Use of lentivirus overexpressing Wnt4 gene or Cre-dependent expression of adeno-associated virus in preparation of drugs for treating myocardial fibrosis

Abstract

The application discloses application of Wnt4 in preparation of drugs for treating fibrosis. The application provides application of a substance for promoting expression of a Wnt4 gene or increasing content and / or activity of Wnt4 in preparation of products; the products are used for treating myocardial fibrosis, promoting blood vessel regeneration and the like. The application proves by experiments that overexpression of the Wnt4 gene can induce transformation of fibroblasts into endothelial cells, make the fibroblasts have the ability of forming blood vessels, increase heart function, increase blood vessel density and the like. The Wnt 4 protein is expected to become one of effective treatment methods for targeting cardiac fibroblasts, inhibiting myocardial fibrosis and promoting angiogenesis. The Wnt 4 protein can produce transformation for fibrosis formed by different mechanisms, and has considerable development prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of Wnt4 in the preparation of drugs for treating fibrosis. Background Technology

[0002] When human organs, such as the heart, lungs, skin, and kidneys, suffer tissue damage, fibroblasts are rapidly activated to replace dead tissue or functional cells, ultimately leading to fibrosis or scarring, which affects organ function and repair. Therefore, improving or eliminating fibrosis is one of the most pressing challenges in the treatment of diseases affecting various organs.

[0003] Cardiovascular disease is one of the leading causes of death worldwide, with high morbidity and mortality. Many pathological factors involved in the development of cardiovascular disease, such as oxidative stress, stress and / or volume overload, certain cytokines, ischemia and hypoxia, and neuroendocrine disorders, can induce cardiomyocyte death. The adult heart lacks regenerative capacity; dead myocardium is replaced by activated fibroblasts, leading to fibrosis, which in turn causes a decline in cardiac function, ultimately resulting in a vicious cycle and heart failure.

[0004] Myocardial fibrosis refers to the excessive accumulation of collagen fibers in the normal tissue structure of the myocardium, a significant increase in collagen concentration in the heart tissue, or changes in collagen composition. This pathological change exists in various cardiovascular diseases and is considered to be closely related to arrhythmias, cardiac dysfunction, and even sudden cardiac death. Vascular weakening is a pathophysiological mechanism accompanying myocardial fibrosis. Vascular weakening reduces blood supply to the heart tissue, further inducing myocardial cell death.

[0005] In recent years, myocardial fibrosis and cardiac angiogenesis have received increasing attention from cardiovascular experts both domestically and internationally, but clinical and experimental studies on these conditions are still relatively limited. Currently, angiotensin-converting enzyme inhibitors (ACEIs) are widely used clinically, but there is no clear evidence that they can improve myocardial fibrosis. Recent studies have found that the H1 histamine antagonist tranilast also has anti-fibrotic, hair growth-stimulating, and collagen-inhibiting effects. Although it has been used in scar treatment, whether this drug can improve myocardial fibrosis requires further confirmation.

[0006] In conclusion, there are currently no effective drugs in clinical practice to inhibit myocardial fibrosis and promote angiogenesis. Therefore, the development of novel myocardial fibrosis inhibitors / inhibitors is not only of great significance for the treatment of cardiovascular diseases, but also has equally important reference value for the treatment of fibrosis after damage to other organs.

[0007] Myocardial fibrosis often occurs secondary to various cardiovascular diseases. It can cause myocardial structural disorder and increased tissue heterogeneity, forming the structural basis for arrhythmias and serving as a potential risk factor for sudden death and chronic heart failure. Currently, commonly used drugs for treating cardiovascular diseases mainly include beta-blockers, angiotensin-converting enzyme inhibitors, and diuretics, which increase myocardial contractility and reduce myocardial oxygen consumption. Myocardial fibrosis is a complex pathological process involving multiple autoregulatory systems and can occur in different heart diseases and even different types of the same disease. Currently, there are no effective drugs in clinical treatment specifically targeting and inhibiting fibrosis. Summary of the Invention

[0008] The purpose of this invention is to provide the application of Wnt4 in the preparation of drugs for treating fibrosis.

[0009] The technical solution adopted in this invention is:

[0010] In a first aspect, the invention provides the use of the Wnt4 protein or a substance regulating Wnt4 gene expression in the preparation of a product having the function described in any one of (I) to (VI):

[0011] (I) Treatment of fibrosis;

[0012] (II) Treatment of cardiovascular diseases;

[0013] (III) Promotes angiogenesis;

[0014] (IV) Enhances cardiac function;

[0015] (V) Increases blood vessel density;

[0016] (VI) Promotes the transformation of fibroblasts into endothelial cells.

