Use of recombinant proteins in the preparation of drugs for inhibiting aneurysms

Abstract

The present invention relates to the use of recombinant proteins in the preparation of drugs for inhibiting aneurysms, and belongs to the field of biopharmaceuticals. The present invention discloses the use of recombinant AGGF1 protein in the preparation of drugs for inhibiting aneurysms. The recombinant AGGF1 protein can inhibit the TGF-β1 signaling pathway and vascular remodeling through its receptor integrin α7. Therefore, the recombinant AGGF1 protein inhibits aneurysms by inhibiting the synthesis of TGF-β1 and its signaling pathway, and the recombinant protein achieves aneurysm inhibition by improving vascular structure and function.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to the use of recombinant proteins in the preparation of drugs for inhibiting aneurysms. [Background technology]

[0002] Aneurysms are diseases characterized by focal or diffuse dilation or bulging of blood vessels due to lesions or damage to the blood vessel wall. Their main characteristics include phenotypic transformation of vascular smooth muscle cells, vasodilation, and altered hemodynamics. Aneurysms can occur anywhere, but they are most common and most severe in major arteries, such as the cerebral arteries and the aorta. Rupture of aneurysms in these locations carries a very high mortality rate. The causes of aneurysms are diverse, and chronic diseases such as atherosclerosis and hypertension can cause deterioration of the blood vessel wall. However, treatment options for aneurysms are limited. Surgical treatment can have side effects such as rupture and secondary infection, and there is no effective pharmacological treatment. Summary of the Invention [Problem to be solved by the invention]

[0003] In view of the above-mentioned shortcomings or needs for improvement in the prior art, the present invention provides a use of recombinant protein, the purpose of which is to use AGGF1 recombinant protein to prepare anti-aneurysm drugs, effectively inhibit the progression of aneurysms, and carry out clinical intervention. [Means for solving the problem]

[0004] According to the object of the present invention, there is provided a use of a recombinant protein in preparing a drug for inhibiting aneurysms, the amino acid sequence of which is as follows: (1) the amino acid sequence set forth in SEQ ID NO: 1; or (2) an amino acid sequence that has 80% to 100% identity with the amino acid sequence identified in SEQ ID NO: 1, retains amino acids 606 to 608, and encodes a protein with the same function; or (3) An amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence identified by SEQ ID NO: 1, retaining amino acids 606 to 608, and having activity equivalent to that of the protein shown in sequence SEQ ID NO: 1.

[0005] According to another aspect of the present invention, there is provided use of a composition of a recombinant protein and a TGF-β inhibitor in the preparation of a medicament for inhibiting aneurysms, the amino acid sequence of which is as follows: (1) the amino acid sequence set forth in SEQ ID NO: 1; or (2) an amino acid sequence that has 80% to 100% identity with the amino acid sequence identified in SEQ ID NO: 1, retains amino acids 606 to 608, and encodes a protein with the same function; or (3) An amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence identified by SEQ ID NO: 1, retaining amino acids 606 to 608, and having activity equivalent to that of the protein shown in sequence SEQ ID NO: 1.

[0006] Preferably, the TGF-β inhibitor is pirfenidone.

[0007] Preferably, the aneurysm is an aortic aneurysm or a carotid aneurysm.

[0008] Preferably, the aneurysm is an aneurysm caused by coarctation of the aortic arch.

[0009] Preferably, the aneurysm is an aneurysm caused by a mutation in the FBN1 gene.

[0010] Preferably, the aneurysm is a thoracic aortic aneurysm.

[0011] Preferably, the recombinant protein suppresses aneurysms by inhibiting the synthesis of TGF-β1 and its signaling pathway.

[0012] Preferably, the signal transduction pathway is TGFβ1-TGFβR1-Smad2 / 3 and TGFβ1-TGFβR1-ERK1 / 2.

[0013] Preferably, the recombinant protein achieves aneurysm inhibition by improving vascular structure and function.

[0014] Preferably, the deletion of the amino acids at positions 606 to 608 disrupts the interaction between the AGGF1 protein and its receptor integrin α7, resulting in the loss of the action of the AGGF1 protein. [Effects of the Invention]

[0015] Compared with the prior art, the above technical means of the present invention mainly have the following technical advantages: (1) The administration method of the present invention is simple. In mouse experiments, the protein can be effectively administered by intraperitoneal injection. (2) The present invention has remarkable effects. In the protein treatment experiment, the therapeutic effect of the protein administration group was very obvious, with vascular lesions almost completely restored to normal levels and aneurysms caused by vascular lesions effectively improved. (3) The present invention avoids post-operative complications. Protein therapy is administered by injection only and does not require surgical repair, which avoids side effects such as vascular inflammation and aneurysm rupture. (4) The recombinant AGGF1 protein provided by the present invention can improve vascular smooth muscle, suppress vascular inflammation, and treat aortic aneurysms and carotid aneurysms by inhibiting the synthesis of TGF-β1 and the TGF-β1 signaling pathway (Smads and ERK1 / 2). (5) In the present invention, AGGF1 is essential for pirfenidone to exert its therapeutic effect, which not only demonstrates the importance of AGGF1 but also expands the use of AGGF1 and pirfenidone in the treatment of aneurysms. Therefore, the recombinant AGGF1 protein containing a His tag of the present invention can be used to prepare antianeurysmal drugs for the treatment of aneurysmal diseases. (6) In this invention, the therapeutic effect of AGGF1 on thoracic aortic aneurysm-Marfan syndrome (Marfan syndrome) mice caused by FBN1 gene mutation and on a β-aminopropionitrile-induced aneurysm model has also been verified, demonstrating good application effects and a wide range of applications. [Brief explanation of the drawings]

