Patent Application
20240139283
The present invention relates to the field of treatment of cardiovascular disease, particularly the use of TBX1 in repairing cardiac tissue injury.
Cardiovascular disease is one of the leading causes in China, and the mortality rate has been increasing in recent years. Myocardial cells in mammals can only proliferate for a short period after birth. Once the myocardium matures, it exits the cell cycle, making myocardial damage typically irreversible and associated with poor prognosis. This can lead to serious consequences; for instance, up to 60% of ischemic myocardial infarction patients develop heart failure within six years of their initial event. Current treatments for myocardial infarction primarily focus on restoring blood flow to reduce myocardial ischemic injury and regulating neural and humoral mechanisms through medication to slow adverse ventricular remodeling. However, these treatments cannot entirely prevent ventricular remodeling after myocardial infarction and a considerable number of patients progress to heart failure within a short period even without ongoing ischemic injury.
Therefore, there is an urgent need in this field to develop new therapies for heart diseases.
The purpose of this invention is to provide an active ingredient for effectively treating heart diseases and/or lymphatic vessel abnormalities associated with cardiac injury and the use thereof.
In the first aspect of the invention, it provides a use of an active ingredient for preparing a formulation or drug, which is used for:
In another preferred embodiment, the encoding sequence includes DNA, cDNA, mRNA.
In another preferred embodiment, “promoting the transformation of M1-type macrophages into M2-type macrophages” refers to promoting the transformation of M1-type macrophages (promoting inflammatory responses) into M2 macrophages (promoting tissue repair) in myocardial tissue.
In another preferred embodiment, “heart diseases associated with cardiac injury” refers to heart diseases related to immune responses, especially autoimmune responses.
In another preferred embodiment, the heart diseases associated with cardiac injury are selected from the group consisting of myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, heart damage caused by coronavirus disease 2019 (COVID-19), and a combination thereof.
In another preferred embodiment, the diseases associated with lymphatic abnormalities are selected from the group consisting of: primary lymphedema caused by lymphatic vessel developmental defects, secondary lymphedema caused by lymphatic vessel rupture or obstruction, Turner syndrome, yellow nail syndrome, Hennekam syndrome, and other genetic syndromes presenting with lymphatic vessel edema, and a combination thereof.
In the second aspect of the invention, it provides a use of TBX1 protein, an encoding sequence thereof, or a diagnostic reagent thereof for preparing a diagnostic reagent or diagnostic kit, which is used for prognostic assessment of heart diseases associated with cardiac injury or diseases associated with lymphatic abnormalities.
In another preferred embodiment, the diagnostic reagent is selected from the group consisting of primers, probes, chips, antibodies, and a combination thereof.
In another preferred embodiment, compared to normal individuals or controls, an increase in TBX1 expression level and/or activity suggests a favorable prognosis for the diagnosed subject.
In another preferred embodiment, compared to normal individuals or controls, a decrease in TBX1 expression level and/or activity suggests a poor prognosis for the diagnosed subject.
In another preferred embodiment, the diagnosed subject has or is suspected to have heart diseases associated with cardiac injury or diseases associated with lymphatic vessel abnormalities.
In another preferred embodiment, the method for prognostic assessment of heart diseases associated with cardiac injury or diseases associated with lymphatic abnormalities includes: providing a subject sample, detecting the expression level of TBX1 protein (T1) in the subject sample, and comparing it with the average expression level of TBX1 protein (T0) in cardiac lymphatic vessels of myocardial infarction patients;
In another preferred embodiment, the method for prognostic assessment of heart diseases associated with cardiac injury or diseases associated with lymphatic abnormalities includes providing a subject sample, and detecting whether the subject sample has TBX1 encoding and/or regulatory sequence variants. If the TBX1 variant leads to loss or reduction of TBX1 function, it suggests a poor prognosis for the subject. If the TBX1 variant enhances TBX1 function, it suggests a favorable prognosis for the subject.
In another preferred embodiment, the TBX1 function refers to promoting lymphatic vessel regeneration and forming an immunosuppressive microenvironment.
In another preferred embodiment, the heart diseases associated with cardiac injury include myocardial infarction.
In another preferred embodiment, the subject has a myocardial infarction.
In another preferred embodiment, the subject is a human or a non-human mammal.
