164.88-degree recombinant humanized type III collagen for vascular repair

Abstract

Provided is a 164.88-degree recombinant humanized type III collagen for use in vascular repair. This application relates to the use of collagen in the preparation of medical devices, drugs, or kits for preventing and / or treating cardiovascular disease or repairing vascular damage. The collagen comprises n repeating units, each containing the sequence shown in SEQ ID No. 1. The collagen herein can be used to treat or prevent cardiovascular disease.

Description

[0001] This application claims the benefit of priority to Chinese invention patent application No. 202310890232.8, filed July 19, 2023, entitled “164.88-degree recombinant humanized type III collagen for vascular repair.” This patent application is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the application field of collagen, and in particular to the application of 164.88 degree recombinant type III humanized collagen for vascular repair and reconstruction or anti-hypertension. Background Art

[0003] Collagen is the primary structural protein in human tissues and organs, accounting for approximately 30–40% of total body protein, primarily located in skin, bone, and muscle tissue. As a major component of the extracellular matrix, it meets the requirements of a desirable biomaterial, such as good biocompatibility, adequate mechanical strength, moderate flexibility, and cell adhesion activity, making it a structural protein of biotechnological interest. Currently, at least 28 types of collagen have been described, varying in their location in the body and designated by their time of discovery, from type I to type XXVIII. Their distinctive hallmark is a right-handed triple-helical domain characterized by repeated Gly-XY triplets, with proline and lysine typically occurring at the X and Y positions. However, concerns about the purity, disease transmission, and reproducibility of animal-derived collagen limit its application and necessitate alternative recombinant sources.

[0004] With the advancement of genetic engineering and synthetic biology, recombinant collagen with specific structure and function can be developed and shows potential as starting materials for surgical implants and matrices for regenerative medicine. Generally speaking, recombinant collagen is prepared by transcribing specific gene fragments and expressed in yeast, bacteria or animal cells, with consistent formula yield and high productivity. In order to be biocompatible with the human body, a human collagen gene is mainly selected to encode a specific type of human collagen to produce predictable and reliable recombinant collagen with broad medical application prospects. In March 2021, the National Medical Products Administration (NMPA) of China issued a standard guide for the definition of recombinant collagen. Among them, recombinant humanized collagen is defined as a full-length or fragment functional amino acid sequence encoded by a specific type of human collagen gene, or a combination of functional fragments of human collagen.

[0005] Recombinant humanized type III collagen (Rh COL III) was developed by Shanxi Jinbo Biopharmaceutical Co., Ltd. This technology is based on the original human type III collagen gene sequence. By optimizing and recombining selected sections with high water solubility and bioactivity, a new recombinant human collagen sequence was generated. Experimental studies have confirmed that this collagen has high expression levels, good water solubility, and high bioactivity, surpassing natural human collagen. Studies have shown that Rh COL III has anti-photoaging, wound repair, and proliferation-promoting effects. However, the antihypertensive activity of Rh COL III has not been reported.

[0006] This field needs to further expand the application of recombinant humanized type III collagen. Summary of the Invention

[0007] The inventors previously discovered a recombinant type III collagen with high cell adhesion properties; high stability in aqueous solution, easy preparation and purification, and other advantages (see CN 201811438582.6, which is incorporated herein by reference). In order to expand the application of this recombinant type III collagen, the inventors conducted a large number of biological activity studies. Surprisingly, the inventors found that this recombinant type III collagen may have vascular damage repair activity and can be used to prevent and / or treat cardiovascular diseases. The preparation of this protein refers to the inventor's patent 201811438582.6, which mentions that the crystal structure of this protein has been included in the PDB database, labeled 6A0A, 6A0C. The protein structure analysis article: Hua C, Zhu Y, Xu W, Ye S, Zhang R, Lu L, 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, which describes that the protein has a 164.88° curved structure.

[0008] In a first aspect, the present application provides a use of collagen (also referred to as a polypeptide or protein), a fusion protein comprising collagen, a nucleic acid encoding the collagen or fusion protein, a vector comprising the nucleic acid, a host cell comprising the vector, or a composition comprising collagen, fusion protein, nucleic acid, vector and / or host cell in the preparation of a medical device or drug or drug-device combination product or kit, wherein the medical device or drug or drug-device combination product or kit is used to prevent and / or treat cardiovascular disease in a subject or to repair or prevent and / or treat vascular damage, wherein the collagen comprises n repeating units, and the repeating units comprise:

[0009] (1) the sequence shown in SEQ ID No. 1 (GERGAPGFRGPAGPNGIPGEKGPAGERGAP);

[0010] (2) a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID No. 1; or

[0011] (3) Sequences in which 1 to 5 amino acid residues are substituted, added, deleted, or inserted into the sequence shown in SEQ ID No. 1;

[0012] Wherein n is an integer greater than or equal to 1.

[0013] In one embodiment, the collagen is recombinant humanized type III collagen, preferably 164.88 degree recombinant humanized type III collagen. Preferably, the recombinant humanized type III collagen is in the form of a trimer.

[0014] In one embodiment, the subject has ultraviolet photoaging-induced damaged skin or a diabetic infected wound.

[0015] In one embodiment, collagen has an anti-endothelial cell damage effect.

[0016] In one embodiment, the fusion protein comprises collagen and a protein for promoting collagen secretion, isolation and / or purification.

[0017] In one embodiment, the protein is selected from the group consisting of an enzyme cleavage site sequence, a signal peptide, and a purification tag sequence.

[0018] In one embodiment, the cleavage site sequence is a TEV protease cleavage site sequence.

[0019] In one embodiment, the protein tag sequence is a His tag, a GST tag, an MBP tag, a SUMO tag, a Cytiva Protein Select tag, or a NusA tag.

[0020] In one embodiment, the collagen and the protein are linked directly or through a linker, which is a flexible linker such as (G) a or (GGGGS) b , wherein a and b are each independently an integer of 1-10 or 1-5 or 1-3.

