Patent Application
20230104671
The contents of the electronic sequence listing (702581.02232.xml; Size: 11,485 bytes; and Date of Creation: Oct. 3, 2022) is herein incorporated by reference in its entirety.
The field of the invention relates to methods and compositions for treating cardiovascular diseases including cardiomyopathy and heart failure. In particular, the field of the invention relates to methods and compositions that utilize or comprise inhibitors of hypoxia-inducible factor (HIF)-2α or prolyl hydroxylase domain-2 (PHD2) agonists or inducers for treating cardiomyopathy and heart failure.
During development, the heart grows by cardiomyocyte proliferation and hypertrophy while after birth, the cardiomyocytes lose proliferative potential and heart growth is mainly via cardiomyocyte hypertrophy. Cardiac hypertrophy can happen in physiological and pathophysiological conditions. Pathological hypertrophy induced by hypertension, myocardial infarction, and/or cardiomyopathy results in ventricular remodeling which is associated with systolic and diastolic dysfunction and interstitial fibrosis, and finally leads to deleterious outcomes such as heart failure1,2.
There are multiple cell types in the heart including cardiomyocytes, endothelial cells (ECs), fibroblasts and smooth muscle cells. ECs lining of the inner layer of the blood vessels account for the greatest number of cells in the heart. One of the major functions of cardiac vascular ECs is to supply oxygen and nutrients to support cardiomyocytes3,4. Previous studies have demonstrated that angiogenesis stimulated by VEGF-B or PIGF induces a marked increase of cardiac mass in rodents, whereas inhibition of angiogenesis results in decreased capillary density, contractile dysfunction, and impaired cardiac growth5,6. Cardiac vasculature rarefaction is associated with pathological cardiac hypertrophy and heart failure7. Recent studies have also demonstrated the important role of EC-derived paracrine factors such as Endothelin-1, Apelin, Neuregulin and Agrin in regulating cardiac hypertrophy, regeneration, and repair8,9. However, how ECs crosstalk with cardiomyocytes in the pathogenesis of cardiac hypertrophy and dysfunction is not fully understood.
Disclosed herein are compositions and methods useful for the treatment of cardiovascular disease, including cardiomyopathy, heart failure, or hypertrophy, in a subject in need thereof. In some embodiments the methods comprise administering to the subject in need an effective amount of a HIF-2α inhibitor. In particular embodiments, the HIF-2α inhibitor includes ethacridine, quinidine or an analog of quinidine such as hydroxyethylapoquinine (HEAQ), methantheline, propantheline, or their analogs or any combination thereof. In particular embodiments, the HIF-2α inhibitor includes PT2977, PT2385, PT2399, NKT2152 or C76 or their analogs or any combination thereof.
Pharmaceutical compositions for use in the treatment of cardiovascular disease, including cardiomyopathy, heart failure, and hypertrophy, may include a HIF-2α inhibitor. In particular embodiments, the HIF-2α inhibitor includes ethacridine, quinidine or an analog of quinidine such as hydroxyethylapoquinine (HEAQ), methantheline, propantheline, or their analogs or any combination thereof. In particular embodiments, the HIF-2α inhibitor includes PT2977, PT2385, PT2399, NKT2152 or C76 or their analogs or any combination thereof. In some embodiments, the HIF-2α inhibitor is PHD2. In some embodiments, the method further comprising administering an agent to activate PHD2 or induce PHD2 expression.
Another aspect of the invention provides for a method for treating cardiovascular disease, including cardiomyopathy, heart failure, or hypertrophy, in a subject by administering an effective amount of an agent to activate PHD2 or to induce PHD2 expression.
Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.
Cardiovascular diseases, including cardiomyopathy and hypertrophy are common adaptive responses to injury and stress. Hypoxia signaling is important to the (patho)physiological process of cardiac remodeling. However, the role of endothelial Prolyl-4 hydroxylase 2 (PHD2)/hypoxia inducible factors (HIFs) signaling in the pathogenesis of cardiac hypertrophy and heart failure is unknown. Presented herein is an unexpected role of endothelial PHD2 deficiency in inducing cardiac hypertrophy and fibrosis in a HIF-2a dependent manner.
Compositions and methods for treating cardiovascular disease, including cardiomyopathy and hypertrophy in a subject in need thereof with compounds disclosed herein are provided. As used herein, a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment. In particular embodiments, the subject is a human subject.
A “subject in need of treatment” may include a subject having a disease, disorder, or condition that may be characterized as a cardiovascular disease, including but not limited to cardiomyopathy, left ventricular hypertrophy or heart failure.