In another preferred embodiment, the sample includes a cardiac tissue sample, a cardiac cell sample, a lymphatic endothelial cell sample, a lymph fluid sample, a peripheral blood cell sample, or a combination thereof.
In another preferred embodiment, the sample include a cell sample, a DNA sample, a RNA sample, or a combination thereof.
In another preferred embodiment, the kit further includes detection reagents for additional biomarkers detection, wherein the additional biomarkers are selected from the group consisting of: Top2a, Mki67, Cenpe, Aurkb, and a combination thereof.
In another preferred embodiment, if the expression levels (or abundance) of these additional biomarkers increase, it suggests that the TBX1 variant leads to enhanced TBX1 functionality.
In another preferred embodiment, compared to the normal population or control group, an increase in the expression levels (or abundance) of these additional biomarkers suggests a favorable prognosis for the diagnosed subject.
In another preferred embodiment, compared to the normal population or control group, a decrease in the expression levels (or abundance) of these additional biomarkers suggests a poor prognosis for the diagnosed subject.
In the third aspect of the present invention, it provides an active ingredient capable of being used for repairing cardiac injury and treating diseases associated with lymphatic abnormalities, wherein the active ingredient comprises TBX1 protein, an encoding sequence thereof, a promoting agent thereof, or a combination thereof.
In another preferred embodiment, the active ingredient is a promoting agent of TBX1 protein.
In another preferred embodiment, the promoting agent promotes the expression of TBX1 protein within the lymphatic vessels of the heart and other parts of the body.
In another preferred embodiment, the promoting agent upregulates the functionality of TBX1 protein within the lymphatic vessels of the heart and other parts of the body.
In the fourth aspect of the present invention, it provides an expression vector, wherein the expression vector contains an expression cassette for expressing the TBX1 protein.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, a plasmid, an eukaryotic expression vector, a prokaryotic expression vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a transposon, and a combination thereof.
In another preferred embodiment, the expression vector specifically transfects lymphatic endothelial cells.
In another preferred embodiment, the expression cassette has a structure shown in the Formula I from the 5′ end to the 3′ end:
Z0-Z1-Z2 (I)
wherein,
In another preferred embodiment, the expression cassette further includes a lymphatic endothelial-specific promoter operably linked to the nucleotide sequence encoding the TBX1 protein.
In another preferred embodiment, the length of each nucleotide connecting sequence is 1-30 nt, preferably 1-15 nt, and more preferably 3-6 nt.
In another preferred embodiment, the nucleotide sequence encoding the TBX1 protein is derived from humans or non-human mammals.
In another preferred embodiment, the nucleotide sequence is as shown in SEQ ID NO. 1.
In another preferred embodiment, the nucleotide sequence is as shown in SEQ ID NO. 2.
In the fifth aspect of the present invention, it provides a host cell containing the expression vector of the fourth aspect of the present invention.
In another preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: a bacterial cell, a yeast cell, and a mammalian cell.
In another preferred embodiment, the host cell is a lymphatic endothelial cell.
In another preferred embodiment, the host cell is a cardiac lymphatic endothelial cell.
In the sixth aspect of the present invention, it provides a pharmaceutical formulation comprising (a) the active ingredient of the third aspect of the present invention, or the expression vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention, and (b) a pharmaceutically acceptable carrier, excipient, or diluent.
In another preferred embodiment, the pharmaceutical formulation is in the form of a lyophilized preparation, liquid preparation, or a combination thereof.
In another preferred embodiment, the pharmaceutical formulation is an injectable solution.
In another preferred embodiment, the pharmaceutical formulation further contains additional active ingredients.
Preferably, the additional active ingredients include BMP4, CCL28, anti-CD8 antibodies, or a combination thereof.
In the seventh aspect of the present invention, it provides a use of the active ingredient of the third aspect of the present invention, or the expression vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention, or the pharmaceutical formulation of the sixth aspect of the present invention, in the preparation of a drug for treating heart diseases associated with cardiac injury and/or diseases associated with lymphatic abnormalities.
In another preferred embodiment, heart diseases associated with cardiac injury are selected from the group consisting of: myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, heart damage caused by coronavirus disease 2019 (COVID-19), a combination thereof.
