Application of N-acetylneuraminic acid pyruvate lyase regulating agent in preparation of blood vessel or lymphatic vessel cavity regulating medicine

Abstract

The invention discovers that N-Acetylneuraminic acid pyruvate lyase (N-Acetylneuraminic acid pyruvate lyase, NPL) is highly related to the neogenesis and development of vascular or lymphatic endothelial cells for the first time. Research results show that up-regulation of expression of N-acetylneuraminic acid pyruvate lyase can promote cavitalization of blood vessels or lymphatic vessels and relieve lumen stenosis; by down-regulating the expression of N-acetylneuraminic acid pyruvate lyase or inhibiting the activity of N-acetylneuraminic acid pyruvate lyase, cavitalization of blood vessels or lymphatic vessels can be inhibited, so that the blood vessels are closed or even deleted. Therefore, the N-acetylneuraminic acid pyruvate lyase can be used as a treatment target of the lumen stenosis disease and the hyperplasia of the blood vessel to guide the development of related medicines.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to the application of N-acetylneuraminic acid pyruvate lyase regulator in the preparation of vascular or lymphatic vessel lumen regulation drugs. Background Art

[0002] Endothelial cells constitute important material circulation channels in the body, among which vascular endothelial cells and lymphatic endothelial cells are the two most important types of endothelial cells, which are the structural basis of blood circulation and lymphatic circulation in the body. The formation of the endothelial cell network and the stability of its function play an important role in the maintenance of the circulatory system and the immune system. The establishment of the endothelial network system includes the differentiation, migration, polarization, and luminalization of endothelial cells, among which luminalization is the basis for the functionality of endothelial cells. The material basis for the luminalization of endothelial cells is the polarization of endothelial cells and the formation of the luminal glycocalyx layer. The glycocalyx layer contains a large number of polysialic acid chains and glycoproteins and glycosaminoglycans with terminal sialic acid modifications, which make the luminal side of endothelial cells negatively charged, forming electrostatic repulsion on the luminal side, and plays a decisive role in maintaining the luminal structure and corresponding functions of endothelial cells. The correct glycosylation and sialylation of the luminal surface membrane proteins are the basis for maintaining correct cell molecular recognition and maintaining the permeability of endothelial cells.

[0003] Abnormal cavitation of endothelial cells often causes different types of diseases depending on the tissue where the lesions occur. For example, congenital or chemotherapy-induced intracranial carotid artery stenosis can lead to ischemic stroke; renal artery stenosis caused by factors such as aging can lead to hypertension; diabetes is accompanied by excessive angiogenesis, abnormal proliferation of retinal endothelial cells, and cavitation malformations, often causing varying degrees of visual impairment; the growth of tumor tissues and the migration of tumor cells are accompanied by the regeneration of lymphatic vessels, and the regeneration of lymphatic vessels in tumor tissues promotes tumor metastasis. Therefore, effective regulation of the regeneration and cavitation of blood vessels and lymphatic vessels has important clinical significance for the treatment of diseases such as cranial artery stenosis, renal artery hypertension, diabetes, and solid tumors.

[0004] There is a serious lack of existing specific targeted drugs for endothelial cells, especially drugs for regulating the luminalization process of newborn endothelial cells. The purpose of the present invention is to provide such specific targeted genes and drugs, and to provide drugs with corresponding regulatory activity in vivo. Summary of the invention

[0005] The present invention provides a method for regulating endothelial cell regeneration and luminalization by regulating the expression level of the npl gene or the level of its corresponding protein product. The specific technical scheme of the present invention is as follows: Application of N-acetylneuraminic acid pyruvate lyase regulator in the preparation of vascular or lymphatic vessel lumen regulation drugs.

[0006] The N-acetylneuraminic acid pyruvate lyase of the present invention has a sequence as shown in the npl gene (Gene ID: 100148292, NCBI Reference Sequence: NM_207051.2) in the current international public nucleic acid database GeneBank. The full-length NPL nucleotide sequence or its fragment of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0007] The N-acetylneuraminic acid pyruvate lyase regulator of the present invention is a positive regulator or a negative regulator of N-acetylneuraminic acid pyruvate lyase or its encoding gene.

