmic gels, eye patches, intraocular injections, ophthalmic microspheres, ophthalmic implants, periocular injections, and sustained-release ophthalmic preparations.

[0018] The second objective of this invention is to provide a drug for treating wet age-related macular degeneration, comprising a TPM1 inhibitor as the active ingredient.

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

[0020] This study found that TPM1 promotes angiogenesis, inflammation, and fibrosis in the eye. Inhibition of TPM1 showed similar anti-angiogenic effects to inhibition of VEGF-A, but its effect in reducing inflammation and fibrosis was more significant, thus making it a potential target for the treatment of wAMD. Furthermore, single-cell sequencing revealed that TPM1 was specifically highly expressed in pericytes of CNV lesions in wAMD patients; functional experiments confirmed that knockdown of TPM1 significantly inhibited pericyte proliferation and migration, suggesting that it participates in wAMD progression and unresponsiveness to anti-VEGF therapy by regulating pericyte function. Attached Figure Description

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

[0022] Figure 2 To construct a laser-induced CNV model and detect TPM1 expression in the choroid on day 3 post-surgery. (A) is a schematic diagram of the CNV model. (BC) shows the TPM1 expression level in the choroid of the CNV model on day 3 post-surgery, detected by Western blot and qPCR (n=3). All data are presented as mean ± standard deviation (SD). *: p<0.05, ***: p<0.001.

[0023] Figure 3 To construct a laser-induced CNV model and detect choroidal TPM1 expression on day 7 after intraocular injection of shVEGF-A. (A) is a schematic diagram of the CNV model. (BC) show Western blot and qPCR detection of choroidal TPM1 expression on day 7 after intraocular injection of shVEGF-A in CNV model mice (n=4). All data are presented as mean ± standard deviation (SD). *: p<0.05, ***: p<0.001.

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

[0025] Figure 5 This study examines the functional changes of HBVP after TPM1 knockdown in primary human cerebral perivascular cells (HBVP). (AB) shows single-cell RNA sequencing analysis of choroidal tissue from wAMD patients. (C) shows quantitative CCK-8 analysis 72 h after TPM1 knockdown in HBVP (n=3). (DE) shows quantitative analysis of HBVP scratch patterns and healing rates at 0 h, 24 h, and 48 h after TPM1 knockdown (n=3). Scale bar: 200 μm. All data are presented as mean ± standard deviation (SD). *: p<0.05, ***: p<0.001. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof.

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

[0028] Example 1 I. Materials 1) Plasmid The shRNAs targeting TPM1 and VEGF-A were designed using pLKO.1 as a vector via the Sigma website.

[0029] Tsingke Biotechnology synthesized the designed sequences and ligated them to the pLKO.1 vector using T4 DNA ligase. All plasmids were transformed with DH5α and extracted using a plasmid extraction kit (TIANGEN, DP117TA).

[0030] Table 1 shRNA sequence information .

[0031] 2) Proteins, antibodies, and primers Table 2 Proteins and Antibodies .

[0032] Table 3 Primers .

[0033] Table 4 siRNA oligo sequences .

[0034] II. Methods 1. Laser-induced CNV mouse model 1) Within an area 1.5–2 mm in diameter from the optical disc, four laser photocoagulation spots (spot size 75 μm, power 90 mw, duration 75 ms) were applied to the eyes of adult mice. Laser photocoagulation was performed at positions 12, 3, 6, and 9, avoiding large retinal vessels; 2) After laser photocoagulation, inject a complex containing shTPM1, shVEGF-A or shNC plasmid-PEI (1µg / µL) into the vitreous cavity. 3) Eyes were collected on day 7. The eyeballs were fixed in 4% PFA for 2 hours. The cornea, lens, lenticule, and vitreous humor were removed, and the choroid-RPE complex was separated. The following experiments were performed: 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. It was then incubated overnight at 4°C with allogeneic proteins GS-IB4, α-SMA, and Iba-1 (1:100) bound to primary antibody Alexa Fluor 488 (1:500). The next day, after rinsing with PBS, it was incubated with the corresponding species' secondary antibody at room temperature for 2 hours. The choroid-RPE complex was then rinsed with PBS and laid flat with the RPE side facing up. Images were taken using a fluorescence microscope, and the CNV region was analyzed using image analysis software.

[0035] 2. RNA isolation and extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR) Mouse choroidal tissue was collected, and total RNA was isolated from cells using TRIzol reagent (TIANGEN). cDNA was synthesized from this RNA using the DNase-containing FastKing RT kit (TIANGEN), and qRT-PCR was performed using a SYBR Green (ROCHE) and ABI QuantStudio 6 Flex instrument (Life Technologies). Results were normalized to GAPDH transcripts. Relative folding changes in gene expression were calculated using the delta-delta Ct method.

[0036] 3. Western blot experiment 1) Preparing liquids: ①10×Running Buffer: Add 144 g glycine, 10 g SDS powder, and 30.3 g Tris powder sequentially, using ddH2O as the solvent, and bring the volume to 1 L to prepare a storage solution. Dilute to 1×Running Buffer to prepare the electrophoresis working solution for use. ②5×SDS Loading Buffer: Add 4 g of SDS powder, 20 mg of bromophenol blue, 3.085 g of DTT, 10 mL of Tris-HCl (1M pH 6.8), and 20 mL of glycerol in sequence, and use ddH2O as a solvent to make up to 40 mL. ③ 10× Transfer Buffer: Add 30.3 g Tris powder and 144 g glycine sequentially, using ddH2O as the solvent, and bring the volume to 1 L to prepare a storage solution. Dilute with 10× Transfer Buffer 100 mL + methanol 200 mL + ddH2O 700 mL to prepare 1× Transfer Buffer as the working solution for use. 2) Preparation of separating gel and stacking gel: ① Prepare 10% separating gel (10 mL): Table 5 shows the preparation of 10% separating gel. .