[0016] [Figure 1]Aggf1 haploinsufficiency (heterozygous Aggf1+ / -) exacerbates transverse aortic constriction (TAC)-induced dilatation, remodeling, and inflammation in the right carotid artery. A model of aortic aneurysm-associated arterial remodeling was established by transverse aortic constriction (TAC). (A) Echocardiograms showing blood flow velocity in the left carotid artery (LCA) and right carotid artery (RCA) of Aggf1+ / + (WT) or Aggf1+ / - (KO) mice 3 weeks after TAC or sham surgery. The bar graph on the right side of the figure shows the flow velocity ratio between the RCA and LCA. (B) Echocardiograms of the RCA of WT or KO mice 3 weeks after surgery. The bar graph on the right side shows the lumen diameter of the RCA. After 21 days, the RCA was harvested and used for histological staining and Western blot analysis. (C) H&E staining of RCA cross sections. Bar graphs show media thickness (first panel), adventitia thickness (second panel), common carotid artery wall thickness (third panel), medial cell density (fourth panel), and adventitial cell density (fifth panel). (D) Western blot analysis of AGGF1, phosphorylated ERK1 / 2 (p-ERK1 / 2), total ERK1 / 2 (T-ERK1 / 2), and phosphorylated Smad3 (p-Smad3). GAPDH was used as a loading control. Blots were quantitatively analyzed, and the data are plotted as bar graphs on the right. (E) Sirius red staining and α-smooth muscle actin (α-SMA) immunostaining of RCA cross sections. Bar graphs on the right show quantification of collagen area and the percentage of α-SMA-positive area. (F) Immunostaining of the inflammatory marker MCP-1 and macrophage marker CD68 in RCA sections. The bar graphs on the right show quantification of the percentage of MCP1-positive cell area and CD68-positive cell area. *, P<0.05; **, P<0.01; ***, P<0.001; mouse sample size n=5 / group, other sample size n=4 / group. [Figure 2]Vascular smooth muscle-specific knockout of Aggf1 (SmcKO) exacerbates TAC-induced dilation, remodeling, and inflammation in the right carotid artery. (A) Echocardiograms showing blood flow velocities in the LCA and RCA of Aggf1flox / flox (CTL) and Aggf1smcKO (SmcKO) mice 3 weeks after TAC or sham surgery. The bar graph on the right shows the flow velocity (FV) ratio of the RCA to the LCA. (B) Echocardiograms of the RCA of CTL or SmcKO mice 3 weeks after surgery. The bar graph on the right shows the lumen diameter of the RCA. After 21 days, RCAs were harvested and used for histological staining and Western blot analysis. (C) H&E staining of RCA cross sections. The bar graphs at the bottom show the medial thickness (first panel), adventitia (second panel), total arterial wall thickness (third panel), medial cell density (fourth panel), and adventitial cell density (fifth panel). (D) Protein blot analysis of RCA for AGGF1, p-ERK1 / 2, T-ERK1 / 2, and p-Smad3. β-actin was used as a loading control. (E) Protein blot analysis of RCA for mature TGF-β1. β-actin was used as a loading control. (F) Sirius red staining and immunostaining of α-SMA, MCP-1, and CD68 in RCA cross sections. The bar graphs at the bottom show the quantified percentage of positive areas for the corresponding immunostaining. *, P<0.05; **, P<0.01; ***, P<0.001; mouse sample size n=5 / group, others sample size n=4 / group. [Figure 3]We demonstrate that AGGF1 protein expression is significantly reduced in vascular tissues of mouse aneurysm models and patients with thoracic aortic aneurysms. Intraperitoneal injection of purified human AGGF1 protein attenuates TAC-induced dilation and remodeling of the mouse right carotid artery in a manner dependent on the RDD sequence. (A) The upper panel shows AGGF1 immunostaining of vascular samples from eight healthy subjects and eight patients with thoracic aortic aneurysms. The lower panel shows AGGF1 immunostaining of sham and TAC-treated vessels. (B) Echocardiograms showing blood flow velocities in the LCA and RCA of mice treated with or without wild-type human AGGF1 or mutant AGGF1-RDDdel for 3 weeks after TAC or sham surgery. The bar graph on the right shows the flow velocity (FV) ratio between the RCA and LCA. (C) The lumen diameter of the RCA is shown. After 21 days, RCAs were harvested and used for histological staining. (D) H&E staining of RCA cross sections. The bar graphs at the bottom show the intimal thickness (first panel), adventitia thickness (second panel), total arterial wall thickness (third panel), medial cell density (fourth panel), and adventitial cell density (fifth panel). (E) Sirius red staining and α-SMA immunostaining of RCA cross sections. The bar graphs on the right show the percentage of collagen area and the percentage of α-SMA-positive area. *, P<0.05; **, P<0.01; ***, P<0.001; sample size n≥6 mice / group. [Figure 4]This shows that intraperitoneal injection of purified human AGGF1 protein attenuates TAC-induced inflammation in the right cervical arteries of mice, TGF-β1 maturation and signaling, and ERK1 / 2 signaling, depending on the RDD sequence. After 21 days, RCAs were harvested for histological staining and Western blot analysis. (A) MCP-1 and CD68 immunostaining of RCA sections from the model shown in Figure 3. The bar graphs below show the percentage of MCP1- and CD68-positive cell areas. (B) Western blot analysis of p-ERK1 / 2 and p-Smad3 in RCAs. β-actin was used as a loading control. (C) Protein blot analysis of mature TGF-β1 in RCAs. β-actin was used as a loading control. *, P<0.05; **, P<0.01; ***, P<0.001; sample size of mice n≧6 / group, other experiments sample size n=4 / group. [Figure 5] Intraperitoneal injection of purified human AGGF1 protein attenuates TAC-induced dilation and remodeling of the mouse ascending aorta in a manner dependent on the RDD sequence. (A) Echocardiograms of the ascending aorta of mice treated with or without wild-type human AGGF1 or mutant AGGF1-RDDdel for 3 weeks after TAC or sham surgery. The bar graph on the right shows the luminal diameter of the ascending aorta. 