Application of chemokine CCL17 as a therapeutic target in the preparation of products that inhibit or delay aging

Abstract

The present invention discloses the use of the chemokine CCL17 as a therapeutic target in the preparation of products that inhibit or delay aging. The present invention also discloses the use of substances that inhibit the activity of CCL17 protein or substances that reduce the content of CCL17 protein in inhibiting or delaying aging, promoting angiogenesis, treating or improving vascular dysfunction and reducing vascular remodeling. The present invention demonstrates through experiments that the increased level of CCL17 during aging is a key regulatory factor for vascular remodeling and aging, and also demonstrates that CCL17 can be used as a target for reducing vascular remodeling. The present invention reveals for the first time that CCL17 can be used as a new therapeutic target in aging and Ang II-induced vascular dysfunction. Targeting CCL17 will be a novel and promising strategy for angiogenesis based on anti-inflammatory aging.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of chemokine CCL17 as a therapeutic target in the preparation of products that inhibit or delay aging. Background Art

[0002] Aging leads to increased susceptibility to age-related diseases, particularly cardiovascular disease, which has become a public health priority. Recent studies have increasingly revealed the critical role of the vasculature as a gatekeeper to lifespan and healthspan. From this perspective, angiogenesis has anti-aging potential.

[0003] Circulating proteomic signatures are closely associated with aging and aging-related diseases. However, no drug targeting circulating proteins has a definitive therapeutic effect on aging-induced vascular dysfunction. Chronic inflammation is an important contributor to multi-organ regeneration throughout the body. For example, a modest increase in circulating vascular endothelial growth factor (VEGF) can reduce chronic inflammation, thereby improving overall health and extending lifespan in mice. Circulating cytokines and chemokines are important components of the immune system and are implicated in cardiovascular homeostasis and aging-related cardiovascular diseases.

[0004] The vascular system is considered one of the major determinants of aging and lifespan. Vascular regeneration has recently been shown to promote healthy aging and extend the healthy lifespan of animals. Therefore, identifying the drivers of vascular aging is crucial for delaying aging. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to treat or improve vascular dysfunction, alleviate vascular remodeling, regenerate blood vessels, and inhibit or delay aging.

[0006] In order to solve the above technical problems, the present invention provides a novel use of a substance for inhibiting the activity of CCL17 protein or a substance for reducing the content of CCL17 protein.

[0007] The present invention provides the use of a substance that inhibits CCL17 protein activity or reduces CCL17 protein content in any of the following a1) to a8):

[0008] a1) Preparation of products that inhibit or delay aging;

[0009] a2) Inhibit or delay aging;

[0010] a3) preparing products that promote angiogenesis;

[0011] a4) Promote angiogenesis;

[0012] a5) Preparation of products for treating or improving vascular dysfunction;

[0013] a6) Treat or improve vascular dysfunction;

[0014] a7) preparing a product that attenuates vascular remodeling;

[0015] a8) Attenuate vascular remodeling.

[0016] In order to solve the above technical problems, the present invention also provides a product.

[0017] The active ingredient of the product is a substance that inhibits the activity of CCL17 protein or a substance that reduces the content of CCL17 protein; the function of the product is any one of the following b1) to b4):

[0018] b1) Inhibit or delay aging;

[0019] b2) Promote angiogenesis;

[0020] b3) Treat or improve vascular dysfunction;

[0021] b4) Attenuate vascular remodeling.

[0022] In any of the above-mentioned uses or products, the vascular dysfunction is vascular dysfunction induced by aging or Ang II.

[0023] The vascular remodeling is vascular remodeling induced by aging or Ang II.