In another preferred embodiment, diseases associated with lymphatic abnormalities are selected from the group consisting of: primary lymphedema caused by lymphatic malformation, secondary lymphedema caused by lymphatic rupture or obstruction, Turner syndrome, yellow nail syndrome, Hennekam syndrome, and a combination thereof.
In the eighth aspect of the present invention, it provides a method for screening promoting agents of TBX1 protein, comprising the steps of: administering a candidate drug to lymphatic endothelial cells cultured in vitro and/or experimental animals; if the TBX1 mRNA level or TBX1 protein level in the lymphatic endothelial cells cultured in vitro or the lymphatic vessels of the experimental animals is higher relative to the control group that did not receive the candidate drug, or if the function of TBX1 protein is enhanced relative to the control group that did not receive the candidate drug, then the candidate drug can be considered a promoting agent of TBX1 protein.
In another preferred embodiment, the promoting agent of TBX1 protein can be used to treat heart diseases associated with cardiac injury.
In another preferred embodiment, the promoting agent of TBX1 protein can be used to treat diseases associated with lymphatic abnormalities.
In the ninth aspect of the present invention, it provides a method for treating heart diseases associated with cardiac injury, comprising the steps of: administering the active ingredient of the third aspect of the present invention or the pharmaceutical formulation of the sixth aspect of the present invention to a subject in need thereof.
In another preferred embodiment, the subject in need thereof has heart diseases associated with cardiac injury.
In another preferred embodiment, heart diseases associated with cardiac injury are selected from the group consisting of: myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, heart damage caused by coronavirus disease 2019 (COVID-19), and a combination thereof.
In the tenth aspect of the present invention, it provides a method for treating diseases associated with lymphatic abnormalities, comprising the steps of: administering the active ingredient of the third aspect of the present invention or the pharmaceutical formulation of the sixth aspect of the present invention to a subject in need thereof.
In another preferred embodiment, the subject in need thereof has diseases associated with lymphatic abnormalities.
In another preferred embodiment, diseases associated with lymphatic abnormalities are selected from the group consisting of: primary lymphedema caused by lymphatic malformation, secondary lymphedema caused by lymphatic rupture or obstruction, Turner syndrome, yellow nail syndrome, Hennekam syndrome, and a combination thereof.
It should be understood that within the scope of the present invention, the various technical features described above and those specifically described in the following examples can be combined with each other to form new or preferred technical solutions. Due to limitations in space, these combinations are not exhaustively listed here.
Through extensive and in-depth research, the inventors have unexpectedly developed an active ingredient for the treatment of heart diseases related to cardiac injury for the first time. The inventors discovered that overexpression of the TBX1 protein in cardiac lymphatic endothelial cells promotes lymphatic endothelial cell proliferation. More important, TBX1 plays an immunomodulatory role in promoting the establishment of an immunosuppressive microenvironment in the post-myocardial infarction (MI) heart, thereby inhibiting autoimmune reactions after cardiac injury, alleviating concurrent cardiac tissue inflammation, and facilitating the repair and regeneration of damaged cardiac tissue. Experimental evidence demonstrates that overexpressing TBX1 in cardiac lymphatic endothelial cells significantly improves cardiac function after MI and promotes the repair of post-MI cardiac tissue. Based on these findings, the present invention has been completed.
To facilitate a better understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise explicitly stated, each term below should have the meaning described below. Other definitions are provided throughout the specification.
The term “approximately” can refer to a value or composition within an acceptable range of error determined by ordinarily skilled technicians in this field, which partly depends on how the value or composition is measured or determined. For example, as used in this specification, “approximately 100” includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used in this specification, the terms “comprising” or “including (containing)” can be open, semi-closed, or closed. In other words, the terms also encompass “essentially consisting of” or “consisting of.”
As used in this specification, the terms “subject” and “individual in need” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, and goats.
TBX1
The present invention provides an active ingredient for repairing cardiac injury, the active ingredient comprising: the TBX1 protein, its encoding sequence, its enhancers, or a combination thereof.
