Suppressing GATA4 in chondrocytes

Abstract

A method of reducing at least one of expression of GATA4 and activity of GATA4 in chondrocytes in vivo includes introducing an inhibitor agent comprising at least one of siRNA and a small molecule compound active to reduce at least one of the expression of GATA4 and activity of GATA4.

Description

TITLE OF INVENTIONSuppressing GATA4 in ChondrocytesCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application Serial No. 63 / 754,648, filed February 6, 2025, the disclosure of which is incorporated herein by reference.BACKGROUND

[0002] The following information is provided to assist the reader in understanding technologies disclosed below and the environment in which such technologies may typically be used. The terms used herein are not intended to be limited to any particular narrow interpretation unless clearly stated otherwise in this document. References set forth herein may facilitate understanding of the technologies or the background thereof. The disclosure of all references cited herein are incorporated by reference.

[0003] Aging is an inevitable phenomenon resulting in limited functionality, loss of structural integrity, and inability to effectively resist injury and disease. Osteoarthritis (OA), the world’s most common form of degenerative disease, has been demonstrated to be associated with the advancement of age. Specifically, OA is estimated to affect 32.5 million Americans, with most cases occurring in adults over the age of 45. In fact, 1 in 3 people over the age of 65 are suffering from OA. There are many contributors linked to the onset and progression of aging, such as DNA damage, organelle dysfunction, and telomere shortening, but the mechanism underlying age-related OA has not been fully understood. Evidence suggests that mitochondrial dysfunction leads to oxidative stress, resulting in an excess of reactive oxygen species (ROS). The accumulation of ROS is linked to DNA damage and telomere shortening, contributing to an aged phenotype in cells. Furthermore, permanent cell cycle arrest, known as cellular senescence, contributes to chondrocyte aging and OA through the senescence-associated secretory phenotype (SASP) expression. The low-grade chronic state of inflammation that is commonly associated with aging, also known as “inflammaging,” is thought to contribute to OA along with the accumulation of ROS.

[0004] OA is now considered as a whole joint disease, and cartilage degradation represents the central feature. The physiological role of articular cartilage is to support and protect the bones of diarthrodial joints through absorbing mechanical loads and facilitating frictionless movements. The extracellular matrix (ECM) of the cartilage is composed primarily of collagen type II and glycosaminoglycans, which function to hydrate the cartilage, providing frictionless movement between bones. The gradual breakdown and rebuilding of ECM components in the cartilage is common in healthy adults. As we age, the regenerative response of chondrocytes begins to decline. The whole joint nature of OA and the limited regenerative capacity of chondrocytes have contributed to the difficulty in developing disease-modifying osteoarthritis drugs (DMOADs). To date, no DMOADs have reached FDA approval.

[0005] Currently, OA-associated changes in chondrocytes have been widely examined, which have significantly enhanced the understanding of this disease and promoted the development of potential treatments. However, OA is a combined consequence of different physiological stressors, including aging, injuries, obesity, etc. Therefore, aged chondrocytes in healthy humans without OA do not necessarily exhibit all the features of OA chondrocytes.SUMMARY OF PREFERRED EMBODIMENTS

[0006] A method of reducing at least one of expression of GATA4 and activity of GATA4 in chondrocytes in vivo includes introducing an inhibitor agent comprising at least one of siRNA and a small molecule compound active, wherein the inhibitor agent is active to reduce at least one of the expression of GATA4 and the activity of GATA4 (for example, transcriptional activity). The inhibitor agent may, for example, be injected into a joint of a patient to reduce at least one of the expression of GATA4 and the activity of GATA4 in the chondrocytes of the joint. The reduction of expression of GATA4 and / or the reduction activity of GATA4 in the chondrocytes may, for example, be used in treating or preventing osteoarthritis. In a number of embodiments, the inhibitor agent includes a small-molecule compound. The inhibitor agent may, for example, include 2-[(2H-1,3-Benzodioxol-5-yl)methyl]butanedioic acid, which has the formula:The inhibitor agent may, for example, include N-4-(diethylamino)phenyl)-5-methyl-3phenylisoxazole-4-carboxamide (also known as 3i-1000), which has the formula:

[0007] At least one of GATA4 siRNA and a small-molecule compound, which is active to reduce at least one of expression of GATA4 and activity of GATA4, may be used in the manufacture of a medicant for the treatment of or preventing of osteoarthritis. The medicant may be manufactured to be injectable into a joint of a patient to reduce the expression of GATA4 in the chondrocytes of the joint. In a number of embodiments, the medicant include the small molecule compound. The medicant may, for example, include 2-[(2H-1,3-Benzodioxol-5-yl)methyl]butanedioic acid. The medicant may, for example, include N-4-(diethylamino)phenyl)-5-methyl-3phenylisoxazole-4-carboxamide.

[0008] A method of treating or preventing osteoarthritis includes injecting an inhibitor agent into a joint of a patient to at least one of reduce expression of GATA4 and reduce activity of GATA-4. The inhibitor agent may include at least one of siRNA and a small molecule compound, wherein the inhibitor agent is active to reduce at least one of the expression and the activity of GATA4. In a number of embodiments, the inhibitor agent is injectable into ajoint of a patient to reduce at least one of expression of GATA4 and activity of G ATA4 in the chondrocytes of the joint. In a number of embodiments, the inhibitor agent includes a small molecule compound. The inhibitor agent may, for example, include 2-[(2H-1,3-Benzodioxol-5-yl)methyl]butanedioic acid. The inhibitor agent may, for example, include N-4-(diethylamino)phenyl)-5-methyl-3phenylisoxazole-4-carboxamide.

[0009] A system for reducing the expression of GATA4 in chondrocytes in a joint includes an injector system and an inhibitor agent including at least one of siRNA and a small molecule compound active to reduce expression or activity of GATA4 within a reservoir of the injector system. In a number of embodiment, the injector system includes a syringe.

[0010] A method of determining targets for treatment of chondrocyte agent includes conducting one or more transcriptomic comparisons between chondrocytes from young donors and chondrocytes from old donors to determine genes which are upregulated genes or are downregulated in the chondrocytes of old donors compared to the chondrocytes of young donors. The method may further include determining transcriptional regulators that may mediate one or more of (i) the genes which are upregulated and (ii) the genes which are downregulated in the one or more transcriptomic comparisons.

[0011] The present devices, systems, and methods, along with the attributes and attendant advantages thereof, will best be appreciated and understood in view of the following detailed description taken in conjunction with the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A illustrates schematically the isolation of healthy chondrocytes from knee joint cartilage from young (Y) and old (O) donors without osteoarthritis (as assessed by experienced surgeons), wherein directly isolated, passage 0 or P0 cells were used for RNA sequencing analysis.

[0013] FIG. IB illustrates the top 50 genes that were significantly differently expressed in young and old chondrocytes.

[0014] FIG. 1C illustrates the activation Z score of the top 10 transcription regulators that are activated (positive) or inhibited (negative) in old versus young chondrocytes.

[0015] FIG. ID illustrates photographs of GATA4 Immunohistochemistry (IHC) of healthy human cartilage tissue from young and aged donors, wherein the bar=50 pm

[0016] FIGS. 1E(i) and 1E(ii) illustrate Table 1, which is a comprehensive list of transcription regulator names and acronyms.

[0017] FIG. IF illustrates relative protein levels of GATA4 in Pl chondrocytes from individual human donors, which were analyzed by western blot.

[0018] FIG. 2 A illustrates schematically a timeline depicting studies in which GATA4 was caused to be overexpressed in healthy young human chondrocytes via infection with GATA4 lentivirus to study whether overexpressing GATA4 impairs the hyaline cartilage formation capacity of young chondrocytes.

[0019] FIG. 2B illustrates RT-qPCR analysis of GATA4 gene expression in two groups.

[0020] FIG. 2C illustrates Western blot results to measure GATA4 protein levels.

[0021] FIG. 2D illustrates Safranin O staining.

