164.88° Recombinant Type III Humanized Collagen for Blood Vessel Repair

Abstract

This invention provides 164.88° recombinant type III humanized collagen for vascular repair. It relates to the use of collagen containing n repeating units with the sequence shown in Sequence ID No. 1 in the manufacture of medical devices, pharmaceuticals, or kits for the prevention and / or treatment of cardiovascular disease or the repair of vascular damage. Collagen can be used for the treatment or prevention of cardiovascular disease.

Description

Technical Field

[0001] This application claims the priority of a Chinese patent application with an application date of July 19, 2023, an application number of 202310890232.8, and an invention title of "164.88-degree recombinant type III humanized collagen for blood vessel repair". This patent application is hereby incorporated herein by reference in its entirety.

[0002] The present invention relates to the application field of collagen, specifically to the application of 164.88-degree recombinant type III humanized collagen in blood vessel repair and reconstruction or anti-hypertension.

Background Art

[0003] Collagen is the main structural protein of human tissue organs, accounting for about 30% - 40% of the total body proteins, and is mainly located in the skin, skeleton, and muscle tissues. As the main component of the extracellular matrix, collagen can meet the requirements of good biomaterials, such as good biocompatibility, appropriate mechanical strength, moderate flexibility, and cell adhesion activity, so it is a structural protein with biotechnological value. Currently, at least 28 types of collagen have been reported, and their in vivo locations are different. They are gradually named from type I to type XXVIII according to the discovery time. Their unique mark is the right-handed triple helix domain, that is, a protein with repeated Gly-X-Y trimers, where proline and lysine usually appear at the X and Y positions. However, concerns about the purity, disease transmission, and reproducibility of animal-derived collagen limit its application and an alternative recombinant source is needed.

[0004] With advances in genetic engineering and synthetic biology, recombinant collagen with specific structures and functions has been developed, demonstrating its potential as a starting material for surgical implants and a matrix for regenerative medicine. Generally, recombinant collagen is produced by transcribing specific gene fragments and expressing them in yeast, bacteria, or animal cells, with consistent yields depending on the formulation method and high yields. To ensure biocompatibility with the human body, mainly human collagen genes are selected to encode specific types of human collagen, producing predictable and reliable recombinant collagen with broad potential for medical applications. In March 2021, the National Medical Products Administration of China (NMPA) published guidelines defining recombinant collagen. Recombinant humanized collagen is defined as a functional amino acid sequence of full length or fragments encoded by a specific type of human collagen gene, or a combination of functional fragments containing human collagen.

[0005] Recombinant humanized type III collagen (Rh COL III) was developed by Shanxi Jinbo Biomedical Co., Ltd. This technology involved optimizing and selecting highly water-soluble and bioactive regions based on the original gene sequence of type III human collagen, followed by codon optimization and splicing recombination to obtain a completely new recombinant human collagen sequence. Experiments have demonstrated that this collagen exhibits high expression levels, good water solubility, and high bioactivity, demonstrating superior performance compared to natural human collagen. Studies have shown that Rh COL III possesses effects such as photoaging resistance, wound repair, and proliferation promotion. The antihypertensive activity of Rh COL III has not yet been reported.

[0006] In this field, there is a need to further expand the applications of recombinant humanized type III collagen. [Overview of the Initiative]

[0007] The inventors previously discovered that recombinant type III collagen has advantages such as high cell adhesion, high stability in aqueous solutions, and ease of production and purification (see CN201811438582.6, which is incorporated herein by reference). To broaden the applications of the recombinant type III collagen, the inventors conducted extensive bioactivity studies. Surprisingly, the inventors discovered that the recombinant type III collagen has vascular damage repair activity and can be used for the prevention and / or treatment of cardiovascular disease. For the production of the protein, refer to the inventors' patent 201811438582.6, in which the protein crystal structure is listed in the PDB database with the numbers 6A0A and 6A0C. Furthermore, the protein structure analysis article by Hua C, Zhu Y, Xu W, Ye S, Zhang R, Lu L, and Jiang S. Characterization by high-resolution crystal structure analysis of a triple-helix region of human collagen type III with potent cell adhesion activity. Biochem Biophys Res Commun. 2019 Jan 22;508(4):1018-1023. doi: 10.1016 / j.bbrc.2018.12.018. Epub 2018 Dec 11. PMID: 30545625; PMCID: PMC7092849 states that the protein has a curved structure of 164.88°.

[0008] In the first aspect, the use of the present invention in the manufacture of a medical device, pharmaceutical, pharmaceutical medical device combination product or kit of collagen (also called polypeptide or protein), a collagen-containing fusion protein, a nucleic acid encoding the collagen or fusion protein, a vector containing the nucleic acid, a host cell containing the vector, or a composition containing collagen, a fusion protein, a nucleic acid, a vector and / or a host cell, wherein the medical device, pharmaceutical, pharmaceutical medical device combination product or kit is used for the prevention and / or treatment of cardiovascular disease in a subject, the repair of vascular damage, or the prevention and / or treatment of vascular damage, and the collagen comprises n repeating units, the repeating units are, (1) The sequence indicated by sequence number 1 (GERGAPGFRGPAGPNGIPGEKGPAGERGAP), (2) A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in Sequence ID No. 1, or (3) A sequence comprising one to five amino acid residues substituted, added, deleted, or inserted into the sequence shown in Sequence ID No. 1, Here, n is an integer greater than or equal to 1.

[0009] In one embodiment, the collagen is recombinant type III humanized collagen, preferably 164.88° recombinant type III humanized collagen. Preferably, the recombinant type III humanized collagen is in trimer form.

[0010] In one embodiment, the subject has damaged skin induced by photoaging due to ultraviolet light or a diabetic infection wound.

[0011] In one embodiment, collagen has an anti-endothelial cell damage effect.

[0012] In one embodiment, the fusion protein comprises collagen and a protein for promoting the secretion, separation, and / or purification of collagen.

[0013] In one embodiment, the protein is selected from an enzyme cleavage site sequence, a signal peptide, and a purified tag sequence.

[0014] In one embodiment, the enzyme cleavage site sequence is the TEV protease enzyme cleavage site sequence.

[0015] In one embodiment, the protein tag sequence is selected from His tag, GST tag, MBP tag, SUMO tag, Cytiva Protein Select tag, or NusA tag.

[0016] In one embodiment, the collagen is linked to the protein directly or via a linker, the linker being a flexible linker, for example (G) a or (GGGGS) b Here, a and b are independently integers between 1 and 10, 1 and 5, or 1 and 3.

[0017] In one embodiment, a medical device, pharmaceutical, pharmaceutical-medical device combination product, or kit is used to prevent and / or treat skin damage or diabetic infection wounds induced by photoaging due to ultraviolet light.

[0018] In one embodiment, the medical device is a gel, a dressing, an invasive device, or an implantable device.

[0019] In one embodiment, n is an integer from 1 to 32. In one embodiment, if n is an integer greater than or equal to 2, each repeating array is directly concatenated. In one embodiment, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31.

[0020] In one embodiment, the collagen may or may not contain the sequence shown in SEQ ID NO: 2 (GPPGPCCGGG).