[0021] In one embodiment, the medical device, drug, drug-device combination product, or kit is also used to prevent and / or treat skin damage induced by ultraviolet light aging or diabetic infected wounds.

[0022] In one embodiment, the medical device is a gel, a dressing, an invasive device, or an implantable device.

[0023] In one embodiment, n is an integer from 1 to 32. In one embodiment, when n is an integer greater than or equal to 2, each repeating sequence is directly connected. 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.

[0024] In one embodiment, the collagen contains the sequence shown in SEQ ID No. 2 (GPPGPCCGGG) or does not contain the sequence shown in SEQ ID No. 2.

[0025] In one embodiment, the collagen comprises:

[0026] a) the amino acid sequence of SEQ ID No. 3;

[0027] b) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID No. 3, which retains the anti-endothelial cell injury effect of the amino acid sequence of SEQ ID No. 3; or

[0028] c) an amino acid sequence in which 1-80 (e.g., 1-70, 1-60, 1-50, 1-40, 1-30, 1-10, 1-8 or 1-5) amino acid residues are added, substituted, deleted or inserted into the amino acid sequence of SEQ ID No. 3, which retains the anti-endothelial cell damage effect of the amino acid sequence of SEQ ID No. 3.

[0029] In one embodiment, the amino acid sequence of SEQ ID No. 3 is

[0030] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP. Herein, the collagen having the amino acid sequence of SEQ ID No. 3 is also referred to as RhCOLIII.

[0031] 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.

[0032] In one embodiment, the cardiovascular disease is selected from the group consisting of arteriosclerosis, atherosclerosis, 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.

[0033] In one embodiment, the medical device or drug or kit is a medical device or drug or kit for lowering blood pressure.

[0034] In one embodiment, the composition comprises a lipid-lowering and / or blood pressure-lowering drug, such as a statin, an angiotensin-converting enzyme inhibitor, a calcium channel blocker, and / or a beta-blocker.

[0035] In a second aspect, the present application provides a pharmaceutical composition or a medical device or a drug-device combination product. In one embodiment, the pharmaceutical composition comprises the collagen described in the first aspect above, a fusion protein comprising collagen, a nucleic acid encoding the collagen or fusion protein, a vector comprising the nucleic acid, a host cell comprising the vector, and a drug for preventing and / or treating cardiovascular disease or for repairing vascular damage. In one embodiment, the drug is a lipid-lowering drug and / or a blood pressure-lowering drug, such as a statin, an angiotensin-converting enzyme inhibitor, a calcium channel blocker, and / or a beta-blocker. In another embodiment, the medical device or drug-device combination product comprises the collagen described in the first aspect above, a fusion protein comprising collagen, a nucleic acid encoding the collagen or fusion protein, a vector comprising the nucleic acid, a host cell comprising the vector, and a material or device for repairing vascular damage. Preferably, the medical device is a gel, a dressing, an invasive device, or an implantable device. In one embodiment, the material or device is a material or device used as a medical device.

[0036] In a third aspect, the present application provides a method for blocking or eliminating angiotensin II-induced vascular endothelial cell damage in vitro, comprising the step of contacting vascular endothelial cells with the collagen described in the first aspect of this invention. In one embodiment, the vascular endothelial cells are umbilical vein vascular endothelial cells, preferably human umbilical vein vascular endothelial cells.

[0037] In a fourth aspect, the present application provides an in vitro method for (1) reducing ROS generation induced by angiotensin II / or (2) alleviating microtubule damage in a cell, the method comprising the step of contacting the cell with the collagen described in the first aspect of the present invention. In one embodiment, the cell is a vascular endothelial cell, preferably an umbilical vein endothelial cell, preferably a human umbilical vein endothelial cell. In one embodiment, the microtubule damage is Ang II-induced microtubule damage.

[0038] In the fifth aspect, the present application provides a method for preventing and / or treating cardiovascular diseases or for repairing or preventing and / or treating vascular damage, which comprises administering to a subject the collagen described in the first aspect above, a fusion protein comprising collagen, a nucleic acid encoding the collagen or fusion protein, a vector comprising the nucleic acid, a host cell comprising the vector, or a composition comprising collagen, fusion protein, nucleic acid, vector and / or host cell, or administering to a subject the pharmaceutical composition or medical device or drug-device combination product of the second aspect of this invention.

[0039] 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.

[0040] In one embodiment, the cardiovascular disease is selected from the group consisting of arteriosclerosis, atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation and thrombotic disease.

[0041] In one embodiment, the composition comprises a lipid-lowering and / or blood pressure-lowering drug, such as a statin, an angiotensin-converting enzyme inhibitor, a calcium channel blocker, and / or a beta-blocker.

[0042] In one aspect, a method for screening an active ingredient for preventing and / or treating cardiovascular disease or repairing vascular damage in a subject is provided, comprising

[0043] (i) providing collagen, wherein the collagen comprises n repeating units, wherein the repeating units comprise:

[0044] (1) the sequence shown in SEQ ID No. 1;

[0045] (2) a sequence that is 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% identical to the sequence shown in SEQ ID No. 1; or

[0046] (3) Sequences in which 1 to 5 amino acid residues are substituted, added, deleted, or inserted into the sequence shown in SEQ ID No. 1;

[0047] wherein n is an integer greater than or equal to 1, preferably an integer from 1 to 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, wherein when n is an integer greater than or equal to 2, the repeated sequences are directly connected;

[0048] (ii) adding the collagen to HUVEC cells treated with angiotensin II;

[0049] (III) Collagen with the following properties is selected as the active ingredient: (a) reducing ROS / or (b) alleviating microtubule damage.

[0050] In one embodiment, the subject has UV photoaging-induced damaged skin or a diabetic infected wound.

[0051] In one embodiment, collagen has an anti-endothelial cell damage effect.

[0052] In one embodiment, the active ingredient is also used to prevent and / or treat skin damage induced by ultraviolet photoaging or diabetic infected wounds.