21 days later, the ascending aorta was harvested and used for histological staining. (B) H&E staining of RCA cross sections. The bar graphs at the bottom show the intimal thickness (first panel), adventitia thickness (second panel), total arterial wall thickness (third panel), medial cell density (fourth panel), and adventitial cell density (fifth panel). *, P<0.05; **, P<0.01; ***, P<0.001; sample size n≥6 mice per group. [Figure 6]This shows that intraperitoneal injection of purified human AGGF1 protein can attenuate TAC-induced remodeling and inflammation in the mouse ascending aorta in a manner dependent on the RDD sequence. Twenty-one days after surgery, ascending aortas were harvested and subjected to histological staining. From top to bottom, the ascending aorta cross-sections were stained with Sirius Red, immunostained with α-SMA, CD68, and MCP-1. The bottom bar graphs show the percentage of positive areas for the corresponding staining. *, P<0.05; **, P<0.01; ***, P<0.001; sample size: n≥6 mice per group. [Figure 7] This shows that intraperitoneal injection of purified human AGGF1 protein attenuates TAC-induced TGF-β1 maturation, signal transduction, and ERK1 / 2 signal transduction in the ascending aorta in a manner dependent on the RDD sequence. After 21 days, RCAs were harvested and subjected to Western blot analysis. (A) Western blot analysis of p-ERK1 / 2 and p-Smad3 in the ascending aorta; β-actin was used as a loading control. (B) Protein blot analysis of mature TGF-β1 in the ascending aorta; β-actin was used as a loading control. *, P<0.05; **, P<0.01; ***, P<0.001; sample size n=4 / group. [Figure 8]AGGF1 suppresses TGF-β1 and ERK1 / 2 signaling and TGF-β1-induced VSMC inflammation. (A) VSMC cells were pre-starved for 12 hours, treated with or without TGF-β1 for 15 minutes, and then treated with AGGF1 for 10 minutes. Cells were then lysed and samples were collected for Western blot analysis of p-ERK1 / 2, T-ERK1 / 2, and p-Smad3. GAPDH was used as a loading control. The bar graph on the right shows quantitative data. (B) VSMC cells were downregulated with siRNA for Aggf1 (SiAggf1) or negative control siRNA (SiNC). After 48 hours, cells were lysed and samples were collected for Western blot analysis of p-ERK1 / 2, T-ERK1 / 2, and p-Smad3. The bar graph on the right shows quantitative data. (C) VSMC cells were overexpressed with AGGF1-FLAG or FLAG (control). After 48 hours, cells were lysed and samples were collected for Western blot analysis of p-ERK1 / 2, T-ERK1 / 2, and p-Smad3. GAPDH was used as a loading control. The bar graph on the right shows quantitative data. (D) VSMC cells were treated with TGF-β1 for 24 hours, followed by AGGF1 for 24 hours. mRNA was then extracted using the Trizol method, reverse-transcribed to obtain cDNA, and used to detect the mRNA levels of Il-6, McP1, Mmp2, and Mmp9 by real-time RT-PCR. (E) VSMC cells were overexpressed with AGGF1-FLAG or FLAG (control). After 24 hours, cells were stimulated with TGF-β1 for 48 hours. mRNA was then extracted using the Trizol method, reverse-transcribed to obtain cDNA. The mRNA levels of Il-6, Mcp1, Mmp2, and Mmp9 were analyzed by real-time RT-PCR. *, P<0.05; **, P<0.01; ***, P<0.001; n≥3 / group. [Figure 9]AGGF1 inhibits TGF-β1 maturation, while knockdown of Aggf1 or Itga7 induces TGF-β1 maturation. (A) VSMC cells were overexpressed with wild-type AGGF1 or mutant AGGF1-RDDdel, and after 48 hours, Western blot analysis of LAP-TGF-β1 and mature TGF-β1 was performed. β-actin (β-actin) was used as a loading control. (B) TGF-β1 levels in the culture medium of VSMC cells overexpressing AGGF1 or AGGF1-RDDdel were measured by ELISA. (C) VSMC cells were treated with AGGF1 or AGGF1-RDDdel for 48 hours, and then Western blot analysis of LAP-TGF-β1 and mature TGF-β1 was performed. GAPDH was used as a loading control. (D) TGF-β1 levels in the culture medium of VSMC cells treated with AGGF1 or AGGF1-RDDdel for 48 hours were measured by ELISA. (E) VSMC cells were transfected with SiAggf1 or SiNC, and 48 hours later, Western blot analysis was performed for LAP-TGF-β1 and mature TGF-β1. (F) TGF-β1 levels in the culture medium of VSMCs transfected with SiAggf1 or SiNC were measured by ELISA. (G) VSMC cells were transfected with SiItga7 or SiNC, and 48 hours later, Western blot analysis was performed for integrin α7, LAP-TGF-β1, and mature TGF-β1. (H) TGF-β1 levels in the culture medium of VSMCs transfected with SiItga7 or SiNC were measured by ELISA. (I) VSMC cells were transfected with SiItga7 or SiNC, and 48 hours later, mRNA was extracted using the Trizol method, reverse transcribed to cDNA, and real-time RT-PCR analysis of Itga7, Tgfb1, and Tgfb2 was performed. (J) TGF-β1 levels in the culture medium of HEK293T cells were measured by ELISA. The transfection plasmids contained LAP-TGF-β1-FLAG, integrin α7-FLAG, AGGF1-FLAG, or AGGF1-RDDdel-FLAG, and the groupings are shown in Figure J. (K) TGF-β1 levels in the culture medium of HEK293T cells were measured by ELISA.Cells were transfected with different plasmids of LAP-TGF-β1-FLAG and integrin α7-FLAG and treated with purified AGGF1 or AGGF1-RDDdel for 24 hours. The groups are shown in Figure K. *, P<0.05; **, P<0.01; ***, P<0.001; n≥3 / group. [Figure 10] Integrin α7 interacts with LAP-TGF-β1 via the RGD sequence, and AGGF1 enhances this interaction. (A) Co-IP analysis of HEK293T cells overexpressing LAP-TGF-β1-GFP and integrin α7-FLAG. (B) Co-IP analysis of VSMC cells overexpressing LAP-TGF-β1-GFP and endogenous integrin