Application of IQGAP3 in the preparation of drugs for preventing and treating atherosclerosis

Abstract

The present invention belongs to the technical field of atherosclerosis treatment, and discloses the application of IQGAP3 in the preparation of drugs for preventing and treating atherosclerosis. In in vitro experiments, IQGAP3 and its GRD domain can inhibit the expression of adhesion factors caused by oxidized low-density lipoprotein (ox-LDL), and can reduce the breakage of endothelial cell connections and the adhesion of monocytes. And endothelial-specific overexpression of IQGAP3-GRD can reduce the plaque area of the aortic root of mice caused by a high-fat diet, and reduce the expression of adhesion factors ICAM1 / VCAM1 in vascular tissue. The present invention opens up a new application field of IQGAP3 and its GRD domain, and provides a meaningful reference for preventing and treating endothelial cell damage and atherosclerosis, and improving vascular pathological conditions.

Description

Technical Field

[0001] The present invention belongs to the technical field of atherosclerosis treatment, and particularly relates to the application of IQGAP3 in the prevention and / or treatment of atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Impaired lipid metabolism underlies the pathological basis of atherosclerosis. Its characteristic manifestation is that lesions in the affected arteries begin in the intima, typically with accumulation of lipids and complex carbohydrates, bleeding, and thrombosis, followed by fibrosis and calcification, and gradual degeneration and calcification of the arterial media, leading to thickening and hardening of the arterial wall and narrowing of the vessel lumen. The lesions often affect large and medium-sized muscular arteries. Once they progress to obstruct the arterial lumen, the tissues or organs supplied by these arteries will suffer ischemia or necrosis, seriously endangering human health. However, its pathogenesis remains incompletely understood, and effective and specific drug targets are currently lacking in clinical practice. Therefore, in-depth investigation of the molecular mechanisms underlying AS and identification of corresponding intervention targets have become a hot topic of scientific research both domestically and internationally, and are of great significance for the early diagnosis, prevention, and treatment of AS.

[0003] Endothelial dysfunction is a key factor in the development and progression of atherosclerosis. Endothelial dysfunction manifests as impaired endothelium-dependent vasodilation, enhanced oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Pathological factors disrupt endothelial cell junctions, increase permeability, and enhance the expression of adhesion factors and chemotactic molecules. This promotes the recruitment of circulating monocytes into the intima to form foam cells and can induce the proliferation and migration of adjacent vascular smooth muscle cells, thereby triggering or exacerbating AS. Therefore, clarifying the mechanisms of endothelial dysfunction will help identify new solutions to vascular diseases by preventing endothelial damage, providing new insights into the prevention and treatment of vascular diseases such as atherosclerosis.

[0004] IQ motif-containing GTPase-activating protein 3 (IQGAP3) is a member of the IQGAP family. By binding to its target proteins, it regulates cell proliferation, adhesion, migration, and cytoskeletal dynamics. Furthermore, IQGAP3 is a major regulator of gastric epithelial tissue stability and repair, and is also a key regulator of mitotic progression and genomic stability. The Ras GTPase-activating protein-related domain (GRD) is a key domain in IQGAP3 that selectively binds to active GTP-Cdc42 and GTP-Rac1, thereby stabilizing the active state of GTP-binding proteins. However, whether IQGAP3 can ameliorate endothelial cell damage and atherosclerosis and its underlying mechanisms remain unknown. Summary of the Invention

[0005] The present invention aims to provide a method for treating endothelial cell damage and / or atherosclerosis using IQGAP3. In this study, researchers injected IQGAP3 into the tail vein of atherosclerosis-bearing mice, which reduced plaque size in the aortic root and decreased the expression of the adhesion cytokines ICAM1 / VCAM1 in vascular tissue.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention claims protection for at least one use of an IQGAP3 protein or a GRD domain of an IQGAP3 protein in (a1) and (a2):

[0008] (a1) Use in the preparation of a medicament for preventing and / or treating atherosclerosis;

[0009] (a2) Application in the preparation of drugs for treating endothelial cell damage.

[0010] The amino acid sequence of the IQGAP3 protein is shown in SEQ ID NO.3;

[0011] The amino acid sequence of the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.2.

[0012] In a second aspect, the present invention claims protection for at least one use of a biomaterial related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein in the following (a1) and (a2):

[0013] (a1) Use in the preparation of a medicament for preventing and / or treating atherosclerosis;

[0014] (a2) Application in the preparation of drugs for treating endothelial cell damage.