[0022] FIG. 2E illustrates collagen type II (COLII) IHC to examine the production of cartilage matrix. Scale Bar=50 pm.

[0023] FIG. 2F illustrates RT-qPCR analysis of gene expression of cartilage matrix proteins aggrecan (AC AN) and collagen type II-al (COL2A1) and hypertrophy markers collagen type X- al (COL10A1) and India hedgehog (IHH).

[0024] FIG 2G illustrates RT-qPCR analysis of gene expression of proinflammatory cytokines, including interleukin (IL)-6, IL-8, and tumor necrosis factor- a (TNF-a) (n=6).

[0025] FIG. 2H illustrates concentrations of IL-6, IL-8, and chemokine (C-C motif) ligand 2 (CCL2) in condition medium (n=3).

[0026] FIG. 21 illustrates RT-qPCR analysis of relative gene expression of matrix-degrading enzymes, including matrix metalloproteinases (MMP)- 1,2,3, 12, and 13, and a disintegrin and metalloproteinase (AD AMTS) 4 and 5 (n=6).

[0027] FIG. 2 J illustrates MMP-1 concentration in condition medium (n=3), wherein student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis in FIGS. 2B, 2F, 2E, 2H, 21 and 2J.

[0028] FIG. 3A illustrates schematically studies of whether GATA4 overexpression activates SMAD1 / 5.

[0029] FIG. 3B illustrates western blot to assess protein levels of phosphorylated SMAD1 / 5 (pSMADl / 5), phosphorylated SMAD2 / 3 (pSMAD2 / 3), and GATA4 in pellets derived from young individuals (Yl-3), which were infected with lentiviral vectors carrying GATA4 or control genes and then stimulated with (+) or without (-) TGF- β3 for two hours.

[0030] FIG. 3C illustrates western blot to assess protein levels of phosphorylated SMAD1 / 5 (pSMADl / 5), phosphorylated SMAD2 / 3 (pSMAD2 / 3), and GATA4 in pellets derived from pooled chondrocytes, which were infected with lentiviral vectors carrying GATA4 or control genes and then stimulated with (+) or without (-) TGF- β3 for two hours.

[0031] FIG. 3D illustrates relative protein levels of GATA4, which were semi-quantified using ImageJ (n=3).

[0032] FIG. 3E illustrates relative protein levels of pSMADl / 5, which were semi-quantified using ImageJ (n=3).

[0033] FIG. 3F illustrates relative protein levels of pSMAD2 / 3, which were semi-quantified using ImageJ (n=3).

[0034] FIG. 3G illustrates the ratio of pSMADl / 5 compared to pSMAD2 / 3, wherein statistics were conducted using one-way Analysis of Variance (ANOVA) with Dunnett’s post hoc analysis in FIGS. 3D-3G.

[0035] FIG. 4 A illustrates schematically studies of influence of GATA4 knockdown on in vitro cartilage formation of old chondrocytes.

[0036] FIG. 4B illustrates schematically four different GATA4 siRNAs with corresponding target sequences assessed for GATA4 knockdown in monolayer culture of old pooled chondrocytes.

[0037] FIG. 4C illustrates RT-qPCR assessing GATA4 levels after siRNA treatment (n=3).

[0038] FIG. 4D illustrates safranin O staining to examine the production of cartilage matrix in the Scrambled Control or GATA4 siRNA group. Scale Bar=50 pm.

[0039] FIG. 4E illustrates COLII IHC to examine the production of cartilage matrix in the Scrambled Control or GATA4 siRNA group. Scale Bar=50 pm.

[0040] FIG. 4F illustrates RT-qPCR analysis of relative gene expression of cartilage matrix proteins AC AN and COL2A1 and hypertrophy marker COL10Al(n=6).

[0041] FIG. 4G illustrates RT-qPCR analysis of relative gene expression of proinflammatory cytokines IL-6 and IL-8 (n=6).

[0042] FIG. 4H illustrates concentrations of IL-6, IL-8, and chemokine (CCL2) in condition medium (n=3).

[0043] FIG. 41 illustrates the relative protein levels of pSMADl / 5, pSMAD2 / 3, and phosphorylated p65 (p- P65) in two groups.

[0044] FIG. 4 J illustrates RT-qPCR analysis of relative gene expression of matrix-degrading enzymes, including MMP-1,2,3,12, and 13, and AD AMTS 4 and 5 (n=6).

[0045] FIG. 4K illustrates MMP-1,2 and 13 concentrations in condition medium (n=3), wherein student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis in FIGS. 4C, 4F, 4G, 4H, 4J, and 4K.

[0046] FIG. 5A illustrates schematically studies of the effect of Gata4 overexpression on OA in the knee joints of mice, wherein studied mice received one intraarticular injection of lentiviral vectors that carried mCherry or Gata4 gene one week before DMM surgery was performed, and knee joints were harvested 6 weeks post-surgery.

[0047] FIG. 5B illustrates levels of Gata4 assessed with IHC and mCherry control.

[0048] FIG. 5C illustrates levels of Gata4 assessed using optical density, wherein the staining was semi-quantitated with Image J.

[0049] FIG. 5D illustrates levels of p-P65 assessed with IHC.

[0050] FIG. 5E illustrates level of p-P65 assessed using optical density, wherein the staining was semi-quantitated with Image J.

[0051] FIG. 5F illustrates an assessment of cartilage degradation using Safranin O / fast green (FG) staining.

[0052] FIG. 5G illustrates calculate OARSI scores for the mCherry control and Gata4.

[0053] FIG. 5H illustrates knee hyperalgesia 6 weeks post-surgery, wherein 507 g was the threshold baseline for non-surgery mice (dashed line), and wherein student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis in FIGS. 5C, 5E, 5G, and 5H.

[0054] FIG. 6A illustrates fold change for a control and for the small molecule NSC140905, also known as or HCA42027 (2-[(2H-1,3-Benzodioxol-5-yl)methyl]butanedioic acid).

[0055] FIG. 6B illustrated a study of assessment via RT-qPCR analysis of relative gene expression of matrix-degrading enzymes, including MMP-1,13 and AD AMTS 4 and 5 (n=3) of the effect of GATA4 small molecule inhibitor, NSC 140905, wherein pooled old human chondrocytes were pelleted and treated with the chondrogenic medium with or without supplanting NSC 140905 for 14 days, and wherein student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis (Bar=50 pm).

[0056] FIG. 6C illustrates photomicrographs using safranin-O / Fast green staining for a control and for 100 pM NSC 140905.

[0057] FIG. 6D illustrates the formula of the small molecule N-4-(diethylamino)phenyl)-5-methyl-3phenylisoxazole-4-carboxamide (also known as 3i- 1000).

[0058] FIG. 7 illustrates an embodiment of an injector system for the delivery of an inhibitor agent hereof via injection.

[0059] FIG. 8 illustrates Table 2 A through 2H hereof, which provide information on age and gender of chondrocyte donors.

[0060] FIG. 9 illustrates Table 3 hereof, which identifies antibodies used for immunofluorescence (IF), Immunohistochemistry (IHC), or Western blot (WB) analysis.

[0061] FIG. 10 illustrates Table 4 hereof, which sets forth primer sequences for qRT-PCT studies hereof.

[0062] FIG. 11 illustrates Table 5 hereof, which sets forth information for LUMINEX assay kits used herein.

[0063] FIG. 12 illustrates Table 6 hereof, which sets forth a list of proteins assessed in LUMINEX assays.DESCRIPTION

[0064] It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described representative embodiments. Thus, the following more detailed description of the representative embodiments, as illustrated in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely illustrative of representative embodiments.

[0065] Reference throughout this specification to “one embodiment” or “an embodiment” (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” or the like in various places throughout this specification are not necessarily all referring to the same embodiment.

[0066] Furthermore, described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments. One skilled in the relevant art will recognize, however, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, et cetera. In other instances, well known structures, materials, or operations are not shown or described in detail to avoid obfuscation.