[0008] Specifically, the positive regulator is N-acetylneuraminic acid pyruvate lyase or its expression product or its expression vector; the negative regulator includes one or more of protein inhibitors targeting N-acetylneuraminic acid pyruvate lyase, nucleic acid aptamers, interfering RNA of the N-acetylneuraminic acid pyruvate lyase encoding gene, gRNA, microRNA, gene knockdown system, and small molecule compound inhibitors.

[0009] In a specific example of the present invention, the positive regulator is an expression vector of N-acetylneuraminic acid pyruvate lyase; the negative regulator is a knockdown system of the N-acetylneuraminic acid pyruvate lyase encoding gene, Cas9+gRNA using gene editing technology, and a small molecule compound inhibitor chelidonic acid (CAS No.: 99-32-1).

[0010] The cavitation regulating drug of the present invention is a drug that promotes or inhibits the cavitation of blood vessels or lymphatic vessels. The positive regulator of N-acetylneuraminic acid pyruvate lyase or its encoding gene is used to treat luminal stenosis diseases, such as cranial artery or carotid artery stenosis and ischemic stroke caused by it, and renal artery stenosis and hypertension caused by it, by promoting the cavitation of blood vessels or lymphatic vessels. The negative regulator of N-acetylneuraminic acid pyruvate lyase or its encoding gene is used to treat vascular hyperproliferative diseases, such as excessive tissue angiogenesis caused by diabetes, abnormal proliferation and cavitation malformation of retinal endothelial cells, and lymphangiogenesis caused by tumors by inhibiting the cavitation of blood vessels or lymphatic vessels.

[0011] Advantages of the present invention: The present invention experimentally proves that the npl gene is specifically expressed in developing endothelial cells. Single-cell RNAseq results show that in three species, humans, mice and zebrafish, the transcription level of npl in developing vascular and lymphatic endothelial cells is high, while npl transcription is not detected in the vast majority of mature vascular and lymphatic endothelial cells. Cell metabolic pathway enrichment analysis of npl-positive cells shows that such cells are in the cell nascent stage.

[0012] Normal intracellular sialic acid synthesis is the material basis for the sialylation of intracellular functional molecules. There are two pathways for the synthesis of sialic acid in cells: one is to use UDP-GlcNAc (UDP-N-acetylglucosamine) as a substrate, and catalyze the production of ManNAc (N-acetyl-D-mannosamine) and ManNAc-6-P (6-acetyl-D-mannosamine phosphate) under the action of GNE (UDP-N-acetylglucosamine-2-epimerase / N-acetylmannosamine kinase). ManNAc-6-P and PEP (phosphoenolpyruvate) synthesize Neu5Ac-9-P (9-phosphate N-acetylneuraminic acid) under the action of NANS (N-acetylneuraminic acid phosphate synthase), and further dephosphorylate by NANP (N-acylneuraminic acid-9-phosphatase) to obtain Neu5Ac (N-acetylneuraminic acid), i.e., sialic acid. The other is to use ManNAc as a substrate and directly synthesize Neu5Ac with PE (pyruvate) under the catalysis of the reversible enzyme NPL (N-acetylneuraminic acid pyruvate lyase).

[0013] The experiments of the present invention prove that the npl cavitation process in the new blood vessel or lymphatic endothelial cells is only related to the NPL-mediated sialic acid synthesis pathway, while the related genes that mediate the conversion of UDP-GlcNAc (UDP-N-acetylglucosamine) to Neu5Ac via ManNAc (N-acetyl-D-mannosamine), such as gne, nansa, nanp, and cmasa, are basically not expressed, suggesting that the NPL protein or npl gene is a specific target for regulating the development of new blood vessels or lymphatic vessels.

[0014] The experiments of the present invention prove that knocking out and knocking down the zebrafish npl gene can lead to vascular lumen occlusion and delayed lymphatic vessel development in zebrafish. The NPL blocker chelidonic acid inhibits the cavitation of new blood vessels by inhibiting the activity of NPL protein. Therefore, based on the above research results, the hyperproliferative vascular lesions can be treated by inhibiting the activity of npl gene or NPL protein.