The TBX1 gene encodes a T-box transcription factor and is located in the human chromosome 22q11.2 region. It is the major disease gene of 22q11.2 microdeletion syndrome. The disease has a prevalence of up to 1:2000 in newborns. 75% of affected children have congenital heart disease, accompanied by craniofacial developmental abnormalities, thymic aplasia, and other congenital defects. In early embryonic development, Tbx1 is expressed in a subset of differentiating cardiac progenitor cells and is also crucial for the development of the cardiac lymphatic system. However, the expression level of Tbx1 in the heart is extremely low in adulthood.
The gene ID of the TBX1 gene in the NCBI database is 6899, and its expressed mRNA sequences have four isoforms: NM_005992.1, NM_080646.2, NM_080647.1, and NM_001379200.1. The mouse Tbx1 gene in the NCBI database has a gene ID of 21380, and its expressed mRNA sequences have four isoforms: NM_011532.2, NM_001285472.1, NM_001285476.1, and NM_001373938.1. As used herein, the encoding sequence of the TBX1 protein can be any of the above sequences or their variants, as long as the sequence can effectively express a functionally normal or enhanced TBX1 protein. The function is to promote lymphangiogenesis and the formation of an immunosuppressive microenvironment.
In another preferred embodiment, the encoding sequence of the TBX1 protein is as shown in SEQ ID NO. 1.
In another preferred embodiment, the encoding sequence of the Tbx1 protein is as the CDS sequence used in the examples:
Cardiac Injury
As used herein, the term “cardiac injury” refers to damage to cardiac tissue that includes degeneration and necrosis of myocardial cells, as well as degeneration and necrosis of cardiac blood vessels and lymphatic vessels. Following cardiac injury, inflammation and autoimmune reactions occur.
The present invention has discovered that TBX1 protein, TBX1 mRNA, or its promoting agents can be used to repair cardiac injuries. Overexpression of TBX1 in cardiac lymphatic endothelial cells not only promotes lymphatic endothelial cell proliferation but also regulates autoimmune reactions, thereby alleviating cardiac inflammation. Thus, these can be employed to treat heart diseases associated with cardiac injury.
The term “heart diseases associated with cardiac injury” encompasses ischemia, acute myocardial injury caused by hypoxia, autoimmune cardiac diseases, and other diseases leading to myocardial damage. In another preferred example, heart diseases associated with cardiac injury are selected from the group consisting of: myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, heart damage caused by coronavirus disease 2019 (COVID-19), and a combination thereof.
Expression Vectors and Host Cells
The present invention provides an expression vector for expressing the TBX1 protein, which contains the encoding sequence of the TBX1 protein of the present invention.
With the provided sequence information, skilled technicians can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transfection/transduction into cells.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, plasmids, eukaryotic expression vectors, prokaryotic expression vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, transposons, and a combination thereof.
Preferably, a nucleic acid sequence encoding the TBX1 protein is included in the vector, preferably provided as an expression vector. It is preferably provided as a gene therapy vector that is optimally suitable for transfection/transduction and expression in target cells (e.g., cochlear supporting cells). The vector can be viral (e.g., adenovirus) or non-viral (e.g., plasmid). Viral vectors include those derived from adenoviruses, including mutated forms of adeno-associated viruses (AAV), retroviruses, lentiviruses, herpesviruses, vaccinia viruses, bovine poxviruses, MMLV, GaLV, simian immunodeficiency viruses (SIV), HIV, poxviruses, and SV40. Preferably, the viral vector is replication-defective or replication-deficient, capable of replicating or conditionally replicating. The viral vector can typically maintain an extrachromosomal state without integrating into the target cell genome. An AAV vector is a preferred viral vector for introducing nucleic acid sequences encoding the TBX1 protein into target cells. The use of specific AAV serotypes (AAV serotypes 2 to AAV serotype 12) or modified versions of these serotypes can achieve selective targeting.
Viral vectors possess the ability to enter cells. However, non-viral vectors such as plasmids can be complexed with reagents to facilitate uptake by target cells. Such reagents include cationic polymers. Optionally, delivery systems such as lipid-based delivery systems can be used. The vectors used in the present invention are preferably suitable for in vivo or ex vivo use and are preferably suitable for use in humans.
Vectors will preferably include one or more regulatory sequences to guide the expression of nucleic acid sequences in target cells. Regulatory sequences can include promoters, enhancers, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, and homologous recombination sites associated with nucleic acid sequences operably linked to them. Vectors may also include selective markers, for example, to determine the expression of vectors in growth systems (e.g., bacterial cells) or target cells.
