iPSC-induced vascular endothelial cells and peptides combined for the treatment of cardiovascular and cerebrovascular diseases

Abstract

The present invention provides a combination of iPSC-induced vascular endothelial cells and polypeptides for treating cardiovascular and cerebrovascular diseases. The present invention also provides an inhibitory peptide that specifically inhibits CCL21 activity, which can treat acute myocardial infarction by inhibiting the activity of CCL21 and reducing the inflammatory regulation of CCL21 / CCR7 signals. The combined use of CCL21 inhibitory peptides and iPSC-induced vascular endothelial cells can significantly reduce the area of ​​myocardial infarction and increase the vascular surface density of myocardial tissue, and has a good therapeutic effect.

Description

Technical Field

[0001] The present application relates to the biological field, and more specifically to the combined treatment of cardiovascular and cerebrovascular diseases by iPSC-induced vascular endothelial cells and polypeptides. Background Art

[0002] Endothelial cells are mainly involved in regulating the vascular function of the human body, and can ensure the normal metabolism and renewal of endothelial cells. Endothelial cells are a single layer of cells, which are highly active cells. They can maintain the tension and structure of blood vessels by regulating vasodilation, contraction, growth inhibition and growth promotion, as well as the balance of anti-inflammatory and pro-inflammatory effects. At the same time, they can regulate the tension of blood vessels by synthesizing and releasing vasoactive substances to stimulate the body fluid and nervous system, especially the blood flow chemistry, thereby regulating platelet function, inflammatory response and the growth and migration of vascular smooth muscle cells. Endothelial cells play an important role in the pathological progression of vascular diseases. If endothelial cells are dysfunctional, it will lead to arteriosclerosis and cardiovascular diseases, which are closely related to cardiovascular risk factors. Unlike atherosclerosis, endothelial dysfunction can be reversed, so vascular diseases should be prevented and treated early.

[0003] Studies have shown that endothelial cells are damaged during hypertension, and the degree of damage is positively correlated with the severity of hypertension. Endothelial function can be improved through antihypertensive treatment. The imbalance between ET and NO secreted by endothelial cells plays an important role in the occurrence and development of hypertension. In patients with essential hypertension (EH) and experimental hypertensive animals, plasma ET levels were found to be higher than those in the control group, and the increased level was related to the degree of hypertension. In the resistance vessels of patients with moderate and severe hypertension, the expression of ET-1 gene in endothelial cells was enhanced, indicating that the synthesis, release and generation of ET increased during hypertension. In recent years, many studies have shown that the large expression of eNOS in endothelial cells and myocardium can improve left ventricular function after myocardial infarction (MI). Moreover, statins can improve left ventricular function and increase survival rate in rats without eNOS functional defects after MI, further indicating the important role of eNOS in left ventricular function after MI. The analysis of eNOS gene polymorphism also shows the clinical relevance of decreased eNOS function. A recent study confirmed that in the eNOS promoter sequence that can cause a decrease in the sensitivity of eNOS stress expression, the eNOS gene polymorphism is much more likely to appear in patients with coronary heart disease, further indicating that the decrease in eNOS activity is related to coronary heart disease. The precursor cells of VECs, endothelial progenitor cells (EPCs), were isolated from umbilical cord venous blood and studied, proving that vascular endothelial cells can secrete a variety of cell growth factors, thereby promoting the angiogenesis process and facilitating the formation of new blood vessels. Some people have explored the transplantation of vascular endothelial cells to treat cardiovascular diseases and enhance the growth of collateral vessels and angiogenesis in ischemic diseases.

[0004] Since endothelial cells can only be obtained by isolating them from living organisms, and human resources are limited, how to obtain a large number of endothelial cells is an important research direction. Since the advent of induced pluripotent stem cell (iPSC) technology, stem cell biology and regenerative medicine have made great progress. Human iPSCs have been widely used in cell therapy development. The endothelial cells induced by iPSCs are derived from humans, are easy to obtain, scalable, can produce almost any desired cell type, avoid ethical issues related to human ESCs, and have good application prospects.