[0024] The treatment or improvement of vascular dysfunction or the reduction of vascular remodeling is specifically embodied in any one of the following c1) to c5):

[0025] c1) inhibit the increase of arterial stiffness;

[0026] c2) reduce pulse wave velocity (PWV);

[0027] c3) improve phenylephrine-induced vasoconstriction and endothelium-dependent / independent relaxation;

[0028] c4) reduce the ratio of aorta weight to body weight;

[0029] c5) reduce the ratios of aortic media thickness / vascular lumen and media area / vascular lumen;

[0030] In any of the above-mentioned applications or products, the substance that inhibits the activity of the CCL17 protein may be a protein, polypeptide or small molecule compound that inhibits the function of the CCL17 protein.

[0031] The substance that reduces the CCL17 protein content may be a substance that inhibits CCL17 protein synthesis, promotes CCL17 protein degradation, or knocks down or knocks out the CCL17 gene.

[0032] The substance that inhibits the synthesis of CCL17 protein can be a substance that inhibits the expression of the gene encoding CCL17 protein, such as a substance that silences the gene encoding CCL17 protein in animals (such as miRNA, siRNA, dsRNA, shRNA, etc.).

[0033] The knockout means that the host cell carrying the knockout substance does not produce the functional protein product of the gene. The knockout substance can be a substance that achieves the host cell not producing the functional protein product of the gene in any way, such as removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or changing the regulatory component (such as promoter editing) so that the coding gene sequence is not transcribed, preventing translation by binding to mRNA, etc. Usually, the knockout is performed at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0034] In a specific embodiment of the present invention, the substance that inhibits the activity of CCL17 protein or reduces the content of CCL17 protein is a CCL17 antibody or a CCL17 gene knockout system.

[0035] The use of CCL17 as a target in any of the following d1) to d4) also falls within the scope of protection of the present invention:

[0036] d1) Developing or preparing products that inhibit or delay aging;

[0037] d2) Developing or preparing products that promote angiogenesis;

[0038] d3) Developing or preparing products for treating or improving vascular dysfunction;

[0039] d4) Develop or prepare products that reduce vascular remodeling.

[0040] The use of CCL17 as a target in constructing an aging animal model or screening products that inhibit or delay aging also falls within the scope of protection of the present invention.

[0041] The amino acid sequence of any of the above-mentioned CCL17 proteins is shown in SEQ ID NO: 1.

[0042] The nucleotide sequence of any of the above-mentioned CCL17 genes is shown in Sequence 2.

[0043] The present invention constructed whole-body CCL17 knockout mice (Ccl17-KO). Ccl17-KO mice and their littermate control group (WT) mice were maintained for 21 months to obtain aged Ccl17-KO and WT mice. There was no difference in weight, heart rate, and blood pressure between aged Ccl17-KO and WT mice. However, compared with aged WT mice, the pulse wave velocity (PWV, an indicator of arterial stiffness in the elderly) of aged Ccl17-KO mice was reduced ( Figure 1 A). In addition, in vitro functional studies analyzed vascular contraction and relaxation, and found that CCL17 knockout improved phenylephrine-induced vasoconstriction and endothelium-dependent / independent relaxation in aged mice ( Figure 1 B). These findings suggest that CCL17 is involved in aging-induced vascular remodeling. Further pathological analysis then showed that the aortic weight / body weight ratio was increased in aged WT mice, while that in aged Ccl17-KO mice was decreased compared with that in young WT mice ( Figure 1 C). Hematoxylin-eosin (H&E) staining showed that the medial thickness and medial area / vascular lumen ratio of the thoracic aorta in aged WT mice were increased compared with those in young WT mice, but the pathological vascular remodeling in aged mice was attenuated by the loss of CCL17 ( Figure 1 D). In addition, CCL17 knockout was observed to alleviate vascular arterial stiffness, vasomotor dysfunction, and pathological vascular remodeling in a model of vascular dysfunction induced by the aging-related vasoconstrictor angiotensin II (Ang II). Figure 1 EH). Collectively, these findings suggest that CCL17 deficiency has a protective effect on aging arteries.