[0053] In one embodiment, the cardiovascular disease is a cardiovascular disease associated with endothelial cell damage, preferably, 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.

[0054] In one embodiment, the cardiovascular disease is selected from the group consisting of atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation and thrombotic disease.

[0055] In one embodiment, the selected collagen comprises:

[0056] a) the amino acid sequence of SEQ ID No. 3;

[0057] b) an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID No. 3; or

[0058] c) an amino acid sequence in which 1-80, for example, 1-70, 1-60, 1-50, 1-40, 1-30, 1-10, 1-8 or 1-5 amino acid residues are added, substituted, deleted or inserted into the amino acid sequence of SEQ ID No. 3.

[0059] In one embodiment, the active ingredient is for use as a medical device or as a drug or drug-device combination product.

[0060] Advantages of this application include:

[0061] 1. RhCOLⅢ collagen does not affect HUVEC cell viability.

[0062] 2. RhCOLⅢ collagen significantly inhibited the production of ROS in HUVEC.

[0063] 3. RhCOLⅢ treatment of cells can significantly alleviate microtubule damage.

[0064] 4. RhCOLⅢ collagen can lower blood pressure in vivo, as demonstrated in spontaneously hypertensive rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The results showed that RhCOLⅢ interacted with HUVEC cells.

[0066] Figure 2 The effect of RhCOLⅢ on HUVEC viability is shown. Compared with the control group (Ang II-induced group), ** indicates p < 0.05, and *** indicates p < 0.001.

[0067] Figure 3 The total intracellular ROS level in HUVECs was detected using the fluorescent probe DCFH-DA. Compared with the control group (Ang II-induced group), *** indicates p < 0.001.

[0068] Figure 4 The results show the effect of RhCOLⅢ on microtubule formation in HUVEC cells.

[0069] Figure 5 Shown are the effects of RhCOL III on blood pressure, body weight, heart rate, and heart weight ratio in SHR. (A) Systolic blood pressure, (B) heart rate, (C) body weight, and (D) heart weight ratio were measured. * indicates P < 0.01, ** indicates P < 0.05, and *** indicates P < 0.001 compared with the control group.

[0070] Figure 6 Shown are the effects of RhCOL III on blood biochemical parameters in SHR. (A) Ang II, (B) LDH, (C) UA, (D) ALT, (E) CERA, (F) LAC. *** indicates p < 0.001 compared with the control group. DETAILED DESCRIPTION

[0071] The following further description is provided to facilitate understanding of the present invention.

[0072] As used in this article, "vascular endothelial cells (VECs)", also known as endothelial cells, are a layer of flat mononuclear cells between the blood flow and the blood vessel wall. Damage to VECs plays a very important role in the pathogenesis and pathological processes of atherosclerotic heart disease, acute myocardial infarction, acute myocardial infarction with no reflow, various cerebrovascular diseases, and renal vascular diseases. When conducting vascular endothelial cell experiments, the cell model usually selected is "human umbilical vein endothelial cells" (Human umbilical vein endothelial cells, abbreviated as HUVECs). The vascular endothelium covers the inner wall of the blood vessel and is in direct contact with the blood in the vascular lumen. Studies have shown that impaired endothelial function and NO metabolism disorders are the initiating factors and central links of atherosclerosis (AS). Endothelial damage is the first step in macroscopic and microvascular dysfunction in diabetic patients and is a high incidence cause of diabetic vascular complications (such as atherosclerosis, nephropathy, retinopathy, and neuropathy). Endothelial cells, as the main cell type in endothelial tissue, play a vital role in regulating vascular structure and function by releasing vasoactive factors such as prostacyclin (PGI2), reactive oxygen species (ROS), endothelin-1 (ET-1), nitric oxide (NO), angiotensin II (Ang II), etc. After long-term exposure to a hyperglycemic environment, endothelial cells secrete increased vasoconstrictor and inflammatory factors (such as TNF-α, Ang II, ROS, ET-1), and reduced NO production and high-density lipoprotein (HDL) uptake, leading to vascular fragility, enhanced oxidative stress, and impaired endothelial repair, a state that promotes atherosclerosis.

[0073] As used herein, "angiotensin (Ang) II" is a crucial vasoactive substance in the renin-Ang-aldosterone system (RAAS) and a key contributor to various cardiovascular diseases. Secreted primarily by vascular smooth muscle cells (VECs) and with the ability to constrict blood vessels, it can damage the structure and function of VECs through various mechanisms, significantly impacting their ultrastructure, barrier function, and secretory function. It can also cause cellular senescence and induce apoptosis.

[0074] As used herein, "medical device" refers to instruments, equipment, appliances, in vitro diagnostic reagents and calibrators, materials, and other similar or related items that are used directly or indirectly on the human body. The collagen herein can be used as a medical device and can therefore be used as a medical device. In some embodiments, the collagen of the present invention exerts a pharmacological effect and can be used as a drug. In some embodiments, the collagen of the present invention is used as a drug-device combination product. The drug-device combination product that mainly acts as a drug or as a medical device can be determined based on the main mechanism of action of the collagen. The collagen of the present invention can be used as a medical device. For example, after injection, the triple helical structure of collagen self-assembles through intermolecular interactions to form a collagen fiber network. The network structure supports cells and tissues, physically connects damaged blood vessels, improves the mechanical properties and elasticity of blood vessels, and ultimately achieves repair of vascular damage.

[0075] "Pharmaceutical composition" means a composition comprising the collagen of the present invention in combination with at least one additional pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering bioactive agents to animals (particularly mammals), including adjuvants, excipients or vehicles, such as diluents, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants.