[0005] Acute myocardial infarction (AMI) is a common clinical emergency. AMI has clinical characteristics such as rapid onset, rapid changes, and high mortality, which requires every second to be saved when treating it. Clinically, the main treatment principles are to restore myocardial blood perfusion, reduce the scope of myocardial ischemia, avoid the expansion of infarction, and protect and maintain cardiac function. Drug treatment of AMI includes thrombolytic therapy, the use of nitrates, anticoagulation and antiplatelet therapy, etc. Studies have also found that mesenchymal stem cells can differentiate into vascular endothelial cells and vascular smooth muscle cells, directly form new blood vessels to restore the blood supply of ischemic myocardium, and rescue myocardial cells on the verge of apoptosis for the treatment of acute myocardial infarction. Studies have also found that after endothelial progenitor cells or vascular endothelial cells are transplanted into mice, the generated vascular endothelial cells secrete cytokines such as VEGF, which can promote capillary proliferation, improve local blood supply, and promote differentiation to form new blood vessels to achieve the treatment of myocardial infarction. However, at present, the therapeutic effect of simply inhibiting vascular endothelial cells is not high enough, and combined use of drugs to achieve rapid and effective treatment of myocardial infarction is an important research direction.

[0006] Inflammatory response is the key to myocardial tissue degradation and cell apoptosis. Therefore, timely and effective inhibition of inflammatory response after myocardial infarction is important for preventing ventricular remodeling and protection after myocardial infarction. CCL21 / CCR7 is widely expressed in non-lymphoid tissues such as fibroblasts, smooth muscle cell nuclei and endothelial cells, and is involved in biological effects such as inflammation regulation, smooth muscle cell proliferation and matrix remodeling. Treatment with anti-CCL21 monoclonal antibodies can significantly inhibit the increase in myocardial MMP-9 expression and inflammatory response after acute myocardial infarction, playing a role in protecting post-infarction cardiac remodeling and improving cardiac function. However, due to the high cost and strict conditions for use of CCL21 monoclonal antibodies, it is not suitable for large-scale promotion and use. Summary of the invention

[0007] The invention provides a novel CCL21 inhibitory peptide, which is obtained by screening from a peptide library through molecular docking.

[0008] Furthermore, the CCL21 inhibitory peptide is CCL21-IN-2, and its amino acid sequence is shown in SEQ ID NO:1.

[0009] Specifically, the present invention provides use of the CCL21 inhibitory peptide CCL21-IN-2 in the preparation of a drug for inhibiting the activity of CCL21.

[0010] Furthermore, the present invention provides use of the CCL21 inhibitory peptide CCL21-IN-2 in preparing a drug for treating acute myocardial infarction.

[0011] Furthermore, the present invention also provides iPSC-induced vascular endothelial cells.

[0012] Specifically, the iPSC-induced vascular endothelial cells are purchased commercially.

[0013] Alternatively, the iPSC-induced vascular endothelial cells are prepared by inducing iPSC cells using a commercial vascular endothelial induction kit. Specifically, the iPSC cells are induced using DEF-CS TM iPSC cell directed differentiation reagent to vascular endothelial cells is used for the directed differentiation of human iPSC cells to vascular endothelial cells. The kit contains 2 coating agents, 2 basal culture media for differentiation and 6 culture media additives. The specific differentiation induction is carried out according to the instructions. Take 3 single wells of 6-well plates (2×10 5 cels) IPS cells were first treated with Celartis@DEF-CS TM 500CutureSystem (Y30010) was used for 4 generations, followed by mesodermal differentiation, endothelial cell differentiation, maturation and expansion culture according to the kit, with a differentiation cycle of 12 days (Day 0 to Day 12). The vascular endothelial cells obtained by induced differentiation were identified to express vascular endothelial markers such as CD31, CD144, Tie2 and VEGFR2. The positive cells were screened and expanded using MiraCell EC Culture Medium (Y50053) and then frozen for future use.

[0014] Furthermore, the present invention provides use of CCL21 inhibitory peptide CCL21-IN-2 and iPSC-induced vascular endothelial cells in preparing a drug kit for treating acute myocardial infarction.

