Extracellular vesicle for treating atherosclerosis and application thereof

Abstract

The invention discloses an extracellular vesicle for treating atherosclerosis and application of the extracellular vesicle, and belongs to the field of biological medicine. The extracellular vesicle with a treatment function is prepared by taking a pig aorta blood vessel as a raw material and carrying out collagenase and elastin enzymolysis and ultracentrifugation. Experimental results show that the vascular tissue-derived extracellular vesicles provided by the invention can significantly enhance the proliferation, migration and tube formation capabilities of human umbilical vein endothelial cells in a physiological state; for human umbilical vein endothelial cells in a pathological state subjected to atherosclerosis modeling, the extracellular vesicle provided by the invention can also effectively repair the proliferation, migration and tubulation capabilities of the human umbilical vein endothelial cells and up-regulate the expression of endothelial functional genes. The invention provides a new biological material for developing medicines for treating atherosclerosis, and has outstanding clinical application value and wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an extracellular vesicle for treating atherosclerosis and its application. Background Technology

[0002] Cardiovascular disease (CVD) refers to a group of diseases affecting the heart and blood vessels, encompassing a wide range of conditions affecting the circulatory system, including coronary artery disease, heart failure, and arrhythmias. Atherosclerosis (AS) is a chronic inflammatory disease driven by the accumulation of lipids in the arteries, leading to arterial narrowing and thrombosis, which can potentially result in death. Emerging evidence confirms that the incidence of atherosclerosis is increasing year by year and is gradually and seriously affecting young people. Vascular inflammation promotes the formation of atherosclerotic plaques by activating molecular and cellular pathways and plays a key role in atherosclerotic plaques, which are unstable and can rupture at any time, leading to adverse events such as stroke or myocardial infarction.

[0003] Extracellular vesicles (EVs) are membrane-sealed microparticles released from cells into the extracellular space, serving as crucial messengers for intercellular communication. These diverse vesicles, including exosomes, microvesicles, and apoptotic bodies, originate from different cellular compartments and are released through various mechanisms. EVs are complex microparticles carrying proteins, lipids, DNA, mRNA, microRNA, and lncRNA, reflecting the identity and physiological state of their parent cell. Dysregulation of EV communication within the cardiovascular system is associated with various diseases, including atherosclerosis, myocardial infarction, heart failure, and arrhythmias. EVs can promote inflammation, thrombosis, and adverse remodeling, leading to disease progression.

[0004] However, current research is limited to extracting extracellular vesicles from cell culture supernatants and body fluids, and then studying the bioactivity of extracellular vesicles. There are no reports on the use of extracellular vesicles extracted from physiological vascular tissue to treat cells with impaired vascular function. Summary of the Invention

[0005] The purpose of this invention is to provide an extracellular vesicle for treating atherosclerosis and its application, in order to solve the problems existing in the prior art. The extracellular vesicles extracted from vascular tissue by this invention have a significant enhancing effect on the proliferation, migration and tube formation ability of human umbilical vein endothelial cells under physiological or pathological (atherosclerosis modeling) conditions.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an extracellular vesicle for the prevention and / or treatment of atherosclerosis, wherein the preparation method of the extracellular vesicle includes the following steps:

[0008] The aorta was taken, cut into pieces, washed, and then enzymatically hydrolyzed. After enzymatic hydrolysis, the sample was centrifuged, the supernatant was collected, and filtered to obtain the filtrate.

[0009] The filtrate was centrifuged at high speed, the precipitate and the top oil film were collected, resuspended, and centrifuged again at high speed to collect the precipitate, thus obtaining the extracellular vesicles.

[0010] Furthermore, the enzymes used in the enzymatic hydrolysis include collagenase and elastase.

[0011] Furthermore, the collagenase is collagenase I, collagenase II, collagenase IV, or collagenase V.