“Operably linked” means that the nucleic acid sequence is functionally associated with the sequence it is operably linked to, such that they are connected in a manner that affects their expression or function in relation to each other. For example, a nucleic acid sequence operably linked to a promoter will have an expression pattern influenced by the promoter.
The present invention also provides a host cell for expressing the TBX1 protein. Host cells can be prokaryotic cells such as bacterial cells, or lower eukaryotic cells such as yeast cells, or higher eukaryotic cells such as mammalian cells. Representative examples include Escherichia coli, Bacillus subtilis; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, 293 cells, T cells, and NK cells.
Pharmaceutical Formulation
The present invention provides a pharmaceutical formulation or composition comprising: (a) the active ingredient as described in the second aspect of the present invention, or the vector as described in the third aspect of the present invention, or the host cell as described in the fourth aspect of the present invention, and (b) a pharmaceutically acceptable carrier, excipient, or diluent.
In another preferred embodiment, the pharmaceutical formulation is used for repairing heart tissue injuries.
In another preferred embodiment, the pharmaceutical formulation is used for treating heart diseases associated with cardiac injury.
In another preferred embodiment, the pharmaceutical formulation is used for treating diseases associated with lymphatic abnormalities.
The “active ingredient” in the pharmaceutical formulation of the present invention refers to TBX1 protein, its encoding sequence, or its promoting agent, or a combination thereof, or the expression vector of the present invention, such as a viral vector. The “active ingredient”, formulation, and/or composition of the present invention can be used to treat heart diseases associated with cardiac injury and/or diseases associated with lymphatic vessel abnormalities. The term “safe and effective dose” refers to an amount of the active ingredient sufficient to significantly improve the condition or symptoms without causing severe side effects. “Pharmaceutically acceptable carrier or excipient” refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. “Compatibility” here refers to the ability of the components in the composition to mix with each other and with the active ingredient of the present invention without significantly reducing the efficacy of the active ingredient.
The formulations and/or compositions of the present invention may also include BMP4, CCL28, anti-CD8 antibodies, and other active ingredients similar in function to the TBX1 molecule. The formulations and/or compositions can be used in combination with other known drugs for treating heart diseases associated with cardiac injury and/or diseases associated with lymphatic abnormalities in the field.
The composition can be in liquid or solid form, such as powder, gel, or paste. Preferably, the composition is in liquid form, preferably injectable liquid. Suitable excipients will be known to those skilled in the field.
In any administration mode, preferably, the carrier is provided as an injectable liquid. Preferably, the injectable liquid is provided as capsules or syringes.
Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium dodecyl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, and deionized water.
The composition can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable or dispersible solutions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.
Method for Screening Promoting Agents of TBX1 Protein:
The present invention also provides a method for screening promoting agents of the TBX1 protein, comprising the steps of: administering candidate drugs to in vitro cultured lymphatic endothelial cells and/or experimental animals. If the TBX1 mRNA level, TBX1 protein level, or TBX1 protein function in the in vitro cultured lymphatic endothelial cells or lymphatic vessels in experimental animals is elevated relative to the control group that did not receive the candidate drug, then the candidate drug can be considered a promoting agent of the TBX1 protein.
In the present invention, the term “candidate drug” includes, but is not limited to: small molecule compounds, nucleic acid drugs, or protein drugs. The candidate drug may directly or indirectly affect TBX1, but ultimately leads to an increase in TBX1 protein function, level, or TBX1 mRNA level in in vitro cultured lymphatic endothelial cells or lymphatic vessels in experimental animals.
In another preferred example, the promoting agent of the TBX1 protein can be used for treating heart diseases associated with cardiac injuries.
The main advantages of the present invention are as follows:
Next, specific examples are provided to further illustrate the present invention. It should be understood that these examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental methods without specific conditions in the following examples are generally carried out under conventional conditions as described in references like “Molecular Cloning: A Laboratory Manual” by Sambrook et al., Cold Spring Harbor Laboratory Press, 1989, or according to the conditions recommended by manufacturers. Unless otherwise indicated, percentages and ratios are calculated by weight.
Materials, reagents, instruments, etc., used in the examples, if not specifically mentioned, can be obtained through commercial sources.
