Adipocyte enhancer binding protein 1 (AEBP1) as a potential therapeutic target to combat cardiac fibrosis

Fibrosis is one of the major hallmarks of heart failure (HF) progression and is characterized by fibroblast activation and excess extracellular matrix (EMC) deposition. RNA sequencing of myocardial tissue acquired from HF patients showed a significant upregulation of adipocyte enhancer binding protein (Aebp1) and increased AEBP1 protein expression was observed in regions around the infarct. AEBP1 is a secreted protein known to bind to TGFß-receptor and activate fibroblasts. AEBP1 has been identified in lung and liver fibrosis as a potential therapeutic target to combat fibrosis. However, the role of AEBP1 in cardiac fibrosis is not well understood and requires further investigation. Cultured human cardiac fibroblasts stimulated with TGFß showed a significant increase in activated fibroblast markers like α-smooth muscle actin (⍺SMA) and transgelin (SM22) indicating fibroblast activation. AEBP1 also showed a significant increase in activated fibroblasts when compared to quiescent fibroblasts. Further investigation showed that overexpressing AEBP1 in fibroblasts leads to its activation, evident from significant upregulation of fibroblast activation markers (⍺SMA & SM22) and ECM proteins like collagen (Col1A1) and osteopontin (OSPN), independent of TGFß signaling. In contrast, AEBP1 knockdown prevented active myofibroblast proliferation (evident from decrease in SM22) and a significant reduction in ECM production. In-vivo myocardial infarction (MI) mice model studies also showed a significant increase in serum AEBP1, 4-days post MI and immunohistochemistry analysis showed increased localization of AEBP1 in periinfarct regions making AEBP1 a potential target to prevent active cardiac fibrosis.

ABSTRACT Fibrosis is one of the major hallmarks of heart failure (HF) progression and is characterized by fibroblast activation and excess extracellular matrix (EMC) deposition. RNA sequencing of myocardial tissue acquired from HF patients showed a significant upregulation of adipocyte enhancer binding protein (Aebp1) and increased AEBP1 protein expression was observed in regions around the infarct. AEBP1 is a secreted protein known to bind to TGFß-receptor and activate fibroblasts. AEBP1 has been identified in lung and liver fibrosis as a potential therapeutic target to combat fibrosis. However, the role of AEBP1 in cardiac fibrosis is not well understood and requires further investigation. Cultured human cardiac fibroblasts stimulated with TGFß showed a significant increase in activated fibroblast markers like α-smooth muscle actin (⍺SMA) and transgelin (SM22) indicating fibroblast activation. AEBP1 also showed a significant increase in activated fibroblasts when compared to quiescent fibroblasts. Further investigation showed that overexpressing AEBP1 in fibroblasts leads to its activation, evident from significant upregulation of fibroblast activation markers (⍺SMA & SM22) and ECM proteins like collagen (Col1A1) and osteopontin (OSPN), independent of TGFß signaling. In contrast, AEBP1 knockdown prevented active myofibroblast proliferation (evident from decrease in SM22) and a significant reduction in ECM production. In-vivo myocardial infarction (MI) mice model studies also showed a significant increase in serum AEBP1, 4-days post MI and immunohistochemistry analysis showed increased localization of AEBP1 in periinfarct regions making AEBP1 a potential target to prevent active cardiac fibrosis. ii TABLE OF CONTENTS Abstract ii Introduction 1 Materials and Methods 4 Results 9 Discussion 18 References 22 iii 1 INTRODUCTION In the United States, approximately 6 million individuals suffer from Heart Failure (HF) with annual healthcare cost associated with HF patients around $43 million. This number is predicted to increase to $69.7 million by 2030 barring significant improvements in health outcomes.1 HF is a pathophysiological condition generally originating from cardiac stress, such as myocardial infarction (MI), which is characterized by death of cardiac muscle cells due to prolonged lack of oxygen and nutrients.2,3,4 This leads to decreased cardiac output and ejection