Cardiomyopathy represents a collection of diverse conditions of the heart muscle. Cardiomyopathy is a disease of the heart muscle that makes it harder for the heart to pump blood to the rest of the body and can lead to heart failure. Cardiomyopathy can be acquired, e.g., developed because of another disease, condition, or factor, or inherited. The cause is not always known. The main types of cardiomyopathy include dilated, hypertrophic, arrhythmogenic and restrictive cardiomyopathy.
Left ventricular hypertrophy (LV hypertrophy or LVH) is a thickening of the walls of the lower left heart chamber. The left ventricle is the heart's main pumping chamber and this chamber pumps oxygenated blood into the aorta, the large blood vessel that delivers blood to the body's tissues. If the left ventricle has to work too hard, its muscle hypertrophies (enlarges) and becomes thick. This is called left ventricular hypertrophy (LVH). The thickened heart wall can become stiff and blood pressure in the heart increases, making it harder for the heart to effectively pump blood. Because of the increased thickness, blood supply to the muscle itself may become inadequate. This can lead to cardiac ischemia (not enough blood and oxygen at the tissue level), myocardial infarction (heart attack), or heart failure. Uncontrolled high blood pressure is the most common cause of LV hypertrophy. Complications include irregular heart rhythms, called arrhythmias, and heart failure.
Heart failure occurs when the heart muscle doesn't pump blood as well as it should. Heart failure develops when the heart, via an abnormality of cardiac function (detectable or not), fails to pump blood at a rate commensurate with the requirements of the metabolizing tissues or is able to do so only with an elevated diastolic filling pressure. Heart failure can occur if the heart cannot pump (systolic) or fill (diastolic) adequately. Congestive heart failure is a type of heart failure.
The Examples show that cardiomyopathy patients have decreased levels of PHD2, and that loss of endothelial PHD2 induces LV hypertrophy, including increased LV wall thickness, reduced LV chamber size, and increased expression of hypertrophy markers including the genes Ano, Bnp and Myh7. Further, a causal role for endothelial HIF-2a in mediating LV hypertrophy and fibrosis is shown. Thus compositions that inhibit HIF-2a or increase PHD2 may be therapeutic in cardiomyopathy.
In some embodiments, a subject may be in need of treatment, for example, treatment may include administering a therapeutic amount of one or more agents that inhibits, alleviates, or reduces the signs or symptoms of cardiomyopathy.
Pharmaceutical or therapeutic compositions disclosed herein may be formulated for administration by any suitable route of delivery, such as oral, parenteral, rectal, nasal, topical, or ocular routes, or by inhalation.
In some embodiments the compositions and methods for treating cardiovascular disease, including cardiomyopathy and LV hypertrophy comprises administering effective amount of an inhibitor of hypoxia-inducible factor (HIF)-2a (HIF-2a). HIFs are transcription factors that respond to decreases in available oxygen in the cellular environment, or hypoxia. Members of the HIF family include HIF-1α, HIF-1β, HIF-2α, and HIF-3α. The α-subunit of HIF is delicately controlled by HIF-prolyl hydroxylases (PHD) 1-3.
HIF-2α (or HIF-2alpha or HIF-2a) is, encoded by HIF2A, the endothelial PAS domain-containing protein 1 in mammals. HIF-2α is active under hypoxic conditions and is important for maintaining the catecholamine balance required for protection of the heart. Hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2), or prolyl hydroxylase domain-containing protein 2 (PHD2), is an enzyme encoded by the EGLN1 gene. It is also known as Egl nine homolog 1. PHD2 is a α-ketoglutarate/2-oxoglutarate-dependent hydroxylase, a superfamily non-heme iron-containing proteins. In humans, PHD2 is one of the three isoforms of hypoxia-inducible factor-proline dioxygenase, which is also known as HIF prolyl-hydroxylase.
Under hypoxia condition, PHD activities are inhibited leading to stabilization of HIF-α proteins which activate downstream target gene expression. In normoxia, HIF alpha subunits are marked for the ubiquitin-proteasome degradation pathway through hydroxylation of proline-564 and proline-402 by PHD2. Both HIF-1α and HIF-2α share similar DNA binding site or hypoxia response element (HRE). Thus, some genes are co-regulated by HIF-1α and HIF-2α. However, HIF-1α and HIF-2α also control some sets of unique downstream targets, for example, LDHA, is a HIF-1α target while OCT4 is a HIF-2α target.
The pharmaceutical compositions of the present disclosure may include one or more compounds that inhibit HIF-2α. By way of example, but not by way of limitation, such compounds include quinidine and analogs thereof, and ethacridine and analogs thereof, propantheline/methantheline and analogs thereof. Additional HIF-2α inhibitors include, without limitation, PT2977 (also known as belzutifan, Welireg, MK-6482), PT2385, PT2399, NKT2152, C76, and analogs thereof. PHD2 also inhibits HIF2a through hydroxylation of proline-564 and proline-402 and thereby marking HIF for the ubiquitin-proteasome degradation pathway. In some embodiments, the pharmaceutical composition of the present disclosure includes one or more HIF-2α inhibitors wherein at least one of the HIF-2α inhibitors is PHD2.