[0015] The present invention proves through experiments that upregulating the expression of npl gene in endothelial cells can increase the diameter of the vascular lumen. Therefore, based on the above research results, the lumen of blood vessels or lymphatic vessels can be promoted by promoting the expression of npl gene or increasing the activity of NPL protein, which can be used to treat lumen stenosis diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Single-cell RNA-Seq data analysis results of npl during endothelial cell development.

[0017] A shows the expression of npl in juvenile and adult human, mouse and zebrafish endothelial cells; B shows the results of cell pathway enrichment analysis of npl-positive cells; C shows the expression of enzyme genes related to the sialic acid synthesis pathway in npl-positive cells.

[0018] Figure 2 Transcription of npl gene during endothelial cell development.

[0019] Figure 3 Effects of inhibiting npl expression on vascular development. A is the laser confocal microscopic imaging result of blood vessels at 48hpf and 72hpf after Mo injection. B is the statistical result of blood vessel length at 48hpf and 72hpf after Mo injection. C is the confocal imaging result of blood vessels after Mo injection and nplmRNA complementation, and development to 48hpf.

[0020] Figure 4 Laser confocal microscopy imaging and statistical results of the effects of different N-acetylglucosides replenishment on lymphatic vessel development after Mo treatment.

[0021] Figure 5 Effect of knocking out the npl gene in the zebrafish genome on vascular lumen development. A is a comparison of the homology of the NPL protein domains of humans (NP_110396.1), mice (NP_083025.1) and zebrafish (NP_996934.2). B is the design of the targeting sequence gRNA and the sequencing results and translation mutation results of the -16bp homozygous mutant individuals obtained after gene knockout. C is the laser confocal microscopy imaging and statistical results of the blood vessels after knocking out the npl gene in the zebrafish genome.

[0022] Figure 6 Effects of chelidonic acid (CA), a small molecule blocker of NPL, on endothelial cell development.

[0023] Figure 7 Effects of overexpression of the npl gene in zebrafish vascular endothelial cells on blood vessels. A is the imaging result of zebrafish embryos with chimeric expression of npl vascular endothelial cells at 72 hpf. B is the imaging result of zebrafish embryos with chimeric expression of npl vascular endothelial cells at 5 dpf. DETAILED DESCRIPTION

[0024] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0025] Example 1 The npl gene is specifically expressed during endothelial cell development This example uses single-cell RNAseq analysis and zebrafish in situ hybridization technology to confirm that the npl gene is specifically expressed during endothelial cell development. The results are as follows Figure 1 , 2 shown.

[0026] The single cell data sources refer to GSE134355 and GSE198832. Cell dimensionality reduction analysis and specific gene expression level analysis were performed using R language, and further GO pathway enrichment analysis was performed to obtain the metabolic pathway enrichment analysis of npl-positive cells. Figure 1 shown. Figure 1 A is the result of RNA-Seq data analysis, showing that the expression level and expression ratio of npl gene are low in mature endothelial cells of human, mouse and zebrafish; while in lymphatic endothelial cells and / or vascular endothelial cells at the developmental stage, npl gene is expressed at a high ratio and high level. The results of pathway enrichment analysis of cells with upregulated npl gene are shown in Figure 2. Figure 1 B shows that the metabolic pathways of npl-positive cells are enriched in endothelial cell regeneration-related pathways, and these cells are in the endothelial cell regeneration stage.

[0027] In order to study the relationship between endothelial cell development and sialic acid synthesis, the cell expression characteristics of npl-positive cells were further analyzed. The results showed that during the luminalization of endothelial cells, genes related to the conversion of UDP-GlcNAc (UDP-N-acetylglucosamine) to Neu5Ac via ManNAc (N-acetyl-D-mannosamine), such as gne, nansa, nanp, and cmasa, were basically not expressed, suggesting that the luminalization of endothelial cells is only related to the sialic acid synthesis pathway mediated by NPL.

[0028] Specific probe primers were designed for the mRNA sequence of the zebrafish npl gene, and the probe fragment was amplified and linked to the pGEM-T vector. After the specific restriction enzyme was selected for digestion, the DIG-labeled probe was obtained by in vitro transcription. The probe was used for zebrafish in situ hybridization experiments, and the results were as follows: Figure 2 As shown, the results showed that at different developmental stages of zebrafish, npl was expressed in the early stages of endothelial cell development, and only a small proportion of mature endothelial cells expressed npl.