fraction (EF) which in turn negatively affects other body systems. In addition to decreased EF, HF is also accompanied by structural remodeling, such as fibrosis progression, cardiomyocyte death, altered cardiomyocyte surface area, thickening/thinning of chamber walls, etc.5,6 The extent of this remodeling is contingent upon the strength needed to counterbalance the increased load experienced by surviving myocytes in the peri-infarct (or border) zone. Following MI, circulating or tissue-specific fibroblasts are activated to form activated fibroblasts, or myofibroblast, and these myofibroblasts secrete ECM proteins, mainly collagen.6,7,8 Initially, this increase in ECM production is beneficial as it strengthens the otherwise weakened, thinning necrotic region of cardiac muscle and prevents cardiac rupture. However, uncontrolled fibroblast activation and ECM production can lead to detrimental cardiac outcomes such as LV wall stiffening and a decline in mechanoelectrical coupling, both of which significantly impairs the normal physiological function of the heart.6,9,10 It is well known that the TGFß signaling pathway predominantly 2 drives fibroblast activation.11,12 However, there are no clinically approved therapeutics to modulate the TGFß pathway, as this molecule possesses many biological functions, making it a poor therapeutic target.13 However, recent literature shows that other pathways surrounding TGFß mediated fibrosis are beginning to be understood.14 As fibroblasts become activated and begin to differentiate to myofibroblasts they exhibit a phenotypic change, namely increased expression of α-smooth muscle actin (⍺SMA) and transgelin (SM22).15 ⍺SMA is considered a marker of myofibroblasts which, in addition to collagen production, also produces other contractile proteins. These myofibroblasts are distinct from smooth muscle cells in their lack of desmin, smoothelin, and other proteins characteristic of muscle cells.14,16,17,18 SM22 is an early marker of smooth muscle differentiation and in fibroblasts serves as a marker of the phenotypic change characteristic of activation. Myocardin-related transcription factor (MRTF) is another protein that has been shown to contribute to myofibroblast differentiation by inducing transcriptional activity in fibroblasts.19 RNA sequencing of fibrotic myocardial tissue taken from HF patients showed upregulation of Adipocyte enhancer binding protein (Aebp1). Aebp1 codes for a secreted protein (AEBP1) that is involved in protein-protein interaction and Aclp mRNA that acts as a transcriptional repressor. Therefore, there’s a dual role of AEBP1 in the nucleus and in the cytoplasm.19,20 The role of AEBP1 in fibrosis progression has been researched in several organs, such as the liver, skin, white adipose tissue (WAT) and the lungs where it has been reported to promote fibroblast differentiation through activation of TGFß and SMAD signaling pathways.17,18,19,24 Tumelty et al. showed that AEBP1 binds to TGFß 3 receptor and activates the TGFß signaling pathway.20 When activated, TGFß receptors phosphorylate SMAD proteins (SMAD2/3), which in turn form a complex that translocate into the nucleus and promotes the transcription of ECM proteins. This study reported AEBP1 induced phosphorylation of SMAD proteins only in the presence of TGFß. However, this study also suggested that AEBP1 may modulate the production of collagen through a TGFß independent pathway. In another study by Gerhard et al., AEBP1 was shown to be a central regulator of fibrosis progression in nonalcoholic steatohepatitis.20 These novel discoveries in a diverse range of organ systems may present new options for therapeutic targets to combat fibrosis progression both within specific organs and in the context of multi-organ fibrosis. In this study we investigate the roles of AEBP1 and its potential to halt the progression of cardiac fibrosis. Using human cardiac fibroblasts (HCF) we demonstrate that TGFß stimulation activates HCF and induces myofibroblast formation. Myofibroblast phenotype was confirmed by the expression of markers such as SM22 and αSMA.21 These stimulated HCF also display elevated levels of AEBP1 when compared to controls (unstimulated). Further, using