As used herein the term “analogue” or “functional analogue” refer to compounds having similar physical, chemical, biochemical, or pharmacological properties. Functional analogues are not necessarily structural analogues with a similar chemical structure. An example of pharmacological functional analogues are morphine, heroine, and fentanyl, which have the same mechanism of action, but fentanyl is structurally quite different from the other two.
Quinidine (CAS 56-54-2) is a medication that acts as a class I antiarrhythmic agent in the heart. It prolongs cellular action potential and decreases automaticity. Quinidine also blocks muscarinic and alpha-adrenergic neurotransmission. Analogs of quinidine include, but are not limited to hydroxyethylapoquinine (1-[(5Z)-5-ethylidene-1-azabicyclo[2.2.2]octan-2-yl]-1-(6-hydroxyquinolin-4-yl)propane-1,3-diol; dihydrochloride; CAS 5414-52-8; HEAQ), hydroxyethylquinine (HEQ) [Antimicrob Agents Chemother. 2014 February; 58(2): 820-827], and hydroxyethylquinidine (HEQD)[Id.].
Ethacridine (7-ethoxyacridine-3,9-diamine, CAS 442-16-0) is an aromatic organic compound based on acridine. Its primary use is as an antiseptic in solutions of 0.1%. It is effective against mostly Gram-positive bacteria, such as Streptococci and Staphylococci, but ineffective against Gram-negative bacteria such as Pseudomonas aeruginosa. Ethacridine is also used as an agent for second trimester abortion.
Propantheline (methyl-di(propan-2-yl)-[2-(9H-xanthene-9-carbonyloxy)ethyl]azanium, brand name Probanthine among others; CAS 298-50-0) is one of a group of antimuscarinic agents (including methantheline bromide) for the treatment of excessive sweating, cramps or spasms of the stomach, intestines (gut) or bladder, and involuntary urination. It can also be used to control the symptoms of irritable bowel syndrome and similar conditions.
Methantheline (diethyl-methyl-[2-(9H-xanthene-9-carbonyloxy)ethyl]azanium; CAS 5818-17-7) is a synthetic quaternary ammonium antimuscarinic used to relieve cramps or spasms of the stomach, intestines, and bladder.
PT2977 (3-[[(1S,2S,3R)-2,3-difluoro-1-hydroxy-7-methylsulfonyl-2,3-dihydro-1H-inden-4-yl]oxy]-5-fluorobenzonitrile; CAS 1672668-24-4), also known as Belzutifan or MK-6482, the brand name Welireg, is an orally active, small molecule inhibitor of hypoxia inducible factor HIF-2a with potential antineoplastic activity. Upon oral administration, belzutifan binds to and blocks the function of HIF-2α, thereby preventing HIF-2α/HIF-β heterodimerization and its subsequent binding to DNA. This results in decreased transcription and expression of HIF-2α downstream target genes, many of which regulate hypoxic signaling.
PT2385 (3-[[(1S)-2,2-difluoro-1-hydroxy-7-methyl sulfonyl-1,3-dihydroinden-4-yl]oxy]-5-fluorobenzonitrile; CAS 1672665-49-4) is a HIF-2α inhibitor which causes an allosteric disruption of a protein-protein interaction between HIF-2α and Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT).
PT2399 (3-[[(1S)-7-(difluoromethylsulfonyl)-2,2-difluoro-1-hydroxy-1,3-dihydroinden-4-yl]oxy]-5-fluorobenzonitrile; CAS 1672662-14-4) is a selective HIF-2α antagonist, which directly binds to HIF-2α PAS B domain.
NKT2152 (CAS 2511247-29-1) is a selective HIF-2α inhibitor that blocks HIF herterodimerization and inhibits HIF-2α function. [WO 2022/187528; NCT05119335]
Compound 76 or C76 (methyl 3-[(2E)-2-[cyano(methylsulfonyl)methylidene]hydrazinyl]thiophene-2-carboxylate; CAS 882268-69-1) is a cell-permeable thienylhydrazone compound that suppresses the translation of cellular HIF-2α message by enhancing the binding of IRP1 (Iron-Regulatory Protein 1) to the IRE (Iron-Responsive Element) region at the 5′ UTR of the HIF-2α mRNA.