[0012] Furthermore, the enzymatic hydrolysis was performed at a temperature of 37°C, a rotation speed of 300 rpm, and a time of 6 hours.

[0013] Furthermore, the ultracentrifugation temperature is 4℃, the relative centrifugal force is 120000×g, and the time is 70min.

[0014] The present invention also provides the use of the above-mentioned extracellular vesicles in the preparation of medicaments for the prevention and / or treatment of cardiovascular diseases.

[0015] Furthermore, the cardiovascular disease mentioned is atherosclerosis.

[0016] The present invention also provides a drug for the prevention and / or treatment of atherosclerosis, with the above-mentioned extracellular vesicles as the main active ingredient.

[0017] Furthermore, it also includes pharmaceutically acceptable excipients.

[0018] Furthermore, the dosage form of the drug includes an injection.

[0019] The present invention discloses the following technical effects:

[0020] This invention utilizes porcine aortic blood vessels as raw material, and prepares extracellular vesicles with therapeutic functions through enzymatic hydrolysis with collagenase and elastase followed by ultracentrifugation. Experimental results show that the extracellular vesicles derived from vascular tissue provided by this invention can significantly enhance the proliferation, migration, and tube-forming ability of human umbilical vein endothelial cells under physiological conditions. For human umbilical vein endothelial cells under pathological conditions induced by atherosclerosis, the extracellular vesicles provided by this invention can also effectively repair their proliferation, migration, and tube-forming abilities, and upregulate the expression of endothelial functional genes. This invention provides new biomaterials for the development of drugs to treat atherosclerosis, possessing outstanding clinical application value and broad application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The graph shows the comparison of cell proliferation capacity among different experimental groups at 24h, 48h, and 72h; where ns indicates P>0.05, * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001.

[0023] Figure 2 The figure shows the comparison results of cell migration ability in different experimental groups; where (A) is the observation result of migration experiment; (B) is the statistical result of migration rate; ns indicates P>0.05, * indicates P<0.05; *** indicates P<0.001; **** indicates P<0.0001;

[0024] Figure 3 The figure shows the comparison results of cell tube-forming ability in different experimental groups; where (A) is the observation result of tube-forming ability experiment; (B) is the statistical result of tube-forming ability; ns indicates P>0.05; ** indicates P<0.01;

[0025] Figure 4 Figure 1 shows the results of qPCR determination of the expression levels of endothelial cell functional genes CD31, vWF, and FLk1; where ** indicates P < 0.01 and *** indicates P < 0.001. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] This invention provides an active substance capable of treating atherosclerosis, namely extracellular vesicles derived from vascular tissue. This substance participates in the pathophysiological processes of blood vessels through intercellular communication via horizontal transfer of lipids, proteins, and nucleic acids.

[0032] This invention investigated the biological activity of extracellular vesicles derived from vascular tissue on blood vessels using biological experimental techniques such as the CCK8 assay, scratch healing assay, matrix gel tube formation assay, and quantitative polymerase chain reaction (RT-qPCR). The results showed that, compared to untreated human umbilical vein endothelial cells, the group with added extracellular vesicles from vascular tissue exhibited stronger proliferation, migration, and tube formation capabilities. Furthermore, when constructing an atherosclerosis model using human umbilical vein endothelial cells stimulated with oxidized low-density lipoprotein, the addition of extracellular vesicles from vascular tissue could restore their proliferation, migration, and tube formation capabilities.

[0033] Example 1

[0034] 1. Preparation of extracellular vesicles (VEVs) derived from vascular tissue

[0035] 1.1 Experimental Materials

[0036] 1.1.1 Reagents

[0037] Collagenases I, II, IV, and V were purchased from Beijing Solarbio Science & Technology Co., Ltd.; elastase was purchased from MedChemExpress; Ham's F-12k (Kaighn's) medium and phosphate-buffered saline (PBS) (1×) were purchased from Wuhan Sewell Biotechnology Co., Ltd.