α7. (C) Co-IP analysis of HEK293T cells overexpressing wild-type LAP-TGF-β1-GFP or mutant LAP-TGF-β1-RGDdel-GFP and integrin α7-FLAG. (D) Co-IP analysis of VSMC cells overexpressing LAP-TGF-β1-GFP or LAP-TGF-β1-RGDdel-GFP. (E) Co-IP analysis of HEK293T cells overexpressing LAP-TGF-β1-GFP, α7-FLAG, wild-type AGGF1-GFP, or mutant AGGF1-RDDdel-GFP. Quantitative data for the LAP-TGF-β1-GFP / α7-FLAG ratio are plotted in the bar graph on the right. (F) Co-IP analysis of HEK293T cells overexpressing LAP-TGF-β1-GFP and integrin α7-FLAG and stimulated with AGGF1 or AGGF1-RDDdel for 24 hours. Quantitative data for the LAP-TGF-β1-GFP / α7-FLAG ratio are plotted in the bar graph on the right. *, P<0.05; **, P<0.01; ***, P<0.001; n=3 / group. [Figure 11]This shows that TGF-β1 inhibits AGGF1 expression and pirfenidone (PFD) increases AGGF1 expression. (A) Protein blot analysis of AGGF1 was performed after VSMC cells were treated with TGF-β1 for 2 and 4 days. VSMCs were starved for 12 hours and then stimulated with TGF-β1. (B) Protein blot analysis of LAP-TGF-β1 and mature TGFβ1 was performed after VSMC cells were treated with PFD for 2 and 4 days. (C) Mature TGF-β1 levels in the medium of VSMCs treated with PFD for 2 days were measured by ELISA. (D) Protein blot analysis of AGGF1 in VSMCs treated with PFD for 2 and 4 days. (E) Real-time RT-PCR analysis of Tgfb1 and Tgfb2 mRNA levels from VSMCs treated with PFD for 2 days was normalized to β-actin. *, P<0.05; **, P<0.01; ***, P<0.001; n≥4 / group (in the study). [Figure 12] Pirfenidone attenuates TAC-induced carotid artery dilation, remodeling, and inflammation by inhibiting phosphorylation of Smad3 and ERK1 / 2 in Aggf1+ / + (WT) mice, but not in Aggf1+ / - (KO) mice. (A) Echocardiograms of the LCA and RCA in WT or KO mice 3 weeks after TAC. The bar graph on the right shows the flow velocity (FV) ratio of the RCA to the LCA. (B) The mean luminal diameter of the RCA 3 weeks after TAC is shown. (C) H&E staining of RCA cross sections. The bar graphs at the bottom show the intimal thickness (first panel), adventitia thickness (second panel), common carotid artery wall thickness (third panel), medial cell density (fourth panel), and adventitial cell density (fifth panel). (D) Sirius red staining and immunostaining of α-SMA, MCP-1, and CD68 in RCA cross sections. The bar graphs on the right show the quantified percentage of positive areas for the corresponding immunostaining. (E) Immunoblot analysis of AGGF1, p-ERK1 / 2, p-Smad3, and T-ERK1 / 2 in RCA. GAPDH was used as a loading control. *, P<0.05; **, P<0.01; ***, P<0.001; n=5 / group (in mouse studies), other sample sizes n=4 / group. [Figure 13] Treatment of spontaneous aortic aneurysms in FbnC1041G / + mutant mice with AGGF1. (A) Echocardiograms of the aorta from WT or FbnC1041G / + mutant male mice at 18 weeks of age. The bar graph on the right shows the luminal diameter of the ascending aorta. (B) Immunoblot analysis of p-Smad2, p-Smad3, p-ERK, and mature TGF-β1 in the ascending aorta from WT or FbnC1041G / + mutant male mice. GAPDH was used as a loading control. (C) Sirius red staining and immunostaining of α-SMA, MCP-1, and CD68 in ascending aortic cross-sections. The bar graph on the right shows the quantified percentage of positive area for the corresponding immunostaining. *, P<0.05; **, P<0.01; ***, P<0.001; n=8 / group (in mouse studies); other sample sizes, n=4 / group. [Figure 14] Treatment of β-aminopropionitrile (BAPN)-induced aortic aneurysms with AGGF1. (A) Echocardiograms of the aorta after 28 days of water or BAPN administration in 3-week-old mice. The bar graph on the right shows the luminal diameter of the ascending aorta. (B) Survival rates in four groups of mice in the induced aneurysm model. (C) The incidence of aortic dissection in four groups of mice in the induced aneurysm model. (D) H&E staining of ascending aortic cross-sections. The bar graphs at the bottom show the intimal thickness (first panel), adventitia thickness (second panel), and common carotid artery wall thickness (third panel). (E) Sirius red staining of ascending aortic cross-sections and immunostaining of α-SMA, MCP-1, and CD68. The bar graphs on the right show the quantified percentage of positive areas for the corresponding immunostaining. (F) Immunoblot analysis of p-Smad2, p-Smad3, p-ERK, and mature TGFβ1 in the ascending aorta of four groups of model mice, with GAPDH used as a loading control. *, P<0.05; **, P<0.01; ***, P<0.001; n≥8 / group (in mouse studies), other sample sizes n=4 / group. [Figure 15]This diagram illustrates the mechanism by which AGGF1 acts on smooth muscle cells via integrin α7. Diseases such as hypertension and vascular aging stimulate cells to synthesize TGFβ1, secrete it extracellularly, and cleave it into mature TGFβ1. Mature TGFβ1 binds to a type II receptor dimer (TβRII) on the cell membrane, which then recruits a type I receptor dimer (TβRI) to form a heterotetrameric complex. The type I receptor is a serine / threonine kinase receptor that can stimulate the phosphorylation of Smad2 / 3 and ERK1 / 2. Smad2 / 3 recruits and binds to Smad4, which then enters the nucleus and regulates target gene expression. Meanwhile, ERK1 / 2 can directly enter the nucleus and regulate gene expression. These genes promote vascular remodeling, ultimately leading to diseases such as aneurysms and atherosclerosis. AGGF1 promotes the binding of integrin α7 to LAP-TGFβ1, inhibiting its cleavage, and inhibits the phosphorylation of Smad2 / 3 and ERK1 / 2, thereby blocking the TGFβ1 signaling pathway. In the development and progression of diseases such as aneurysms, TGFβ1 acts as the "accelerator," while AGGF1 acts as the "brake" for TGFβ1. DETAILED DESCRIPTION OF THE INVENTION