[0029] Example 2 Effect of inhibiting npl expression on vascular development Morpholino (hereinafter referred to as Mo, synthesized by Gene Tools, working concentration 8μg / μL, sequence 5'-CGCGTTGAGACATTTCTTCCTTTCA-3') that inhibits npl translation was treated at 65℃ for 10 min before use. About 3nl was injected into the one-cell stage Tg(fli1ep:EGFP-CAAX) zebrafish fertilized eggs, and the treated embryos and the control group were cultured at 28.5℃. Ten embryos were randomly selected at 48hpf and 72hpf, respectively, and after being anesthetized with MS222, they were fixed in low-melting point agarose gel, and the vascular development was photographed under a laser confocal microscope. The results are shown in Figure 3 shown. Figure 3 A is the microscopic imaging result of vascular endothelial cell development at 48hpf and 72hpf after Mo injection. Figure 3 B is the statistical result of blood vessel length at 48hpf and 72hpf after Mo injection. The results showed that injection of Mo delayed blood vessel development by inhibiting npl translation. The blood vessel length at 48 hours was shorter than that in the control group. After 72 hours of development, there was no significant difference in blood vessel length between the control group and the control group, but there were defects in vascular lumen development, and the number of lumen stenosis was significantly different from that in the control group (p<0.001). The results showed that inhibiting the transcription of npl during development shortened the length of intersegmental vessels (ISV) and inhibited lumen development.

[0030] The coding sequence of the amplified npl gene was connected to the pCS2+ vector. After the vector was linearized with restriction endonucleases, the in vitro transcription kit (mMESSAGE mMACHINE TM SP6 transcription kit, AM1340, Invitrogen TM ) was transcribed to obtain npl mRNA fragments. npl mRNA (300ng / μL) was co-injected with Mo and cultured at 28.5℃. When the embryos developed to 48hpf, the vascular development was photographed using a laser confocal microscope. The experimental results showed that npl mRNA complementation could rescue the inhibitory effect of npl Mo knockdown on endothelial cell development.

[0031] The above experimental results suggest that knocking down the expression of npl can cause delayed vascular lumen development.

[0032] Example 3 Effect of inhibiting npl expression on lymphatic vessel development The experimental method of using Mo to knock down zebrafish npl is as described in Example 2, and the treated fish line is Tg (kdrl:ras-mCherry::lyve1b:topza-YFP). The Mo-injected embryos were randomly divided into 4 groups, with 10 embryos in each group. Starting from 8 hours, they were treated with 0.8mM N-acetyl-neuraminic acid (NeuAc, i.e. sialic acid, purchased from sigma, item number A2388) or the related intermediate product of sialic acid step-by-step synthesis N-acetyl-D-glucosamine (GlcNAc, purchased from sigma, item number A8625) or N-acetyl-D-mannosamine (ManNAc, purchased from sigma, item number A8176), respectively. The new treatment solution was replaced every 12 hours to carry out a rescue experiment. On the 5th day, laser confocal microscopy was used to photograph the development of lymphatic vessels to evaluate the effects of Mo treatment and different N-acetylglucosides immersion treatments on lymphatic vessel development. Figure 4 As shown, the experimental results showed that Mo treatment significantly delayed the formation of lymphatic vessels (p<0.001). Among the three N-acetylated sugars, only the supplementation of sialic acid (NeuAc) had a rescue effect on the delayed lymphatic vessel development caused by Mo, mainly manifested in the significant increase in the migration of the thoracic duct (TD) and the development of the intersegmental lymphatic vessels (ISLV) compared with the Mo-treated group (p<0.005 and p<0.001). The supplementation of N-acetyl-D-glucosamine or N-acetyl-D-mannosamine (another metabolite in the sialic acid synthesis pathway) could not rescue the delayed lymphatic vessel development phenotype caused by npl deficiency. The above results suggest that knocking down the expression of npl can cause delayed lymphatic vessel lumen development.