[0038] 1.1.2 Consumables / Instruments

[0039] The Heareus Pico 17 centrifuge was purchased from Thermo Fisher Scientific, USA; the Optima L 100-XP ultracentrifuge was purchased from Beman Corporation, USA.

[0040] 1.2 Experimental Methods

[0041] 1.2.1 Extraction of extracellular vesicles (VEVs) from vascular tissue

[0042] 1.2.1.1 Take 0.5g of porcine aortic blood vessel under normal physiological conditions and place it in a centrifuge tube. Add an appropriate amount of PBS and cut the blood vessel into 1mm×1mm fragments. After cutting, wash the blood vessel tissue by centrifugation with PBS (4℃, 500×g, 5min), repeating twice. Add 10mL of different collagenases (I, II, IV, V) + elastase at a concentration of 2mg / mL dissolved in Ham's F-12k (Kaighn's) medium to each 0.5g tissue sample. Place the sample on a shaker (37℃, 300rpm) for enzymatic hydrolysis and record the reaction start time. Observe the enzymatic hydrolysis every 2 hours and mark the results. The enzymatic hydrolysis reaction is terminated after 6 hours.

[0043] 1.2.1.2 Centrifuge the enzymatically digested sample for 10 min (4℃, 500×g), collect the supernatant, and centrifuge again for 60 min (4℃, 4000×g). After centrifugation, filter the supernatant through a 0.22 μm filter. Store the sample at 4℃.

[0044] 1.2.1.3 Transfer the sample to an ultracentrifuge tube and balance with PBS (error within 10 mg). Perform the first centrifugation for 70 min (4℃, 120000×g, SW32Ti). After centrifugation, remove the vacuum from the evacuation chamber, discard the supernatant, retaining the oil film and precipitate at the top. Gently agitate the precipitate at the bottom with PBS, add 10 mL of PBS, balance, and centrifuge again for 70 min (4℃, 120000×g, SW41Ti). After centrifugation, discard the supernatant and gently agitate the precipitate. Transfer the sample to a 1.5 mL centrifuge tube and store in a -80℃ freezer.

[0045] 2. Studies on the vascular biological activity of extracellular vesicles derived from vascular tissue.

[0046] 2.1 Experimental Materials:

[0047] 2.1.1 Reagents

[0048] ECM medium was purchased from ScienCell Research Laboratories; Phosphate-Buffered Saline (PBS) (1×) was purchased from Wuhan Sewell Biotechnology Co., Ltd.; Trypsin was purchased from Gibco; Ham's F-12k (Kaighn's) medium was purchased from Wuhan Sewell Biotechnology Co., Ltd.; Cell Counting Kit-WST-8 Kit was purchased from Guangzhou Yiyuan Biotechnology Co., Ltd.; Oxidized Low-Density Lipoprotein (ox-LDL) was purchased from Guangzhou Yiyuan Biotechnology Co., Ltd.; Matrigel was purchased from MedChemExpress; DMSO was purchased from Dalian Meilun Biotechnology Co., Ltd.; Isopropanol was purchased from Shanghai Maclean Biotechnology Co., Ltd.; Chloroform was purchased from Honeywell International; Trizol Reagent was purchased from Thermo Fisher Scientific; cDNA First-Strand Synthesis Kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; MonAmp SYBR Green qPCRMix was purchased from Mona Biotechnology Co., Ltd.

[0049] 2.1.2 Consumables / Instruments

[0050] The BCL-1360A clean bench, 370 series Steri-Cycle CO2 cell culture incubator, C1 electric pipette, and QuantStudio 3 real-time quantitative PCR system were purchased from Thermo Fisher Scientific, USA; the DP74 digital microscope camera was purchased from Olympus Corporation, Japan; the multi-functional microplate reader was purchased from Wuhan Sewell Biotechnology Co., Ltd.; 12, 24, and 96-well cell culture plates were purchased from Thermo Fisher Scientific, USA; and the JULITMStage real-time live cell imaging system was purchased from Beijing Baihuida Co., Ltd.