[0015] The biological material related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein is at least one of the following (b1)-(b2):

[0016] (b1) a gene encoding the IQGAP3 protein;

[0017] (b2) a gene encoding the GRD domain of the IQGAP3 protein;

[0018] (b3) an adenovirus comprising the coding gene described in (b1);

[0019] (b4) An adenovirus comprising the coding gene described in (b2).

[0020] Furthermore, the NCBI sequence number of the nucleotide sequence of the gene encoding the IQGAP3 protein is 128239; the nucleotide sequence of the gene encoding the GRD domain of the IQGAP3 protein is shown in SEQ ID NO.1.

[0021] Furthermore, the active ingredient of the drug in the above application includes the IQGAP3 protein or the GRD domain of the IQGAP3 protein; or includes a biological material related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein.

[0022] In a third aspect, the present invention seeks to protect a drug for preventing and / or treating atherosclerosis or treating endothelial cell damage, wherein the active ingredient of the drug includes an IQGAP3 protein or the GRD domain of an IQGAP3 protein, or a biomaterial related to the IQGAP3 protein or the GRD domain of an IQGAP3 protein.

[0023] Furthermore, the amino acid sequence of the IQGAP3 protein, the amino acid sequence of the GRD domain of the IQGAP3 protein, and the biological materials related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein are as described above.

[0024] The present invention studies the medical use of GTPase activating protein 3 containing an IQ motif and its Ras GTPase activating protein related domain (IQGAP3-GRD), and specifically relates to the use of GTPase activating protein 3 containing an IQ motif and the GRD domain of IQGAP3 protein in the prevention and treatment of atherosclerosis.

[0025] Beneficial effects of the present invention:

[0026] In in vitro experiments, IQGAP3 and its GRD domain inhibited the expression of adhesion factors induced by oxidized low-density lipoprotein (ox-LDL) and reduced the disruption of endothelial cell junctions and monocyte adhesion. Furthermore, endothelial-specific overexpression of IQGAP3-GRD reduced the plaque area in the aortic root of mice induced by a high-fat diet and decreased the expression of the adhesion factors ICAM1 / VCAM1 in vascular tissue. This invention opens up a new application area for IQGAP3 and its GRD domain, providing valuable insights for preventing and treating endothelial cell damage, atherosclerosis, and improving vascular pathology. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The diagrams show the schematic diagrams for constructing full-length IQGAP3, IQGAP3 with GRD domain removed, and adenovirus with GRD domain removed, as well as the overexpression efficiency diagrams of the three adenoviruses.

[0028] Figure 2Human umbilical vein endothelial cells were transfected with adenovirus containing control (Ad-GFP), full-length IQGAP3 (Ad-IQGAP3), or IQGAP3 with the GRD domain removed (Ad-ΔGRD) for 24 hours. Ox-LDL (100 μM, 24 hours) was then administered. (A) Western blot analysis of endothelial cell adhesion cytokines ICAM1, VCAM1, and IQGAP3 expression. (B) Immunofluorescence analysis of monocyte adhesion. *<0.05.

[0029] Figure 3 Human umbilical vein endothelial cells were transfected with control (Ad-GFP) and (IQGAP3-GRD) and then treated with ox-LDL (100 μM, 24 hours). A shows the expression of endothelial cell adhesion cytokines ICAM1 and VCAM1 detected by western blot. B shows the expression of monocyte adhesion by immunofluorescence. *<0.05.

[0030] Figure 4 Six-week-old male ApoE- / - mice were injected with endothelial-specific adeno-associated virus (AAVendo) encoding control (GFP) and IQGAP3-GRD domain (IQGAP3-GAD) through the tail vein. Two weeks later, they were fed a high-fat diet (HFD). After eight weeks of feeding, samples were collected and Oil Red O staining was used to detect gross plaque formation in the aorta.

[0031] Figure 5 The aortic root was paraffin-embedded and sectioned, and histologically stained. A is a schematic diagram of H&E staining to detect aortic root plaque morphology; B is Masson staining to detect collagen deposition in aortic root plaques; and C is Oil Red O staining to detect lipid deposition in the aortic root.