[0021] In one embodiment, collagen is a) Amino acid sequence of SEQ ID NO: 3, b) An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 3, and which retains the anti-endothelial cell damage effect of the amino acid sequence of SEQ ID NO: 3, or c) An amino acid sequence having 1 to 80 amino acid residues (e.g., 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 10, 1 to 8, or 1 to 5) added, substituted, deleted, or inserted into the amino acid sequence of SEQ ID NO: 3, which includes an amino acid sequence that retains the anti-endothelial cell damage effect of the amino acid sequence of SEQ ID NO: 3.

[0022] In one embodiment, the amino acid sequence of SEQ ID NO: 3 is GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP. In this specification, collagen having the amino acid sequence of SEQ ID NO: 3 is also referred to as RhCOL III.

[0023] In one embodiment, the cardiovascular disease is a cardiovascular disease associated with endothelial cell damage. In one embodiment, the endothelial cell damage is endothelial cell damage induced by angiotensin II or ROS, and / or the vascular damage is vascular damage induced by angiotensin II or ROS. In one embodiment, the vascular damage is vascular endothelial damage, preferably diabetic vascular endothelial damage.

[0024] In one embodiment, the cardiovascular disease is selected from arteriosclerosis, atherosclerotic arteriosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation, and thrombotic disease. In one embodiment, the hypertension is essential hypertension.

[0025] In one embodiment, the medical device, pharmaceutical product or kit is a medical device, pharmaceutical product or kit for reducing blood pressure.

[0026] In one embodiment, the composition includes lipid regulators and / or antihypertensive drugs such as statin lipid-lowering drugs, angiotensin-converting enzyme inhibitors, calcium channel blockers, and / or beta blockers. <着

[0027] In a second embodiment, the present application provides a pharmaceutical composition, a medical device, or a pharmaceutical-medical device combination product. In one embodiment, the pharmaceutical composition comprises the collagen described in the first embodiment, a collagen-containing fusion protein, a nucleic acid encoding the collagen or fusion protein, a vector containing the nucleic acid, a host cell containing the vector, and a pharmaceutical for the prevention and / or treatment of cardiovascular disease or the repair of vascular damage. In one embodiment, the pharmaceutical is a lipid regulator and / or antihypertensive agent such as a statin-type lipid-lowering agent, angiotensin-converting enzyme inhibitor, a calcium channel blocker and / or a β-receptor blocker. In another embodiment, the medical device or pharmaceutical-medical device combination product comprises the collagen described in the first embodiment, a collagen-containing fusion protein, a nucleic acid encoding the collagen or fusion protein, a vector containing the nucleic acid, a host cell containing the vector, and a material or device for repairing vascular damage. Preferably, the medical device is a gel, a dressing, an intrusion device, or an implantable device. In one embodiment, the material or device is a material or device used as a medical device.

[0028] In a third embodiment, the method for blocking or removing angiotensin II-induced vascular endothelial cell damage in vitro according to the present invention includes the step of bringing vascular endothelial cells into contact with collagen as described in the first embodiment of this specification. In one embodiment, the vascular endothelial cells are umbilical vein endothelial cells, preferably human umbilical vein endothelial cells.

[0029] In a fourth embodiment, the in vitro method according to the present application includes the steps of (1) reducing angiotensin II-induced ROS production or (2) mitigating microtubule damage in cells and bringing the cells into contact with collagen as described in the first embodiment of this specification. In one embodiment, the cells are vascular endothelial cells, preferably umbilical vein endothelial cells, and preferably human umbilical vein endothelial cells. In one embodiment, the microtubule damage is AngII-induced microtubule damage.

[0030] In the fifth aspect, a method for the prevention and / or treatment of cardiovascular disease, or for the repair, prevention, and / or treatment of vascular damage according to the present application, includes the step of administering to a subject the collagen, collagen-containing fusion protein, nucleic acid encoding the collagen or fusion protein, a vector containing the nucleic acid, a host cell containing the vector, or a composition containing collagen, fusion protein, nucleic acid, vector and / or host cell as described in the first aspect above, or administering to a subject the pharmaceutical composition, medical device or pharmaceutical-medical device combination product as described in the second aspect of this specification.

[0031] In one embodiment, the cardiovascular disease is a cardiovascular disease associated with endothelial cell damage. In one embodiment, the endothelial cell damage is angiotensin II or ROS-induced endothelial cell damage, and / or the vascular damage is angiotensin II or ROS-induced vascular damage.

[0032] In one embodiment, cardiovascular disease is selected from arteriosclerosis, atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation, and thrombotic disease.

[0033] In one embodiment, the composition includes lipid modulators and / or antihypertensive agents such as statins, angiotensin-converting enzyme inhibitors, calcium channel blockers and / or β-receptor blockers.

[0034] A screening method for active ingredients used in the prevention and / or treatment of cardiovascular disease or repair of vascular damage in a subject according to one embodiment is: (i) A step of providing collagen comprising n repeating units, wherein the repeating units are (1) The sequence shown in sequence number 1, (2) A sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in sequence number 1, (3) A sequence comprising one to five amino acid residues substituted, added, deleted, or inserted into the sequence shown in Sequence ID No. 1, Here, n is an integer greater than or equal to 1, preferably an integer between 1 and 32, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, and when n is an integer greater than or equal to 2, each repeating array is directly concatenated, step by step, (ii) The step of adding the collagen to HUVEC cells treated with angiotensin II, (III) The step of selecting collagen as an active ingredient that has the properties of (a) reducing ROS and / or (b) mitigating microtubule damage.

[0035] In one embodiment, the subject has damaged skin induced by photoaging due to ultraviolet light or a diabetic infection wound.

[0036] In one embodiment, collagen has an anti-endothelial cell damage effect.

[0037] In one embodiment, the active ingredient is further used to prevent and / or treat skin damage induced by photoaging due to ultraviolet light or diabetic infection wounds.

[0038] In one embodiment, the cardiovascular disease is a cardiovascular disease associated with endothelial cell damage, preferably the endothelial cell damage is angiotensin II or ROS-induced endothelial cell damage, and / or the vascular damage is angiotensin II or ROS-induced vascular damage.

[0039] In one embodiment, the cardiovascular disease is selected from atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation, and thrombotic disease.

[0040] In one embodiment, the selected collagen is a) Amino acid sequence of SEQ ID NO: 3, b) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of Sequence ID No. 3, or c) An amino acid sequence comprising 1 to 80 amino acid residues, for example, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 10, 1 to 8, or 1 to 5, added, substituted, deleted, or inserted into the amino acid sequence of SEQ ID NO: 3.

[0041] In one embodiment, the active ingredient is used as a medical device, a pharmaceutical product, or a pharmaceutical-medical device combination product.

[0042] This application includes the following advantages:

[0043] 1. RhCOL III collagen does not affect HUVEC cell viability.

[0044] 2. RhCOL III collagen significantly suppresses ROS production in HUVEC.

[0045] 3. RhCOL III-treated cells can significantly mitigate microtubule damage.