Activation of Tbx1 Expression in Cardiac Lymphatic Endothelial Cells after Myocardial Infarction
Following induction of myocardial infarction in adult mice, the expression of Tbx1 in cardiac endothelial cells continued to increase, and 90% of Tbx1-positive cells were identified as lymphatic endothelial cells (
Impaired Recovery after Myocardial Infarction Due to Lymphatic Endothelial-Specific Deletion of Tbx1
A mouse model with tissue-specific deletion of Tbx1 in cardiac lymphatic endothelial cells was generated (
Transcriptome sequencing (
Tbx1 Regulates Autoimmune Response after Myocardial Infarction
Chromatin immunoprecipitation sequencing (ChIP-seq) detected binding sites of the transcription factor Tbx1 on the cardiac endothelial cell genome. Genes related to autoimmune tolerance were significantly enriched, indicating Tbx1's potential role in regulating autoimmune response after myocardial infarction (
Endothelial-specific transcriptome analysis revealed that Tbx1 deficiency led to decreased mRNA levels of immunomodulatory genes, such as Icam1, Ccl21, and Ccl28, in endothelial cells. Icam1 is an adhesion protein involved in leukocyte migration and suppression of leukocyte immune activity. Ccl21 and Ccl28 are cytokines that regulate the immune microenvironment and attract immunosuppressive cells like regulatory T cells to inflammation sites, reducing autoimmune response. Immunofluorescence experiments showed reduced protein levels of Icam1, Ccl21, and Ccl28 in lymphatic endothelial cells of Tbx1-deficient mice seven days after myocardial infarction (
Single-cell RNA sequencing analysis revealed active proliferation of CD8+ T cells in the hearts of Tbx1-deficient mice, resulting in a significant increase in cell numbers (
Tbx1 Deficiency Leads to Increased Macrophage Infiltration after Myocardial Infarction
Myocardial infarction was induced in adult Tbx1-deficient mice, and macrophage subsets were analyzed by flow cytometry at the 7th day after myocardial infarction. Results revealed a significant decrease in M2 macrophages (characterized by high F4/80 expression and low Ly6c expression, promoting repair and inflammation resolution) and a significant increase in M1 macrophages (characterized by low F4/80 expression and high Ly6c expression, indicating inflammation and potential autoimmune activation) in the Tbx1-deficient group compared to controls (
Immunohistochemistry staining also showed reduced M2 macrophage cell counts at the infarct site in the hearts of Tbx1-deficient mice at the 7th day after myocardial infarction (
Overexpression of Tbx1 in Lymphatic Endothelial Cells Activates Lymphatic Endothelial Cell Proliferation
Lymphatic endothelial cells play a critical role in myocardial infarction repair. During the transition from the acute inflammatory phase to the reparative phase, alterations in the transcriptome of lymphatic endothelial cells driven by Tbx1 not only promote cardiac lymphangiogenesis but also confer additional immunomodulatory functions. This helps suppress post-myocardial infarction autoimmune responses and prevent sustained attacks from autoreactive T cells and chronic inflammation. Therefore, in this example, we targeted lymphatic endothelial cells to investigate the feasibility of using Tbx1 overexpression to regulate the cardiac immune microenvironment for myocardial infarction treatment.
Firstly, Rosa26Stop-Tbx1-ZG mice (Tbx1OE mice,
Results revealed that activation of Tbx1 in cardiac lymphatic endothelial cells increased the expression of proliferation-related genes in lymphatic endothelial cells of adult mice hearts. Notably, expression levels of genes such as Top2a, Mki67, Cenpe, and Aurkb were more than doubled (n=3, P<1×10−5) after Tbx1 overexpression (
Effects of Lymphatic Endothelial Tbx1 Overexpression on Post-Myocardial Infarction Recovery
Lymphatic-specific Prox1Cre mice were crossed with Tbx1OE mice to obtain double heterozygous mice. Adult double heterozygous mice were orally administered tamoxifen to induce Tbx1 overexpression in lymphatic endothelial cells. One week later, these mice underwent coronary artery ligation to model myocardial infarction. Cardiac function was measured using echocardiography 7, 14, and 60 days after surgery.