[0015] Specifically, the medicine kit also includes instructions for use. The two components in the medicine kit are stored separately for standby use. The CCL21 inhibitory peptide CCL21-IN-2 exists in the form of a pharmaceutical composition.

[0016] The pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.

[0017] Examples of suitable pharmaceutically acceptable excipients include one or more polymers, wetting agents or surfactants, pH adjusters, isotonicity adjusters, preservatives, buffers, and chelating agents, or any combination thereof.

[0018] Examples of suitable pharmaceutically acceptable pH adjusting agents include, but are not limited to, sodium hydroxide, citric acid, hydrochloric acid, boric acid, acetic acid, phosphoric acid, succinic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium oxide, calcium carbonate, magnesium carbonate, magnesium aluminum silicate, malic acid, potassium citrate, sodium citrate, sodium phosphate, lactic acid, gluconic acid, tartaric acid, 1,2,3,4-butanetetracarboxylic acid, fumaric acid, diethanolamine, monoethanolamine, sodium carbonate, sodium bicarbonate, triethanolamine, or any combination thereof. In one embodiment, the pharmaceutically acceptable pH adjusting agent is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0019] Examples of suitable pharmaceutically acceptable wetting agents or surfactants include, but are not limited to, amphoteric, nonionic, cationic or anionic molecules. Suitable surfactants include, but are not limited to, polysorbates, sodium dodecyl sulfate (sodium lauryl sulfate), dodecyl dimethylamine oxide, docusate sodium, cetyl trimethyl ammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, N, N-dimethyl dodecylamine-N-oxide, cetyl trimethyl ammonium bromide, polyoxyethylene (10) lauryl ether, surfactants (plant oil-based fatty alcohol polyoxyethylene ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol), bile salts (e.g., sodium deoxycholate and sodium cholate), polyoxyethylene Ethylene castor oil, nonylphenol ethoxylate, cyclodextrin, lecithin, methylbenzethonium chloride, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils and fats, sulfated esters, sulfated alkanolamides, alkylphenols (ethoxylated and sulfated), ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylates, polyethylene glycol esters, sorbitan esters and their ethoxylated derivatives, fatty acid glycol esters, carboxamides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, etc.

[0020] Furthermore, iPSC-induced endothelial cells are stored in a freeze-dried form.

[0021] In the heart disease therapeutic agent of the present invention, the amount of iPSC-induced vascular endothelial cells used may vary depending on the patient's state, weight, age, symptoms, physical condition, etc., as well as the dosage form of the heart disease therapeutic agent of the present invention, but from the perspective of fully exerting the therapeutic effect of the heart disease therapeutic agent, the greater the amount of iPSC-induced vascular endothelial cells, the better. Generally, when administering to adults, the number of cells used as iPSC-induced vascular endothelial cells is 1×10 3 -1×10 12 The preferred number / time is 1×10 4 -1×10 11 Better than 1×10 5 -1×10 10 The better one is 5×10 6 -1×10 9 Pieces / times.

[0022] In addition, the dosage of CCL21 inhibitory peptide CCL21-IN-2 is preferably 0.1 mg to 300 mg / time, preferably 5 mg to 100 mg / time, and preferably 10 mg to 50 mg / time. The dosage of CCL21 inhibitory peptide CCL21-IN-2 per patient's body weight is preferably 1 μg to 50 mg / kg, preferably 100 μg to 50 mg / kg, and more preferably 200 μg to 50 mg / kg. In addition, the CCL21 inhibitory peptide CCL21-IN-2 can be administered multiple times as a single dose, or the dosage can be divided into multiple doses. For example, the patient is administered once / day.