[0076] As used herein, "recombinant type III collagen" is a functional amino acid sequence of the full length or fragment encoded by the human type III collagen gene, or a combination of functional fragments of human collagen. In this article, RhCOLⅢ is a plurality of repeating units, which are fragments of human type III collagen. The recombinant type III collagen herein may contain a small amount of non-natural sequence at the N-terminus and / or C-terminus, such as GPPGPCCGGG (SEQ ID No. 2). The sequence of the repeating unit may be derived from humans. The recombinant type III collagen described herein may have vascular endothelial cell repair function, or may block or eliminate Ang II-induced damage to vascular endothelial cells. In some embodiments, the collagen herein is synthetically prepared. In some embodiments, the collagen herein is expressed in prokaryotes, such as Escherichia coli. In some embodiments, the collagen herein is expressed in eukaryotes, such as yeast cells, such as Pichia pastoris cells. In a preferred embodiment, the collagen herein is expressed by Escherichia coli.

[0077] For example, the recombinant type III collagen may comprise: a) the amino acid sequence of SEQ ID No. 3; b) an amino acid sequence that is 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% identical to the amino acid sequence of SEQ ID No. 3, 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 1-80 amino acid residues are mutated (e.g., added, substituted, deleted or inserted) in the amino acid sequence of SEQ ID No. 3, which retains the anti-endothelial cell damage effect of the amino acid sequence of SEQ ID No. 3. 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80.

[0078] As used herein, a repeating unit is a fragment derived from a naturally occurring protein, such as type III collagen. Recombinant type III collagen herein can be formed by linking a certain number of repeating units in a complementary manner. In the present invention, the repeating sequence of SEQ ID No. 1 used is GERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQ ID No. 1). The collagen of the present invention may comprise multiple repeating sequences, wherein there is no linker between the repeating sequences. The repeating sequence may also be a variant of the sequence set forth in SEQ ID No. 1, such as 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 to the sequence set forth in SEQ ID No. 1; or a sequence having 1-5 (1, 2, 3, 4, or 5) amino acid residues substituted, added, deleted, or inserted into the sequence set forth in SEQ ID No. 1. The number of repeats, n, of repeating units may be an integer greater than or equal to 1. For example, n can be an integer from 1 to 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.

[0079] As used herein, host cells can be eukaryotic cells, such as fungi and yeast, or prokaryotic cells, such as Enterobacteriaceae. It should be understood that those skilled in the art can replace the above-mentioned E. coli strains as host cells with other expression strains.

[0080] The present invention also provides nucleic acid molecules comprising nucleic acid sequences encoding the collagen of the present invention. The nucleic acid can be DNA or cDNA. The nucleic acid molecule can be primarily composed of a nucleic acid sequence encoding the peptide of the present invention, or can be composed solely of a nucleic acid sequence encoding the peptide of the present invention. Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, it will be understood by those skilled in the art that nucleic acid molecules with different nucleic acid sequences can encode the same amino acid sequence.

[0081] The present invention also provides a vector comprising the nucleic acid sequence of the present invention. Suitable vectors are known in the art of vector construction and include the selection of promoters and other regulatory elements, such as enhancer elements. The vectors of the present invention include sequences suitable for introduction into cells. For example, the vector can be an expression vector in which the collagen coding sequence is controlled by its own cis-acting regulatory elements, and the vector design facilitates gene integration or gene replacement in host cells.

[0082] It will be understood by those skilled in the art that, as used herein, the term "vector" includes DNA molecules, such as plasmids, phages, viruses, or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences. Suitable phage and viral vectors include, but are not limited to, lambda phage, EMBL phage, simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine mammary cancer virus, lentivirus, and the like.

[0083] The collagen of the present invention comprises a sequence as 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) in the sequence as shown in 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. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80.

[0084] 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-endothelial cell damage effect of the amino acid sequence of SEQ ID No. 3.

[0085] Amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as SEQ ID NO: 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-endothelial cell damage effect of the amino acid sequence of SEQ ID No. 3.

[0086] Amino acid insertion refers to inserting an amino acid residue at an appropriate position in an amino acid sequence, such as SEQ ID NO: 1 or 3. The inserted amino acid residues may be completely or partially adjacent to each other, or none of the inserted amino acids may be adjacent to each other, as long as the collagen protein 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. In this context, the amino acid insertion position is not between the repeated sequences.

[0087] Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted 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-endothelial cell damage effect of the amino acid sequence of SEQ ID No. 3.

[0088] In the present invention, substitutions may be conservative amino acid substitutions, meaning that three, preferably two, or one amino acid is replaced with amino acids having similar or similar properties 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 as substitutions within one or more of the amino acid classes reflected in the following table:

[0089] Conserved amino acid residues:

[0090] Acidic residues D and E

[0091] Basic residues K, R, and H

[0092] Hydrophilic uncharged residues S, T, N, and Q

[0093] Aliphatic uncharged residues G, A, V, L and I

[0094] Nonpolar uncharged residues C, M, and P

[0095] Aromatic residues F, Y and W.

[0096] Physical and functional classification of alternative amino acid residues:

[0097] Containing alcohol residues S and T

[0098] Aliphatic residues I, L, V and M

[0099] Cycloalkenyl-related residues F, H, W, and Y

[0100] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y

[0101] Negatively charged residues D and E

[0102] Polar residues C, D, E, H, K, N, Q, R, S, and T

[0103] Positively charged residues H, K, and R

[0104] Small residues A, C, D, G, N, P, S, T, and V

[0105] Minimal residues A, G, and S

[0106] Residues A, C, D, E, G, H, K, N, Q, R, S, P and T involved in turn formation Flexible residues Q, T, K, S, G, P, D, E and R.