In some embodiments, pharmaceutical composition of the present disclosure includes one or more HIF-2α inhibitors, and at least one additional active agent or procedure. KRH102053 (4-methoxy-6-((4-methoxybenzyl)amino)-2,2-dimethylchroman-3-yl methioninate; CAS 1353254-53-1) or analogs thereof or KRH102140 (N-(2-fluorobenzyl)-2-methyl-2-phenethyl-2H-chromen-6-amine; CAS 864769-01-7) are exemplary molecules that activate PHD2 and inhibit HIF activity.
Exemplary additional active agents and procedures include but are not limited to angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, water pills, also called diuretics, catheter procedure, and surgery.
Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion.
As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration. In some embodiments, the subject is responsive to therapy with one or more of the compounds disclosed herein in combination with one or more additional therapeutic agents.
As used herein the term “effective amount” refers to the amount or dose of the compound that provides the desired effect. In some embodiments, the effective amount is the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. Suitably the desired effect may be improving symptoms of cardiomyopathy.
In some embodiments, improvement in a patient's condition, as compared to an untreated control subject, is noted within about a day, a week, two weeks, within about one month or two months after the first treatment is administered. By way of example, improvements in a patient's condition may include, but is not limited to an improvement in clinical symptoms, test results, histopathological findings, quality of life, LV wall thickness, LV chamber size, LV cell surface measurements, and perivascular or intercardiomyocyte fibrosis.
An effective amount can be readily determined by those of skill in the art, including an attending diagnostician, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
Some embodiments of the present disclosure include methods for treating cardiovascular disease, including cardiomyopathy, LV hypertrophy and heart failure. In some embodiments, the cardiovascular disease is characterized by decreased expression of PHD2 in endothelial cells.
In some embodiments the methods for treating cardiovascular disease include administration to a subject, an effective amount of a HIF-2α inhibitor. Without limitation, the method may include administering ethacridine, quinidine or an analog of quinidine such as hydroxyethylapoquinine (HEAQ), methantheline, and propantheline, and their analogs. Without limitation, the method may include administering PT2977, PT2385, PT2399, NKT2152, C76, combinations thereof or analogs thereof. In particular embodiments, the method may include increasing PHD2 activity or expression.
Some embodiments of the present disclosure include methods for reducing left ventricular hypertrophy wherein the method comprises administering an effective amount of a HIF-2α inhibitor. Without limitation, the method may include administering ethacridine, quinidine or an analog of quinidine such as hydroxyethylapoquinine (HEAD), methantheline, and propantheline, and their analogs. Without limitation, the method may include administering PT2977 (belzutifan Welireg, MK-6482), PT2977, PT2385, PT2399, NKT2152, C76, combinations thereof or analogs thereof. In particular embodiments, the method may include increasing PHD2 activity or expression.
Some embodiments of the present disclosure include methods comprising administering an effective amount of an agent to activate PHD2 or to induce PHD2 expression. Activating or inducing PHD2 causes the transcription or translation of the EGLN1 gene, or alternatively an active form of the PHD2 protein. Based on the Example herein, PHD2 is active under normoxic conditions and PHD2 expression is decreased in cardiomyopathy patients. An agent to activate PHD2 or induce PHD2 expression causes a change in EGLN1 gene expression or PHD2 protein such that there is more EGLN1 or PHD2 protein activity is increased.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
As used herein, the term “decrease” or the related terms “decreased,” “reduce” or “reduced” refers to a statistically significant decrease. For the avoidance of doubt, the terms generally refer to at least a 10% decrease in a given parameter, and can encompass at least a 20% decrease, 30% decrease, 40% decrease, 50% decrease, 60% decrease, 70% decrease, 80% decrease, 90% decrease, 95% decrease, 97% decrease, 99% or even a 100% decrease (i.e., the measured parameter is at zero).
As used herein, the term “increase” or related terms means a change or combination of changes that result in a second measurement exceeding the first measurement.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.
Methods and Compositions for Treatment of Cardiomyopathy and Heart Failure
Hypoxia-inducible factors (HIF) are key transcriptional factors mediating cellular response to oxygen levels. The a-subunit of HIF is delicately controlled by HIF-prolyl hydroxylases (PHD 1-3)10-12. Under normoxia condition, PHDs hydroxylate HIF-α (mainly HIF-1α and HIF-2α), then E3 ligase VHL promotes degradation of hydroxylated HIF-α through proteasome-degradation pathway. Under hypoxia condition, PHD activities are inhibited leading to stabilization of HIF-α proteins which activate downstream target gene expression. Both HIF-1α and HIF-2α share similar DNA binding site or hypoxia response element (HRE)11,13. Thus, some genes are co-regulated by HIF-1α and HIF-2α, such as CXCL12. However, HIF-1α and HIF-2α also control some sets of unique downstream targets, for example, LDHA, is a HIF-1α target while OCT4 is a HIF-2α target14. HIF-1α is ubiquitously expressed whereas HIF-2α is more restricted in certain cell types such as endothelial cells (ECs), and alveolar type 2 epithelial cells15, suggesting that HIF-α have distinct function in different cell types under different (patho)physiological conditions.