an AEBP1 overexpression (O/N) viral vector, we show that fibroblasts differentiate to form myofibroblasts even in the absence of TGFß, indicating a TGFß independent fibrotic significant pathway. Knockdown (KD) experiments were also performed to show that AEBP1 silencing in myofibroblasts prevents active fibroblast differentiation and results in reduced ECM secretion. The ability of AEBP1 to impose fibroblast activation phenotypes in the absence of TGFß stimulation and the apparent 4 requirement for AEBP1 shown by lack of fibroblast activation in AEBP1 KD models suggests AEBP1 may be a strong target for future therapeutics. While our in vitro studies allude to AEBP1 having therapeutic potential, further in vivo testing is necessary to validate these results in a biological system. Using a mouse MI model, we found that AEBP1 expression is significantly elevated in mouse serum 4 days post MI, with ⍺SMA expression being similarly upregulated in the infarct and periinfarct region 4-days post MI. An increase in AEBP1 in peri-infarct regions also indicates that AEBP1 possibly contributes towards fibrosis progression in vivo. However, we will perform experiments like AEBP1 KD in vivo to test our hypothesis. Overall, we show that AEBP1 plays a crucial role in cardiac fibrosis progression and can be used as a potential therapeutic target to combat cardiac fibrosis. Therefore, AEBP1 may plausibly emerge as a potential target to prevent multisystem fibrosis. MATERIALS AND METHODS Masson's trichrome/ Fibrosis analysis - Human myocardial tissue was used for this experiment and formalin fixed for 48h followed by paraffin embedding. 5μm thick sections were cut and stained with Trichrome for fibrosis analysis using the Dako automated special strainer. The slides for fibrosis analysis were scanned under 20x and analyzed using Aperio Image Scope software (version12.3.2.8013) (using the colocalization v9 algorithm). A ratio of the total stained area to collagen-stained area was reported. 5 Human cardiac fibroblast (HCF) culture - stimulation, overexpression (Ad-CMVhAEBP1-GFP) and knockdown (Ad-CMV-shAEBP1-GFP). HFC were stored in liquid nitrogen until they were thawed and consequently plated. HCF were given 24 hours to recover from freeze-thaw cycle before any experimentation. HCF (PromoCell #c-12375) were cultured in HCF media (PromoCell #C-23010) on 12-well plates with 50,000cells/well for 48h. Cells were stimulated with 10ng/ml TGFß at for 48h with media change every 24h. Viral infection (overexpression or knock-down) was performed at 250 MOI for 72h before or after stimulation. The cells were infected with virus for 24h and then the media changed every 24h. Cells were then harvested to perform respective experiments. Immunocytochemistry (ICC) - Cells were cultured on round coverslips for this experiment. Cells were rinsed with 1X PBS twice and fixed in 10% formalin for 20 mins at room temperature (RT). After an additional PBS rinse, 0.5% Triton in PBS was added for 10 mins at RT. After removing the solution, 800 μl of fetal bovine serum (FBS) was added to the plate and cells were blocked for 60 mins at RT. Cells were then incubated overnight at 4C with the primary antibodies in FBS at a concentration of 1:200. The list of antibodies used are listed in Table 1. Cells were then washed in 1X PBS thrice for 10 mins each. Cells were incubated in secondary antibodies for 1h at a concentration of 1:200 at RT. Cells were washed twice with 1xPBS and DAPI was added at a concentration of 1:1000 in PBS for 20 mins. After a final PBS rinse, coverslips were mounted onto the slides using mounting medium (Sigma, #F4680). All images were taken in the Leica SP8 system using the same settings. 