[0023] Beneficial Effects

[0024] The present invention provides a combination of iPSC-induced vascular endothelial cells and polypeptides for treating cardiovascular and cerebrovascular diseases. The present invention also provides an inhibitory peptide that specifically inhibits CCL21 activity, which can treat acute myocardial infarction by inhibiting the activity of CCL21 and reducing the inflammatory regulation of CCL21 / CCR7 signals. The combined use of CCL21 inhibitory peptides and iPSC-induced vascular endothelial cells can significantly reduce the area of ​​myocardial infarction and increase the vascular surface density of myocardial tissue, and has a good therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Effect of peptides on relative chemotactic activity of cells

[0026] Figure 2 Results of infarct area in each group DETAILED DESCRIPTION

[0027] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0028] Example 1 Screening of CCL21 inhibitory peptides

[0029] Enter "CCL21" in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and retrieve the amino acid sequence and protein three-dimensional structure data of human CCL21 protein and mouse CCL21 protein in combination with the Protein Crystal Database (PDB). Molecular docking was performed using the 9-peptide compound database constructed by the applicant, and flexible molecular docking was performed using the Molecular Operating Environment software. The London free energy algorithm was used for preliminary screening, and then the GBVI / WSA free energy algorithm was used to select and sort the peptide structures that can antagonize the binding of human and mouse CCL21 to CCR7 from the preliminary screening results. The top 10 peptides were selected for HTRF detection, and the interaction between Tag1-CCL21 and its receptor Tag2-CCR7 was detected by using Anti-Tag1 Eu Cryptate reagent (donor) and Anti-Tag2XL665antibody (receptor). When CCL21 and CCR7 are tightly bound, the donor and the acceptor are brought close together. When the donor is excited by 320nm excitation light, the emitted fluorescence will be absorbed by the acceptor as its excitation light, and the acceptor will emit 665nm fluorescence. This phenomenon is called fluorescence resonance energy transfer. When the binding of CCL21 and CCR7 is blocked by a peptide inhibitor, the distance between the donor and the acceptor becomes larger. When the donor is excited by 320nm excitation light, fluorescence resonance energy transfer will not occur at this time, and only the 620nm fluorescence emitted by the donor can be detected. According to the ratio of Signal665 and Signal620, the peptide inhibitor with better blocking effect is screened. Specifically, prepare a 96-well plate, add 4μL 1×Tag1-CCL21 solution to each well, dilute the peptide to 1mmol / L with PPI Europium Detection Buffer, add 2μL to each well (add 2μL buffer to the positive control and negative control groups), add 4μL 1×Tag2-CCR7 solution (add 4μL buffer to the negative control), and incubate at room temperature for 15min. Add 1:1 premixed Anti-Tag1Eu and Anti-Tag2 XL665 to a 96-well plate, add 10 μL to each well, and treat 3 replicates for each well. Seal the plate and incubate at room temperature for 2 hours. With an excitation light of 320 nm, measure the signal values ​​at 665 nm and 620 nm using a full-wavelength microplate reader. The results are shown in Table 1.

[0030] Table 1 Blocking effect of peptides on CCL21 and CCR7

[0031] Peptide name Inhibition rate / % FKRWSVRWW 26.5±0.6 FGTALAYAG 85.6±1.2 ICQLMVK 13.8±0.3 QEDFFAIHD 33.1±0.8 QAGLHRATA 40.5±1.0 CPIIVHKHT 19.5±0.3 TNFGRVSYK 66.4±1.7 VWANEMWNF 38.5±0.4 AWVPANKLW 50.3±0.8 MQPGWGAPK 26.7±0.2

[0032] The protein level activity detection used HTRF experiment to evaluate the blocking effect of peptides on the in vitro binding of CCL21 and CCR7. The results are shown in Table 1. Two peptides had an inhibition rate of more than 60% on the binding of CCL21 and CCR7. Among them, peptide No. 2 (FGTALAYAG) had an inhibition rate of 85.6% on the binding of CCL21 and CCR7. It can effectively inhibit the binding of CCL21 and CCR7 in vitro. This peptide was named CCL21-IN-2 for subsequent experiments.