[0051] 2.2 Experimental Methods

[0052] 2.2.1 Cell Culture

[0053] 2.2.1.1 Cell Culture

[0054] The cells used in this experiment were primary human umbilical vein endothelial cells, all purchased from Wuhan Pronosei Life Sciences Co., Ltd., and were at passage 8-12 at the time of use. All cells were cultured at 37℃ and 5% CO2. The experimental groups were: control group, extracellular vesicles (VEVs) derived from vascular tissue prepared with type I collagenase group, and oxidized low-density lipoprotein (ox-LDL) group.

[0055] Group 1: Extracellular vesicles derived from vascular tissue prepared with type I collagenase + oxidized low-density lipoprotein (VEVs + ox-LDL).

[0056] 2.2.1.2 Cell passage

[0057] Remove the culture flask filled with cells, discard the old culture medium, wash twice with PBS, add 0.5 mL of trypsin, observe under a microscope that the cells have finished digesting, quickly add culture medium to stop digestion, thoroughly pipette, aliquot the cells into 3 culture flasks, and place them in an incubator to continue cell culture.

[0058] 2.2.1.3 Cell resuscitation

[0059] Remove the frozen cells from liquid nitrogen, place them in a water bath and heat rapidly, then shake rapidly until thawed. Add the prepared culture medium and centrifuge (1100 rpm, 5 min). After centrifugation, aspirate the supernatant, add 5 mL of culture medium and mix thoroughly. Add the mixed cell suspension to the culture flask.

[0060] 2.2.1.4 Cell cryopreservation

[0061] Remove the culture flask filled with cells, discard the old culture medium, wash twice with PBS, add 0.5 mL of trypsin, observe under a microscope that the cells have been digested, and quickly add culture medium. Centrifuge the cell suspension (1100 rpm, 5 min), discard the supernatant, add culture medium and cryopreservation solution (9:1), mix thoroughly, add the cell cryopreservation suspension to cryovials, place the cryovials in a -80℃ freezer overnight, and then transfer to liquid nitrogen.

[0062] 2.2.2 CCK-8 assay for cell proliferation

[0063] Primary human umbilical vein endothelial cells were used at a concentration of 4 × 10⁻⁶ 3 Seeds were inoculated at a density of 1×10⁶ cells / well in 96-well plates, following the control group and 1×10⁶ cells / well. 8 particles / mL VEVs, 2×10 8 particles / mL VEVs, 5×10 8 particles / mL VEVs, 1×10 9 particles / mL VEVs, 50μg / mL ox-LDL, 1×108 particles / mL VEVs+50μg / mL ox-LDL, 2×10 8 particles / mL VEVs+50μg / mL ox-LDL, 5×10 8 particles / mL VEVs+50μg / mL ox-LDL, 1×10 9 The grouping conditions for particles / mL VEVs + 50μg / mL ox-LDL were as follows: incubation at 37℃ in a 5% CO2 incubator for 24, 48, and 72 h. After incubation, 10μL of CCK-8 solution was added to each well, and the wells were incubated for another 1 h. The absorbance was measured at 450nm wavelength using a microplate reader.

[0064] 2.2.3 Scratch assay to determine cell migration

[0065] In a 12-well plate, each well is inoculated with 1×10⁻⁶. 5 Cells. Once the cells reached a confluent monolayer, a 10 μL pipette tip was used to scratch the cell layer perpendicular to the cell plane. After scratching, the cells were washed three times with PBS to remove suspended cells. The control group was given serum-free Ham's F-12k medium; the vascular tissue-derived extracellular vesicle (VEV) group was given 2e 8 particles / mL VEVs; oxidized low-density lipoprotein (ox-LDL) group added 10 μg / mL ox-LDL; vascular tissue-derived extracellular vesicles + oxidized low-density lipoprotein (VEVs + ox-LDL) group added 2e 8 Particles / mL VLVEVs + 10 μg / mL ox-LDL. Cells from each group were placed in a suitable field of view using a real-time live-cell imaging system and cultured at 37°C and 5% CO2 for 48 h. After culture, the scratch area at 0 h, 12 h, 24 h, and 48 h was calculated using ImageJ software.