6 RNA Extraction - Human myocardial tissue and HCF were used for this experiment. miRNeasy Mini kit (Qiagen) was used for RNA extraction. The extracted RNA was used for total RNA sequencing (RNA Seq). Agilent RNA ScreenTape Assay was used for QC experiments. RNA sequencing & Real-Time Quantitative Polymerase Chain Reaction (qRT PCR) - Illumina TruSeq Stranded RNA kit was used for library preparation and Ribo-Zero Gold was used to remove rRNA and the sequencing was performed on an Illumina HiSeq 2500 with 50bp single-end reads. The same RNA was used for cDNA synthesis (NEB #E3010S) and qRT was performed using Aebp1 and Vinculin primers (Table 1). Protein Extraction - HCF were lysed in 1X RIPA buffer (Cell Signaling Technology #9806S) containing 2X protease and phosphatase inhibitor (Thermo Scientific #78440) and was allowed to sit for 20 mins. 5μl of 100 mmol PMSF was added to the homogenate and allowed to rotate for 30 mins at 4°C, followed by 10 mins of centrifugation at 4°C at 18407 rcf. Supernatant was transferred to a new tube and Pierce BCA Protein Assay kit (Thermo Scientific, #23225) was used for protein estimation. Equal volume of 2X Laemmli buffer with 10% DTT was added to the supernatant and boiled for 10 mins at 98°C. Western Blotting (WB) - Using 30 μg of protein, gels were run at constant volts (25V/gel) and then transferred to a nitrocellulose membrane at constant current (350 mA) for 1h. Membranes were then blocked for 1h using Odyssey Blocking Buffer (LiCor #927-50000) and subsequently probed with primary antibodies overnight. The antibodies used and their concentration are listed in Table 1. Blots were then washed with 1X TBS-tween thrice, 7 followed by incubation with secondary antibodies (anti-mouse or anti-rabbit 1:10000) for 30 mins in the dark. An additional three washes with 1X TBS-tween were performed to prepare blots for scanning using Odyssey Infrared Imager. Image Studio Lite software version 5.2 was used to analyze the blots. Lane loading controls were used in each blot and when possible, gels were run simultaneously. Total Protein Staining (TPS) was used to normalize the expression of protein in each blot. The following antibodies were used: AEBP1 (Santa Cruz Bio., sc-271374),), ⍺SMA (Abcam, ab5694), SM22 (Cell Signaling, 62567s), VIN (Cell Signaling, 13901s), PAI1 (Invitrogen, MA3-012), OSPN (Cell Signaling, 8828s), Col1a1 (Cell Signaling, 8685s), MRTF (Cell Signaling, 66562s). Animals and Animal care - All animal studies were performed in accordance with the University of Iowa Animal Care and Use Committee (IACUC). All procedures involving animals were approved by the Animal Care and Use Committee of the University of Utah and complied with the American Physiological Society’s Guiding Principles in the Care and Use of Animals and the UK Animals (Scientific Procedures) Act 1986 guidelines. The mice were housed in 12 h dark/light cycle at 70 °F and 40% humidity. Mouse myocardial infarction (MI) model - 12-week-old C57BLJ mice were used for this study. Both male and female mice were used for all experiments. Mice were anesthetized with 3% isoflurane mixed with oxygen, naired and intubated. A thoracotomy was performed and the muscle between the 4th and 5th intercostal muscle was cut to expose the heart. A 6-0 suture was used to ligate the proximal left anterior descending artery 8 (LAD). The muscle and skin were sutured back, and the mice allowed to recover in a heating pad. Immunohistochemistry (IHC) - Mice/human heart tissue was used for this study. Hearts were paraffinized and sectioned at 5μm thickness. Slides were deparaffinize by serial washing in xylene twice for 5 mins each followed by washing in 95% EtOH twice for 5 mins each. Slides were washed once in 70% EtOH for 5 mins and once in ddH2O for 5 mins. Slides were finally washed once in 1X PBS 5 mins followed by 30mins incubation at 70°C in epitope retrieval solution (IHC-Tek, Cat# IW-1100). The slides were then cooled at RT for 30 mins. After a 5 min PBS wash, a circle section with PAP pen was drawn. 500μl of 5% serum (diluted in 0.2% PBS-Triton) was added to the sections and incubated for 30 mins. Sections were incubated in primary antibodies prepared in 5% serum at a 1:200 dilution, overnight at 4°C. The slides were then placed in a dark room where the rest of the protocol was performed. Slides were washed thrice for 5 mins with 1xPBS and incubated for 1.5h in secondary antibodies in 5% serum at a dilution of 1:200. Slides were then washed twice in 1xPBS and incubated in WGA (1:1000 in 1xPBS) for 1 hr at RT. After a final PBS wash for 10 mins, the slides were cleaned and dried carefully. Mounting medium was placed on the tissue and coverslips were then placed on slides