[0044] To determine whether CCL17 inhibition could be used as a new therapeutic strategy, mice were treated with Ang II and CCL17 antibodies or isotype IgG antibodies for 4 weeks. It was found that serum CCL17 levels increased after Ang II exposure, while CCL17 antibodies almost completely offset the above effects without affecting blood pressure and heart rate. Compared with IgG-treated controls, mice treated with CCL17 antibodies showed a significant reduction in Ang II-induced vascular arterial stiffness ( Figure 1 I). In addition, the present invention also found that after administering therapeutic CCL17 neutralizing antibodies to mice treated with Ang II, systolic and diastolic function was improved ( Figure 1 J). Vascular remodeling was also assessed, and it was observed that treatment with a CCL17 neutralizing antibody blocked the effects of Ang II on aortic weight increase and pathological vascular remodeling ( Figure 1 K and Figure 1These studies demonstrate that CCL17 exacerbates aging- and Ang II-induced pathological vascular remodeling. Therapeutic administration of a CCL17-neutralizing antibody inhibits vascular remodeling.

[0045] This study experimentally demonstrates that elevated CCL17 levels during aging are a key regulator of vascular remodeling and aging, and also demonstrates that CCL17 can serve as a target for attenuating vascular remodeling. This study reveals for the first time that CCL17 can serve as a novel therapeutic target for aging and Ang II-induced vascular dysfunction. Targeting CCL17 is a novel and promising strategy for anti-inflammatory aging-based angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Knockout or inhibition of CCL17 can suppress aging- or Ang II-induced vascular dysfunction in mice. *P<0.05, **P<0.01, ***P<0.001. One-way analysis of variance was used for statistical analysis. Figures a and b show the results of studies on "CCL17 knockout suppresses aging-induced vascular dysfunction"; Figures eh show the results of studies on "CCL17 knockout suppresses Ang II-induced vascular dysfunction"; and Figure il shows the results of studies on "CCL17 neutralizing antibodies suppress Ang II-induced vascular dysfunction." DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0048] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0049] The wild-type C57BL / 6J mice (male) in the following examples are products of Beijing Weitonglihua Animal Company.

[0050] The Ccl17 knockout mice (Ccl17-KO) in the following examples are described in the document “Zhang Y, Ye Y, Tang X, Wang H, Tanaka T, Tian R, et al. CCL17 acts as a novel therapeutic target inpathological cardiac hypertrophy and heart failure. J Exp Med 2022; 219. DOI: 10.1084 / jem.20200418.”

[0051] The experimental animals used in the following examples were housed as follows: Mice were housed in a pathogen-free facility with free access to food and maintained under a standard 12-hour light / dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Chinese Academy of Medical Sciences and Peking Union Medical College. To analyze the role of Ccl17 in vascular aging, wild-type C57BL / 6J mice and Ccl17-KO mice were maintained for 21 months.

[0052] The pulse wave velocity (PWV) measurement steps in the following embodiment are as follows: Pulse wave Doppler ultrasound velocity measurements are continuously performed at the distal and proximal positions of the carotid artery, while the electrocardiogram (ECG) signal is recorded for a short period of time, during which the mouse is observed to have a stable heart and respiratory rate. The conduction time is similar to the most recent arrival time determined by subtracting the distal arrival time between the peak of the ECG R wave and the foot of the velocity upstroke. For consistency of detection, the arrival time is fixed to the time to reach 20% of the peak. The left common carotid artery (LCCA) was selected as the measurement target because it easily provides a uniform and branchless measurement path of more than about 10 mm between the aortic arch and the aortic arch. The distance between the measurement points, including the distal and proximal positions, is determined by B-ultrasound. The proximal velocity measurement portion is 1 mm downstream of the aortic arch. The distal velocity measurement is performed at the common carotid artery bifurcation 1.5 mm upstream.