Egln1 null mutant mice exhibit polycythemia and congestive heart failure18. However, cardiomyocyte specific-disruption of Egln1 causes only mild abnormality with the presence of occasional myocytes with increased hypereosinophilia and blurring of the cross-striations19, indicating loss of PHD2 cells other than cardiomyocytes causes congestive heart failure. Fan et al. show that EC deletion of both PHD2 and 3 induces spontaneous cardiomegaly due to enhanced cardiomyocyte proliferation and also prevents heart failure induced by myocardial infarction. However, it is unknown whether these phenotypes are mediated by endothelial loss of either PHD2 or PHD3 alone. Studies have shown loss of both PHD2 and 3 but not PHD2 alone in cardiomyocyte induces ischemic cardiomyopathy20. To our surprise, we observed spontaneous left ventricular (LV) hypertrophy and cardiac fibrosis in tamoxifen-inducible EC-specific Egln1 knockout adult mice. Using the mice with endothelial deletion of Egln1 alone, Egln1 and Hif1a, or Egln1 and Hif2a, we found that loss of endothelial PHD2-induced cardiac hypertrophy and fibrosis was mediated by HIF-2α activation and pharmacological inhibition of HIF-2α inhibited LV hypertrophy and cardiac fibrosis. Analysis of single cell RNA sequencing datasets revealed EGLN1 mainly expressed in vascular ECs of the human heart under normal condition. In patients with hypertrophic cardiomyopathy, EGLN1 expression in LV heart tissue (mRNA) and cardiac vascular ECs (protein) was markedly deceased, which validate the clinical relevance of our findings that endothelial PHD2 deficiency induces LV hypertrophy and cardiac fibrosis. Thus, targeting PHD2/HIF-2α signaling can be a therapeutic approach for pathological cardiac hypertrophy leading to cardiomyopathy and heart failure.
The patient heart samples were collected from six cardiomyopathy patients including four hypertrophic cardiomyopathy, one dilated cardiomyopathy, and one hypertensive cardiomyopathy. Control samples were collected from similar age range individuals (five males, one female) without heart disease. The cause of death for controls was mechanical injuries. Failing heart samples were obtained from the left ventricular anterior wall during heart transplantation or implantation of LVAD (LV assistant device)21,22. The non-failing heart samples were obtained from the LV free wall and procured from National Disease Research Interchange and University of Pennsylvania. Non-failing heart donors showed no laboratory signs of cardiac disease.
Egln1Tie2Cre mice, Egln1/Hif1aTie2Cre and Egln1/Hif2aTie2Cre double knockout mice were generated as described previously16. Egln1EndoCreERT2 mice were generated by crossing Egln1 floxed mice with EndoSCL-CreERT2 transgenic mice expressing the tamoxifen-inducible Cre recombinase under the control of the 5′ endothelial enhancer of the stem cell leukemia locus23. At 8 weeks of age, Egln1EndoCreERT2 and Egln1f/f mice were treated with 2 mg tamoxifen intraperitoneally for 5 days to induced Egln1 deletion only in ECs. Mice were scarified at the age of ˜9 months. Both male and female mice were used in these studies. The use of animals was in compliance with the guidelines of the Animal Care and Use Committee of the Northwestern University and of the University of Arizona.
Echocardiography was performed on a Fujifilm VisualSonics Vevo 2100 using an MS550D (40 MHz) transducer as described previously. Briefly, mice were anesthetized in an induction chamber filled with 1% isoflurane. The left ventricle anterior/posterior wall thickness during diastole (LVAW and LVPW), the LV internal dimension during diastole (LVID), the LV fractional shortening (LVFS) and the cardiac output (CO) were obtained from the parasternal short axis view using M-mode. Results were calculated using VisualSonics Vevo 2100 analysis software (v. 1.6) with a cardiac measurements package and were based on the average of at least three cardiac cycles.
We used the publicly available metadata from two human fetal hearts (GSM4008686 and GSM4008687)24. The metadata was processed in R (version 4.0.2) via Seurat package V3.2.325. Briefly, cells that expressed fewer than 100 genes, and cells that expressed over 4,000 genes, and cells with unique molecular identifiers (UMIs) more than 10% from the mitochondrial genome were filtered out. The data were normalized and integrated in Seurat, followed by Scaled and summarized by principal component analysis (PCA), and then visualized using UMAP plot. FindClusters function (resolution=0.5) in Seurat was used to cluster cells based on the gene expression profile. Cardiomyocyte (NPPA, TNNT2), endothelial cells (CDH5), fibroblasts (FN1, VIM), smooth muscle cells (ACTA2), and macrophages (CD68, CD14) clusters were annotated based on the expression of known markers.