[0046] 4. RhCOL III collagen can lower blood pressure in vivo, as demonstrated in rats with spontaneous hypertension. [Brief explanation of the drawing]

[0047] [Figure 1]This shows the interaction between RhCOL III and HUVEC cells. [Figure 2] This shows the effect of RhCOL III on HUVEC survival. Compared to the control group (AngII induction group), ** represents p<0.05 and *** represents p<0.001. [Figure 3] This shows the detection of total intracellular ROS levels in HUVEC using the fluorescent probe DCFH-DA. Compared to the control group (AngII-induced group), *** indicates p<0.001. [Figure 4] This shows the effect of RhCOL III on microtubule formation in HUVEC cells. [Figure 5] This shows the effects of RhCOL III on blood pressure, body weight, heart rate, and cardiac weight ratio in SHR. (A) Systolic blood pressure, (B) heart rate, (C) body weight, and (D) cardiac weight ratio were measured. Compared to the control group, * represents p<0.01, ** represents p<0.05, and *** represents p<0.001. [Figure 6] The effect of RhCOL III on SHR blood biochemical indices is shown. (A)AngII, (B)LDH, (C)UA, (D)ALT, (E)CERA, (F)LAC. Compared to the control group, *** indicates p<0.001. [Modes for carrying out the invention]

[0048] Further explanations will be provided below to facilitate understanding of the present invention.

[0049] As used herein, “vascular endothelial cells (VECs),” also known as endothelial cells, are a single layer of flattened monocytes that interpose between the blood flow and the vessel wall. VEC damage is involved in atherosclerotic heart disease, acute myocardial infarction, acute myocardial infarction without reperfusion, and various cerebrovascular diseases. The onset and pathological processes of renal vascular diseases, among others, play a very important role. When conducting vascular endothelial cell experiments, the cell model commonly used is “human umbilical vein endothelial cells” (abbreviated as HUVECs). The vascular endothelium lines the inner wall of vessels and is in direct contact with the blood in the lumen of the vessel. As is evident from research, vascular endothelial dysfunction and NO metabolic disorders are initiators and central components of atherosclerosis (AS). Endothelial damage is the first symptom of macrovascular and microvascular dysfunction in diabetic patients and is a major cause of diabetic vascular complications (e.g., atherosclerosis, renal disease, retinal lesions, and neuropathy). Endothelial cells, as the main cell type in endothelial tissue, play a crucial role in regulating vascular structure and function by releasing vascular activating factors such as prostacyclin (PGI2), reactive oxygen species (ROS), endothelin-1 (ET-1), nitric oxide (NO), and angiotensin II (AngII). After prolonged exposure to a hyperglycemic environment, vasoconstrictive and inflammatory factors secreted by endothelial cells (e.g., TNF-α, AngII, ROS, ET-1) increase, NO production and high-density lipoprotein (HDL) uptake decrease, the vascular system becomes fragile, oxidative stress is enhanced, and endothelial repair is impaired, which is a state that promotes atherosclerosis.

[0050] As used herein, "angiotensin (Ang) II" is a particularly important vasoactive substance in the renin-Ang-aldosterone system (RAAS) and a significant promoter of various cardiovascular diseases. Angiotensin (Ang) II has a vasoconstrictive function and is secreted mainly from vascular endothelial cells (VECs) and vascular smooth muscle cells. Through various mechanisms, it causes structural and functional damage to VECs, severely impacting their ultrastructure, barrier function, and secretory function, and can also induce cellular senescence and apoptosis.

[0051] As used herein, “medical device” means any device, equipment, instrument, in vitro diagnostic reagent and calibrator, material, and other similar or related articles used directly or indirectly in the human body. Collagen as used herein can be used as a medical device because it enables its use as a medical device. In some embodiments, the collagen of the present invention can exert pharmacological effects and can be used as a pharmaceutical. In some embodiments, the collagen of the present invention is used as a pharmaceutical-medical device combination product. Based on the main mechanism of action of collagen, it can be determined whether the pharmaceutical-medical device combination product is primarily pharmaceutical or primarily medical device. The collagen of the present invention can be a medical device. For example, after injection, collagen's triple helix structure self-organizes through intermolecular interactions to form a collagen fiber web, which provides support to cells and tissues, physically connects damaged blood vessels, improves the mechanical properties and vascular elasticity of blood vessels, and ultimately enables the repair of vascular damage.

[0052] "Pharmaceutical composition" means a composition comprising a combination of the collagen of the present invention and at least one other pharmaceutically acceptable vector. "Pharmaceutically acceptable vector" means a medium for delivering a bioactive agent to animals (particularly mammals) that are generally accepted in the art, and includes adjuvants, excipients or vehicles, such as diluents, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants.

[0053] As used herein, “recombinant type III collagen” is a full-length or fragmentary functional amino acid sequence encoded by the human type III collagen gene, or a combination of human collagen-containing functional fragments. Herein, RhCOL III is a plurality of repeating units that are human type III collagen fragments. The recombinant type III collagen described herein may contain small amounts of non-natural sequences at the N-terminus and / or C-terminus, e.g., GPPGPCCGGG (SEQ ID NO: 2). The repeating unit sequence may be of human origin. The recombinant type III collagen described herein has vascular endothelial cell repair function or can block or eliminate vascular endothelial cell damage induced by AngII. In some embodiments, the collagen described herein is produced by synthesis. In some embodiments, the collagen described herein is produced by expression in prokaryotes, e.g., Escherichia coli. In some embodiments, the collagen described herein is produced by expression in eukaryotes, e.g., yeast cells such as Pichia cells. In preferred embodiments, the collagen described herein is expressed in Escherichia coli.

[0054] For example, recombinant type III collagen may include a) the amino acid sequence of SEQ ID NO: 3, b) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 3, and which retains the anti-endothelial cell damage effect of the amino acid sequence of SEQ ID NO: 3, or c) an amino acid sequence in which 1 to 80 amino acid residues are mutated (e.g., added, substituted, deleted, or inserted) from the amino acid sequence of SEQ ID NO: 3, and which retains the anti-endothelial cell damage effect of the amino acid sequence of SEQ ID NO: 3. The number of mutations may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80.

[0055] As used herein, repeating units are fragments derived from naturally occurring proteins, such as type III collagen. Recombinant type III collagen can be formed herein by complementary linking of repeating units in a fixed number. In the present invention, the repeating sequence of Sequence ID No. 1 used is GERGAPGFRGPAGPNGIPGEKGPAGERGAP (Sequence ID No. 1). The collagen of the present invention may contain multiple repeating sequences that do not contain linkers. The repeating sequence may be a variant of the sequence shown in Sequence ID No. 1, for example, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in Sequence ID No. 1, or a sequence in which 1 to 5 (1, 2, 3, 4, or 5) amino acid residues are substituted, added, deleted, or inserted into the sequence shown in Sequence ID No. 1. The number of repeats n in a repeating unit may be an integer of 1 or more. For example, n can be an integer between 1 and 32, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0056] As used herein, the host cell may be a eukaryotic cell such as a fungus or yeast, or a prokaryotic cell such as an enteromycete. Those skilled in the art will understand that the host cell can be made by replacing the above-mentioned Escherichia coli strain with another expressing strain.