[0067] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a molecule” includes a plurality of such molecules and equivalents thereof known to those skilled in the art, and so forth, and reference to “the molecule” is a reference to one or more such molecules and equivalents thereof known to those skilled in the art, and so forth. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value, as well as intermediate ranges, are incorporated into the specification as if individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contraindicated by the text.

[0068] As used herein, the terms “small molecule” refers to a molecule having a molecular weight no greater than IkDa. As used herein, the term “approximately” when used in connection with a value means within 5%, within 2%, or within 1% of the value unless otherwise indicated herein or otherwise clearly contraindicated by the text. As used herein the term “and / or” means one of or both of an entity.

[0069] Although the causal association between aging and osteoarthritis (OA) has been documented, the understanding of the underlying mechanism remains incomplete. To define the regulatory molecules governing chondrocyte aging, transcriptomic analysis of young and old human chondrocytes from healthy donors were performed in studies hereof. The data predicted that GATA binding protein 4 (GATA4) may play an important role in mediating the difference between young and old chondrocytes. Results from immunostaining and western blot showed significantly higher GATA4 levels in old human or mouse chondrocytes when compared to young cells. Moreover, overexpressing GATA4 in young chondrocytes remarkably reduced their cartilage-forming capacity in vitro and induced the upregulation of proinflammatory cytokines. Conversely, suppressing GATA4 expression in old chondrocytes, through either siRNA or a small-molecule inhibitor NSC140905, increased the production of aggrecan and collagen type II, and also decreased levels of matrix-degrading enzymes, In OA mice induced by surgical destabilization of the medial meniscus, intraarticular injection of lentiviral vectors carrying mouse Gata4 resulted in a higher OA severity, synovial inflammation, and pain level when compared to control vectors. Mechanistically, it was found that overexpressing GATA4 significantly increased the phosphorylation of SMAD1 / 5.

[0070] The GATA family includes several proteins (GATA1-6) with different variations of DNA-binding domains composed of zinc finger structures. Particularly, GATA4, 5, and 6 are involved in the development of the mesoderm and endoderm tissues, in which GATA4 has been detected in the development of structures such as the heart, pancreas, lung, and liver. GATA4 and GATA6 are the only members of the GATA family that have been determined to be associated with aging. GATA4 is unique in that it regulates tissues in a context-dependent manner and adopts a multifaceted role in the body, contributing to other age-related diseases such as atherosclerosis and heart failure.

[0071] GATA4 may also be associated with metabolic regulation. Studies have demonstrated that GATA4 may regulate obesity. Gata4 knockout mice were found to be resistant to the high-fat diet, and that glucagon-like peptide- 1 (GLP-1) release was increased. A decreased risk for the development for insulin resistance in the knockout mice was indicated. Further studies showed that knockdown of Gata4 increases GLP-1 release, in turn suppressing the development of hepatic steatosis and fibrosis, ultimately blocking hepatic de novo lipogenesis. GLP-1 based therapies are being studied for the treatment of OA.

[0072] GATA4 was introduced as a senescence regulator in studies demonstrating the role of GATA4 in human fibroblasts wherein it was determined that the inhibition of autophagy caused GATA4 accumulation following DNA damage. Further studies have shown that GATA4 regulates angiogenesis and inflammation in fibroblast-like synoviocytes in rheumatoid arthritis, indicating that GATA4 is required for the inflammation induced by IL-1b. This study also demonstrated that GATA4 binds to promoter regions on Vascular Endothelial Growth Factor (VEGF)-A and VEGFC to enhance transcription and regulate angiogenesis.

[0073] In studies specific to chondrocytes, it has been demonstrated that increased GATA4 levels are associated with chondrocyte senescence, an important change often observed in OA chondrocytes. Moreover, suppressing GATA4 with siRNA was shown to effectively abolish ionizing radiation-induced senescent phenotype in chondrocytes. Previous studies relevant to chondrocytes were conducted in vitro. The studies hereof are the first to demonstrate the role of GATA4 in regulating chondrocyte aging.

[0074] In a number of studies hereof, the transcriptome of young and old human chondrocytes isolated from healthy donors, without joint diseases, were compared to identify key regulators that contribute to an “aged” state in chondrocytes prior to OA onset. It is believed that use of transcriptomic comparisons between young and old chondrocytes, whichis informative in defining targets to stop or reverse chondrocyte aging, is a novel method for defining such targets.

[0075] Unbiased analysis was used to define the key regulatory molecules that mediate chondrocyte aging. Assessment of the chondrocyte genome demonstrated the upregulation of several factors in aged chondrocytes, and transcription factor GATA binding protein 4 (GATA4) specifically drew attention since it is associated with DNA damage and cellular senescence. Without limitation to any mechanism, upregulation of GATA4 was shown to increase nuclear factor-kappa-B (NF-KB) pathway activation. NF-KB is thought to amplify and potentially propagate cellular senescence during the aging process through the senescence-associated secretory phenotype (SASP), which could contribute to a low-grade state of chronic inflammation. Furthermore, the upregulation of GATA4 in OA chondrocytes has been reported.

[0076] It was hypothesized in the studies hereof that the increased GATA4 level contributes to chondrocyte aging and accelerated OA progression, for example, upon injuries. The hypothesis was first tested by increasing or suppressing GATA4 expression in healthy chondrocytes and examining their cartilage-forming capacity. When GATA4 was overexpressed, alterations to the TGF-b signaling pathway and activation of the NF-kB signaling pathway were observed. The role of GATA4 in vivo was also assessed by injecting lentiviral vectors carrying control or mouse Gata4 genes into the knee joints of OA mice induced by surgically created destabilization of the medial meniscus (DMM). Further, a mechanistic study was conducted to explore how GATA4 impacts chondrocyte phenotypes and functions.

[0077] To limit the influence of in vitro expansion on cell phenotype, P0 human chondrocytes were used for RNA sequencing (see FIG. 1 A). It was found that 303 genes were upregulated and 163 genes were downregulated in aged chondrocytes compared to young cells. The top 50 most changed genes are listed in FIG. IB. The osteoarthritis pathway was identified to be activated in old chondrocytes.

[0078] Through Ingenuity Pathway Analysis (IPA) Upstream Regulator Analysis, transcriptional regulators that may mediate the difference between young and old cells were predicted, and the top 20 transcriptional regulators are shown in FIG. 1C. Given that GATA4 has been shown to regulate chondrocyte senescence and be involved in the activation of Nuclear Factor Kappa B (NF-KB) pathway in tissues like the nucleus pulposus and synovium, it was selected for further investigation in the studies hereof. Other molecules may also beinvolved in chondrocyte aging. For example, hypoxia-inducible factor la (HIF1A) was predicted to be the most active transcription regulator. Indeed, all the molecules depicted in FIG. 1C can be candidates for validation and examination in further studies such as performed herein for GATA4. FIGS. 1E(i) and 1E(ii) illustrate Table 1 which provides a comprehensive list of transcription regulator names and acronyms from the studies hereof.

[0079] Initial data analysis predicted that GATA4 was upregulated in aged chondrocytes compared to young donors. Immunohistochemistry (IHC) results indicated that GATA4 was more abundant in articular cartilage harvested from aged individuals (FIG. ID). Analyzing knee joints collected from young and old mice also demonstrated that GATA4 levels were higher in aged cartilage tissues compared to young. To further confirm the findings, western blot was used to examine GATA4 levels in isolated chondrocytes, and the results indicated that aging correlates with the increase of GATA4 in chondrocytes (FIG. IF).