[0107] The degree of association between two amino acid sequences or between two nucleotide sequences is described by parameter " sequence identity ".For purposes of the present invention, use as in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, people such as Rice, 2000, Trends Genet. [genetics trend] 16:276-277) (preferred 5.0.0 version or updated version) Ni Deer program implemented Ni Deerman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [J.Molecular Biology] 48:443-453) determine the sequence identity between two amino acid sequences.The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using non-simplified option to obtain) of Ni Deer, who is labeled as " longest identity ", is used as identity percentage and is calculated as follows:

[0108] (number of identical residues × 100) / (length of alignment - total number of gaps in the alignment)

[0109] For purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented by the Needleman program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferred 5.0.0 version or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needleman, labeled "longest identity," (obtained using the non-simplified option), which is labeled "longest identity," is used as percent identity and is calculated as follows:

[0110] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment)

[0111] The term "subject" refers to any human or other animal, particularly other mammals, that is being treated for prevention, treatment, or diagnosis. Other mammals may include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like. In some embodiments, the subject includes humans of any age who have risk factors for cardiovascular disease. Common risk factors include age, sex, weight, family history, sleep apnea, alcohol or smoking, arrhythmias due to lack of exercise, or signs of insulin resistance.

[0112] The term "treatment" refers to the medical management of a patient intended to cure, improve, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to the improvement of a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed to removing the cause of the disease, pathological condition or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition or disorder; preventive treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a disease, pathological condition or disorder; and supportive treatment, i.e., treatment that supplements another specific therapy for improving a disease, pathological condition or disorder. In some embodiments, the recombinant collagen of the present invention can treat cardiovascular disease, be used to repair vascular damage, or treat vascular damage. In some embodiments, the recombinant collagen of the present invention can prevent (i.e., preventive treatment) cardiovascular disease or vascular damage. In some embodiments, the recombinant collagen of the present invention can be used as supportive treatment, that is, the collagen of the present invention can be used to supplement the treatment of another specific therapy for improving a disease, pathological condition or disorder.

[0113] Ang II-induced human umbilical vein endothelial cell model

[0114] Damage to vascular endothelial cells is an important cause of cardiovascular diseases such as hypertension, and the degree of damage is positively correlated with the severity of hypertension. Damage to cells will lead to changes in their secretory function, such as increased vascular permeability, increased vasoconstrictor factors and decreased vasodilation factors, leading to increased vascular pressure and the formation of hypertension. Hypertension further causes endothelial cell damage, forming a vicious circle. Human umbilical vein endothelial cells (HUVECs) have similar biological characteristics to vascular endothelial cells, are close to the physiological state of the human body, have no species differences, are easy to obtain, have abundant sources and are ethical, and are widely used by researchers at home and abroad to establish cell models simulating hypertensive damage. Liu Guoyan et al. (Food Science, 174, vol. 38, No. 13, 2017) used Ang II to induce human umbilical vein endothelial cells to establish a hypertensive injury model.

[0115] Treatment

[0116] Provided herein are methods for preventing and / or treating cardiovascular disease in a subject or for repairing or preventing and / or treating vascular damage, comprising administering to the subject a collagen as described herein, a fusion protein comprising the collagen, a nucleic acid encoding the collagen or fusion protein, and / or a vector comprising the nucleic acid, or a composition comprising the collagen, fusion protein, nucleic acid, and / or vector. The collagen may be any of the collagens described in the first aspect herein. The subject may also have damaged skin induced by ultraviolet light aging or a diabetic infection wound. Therefore, the treatment methods of the present invention are also used to prevent and / or treat damaged skin induced by ultraviolet light aging or a diabetic infection wound. The vascular damage may be endothelial damage.

[0117] The cardiovascular disease may be a cardiovascular disease associated with endothelial cell damage. Preferably, the endothelial cell damage is angiotensin II-induced endothelial cell damage, and / or the vascular damage is angiotensin II-induced vascular damage. The cardiovascular disease may be selected from the group consisting of 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 comprise administering to the subject a lipid-lowering drug and / or a blood pressure-lowering drug, such as a statin, an angiotensin-converting enzyme inhibitor, a calcium channel blocker, and / or a beta-blocker.

[0118] use

[0119] Provided herein is the use of collagen, a fusion protein comprising collagen, a nucleic acid encoding the collagen or fusion protein, a vector comprising the nucleic acid, a host cell comprising the vector, or a composition comprising collagen, fusion protein, nucleic acid, vector and / or host cell in the preparation of a medical device or drug or kit, wherein the medical device or drug or kit is used to prevent and / or treat cardiovascular disease in a subject or to repair or prevent and / or treat vascular damage. The collagen can be any of the collagens described in the first aspect of this invention. The subject can also have damaged skin induced by ultraviolet light aging or a diabetic infection wound. Therefore, the medical device or drug is also used to prevent and / or treat damaged skin induced by ultraviolet light aging or a diabetic infection wound. The vascular damage can be endothelial damage.

[0120] The present invention also provides collagen, a fusion protein comprising collagen, a nucleic acid encoding the collagen or fusion protein, a vector comprising the nucleic acid, a host cell comprising the vector, or a composition comprising collagen, fusion protein, nucleic acid, vector and / or host cell, which is used to prevent and / or treat cardiovascular disease in a subject or to repair or prevent and / or treat vascular damage. The vascular damage may be endothelial damage. The collagen may be any of the collagens described in the first aspect of the present invention. The collagen, a fusion protein comprising collagen, a nucleic acid encoding the collagen or fusion protein, a vector comprising the nucleic acid, a host cell comprising the vector, or a composition comprising collagen, fusion protein, nucleic acid, vector and / or host cell can also be used to prevent and / or treat skin damage induced by ultraviolet light aging or diabetic infection wounds.

[0121] The cardiovascular disease may be a cardiovascular disease associated with endothelial cell damage. Preferably, the endothelial cell damage is angiotensin II-induced endothelial cell damage, and / or the vascular damage is angiotensin II-induced vascular damage. The cardiovascular disease may be selected from the group consisting of 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 comprise administering to the subject a lipid-lowering drug and / or a blood pressure-lowering drug, such as a statin, an angiotensin-converting enzyme inhibitor, a calcium channel blocker, and / or a beta-blocker.

[0122] In vitro methods

[0123] The present invention relates to a method for blocking or eliminating angiotensin II-induced vascular endothelial cell damage in vitro, which comprises the step of contacting vascular endothelial cells with the collagen described herein. Preferably, the vascular endothelial cells are umbilical vein vascular endothelial cells, preferably human umbilical vein vascular endothelial cells.