[0033] Example 2 Identification of biological activity of CCL21 inhibitory peptide CCL21-IN-2

[0034] The chemotaxis inhibition activity was used to detect the biological activity of CCL21 inhibitory peptide CCL21-IN-2. 1640 medium containing 3% FBS and 10nmol / L recombinant CCL21 protein was added to the lower chamber of Transwell, in which different final concentrations of CCL21 inhibitory peptide CCL21-IN-2 polypeptide 1μg / mL, 50μg / mL, 100μg / mL were added, and no inhibitory peptide was added as a control group. Human colon cancer cell SW480 was cultured for 24h without serum and the density was 5x10 3 / mL of cell suspension was added to the upper chamber of Transwell (1.5mL / well); cultured at 37℃, 5% CO2 for 3h, the Transwell was removed, the cells were fixed with 4% paraformaldehyde, and then stained with hematoxylin-eosin. Ten fields of view (10×25 times) were randomly selected under a microscope to count the number of cells on the back of the Transwell membrane. The relative chemotactic activity of the cells was expressed as the ratio (%) of the mean number of cells in the experimental group to the mean number of cells in the control group. The blank control group was a cell without any peptide treatment or recombinant CCL21 protein. The results are shown in Figure 1 shown.

[0035] The results of detecting the antagonistic effect of inhibitory peptide CCL21-IN-2 on CCL21-mediated SW480 cell migration are as follows Figure 1 As shown in the figure, the number of SW480 cells that migrated to the lower chamber in the CCL21+inhibitory peptide CCL21-IN-2 group was significantly reduced compared with the CCL21 group (P<0.05), and the antagonistic effect showed a dose-dependency. Under the condition of 100μg / mL peptide treatment, the relative chemotactic activity of the cells was only (29.5±1.3)%, and the inhibitory activity was good. This fully demonstrates that the inhibitory peptide CCL21-IN-2 can have a good inhibitory effect on the migration of colon cancer cells induced by CCL21, and can be used as an ideal CCL21-CCR7 antagonist.

[0036] Example 3 Therapeutic effects of CCL21 inhibitory peptide CCL21-IN-2 and iPSC-induced vascular endothelial cells on myocardial infarction

[0037] iPSC cells were purchased from CellArtis, CellArtis 00185 Human iPs cell line P11025 product number: Y00185.

[0038] iPSC cells were cultured using DEF-CS (Takara Cellarits catalog number Y50301) TM iPSC cell directed differentiation reagent to vascular endothelial cells is used for the directed differentiation of human iPSC cells to vascular endothelial cells. The kit contains 2 coating agents, 2 basal culture media for differentiation and 6 culture media additives. The specific differentiation induction is carried out according to the instructions. Take 3 single wells of 6-well plates (2×10 5 cels) IPS cells were first treated with Celartis@DEF-CS TM 500CutureSystem (Y30010) was used for 4 generations, followed by mesodermal differentiation, endothelial cell differentiation, maturation and expansion culture according to the kit, with a differentiation cycle of 12 days (Day 0 to Day 12). The vascular endothelial cells obtained by induced differentiation were identified to express vascular endothelial markers such as CD31, CD144, Tie2 and VEGFR2, of which CD31+CD144+ positive cells accounted for 96.3%. After screening, the positive cells were expanded and cultured using MiraCell EC Culture Medium (Y50053) and then frozen for future use.