[0066] 2.2.4 Matrigel matrix tube forming experiment

[0067] Remove the Matrigel and thaw overnight at 4°C. After thawing, place on ice and aliquot into 400 μL portions. Dilute Matrigel with Ham's F-12k medium 2:1, add 50 μL of Matrigel to a 96-well plate, and incubate at 37°C for 40 min to allow the Matrigel to solidify. Spread cells at a rate of 1.2 × 10⁶ cells / well. 4Tubular structures were seeded onto cured matrix gel and divided into three groups: control group, vascular tissue-derived extracellular vesicles (VEVs) group, oxidized low-density lipoprotein (ox-LDL) group, and vascular tissue-derived extracellular vesicles + ox-LDL group. After incubation at 37°C and 5% CO2 for 6 hours, the tubular structures were observed under a microscope. Three parallel wells were set up for each experimental group, and three fields of view were randomly captured from each well. The branching, region, and number of tubular structures were measured using ImageJ software.

[0068] 2.2.5 RT-qPCR

[0069] 2.2.5.1 mRNA extraction

[0070] 1) When endothelial cells are cultured in 24-well plates according to their groups, after the culture is completed, the old culture medium in the well plate is removed, washed twice with PBS, 200 μL of Trizol reagent is added to each well, let stand for 5 seconds, and continuously pipet to transfer to 1.5 mL centrifuge tubes.

[0071] 2) Add 1 / 5 volume of Trizol to the obtained sample in chloroform, shake up and down 20 times, and let stand on ice for 5 minutes.

[0072] 3) Centrifuge for 15 min (4℃, 12000×g), transfer the colorless supernatant to a new 1.5 mL centrifuge tube, add the same volume of isopropanol as the supernatant, shake well to mix thoroughly, place on ice and let stand for 10 min, then centrifuge at 4℃, 12000×g for 10 min, and discard the supernatant.

[0073] 4) Add an equal volume of 75% ethanol to the centrifuge tube containing the precipitate, vortex to mix, and centrifuge at 4°C and 12000×g for 5 min. Discard the supernatant, open the centrifuge tube cap, and allow it to air dry. Then add 10 μL of enzyme-free water to fully dissolve the precipitate to obtain the mRNA solution.

[0074] 5) Measure the absorbance values ​​of the sample at 260nm and 280nm using a spectrophotometer, and calculate the A260 / A280. Samples with an absorbance in the range of 1.8-2.0 are usable.

[0075] 2.2.5.2 Reverse Transcription

[0076] 1) Prepare the first reverse transcription system according to the cDNA kit instructions:

[0077] 1 ng - 2 μg Total RNA, 2 μL LOligo-dT (10 mM), 1 μL Super Pure dNTPs (10 mM), and RNase-free H2O to bring the total to 14.5 μL.

[0078] 2) Incubate the above mixture at 65°C for 5 minutes.

[0079] 3) After incubation, place the above reaction solution on ice for 2 minutes and add the second reaction solution.

[0080] The second reaction system (total volume 20 μL) consisted of 14.5 μL of the above reaction solution, 4 μL of 5×TIANScriptⅡRTaseBuffer, 0.5 μL of RNasin (40 U / μL), and 1 μL of TIANScriptⅡRTase (200 U / μL).

[0081] 4) The above reaction solution was reacted sequentially at 42℃ for 60 min and 85℃ for 5 min, and then placed on ice for subsequent operations.