[0032] Figure 6 Aortic tissue protein and RNA were extracted, and the expression of adhesion factors (icam1, vcam1, sele, selp) and chemotactic molecules (ccl2, cxcl2) in aortic tissue was detected by western blot and qPCR, *<0.05. DETAILED DESCRIPTION

[0033] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0034] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The reagents and materials used in the following examples are all commercially available products. The full-length IQGAP3, IQGAP3 with GRD domain removed, and adenovirus encoding the GRD domain of IQGAP3 used in the examples were purchased from GeneCare, and the sequences are as follows: IQGAP3-GRD encoding gene sequence (933-1286aa)

[0035] ATGGTTCTGGACAAGCAGAAGGGTTTTAAAGTCGCTGAGCAAAGAGAAACGGCAGAA

[0036] ACTAGAAGCATACCAACACCTCTTCTACCTGCTCCAGACTCAGCCCATCTACCTGGCC

[0037] AAGCTGATCTTTCAGATGCCACAGAACAAAACCACCAAGTTCATGGAGGCAGTGATT

[0038] TTCAGCCTGTACAACTATGCCTCCAGCCGCCGAGAGGCCTATCTCCTGCTCCAGCTGT

[0039] TCAAGACAGCACTCCAGGAGGAAATCAAGTCAAAGGTGGAGCAGCCCCAGGACGTG

[0040] GTGACAGGCAACCCAACAGTGGTGAGGCTGGTGGTGAGATTCTACCGTAATGGGCGG

[0041] GGACAGAGTGCCCTGCAGGAGATTCTGGGCAAGGTTATCCAGGATGTGCTAGAAGAC

[0042] AAAGTGCTCAGCGTCCACACAGACCCTGTCCACCTCTATAAGAACTGGATCAACCAG

[0043] ACTGAGGCCCAGACAGGGCAGCGCAGCCATCTCCCATATGATGTCACCCCGGAGCAG

[0044] GCCTTGAGCCACCCCGAGGTCCAGAGACGACTGGACATCGCCCTACGCAACCTCCTC

[0045] GCCATGACTGATAAGTTCCTTTTAGCCATCACCTCATCTGTGGACCAAATTCCGTATGG

[0046] GATGCGATATGTGGCCAAAGTCCTGAAGGCAACTCTGGCAGAGAAATTCCCTGACGC

[0047] CACAGACAGCGAGGTCTATAAGGTGGTCGGGAACCTCCTGTACTACCGCTTCCTGAA

[0048] CCCAGCTGTGGTGGCTCCTGACGCCTTCGACATTGTGGCCATGGCAGCTGGTGGAGC

[0049] CCTGGCTGCCCCCCAGCGCCATGCCCTGGGGGCTGTGGCTCAGCTCCTACAGCACGC

[0050] TGCGGCTGGCAAGGCCTTCTCTGGGCAGAGCCAGCACCTACGGGTCCTGAATGACTA

[0051] TCTGGAGGAAACACACCTCAAGTTCAGGAAGTTCATCCATAGAGCCTGCCAGGTGCC

[0052] AGAGCCAGAGGAGCGTTTTGCAGTGGACGAGTACTCAGACATGGTGGCTGTGGCCAAACCCATGGTGTACATCACCGTGGGGGAGCTG(SEQ ID NO.1).

[0053] IQGAP3 - GRD amino acid sequence:

[0054] MMVLDKQKGLKSLSKEKRQKLEAYQHLFYLLQTQPIYLAKLIFQMPQNKTTKFMEAVIF

[0055] SLYNYASSRREAYLLLQLFKTALQEEIKSKVEQPQDVVTGNPTVVRLVVRFYRNGRGQS

[0056] ALQEILGKVIQDVLEDKVLSVHTDPVHLYKNWINQTEAQTGQRSHLPYDVTPEQALSHP

[0057] EVQRRLDIALRNLLAMTDKFLLAITSSVDQIPYGMRYVAKVLKATLAEKFPDATDSEVY

[0058] KVVGNLLYYRFLNPAVVAPDAFDIVAMAAGGALAAPQRHALGAVAQLLQHAAAGKAFSGQSQHLRVLNDYLEETHLKFRKFIHRACQVPEPEERFAVDEYSDMVAVAKPMVYI(SEQ ID NO.2).