avoiding any bubbles. They were then stored in the dark at 4°C and imaged the following day. All images were taken in the Leica SP8 system using the same settings. Enzyme-linked immunosorbent assay (ELISA)– Blood was collected from the tail vein following 2-, 4-, and 8-days post MI. Samples were centrifuged for 15 minutes at 3500 9 RPM at 4°C. Serum supernatant was collected and assayed with Human AEBP1 ELISA Kit (Biorbyt Cat#: orb437774). Assay was performed as per manufacturer’s specifications. RESULTS RNA sequencing data of human HF myocardial tissue correlates AEBP1 with increased fibrosis Myocardial tissue from HF patients were analyzed using Masson’s trichrome to quantify percent fibrosis. Percent fibrosis was measured and found to be significantly higher in HF myocardium when compared to non-failing donor myocardium (Figure 1a). RNA sequencing was performed on myocardial tissue from HF patients to identify genes that were differentially expressed in the failing heart. Among many other genes shown to 10 be upregulated, Aebp1 was significantly overexpressed in HF patients compared to donors, suggesting a plausible role of AEBP1 in HF (Figure 1b). IHC performed on fibrotic and 11 non-fibrotic regions showed more AEBP1 expression in regions of fibrosis compared to normal myocardium (Figure 1c). Taken together, elevated AEBP1 expression correlates with regions of fibrosis in HF myocardium. Increased AEBP1 expression observed in activated cardiac fibroblasts HCF were stimulated for two days with TGFβ, and cells were harvested and processed for WB analysis (Figure 2a). HCF stimulated with TGFβ showed a significant upregulation of ⍺SMA OSPN, and SM22 (Figure 2b-2e), suggesting activation of myofibroblasts and secretion of ECM. qRT-PCR data gathered from the same treatment group showed significant increase in Aebp1 mRNA (Figure 2f). Additionally, ELISA performed on the media of these cells showed a significant increase in AEBP1 in 12 the stimulated population when compared to the unstimulated population (Figure 2g). These results show that activated cardiac fibroblasts express AEBP1 upon activation and differentiation. Using the HCF stimulation model described in Figure 2a, both unstimulated (US) and stimulated (S) samples were stained for DAPI, ⍺SMA and AEBP1 (Figure 2h). Confocal imaging of HCFs showed a significant increase in both AEBP1 and ⍺SMA in the TGFβ stimulated cells but not in the unstimulated cells. This joint expression of AEBP1 and ⍺SMA in the stimulated samples, and the lack thereof in the unstimulated samples, is further suggestive of AEBP1’s role in fibroblast activation in-vitro. AEBP1 overexpression in HCF leads to increased fibroblast activation and ECM secretion. TGFβ stimulation of HCF showed protein expression consistent with activated fibroblasts. In order to understand AEBP1’s relationship to other fibrosis mediators and its position in the pathways responsible for HCF activation, experiments were conducted using an adeno virus 9 (AdV)-mediated AEBP1 overexpression (O/N) model. Cells were 13 treated with adenovirus containing either AEBP1 O/N vector in the experimental group or GFP vector in the control group as shown in Figure 3a. qRT PCR data shows a significant upregulation of Aebp1 expression indicating efficacy of adenovirus overexpression (Figure 3b). Based on this confirmation, we analyzed the expression levels of ⍺SMA and MRTF by WB and found that expression levels of these proteins were also significantly upregulated in cells that received AEBP1 adenovirus treatment (Figure 3c-e). 15 O/N (Figure 5a-d). This data further suggests AEBP1 can independently initiate fibroblast activation by interacting with transcription factors, such as MRTF, and inducing nuclear translocation and consequential transcription and ECM secretion. In summary, our data indicates that AEBP1 closely modulates the activation of fibrotic pathways consistent with expression characteristic of TGFβ stimulation. AEBP1 could possibly be upstream of TGFβ signaling pathway, as AEBP1 seems to enact the same action without the presence of TGFβ. AEBP1 knockdown in activate fibroblasts leads to decreased myofibroblast differentiation and results in reduced ECM deposition