[0057] The present invention further provides nucleic acid molecules comprising a nucleic acid sequence encoding the collagen of the present invention. The nucleic acid may be DNA or cDNA. The nucleic acid molecule may consist mainly of a nucleic acid sequence encoding the peptide described in the present invention, or it may consist only of nucleic acids encoding the peptide described in the present invention. Such nucleic acid molecules can be synthesized by methods known in the art. Due to the degenerate nature of the genetic code, as will be understood by those skilled in the art, nucleic acid molecules with different nucleic acid sequences can encode the same amino acid sequence.

[0058] The present invention further provides vectors comprising nucleic acid sequences described in the present invention. Suitable vectors are known in the field of vector construction and include promoter selection and other regulatory elements such as enhancer elements. Vectors described in the present invention include sequences suitable for cell introduction. For example, the vector may be an expression vector, in which the collagen coding sequence is controlled by its own cis-acting regulatory element, and the vector design facilitates gene matching or gene replacement in host cells, etc.

[0059] As those skilled in the art will understand, in this invention, the term “vector” includes DNA molecules, such as plasmids, phages, viruses, or other vectors, and comprises one or more heterologous or recombinant nucleic acid sequences. Suitable phage and viral vectors include, but are not limited to, lambda phage, EMBL phage, monkey viruses, bovine papillomavirus, Epstein-Barr virus, adenovirus, herpesvirus, mouse sarcoma virus, mouse mammary cancer virus, lentivirus, and the like.

[0060] The collagen of the present invention includes the sequence shown in SEQ ID NO: 1 or 3, or a sequence in which one or more amino acids are mutated (e.g., substituted, deleted, inserted and / or added) to the sequence shown in SEQ ID NO: 1 or 3, insofar as the collagen of the present invention retains the cardiovascular disease therapeutic effect or anti-vascular endothelial cell damage effect of the amino acid sequence of SEQ ID NO: 3. "Multiple" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80.

[0061] Amino acid addition refers to adding amino acids to the C-terminus or N-terminus of an amino acid sequence, such as SEQ ID NO: 1 or 3, as long as the collagen of the present invention retains the cardiovascular disease therapeutic effect or anti-vascular endothelial cell damage effect of the amino acid sequence of SEQ ID NO: 3.

[0062] Amino acid substitution refers to the substitution of a certain amino acid residue at a certain position in an amino acid sequence, such as in sequence number 1 or 3, with another amino acid residue, as long as the collagen of the present invention retains the cardiovascular disease therapeutic effect or anti-vascular endothelial cell damage effect of the amino acid sequence of Sequence ID No. 3.

[0063] Amino acid insertion refers to inserting amino acid residues at appropriate positions in an amino acid sequence, such as sequence SEQ ID NO: 1 or 3, as long as the collagen of the present invention retains the cardiovascular disease therapeutic effect or anti-vascular endothelial cell damage effect of the amino acid sequence SEQ ID NO: 3. All or part of the inserted amino acid residues may be adjacent to each other, or the inserted amino acids may not be adjacent to each other. In this specification, the amino acid insertion sites are not located between repeat sequences.

[0064] Amino acid deletion refers to the ability to delete one, two, or three or more amino acids from an amino acid sequence, such as the sequence of SEQ ID NO: 1 or 3, as long as the collagen of the present invention retains the cardiovascular disease therapeutic effect or anti-vascular endothelial cell damage effect of the amino acid sequence of SEQ ID NO: 3.

[0065] In the present invention, substitutions may be conservative amino acid substitutions, meaning that three, more preferably two, or one amino acid are substituted with similar or adjacent amino acids to form a peptide compared to the amino acid sequence of SEQ ID NO: 1 or 3. In the context of the present invention, conservative substitutions may be defined by substitutions within one or more amino acid types as reflected below.

[0066] Conservative amino acid residues: Acidic residues D and E Basic residues K, R, and H Hydrophilic uncharged residues S, T, N and Q Aliphatic uncharged residues G, A, V, L and I Nonpolar uncharged residues C, M, and P Aromatic residues F, Y, and W.

[0067] Physical and functional classification of candidate amino acid residues: Alcohol group-containing residues S and T Aliphatic residues I, L, V, and M Cycloalkenyl group-related residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Loaded electrical residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T and V Tiny residues A, G, and S Residues involved in reverse turn formation: A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, P, D, E, and R.

[0068] The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". For the purposes of this invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453), which is implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later). The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as a percentage of identity. It is calculated as (same residue × 100) / (alignment length - total number of gaps in the alignment).

[0069] For the purposes of this invention, sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above), which is implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later). The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as a percentage of identity. It is calculated as (same deoxyribonucleotide × 100) / (alignment length - total number of gaps in the alignment).

[0070] The term “subject” refers to any human or other animal, in particular other mammals, receiving prevention, treatment, and diagnosis. Other mammals may include, for example, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, etc. In some embodiments, subjects include humans of any age with risk factors for cardiovascular disease. Common risk factors include age, sex, weight, family history, sleep apnea, alcohol consumption or smoking, absence of exercise arrhythmias, or signs of insulin resistance.

[0071] The term “treatment” refers to medical management for a patient aimed at curing, improving, stabilizing or preventing a disease, pathological condition, or disorder. The term includes active treatment aimed at improving a disease, pathological condition, or disorder, and causal treatment aimed at eliminating the cause of the associated disease, pathological condition, or disorder. The term also includes palliative treatment, i.e., treatment designed to alleviate symptoms without curing the disease, pathological condition, or disorder; prophylactic treatment, which aims to minimize, partially or completely suppress the onset of the associated disease, pathological condition, or disorder; and supportive treatment, which is treatment to complement another specific therapy aimed at improving the associated disease, pathological condition, or disorder. In some embodiments, the recombinant collagen of the present invention can be used to treat cardiovascular disease, repair vascular damage, or treat vascular damage. In some embodiments, the recombinant collagen of the present invention can prevent (i.e., provide prophylactic treatment) cardiovascular disease or vascular damage. In some embodiments, the recombinant collagen of the present invention can be used as supportive treatment, i.e., the collagen of the present invention may be used to complement another specific therapy aimed at improving the associated disease, pathological condition, or disorder.

[0072] Human umbilical vein endothelial cell model induced by AngII Endothelial cell damage is a significant cause of cardiovascular diseases such as hypertension, and the degree of damage positively correlates with the severity of hypertension. When cells are damaged, their secretory function changes, for example, vascular permeability increases, vasoconstrictive factors increase and vasodilating factors decrease, leading to increased vascular pressure and hypertension. Hypertension further exacerbates endothelial cell damage, creating a vicious cycle. Human umbilical vein endothelial cells (HUVECs) have biological characteristics similar to vascular endothelial cells, are close to the physiological state of humans, have no species differences, are easily available, have a rich variety of origins, and are ethically compliant. They are widely used by researchers both domestically and internationally to construct cell models that simulate hypertension damage. Liu Guoyan et al. (Food Science, 174, vol.38, No.13, 2017) constructed a hypertension damage model by inducing human umbilical vein endothelial cells using AngII.