[0082] 2.2.5.3 qPCR

[0083] 1) Prepare the reaction system (total volume 20 μL) according to the qPCR instructions:

[0084] MonAmp SYBR Green qPCR Mix 10 μL, Forward Primer (10 μM) 0.4 μL, Reverse Primer (10 μM) 0.4 μL, cDNA 9.2 μL.

[0085] 2) Set up the reaction program, which consists of three steps: pre-denaturation (95℃, 30s), denaturation (95℃, 10s), and annealing / extension (60℃, 30s). The denaturation and annealing / extension steps are repeated 40 times before the reaction begins.

[0086] 2.3 Experimental Conclusions

[0087] 2.3.1 Vascular tissue-derived extracellular vesicles (VEVs) can promote endothelial cell proliferation under physiological and pathological conditions.

[0088] like Figure 1 As shown, the cell group with added VEVs had stronger proliferative capacity compared with the untreated cell group; and VEVs were able to repair endothelial cell proliferation compared with the inflammatory cell injury group constructed with ox-LDL.

[0089] 2.3.2 EVs have the ability to promote endothelial cell migration under physiological and pathological conditions.

[0090] like Figure 2 As shown, over time, the cell group with added VEVs exhibited stronger migration ability compared to the untreated cell group, and VEVs were able to alleviate the inhibition of ox-LDL on endothelial cell migration.

[0091] 2.3.3 VEVs have the ability to promote endothelial cell tubulation under physiological and pathological conditions.

[0092] like Figure 3 As shown, at 6 h, compared with the untreated cell group, the cells with added VEVs had a stronger tube-forming ability (generating nodes, regions, branches, especially the number of branches) and were able to improve the reduction in endothelial cell tube-forming caused by ox-LDL.

[0093] 2.3.4 VEVs upregulated the expression of endothelial function genes CD31, vWF, and FLk1.

[0094] like Figure 4 As shown, VEVs improved the expression of the CD31 gene in endothelial cells under ox-LDL conditions; under physiological and pathological conditions (ox-LDL-based cell damage model), VEVs promoted the expression of the vWF gene; when ox-LDL caused cell damage, the expression of the FLk1 gene was downregulated, but the addition of VEVs increased its expression.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An extracellular vesicle for preventing and / or treating atherosclerosis, characterized in that, The preparation method of the extracellular vesicles comprises the following steps: Take the aorta blood vessels, cut them into pieces, wash them, and then perform enzymatic hydrolysis, centrifuge the hydrolyzed product, collect the supernatant, filter it, and obtain the filtrate; The filtrate is subjected to ultracentrifugation, the precipitate and the top oil film are collected, resuspended, subjected to ultracentrifugation again, and the precipitate is collected, thereby obtaining the extracellular vesicles.

2. Extracellular vesicles according to claim 1, characterized in that, The enzyme used in the enzymatic hydrolysis comprises collagenase and elastase.

3. Extracellular vesicles according to claim 2, characterized in that, The collagenase is collagenase I, collagenase II, collagenase IV or collagenase V.

4. The extracellular vesicle of claim 1, wherein, The temperature of the enzymatic hydrolysis is 37℃, the rotation speed is 300 rpm, and the time is 6 h.

5. The extracellular vesicle of claim 1, wherein, The temperature of the ultracentrifugation is 4℃, the relative centrifugal force is 120000xg, and the time is 70 min.

6. Use of the extracellular vesicles according to any one of claims 1-5 in the preparation of a drug for preventing and / or treating cardiovascular diseases.

7. Use according to claim 6, wherein The cardiovascular diseases are atherosclerosis.

8. A medicament for preventing and / or treating atherosclerosis, characterized by, The extracellular vesicles according to any one of claims 1-5 are used as the main effective component.

9. The medicament according to claim 8, wherein Pharmaceutically acceptable adjuvants are further included.

10. The medicament according to claim 8, wherein The dosage form of the drug comprises injections.

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