In order to investigate the importance of AEBP1 in the process of HCF activation and determine its effects on downfield protein expression, AEBP1 expression was knocked down by shRNA transduction. Cells were stimulated to induce the myofibroblast 16 phenotype before AEBP1 knockdown and following TGFβ stimulation, HCF cultures were transduced with shAEBP1/GFP and subsequently harvested as shown in Figure 6a. qRT PCR results show a significant reduction in Aebp1 in KD samples when comparted to cells treated with GFP (Figure 6b). WB analysis showed significant downregulation of SM22, MRTF, and Col1a1 but not ⍺SMA. (Figure 6c-h) This led us to hypothesize that the effect of TGFβ stimulation on cells cannot be reversed, but that, AEBP1 KD prevents active fibroblast differentiation, evident from the significant reduction in SM22. Col1A1 similarly show significant reduction in expression, suggesting AEBP1 KD leads to an inhibition of ECM secretion and arrests fibrosis progression. Therefore, AEBP1 KD after myofibroblast differentiation significantly prevents active fibroblast differentiation and ECM deposition but does not reverse myofibroblast differentiation. 17 Increased AEBP1 expression in infarct and per-infarct regions in mice model of myocardial infarction AEBP1 O/N and KD experiments in HCF were helpful in ascertaining the role of AEBP1 in cardiac fibroblast activation and prevention of myofibroblast differentiation respectively. However, in order to understand the mechanism in vivo, we induced HF in mice by ligating the left anterior descending coronary artery (Figure 7). Mice were sacrificed at 2-, 4-, and 8-days post MI. WB analysis on mice myocardial tissue showed a significant increase in ⍺SMA 4- and 8-days post MI and (Figure 8a-b). 18 ELISA performed on serum samples showed a significant increase in AEBP1 4-days post MI (Figure 8c). IHC staining of the mice myocardium showed an increase in AEBP1 expression in infarct and particularly the peri-infarct zone suggesting a strong role of AEBP1 in active fibrosis progression. (Figure 8d). 19 DISCUSSION Heart failure (HF) has been and continues to be a national and international health concern. While the best antidote is prevention, HF will continue to be a problem that merits a remedy. Following an insult to the heart, fibrotic scarring develops to replace necrotic regions of myocardium and ultimately preserves the structural integrity of the heart. However, if not managed, the newly forming scar exceeds its benefits and becomes detrimental to the function of the heart, ultimately leading to myocardial rigidity, decreased functionality both structurally and on a cellular level and decreases mechanoelectrical coupling. These physiological changes are what leads to HF and if untreated, ultimately leads to death. If this process of active fibrosis progression could be guided or controlled in order to maximize benefit and minimize detriment to the heart, many patients could potentially show great improvements in cardiac health following a myocardial stress or injury. Myocardial tissue taken from HF patients at the time of transplant show large percentages of fibrosis. RNA sequencing performed on these samples showed increased expression of Aebp1 in fibrotic tissue from failing hearts. Myocardial tissues from these hearts were also stained for AEBP1 and we observed an increase in AEBP1 in infarct and peri-infarct myocardium when compared to nonfibrotic myocardium. TGFβ was used to stimulate fibroblast differentiation which led to increased ECM protein production and differential protein expression consistent with the activated fibroblast phenotype. In stimulated HCF, AEBP1 was also shown to be significantly 20 upregulated, a result that was further confirmed by ICC and confocal imaging. We showed that fibroblasts stimulated with TGFβ expressed ⍺SMA and AEBP1 while fibroblasts that were not stimulated did not express these proteins. AEBP1 overexpression was performed in HCF, and we observed that AEBP1 can independently drive fibroblast activation and more importantly, in the absence of TGBβ. We found a significant increase in ⍺SMA and MRTF (markers of fibroblast activation) and Col1A1 and OSPN (ECM proteins) upon