[0073] Treatment method A method for preventing and / or treating cardiovascular disease or repairing, preventing and / or treating vascular damage in a subject according to this specification comprises the step of administering to a subject a collagen, a collagen-containing fusion protein, a nucleic acid encoding the collagen or fusion protein and / or a vector containing the nucleic acid, or a composition containing collagen, a fusion protein, a nucleic acid and / or a vector as described herein. The collagen may be any of the collagens described in the first aspect of this specification. The subject may further have damaged skin induced by photoaging due to ultraviolet light or a diabetic infection wound. Therefore, the therapeutic method of the present invention can also be used to prevent and / or treat skin damage induced by photoaging due to ultraviolet light or a diabetic infection wound. The vascular damage may be vascular endothelial damage.

[0074] The cardiovascular disease may be a cardiovascular disease associated with endothelial cell damage, preferably the endothelial cell damage being angiotensin II-induced endothelial cell damage and / or the vascular damage being angiotensin II-induced vascular damage. The cardiovascular disease may be selected from atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation and thrombotic disease. The method of the present invention may further include administering a lipid modifier and / or antihypertensive agent, such as a statin-type lipid-lowering agent, angiotensin-converting enzyme inhibitor, calcium channel blocker and / or β-receptor blocker, to a subject.

[0075] use This specification provides for the use of collagen, collagen-containing fusion proteins, nucleic acids encoding the collagen or fusion protein, vectors containing the nucleic acid, host cells containing the vector, or compositions containing collagen, fusion proteins, nucleic acids, vectors and / or host cells in the manufacture of medical devices, pharmaceuticals or kits for the prevention and / or treatment of cardiovascular disease in a subject, or for the repair, prevention, and / or treatment of vascular damage. The collagen may be any of the collagens described in the first aspect of this specification. The subject may further have damaged skin induced by photoaging due to ultraviolet light or diabetic infection wounds. Therefore, the medical device or pharmaceutical may further be used for the prevention and / or treatment of skin damage induced by photoaging due to ultraviolet light or diabetic infection wounds. The vascular damage may be vascular endothelial damage.

[0076] This specification provides collagen, collagen-containing fusion proteins, nucleic acids encoding the collagen or fusion protein, vectors containing the nucleic acid, host cells containing the vector, or compositions comprising collagen, fusion proteins, nucleic acids, vectors, and / or host cells for the prevention and / or treatment of cardiovascular disease in subjects, or for the repair, prevention, and / or treatment of vascular damage. The vascular damage may be vascular endothelial damage. The collagen may be any of the collagens described in the first aspect of this specification. Compositions comprising collagen, collagen-containing fusion proteins, nucleic acids encoding the collagen or fusion protein, vectors containing the nucleic acid, host cells containing the vector, or collagen, fusion proteins, nucleic acids, vectors, and / or host cells may be used for the prevention and / or treatment of skin damage induced by photoaging due to ultraviolet light or diabetic infection wounds.

[0077] The cardiovascular disease may be a cardiovascular disease associated with endothelial cell damage, preferably the endothelial cell damage being angiotensin II-induced endothelial cell damage and / or the vascular damage being angiotensin II-induced vascular damage. The cardiovascular disease may be selected from atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation and thrombotic disease. The method of the present invention may further include administering a lipid modifier and / or antihypertensive agent, such as a statin-type lipid-lowering agent, angiotensin-converting enzyme inhibitor, calcium channel blocker and / or β-receptor blocker, to a subject.

[0078] In vitro method This specification relates to a method for in vitro blocking or removing angiotensin II-induced vascular endothelial cell damage, comprising the step of bringing the vascular endothelial cells into contact with collagen as described herein, wherein the vascular endothelial cells are umbilical vein endothelial cells, and more preferably human umbilical vein endothelial cells.

[0079] This specification relates to an in vitro method for (1) reducing angiotensin II-induced ROS production and (2) mitigating AngII-induced microtubule damage in cells, comprising the step of bringing cells into contact with collagen as described herein, wherein the cells are preferably vascular endothelial cells, preferably umbilical vein endothelial cells, and preferably human umbilical vein endothelial cells.

[0080] composition The composition of the present invention may also be a pharmaceutical composition. The pharmaceutical composition may contain pharmaceutically acceptable vectors such as adjuvants, preservatives, wetting agents, emulsifiers, and dispersants. To ensure the absence of microorganisms, the above sterilization procedure may be utilized, and various antimicrobial and antifungal agents (e.g., parahydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc.) may be added. The composition may further preferably contain isotonic agents such as sugars and sodium chloride. Furthermore, extended absorption in injectable pharmaceutical forms can be achieved by including absorption retarders (e.g., aluminum monostearate and gelatin). Pharmaceutically acceptable vectors are manufactured based on many factors within the scope of knowledge of those skilled in the art. These factors include, but are not limited to, the type and nature of the manufactured activator, the subjects to whom the composition containing the drug will be administered, the desired route of administration of the pharmaceutical composition, and the target therapeutic indication. Pharmaceutically acceptable vectors include both aqueous and non-aqueous liquid media, as well as various solid and semi-solid dosage forms. Such vectors may further contain many different components and additives other than the activator, and such other components are included in the formulation for various reasons well known to those skilled in the art (e.g., stabilization of activators, binders, etc.). Descriptions of appropriate pharmaceutically acceptable vectors and factors relating to their selection can be found in many readily available sources, for example, Allen, LV, Jr., et al., Remington: The Science and Practice of Pharmacy (Vol. 2), 22nd edition, Pharmaceutical Press (2012).

[0081] Collagen or pharmaceutical compositions may be administered orally or parenterally, including, but not limited to, intravenous, intramuscular, intraarterial, intrasacral, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intravertebral, epidural, and intrasternal injections and infusions.

[0082] In particular, when provided as a single dose unit, chemical interactions may exist between the combined active ingredients. Therefore, when the collagen of the present invention and the second therapeutic agent are combined as a single dose unit, the active ingredients are combined as a single dose unit, but the formulation is such that physical contact between the active ingredients is minimized (i.e., reduced). For example, the active ingredients may be enterically coated. By enterically coating one of the active ingredients, it is possible not only to minimize contact between the combined active ingredients, but also to control the release of one of these ingredients in the gastrointestinal tract, so that one of these ingredients is released in the intestines instead of the stomach. Alternatively, the active ingredients may be coated with a material that affects sustained release throughout the gastrointestinal tract and minimizes physical contact between the combined active ingredients. In some embodiments, the collagen may be coated to protect it from gastric juice digestion.

[0083] Another method relates to the production of a composition in which the active ingredient is further isolated by coating one component with a sustained-release and / or enteric-release polymer and another component with a polymer (e.g., low-viscosity grade hydroxypropyl methylcellulose (HPMC)) or other suitable material known in the art. The polymer coating is used to form an additional barrier to prevent interaction with other components.