[0124] The present invention relates to an in vitro method for (1) reducing ROS generation induced by angiotensin II and (2) alleviating Ang II-induced microtubule damage in cells, the method comprising the step of contacting the cells with the collagen described herein, preferably, the cells are vascular endothelial cells, preferably umbilical vein endothelial cells, preferably human umbilical vein endothelial cells.

[0125] Composition

[0126] The composition of the present invention can be a pharmaceutical composition. The pharmaceutical composition may contain pharmaceutically acceptable carriers, such as adjuvants, preservatives, wetting agents, emulsifiers and dispersants. The above-mentioned sterilization procedures and the addition of various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, etc.) can ensure that there are no microorganisms. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, extended absorption of injectable drug forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin). Pharmaceutically acceptable carriers are formulated based on many factors within the knowledge of those of ordinary skill in the art. These include, but are not limited to, the type and properties of the active agent being formulated; the subject to whom the composition containing the agent is to be administered; the intended route of administration of the pharmaceutical composition; and the target therapeutic indication. Pharmaceutically acceptable carriers include both aqueous and non-aqueous liquid media, as well as various solid and semi-solid dosage forms. Such carriers may also include many different ingredients and additives in addition to the active agent, such additional ingredients being included in the formulation for a variety of reasons well known to those of ordinary skill in the art (e.g., stabilization of the active agent, binders, etc.) Descriptions of suitable pharmaceutically acceptable carriers and factors involved in their selection can be found in a variety of readily available sources, such as, for example, Allen, LV, Jr. et al., Remington: The Science and Practice of Pharmacy (Volume 2), 22nd Edition, Pharmaceutical Press (2012).

[0127] The collagen or pharmaceutical composition can be administered orally or parenterally, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0128] Particularly when provided as a single dose unit, there is the possibility of chemical interaction between the active ingredients of the combination. For this reason, when the collagen of the present invention and the second therapeutic agent are combined in a single dose unit, they are formulated so that although the active ingredient is combined in a single dose unit, the physical contact between the active ingredients is minimized (that is, reduced). For example, the active ingredient can be enteric coated. By carrying out enteric coating to one of the active ingredients, not only the contact between the active ingredients of the combination can be minimized, but also the release of one of these components in the gastrointestinal tract can be controlled so that one of these components is not released in the stomach, but released in the intestinal tract. One of the active ingredients can also be coated with a material, which affects sustained-release in the whole gastrointestinal tract and is also used to minimize the physical contact between the active ingredients of the combination. In some embodiments, in order to prevent collagen from being digested by gastric juice, collagen can be coated.

[0129] Another approach involves formulating a combination product in which one component is coated with a sustained-release and / or enteric-release polymer and the other component is also coated with a polymer such as a low viscosity grade of hydroxypropylmethylcellulose (HPMC) or other suitable materials as known in the art to further separate the active ingredients. The polymer coating serves to form an additional barrier to interaction with the other component.

[0130] The dosage regimen of the collagen of the present invention will vary depending on known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition and weight of the recipient; the nature and extent of the symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and hepatic function and the desired effect.

[0131] medical devices

[0132] "Medical device" refers to instruments, equipment, apparatus, in vitro diagnostic reagents and calibrators, materials, and other similar or related items for direct or indirect use on the human body. The collagen herein can be used as a medical device. In some embodiments, the collagen of the present invention exerts a pharmacological effect and can be used as a medication. In some embodiments, the collagen of the present invention is used as a drug-device combination product.

[0133] Collagen or recombinant collagen as described herein are suitable for a variety of purposes. In some embodiments, collagen or recombinant collagen is directly applied to or introduced into a subject's body, particularly a vascular injury site, as a medical device. In some embodiments, collagen or recombinant collagen can be used as a medical device in a variety of therapeutic applications or preventive applications. Such applications can be used to prevent or treat vascular injury, for example, in vascular injury involving skin and other collagen-containing structures and organs. Collagen or recombinant collagen as described herein can also be used to provide a biocompatible coating for certain medical devices to promote healing of injuries and disorders in body regions. In this region, such devices are used to treat or prevent cardiovascular disease in a subject.

[0134] The following examples are provided to further illustrate the present invention.

[0135] Example

[0136] The objectives, technical features and beneficial effects of the present invention are further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

[0137] The inventors conducted a cytotoxicity experiment and verified that Rh COLⅢ had no effect on the viability of human umbilical vein endothelial cells (HUVEC) within the concentration range of 0.1-2 mg / mL.

[0138] The inventors then evaluated the protective effect of Rh COLⅢ on angiotensin II (AngII)-induced HUVEC endothelial injury. The results showed that Rh COLⅢ inhibited the fluorescence intensity of intracellular reactive oxygen species (ROS).

[0139] The RhCOLⅢ used in this article was prepared by the inventors. The sequence is GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERG APGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGA PGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP. The specific preparation process can be found in CN 201811438582.6.

[0140] Example 1: RhCOLIII exerts its effects by attaching to the cell surface.

[0141] Prepare recombinant collagen RhCOLⅢ solution with 0.1M sodium carbonate buffer (pH 9.0) at a concentration of ≥2mg / mL in a volume of 20mL. Add 50μL of FITC (manufacturer MCE, catalog number HY-66019) solution to the recombinant collagen solution in a ratio of 1mL of recombinant collagen solution. Add FITC in small amounts (5μL each time) to the recombinant collagen solution while gently stirring. After the required FITC is added, incubate the reaction solution at 4℃ in the dark for 8h. Add 1.05mL of NH4Cl solution to the reaction solution and shake gently to make the final concentration of NH4Cl 50mM. Terminate the reaction at 4℃ for 2h. Dialyze overnight. 4 The cells were seeded at a density of 100 μg / cm2 in a 24-well plate, and after attachment, the prepared recombinant collagen-FITC marker was added. After incubation in the dark for 30 min, the cells were fixed with 4% paraformaldehyde for 10 min, and anti-fade mounting medium was added before observation and photography were performed using a fluorescence inverted microscope.