WT or Egln1Tie2Cre female mice at the age of 3 weeks were delivered at a dose of 750 cGy/mouse. At 3 hours following irradiation, mice were transplanted with 1×107 bone marrow cells (in 150 μl of HBSS) isolated from male Egln1Tie2Cre or WT mice through tail vein injection. Mice were used for heart dissection at the age of 3.5 months as described previously16
Following PBS perfusion, heart tissue was embedded in OCT for cryosectioning for immunofluorescent staining. Heart sections (5 μm) were fixed with 4% paraformaldehyde, followed by blocking with 0.1% Triton X-100 and 5% normal goat serum at room temperature for 1 h. After 3 washes, they were incubated with anti-Ki67 (1:25, Abcam, Cat #ab 1667), anti-CD31 antibody (1:25, BD Biosciences, Cat #550274) at 4° C. overnight and were then incubated with either Alexa 647-conjugated anti-rabbit IgG (Life Technology), or Alexa 594-conjugated anti-rabbit IgG or Alexa 488 conjugated anti-rat IgG at room temperature for 1 h after 3 washes. Nuclei were counterstained with DAPI contained in Prolong Gold mounting media (Life Technology).
For Wheat Germ Agglutinin (WGA) staining, WGA conjugated with FITC or WGA conjugated with Alexa 647 were stained with cryosectioned slides at room temple for 10 mins.
For immunohistochemistry staining on paraffin sections, the tissues were cut into 5 μm thick sections after paraffin processing. Heart sections were then dewaxed and dehydrated. Antigen retrieval was performed by boiling the slides in 10 mmol/L sodium citrate (pH 6.0) for 10 minutes. After blocking, slides were incubated with anti-PHD2 antibody (1:200, Cell Signaling Technology, Cat #4835), at 4° C. overnight. Slides were incubated with 6% H2O2 for 30 min after primary antibody incubation and were then biotinylated with a rabbit IgG and ABC kit (Vector Labs) for immunohistochemistry. The nucleus was co-stained with hematoxylin (Sigma-Aldrich).
For histological assessment, hearts were harvested and washed with PBS, followed by fixation in 4% formalin and dehydrated in 70% ethanol. After paraffin processing, the tissues were cut into semi-thin 5 m thick sections. Sections were stained with H & E staining or Masson's trichrome kit as a service of charge at Core Facility.
Total RNA was isolated from frozen left ventricular tissues with Trizol reagents (Invitrogen) followed by purification with the RNeasy Mini kit including DNase I digestion (Qiagen). One microgram of RNA was transcribed into cDNA using the high-capacity cDNA reverse transcription kits (Applied Biosystems) according to the manufacturer's protocol. Quantitative RT-PCR analysis was performed on an ABI ViiA 7 Real-time PCR system (Applied Biosystems) with the FastStart SYBR Green Master kit (Roche Applied Science). Target mRNA was determined using the comparative cycle threshold method of relative quantitation. Cyclophilin was used as an internal control for analysis of expression of mouse genes. The primer sequences are provided in Table 1.
AAAGACTGG
CTCGTGCTC
GATGCCAA
TCTCATCT
GGT
GCA
GTCTCCTCTG
ACCACCCTG
ACTTCAAC
TTGCTGTA
AGCG
GCCAA
GATAGATGAA
AGGATTGGA
GGCAGGA
GCCCAGAGT
AGCCGC
GGACTAGG
TGTTTCTGCT
CTCCGACTT
TTTCCTTTA
TTCTCTTAT
TCTGTC
CAGCTC
TGCAAAGGCT
GCCAACACC
CCAGGTC
AACCTGTC
TGAGGGC
CAAGTTC
TCACCAAACT
GACCAGGAG
CAGAAGAT
GACCAGG
GTAGGA
AAG
Total RNA was isolated from left ventricular tissues with Trizol reagents (Invitrogen) followed by purification with the RNeasy Mini kit including DNase I digestion (Qiagen). RNA sequencing was carried out by Novogene corporation on Illumina Hiseq platform. The original sequencing data were trimmed using FASTX and aligned to the reference genome using TopHat2. The differential expression analysis was performed using Cuffdiff software26.
Statistical analysis of data was done with Prism 7 (GraphPad Software, Inc.). Statistical significance for multiple-group comparisons was determined by one-way ANOVA with Tukey post hoc analysis that calculates corrected P values. Two-group comparisons were analyzed by the unpaired two-tailed Student's t test. As the covariates may affect the response, two-group comparisons of data from samples of cardiopathy patients and controls were examined by linear regression models where the covariates including age were included/adjusted. All bar graphs represent means±SD.