[0017] In order to clarify the objectives, technical means, and advantages of the present invention, the present invention will be described in more detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described in this specification are used only to illustrate the present invention and are not intended to limit the present invention. In addition, the technical features of the various embodiments of the present invention described below can be combined with each other as long as they are not mutually inconsistent. Various equivalent changes, substitutions, modifications, or alterations made based on ordinary knowledge and conventional means in the art without departing from the above technical idea and disclosed spirit of the present invention are all included in the protection scope of the present invention.

[0018] In order to effectively inhibit the occurrence of aneurysms, perform clinical intervention, clarify the pathogenesis of aneurysms, and find effective intervention methods, the present invention provides an in vitro purified recombinant protein, i.e., AGGF1 protein, for preparing an anti-aneurysm drug effective for treating aortic aneurysms and carotid aneurysms. The recombinant protein provided by the present invention is a protein encoded by the nucleic acid sequence shown in SEQ ID NO. 2, which is the nucleic acid sequence encoding the recombinant protein.

[0019] The recombinant protein was named AGGF1 protein, and the gene encoding it was discovered and cloned in congenital venous malformation-osseous hypertrophy syndrome (Klippel-Trenaunay syndrome, abbreviated as KTS) through genetic linkage mapping analysis. AGGF1 protein is highly expressed in endothelial cells, smooth muscle cells, and osteoblasts.

[0020] The method for cloning the recombinant protein gene that suppresses aneurysms in the present invention involves cloning the AGGF1 gene from human cDNA by PCR using the forward primer shown in SEQ ID NO:3 and the reverse primer shown in SEQ ID NO:4.

[0021] In the recombinant plasmid of the present invention that expresses the recombinant protein gene that suppresses aneurysms, the AGGF1 gene is linked to a His tag sequence and inserted into the multiple cloning enzyme cleavage site of the expression vector pet28a to obtain the recombinant plasmid pet28a-AGGF1 plasmid.

[0022] In the recombinant protein for suppressing aneurysms of the present invention, the amino acid sequence of the protein is as follows: (1) the amino acid sequence set forth in SEQ ID NO: 1; or (2) an amino acid sequence that has 80% to 100% identity with the amino acid sequence identified in SEQ ID NO: 1, retains amino acids 606 to 608, and encodes a protein with the same function; or (3) An amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence identified by SEQ ID NO: 1, retaining amino acids 606 to 608, and having activity equivalent to that of the protein shown in sequence SEQ ID NO: 1.

[0023] The method for preparing a recombinant protein for inhibiting aneurysms according to the present invention comprises the following steps: (1) The human AGGF1 gene was linked to a His tag sequence and then inserted into the multiple cloning enzyme cleavage site of the prokaryotic expression vector pet28a to construct a recombinant plasmid containing the AGGF1 gene, pet28a-AGGF1. (2) The recombinant plasmid from step (1) is transfected into E. coli BL21(DE3), and kanamycin resistance screening is performed to obtain positive cell clones having antibiotic resistance. (3) The positive cell clone obtained in step (2) is expanded and then separated and purified to obtain the recombinant protein. [Example]

[0024] Example 1 Preparation of AGGF1 recombinant protein: The AGGF1 protein expression plasmid pet28-AGGF1 was expressed in E. coli BL21(DE3). The expression strain was cultured overnight in 20 ml of kanamycin-resistant LB medium. The next day, this 20 ml medium was added to 1 L of kanamycin-resistant LB medium and cultured on a shaker at 37°C for 2.5 hours. Protein expression was then induced by adding 1 mM IPTG. IPTG is a highly stable lactose analog that can inhibit the lac repressor protein and induce the synthesis of β-galactosidase. This enzyme can promote lactose utilization. IPTG can be used to induce the expression of target genes controlled by the lac operon. After induction with IPTG, E. coli was allowed to grow in the medium for 5.5 hours. The medium was centrifuged at 4000 rpm for 10 minutes at 4°C. If protein isolation is not performed immediately, the E. coli pellet can be stored at -20°C. Purification of the recombinant protein was performed according to the QIAexpressionist High-Level Expression and 6xHis-Tagged Protein Purification Manual (Qiagen). Six ml of buffer (50 mM NaHPO, 300 mM NaCl, 20 mM imidazole, 0.05% Tween-20, pH 8.0) was prepared and supplemented with a protease inhibitor mixture (6 μg / ml chymostatin, 1 μg / ml E64, 2 μg / ml aprotinin, 0.5 μg / ml phosphoramidon, 1 μg / ml pepstatin A, 5 μg / ml leupeptin, 5 μg / ml antipain, and 0.1 mM benzamidine (Sigma)). One liter of medium was centrifuged, and the resulting E. coli pellet was resuspended in a buffer solution. 1 mg / ml lysozyme (Invitrogen) was added to the suspension, which was then placed in an ice bath for 30 minutes. The suspension was disrupted using the ultrasonic probe of an ultrasonic homogenizer. Six cycles were performed at 300 W output, 10 seconds per cycle, with 10-second intervals between each cycle. The disrupted product was transferred to an EP tube and centrifuged at 12,000 rpm, 4°C, and 50 minutes. The supernatant was aspirated and stored on ice.His-tagged proteins were purified from the supernatant using Ni-NTA agarose beads. Ni-NTA metal chelate affinity chromatography exhibits high affinity for 6xHis-tagged biomolecules. The Ni-NTA beads were washed twice with 5 ml of EB, and the supernatant was added to the beads (1 ml of supernatant added to each bead). The mixture was incubated overnight at 4°C on a shaker. The next day, the beads were centrifuged and the supernatant discarded. To reduce nonspecifically bound proteins, the beads were washed three times with buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 0.05% Tween-20, pH 8.0). The 6xHis-tagged proteins were eluted with 1 ml of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 0.05% Tween-20, pH 8.0). The eluted protein was dialyzed twice against 1×EB at 4° C. using Spectra / Por dialysis tubing (Spectrum Laboratories, Inc., USA). After dialysis, the purified protein was aliquoted and stored at −80° C. This is the recombinant protein provided by the present invention.