[0142] Experimental results:

[0143] To study the interaction mechanism between RhCOLⅢ and HUVEC, we used FITC to label RhCOLⅢ. Figure 1 It can be seen that a large amount of RhCOL III collagen is attached to the surface of HUVEC cells, indicating that RhCOL III does not enter the cells but exerts its effect by attaching to the cell surface.

[0144] Example 2: CCK8 assay to determine the effect of RhCOLⅢ on HUVEC cells

[0145] HUVEC cells were cultured in ECM medium (Sclencell, catalog number 1001) (1% penicillin-streptomycin, 5% fetal bovine serum) at 37°C in an incubator containing 5% carbon dioxide. 3 Cells were evenly plated in a 96-well plate and cultured in an incubator containing 5% carbon dioxide at 37°C. After attachment, cells were starved for 12 hours. RhCOLⅢ (dissolved in ECM medium) at different concentrations (0.1, 0.5, 1, and 2 mg / mL) or 8% NaCl (as a control) were then added and incubated for 24 hours. After washing, 100 μL of 1 mg / mL CCK-8 (Vazyme, catalog number A311-01) was added and incubated for 1 hour. The absorbance was measured at 450 nm using a microplate reader. The formula for calculating relative cell viability is:

[0146] Cell viability (%) = (measured value - blank value) / (control value - blank value) × 100%

[0147] Implementation results:

[0148] The effect of adding different concentrations (0.1, 0.5, 1 and 2 mg / mL) of RhCOLⅢ on the viability of HUVEC cells was detected. Figure 2 The results showed that the sample did not affect HUVEC cell viability when the concentration was lower than 2 mg / mL and could be used for subsequent experiments.

[0149] Example 3: Determination of intracellular reactive oxygen species (ROS)

[0150] Abnormal vascular function is the key feature of hypertension, atherosclerosis, diabetes and aging, and is therefore the main contributing factor to morbidity and mortality. The common factor driving the vascular dysfunction in these disease states is the excessive production of reactive oxygen species (ROS). Superoxide anion and related ROS not only quench nitric oxide (NO), but they also directly damage the function of cellular proteins by oxidative post-translational modification, driving the infringement in inflammation, cell proliferation, fibrosis, atherosclerosis and membrane transport. The main contributing factor of the ROS that should rise in vascular diseases is the activation of the renin-angiotensin system, which produces angiotensin II (Ang II)-activated NADPH oxidase, and the uncoupling of endothelial nitric oxide synthase (eNOS).

[0151] 2x10 4 HUVEC cells were evenly plated in 24-well plates and cultured in ECM medium in an incubator with 5% carbon dioxide at 37°C. After attachment, cells were starved for 12 hours. The remaining medium was discarded from the 24-well plates, and RhCOL III (prepared in ECM medium) at various concentrations (0.1, 0.5, 1, and 2 mg / mL) was added and incubated for 2 hours. Ang II (prepared in ECM medium) was then added to each well and incubated for 24 hours. The remaining medium was discarded, and the cells were carefully washed three times with PBS buffer. 200 μL of 10 μM DCFH-DA reagent (Beyotime, catalog number S0033M) was added and incubated in a 37°C incubator for 30 minutes. The cells were then carefully washed three times with PBS buffer. Throughout the experiment, wells without RhCOL III or Ang II served as blank controls. Cells were observed and photographed under an inverted fluorescence microscope, and the mean optical density was calculated using ImageJ.

[0152] Experimental results:

[0153] The effect of adding different concentrations (0.1, 0.5, 1 and 2 mg / mL) of RhCOLⅢ on the production of ROS in HUVEC cells was detected. Figure 3As shown, the blank group had no obvious fluorescence. Compared with the blank group, the Ang II group had a significantly enhanced ROS fluorescence level. RhCOLⅢ treatment reduced the increase in ROS production induced by Ang II. RhCOLⅢ significantly inhibited the production of ROS in HUVEC in a dose-dependent manner. Ang II activates NADPH oxidase by binding to angiotensin type I receptor (AT1-R), promoting the production of ROS 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. NADPH redox reaction platform and its regulatory role in the Ang II-mediated ROS signaling pathway [J]. Advances in Physiological Sciences, 2012(06):41-46.). Treatment of HUVEC with RhCOLⅢ can eliminate Ang II-induced ROS production and alleviate Ang II-induced microtubule damage. RhCOLⅢ can be used to treat or prevent vascular damage and cardiovascular disease.

[0154] Example 4: RhCOLⅢ treatment can significantly alleviate Ang II-induced microtubule damage.

[0155] HUVEC cells were evenly plated in 24-well plates and starved for 12 hours after attachment. The remaining medium was discarded from the 24-well plates. RhCOL III at varying concentrations (0.1, 0.5, 1, and 2 mg / mL) was added and incubated for 2 hours. Ang II (1 μM) was then added to each well and incubated for 24 hours. The remaining medium was discarded, and the cells were carefully washed three times with PBS buffer. The cells were fixed with 4% formaldehyde for 15 minutes at room temperature, followed by incubation in PBS containing 0.1% Triton X-100 for 10 minutes. Finally, Tubulin-Tracker Red (Beyotime, Catalog No. C1050) staining solution was added and incubated in the dark for 30-60 minutes at room temperature. The cells were then carefully washed three times with PBS buffer. Antifade mounting medium containing DAPI was added, and the cells were observed and photographed under an inverted fluorescence microscope.

[0156] Experimental results:

[0157] The effect of adding different concentrations (0.1, 0.5, 1 and 2 mg / mL) of RhCOLⅢ on the microtubule formation of HUVEC cells was detected. Figure 4 As shown in Figure 3, compared with the control group, Ang II inhibited the formation of microtubules. RhCOLⅢ treatment significantly alleviated Ang II-induced microtubule damage.