[0053] The specific steps of the vasodilation test method in the following examples are as follows: After sacrificing mice, the aorta was removed and suspended in a wire myograph (Danish Myo Technology, Aarhus, Denmark) to record changes in isometric force. The operating conditions were Krebs solution (solvent: water; solutes: 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, and 11 mM D-glucose) with 95% O2-5% CO2 ventilation support. Once stable tension was induced by phenylephrine (Phe, 1 μmol / L), acetylcholine (ACh, an endothelium-dependent agonist) was gradually added (3 nmol / L-10 μmol / L) to induce endothelium-dependent relaxation. After the decline stabilized, 100 mM of NOS inhibitor L-Name was added, and after waiting for 5-10 minutes, sodium nitroprusside (SNP, endothelium-independent agonist) was added to induce endothelium-independent relaxation.

[0054] The specific steps of the histopathological analysis method in the following examples are as follows: For histological analysis, mouse aortas were fixed in 4% paraformaldehyde. The fixed aortas were embedded in paraffin, cut into 5 μm sections, and stained with a hematoxylin and eosin (H&E) kit (Servicebio, G1005). Medial thickness and medial area / lumen ratio were analyzed using Image-Pro Plus 6.0.

[0055] Ang II in the following examples is a product of Sigma-Aldrich, with the product number being #A9525.

[0056] The CCL17 antibody used in the following examples is a product of R&D Systems, with the catalog number #MAB529.

[0057] The IgG antibody used in the following examples is a product of R&D Systems, catalog number #MAB006.

[0058] Example 1: Knockout or inhibition of CCL17 can inhibit aging or Ang II-induced vascular dysfunction

[0059] The test animals are as follows:

[0060] Young group (Young): 4-month-old wild-type C57BL / 6J mice (Young+WT) and 4-month-old Ccl17 knockout C57BL / 6J mice (Young+Ccl17-KO).

[0061] Aged group: 21-month-old wild-type C57BL / 6J mice (Aged+WT) and 21-month-old Ccl17 knockout C57BL / 6J mice (Aged+Ccl17-KO).

[0062] All mice were male.

[0063] 1. CCL17 knockout can inhibit vascular dysfunction caused by aging

[0064] 1. Pulse Wave Velocity (PWV) Measurement

[0065] The pulse wave velocity (PWV), an indicator of arterial stiffness in the elderly, was measured in the aorta of young and old mice. Each group contained 11 or 12 wild-type and Ccl17-KO mice.

[0066] The results are as follows Figure 1 As shown in A, the results showed that the pulse wave velocity (PWV) of aged Ccl17-KO mice was reduced compared with that of aged WT mice, indicating that CCL17 knockout can inhibit the increase in arterial stiffness caused by aging.

[0067] 2. In vitro analysis of aortic vasomotor function

[0068] Vasomotor function in the aorta of young and old mice was analyzed in vitro. Each group contained six wild-type and Ccl17-KO mice.

[0069] The results are as follows Figure 1 Figure B shows the left panel showing arterial vasoconstriction mediated by phenylephrine; the middle panel shows the response to acetylcholine, reflecting endothelium-dependent relaxation; and the right panel shows the response to the nitric oxide (NO) donor sodium nitroprusside, reflecting endothelium-independent relaxation. This indicates that CCL17 knockout improves phenylephrine-induced vasoconstriction and endothelium-dependent / independent relaxation in aged mice.

[0070] 3. Aortic weight / body weight ratio

[0071] The aorta weight and body weight of young and old mice were measured, and the ratio of aorta weight to body weight was calculated. The number of wild-type and Ccl17-KO mice in each group was 11 or 12.

[0072] The results are as follows Figure 1 As shown in Figure C, the results show that compared with young WT mice, the aorta weight / body weight ratio of old WT mice increased, while that of old Ccl17-KO mice decreased, indicating that CCL17 knockout reduced the aorta weight / body weight ratio of old mice.

[0073] 4. Aortic media thickness and media area / vascular lumen ratio

[0074] Thoracic aortas from young and aged mice were stained with hematoxylin and eosin (H&E), and the medial thickness and medial area / lumen ratio were quantified. Each group contained 7 or 8 wild-type and Ccl17-KO mice.