[0033] Example 4 Effect of knocking out npl on angiogenesis NPL protein is highly conserved in humans, mice and zebrafish. Based on the results of protein conservation analysis and protein domain function prediction, the gene sequence corresponding to the substrate binding position of NPL protein was selected, and a specific targeting gRNA (5'-GTTAATGGC ACAACAGGTGA-3'), which was synthesized and mixed with in vitro transcribed mRNA of cas9 (the transcription steps of Cas9 mRNA were the same as those of npl mRNA in Example 3, cas9 mRNA was 300 ng / μL, and the final concentration of gRNA was 100 ng / μL) and then co-injected into one-cell stage zebrafish Tg (fli1ep:EGFP-CAAX::gata1:dsRed) fertilized eggs. After being raised to adult fish, the tail fin was cut and the tissue was lysed (DNA-EZ Reagents V universal one-step DNA extraction solution, B642315-0010, Shanghai Bioengineering), PCR technology was used to amplify the mutation region (amplification primers: npl-F: 5'-AAATGCTTTACAGAATCATGCCT-3', npl-R: 5'-CGTGTGGTTTTAGGTTAGAATACAG-3', Tm=53°C, product length is about 500bp), connected to the pUCm-T vector (B620433-0001, Shanghai Bioengineering) and sequenced to identify the -16bp heterozygous mutant individual. After the mutant genotype was inbred, the offspring was further identified to obtain homozygous mutant individuals. After the homozygotes were inbred, the embryos were collected, cultured at 28.5°C, and laser confocal photography was performed at 48hpf. The implementation process is as described in Example 2. The experimental results are shown in Figure 5 As shown, Figure 5 A is a comparison of the homology of NPL proteins in humans (NP_110396.1), mice (NP_083025.1) and zebrafish (NP_996934.2). The results show that the functional domains of NPL are well conserved among the three species. The TT position of the sequence in the dotted box is the substrate binding position, which is highly conserved among the three species. The conserved sequence corresponds to the fourth exon of the npl mRNA coding region. Figure 5 B is the gRNA designed to target the exon position, and the sequence is the WT (wildtype) sequence underlined in black. The sequencing results of the mutant showed that the relevant sequence of the substrate binding site was missing after the mutation, and a frameshift mutation was caused. Figure 5 C laser confocal microscopy imaging and statistical results showed that although the length of intersegmental vessels in mutant zebrafish with npl knockout was not significantly different from that in the control group (p=0.41), the lumen variation range of intersegmental vessels in mutant zebrafish was significantly different from that in the control group, and some vessels had intermediate closure (p<0.005). The above results suggest that knockout of the npl gene can lead to delayed development of new blood vessels and narrow lumen.

[0034] Example 5 Effect of npl negative regulator on angiogenesis Chelidonic acid (CA) is a specific inhibitor of NPL. In this example, 0.1 mg / mL, 0.2 mg / mL and 0.5 mg / mL CA (382272, Sigma) were used to immerse zebrafish embryos of Tg (fli1ep:EGFP-CAAX) and Tg (kdrl:ras-mCherry::lyve1b:topza-YFP) that developed to 24 hpf. The inhibitory effects were observed at 48 hpf and 5 dpf, respectively. The results are shown in the figure. Figure 6 As shown. Compared with the control group, with the increase of chelidonic acid concentration, the length of ISV did not change significantly, but the number of ISVs with closed lumen increased significantly. When the CA concentration was 0.5 mg / mL, the longitudinal growth of ISV was significantly inhibited. When the CA concentration increased to 5 dpf, the developmental inhibition of lymphatic vessel differentiation and migration was dose-dependent. When the concentration was greater than 0.2 mg / mL, the separation of DA and PV could be inhibited, resulting in the delay of lymphatic vessel development. Laser confocal imaging results showed that CA could inhibit the cavitation of new blood vessels in zebrafish larvae and inhibit the differentiation and migration of lymphatic vessels, and it was concentration-dependent.