[0080] To examine the functions of GATA4 in chondrocytes, GATA4 was caused to be overexpressed in healthy young human chondrocytes via infection with GATA4 lentivirus (see FIG. 2 A). Real-time quantitative PCR (RT-qPCR), immunostaining, and western blot confirmed the significantly increased GATA4 expression after infection, which did not impact the expression levels of collagen type II (COLII)-al (COL2A1) and aggrecan (ACAN), but significantly upregulated the expression of Collagen type X (COLX)-al (COL10A1) and India hedgehog (IHH), two representative chondrocytic hypertrophy markers. The cartilage formation capacity of cells was examined by culturing cells in chondrogenic medium for 7 days. Immunohistochemical (IHC) staining, RT-qPCR (FIG 2B), and western blot (FIG. 2C) demonstrated the continuous overexpression of GATA4 in newly formed tissues. Significantly reduced GAG and COLII production was found in the overexpression GATA4 group (GATA4 group) compared to the GFP control group (FIGS. 2D-E). Similar results were observed in the study testing chondrocytes from individual donors. GATA4 overexpression did not impact the expression of COL10A1 and IHH in the cartilage pellets (FIG. 2F).Additionally, tissues from the GATA4 group secreted more proinflammatory cytokines, including IL-6, IL-8, TNF-a, and chemokine CCL2 (FIGS. 2G and 2H), as well as enzymes that can break down cartilage, including matrix metalloproteinases (MMP)-l, 2, 12, and a disintegrin and metalloproteinases (ADAMTS) 5 (FIGS. 21 and 2 J). Although the expression levels of MMP-13 gene decreased in GATA4 group, the protein levels showed no difference between the two groups.

[0081] Increased S MAD 1 / 5 phosphorylation represents an important feature of aged chondrocytes. It was thus examined whether increased GATA4 levels are associated with SMAD1 / 5 activation. Such methodology of such studies is illustrates schematically in FIG.3A. In the studies testing chondrocytes from individual donors (FIGS. 3B, and 3D-3G), overexpression of GATA4, even without the stimulation of TGF-|33, was sufficient to activate SMAD1 / 5, but not SMAD2 / 3. In addition, the group that was co-treated with GATA4 overexpression and TGF-[33 displayed the highest phosphorylated SMAD1 / 5 (pSMADl / 5) levels in all tested groups. Activation of SMAD2 / 3 was not impacted by GATA levels. A similar trend was also observed in the study using pooled chondrocytes (FIG. 3C).

[0082] The potential of suppressing GATA4 in reversing chondrocyte aging were then tested as illustrated schematically in FIG. 4A. Several GATA4 siRNAs (see FIG. 4B) were tested to examine their capacity to suppress GATA4. Based on RT-qPCR results, siRNA2 was selected to be used in all following studies because it induced the lowest expression of GATA4. FIG.4B illustrates schematically four different GATA4 siRNAs tested with corresponding target sequences assessed for GATA4 knockdown in monolayer culture of old pooled chondrocytes. FIG. 4C illustrates RT-qPCR assessing GATA4 levels after siRNA treatment (n=3).

[0083] GATA4 knockdown resulted in increased cartilage formation from old chondrocytes, which did not influence the expression of hypertrophy marker COL10A1 (see FIGS. 4D-4F). Moreover, although no difference was observed between the scrambled control and GATA4 siRNA groups regarding the expression of IL-6 and IL-8, the protein level of IL-8 was higher in the GATA4 group. The protein level of CCL2 was significantly decreased after GATA4 knockdown (FIGS. 4G, 4H). The expression levels of MMPs and ADAMTSs were also tested (FIGS. 4J-4K). In general, suppressing GATA4 either decreased or caused no significant changes to the levels of those enzymes. In particular, MMP-13 levels were reduced, in both gene and protein levels (in condition medium), after GATA4 knockdown. Mechanistically, GATA4 siRNA treatment also lowered the phosphorylation of SMAD1 / 5 and p-P65 (FIG. 41)-

[0084] The physiological functions of GATA4 were further examined using a mouse model as illustrated schematically in FIG. 5A. Specifically, lentiviral vectors carrying Gata4 or mCherry genes were injected into the knee joints of young mice. One week after the injection, DMM surgery was conducted to induce OA. Because it was expected that Gata4 overexpression would accelerate OA progression, samples were harvested for analysis 6 weeks after DMM.

[0085] Results from IHC indicated a successful Gata4 overexpression in hyaline cartilage even 6 weeks after injection (see FIGS. 5B, 5C). Mice overexpressing Gata4 in the knee joint resulted in the elevation of p-P65 (see FIGS. 5D, 5E), indicating increased inflammation. Moreover, the mice in the Gata4 group displayed more severe OA and higher knee hyperalgesia than the control group, as demonstrated by a lower withdrawal threshold, (see FIGS. 5F-5H). Mice from the Gata4 group displayed higher synovial inflammation.

[0086] As described above, understanding the molecular mechanisms of OA is important for the development of treatment methods. In the studies hereof, the present inventors discovered the critical roles of aging-associated increases in GATA4 levels. Specifically, overexpressing GATA4 in young chondrocytes impaired their capacity to form normal hyaline cartilage, while suppressing GATA4 in old chondrocytes restored their chondrogenic potential. The studies hereof also demonstrated that GATA4 functions partially by promoting the activation of SMAD1 / 5. The in vivo studies hereof further confirmed that high Gata4 expression accelerated OA progression in mice. Moreover, as discussed further below a small-molecule GATA4 inhibitor was identified that can partially restore the capacity of old chondrocytes to create healthy cartilage, representing a potential disease-modifying osteoarthritis drug (DMOAD).

[0087] Given the recognized challenges in harvesting healthy cartilage tissues from donors without arthritis, there are limited reports investigating chondrocyte aging per se. The current findings include proliferation and post-expansion chondrogenic capacity reduction with aging, increased MMP-13 production in response to catabolic stimuli, and altered response to TGF-b. It has been shown that aged chondrocytes displayed a reduced proliferation potential compared to young cells. Additionally, cartilage tissues generated by old chondrocytes contained more senescent cells than those from young cells.

[0088] Transcriptomic analysis hereof enabled assessment of the expression of different genes and genetic pathways that occur as chondrocytes age. Of note, Hypoxia-Inducible Factor la (HIFl ) was the most differentially expressed gene predicted to regulate chondrocyte aging. The connection between HIFla and aging has been previously reported. Furthermore, additional studies have investigated HIFla in association with OA and assessed its use as a therapeutic target. GATA4 has been less studied in chondrocytes but is highly associated with cellular senescence, an aging hallmark. As set forth above, HIFla and other molecules listed in, for example, FIG. 1C may be further studied using the same strategy employed in the studies hereof.

[0089] Without limitation to any mechanism, a theory of chondrocyte aging suggests that alterations to the TGF- P pathway induce chondrocyte hypertrophy and result in articular cartilage that is prone to OA development. The studies hereof, in investigating the function of GATA4 in chondrocytes, assessed how GATA4 levels contribute to TGF-b alterations in chondrocytes and found that GATA4 levels negatively correlated with the anabolic potential of chondrocytes (see, for example, FIGS. 2A-2J and 4A-4K). There was an observed decrease in chondrogenesis and an increase in hypertrophy-related genes following GATA4 overexpression (see FIG. 2G). To maintain healthy cartilage homeostasis, numerous pathways are involved. In particular, TGF- is an important cytokine necessary for cartilage homeostasis during OA, and aged chondrocytes respond differently to TGF-P compared to their young counterparts.

[0090] Mechanistically, in the TGF-b pathway, TGF-P binds to the heterotetrameric receptor complex, which can be grouped into three receptor types (type I, type II, and type III). When TGF-p binds to its corresponding receptor, the activin-receptor-like kinases (ALKs) are activated. Typically, the anabolic binding of TGF-P to ALK4 / 5 results in the phosphorylation of SMAD2 / 3, protecting chondrocytes from hypertrophy. However, there can also be catabolic effects associated with the expression of SMAD1 / 5, typically expressed when TGF-P binds to ALK1 / 2 / 3 / 6. The phosphorylation of SMAD1 / 5 results in the promotion of ECM degrading proteins such MMP-13. As mentioned previously, research has demonstrated that aged chondrocytes respond differently to TGF-P compared to young chondrocytes.Chondrocyte aging has been linked to the increase of pSMADl / 5 signaling.