[0017] A second aspect of the present invention provides the use of a substance that regulates the activity of the Wnt4 protein described in the first aspect of the present invention in the preparation of a product having the function described in any one of (I) to (VI):

[0018] (I) Treatment of fibrosis;

[0019] (II) Treatment of cardiovascular diseases;

[0020] (III) Promotes angiogenesis;

[0021] (IV) Enhances cardiac function;

[0022] (V) Increases blood vessel density;

[0023] (VI) Promotes the transformation of fibroblasts into endothelial cells.

[0024] A third aspect of the present invention provides the use of a substance that regulates the content of the Wnt4 protein described in the first aspect of the present invention in the preparation of a product having any one of the functions described in (I) to (VI):

[0025] (I) Treatment of fibrosis;

[0026] (II) Treatment of cardiovascular diseases;

[0027] (III) Promotes angiogenesis;

[0028] (IV) Enhances cardiac function;

[0029] (V) Increases blood vessel density;

[0030] (VI) Promotes the transformation of fibroblasts into endothelial cells.

[0031] In some embodiments of the present invention, the substance regulating Wnt4 gene expression is a substance that increases or enhances the expression of the Wnt4 gene; the substance regulating the activity of the Wnt4 protein in the first aspect of the present invention is a substance that increases or enhances the activity of the Wnt4 protein; and the substance regulating the content of the Wnt4 protein in the first aspect of the present invention is a substance that increases or enhances the content of the Wnt4 protein.

[0032] In some embodiments of the present invention, the substance that increases or enhances the expression of the Wnt4 gene, the substance that increases or enhances the activity of the Wnt4 protein, or the substance that increases or enhances the content of the Wnt4 protein is a biological material related to the Wnt4 protein, and the biological material is any one of B1) to B4) below:

[0033] B1) The nucleic acid molecule encoding Wnt4;

[0034] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0036] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or containing

[0037] B3) The recombinant microorganisms of the recombinant vector.

[0038] In a fourth aspect, the present invention provides a product, the active ingredient of which is a substance that increases or enhances the expression of the Wnt4 gene and / or a substance that increases or enhances the activity of the Wnt4 protein and / or a substance that increases or enhances the content of the Wnt4 protein; the use of the product is as shown in any one of (I) to (VI) below:

[0039] (I) Treatment of fibrosis;

[0040] (II) Treatment of cardiovascular diseases;

[0041] (III) Promotes angiogenesis;

[0042] (IV) Enhances cardiac function;

[0043] (V) Increases blood vessel density;

[0044] (VI) Promotes the transformation of fibroblasts into endothelial cells.

[0045] In some embodiments of the present invention, the substance that increases or enhances the expression of the Wnt4 gene, the substance that increases or enhances the activity of the Wnt4 protein, or the substance that increases or enhances the content of the Wnt4 protein is a biological material related to the Wnt4 protein, and the biological material is any one of B1) to B4) below:

[0046] B1) The nucleic acid molecule encoding Wnt4;

[0047] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0048] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0049] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or containing

[0050] B3) The recombinant microorganisms of the recombinant vector.

[0051] In some embodiments of this invention, the fibrosis refers to myocardial fibrosis, skin fibrosis (scars), renal fibrosis, pulmonary fibrosis, liver fibrosis, glial scars formed by astrocytes in brain injury, gastrointestinal fibrosis, muscle fibrosis, etc. Since the principle of fibrosis in different organs is the same—it is formed by the activation and proliferation of fibroblasts after organ injury—it can be inferred that adding Wnt4 to skin scar treatment products can enhance the scar treatment effect or address related fibrotic diseases such as renal fibrosis, pulmonary fibrosis, liver fibrosis, glial scars formed by astrocytes in brain injury, gastrointestinal fibrosis, and muscle fibrosis.

[0052] In some embodiments of the present invention, the fibroblasts are myocardial fibroblasts.

[0053] A fifth aspect of the present invention provides a method for promoting the transformation of cardiomyocytes into endothelial cells by increasing or enhancing the expression of the Wnt4 gene and / or increasing or enhancing the activity of the Wnt4 protein and / or increasing or enhancing the content of the Wnt4 protein.

[0054] In some embodiments of the present invention, the expression of the Wnt4 gene can be improved or enhanced by means of viral vectors containing the Wnt4 gene, activators of upstream genes that regulate Wnt4 expression and their molecular signaling pathways, potential activators that promote Wn4 protein expression, etc.; the content of the Wnt4 protein can be improved or enhanced by means of direct administration of exogenous Wnt4 protein, Wnt4 functional mimics, etc.