[0035] Example 6 Effect of overexpression of npl gene on neovascularization morphology The npl coding sequence on the pCS-npl plasmid constructed in Example 2 was used to replace the EGFP-CAAX segment on the existing fli1ep:EGFP-CAAX-T2A-nls-mCherry plasmid to obtain a plasmid for overexpressing npl in endothelial cells, named fli1ep:npl-T2A-nls-mCherry. nls-mCherry marks the nuclei of cells overexpressing npl in red. The plasmid (final concentration 75ng / uL) was mixed with transposase mRNA (final concentration 50ng / μL) and injected into Tg (fli1ep:EGFP-CAAX) one-cell stage fertilized eggs. The injection volume was about 3nl. The treated embryos were cultured at 28.5°C, and the juveniles with red endothelial cell nuclei were picked at 72hpf and 5dpf, and the vascular development status was photographed using laser confocal microscopy. The results are shown in Figure 7 shown. Figure 7A is the imaging result of zebrafish embryos with chimeric expression of npl vascular endothelial cells that developed to 72 hpf. a and b are cross-sectional views of trunk blood vessels. a is a control cross-sectional view. The nucleus of the cell indicated by the arrow in b is red, which is an npl overexpressing cell. The vascular diameter of the PV in b is larger than that in a. c is a local magnification of the intersegmental blood vessels. c' shows npl overexpressing cells alone. The arrow indicates a cell overexpressing npl. The lumen diameter at the position corresponding to the arrow is larger than that of the control blood vessel. B is the imaging result of zebrafish embryos with chimeric expression of npl endothelial cells that developed to 5 dpf. d' is a local magnification of the area indicated in d in the zebrafish trunk. npl overexpression increases the diameter of the lumen of the intersegmental blood vessels at the corresponding position. The arrow indicates the red position of the endothelial cell nucleus, and * indicates the control site. d" is the local magnification result of the position indicated by the dotted box d'. The arrow position shows that overexpression of npl can promote the enhancement of new blood vessels. * shows endothelial cells that do not overexpress npl, and the endothelial cells at the corresponding position are not luminal. The results show that npl-overexpressing cells exist in the vascular endothelial cells at PV, ISV, DLAV and PLA, and the lumen diameter at the corresponding position of the vascular endothelial cells overexpressing npl is significantly increased. The above results suggest that overexpression of the npl gene can increase the inner diameter of the vascular lumen.

Claims

1. Application of N-acetylneuraminic acid pyruvate lyase regulators in the preparation of drugs for regulating vascular or lymphatic cavitation.

2. The use according to claim 1, characterized in that The N-acetylneuraminic acid pyruvate lyase regulator is a positive regulator or a negative regulator of N-acetylneuraminic acid pyruvate lyase or its encoding gene.

3. The use according to claim 2, characterized in that The positive regulator is an N-acetylneuraminic acid pyruvate lyase gene or its expression product or its expression vector; the negative regulator includes one or more of a protein inhibitor targeting N-acetylneuraminic acid pyruvate lyase, a nucleic acid aptamer, an interfering RNA of the N-acetylneuraminic acid pyruvate lyase encoding gene, a gRNA, a microRNA, a gene knockdown system, and a small molecule compound inhibitor.

4. The use according to claim 3, characterized in that The negative regulator is selected from gRNA or chelidonic acid.

5. The use according to any one of claims 1 to 4, characterized in that Drugs for regulating vascular and lymphatic vessel luminalization are drugs that promote or inhibit vascular or lymphatic vessel luminalization.

6. The use according to claim 5, characterized in that The positive regulator of N-acetylneuraminic acid pyruvate lyase or its encoding gene is a drug that promotes the cavitation of blood vessels or lymphatic vessels, and the negative regulator of N-acetylneuraminic acid pyruvate lyase or its encoding gene is a drug that inhibits the cavitation of blood vessels or lymphatic vessels.

7. The use according to claim 6, characterized in that The drug promoting the luminalization of blood vessels or lymphatic vessels is used to treat luminal stenosis diseases, and the drug inhibiting the luminalization of blood vessels or lymphatic vessels is used to treat vascular hyperproliferation diseases.

8. The use according to claim 7, characterized in that The lumen stenosis disease includes cranial artery or carotid artery stenosis and ischemic stroke caused by it, renal artery stenosis and hypertension caused by it; the vascular hyperproliferation disease includes excessive tissue angiogenesis caused by diabetes, abnormal proliferation and cavitation of retinal endothelial cells, and lymphangiogenesis caused by tumors.

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