[0091] The present studies indicated that GATA4 resulted in the phosphorylation of SMAD1 / 5 even without TGF-P stimulation, which however did not alter the phosphorylation of SMAD2 / 3. The relationship of GATA4 and SMAD1 / 5 in the chondrocytes and the involvement of GAGA 4 in the TGF-p signaling pathway is complex and not fully understood, a prior study reported that GATA4 was regulated by SMAD1 / 5.

[0092] Additionally, a common hallmark of chondrocyte aging is the alternation of ECM, including composition change and stiffening. However, there is relatively little known of the mechanism by which aging impacts the ECM and its reciprocal interplay with resident cells. Investigating ECM alterations in conjunction with cellular senescence and TGF-P signaling could provide further insights into cartilage aging. A recent study associated matrix stiffening with the promotion of chondrocyte senescence. Furthermore, matrix stiffening has been associated with modulating the TGF- p signaling pathway. Further studies may investigatethe potential of matrix stiffening and the effect of GATA4 on pericellular matrix proteins such as decorin, biglycan, collagen VI and XV, as these proteins assist with the regulation of biochemical interactions and assist with the maintenance of the chondrocyte microenvironment. Herein, the TGF- [3 signaling pathway can further alter the extracellular microenvironment, which could promote cellular senescence and subsequently NF-kB pathway activation. Further investigation of ECM alterations and aging could elucidate the molecular events governing those changes, providing insights on the interplay between chondrocytes and their environment in the context of aging.

[0093] While the TGF- [3 pathway is closely associated with cartilage matrix remodeling, the present studies further demonstrated increases in the levels of multiple proinflammatory cytokines after GATA4 overexpression. The NF-KB pathway was investigated since it is also activated in aged tissues. During the aging process, NF-KB is thought to amplify and potentially propagate cellular senescence through the senescence-associated secretory phenotype (SASP). A study indicated that GATA4 regulates NF-KB in dental pulp cells and fibroblasts. Specifically, using siRNAs, they determined that the knockdown of GATA4 decreased p65 production induced by lipopolysaccharide (LPS). Other studies have investigated the role of GATA4 in the synovium and the progression of the disease state of rheumatoid arthritis and OA. It has been shown that the knockdown of GATA4 attenuated synovial inflammation and joint damage in a collagenase-induced arthritis mouse model. Another study investigated the roles of GATA4 in fibroblast-like synoviocytes (FLS) and determined that GATA4 induced cellular senescence in FLSs in OA progression.

[0094] The studies of the present invention show that the siRNA knockdown of GATA4 decreased the phosphorylation of p65 in aged chondrocytes (FIG. 4A-4K). The studies hereof also found that MMP-13 expression levels decreased in both GATA4 overexpression and knockdown experiments. MMP-13 is constitutively produced in human chondrocytes but is only activated under pathological conditions. Given that MMP-13 is regulated by different transcriptional factors and cytokines, as well as RNAs, its associations with GATA4 require further investigation. Collectively, and without limitation to any mechanism, such findings further indicate that GATA4 might regulate aging partially through TGF-(3 and NF-KB pathways.

[0095] While the present studies have not fully explored why aging promotes GATA4 expression, another study indicated that DNA damage contributed to GATA4 accumulation. It is known that DNA damage response (DDR) is regulated by ataxia telangiectasia mutated(ATM) and ataxia telangiectasia and Rad3-related (ATR) signaling. The activation of these signaling pathways inhibits autophagy-related protein p62. In addition, increased DNA damage has been reported in old chondrocytes. DNA damage may be a reason for GATA4 upregulation. The data hereof this supports that possibility. The DNA damage-inducing agent, doxorubicin, promoted the upregulation of GATA4 in chondrocytes. In another study, Tributyltin (TBT), a well-known endocrine-disrupting chemical, was used to induce DNA damage in articular chondrocytes. After 24 hours of TBT treatment, there was a significant increase in GATA4 expression and expression of senescence markers. It has also been demonstrated that the suppression of p62 following DNA damage leads to GATA4 accumulation due to the lack of autophagy. DNA damage is known to increase with age.

[0096] Therefore, and without limitation to any mechanism, the present inventors hypothesized that DNA damage due to aging is a key driver of the upregulation of GATA4 in old chondrocytes. In summary, the studies hereof determined that GATA4 is increased in aged chondrocytes compared to young in both humans and mice, which may be induced by increased DNA damage observed in aged cells. The studies hereof also demonstrated that GATA4 overexpression impaired the quality and quantity of cartilage created by chondrocytes and accelerated OA progression in mice. Conversely, suppressing GATA4 with siRNA or small-molecule inhibitors partially restores the capacity of old chondrocytes to form cartilage. Additionally, the studies hereof found that GATA4 can activate SMAD1 / 5 and change chondrocyte response to TGF-p. Overall, GATA4 may be a contributor to OA onset and progression in aged individuals, which can also serve as a potential target to prevent aging-associated OA.

[0097] In further studies hereof, a small-molecule GATA4 inhibitor NSC 140905 was shown to significantly reduce expression of GATA4 and to promote cartilage formation from old chondrocytes and reduce the expression of proinflammatory cytokines (see FIGS. 6A-6C). Taken together, suppressing GATA4 partially restored the capacity of old chondrocytes to create new cartilage.

[0098] In a treatment protocol, the expression of GATA4 in chondrocytes may be decreased via administration of GATA4 siRNA as described above. Moreover, as further demonstrated by the studies hereof, small molecules such as NSC 140905, also known as, HCA42027 (2-[(2H-l,3-Benzodioxol-5-yl)methyl]butanedioic acid) may be used to reduce the expression of GATA4 in aged chondrocytes, ameliorate the senescence levels, and restore their regeneration capacity. In the studies of FIG. 6 A, old-pooled chondrocytes (6 donors >65 years old) werepassaged to passage 5 (P5) and then pelleted. The control group was treated with chondrogenic medium containing: DMEM, lx Antibiotics-Antimycotics, lx Insulin-Transferrin-Selenium, lx sodium pyruvate, 10-7 M dexamethasone, 40 pg / mL L-proline, supplemented with 10 ng / mL transforming growth factor- 133 (TGF- (33), and 50 pg / mL ascorbic acid-2-phosphate. For the treatment group, the same media was used and the small molecule, NSC140905, was supplemented in the media at 100 pM. The pellets were treated for 14 days, with daily media changes. Upon day 14 pellets were collected for immunohistochemistry, RT-qPCR, and western blot. FIG. 6B illustrates a study of assessment via RT-qPCR analysis of the effect of GATA4 small molecule inhibitor, NSC 140905, on relative gene expression of matrix-degrading enzymes, including MMP-1,13 and AD AMTS 4 and 5 (n=3). FIG. 6C illustrates photomicrographs using safranin-O / Fast green staining for a control and for 100 pM NSC140905.

[0099] Given that GATA4 upregulation is observed in both synovial membrane and cartilage in patients with osteoarthritis, the treatments hereof represent a new method to treat osteoarthritis. Suppressing GATA4 for treating chondrocyte aging and osteoarthritis is a novel and promising strategy. The present inventors are the first to report that small molecules such as NSC 140905 can be used to treat osteoarthritis. Other suitable small molecules for use herein may be identified via literature, theory, and screening as known in the chemical and pharmaceutical arts, including, for example, high throughput virtual screening. See, for example, El-Hachem, N., and Nemer, G., Identification of new GAT Ad-small molecule inhibitors by structure-based virtual screening, Bioorg. Med. Chem. 19, 1734-1742 (2011). SiRNA for use herein may be identified as described herein. N-4-(diethylamino)phenyl)-5-methyl-3phenylisoxazole-4-carboxamide has, for example, been shown to be a GATA4 inhibitor. Kinnunen, S. M., et al., Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4- NKX2-5 Interaction. Sci Rep, 8, 4611 (2018).