[0037] ②Prepare a 5% concentrate (5 mL): Table 6 shows the preparation of 5% concentrated gel. .

[0038] 3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution to the tank. Check for leakage. Remove the comb and add the marker and sample to the gel wells in sequence. Add electrophoresis solution to the electrophoresis tank and set the program to 80 V for 30 min constant voltage electrophoresis. Then switch to 120 V for 1 h constant voltage electrophoresis. 4) Transfer: First, activate the PVDF membrane with methanol, and remove the gel from step 3). Assemble the transfer clamps in a "sandwich" structure, taking care to avoid leaving air bubbles during assembly. Assemble the transfer apparatus, add the transfer working solution, set the program to a constant current of 250 mA for 2 h, and place it on ice for transfer. 5) Blocking: Take out the membrane after the transfer is completed, add 1×TBST and place it on a shaker at room temperature for 5 min to clean it, discard it, add 5% skim milk as the blocking solution and place it on a shaker at room temperature for blocking for 1 h. 6) Primary antibody incubation: Discard the blocking solution, add 1×TBST and place on a shaker at room temperature to wash until the blocking solution is clean. Discard the liquid, add the primary antibody prepared with 5% BSA as antibody dilution solution, and incubate overnight on a shaker in a 4°C cold storage. 7) Membrane washing: Wash the membrane three times with 1×TBST, 10 min each time; 8) Secondary antibody incubation: Discard the solution, add secondary antibody prepared with 5% skim milk at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 h; 9) Same as step 7). 10) Exposure and development: Prepare the developer (A solution: B solution = 1:1 ratio, prepare and use immediately). Place the film on the plate, add developer to the film, shake well, expose and develop on the machine, and save the results.

[0039] 4. CCK-8 cell proliferation experiment HBVP at 5×10 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight. 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 medium, and CCK8 assay reagent was added at a rate of 10 μL / well. The cells were then mixed and returned to 37°C for another h of culture. The wavelength at 450 nm was then measured using a microplate reader. The ratio of the experimental group to the control group was calculated as the statistical result.

[0040] 5. Scratch test HBVP at 1.5×10 5 Inoculated at a density of cells / well in 6-well plates, and after reaching approximately 85% confluence the following day, transfection experiments with siTPM1 and si-Control were performed. After 24 h, the medium was replaced with serum-free PM medium, and mitomycin C (1 µM) was added for 1 h. Using a 200 μL pipette tip, scratches were made at the center of the wells. The medium was discarded, the plates were washed once, and fresh serum-free PM medium was added. The scratch status was photographed and recorded at 0 h, and again at 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).

[0041] III. Results 1. To explore novel targets in the process of angiogenesis, we constructed a laser-induced mouse choroidal angiogenesis (CNV) model and performed RNA sequencing. Sequencing analysis revealed that TPM1 is upregulated after CNV formation. Figure 1 (AB in the middle).

[0042] 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).

[0043] 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.

[0044] 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.

[0045] 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 5 Therefore, we knocked down TPM1 in human brain perivascular cells (HBVP) and performed functional experiments to verify this. CCK-8 assay results showed that, compared with the control group, TPM1 knockdown significantly inhibited the proliferation of HBVP (AB). Figure 5 (C in the text); Scratch assays further confirmed that TPM1 knockdown significantly reduced HBVP migration ability ( Figure 5 The results indicate that TPM1 plays an important role in maintaining pericyte proliferation and migration, suggesting that it may affect pericyte function during wAMD pathology and thus participate in the formation of the anti-VEGF therapy non-response mechanism.

[0046] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of TPM1 inhibitors in the preparation of drugs for the treatment of wet age-related macular degeneration.

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

3. The application according to claim 1, characterized in that, The TPM1 inhibitor is used to inhibit or silence the TPM1. TPM1 Gene reagents.

4. The application according to claim 3, characterized in that, The suppression or silence TPM1 Gene-related agents include methods such as RNA interference, gene knockout, chemical repression, miRNA-mediated silencing, transcription factor repression, or epigenetic modification to suppress or silence genes. TPM1 Gene reagents.

5. The application according to claim 4, characterized in that, The RNA interference reagents include those targeting TPM1 The gene's siRNA, dsRNA, or shRNA.

6. The application according to claim 5, characterized in that, The target TPM1 The shRNA sequence of the gene is shown in SEQ ID NO. 1-2; the target TPM1 The sequence of the gene's siRNA is shown in SEQ ID NO.

11.

7. The application according to any one of claims 1-6, characterized in that, The drug is an ophthalmic preparation.

8. The application according to claim 7, characterized in that, The ophthalmic preparations include eye drops, eye ointments, eye sprays, ophthalmic gels, eye patches, intraocular injections, ophthalmic microspheres, ophthalmic implants, periocular injections, and sustained-release ophthalmic preparations.

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