AEBP1 ON. AEBP1 KD experiments were performed on HCF that were first stimulated with TGFβ (to induce fibroblast differentiation) and subsequently transduced with AEBP1 shRNA which inhibits the translation of AEBP1. As AEBP1 expression is inhibited, fibroblast activation and ECM production is significantly downregulated. Interestingly, ⍺SMA is not significantly affected by AEBP1 inhibition, but MRTF and SM22 show significant downregulation. Additionally, even though some myofibroblast markers may not be significantly affected, if AEBP1 is absent, even after TGFβ stimulation, the process of fibrosis progression is halted. Hence, fibroblasts stimulated by TGFβ maintain their smooth muscle-like phenotype but do not proliferate nor secrete ECM proteins. This inhibition-based fibrosis modulation suggests AEBP1 is a potential therapeutic target for management of fibrosis progression. Further experiments were conducted in vivo to understand the temporal aspects of fibrosis progression in connection with AEBP1 expression and activation of fibroblasts. Our results showed that 4-days after cardiac injury, AEBP1 expression increases in the 21 serum. These temporal results were confirmed in the infarcted myocardium using WB analysis and showed a similar peak in ⍺SMA 4-days after MI. Staining of infarcted areas at different time points after MI also suggests the deposition of ECM proteins follows the same pattern, further confirming the modulating role of AEBP1 in the formation of fibrotic scars in the ischemic heart. We also observed that AEBP1 was expressed in the peri-infarct regions in the mice myocardium, re-iterating its potential in fibrosis progression. Overall, we show that AEBP1 independently contributes to the activation of cardiac fibroblasts, both in vitro and in vivo. The presence of AEBP1 alone is sufficient to activate the pathways responsible for phenotypic change in fibroblasts that result in increased deposition of ECM proteins. In the absence of TGFβ, HCF over-expressing AEBP1 experienced activation, suggesting AEBP1 activates fibroblasts in a TGFβ-independent pathway. Even when stimulated by TGFβ, HCF in the AEBP1 KD model showed reduced ECM production and inhibition of active fibroblast differentiation. Hence, not only is AEBP1 sufficient to activate cardiac fibroblasts through alternative pathways, but it is also a necessary modulator of the normal fibroblast activation pathway. The imperative nature of the role of AEBP1 makes it an excellent therapeutic target in controlling active fibrosis and cardiac remodeling following MI. This also applies to a larger multisystem model in which AEBP1 could be used as a fibrosis modulator in other organs, either in conjunction or singularly. There is still much research to conduct to determine the efficiency of an AEBP1 inhibition therapeutic in a complex biological system, but the results of the current study suggests AEBP1 as a potential target to combat cardiac fibrosis. 22 REFERENCES 1 Urbich, M., Globe, G., Pantiri, K. et al. A Systematic Review of Medical Costs Associated with Heart Failure in the USA (2014–2020). PharmacoEconomics 38, 1219– 1236 (2020). https://doi.org/10.1007/s40273-020-00952-0 2 Torabi, A., Cleland, J. G., Rigby, A. 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Journal of molecular and cellular cardiology, 35(12), 1407-1420. 25 Acknowledgements Thank you to all of my many teachers and mentors for all the support and guidance they have provided me. Thank you to all my lab mates for humanizing an otherwise numerically and procedurally dominated environment. Thank you Sutip for helping me develop my “why” for doing research and for believing in my potential. Special thanks to Dr. Stavros Drakos for providing me the opportunity to learn through research and for the strongly supportive encouragement to shoot for the stars. Lastly, thank you Anu for helping me grow from a clueless undergraduate into a capable researcher. Thank you for taking time to teach me and reteach me and for having the patients to let me fail. Most of all, thank you for being my close friend and sharing in the ups and downs that life has to offer. 26 Name of Candidate: Dallen Calder Date of Submission: May 09, 2022