[0100] In representative studies with mice, siRNA in a concentration of approximately 1x10-13to 1X10-11mol may be effective. Small molecules in the range of approximately lOuM to approximately ImM may be effective.

[0101] Small-molecule compounds, siRNA, and other treatments may, for example, be intraarticularly injected in solution, which is a simple and straightforward strategy to apply drugs to influence chondrocytes and cartilage, in pharmaceutically effective amounts.Alternatively, clinical and preclinical studies suggest that sustained and controlled drug release, such as through the polymeric implantable drug delivery systems, can avoidcomplications due to repeated injections, such as infection and unnecessary systemic exposure to high doses of drugs. Therefore, one can encapsulate small-molecule compounds and / or other treatments hereof into a carrier, such as polymer-based (for example, poly(lactic-co-glycolic acid) (PLGA)) microparticles or nanoparticles, which can provide long-term and controlled release through only one intraarticular injection. For example, 1-10 mg of compounds can be loaded into 3-30 mg microparticles, which will be intraarticularly injected based on 0.1 -Img compounds / 1kg body weight.

[0102] Small-molecule compounds may be administered in a pharmaceutically effective amount of the compound, a pharmaceutically acceptable salt of the compound, or a pharmaceutically effective prodrug. In general, treatments for decreasing the expression of GATA4 in chondrocytes may be administered by any conventional route of localized administration. In general, a pharmaceutically effective amount or dosage contains an amount of one of the treatments effective to reduce expression of GATA4 and display antiosteoarthritis behavior. Pharmaceutical compositions containing an active ingredient to decrease GAGA4 expression, a pharmaceutically acceptable salt thereof, or a prodrug in association with a pharmaceutically acceptable carrier or diluent are also within the scope hereof. In general, treatments hereof for decreasing expression GATA4 may be constituted into any form suitable for the mode of administration.

[0103] In a number of embodiments, an inhibitor agent hereof is delivered locally to a region of interest such as to affected cartilage (for example, by injection). FIG. 7 illustrates schematically a delivery system such as an injector system 100 (which may be manually operated or powered) to deliver an inhibitor agent 200 hereof to the region of interest.

[0104] Experimental

[0105] Cell isolation and expansion. Healthy human knee cartilage tissues were harvested from arthritis-free donors through an established protocol with the National Disease Research Interchange (NDRI). This study was approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents. Cartilage was diced into ~ ~ 1 mm3pieces with a scalpel and incubated with a dissociation medium that was composed of high glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco / Thermo Fisher Scientific, Waltham, MA, United States), 2% Antibiotics- Antimycotics (Life Technologies, Carlsbad, CA, United States), and collagenase type II (1 mg / mL(w / v), Worthington Biochemical Corporation, Lakewood, NJ, United States). 10 mL medium was used for 1g of cartilage, and the treatment lasted for 16 hours in a shaker at 37°C. The mixture was then passed through a70 μm strainer to collect single chondrocytes. Isolated chondrocytes were seeded in tissue culture flasks at 1×104cells / cm2and maintained in growth medium (GM, DMEM containing 10% fetal bovine serum (FBS, Life Technologies) and 1% Antibiotics-Antimycotics). After cells were fully attached to the culture substrate, the medium was changed every three days until cells reached 70-80% confluency. Cells were detached with Trypsin / EDTA (Gibco / Thermo Fisher Scientific) and passaged.

[0106] Individual Chondrocyte Monolayer Culture. Chondrocytes isolated from healthy human cartilage were expanded to passage 1 (Pl) and plated in tissue culture 6 well plates at P2, treated with GM, and cultured until cells reached 80% confluency. Media changes occurred every two days until cell collection. Tables 2 A through 2H of FIG. 8 lists the chondrocyte donor ages and genders used for different experiments.

[0107] Young and Old Chondrocyte Pools. Chondrocytes at passage 0 (P0) were pooled with three to four other young chondrocyte donors (< 45 years; Table 2D of FIG. 8) and grown in tissue culture flasks with GM at a cell seeding density of 1x106cells per flask. Media was changed weekly. Once cells reached 80% confluency, cells were collected using Trypsin / EDTA. Some cells were frozen using Recovery Cell Culture Freezing Medium (Gibco / Thermo Fisher Scientific), and other cells were used for subsequent passaging. The same methods were used for the old (> 65 years) chondrocyte pool. Tables 2E and 2F of FIG.8 lists the chondrocyte ages and genders used in each pool. As a result of the extensive amount of work conducted using these pools, two separate chondrocyte pools were made.

[0108] RNA sequencing (RNA-Seq) and bioinformatics analysis. The transcriptomic differences among three young and three old chondrocyte donors (Table 2A of FIG. 8) were assessed through RNA-Seq. Individual chondrocyte cultures (PO) were lysed with the QIAzol reagent (Qiagen, German Town, MD, United States), and RNA was isolated from the lysate using an RNA Easy Plus Universal Kit (Qiagen). Extracted RNA were quantitated Qubit™ RNA BR Assay Kit (Thermo Fisher Scientific) followed by the RNA quality check using Fragment Analyzer (Agilent Technologies, Santa Clara, CA). For each sample, RNA libraries were prepared from 500ng RNA using the KAPA mRNA HyperPrep Kit (Roche, Indianapolis, IN) according to manufacturer’s protocol, followed by quality check using Fragment Analyzer (Agilent Technologies) and fluorescent quantification on the Infinite F Nano + (Tecan, Mannedorf, Switzerland). The libraries were normalized and pooled, and then sequenced using NovaSeq6000 platform (Illumina, San Diego, CA,) to an average of 50M 100PE reads.

[0109] Quality control was first applied to raw RNA sequencing reads by tool FastQC. Low-quality reads and adapter sequences were filtered out by the Trimmomatic tool. Surviving reads were then aligned to human reference genome hg38 using STAR aligner, and gene counts were quantified. Differential expression analysis was performed based on gene counts by R package “DESeq2” and DEGs were selected by adjusted p-value < = 0.05 and fold change > = 1.5. These DEGs were then applied to Ingenuity Pathway Analysis (IPA) to detect enriched pathways. This software employs databases of prebuilt pathways with known genes summarized from previous studies, checks the overlap between the DEG list and known pathways and performs statistical tests to determine the enrichment. Significant pathways were defined by p-value < = 0.05. Statistically stringently, FDR = 5% cutoff should be applied to control the false discovery rate. To encourage more gene candidates, this study went by p-value< = 0.05 and fold-change> = 1.5 cutoff. All the tools were run by default parameter settings.

[0110] Immunohistochemistry (IHC) to examine the GATA4 levels in native cartilage tissues. Human cartilage tissues were fixed in 10% buffered formalin (Fisher Chemical, Fair Lawn, NJ) at 4°C overnight and then subjected to a graded ethanol dehydration series, starting from 20% ethanol and progressing to 100% ethanol. Subsequently, they were embedded in Paraplast X-tra (Leica Biosystems Inc. Richmond, IL). The Paraplast-embedded samples were sectioned at a thickness of 6 pm using a rotary Leica microtome (Leica Microsystems Inc., Deerfield, IL, Model RM 2255). Young and old mouse knee joints were gifts from a lab of Albert Einstein College of Medicine, which otherwise were wastes. After the specimens were fixed and decalcified in a formic acid-based bone decalcifier (StatLab, Mckinney, TX, USA) for a period of two weeks, they were then embedded and sectioned as described above.

[0111] For immunohistochemistry (IHC), the formalin-fixed paraffin-embedded sections first underwent antigen retrieval based on different antibodies. Slides were then blocked with 10% goat or horse serum (Abeam, Cambridge, MA) in phosphate buffered saline (PBS, Thermo Fisher Scientific) for Ih, incubated at 4°covernight with the primary antibody against GATA4, then incubated with a biotinylated anti-mouse / rabbit immunoglobulin G(IgG) secondary' antibody for Ih, with signal detection via DAB substrate kit (Abeam). The Nikon Eclipse E800 upright microscope (Melivile, NY, United States) was used to image the stained sections. Antibody specifications can be found in Table 3 of Figure 9.