[0025] Example 2 AGGF1-RDD del Protein preparation: A protein (homologous protein obtained by truncation of the AGGF1 protein sequence) was prepared by deleting three amino acids (positions 606-608, sequence RDD) from the amino acid sequence of the AGGF1 recombinant protein. Using the eukaryotic expression pcDNA3.1-AGGF1 as a template, a DNA fragment in which nine bases were deleted from the AGGF1 gene was amplified by PCR. The resulting fragment was double-digested and then cloned into a pet28 protein expression plasmid. The plasmid was transformed into Ecoli BL21, and expression of the truncated AGGF1 gene fusion protein was induced with IPTG. Expression of the truncated AGGF1 gene fusion protein was confirmed by Western blotting. The preparation method was the same as in Example 1.

[0026] Example 3 Changes in the characteristics of a TAC surgery-induced aneurysm model in Aggf1 knockout mice. In this experiment, two strains of mice were used. One was Aggf1 whole body knockout mice (Aggf1 + / - ), and the control mice were wild-type mice (WT) (Fig. 1). The other mice were Aggf1 smooth muscle-specific knockout mice (Aggf1 smcKO ), and the control mice were mice with only the loxp site (Aggf1 fl / fl Mice in each group were divided into sham and TAC groups depending on whether or not they underwent TAC surgery, with a sample size of ≥5 mice. 21 days after TAC surgery, the vascular lumen diameter was measured using Doppler ultrasound, and the carotid arteries and aortas were collected for histological staining and Western immunoblotting to evaluate the effect of AGGF1. As shown in Figures 1 and 2, the effect of Aggf1 was evaluated. + / - Mice and Aggf1 smcKO In mice, no difference was observed in the carotid artery lumen diameter or histological staining in the sham group, but after TAC surgery, Aggf1 was significantly increased compared to the control group. + / - Mice and Aggf1 smcKO In both mice, significant vasodilation (blood flow velocity and luminal diameter), remodeling (H&E staining, Sirius Red staining, and αSMA), and inflammation (MCP1, CD68) were observed. Meanwhile, Western immunoblotting analysis revealed that after TAC surgery, AGGF1 levels in the carotid arteries of both mouse strains were decreased, while p-Smad3 and p-ERK1 / 2 levels were significantly increased. Furthermore, the Aggf1 levels in Figure 2 were significantly increased. smcKO The levels of mature TGFβ1 in the mice were also significantly increased. These results indicate an important role for AGGF1 in blood vessels.

[0027] Use of the recombinant proteins synthesized in Examples 1 and 2 in the preparation of anti-aneurysmal drugs. This research team obtained vascular samples from the thoracic aorta region of eight healthy individuals and eight patients with thoracic aortic aneurysms from a hospital. As shown in Figure 3A, AGGF1 immunostaining revealed a significant decrease in AGGF1 levels in the aneurysm samples. Consistent changes were also observed in TAC mice. That is, the expression level of AGGF1 protein was significantly decreased in both the aneurysm mouse model and the vascular tissue of patients with thoracic aortic aneurysms. Next, in this study, C57BL / 6 mice were divided into six groups. After sham surgery, they were divided into the buffer group, the AGGF1 group, and the AGGF1-RDD group. del group (Sham+buffer, Sham+AGGF1, Sham+AGGF1-RDD del ), and after TAC surgery, the buffer group, AGGF1 group, and AGGF1-RDD del group (TAC+buffer, TAC+AGGF1, TAC+AGGF1-RDD del ) with ≥6 mice in each group. Buffer is a protein elution solution. 21 days after TAC surgery, the luminal diameter of the blood vessels was measured using Doppler ultrasound, and the carotid arteries and aortas were collected for histological staining and Western immunoblotting to evaluate the effect of AGGF1. From the day after surgery, recombinant AGGF1 protein or AGGF1-RDD was administered. del The protein and the same amount of EB as a control were intraperitoneally injected into mice every other day for 3 weeks at a protein dose of 0.5 mg / kg.

[0028] As shown in Figure 3, in the sham group, AGGF1 protein or AGGF1-RDD del Although the protein had little effect on mouse carotid arteries and aortas, AGGF1 protein significantly suppressed vasodilation and remodeling after TAC surgery compared with control buffer treatment of mouse TAC-induced aneurysms. delNo such effect was observed with AGGF1 protein. Figure 4 shows that AGGF1 protein significantly suppresses vascular inflammation compared with control buffer treatment of mouse TAC-induced aneurysms, and that AGGF1 protein suppresses p-Smad3, p-ERK1 / 2, and mature TGFβ1 levels in both sham and TAC surgery. del The protein had no such effect. Figure 5 shows that the dilatation and vascular thickening of the ascending aorta in mice (H&E staining) were examined. The results show that AGGF1 protein significantly inhibits the dilatation and vascular thickening of the ascending aorta. Figure 6 shows that after TAC surgery, remodeling and inflammation of the ascending aorta were significantly enhanced and inhibited by AGGF1 protein. Figure 7 shows that Western blot analysis showed that AGGF1 protein could inhibit the levels of p-Smad3, p-ERK1 / 2, and mature TGFβ1. AGGF1-RDD del Protein had no such effect.