[0055] The beneficial effects of this invention are:

[0056] This invention reveals that after cardiac injury, some fibroblasts can transdifferentiate into endothelial cells through mesenchymal transition (MEndoT), a novel phenomenon termed mesenchymal-endothelial transition (MEndoT). Simultaneously, increasing fibroblast-to-endothelial cell transition promotes angiogenesis in the periphery of cardiac injury, thereby reducing cardiac damage. Wnt4 is a secreted glycoprotein. This invention finds that Wnt4 can regulate fibroblast-to-endothelial cell transition, thereby inhibiting myocardial fibrosis, promoting angiogenesis, reducing fibrosis, and protecting cardiac function; playing a crucial role in cardiac repair. Wnt4 protein shows promise as an effective treatment method targeting cardiac fibroblasts to inhibit myocardial fibrosis and promote angiogenesis. It can also transform fibrosis formed by different mechanisms and in different diseases, demonstrating considerable developmental potential. Attached Figure Description

[0057] Figure 1 This is a flowchart.

[0058] Figure 2 The expression level of Wnt4 in isolated mouse myocardial fibroblasts was increased, accompanied by an increase in the expression level of endothelial cell-related marker genes.

[0059] Figure 3 In mice, the expression level of Wnt4 in fluorescently labeled fibroblasts in the heart injury area increased over time after cardiac injury.

[0060] Figure 4 Wnt4 is significantly highly expressed in endothelial cells transformed from cardiac fibroblasts in cardiac tissue.

[0061] Figure 5 Overexpression of Wnt4 in mouse fibroblasts significantly increased the expression of endothelial cell-related marker genes. Figure 5 A represents the expression level of Wnt4; Figure 5 B represents the expression levels of endothelial cell-related marker genes VECAD, vWF, and eNOS.

[0062] Figure 6This study demonstrates, through tube formation experiments, that overexpression of Wnt4 enables fibroblasts to develop angiogenesis. Figure 6 A is a morphological diagram; Figure 6 B is a statistical chart of pipe length.

[0063] Figure 7 The low-density lipoprotein (LDL) uptake experiment showed that overexpression of Wnt4 enabled fibroblasts to have endothelial cell function; the white arrows indicate cells that have taken up LDL. Figure 7 A is a morphological diagram; Figure 7 B is a statistical chart of LDL uptake rate.

[0064] Figure 8 Overexpression of Wnt4 in animals was used to improve cardiac function after injury. Left ventricular ejection fraction and left ventricular shortening rate are both indicators of ventricular function; Pre, I / R 7d, and I / R 14d represent pre-injury, 7d and 14d post-injury, respectively. Figure 8 A is a morphological diagram; Figure 8 B is a left ventricular ejection fraction chart; Figure 8 C is a statistical chart of left ventricular shortening rate.

[0065] Figure 9 Overexpression of Wnt4 in mouse cardiac fibroblasts can reduce fibrosis following cardiac injury. Figure 9 A is a morphological diagram; Figure 9 B is a statistical chart of the fibrosis area.

[0066] Figure 10 Overexpression of Wnt4 in mouse cardiac fibroblasts increases angiogenesis after cardiac injury; gray and white areas in the figure represent blood vessels. Figure 10 A is a morphological diagram; Figure 10 B is a statistical graph of blood vessel density.

[0067] Figure 11 Overexpression of Wnt4 activates the phosphorylation JNK signaling pathway, leading to the transformation of cardiomyocytes into endothelial cells. VECAD and Occludin are endothelial cell markers.

[0068] Figure 12 for Figure 11 The statistical chart shows the expression level detection results. Detailed Implementation

[0069] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0070] The method described in this invention for treating heart disease by inducing MEndoT through overexpression of Wnt4 to target fibroblasts is as follows: Figure 1 As shown.

[0071] Example 1: Cell Experiment

[0072] 1. C57 wild-type mice were euthanized, their hearts were removed, and cardiac fibroblasts were obtained by digestion with trypsin and collagenase.

[0073] 2. Wnt4 was overexpressed in isolated cardiac fibroblasts using lentivirus. RNA was extracted from the cells after 48 hours, and the expression levels of Wnt4 and endothelial cell-related marker genes were detected. Figures 2-5 As shown, after transfection with the virus, the expression level of Wnt4 was significantly increased, and the expression of endothelial cell-related marker genes VECAD, vWF and eNOS was also significantly increased, indicating that overexpression of Wnt4 in normal in vitro cultured cardiomyocytes can induce the transformation of fibroblasts into endothelial cells.