Decreased PHD2 Expression in Patients with Cardiomyopathy
PHDs/VHL/HIF signaling have been implicated in many physiological and pathological conditions of heart development and heart diseases. Leveraging the public single-cell RNA-sequencing dataset, we first analyzed the mRNA expression of the key molecules of PHDs/VHL/HIF signaling from fetal and adult hearts. Our data demonstrated that EGLN1 and EPAS1 (encoding HIF-2α) are highly expressed in cardiac ECs in both the fetal and adult hearts (
Constitutive loss of endothelial PHD2 induces left ventricular hypertrophy and fibrosis.
To investigate the role of endothelial PHD2 in the heart in vivo, we generated Egln1Tie2Cre mice by breeding Egln1 floxed mice with Tie2Cre transgenic mice. Dissection of cardiac tissue showed marked increase of the ratio of LV vs body weight (LV/BW), indicative of LV hypertrophy (
As Tie2Cre also expresses in hematopoietic cells in addition to ECs, we next determined whether hematopoietic cell-expressed PHD2 plays a role in inducing LV hypertrophy. We performed bone marrow transplantation via transplanting WT or Egln1Tie2Cre bone marrow cells to lethally irradiated WT or Egln1Tie2Cre mice. Three months post-transplantation, we did not observe any change of LV/BW ratio in WT mice transplanted with Egln1Tie2Cre bone marrow cells compared to WT mice with WT bone marrow cells, indicating loss of PHD2 in bone marrow cells per se didn't induce LV hypertrophy. Similarly, WT bone marrow cell transplantation to Egln1Tie2Cre mice didn't affect LV hypertrophy seen in Egln1Tie2Cre mice transplanted with Egln1Tie2Cre bone marrow cells (
To determine if the cardiac hypertrophy in the Egln1Tie2Cre mice is ascribed to potential developmental defects in mice with Tie2Cre-mediated deletion of Egln1, we generated mice with tamoxifen-inducible endothelial Egln1 deletion via breeding Egln1 floxed mice with EndoSCL-CreERT2 mice27 (
Previous studies demonstrated that stimulation of angiogenesis in the absence of other insults can drive myocardial hypertrophy in mice via overexpression of PR39 or VEGF-B5. PHD2 deficiency has been shown to induce EC proliferation and angiogenesis in vitro and in vivo33,34 To determine if vascular mass is increased in the LV of the Egln1Tie2Cre mice, we performed immunostaining with endothelial marker CD31 and WGA. The capillary/myocyte ratio was drastically increased in the LV of Egln1Tie2Cre mice compared to WT mice (
As Egln1 deletion stabilizes both HIF-1α and HIF-2α, we generated Egln1/Hif1aTie2Cre Egln1/Hif2aTie2Cre double knockout and Egln1/Hif1a/Hif2aTie2Cre triple knockout mice (
To understand the downstream mechanisms of endothelial HIF-2α in regulating cardiac function, we performed whole transcriptome RNA sequencing (RNA-seq) of LV tissue dissected from WT, Egln1Tie2Cre and Egln1/Hif2aTie2Cre mice (
To assess the therapeutic potential of HIF-2α inhibition for cardiomyopathy, we treated 3 week-old Egln1Tie2Cre mice with the HIF-2α translation inhibitor C7631,35 for 11 weeks. C76 treatment inhibited LV hypertrophy of Egln1Tie2Cre mice evident by decreased LV/BW ratio compared to vehicle treatment (
In this study we demonstrate that endothelial PHD2 is markedly decreased in the hearts of patients with cardiomyopathy. Genetic deletion of endothelial Egln1 in mice induces spontaneously severe cardiac hypertrophy and fibrosis. Through genetic deletion of Hif2a or Hif1a in Egln1Tie2Cre mice, we also demonstrate that endothelial HIF-2α but not HIF-1α activation is responsible for PHD2 deficiency-induced LV hypertrophy and fibrosis. Moreover, pharmacological inhibition of HIF-2α reduces cardiac hypertrophy in Egln1Tie2Cre mice. Thus, our data provide strong evidence that endothelial homeostasis is crucial to maintain normal cardiac function.
We demonstrate that PHD2 is mainly expressed in the vascular endothelium in human heart under normal condition, and its expression is markedly reduced in cardiac tissue and ECs of patients with cardiomyopathy.