[0029] These data demonstrate that AGGF1 protein therapy effectively attenuates vascular lesions in a TAC-induced mouse aneurysm model by suppressing vasodilation, remodeling, and inflammation.

[0030] In vitro cell experiments to investigate the molecular mechanism of AGGF1 anti-aneurysm activity. In vitro experiments were performed using aortic smooth muscle cells and HEK293T cells. As shown in Figure 8A-C and Figure 9A-F, Western blot analysis and ELISA experiments confirmed that AGGF1 inhibited the TGFβ1 signaling pathway (Smad3 and ERK1 / 2) and mature TGFβ1 levels in the presence of recombinant AGGF1 protein, AGGF1-FLAG overexpression, or AGGF1 silencing. RT-PCR results (Figure 8D-E) indicated that overexpression of recombinant AGGF1 protein and AGGF1-FLAG suppressed TGFβ1-induced inflammation (Il6, McP1, Mmp2, and Mmp9). Western blot analysis and ELISA results (Figure 9G-H) indicated that mature TGFβ1 levels were significantly elevated after integrin α7 silencing. RT-PCR results (Figure 9I) indicated that integrin α7 silencing did not affect TGFβ1 mRNA levels. The ELISA experiment results J to K in Figure 9 showed that overexpression of AGGF1-FLAG or recombinant AGGF1 protein enhanced the suppression of mature TGFβ1 by integrin α7. del Protein or AGGF1-RDD del -FLAG had no such effect. Western blot analysis in Figure 10 confirmed that LAP-TGFβ1 interacts with integrin α7 via the RGD sequence, which is also the molecular mechanism underlying the inhibition of TGFβ1 by integrin α7.

[0031] AGGF1 is a key mediator of pirfenidone treatment of aneurysms. In Figure 11, VSMCs were incubated in vitro with TGF-β1 and pirfenidone (PFD). Figure A shows Western blot analysis demonstrating that TGF-β1 suppressed AGGF1 levels. Figures B–D show that PFD not only suppressed the synthesis of mature TGF-β1 but also increased AGGF1 levels. On the other hand, Figure E shows RT-PCR results indicating that PFD treatment for 2 days did not affect the mRNA levels of Tgfb1 and Tgfb2. Figure 12 shows Aggf1 knockout mice. In Figures A–B, PFD significantly reduced the dilation of the right carotid artery and the blood flow velocity ratio of the right / left carotid aneurysms in WT mice. Figures C–D also show that PFD significantly reduced vascular remodeling (H&E staining, Sirius Red, and αSMA) and inflammation (MCP1, CD68) in the right carotid artery of WT mice. However, PFD was observed in Aggf1 heterozygous knockout mice (Aggf1 + / - Western immunoblotting experiments showed that PFD significantly increased AGGF1 levels and decreased p-Smad3 and p-ERK1 / 2 levels in the RCA of WT mice after TAC, but not Aggf1. + / - No such effect was observed in mice. These results indicate that TGF-β inhibitors increase the expression level of AGGF1. However, at the same time, studies on the molecular mechanism revealed that maintaining a relatively high level of AGGF1 is necessary for TGF-β inhibitors to exert their therapeutic effect.

[0032] AGGF1 protein is Fbn1 C1041G / + Suppresses mutagenic mouse aortic disease. In the present invention, Fbn1, which has already been published by Tongji Hospital, C1041G / +Mutant mice were obtained. Compared to wild-type mice, these mice were found to have spontaneous aortic dilation and aortic aneurysm formation at 10 weeks. After obtaining these mice, buffer or AGGF1 protein was intraperitoneally injected into the mice from 10 to 18 weeks. At 18 weeks, the mice underwent ultrasound examination, and were then euthanized. The aortas were then harvested for Western blotting and histochemical staining. As shown in Figure 13A, at 18 weeks, Fbn1 mutant mice exhibited significantly higher levels of aortic dilation compared to wild-type mice. C1041G The ascending aorta of mutant mice was dilated by more than 50%, and AGGF1 protein significantly inhibited this dilation. At the same time, the results of Western immunoblotting in Figure 13B showed that Fbn1 C1041G / + The mutant mice showed significantly elevated levels of p-Smad2, p-Smad3, p-ERK, and mature TGFβ1, all of which were suppressed by AGGF1 protein. The immunohistochemistry results in Figure 13C showed that Fbn1 C1041G / + The mutant mice showed marked thickening of the aortic media, with no change in the adventitia, and a marked increase in the area positive for αSMA / CD68 / MCP1 immunohistochemistry. AGGF1 protein significantly suppressed the thickening of the vascular media and the increase in αSMA / CD68 / MCP1 levels.

[0033] AGGF1 protein suppresses β-aminopropionitrile (BAPN)-induced mouse aortic aneurysms. β-Aminopropionitrile is a lysyl oxidase (LOX) inhibitor and is easily soluble in water. Administration of 0.25% BAPN to 3-week-old mice resulted in a very high incidence of thoracic aortic aneurysms after 4 weeks. Three-week-old C57BL / 6 male mice were divided into four groups, and the aortas were measured by ultrasound on day 28. The mice were then euthanized and the aortas were harvested for Western blotting and histochemical staining. As shown in Figure 14A, ultrasound examination revealed that, compared with the control group, BAPN significantly induced dilation of the ascending aorta, and AGGF1 inhibited vasodilation, but AGGF1-RDD significantly reduced the dilation. delIn Figure 14B, the survival rate of the BAPN group mice was only 57%, but AGGF1 protein significantly improved the survival rate of these aneurysm model mice, and AGGF1-RDD del This effect was not observed in the BAPN-induced aortic dissection. Figure 14C shows that AGGF1 does not affect the incidence of BAPN-induced aortic dissection. Figure 14D shows H&E staining of the aorta in the BAPN group, revealing significant thickening of the media and adventitia. Figure 14E shows immunohistochemistry of the aorta in the BAPN group, revealing a significant increase in the αSMA / CD68 / MCP1-positive area. AGGF1 significantly suppressed aortic thickening, remodeling, and inflammation, whereas the effects of AGGF1 protein lacking the RDD sequence were significantly attenuated. Figure 14F shows Western immunoblotting results of the BAPN group, revealing significant increases in p-Smad2, p-Smad3, p-ERK, and mature TGFβ1 levels, all of which were suppressed by AGGF1 protein, but not by AGGF1-RDD. del Proteins were shown to have no such effect.