[0112] Western blot to examine the GATA4 levels in human chondrocytes. Pl young and old chondrocytes were used for individual chondrocyte analysis of GATA4. Cells were washed in pre-cooled PBS (Thermo Fisher Scientific) three times. Using the RIPA buffer (Sigma-Aldrich) supplemented with the protease and phosphatase Inhibitor Single-Use Cocktail (Gibco / Thermo Fisher Scientific) and a cell scraper, monolayer culture samples were collected. A pestle was used to homogenize pellets in the RIPA cocktail for pellet culture samples. The protein concentration of the supernatant was determined by the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Proteins were fractioned electrophoretically on the NuPAGE 4-12%, Bis-Tris Mini Protein Gel (Gibco / Thermo Fisher Scientific) and then transferred to a polyvinylidene fluoride (PVDF) membrane using the iBlot Dry Blotting System (Invitrogen, Waltham, MA, United States). The membrane was blocked with 3% nonfat milk (Bio-Rad, Hercules, CA, USA), diluted with lx Tris-buffered saline (TBS, Gibco / Thermo Fisher Scientific) and 0.1% Tween 20 (Sigma-Aldrich) (TBST) at room temperature for 1.5 h, washed, and incubated with the primary antibody at 4°C overnight on a rotating shaker. The membrane was washed 7 times for 3 min with TBST buffer and incubated with horseradish peroxidase (HRP)-linked secondary antibodies (GE Healthcare Life Sciences, Malborough, MA, United States) for 1.5 h at room temperature. After being washed 5 times with TBST, the membrane was incubated in the chemiluminescence substrate SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Images were acquired using the ChemiDocTM Touch Imaging System (Bio-Rad). Images were quantified using Image J. Antibody information is included in Table 3 of FIG. 9.

[0113] Overexpression of GATA4 in young human chondrocytes. P2 young, pooled chondrocytes or young, individual chondrocytes were transduced with the lentiviral vector containing GATA4 fused with dTomato gene or the control lentivirus carrying EGFP for 10 hours. After that, flasks were rinsed with PBS for 2 times and the medium was replaced by fresh GM. Both vectors were created and packed by VectorBuilder (>108TU / mL, VectorBuilder, Chicago, IL, United States). To detect the number of cells transduced, cultures were imaged with an EVOS M5000 microscope (Thermo Fisher Scientific) after 72 h of initial transduction. After transduction, western blot, RT-qPCR, and IHC were used to verify the stable expression of GATA4 in cells.

[0114] RNA isolation and quantitative real-time polymerase chain reaction (RT-qPCR). For pellet culture, samples were first rinsed with PBS twice and a pestle and electric pulverizer were used to crush pellets. Cells were homogenized in Qiazol (Qiagen). Total RNA wasisolated and purified using the RNAeasy Plus Universal Mini Kit (Qiagen, Cat. NO. 74104) according to the manufacturer’s protocol. The reverse transcription to the complementary DNA (cDNA) was accomplished using the SuperScript IV VILO Master Mix (Invitrogen). RT-qPCR was performed on a real-time PCR instrument (QuantStudio 3, Applied Biosystems, Foster City, CA, United States) using the SYBR Green Reaction Mix (Applied Biosystems) with custom primers ordered from Integrated DNA Technologies (IDT, Newark, NJ, United States). Relative gene expression levels were calculated through the 2-ΔΔCtmethod. Ribosomal protein L13A (RPL13A was used as the housekeeping gene. Full names and abbreviations of genes and their corresponding proteins are listed in Table 1 of FIGS. 1 E(i) and lE(ii), and primer sequences are listed in Table 4 of FIG. 10

[0115] Pellet culture and chondrogenesis of young human Chondrocytes overexpressing GATA4. Following the lentiviral transduction of GATA4 gene in young, pooled chondrocytes, the cells were collected and formed into pellets at a cell seeding density of 3 x 105cells per pellet. Pellets were treated with chondrogenic medium (CM, DMEM with 1% v / v Insulin-Transferrin-Selenium-Ethanolamine (ITS, Gibco / Thermo Fisher Scientific), 1% antibiotic-antimycotics, 10-7M dexamethasone (Sigma- Aldrich), 40 pg / mL L-proline (Sigma- Aldrich), supplemented with 10 ng / mL transforming growth factor- p3 (TGF- P3, Peprotech, Rocky Hill, NJ, United States), and 50 pg / mL ascorbic acid-2 -phosphate (Sigma-Aldrich)). Medium was changed daily for seven days. RT-qPCR, histology, IHC, and western blot were used to characterize the tissues.

[0116] Safranin O / Fast green staining. Pellet samples were fixed in 10% buffered formalin (Fisher Chemical) for 2 h at room temperature and then rinsed with PBS. Pellets then underwent serial dehydration in 30,50,70,95, and 100% ethanol for one hour each. The 100% ethanol was refreshed once for an additional hour prior to sample clearing in Xylene. Pellets were cleared in Xylene (Fisher Chemical) for two hours. Pellets were then placed in Paraplast X-tra (Leica Biosystems Inc.) overnight. The next day, pellets were embedded in Paraplast X-tra blocks and sectioned at a 6-pm thickness using a Leica microtome (Leica Microsystems Inc., Model RM 2255).

[0117] Slides were stained using Safranin O (0.5%, Catalog number: 50240, Sigma-Aldrich), in 1% acetic acid (Catalog number: A6283, Sigma-Aldrich), 0.005% fast green (0.05 g, Catalog number: 104022, Sigma- Aldrich) in 100 mL distilled water (Invitrogen) and counterstained with Hematoxylin QS solution (Catalog number: H3404, Vector Laboratories INC). Imaging was conducted using a Nikon Eclipse E800 upright microscope.

[0118] LUMINEX multiplex assays. Upon 7 days of chondrogenesis, condition medium from pellets was collected and flash frozen in liquid nitrogen and immediately stored in -80oC. LUMINEX assays were accomplished using the Bio-Plex 200 system (Bio-Rad). Data collection and analysis were conducted using the Bio-Plex Manager 6.1 software as established in previous studies.19LUMINEX kit information can be found in Table 5 of FIG. 11 A list of proteins assessed in LUMINEX is provide in TABLE 6 of FIG. 12.

[0119] Knockdown of GATA4 in old human chondrocytes. The siRNA targeting human GATA4 (ON-TARGET plus Human GATA4 (2626) siRNA, J-008244-06-0005, Horizon Discovery Biosciences Limited, Cambridge, UK) was used in this study with a scrambled siRNA (ON-TARGET plus non-targeting siRNA #1, Catalog number: D-001810-01-05, Horizon Discovery Biosciences Limited) as the control. Old, pooled chondrocytes were transfected with the siRNA using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific). After 24 hours of incubation, the transfection medium (Opti-MEM Reduced Serum Medium, Thermo Fisher Scientific) was changed to GM. Transfected cells were collected after 48 hours for RT-qPCR to confirm the knockdown efficiency.

[0120] Pellet culture and chondrogenesis of old human chondrocytes with GATA4 Knockdown. Following the 48h transfection of scrambled control siRNA or siRNA targeting GATA4, cells were collected using Trypsin / EDTA (Thermo Fisher Scientific) and pellets were made at a cell density of 3x105cells per pellet. Pellets were treated with CM supplemented with 10 ng / mL TGF- [33 and 50 ug / mL ascorbic acid-2-phosphate for 7 days. Upon day 7, pellets were collected for RT-qPCR, western blot, and IHC.