[0084] The method of administering collagen according to the present invention varies depending on known factors (for example, the pharmacodynamic characteristics of a particular drug, its method and route of administration, the subject's race, age, sex, health, medical status, weight, the nature and severity of symptoms, the type of parallel treatment, the frequency of treatment, the route of administration, the patient's renal and hepatic function, and the desired effect).

[0085] Medical devices "Medical device" means equipment, facilities, instruments, in vitro diagnostic reagents and calibrators, materials, and other similar or related articles used directly or indirectly on the human body. Collagen as used herein can be used as a medical device because it enables its use in medical devices. In some embodiments, the collagen of the present invention can exert pharmacological effects and can be used as a pharmaceutical. In some embodiments, the collagen of the present invention is used as a pharmaceutical-medical device combination product.

[0086] The collagen or recombinant collagen described herein is suitable for a variety of purposes. In some embodiments, the collagen or recombinant collagen is applied or introduced directly to the body of a subject, particularly to sites of vascular injury, as a medical device. In some embodiments, the collagen or recombinant collagen may be used as a medical device for various therapeutic or preventive applications. Such applications may be used for the prevention or treatment of vascular injury, for example, for vascular injury relating to the skin and other structures and organs containing collagen. The collagen or recombinant collagen described herein may be used to provide biocompatible coatings for specific medical devices to promote the healing of injuries and disorders in areas of the body. In this area, such devices may be used for the treatment or prevention of cardiovascular disease in a subject.

[0087] To further illustrate the present invention, the following embodiments are provided. [Examples]

[0088] The objectives, technical features, and beneficial effects of the present invention will be described in more detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0089] The inventors conducted cytotoxicity experiments and verified that RhCOL III does not affect the cell viability of human umbilical vein endothelial cells (HUVECs) at concentrations within the range of 0.1–2 mg / mL.

[0090] The inventors then evaluated the protective effect of RhCOL III against angiotensin II (AngII)-induced HUVEC endothelial damage. Their results showed that RhCOL III suppressed the fluorescence intensity of intracellular reactive oxygen species (ROS).

[0091] The RhCOL III used in this specification was manufactured and obtained by the inventor. The sequence is GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP. For specific details on the manufacturing process, please refer to CN201811438582.6.

[0092] Example 1: RhCOL III acts by adhering to the cell surface.

[0093] Recombinant collagen RhCOL III solution was prepared in 0.1 M sodium carbonate buffer (pH 9.0) to a concentration of ≥2 mg / mL and a volume of 20 mL. FITC (manufacturer MCE, product catalog number HY-66019) was added in small amounts (5 μL each time) to the recombinant collagen solution at a ratio of 50 μL to 1 mL of the recombinant collagen solution, with gentle stirring during addition. After the desired amount of FITC had been added, the reaction mixture was incubated at 4°C in the dark for 8 hours. 1.05 mL of NH4Cl solution was added to the reaction mixture, and the mixture was gently shaken until the final concentration of NH4Cl reached 50 mM. The reaction was stopped at 4°C for 2 hours. Dialysis was performed overnight. HUVEC was divided into 2 x 10⁻⁶ units. 4 The culture was inoculated into a 24-well plate at the specified density, adhered to the plate walls, then the prepared recombinant collagen-FITC labeled product was added. After incubation for 30 minutes in the dark, the product was fixed with 4% paraformaldehyde for 10 minutes, an anti-quenching mounting agent was added, and the samples were observed and photographed using an inverted fluorescence microscope.

[0094] Experimental results: To study the interaction mechanism between RhCOL III and HUVEC, RhCOL III was labeled with FITC. As can be seen in Figure 1, the large amount of RhCOL III type collagen attached to the surface of HUVEC cells demonstrated that RhCOL III acts by adhering to the cell surface rather than entering the cell.

[0095] Example 2: Measurement of the effect of RhCOL III on HUVEC cells using the CCK8 method HUVEC cells were cultured in ECM medium (manufacturer: Screncell, product catalog number 1001) (1% penicillin-streptomycin, 5% fetal bovine serum) at 37°C in an incubator containing 5% carbon dioxide. 5x10 3The cells were uniformly spread in a 96-well plate and cultured at 37°C in an incubator containing 5% carbon dioxide. After adhesion to the plate wall, the cells were starved for 12 hours, and then incubated for 24 hours with different concentrations (0.1, 0.5, 1, and 2 mg / mL) of RhCOL III (dissolved in ECM medium) or 8% NaCl (as a control). After washing, 100 μL of 1 mg / mL of CCK-8 (manufacturer Vazyme, product catalog number A311-01) was added and incubated for 1 hour. Absorbance was measured at 450 nm using a microplate reader. The formula for calculating cell viability is: Cell viability (%) = (measured value - blank value) / (control value - blank value) × 100%.

[0096] Experimental results: The effect of adding RhCOLIII at different concentrations (0.1, 0.5, 1, and 2 mg / mL) on HUVEC cell viability was detected, and the results are shown in Figure 2. The results showed that at concentrations below 2 mg / mL, the samples did not affect HUVEC cell viability and could be used in subsequent experiments.

[0097] Example 3: Measurement of intracellular reactive oxygen species clusters (ROS) Abnormal vascular function is a major characteristic of hypertension, atherosclerosis, diabetes, and aging, and is therefore a major cause of disease onset and mortality. A common factor driving vascular dysfunction in these disease states is the overproduction of reactive oxygen species (ROS). Superoxide anions and associated ROS not only quench nitric oxide (NO) but also directly impair the function of cellular proteins through post-translational modification, driving inflammation, cell proliferation, fibrosis, atherosclerosis, and damage to membrane transport. The main factors contributing to the elevated ROS in vascular diseases are activation of the renin-angiotensin system, production of angiotensin II (AngII)-activated NADPH oxidase, and decoupling of endothelial nitric oxide synthase (eNOS).

[0098] 2x10 4HUVEC cells were uniformly spread in a 24-well plate and cultured in ECM medium at 37°C in an incubator containing 5% carbon dioxide. After wall adhesion, the cells were starved for 12 hours. The old medium in the 24-well plate was discarded, and RhCOL III (prepared in ECM medium) at different concentrations (0.1, 0.5, 1, and 2 mg / mL) was added and incubated for 2 hours. Then, 1 μmM AngII (prepared in ECM medium) was added to each well and incubated for 24 hours. The old medium was discarded, and the cells were carefully washed with PBS buffer solution. This process was repeated three times. 200 μL of 10 μm DCFH-DA reagent (manufacturer Biyuntian, product catalog number S0033M) was added, and the cells were incubated in a 37°C constant temperature incubator for 30 minutes. The cells were then carefully washed with PBS buffer solution, and this process was repeated three times. Throughout the entire experiment, wells without RhCOL III and AngII were blank groups. The samples were observed and photographed using a fluorescence inverted microscope, and the average optical density was calculated using ImageJ.