[0034] As mentioned above, three aneurysm models were adopted in this invention. The first is an aortic aneurysm-associated arterial remodeling model established by transverse aortic constriction (TAC). The second is an Fbn1 aneurysm model. C1041G / + The first model was a mutation-induced Marfan syndrome-thoracic aortic aneurysm model, and the third was a BAPN-induced thoracic aortic aneurysm model. In both models, it was demonstrated that AGGF1 protein treatment could effectively suppress vascular inflammation and remodeling associated with aortic aneurysms.

[0035] Figure 15 illustrates the mechanism by which AGGF1 acts on smooth muscle cells via its receptor integrin α7. Diseases such as hypertension and vascular aging stimulate cells to synthesize TGF-β1, secrete it extracellularly, and cleave it into mature TGFβ1. Mature TGFβ1 binds to a type II receptor dimer (TβRII) on the cell membrane, which then recruits a type I receptor dimer (TβRI) to form a heterotetrameric complex. The type I receptor is a serine / threonine kinase receptor that can stimulate the phosphorylation of Smad2 / 3 and ERK1 / 2. Smad2 / 3 recruits and binds to Smad4, which then enters the nucleus and regulates target gene expression. Meanwhile, ERK1 / 2 can directly enter the nucleus and regulate gene expression. These genes promote vascular remodeling, ultimately leading to diseases such as aneurysms and atherosclerosis. AGGF1 promotes the binding of integrin α7 to LAP-TGFβ1, inhibiting its cleavage, and inhibits the phosphorylation of Smad2 / 3 and ERK1 / 2, thereby blocking the TGFβ1 signaling pathway.

[0036] AGGF1 interacts with its receptor integrin α7, strengthening the interaction between integrin α7 and LAP-TGF-β1 and inhibiting TGF-β1 maturation, thereby suppressing TGF-β1 signaling and ERK1 / 2 signaling. At the same time, the combined use of AGGF1 and pirfenidone is also necessary for aneurysms. The recombinant AGGF1 protein containing a His tag of the present invention can reduce vascular lesions, improve vascular function in mice, and ultimately suppress aneurysms by inhibiting the synthesis of TGF-β1 and its signaling pathway. Therefore, the recombinant AGGF1 protein containing a His tag of the present invention can be used to prepare anti-aneurysm drugs for the treatment of aneurysm-related diseases.

[0037] It is readily apparent to those skilled in the art that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention are all included in the protection scope of the present invention. [Sequence table] JPEG2026500320000002.jpg244162JPEG2026500320000003.jpg252162JPEG2026500320000004.jpg253162JPEG2026500320000005.jpg253162JPEG2026500320000006.jpg45146

Claims

1. 1. Use of a recombinant protein in the preparation of a medicament for inhibiting aneurysms, comprising: The amino acid sequence of the recombinant protein is (4) the amino acid sequence shown in SEQ ID NO: 1; or (5) an amino acid sequence having 80% to 100% identity with the amino acid sequence identified by SEQ ID NO: 1, retaining amino acids 606 to 608, and encoding a protein with the same function; or (6) An amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence specified by SEQ ID NO: 1, and which retains the amino acids 606 to 608 and has an activity equivalent to that of the protein shown in SEQ ID NO:

1. Use of a recombinant protein, characterized in that:

2. 1. Use of a composition of a recombinant protein and a TGF-β inhibitor in the preparation of a medicament for inhibiting aneurysms, comprising: The amino acid sequence of the recombinant protein is (4) the amino acid sequence shown in SEQ ID NO: 1; or (5) an amino acid sequence having 80% to 100% identity with the amino acid sequence identified by SEQ ID NO: 1, retaining amino acids 606 to 608, and encoding a protein with the same function; or (6) An amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence specified by SEQ ID NO: 1, and which retains the amino acids 606 to 608 and has an activity equivalent to that of the protein shown in SEQ ID NO:

1. and Preferably, the use of a composition of a recombinant protein and a TGF-β inhibitor, wherein the TGF-β inhibitor is pirfenidone.

3. 3. The use according to claim 1 or claim 2, wherein the aneurysm is an aortic aneurysm or a carotid aneurysm.

4. 3. The use according to claim 1 or claim 2, wherein the aneurysm is an aneurysm caused by coarctation of the aortic arch.

5. The use according to claim 1 or 2, wherein the aneurysm is an aneurysm caused by a mutation in the FBN1 gene.

6. 3. The use according to claim 1 or claim 2, wherein the aneurysm is a thoracic aortic aneurysm.

7. The use according to claim 1 or 2, wherein the recombinant protein suppresses aneurysms by inhibiting the synthesis of TGF-β1 and its signaling pathway.

8. The use according to claim 7, wherein the signal transduction pathways are TGFβ1-TGFβR1-Smad2 / 3 and TGFβ1-TGFβR1-ERK1 / 2.

9. The use according to claim 1 or claim 2, characterized in that the recombinant protein achieves the suppression of aneurysms by improving the structure and function of blood vessels.

10. The use according to claim 1 or 2, wherein the deletion of the amino acids 606 to 608 disrupts the interaction between the AGGF1 protein and the receptor integrin α7, resulting in the loss of the action of the AGGF1 protein.

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