[0075] The results are as follows Figure 1 As shown in Figure D, the results showed that compared with young WT mice, the thoracic aorta media thickness and media area / vascular lumen ratio were increased in aged WT mice, but the pathological vascular remodeling in aged mice was attenuated by CCL17 deficiency, indicating that CCL17 knockout attenuated vascular remodeling in aged mice.

[0076] 2. CCL17 knockout inhibits angiotensin II (Ang II)-induced vascular dysfunction

[0077] Young wild-type C57BL / 6J mice and Ccl17-KO mice were subcutaneously implanted with angiotensin II (Ang II) sustained-release pumps for 4 weeks at a dose of 1.3 mg / kg / day to create Ang II model mice. The aortas of these mice were then analyzed. Mice implanted with saline sustained-release pumps served as controls, creating the saline control group.

[0078] 1. Pulse Wave Velocity (PWV) Measurement

[0079] The pulse wave velocity (PWV, an indicator of arterial stiffness in the elderly) of the aorta was measured in the Ang II and Saline groups, respectively. The number of wild-type and Ccl17-KO mice in each group was 10, 11, or 12.

[0080] The results are as follows Figure 1 As shown in Figure E, the pulse wave velocity (PWV) values ​​of Ccl17-KO mice in the Ang II group were reduced compared with those of WT mice in the Ang II group, indicating that CCL17 knockout inhibited the increase in arterial stiffness induced by Ang II.

[0081] 2. In vitro analysis of aortic vasomotor function

[0082] Vasomotor function of the aorta was analyzed in vitro in mice in the Ang II and Saline groups, with six wild-type and six Ccl17-KO mice in each group.

[0083] The results are as follows Figure 1Figure F shows arterial vasoconstriction mediated by phenylephrine (LEP). The middle figure shows the response to acetylcholine, reflecting endothelium-dependent relaxation. The right figure shows the response to the nitric oxide (NO) donor sodium nitroprusside, reflecting endothelium-independent relaxation. This indicates that CCL17 knockout improves NE-induced vasoconstriction and endothelium-dependent / independent relaxation in Ang II mice.

[0084] 3. Aortic weight / body weight ratio

[0085] The aorta weight and body weight of mice in the Ang II and Saline groups were measured, and the aorta weight / body weight ratio was calculated. The number of wild-type and Ccl17-KO mice in each group was 10, 11, or 12.

[0086] The results are as follows Figure 1 As shown in Figure G, the results showed that compared with the WT mice in the Saline group, the aorta weight / body weight ratio of WT mice in the Ang II group was increased, while the aorta weight / body weight ratio of Ccl17-KO mice in the Ang II group was decreased, indicating that CCL17 knockout reduced the aorta weight to body weight ratio of Ang II mice.

[0087] 4. Aortic media thickness and media area / vascular lumen ratio

[0088] Thoracic aortas of mice in the Ang II and Saline groups were stained with hematoxylin and eosin (H&E), and the medial thickness and medial area / vascular lumen ratio were quantified. There were 7 or 8 wild-type and Ccl17-KO mice in each group.

[0089] The results are as follows Figure 1 As shown in Figure H, the results showed that compared with WT mice in the saline group, the thoracic aorta media thickness and media area / vascular lumen ratio of WT mice in the Ang II group were increased, but the pathological vascular remodeling in Ccl17-KO mice in the Ang II group was attenuated due to the loss of CCL17. This indicates that CCL17 knockout inhibits vascular remodeling in Ang II mice.

[0090] 3. CCL17 neutralizing antibodies can inhibit Ang II-induced vascular dysfunction

[0091] Young wild-type C57BL / 6J mice were subcutaneously implanted with angiotensin II (AngII) sustained-release pumps for 4 weeks at a dose of 1.3 mg / kg / day. They were also infused with a CCL17 neutralizing antibody (CCL17 antibody) or a control isotype antibody (IgG antibody) for 4 weeks at a dose of 100 μg / mouse / day. These mice were then subjected to Ang II aorta analysis. Mice implanted with saline sustained-release pumps served as controls, creating the control group (Saline).