[0074] 3. In the culture chamber that has been pre-lined with substrate gel, follow the 6×10... 4 / cm 2 Cardiac fibroblasts overexpressing Wnt4 were seeded at a density and cultured in a cell culture incubator at 37°C with 5% CO2 for 12 hours. Figure 6 As shown, cardiomyocytes overexpressing Wnt4 formed tubules, indicating that overexpression of Wnt4 enables fibroblasts to form blood vessels.

[0075] 4. In a 24-well board pre-coated with gelatin, install 6×10 4 / cm 2 Cardiac fibroblasts overexpressing Wnt4 were seeded at a density and cultured in a cell culture incubator at 37°C with 5% CO2 for 4 hours. Figure 7 As shown, it was observed that the number of myocardial fibroblasts capable of uptake LDL increased after overexpression of Wnt4, and LDL uptake is one of the main functions of endothelial cells.

[0076] The above results indicate that in vitro overexpression of Wnt4 can induce the transformation of cardiomyocytes into endothelial cells, which then possess endothelial cell functions and further promote angiogenesis.

[0077] Example 2 Animal Experiment

[0078] 1. Irradiate Cre-dependent expressed adeno-associated virus at a concentration of 1×10⁻⁶. 11 U / moxifen was injected into Col1a2-Cre mice via the jugular vein, followed by 10 consecutive days of tamoxifen injection to induce specific overexpression of Wnt4 in cardiomyocytes.

[0079] 2. Mice specifically overexpressing Wnt4 in myocardial fibroblasts and control mice underwent thoracotomy under inhalation anesthesia. The left anterior descending coronary artery was ligated 1 mm below the left atrium. After 45 minutes, the ligation was released, and the vascular recanalization was confirmed before thoracotomy. Intraoperative electrocardiography was performed to confirm successful modeling of myocardial ischemia-reperfusion injury. Postoperatively, disinfection, analgesia, liquid diet, and warming were provided.

[0080] 3. Cardiac function in mice was monitored by echocardiography at 7 and 14 days post-heart injury. Overexpression of Wnt 4 in cardiac fibroblasts significantly improved cardiac function in mice after I / R surgery.

[0081] Left ventricular ejection fraction (LVEF) refers to the proportion of blood pumped by the left ventricle with each left ventricular contraction, reflecting the ventricle's ejection function from a volume perspective. Left ventricular shortening rate (LVSC) refers to the proportion of change in the heart's internal diameter with each left ventricular contraction, reflecting the ventricle's ejection function from a length perspective. For example... Figure 8 As shown, overexpression of Wnt4 in cardiac fibroblasts increases ejection fraction and enhances cardiac pumping capacity in mice, resulting in increased cardiac function.

[0082] 4. Mouse hearts were harvested 14 days after cardiac injury surgery for Masson staining and fibrosis area calculation. See [link to results]. Figure 9 ,according to Figure 9 It can be seen that overexpression of Wnt4 in cardiac fibroblasts can significantly reduce postoperative cardiac fibrosis in mice.

[0083] 5. Mouse hearts were harvested 14 days after cardiac injury surgery. Vascular endothelium was fluorescently stained (green fluorescence), and vascular area was calculated. See the results below. Figure 10 ,according to Figure 10 It can be seen that overexpression of Wnt4 in cardiac fibroblasts can significantly increase vascular density.

[0084] Example 3: Molecular mechanism by which Wnt4 promotes the transformation of fibroblasts into endothelial cells

[0085] 1. C57 wild-type mice were euthanized, their hearts were removed, and cardiac fibroblasts were obtained by digestion with trypsin and collagenase.

[0086] 2. Inhibitors of the Wnt4 downstream signaling pathway JNK and β-catenin were added to the isolated cardiac fibroblasts. After inhibiting the downstream signaling pathways, cellular proteins were extracted, and the expression levels of Wnt4 and endothelial cell-related markers were detected. For example... Figures 11-12 As shown: Overexpression of Wnt4 activates the phosphorylated JNK signaling pathway, causing cardiomyocytes to transform into endothelial cells.

[0087] The above detailed embodiments have provided a clear description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. Application of substances that increase Wnt4 gene expression in the preparation of products for treating fibrosis: The substance is a lentivirus overexpressing the Wnt4 gene or a Cre-dependent adeno-associated virus; The fibrosis is myocardial fibrosis.

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