[0121] GATA4 Small Molecule NSC 140905. GATA4 small molecule, NSC140905, also known as HCA 42027( Biosynth Ltd, Compton, United Kingdom), was reconstituted to 7.5 mM stock solution using UltraPure™ DNase / RNase-Free Distilled Water (Invitrogen) on a shaker at 37° until completely dissolved. Old, pooled chondrocytes were pelleted and treated with CM supplemented with 10 ng / mL TGF- [33 and 50 ug / mL ascorbic acid-2-phosphate with 100 pM NSC 140905 for 14 days. Pellets were collected for IHC, western blot, and RT-qPCR.

[0122] Animal model. All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC). Young (8 weeks) male C57BL / 6 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained in pathogen-free conditions, with no more than five mice per cage. Mice were provided ad libitum access to food and water, and a 12-hour light / dark cycle to simulate natural circadianrhythms. To minimize bias, mice were randomly assigned to either control or GATA4 groups, with 8 mice in each group.

[0123] Intraarticular injection. Intraarticular injections were administered to mice between 10-12 weeks of age under general anesthesia to safeguard the well-being of the animals and to minimize procedural discomfort. Under general anesthesia with 2% isoflurane in an oxygen mixture, the mice were placed in a supine position, and the right knee joint was positioned at a 90-degree flexion to facilitate accurate injection into the joint space. The injection site was meticulously identified medial to the patellar tendon. Using a 29-gauge needle, a volume of 10 pL of lentiviral particles encoding either GATA4 or a control vector (at a concentration >108TU / mL, VectorBuilder) was precisely administered into the intraarticular space of the right knee. The precision of the injection was ensured by employing a consistent technique across all animals, thereby reducing variability in the delivery of the viral vectors.

[0124] Destabilization of the medial meniscus (DMM) surgery. One week after viral vector injection, DMM surgery was performed to induce the OA model on mice at 11-13 weeks of age.2021Briefly, a medial parapatellar incision was made to expose the right knee joint, followed by a careful opening of the joint capsule. The anterior medial menisco-tibial ligament was identified and transected using microscissors. The joint capsule and skin were subsequently sutured with 6-0 silk thread. For experimental controls, a sham operation was performed in which the joint capsule was exposed as in the DMM surgery, but the medial menisco-tibial ligament was left intact.

[0125] Knee hyperalgesia. The inventors hereof expected accelerated OA development after Gata4 overexpression. To observe these differences, we used six weeks as the time point at which control mice had begun displaying mild OA symptoms. Knee hyperalgesia was evaluated using a Pressure Application Measurement (PAM) device (Ugo Basile, Varese, Italy)22,23Mechanical stimuli were applied to the mouse's knee joint to assess the degree of hyperalgesia based on the applied pressure and the mouse's response. Six weeks after either DMM or sham surgery, the mice were carefully removed from their cages to ensure they remained calm and unstimulated. The mouse was held securely by its back to maintain a straight posture, with the right knee joint flexed at approximately 90 degrees. The PAM device was placed on the index finger of the right hand of testers, which, along with the thumb, was used to apply pressure to the medial side of the knee joint, ensuring proper contact. Pressure was gradually applied at a constant rate of 30 g / s while monitoring thepressure curve displayed on the computer. The sensor was released immediately when the mouse exhibited a response to the applied stress, such as head movement, vocalization, or knee withdrawal. The pressure value displayed by the software, and the maximum pressures that mice can withstand were recorded. Two measurements were taken per knee, one on the medial side, and one on the lateral side the average was calculated for accuracy.

[0126] Synovial inflammation score. After 6 weeks, knee joints were collected and sectioned as described above. Histology staining and IHC were used to assess OA severity. Synovial inflammation score was evaluated according to a scoring protocol outlined in previous studies24, which relies on the hematoxylin and eosin (H& E)-stained tissue sections.

[0127] Statistical Analysis. Each experiment was carried out with at least three biological replicates. Data is presented as mean ± standard deviation unless otherwise specified.Detailed information of sample size, pre-processing, and statistical methods have been specified in each figure legend. Prism 10 (GraphPad, San Diego, CA) was used for statistical analysis. The significance level was set at 0.05 and indicated by * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

[0128] The foregoing description and accompanying drawings set forth a number of representative embodiments at the present time. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope hereof, which is indicated by the following claims rather than by the foregoing description. All changes and variations that fall within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

CLAIMS1. A method of reducing at least one of expression of GATA4 and activity of GATA4 in chondrocytes in vivo comprising introducing an inhibitor agent comprising at least one of siRNA and a small molecule compound, wherein the inhibitor agent is active to reduce at least one of the expression of GATA4 and die activity of GATA4.

2. The method of claim 1 wherein the inhibitor agent is injected into a joint of a patient to reduce at least one of the expression of GATA4 and the activity of GATA4 in the chondrocytes of the joint.

3. The method of any one of claims 1 and 2 wherein the reduction of at least one of expression of GATA4 and activity of GATA4 in the chondrocytes is used in treating or preventing osteoarthritis.

4. The method of any one of claims 1 through 3 wherein the inhibitor agent comprises a small molecule compound.

5. The method of claim 4 wherein the inhibitor agent comprises 2-[(2H-l,3- Benzodioxol-5-yl)methyl]butanedioic acid, which has the formula:

6. The method of claim 4 wherein the inhibitor agent comprises N-4- (diethylamino)phenyl)-5-methyl-3phenylisoxazole-4-carboxamide, which has theformula:

7. A use of at least one of a GATA4 siRNA and a small-molecule compound, which is active to reduce at least one of expression of GATA4 and activity of GATA4, in the manufacture of a medicant for the treatment of or preventing of osteoarthritis.

8. The used of claim 7 wherein the medicant is manufactured to be injected into a joint of a patient to reduce the expression of GATA4 in chondrocytes of the joint.

9. The use of any one of claims 7 and 8 wherein the medicant comprises the smallmolecule compound.

10. The use of claim 9 wherein the medicant comprises 2-[(2H-l,3-Benzodioxol-5- yl)methyl]butanedioic acid, which has the formula:

11. The use of claim 9 wherein the medicant comprises N-4-(diethylamino)phenyl)-5- methyl-3phenylisoxazole-4-carboxamide, which has the formula:

12. A method of treating or preventing osteoarthritis comprising injecting an inhibitor agent into a joint of a patient to reduce at least one of expression of GATA4 and activity' of GATA4.

13. The method of claim 12 wherein the inhibitor agent comprises at least one of siRNA and a small molecule compound, wherein the inhibitor agent is active to reduce at least one of the expression and the activity of GATA4.

14. The method of claim 12 wherein the inhibitor agent is injected into a joint of a patient to reduce at least one of expression of GATA4 and activity of GATA4 in the chondrocytes of the joint.

15. The method of any one of claims 12 through 14 wherein the inhibitor agent comprises a small molecule compound.

16. The method of claim 15 wherein the inhibitor agent comprises 2-[(2H-l,3- Benzodioxol-5-yl)methyl]butanedioic acid, which has the formula:

17. The method of claim 15 wherein the inhibitor agent comprises N-4- (diethylamino)phenyl)-5-methyl-3phenylisoxazole-4-carboxamide, which has theformula:

18. A system for reducing at least one of the expression of GATA4 and activity of GATA4 in chondrocytes in a joint of a patient comprising an injector system and an inhibitor agent comprising at least one of GATA4 siRNA and a small molecule compound active to reduce the at least one of expression of GATA4 and activity of GATA4 within a reservoir of the injector system.

19. The system of claim 18 wherein the injector system comprises a syringe.

20. A method of determining targets for treatment s, comprising:conducting one or more transcriptomic comparisons between chondrocytes from young donors and chondrocytes from old donors to determine genes which are upregulated genes or are downregulated in the chondrocytes of old donors compared to the chondrocytes of young donors.

21. The method of claim 20 further comprising determining transcriptional regulators that may mediate one or more of (i) the genes which are upregulated and (ii) the genes which are downregulated in the one or more transcriptomic comparisons.

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