[0092] 1. Serum CCL17 level, heart rate, and blood pressure measurement

[0093] The serum CCL17 level, heart rate and blood pressure of mice in the Ang II group and Saline group were measured respectively.

[0094] The results are shown in Tables 1 and 2. It was found that serum CCL17 levels increased after Ang II exposure, while CCL17 neutralizing antibodies almost completely offset the above effects without affecting blood pressure and heart rate.

[0095] Table 1. Serum CCL17 level detection results

[0096] Saline (n=8) Ang II+anti-IgG (n=8) Ang II + anti-CCL17 (n=8) CCL17 (pg / ml) 335.0±62.54 538.1±107.2*** <![CDATA[355.5±54.09 ## ]]>

[0097] Table 2. Blood pressure (diastolic and systolic) and heart rate test results

[0098]

[0099] 2. Pulse Wave Velocity (PWV) Measurement

[0100] The pulse wave velocity (PWV, an indicator of arterial stiffness in the elderly) of the aorta was measured in the Ang II and Saline groups. The number of mice injected with CCL17 antibodies and IgG antibodies in each group was 9, 10, or 11.

[0101] The results are as follows Figure 1 As shown in Figure 1, the pulse wave velocity (PWV) values ​​of mice injected with CCL17 antibodies in the Ang II group were lower than those of mice injected with IgG antibodies in the Ang II group, indicating that CCL17 antibodies inhibited the increase in arterial stiffness induced by Ang II.

[0102] 3. In vitro analysis of aortic vasomotor function

[0103] Aortic vasomotor function was analyzed in vitro in mice in the Ang II and Saline groups. Six mice were injected with either CCL17 antibody or IgG antibody in each group.

[0104] The results are as follows Figure 1 Figure J shows the left panel showing arterial vasoconstriction mediated by phenylephrine; the middle panel shows the response to acetylcholine, reflecting endothelium-dependent relaxation; and the right panel shows the response to the nitric oxide (NO) donor sodium nitroprusside, reflecting endothelium-independent relaxation. This indicates that the CCL17 antibody improves both phenylephrine-induced vasoconstriction and endothelium-dependent / independent relaxation in Ang II mice.

[0105] 4. Aortic weight / body weight ratio

[0106] The aorta weight and body weight of mice in the Ang II and Saline groups were measured, and the aorta weight / body weight ratio was calculated. The number of mice injected with CCL17 antibody and IgG antibody in each group was 9, 10, or 11.

[0107] The results are as follows Figure 1 As shown in Figure K, the results showed that compared with the IgG-injected mice in the Saline group, the aorta weight / body weight ratio of the mice in the Ang II group injected with IgG antibodies increased, while the aorta weight / body weight ratio of the mice in the Ang II group injected with CCL17 antibodies decreased. This indicates that the CCL17 antibody reduced the aorta weight to body weight ratio of the Ang II-treated mice.

[0108] 5. Aortic media thickness and media area / vascular lumen ratio

[0109] The thoracic aorta of mice in the Ang II and Saline groups were stained with hematoxylin-eosin (H&E), and the medial thickness and the ratio of medial area to vascular lumen were quantified. The number of mice injected with CCL17 antibodies and IgG antibodies in each group was 7, 8, or 9.

[0110] The results are as follows Figure 1 As shown in Figure 1, the results showed that compared with the IgG-injected mice in the Saline group, the Ang II-injected mice had increased thoracic aortic media thickness and media area / vascular lumen ratio. However, the pathological vascular remodeling in the Ang II-injected mice was attenuated by the CCL17 antibody. This suggests that the CCL17 antibody can inhibit vascular remodeling in Ang II-induced mice.

[0111] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of substances that inhibit CCL17 protein activity or substances that reduce CCL17 protein content in the preparation of products that promote angiogenesis in aging- or Ang II-induced vascular dysfunction; The substance that inhibits the activity of CCL17 protein or reduces the content of CCL17 protein is a CCL17 antibody.

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