[0059] Amino acid sequence of IQGAP3 protein

[0060] MERRAAGPGWAAYERLTAEEMDEQRRQNVAYQYLCRLEEAKRWMEACLKEELPSPVE

[0061] LEESLRNGVLLAKLGHCFAPSVVPLKKIYDVEQLRYQATGLHFRHTDNINFWLSAIAHIG

[0062] LPSTFFPETTDIYDKKNMPRVVYCIHALSLFLFRLGLAPQIHDLYGKVKFTAEELSNMASE

[0063] LAKYGLQLPAFSKIGGILANELSVDEAAVHAAVLAINEAVERGVVEDTLAALQNPSALLE

[0064] NLREPLAAVYQEMLAQAKMEKAANARNHDDRESQDIYDHYLTQAEIQGNINHVNVHG

[0065] ALEVVDDALERQSPEALLKALQDPALALRGVRRDFADWYLEQLNSDREQKAQELGLVE

[0066] LLEKEEVQAGVAAANTKGDQEQAMLHAVQRINKAIRRRVAADTVKELMCPEAQLPPVY

[0067] PVASSMYQLELAVLQQQQGELGQEELFVAVEMLSAVVLINRALEARDASGFWSSLVNPA

[0068] TGLAEVEGENAQRYFDALLKLRQERGMGEDFLSWNDLQATVSQVNAQTQEETDRVLA

[0069] VSLINEALDKGSPEKTLSALLLPAAGLDDVSLPVAPRYHLLLVAAKRQKAQVTGDPGAVL

[0070] WLEEIRQGVVRANQDTNTAQRMALGVAAINQAIKEGKAAQTERVLRNPAVALRGVVPD

[0071] CANGYQRALESAMAKKQRPADTAFWVQHDMKDGTAYYFHLQTFQGIWEQPPGCPLNT

[0072] SHLTREEIQSAVTKVTAAYDRQQLWKANVGFVIQLQARLRGFLVRQKFAEHSHFLRTWL

[0073] PAVIKIQAHWRGYRQRKIYLEWLQYFKANLDAIKIQAWARMWAARRQYLRRRLHYFQK

[0074] NVNSIVKIQAFFRARKAQDDYRILVHAPHPPLSVVRRFAHLLNQSQQDFLAEAELLKLQE

[0075] EVVRKIRSNQQLEQDLNIMDIKIGLLVKNRITLQEVVSHCKKLTKRNKEQLSDMMVLDK

[0076] QKGLKSLSKEKRQKLEAYQHLFYLLQTQPIYLAKLIFQMPQNKTTKFMEAAVIFSLYNYAS

[0077] SRREAYLLLQLFKTALQEEIKSKVEQPQDVVTGNPTVVRLVVRFYRNGRGQSALQEILG

[0078] KVIQDVLEDKVLSVHTDPVHLYKNWINQTEAQTGQRSHLPYDVTPEQALSHPEVQRRL

[0079] DIALRNLLAMTDKFLLAITSSVDQIPYGMRYVAKVLKATLAEKFPDATDSEVYKVVGNL

[0080] LYYRFLNPAVVAPDAFDIVAMAAGGALAAPQRHALGAVAQLLQHAAAGKAFSGQSQHL

[0081] RVLNDYLEETHLKFRKFIHRACQVPEPEERFAVDEYSDMVAVAKPMVYITVGELVNTHRL

[0082] LLEHQDCIAPDHQDPLHELLEDLGELPTIPDLIGESIAADGHTDLSKLEVSLTLTNKFEGLE

[0083] ADADDSNTRSLLSTKQLLADIIQFHPGDTLKEILSLSASREQEAAHKQLMSRRQACTAQ

[0084] TPEPLRRHRSLTAHSLLPLAEKQRRVLRNLRRLEALGLVSARNGYQGLVDELAKDIRNQH

[0085] RHRHRRKAELVKLQATLQGLSTKTTFYEEQGDYYSQYIRACLDHLAPDSKSSGKGKKQP

[0086] SLHYTAAQLLEKGVLVEIEDLPASHFRNVIFDITPGDEAGKFEVNAKFLGVDMERFQLHYQDLLQLQYEGVAVMKLFNKAKVNVNLLIFLLNKKFLRK(SEQ ID NO.3)

[0087] NCBI sequence number of IQGAP3 gene nucleotide sequence: 128239

[0088] Example 1: Inhibitory effect of IQGAP3 on ox-LDL-induced endothelial cell damage

[0089] To investigate the effect of IQGAP3 on ox-LDL-induced endothelial cell injury, we first transfected human umbilical vein endothelial cells with adenovirus encoding full-length IQGAP3 (Ad-IQGAP3), IQGAP3 with the GRD domain removed (Ad-ΔGRD), or a construct containing only the GRD domain of IQGAP3 (Ad-IQGAP3-GRD) for 24 hours to assess overexpression efficiency. Subsequently, human umbilical vein endothelial cells were transfected with adenovirus encoding a control (Ad-GFP), full-length IQGAP3 (Ad-IQGAP3), IQGAP3 with the GRD domain removed (Ad-ΔGRD), or a construct containing only the GRD domain of IQGAP3 (Ad-IQGAP3-GRD) for 24 hours before treatment with ox-LDL (100 μM, 24 hours). Expression of the endothelial cell adhesion cytokines ICAM1 and VCAM1 was assessed by Western blot, and monocyte adhesion was assessed by immunofluorescence.