[0039] Male C57BL / 6 mice were 10 weeks old and weighed (20-26) g. C57BL / 6 mice were first randomly divided into sham operation group, model group, CCL21 peptide intervention group, iPSC-induced vascular endothelial cells combined with CCL21 peptide intervention group (referred to as combined intervention group), and the positive control group was befozi plus heparin. Mice that died unexpectedly due to anesthesia and surgery during the experiment were not included in the experimental animals, and the surviving mice were further randomly divided into groups, with 10 mice in each group. The process of making the mouse myocardial infarction model is as follows: under inhalation anesthesia, the 3rd and 4th intercostal spaces on the left side of the mouse were incised to expose the heart, and the left anterior descending branch of the coronary artery was permanently ligated; the sham operation group only hung the thread without ligating the coronary artery before and after the chest was opened to record the II lead electrocardiogram. The model was successful when the myocardium turned white and the pulsation weakened below the ligation site and the ST segment was significantly elevated on the electrocardiogram. On the day of modeling, 20,000 u / kg body weight of gentamicin was injected ip once a day for 3 days. After modeling, the sham operation group and the model group were intravenously injected with 1 mL of normal saline every day, and the CCL21 peptide intervention group was intravenously injected with 20 mg / kg of peptide every day. The iPSC-induced vascular endothelial cells combined with CCL21 peptide intervention group were intravenously injected with 20 mg / kg of peptide every day. At the same time, the chest was opened again 24 hours after the operation, and iPSC-induced vascular endothelial cells (1×10 6 / mL, 20μl was injected at each injection point). The positive control group was given Befuji plus heparin 20mg / kg every day. On the 21st day after surgery, the mice were killed. The infarct area was detected: the fixed heart was routinely dehydrated, transparent, paraffin-embedded, sliced, and stained with hematoxylin-eosin (HE), and myocardial necrosis was observed under a light microscope. The IMS cell image analysis system was used to test the myocardial infarction area and left ventricular area under a 10x objective microscope, and the infarction area (%) was expressed as myocardial infarction area / left ventricular area × 100%. The results are shown in Figure 2 shown.

[0040] from Figure 2 It can be seen that the CCL21 polypeptide intervention group, the iPSC-induced vascular endothelial cells combined with the CCL21 polypeptide intervention group and the positive control group all significantly reduced the myocardial infarction area of ​​the model mice (P<0.05). In particular, after the iPSC-induced vascular endothelial cells combined with the CCL21 polypeptide were treated together, the infarction area was reduced to (3.5±0.3)%, which had a good effect in reducing myocardial infarction.

[0041] Furthermore, the sections of myocardial tissue after immunohistochemical staining of factor VIII were processed by the IMS cell image analysis system to measure the number of blood vessels showing positive staining of factor VIII in the edge area of ​​the left ventricular infarction. Five fields of view (×100) were randomly observed for each section, and the average was taken. The number of blood vessels per square millimeter, i.e., the surface density, was calculated based on the measurement window area. The results are shown in Table 2.

[0042] Table 2 Effects of each group on vascular surface density

[0043] Group <![CDATA[Vascular surface density (number / mm 2 )]]> Model Group 325.7±30.5* Sham operation group 130.5±8.9 CCL21 peptide intervention group 594.5±43.7#* Combined intervention group 690.5±53.6#* Positive control group 580.4±48.3#*

[0044] * indicates a significant difference compared with the sham operation group (P<0.01), # indicates a significant difference compared with the model group (P<0.01).

[0045] The vascular surface density of the model group was higher than that of the sham operation group, indicating that compensatory angiogenesis occurred after myocardial infarction. As can be seen from Table 2, each treatment group had a significant effect on promoting angiogenesis compared with the model group, especially the vascular endothelial cells used in the combined treatment group could secrete vascular endothelial cell growth factor VEGF and basic fibroblast growth factor bFGF, which could significantly promote angiogenesis in the myocardial tissue around the infarction.

[0046] Furthermore, ultrasound detection revealed that the CCL21 polypeptide intervention group, especially the combined intervention group, could significantly increase the left ventricular ejection fraction LVEF and left ventricular short-axis shortening fraction LVFS levels of mice, reduce the levels of LVEED and LVESD, and the cardiac function was significantly improved compared with the model group.

[0047] Although the embodiments of the present invention are described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present invention, all of which belong to the protection of the present invention.

Claims

1. A CCL21 inhibitory peptide, named CCL21-IN-2, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the CCL21 inhibitory peptide CCL21-IN-2 according to claim 1 in the preparation of an agent for inhibiting CCL21 activity.

3. Use of the CCL21 inhibitory peptide CCL21-IN-2 according to claim 1 in the preparation of a medicament for treating acute myocardial infarction.

4. Use of the CCL21 inhibitory peptide CCL21-IN-2 according to claim 1 and iPSC-induced vascular endothelial cells in the preparation of a drug kit for treating acute myocardial infarction.

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