[0099] Experimental results: When the effects of adding RhCOL III at different concentrations (0.1, 0.5, 1, and 2 mg / mL) on ROS in HUVEC cells were detected, as shown in Figure 3, there was no significant fluorescence in the blank group, while the ROS fluorescence level was clearly enhanced in the AngII group compared to the blank group. RhCOL III treatment reduced the increase in ROS production induced by AngII. RhCOL III significantly suppressed ROS production in HUVEC cells in a dose-dependent manner. AngII activates NADPH oxidase by binding to the angiotensin type I receptor (AT1-R), promoting ROS production and increasing oxidative stress, which is one of the common mechanisms of hyperlipidemia, hypertension, diabetes, and atherosclerosis (Chen Liyun, Wu Yanqing, Zhang Zhenghong, Luo Qianping, Wang Zhengchao, et al. Regulatory effects on the NADPH redox reaction platform and the ROS signaling pathway mediated by AngII [J]. Progress in Physiological Sciences, 2012(06):41~46). Treatment with RhCOL III in HUVEC eliminated the production of ROS induced by AngII and mitigated AngII-induced microtubule damage. RhCOL III could be used for the treatment or prevention of vascular injury and cardiovascular disease.

[0100] In Example 4, RhCOL III treatment significantly mitigated microtubule damage induced by AngII.

[0101] HUVEC cells were uniformly spread in a 24-well plate, attached to the wall, and starved for 12 hours. The old medium in the 24-well plate was discarded, and RhCOL III at different concentrations (0.1, 0.5, 1, and 2 mg / mL) was added and incubated for 2 hours. Then, 1 μm of AngII was added to each well and incubated for 24 hours. The old medium was discarded, and the cells were carefully washed with PBS buffer solution. This process was repeated three times. The cells were fixed with 4% formaldehyde solution at room temperature for 15 minutes, then PBS containing 0.1% Trbuliton X-100 was added and incubated for 10 minutes. Finally, Tubulin-Tracker Red (manufacturer Biyuntian, product catalog number C1050) staining solution was added, and the cells were incubated at room temperature in the dark for 30-60 minutes. The cells were then carefully washed with PBS buffer solution, and this process was repeated three times. An anti-quenching mounting medium containing DAPI was added, and the cells were observed and photographed using an inverted fluorescence microscope.

[0102] Experimental results: The effects of adding RhCOL III at different concentrations (0.1, 0.5, 1, and 2 mg / mL) on microtubule formation in HUVEC cells were detected. As shown in Figure 4, microtubule formation was suppressed in the AngII group compared to the blank group. RhCOL III treatment significantly mitigated microtubule damage induced by AngII.

[0103] Example 5: Monitoring of blood pressure, heart rate, and body weight in rats with spontaneous hypertension. Male rats with spontaneous hypertension (SHR, 8 weeks old) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China). The SHR rats were randomly divided into three groups: an SHR group, a captopril treatment group (10 mg / kg / day), and a RhCOL III treatment group (10 mg / kg / day), with six rats in each group. All animals were kept at a specific constant temperature (20°C). ~Under pathogen-free conditions of 26°C and relative humidity (40%-70%), rats underwent a 12-hour light-dark cycle (12 hours of light / 12 hours of darkness). Blood pressure was measured in each group of rats using a non-incisional tail artery sphygmomanometer, once a week for four consecutive weeks. First, the non-incisional tail artery sphygmomanometer was preheated and its performance was tested. Then, the rats were placed in a quiet environment to allow them to adapt, the base of the tail was placed on the sensor of the non-incisional tail artery sphygmomanometer, the position relative to the tail artery was adjusted, and the measurement button was pressed. The device was able to automatically measure the rats' systolic blood pressure, heart rate, and body weight.

[0104] Experimental results: To evaluate the antihypertensive effect of RhCOL III on SHR, tail artery blood pressure was measured. As shown in Figure 5, treatment with RhCOL III significantly mitigated the increase in blood pressure. Furthermore, evaluation of the effects of RhCOL III on body weight, heart rate, and cardiac weight ratio in SHR showed that RhCOL III did not affect body weight, heart rate, or cardiac weight ratio. Compared to the control group (SHR group), the RhCOL III-treated group and the captopril-treated group showed a decrease in systolic blood pressure. These results indicate that RhCOL III can lower blood pressure in rats with spontaneous hypertension.

[0105] Example 6: Detection of blood biochemistry in rats with spontaneous hypertension. For each treatment group in Example 5, orbital blood was collected after four weeks of continuous injection. The whole blood samples were left overnight at 4°C, then centrifuged at 3000 rpm (at 2°C to 8°C) for 15 minutes. The supernatant was obtained and could be detected immediately. The content of the detection indicator was measured according to the kit instructions (the kit manufacturer and product number are shown in the table below).

[0106] Table 1 Kit Information [Table 1]

[0107] Experimental results: To further investigate the effects of RhCOL III on SHR, a series of biochemical indicators were measured. As shown in Figures B-F of Figure 6, RhCOL III significantly reduced the expression of lactate dehydrogenase and uric acid, effectively mitigating renal damage, but did not significantly affect the expression of alanine aminotransferase, creatinine, and lactate.

[0108] Example 7: Measurement of plasma AngII concentration For each treatment group in Example 6, after continuous injection for 4 weeks, orbital blood was collected, and 200 μL of the target plasma sample was added to each sample. 200 μL of culture medium (7.40 g of EDTA, 12.11 g of Tris-base, 0.063 g of PMSF, and 0.01 mg of SBTI were weighed, dissolved in 90 mL of distilled water, and the pH was adjusted to 5.4-5.5 with acetic acid) was added, and the mixture was incubated at 37°C for 3 hours. Then, 300 μL of AngII internal standard working solution (storage solution diluted with 20% acetonitrile) was added to obtain the pre-treated sample. In a 96-well positive pressure incubator (Oasis MAX μElution SPE, Waters, USA), the SPE plate (Oasis MAX μElution SPE, Waters, USA) was activated with 200 μL of 50% acetonitrile aqueous solution containing 1% formic acid. A 550 μL pretreatment sample was placed on an SPE plate, and then 200 μL of 10% methanol containing 1% aqueous ammonia was added to wash away impurities. Next, the waste liquid plate at the bottom of the SPE plate was replaced with a 96-well sample plate, and the target substance was eluted with 40 μL of 50% acetonitrile aqueous solution containing 1% formic acid and collected on the sample plate. The sample was then measured by LC-MS / MS, linearity was evaluated using a multiple regression equation, and the linear equation and correlation coefficient (R) were recorded and used for the quantification of sample AngII.

[0109] Experimental results: Angiotensin II (AngII) is the main bioactive peptide in the renin-angiotensin-aldosterone system (RAAS) and a major cause of hypertension. As shown in Figure 6A, RhCOL III was able to significantly reduce AngII expression.

[0110] While the above embodiments represent preferred embodiments of the present invention, the embodiments of the present invention are not limited thereto. Any other modifications, alterations, substitutions, combinations, or simplifications made without departing from the spirit and principles of the present invention are all equivalent substitutions and fall within the scope of protection of the present invention.