[0158] Example 5: Monitoring blood pressure, heart rate and body weight in spontaneously hypertensive rats

[0159] Male spontaneously hypertensive rats (SHRs, 8 weeks old) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China). The SHRs were randomly divided into an SHR group, a captopril-treated group (10 mg / kg / day), or a RhCOL III-treated group (10 mg / kg / day), with six rats in each group. All animals were maintained under pathogen-free conditions with a constant temperature (20 ± 26°C) and relative humidity (40-70%), with a 12-h light-dark cycle (12 h light / 12 h dark). Blood pressure was measured weekly for 4 weeks using a noninvasive tail artery sphygmomanometer. First, the sphygmomanometer was preheated and its performance checked. The rats were then placed in a quiet environment to acclimate. The tail root was placed on the sensor of the sphygmomanometer, which was aligned with the tail artery, and the measurement button was pressed. The instrument automatically measured systolic blood pressure, heart rate, and body weight.

[0160] Implementation results:

[0161] To evaluate the antihypertensive effect of RhCOLⅢ on SHR, we measured the tail artery blood pressure. Figure 5 As shown, RhCOL III treatment significantly alleviated elevated blood pressure. We also evaluated the effects of RhCOL III on body weight, heart rate, and heart weight ratio in SHRs, and the results showed that RhCOL III had no effect on body weight, heart rate, and heart weight ratio. Compared with the control group (SHR group), the RhCOL III-treated group and the captopril-treated group had reduced systolic blood pressure. These results reveal that RhCOL III can lower blood pressure in spontaneously hypertensive rats.

[0162] Example 6: Blood biochemical analysis of spontaneously hypertensive rats

[0163] For each treatment group in Example 5, orbital blood was collected after four consecutive weeks of injection. The whole blood samples were placed at 4°C overnight and centrifuged at 3000 rpm for 15 min (2-8°C). The supernatant was immediately tested. The content of the test indicators was determined according to the kit instructions (the manufacturer and product number of the kit are shown in the table below).

[0164] Table 1 Kit information

[0165]

[0166] Implementation results:

[0167] In order to further study the effect of RhCOLⅢ on SHR, we measured a series of biochemical indicators. Figure 6 As shown in Figure 3B, RhCOLⅢ can significantly reduce the expression of lactate dehydrogenase and uric acid, effectively alleviating renal injury, but has no significant effect on the expression of alanine aminotransferase, creatinine and lactic acid.

[0168] Example 7: Determination of Plasma Ang II Concentration

[0169] For each treatment group in Example 6, orbital blood was collected after four consecutive weeks of injection. 200 μL of plasma sample to be tested was added to 200 μL of incubation solution (7.40 g EDTA, 12.11 g Tris-base, 0.063 g PMSF, and 0.01 mg SBTI, dissolved in 90 mL of distilled water, and adjusted to pH 5.4-5.5 with acetic acid). After incubation at 37°C for 3 h, 300 μL of Ang II internal standard working solution (20% acetonitrile diluted stock solution) was added to obtain pre-treated samples. On a 96-well positive pressure apparatus (Oasis MAX μElution SPE, Waters, USA), a SPE plate (Oasis MAX μElution SPE, Waters, USA) was activated with 200 μL of 50% acetonitrile aqueous solution containing 1% formic acid. 550 μL of the pre-treated sample was then transferred to the SPE plate, and impurities were washed away with 200 μL of 10% methanol containing 1% ammonia. Then, the waste liquid plate under the SPE plate was replaced with a 96-well sample plate, and the analyte was eluted with 40 μL of 50% acetonitrile aqueous solution containing 1% formic acid and collected in the sample plate. It was then measured by LC-MS / MS, and the linearity was evaluated using a multiple regression equation. The linear equation and correlation coefficient (R) were recorded for sample Ang II quantification.

[0170] Implementation results:

[0171] Ang II (angiotensin II) is the main bioactive peptide in the renin-angiotensin-aldosterone system (RAAS) and is the main cause of hypertension. Figure 6 As shown in Figure A, RhCOLⅢ can significantly reduce the expression of Ang II.

[0172] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a collagen protein having the amino acid sequence shown in SEQ ID No. 3, a fusion protein comprising the collagen protein, or a composition comprising the collagen protein and / or the fusion protein in the preparation of a medical device, a drug, a drug-device combination product, or a kit, wherein the medical device, drug, drug-device combination product, or kit is used to prevent and / or treat cardiovascular disease in a subject, wherein the cardiovascular disease is hypertension, and the fusion protein comprises collagen protein and a sequence for promoting collagen secretion, separation, and / or purification.

2. The use according to claim 1, wherein the collagen is recombinant humanized type III collagen.

3. The use according to claim 1, wherein the collagen is 164.88 degree recombinant type III humanized collagen.

4. The use according to claim 1, wherein the collagen is recombinant humanized type III collagen in the form of a trimer.

5. The use according to claim 1, wherein the sequence for promoting collagen secretion, separation and / or purification is selected from enzyme cleavage site sequences, signal peptides and purification tag sequences. The use according to claim 5 , wherein the cleavage site sequence is a TEV protease cleavage site sequence.

7. The use according to claim 6, wherein the tag sequence is a His tag, a GST tag, an MBP tag, a SUMO tag, a Cytiva Protein Select tag or a NusA tag.

8. The use according to claim 1, wherein the medical device is a gel, a dressing, or an invasive device.

9. The use according to claim 1, wherein the medical device is an implantable device.

10. The use according to any one of claims 1 to 9, wherein the hypertension is essential hypertension and the drug is a drug for lowering blood pressure.

11. The use according to claim 1, wherein the composition further comprises a statin lipid-lowering drug, an angiotensin-converting enzyme inhibitor, a calcium channel blocker and / or a beta-receptor blocker.

12. The use according to claim 1, wherein the prevention and / or treatment involves the repair of vascular endothelial cell damage.

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