Results indicated that Tbx1-overexpressing mice exhibited significantly improved cardiac ejection fraction two weeks after myocardial infarction (
Lymphatic Endothelial Tbx1 Overexpression Reduces Cytotoxic CD8+ T Cells in the Heart
Lymphatic-specific Prox1Cre mice were crossed with Tbx1OE mice to obtain double heterozygous mice. Adult double heterozygous mice were orally administered tamoxifen to induce Tbx1 overexpression in lymphatic endothelial cells. One week later, these mice underwent coronary artery ligation to model myocardial infarction. Flow cytometry analysis was used to evaluate CD8+ T cell subsets at the 7th day after myocardial infarction. Results indicated a significant increase in PD-1-expressing exhausted CD8+ T cells and a reduction in TNFα expression levels in CD8+ T cells in the Tbx1OE group (
The occurrence of acute inflammation and injury repair following myocardial infarction is a complex, self-driven process involving various cell types such as immune cells, cardiomyocytes, vascular endothelial cells, and fibroblasts. Most research in the field of myocardial infarction repair and regeneration has focused on these cell types.
The results of the present invention suggest that lymphatic endothelial cells play a significant role in the repair of myocardial infarction damage. During the transition from the acute inflammatory phase to the repair phase, Tbx1-driven changes in the transcriptome of lymphatic endothelial cells not only promote lymphatic endothelial cell proliferation but also confer additional immunomodulatory functions. This helps suppress post-myocardial infarction autoimmune responses and prevent sustained attacks from self-reactive T cells and chronic inflammation. Unexpectedly, this invention revealed that Tbx1 in cardiac lymphatic endothelial cells regulates the intramyocardial microenvironment and coordinates post-myocardial infarction repair and regeneration. Tbx1 also assist in post-infarction repair through lymphatic endothelial cells.
Another distinctive feature of the present invention is the revelation of an immunomodulatory repair mechanism that starts at the beginning of the post-myocardial infarction reparative stage. This mechanism is characterized by its unique stage and cell-type specificity, different from previous studies on immune intervention in myocardial infarction repair and regeneration. Prior research has shown that non-selective suppression of inflammation is detrimental to myocardial infarction repair. Current clinical studies of immune intervention in post-myocardial infarction treatment have not demonstrated clear therapeutic benefits. Therefore, this invention provides a new dimension to the orderly regulation mechanism of post-myocardial infarction repair and regeneration. By enhancing this novel endogenous repair and regeneration mechanism, new strategies and approaches to promote post-myocardial infarction recovery and repair are formed. Additionally, creating a favorable microenvironment through the inhibition of autoimmune reactions can also support cell transplantation for myocardial infarction repair and regeneration.
The heart is an organ prone to autoimmune responses. Certain cardiomyocyte-specific proteins are usually not expressed in the thymus, allowing autoreactive T cells against cardiomyocyte-specific antigens to escape central tolerance mechanisms and be released into the peripheral blood. Following cardiac injury, myocardial self-antigens are released, activate autoreactive T cells and lead to autoimmune reactions. Apart from acute myocardial injury caused by ischemia and hypoxia, various diseases leading to myocardial damage, such as myocarditis, certain idiopathic dilated cardiomyopathies, Chagas' disease, and rheumatic heart disease, are associated with persistent autoimmune reactions. Therefore, this invention's research suggests that overexpression of Tbx1 can also be used to treat other heart diseases related to autoimmune reactions, beyond myocardial infarction.
Furthermore, although the mechanisms presented in this invention are primarily based on a cardiac injury model, the genes regulated by Tbx1 and associated with lymphatic endothelial growth are generally expressed in lymphatic endothelial cells throughout various tissues. Thus, similar mechanisms can be applied to treat lymphatic abnormalities in various tissues, such as primary lymphedema caused by defective lymphatic development or secondary lymphedema resulting from lymphatic rupture or obstruction. It can also be applied to genetic syndromes characterized by lymphatic edema, such as Turner syndrome, hypoparathyroidism, yellow nail syndrome, and Hennekam syndrome.
All references mentioned in the present application are are incorporated by reference herein, as if each reference were cited separately. Furthermore, it should be understood that after reading the aforementioned disclosure of this invention, those skilled in the field can make various modifications or variations of the invention, all of which fall within the scope of the claims attached to the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110231913.4 | Mar 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/094932 | 5/20/2021 | WO |