Claims

1. Use of collagen, a collagen-containing fusion protein, a nucleic acid encoding the collagen or fusion protein, a vector containing the nucleic acid, a host cell containing the vector, or a composition comprising collagen, a fusion protein, a nucleic acid, a vector and / or a host cell, in the manufacture of a medical device, a pharmaceutical, a pharmaceutical-medical device combination product or kit, wherein the medical device, pharmaceutical, pharmaceutical-medical device combination product or kit is used for the prevention and / or treatment of cardiovascular disease in a subject, the repair of vascular damage, or the prevention and / or treatment of vascular damage, wherein the collagen comprises n repeating units, and the repeating units are (1) The sequence shown in sequence number 1, (2) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in Sequence ID No. 1, (3) A sequence comprising one to five amino acid residues substituted, added, deleted, or inserted into the sequence shown in Sequence ID No. 1, Here, n is an integer greater than or equal to 1, preferably an integer between 1 and 32, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, and when n is an integer greater than or equal to 2, each repeating array is directly concatenated. Preferably, the subject has damaged skin induced by photoaging due to ultraviolet light or a diabetic infection wound. Preferably, collagen has an anti-endothelial cell damage effect, Preferably, the vascular injury is endothelial injury. Preferably, the medical device, pharmaceutical, pharmaceutical-medical device combination product, or kit is used for the prevention and / or treatment of skin damage or diabetic infection wounds induced by photoaging due to ultraviolet light. Preferably, the collagen is recombinant type III humanized collagen, preferably 164.88° recombinant type III humanized collagen, and preferably the recombinant type III humanized collagen is in trimer form. Preferably, the fusion protein comprises collagen and a protein for promoting the secretion, separation, and / or purification of collagen. Preferably, the protein is selected from the enzyme cleavage site sequence, the signal peptide and the purified tag sequence. Preferably, the enzyme cleavage site sequence is a TEV protease enzyme cleavage site sequence. Preferably, the protein tag sequence is selected from His tag, GST tag, MBP tag, SUMO tag, Cytiva Protein Select tag, or NusA tag. Preferably, the collagen is linked to the protein directly or via a linker, the linker being a flexible linker, for example (G) ａ or (GGGGS) ｂ Here, a and b are independently integers between 1 and 10, 1 and 5, or 1 and 3. Preferably, the medical device is a gel, dressing, invasive device, or implantable device.

2. The use according to claim 1, wherein the collagen includes the sequence shown in SEQ ID NO: 2, or does not include the sequence shown in SEQ ID NO:

2.

3. The aforementioned collagen is a) Amino acid sequence of Sequence ID No. 3, b) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of Sequence ID No. 3, and which retains the anti-endothelial cell damage effect of the amino acid sequence of Sequence ID No. 3, or c) The use according to claim 1 or 2, comprising an amino acid sequence in which 1 to 80 amino acid residues, for example 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 10, 1 to 8, or 1 to 5, are added, substituted, deleted, or inserted into the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence maintains the anti-endothelial cell damage effect of the amino acid sequence of SEQ ID NO:

3.

4. The use according to any one of claims 1 to 3, wherein the cardiovascular disease is a cardiovascular disease related to endothelial cell damage, preferably the endothelial cell damage is angiotensin II or ROS-induced endothelial cell damage, and / or the vascular damage is angiotensin II or ROS-induced vascular damage.

5. The use according to any one of claims 1 to 4, wherein the cardiovascular disease is selected from arteriosclerosis, atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation, and thrombotic disease, preferably, hypertension is spontaneous hypertension, preferably, the drug is a drug that lowers blood pressure, and preferably, vascular endothelial injury is diabetic vascular endothelial injury.

6. The use according to any one of claims 1 to 5, wherein the composition comprises a lipid modifier and / or antihypertensive agent such as a statin-type lipid-lowering agent, an angiotensin-converting enzyme inhibitor, a calcium channel blocker and / or a β-receptor blocker.

7. A pharmaceutical composition comprising collagen according to any one of claims 1 to 3, a collagen-containing fusion protein, a nucleic acid encoding the collagen or fusion protein, a vector containing the nucleic acid, a host cell containing the vector, and a pharmaceutical for the prevention and / or treatment of cardiovascular disease or the repair of vascular damage, wherein the pharmaceutical composition is preferably a lipid modulator and / or antihypertensive agent such as a statin-type lipid-lowering agent, an angiotensin-converting enzyme inhibitor, a calcium channel blocker and / or a β-receptor blocker.

8. A medical device comprising collagen according to any one of claims 1 to 3, a collagen-containing fusion protein, a nucleic acid encoding the collagen or fusion protein, a vector containing the nucleic acid, a host cell containing the vector, and a material or device for repairing vascular damage, preferably a gel, a dressing, an intrusion device, or an implantable device.

9. A method for in vitro blocking or removing angiotensin II or ROS-induced damage to vascular endothelial cells, comprising the step of bringing vascular endothelial cells into contact with collagen according to any one of claims 1 to 3, wherein the vascular endothelial cells are umbilical vein endothelial cells, and preferably human umbilical vein endothelial cells.

10. An in vitro method for (1) reducing angiotensin II-induced ROS production or (2) mitigating microtubule damage, preferably AngII-induced microtubule damage, comprising the step of bringing cells into contact with collagen according to any one of claims 1 to 3, wherein the cells are preferably vascular endothelial cells, preferably umbilical vein endothelial cells, and preferably human umbilical vein endothelial cells.

11. A method for screening active ingredients used in the prevention and / or treatment of cardiovascular disease or repair of vascular damage in subjects, (i) A step of providing collagen comprising n repeating units, wherein the repeating units are (1) The sequence shown in sequence number 1, (2) A sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in Sequence ID No. 1, or (3) A sequence comprising the sequence shown in Sequence ID No. 1 in which 1 to 5 amino acid residues are substituted, added, deleted, or inserted, Here, n is an integer greater than or equal to 1, preferably an integer between 1 and 32, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, and when n is an integer greater than or equal to 2, each repeating array is directly concatenated, step by step. (ii) The step of adding the collagen to HUVEC cells treated with angiotensin II, (iii) The step of selecting collagen as an active ingredient which has the properties of (a) reducing ROS generation and / or (b) mitigating microtubule damage, Preferably, the subject has damaged skin induced by photoaging due to ultraviolet light or a diabetic infection wound. Preferably, collagen has an anti-endothelial cell damage effect, Preferably, the active ingredient is further used for the prevention and / or treatment of skin damage induced by photoaging due to ultraviolet light or diabetic infection wounds. Preferably, the cardiovascular disease is a cardiovascular disease associated with endothelial cell damage, preferably the endothelial cell damage is angiotensin II or ROS-induced endothelial cell damage, and / or the vascular damage is angiotensin II or ROS-induced vascular damage. Preferably, cardiovascular diseases are selected from atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation, and thrombotic diseases. Preferably, the selected collagen is a) Amino acid sequence of Sequence ID No. 3, b) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of Sequence ID No. 3, or c) An amino acid sequence comprising 1 to 80 amino acid residues, for example, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 10, 1 to 8, or 1 to 5, added, substituted, deleted, or inserted into the amino acid sequence of SEQ ID NO: 3, Preferably, the active ingredient is used as a pharmaceutical, a medical device, or a pharmaceutical-medical device combination product. A method for screening active ingredients.

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