Mice with Tie2Cre-mediated deletion of Egln1 develop LV hypertrophy and also RV hypertrophy associated with severe pulmonary hypertension16,31. The RV hypertrophy is secondary to marked increase of pulmonary artery pressure. Furthermore, we employed the tamoxifen-inducible Egln1 knockout mice to determine the role of endothelial PHD2 deficiency in heart function. Seven months after tamoxifen treatment of 8 weeks old adult mice, we observed only LV hypertrophy in the mutant mice with quite normal RV size. These data provide clear evidence that endothelial PHD2 deficiency in adult mice selectively induces LV hypertrophy. Our study demonstrates that loss of endothelial PHD2 alone is sufficient to induce LV hypertrophy without marked changes in cardiomyocyte proliferation. We also observed marked LV cardiac fibrosis with predominantly in the peri-vascular regions and less in inter-cardiomyocytes area, which may lead to heart dysfunction. Cardiac hypertrophy is associated with fibrosis indicates maladaptive deleterious remodeling36.
PHDs are oxygen sensors, which use molecular 02 as a substrate to hydroxylate proline residues of HIF-α. Deficiency of PHD2 results in stabilization and accumulation of HIF-α, and formation of HIF-α/HIF-β heterodimer, which consequently activates expression of a number of HIF target genes that regulate angiogenesis, inflammation and metabolism11,13,37 The cardiac remodeling phenotype in Egln1Tie2Cre mice is ascribed to activation of HIF-2α but not HIF-1α, as Hif2a deletion in EC protects from Egln1-deficiency induced cardiac hypertrophy and fibrosis, whereas Hif1a deletion in Egln1Tie2Cre mice does not show any protection but increases cardiac hypertrophy. Consistently, previous studies show that endothelial Hif1a deletion in TAC-challenged mice induce myocardial hypertrophy and fibrosis, and rapid decompensation38. Although both HIF-1α and HIF-2α express in ECs and share similar DNA binding site, endothelial HIF-1α and HIF-2α plays quite distinct roles in cardiac homeostasis.
Increased vascular mass and EC proliferation are evident in the LV of Egln1Tie2Cre mice demonstrating that PHD2 deficiency in cardiac vascular ECs induces EC proliferation and angiogenesis in mice. PHD2 deficiency in ECs induces angiogenesis which promotes cardiomyocyte hypertrophy leading to LV hypertrophy. Rarefaction of cardiac microvasculature is associated with pathological hypertrophy40. We in fact observed a marked increase of capillary density in Egln1Tie2Cre hearts, which may help to explain normal cardiac function including fractional shortening and ejection fraction in Egln1Tie2Cre hearts
The heart is highly organized and consists of multiple cell types including cardiomyocytes, ECs and fibroblasts. Cell-cell communication in the heart is important for cardiac development and adaptation to stress such as pressure overload. It is well documented that soluble factors secreted by ECs maintain tissue homeostasis in different physiological and pathological microenvironment including cancer and bone marrow niche4,7,41. To date, a growing number of cardio-active factors derived from ECs have been shown to regulate cardiac angiogenesis contributing to cardiac hypertrophy and/or dysfunction, including Nitric oxide (NO), Neuregulin-1, Basic fibroblast growth factor (FGF2), Platelet-derived growth factor (PDGF), Vascular endothelial growth factor (VEGF)8,41. Other endothelium-derived factors such as Endothelin-1, NO, Neuregulin, Bmp4, Fgf23, Lgals3, Lcn2, Spp1, Tgfb1, can directly promote cardiac hypertrophy and fibrosis42-46. Our data suggest that endothelial-derived factors mediate EC-myocyte crosstalk to induce cardiac hypertrophy and fibrosis through excessive angiogenesis and/or paracrine effects.
Pathological cardiac hypertrophy worsens clinical outcomes and progresses to heart failure and death. Modulation of abnormal cardiac growth is becoming a potential approach for preventing and treating heart failure in patients2. Our study demonstrates that pharmacological inhibition of HIF-2α reduces cardiac hypertrophy in Egln1Tie2Cre mice, which provides strong evidence that targeting PHD2/HIF-2α signaling is a promising strategy for patients with pathological cardiac hypertrophy. The HIF-2α translation inhibitor C76 used in this study selectively inhibits HIF-2α translation by enhancing the binding of iron-regulatory protein 1 to the 5′-untranslated region of HIF2A mRNA without affecting HIF-1α expression31,35.
In conclusion, our findings demonstrate for the first time that endothelial PHD2 deficiency in mice induces spontaneous cardiac hypertrophy and fibrosis via HIF-2α activation but not HIF-1α. PHD2 expression was markedly decreased in cardiovascular ECs of patients with cardiomyopathy, validating the clinical relevance of our findings in mice. Thus, selective targeting the abnormality of PHD2/HIF-2α signaling is a potential therapeutic strategy to treat patients with pathological cardiac hypertrophy and fibrosis.
In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
The present application claims priority to U.S. Provisional Patent Application No. 63/262,008, filed Oct. 1, 2021, the entire contents of which are hereby incorporated by reference.
This invention was made with government support under HL148810, HL140409, and HL133851 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63262008 | Oct 2021 | US |