[0090] like Figure 1 As shown, the schematic diagram of the construction of full-length IQGAP3, IQGAP3 with GRD domain removed, and adenovirus containing GRD domain ( Figure 1 A), immunofluorescence images showed that all three adenoviruses could successfully overexpress human umbilical vein endothelial cells ( Figure 1 B).

[0091] like Figure 2 As shown, western bolt and immunofluorescence results showed that overexpression of IQGAP3 could inhibit the increased expression of adhesion factors and increased monocyte adhesion caused by ox-LDL, while overexpression of IQGAP3-ΔGRD had no improvement effect.

[0092] like Figure 3 As shown, western bolt and immunofluorescence results showed that overexpression of IQGAP3-GRD can inhibit the increased expression of adhesion factors and increased monocyte adhesion caused by ox-LDL.

[0093] Example 2: Inhibitory effect of IQGAP3 on atherosclerotic lesions

[0094] To explore the effects of IQGAP3 and its GRD domain on atherosclerotic lesions, we selected 6-week-old SPF male ApoE - / -Mice (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were injected into the tail vein with a control (GFP) or a Flag-tagged IQGAP3-GRD domain-specific adeno-associated virus (AAVendo) (purchased from Suzhou Jimai Gene Pharmaceutical Biotechnology Co., Ltd.). Two weeks later, they were fed a normal diet (NC) or a high-fat diet (HFD) for 8 weeks. Gross aortas were harvested for Oil Red O staining, and the aortic root was harvested for tissue staining. Aortic tissue protein and RNA were collected.

[0095] like Figure 4 As shown, by collecting the mouse aorta and staining with Oil Red O, it was found that the aortic plaque area of ​​the mice injected with IQGAP3-GRD by the tail vein was significantly smaller than that of the mice injected with the control virus by the tail vein.

[0096] like Figure 5 As shown, the aortic roots of mice were harvested, paraffin-embedded, and sectioned. Plaque morphology and structure were observed using H&E staining, collagen deposition within the plaques was assessed using Masson staining, and plaque area and lipid deposition were assessed using Oil Red O staining. The results showed that mice injected with IQGAP3-GRD by the tail vein had significantly smaller aortic plaque area and lipid deposition than those injected with the control virus, while collagen deposition was significantly higher in the control group.

[0097] like Figure 6 As shown, by collecting mouse aortic tissue proteins and RNA, western blot and qPCR results showed that the expression of adhesion factors and chemotactic molecules in the aortic tissue of mice injected with IQGAP3-GRD through the tail vein was significantly lower than that of mice injected with the control virus through the tail vein.

Claims

1. Use of IQGAP3 protein or the GRD domain of IQGAP3 protein in the preparation of a medicament for preventing and / or treating atherosclerosis; The amino acid sequence of the IQGAP3 protein is as shown in SEQ ID NO.3; The amino acid sequence of the GRD domain of the IQGAP3 protein is as shown in SEQ ID NO.

2.

2. Use of a biomaterial related to the IQGAP3 protein or the GRD domain of IQGAP3 protein in the preparation of a medicament for preventing and / or treating atherosclerosis; The biomaterial related to the IQGAP3 protein or the GRD domain of IQGAP3 protein described in claim 1 is at least one of the following (b1)-(b2): (b1) The coding gene of the IQGAP3 protein; (b2) The coding gene of the GRD domain of the IQGAP3 protein; (b3) An adenovirus containing the coding gene described in (b1); (b4) An adenovirus containing the coding gene described in (b2); The NCBI serial number of the nucleotide sequence of the coding gene of the IQGAP3 protein is 128239; the nucleotide sequence of the coding gene of the GRD domain of the IQGAP3 protein is as shown in SEQ ID NO.

1.

3. The application according to claim 1, wherein The active ingredient of the medicament includes the IQGAP3 protein or the GRD domain of the IQGAP3 protein.

4. The application according to claim 2, characterized in that, The active ingredient of the medicament includes a biomaterial related to the IQGAP3 protein or the GRD domain of the IQGAP3 protein.

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