Defective Remodeling Research Articles

Cancer and lymphatic marker FOXC2 drives wound healing and fibrotic tissue formation.

The FOXC2 transcription factor has been tied to a wide range of disease states, serving as a promising prognostic biomarker associated with aggressive basal-like human breast cancers (increased cancer invasion and metastasis). Dysregulation of FOXC2 expression has also been found to promote defects in lymphatic remodeling and hyperplastic lymphedema-distichiasis (LD). Since chronic lymphedema is a forerunner of several malignancies and cancers have been known to arise from poorly healing chronic wounds (e.g., Marjolin ulcers), we examined the effect of Foxc2 dysfunction on skin wound healing. We used our splinted excisional wounding model that mimics human-like wound healing on wildtype and Foxc2+/- mice (n = 4), which demonstrate incomplete lymphatic vasculature and lymphatic dysfunction. Wound size was measured over the course of 18days. Tissue was explanted from both groups at post-operative day (POD) 14 and 18 and stained with Masson's Trichrome to assess scar formation, Picrosirius Red for dermal integrity, or immunofluorescence to assess lymphatic (LYVE1) cell populations. Wildtype mice completely healed by POD 14, while Foxc2+/-mice did not completely heal until POD18. Scar area of healed Foxc2+/-mice (POD 18) was larger than that of healed wild-type mice (POD 14; p = 0.0294). At POD 14, collagen "bers in the scars of Foxc2+/-mice to be narrower (p = 0.0117) and more highly aligned (p = 0.0110), indicating signi"cantly more "brosis in these mice. Collagen "bers in both groups became longer (p = 0.0116) and wider (p = 0.0020) from POD 14 to 18, indicating a temporal evolution of "brosis. Foxc2+/-mice also had lower numbers of LYVE1+, F4/80+ and CD4+ cells compared to wildtype mice. Individuals over 65 years old are more likely to develop cancer and are highly susceptible to developing chronic wounds. Here, we found that FOXC2, which is tied to cancer metastasis and lymphatic dysregulation, also impairs wound healing and promotes "brotic tissue architecture. With FOXC2 proposed as a potential therapeutic target for cancer metastasis, its downstream systemic effects should be considered against the increased chance of developing nonhealing wounds. Further delineation of the microenvironment, cellular events, and molecular signals during normal and Foxc2-associated abnormal wound healing will improve clinical therapies targeting this important marker.

H3K27me3 timely dictates uterine epithelial transcriptome remodeling and thus transformation essential for normal embryo implantation.

Uterine luminal epithelia (LE), the first layer contacting with the blastocyst, acquire receptivity for normal embryo implantation. Besides the well-accepted transcriptional regulation dominated by ovarian estrogen and progesterone for receptivity establishment, the involvement of epigenetic mechanisms remains elusive. This study systematically profiles the transcriptome and genome-wide H3K27me3 distribution in the LE throughout the preimplantation. Combining genetic and pharmacological approaches targeting the PRC2 core enzyme Ezh1/2, we demonstrate that the defective remodeling of H3K27me3 in the preimplantation stage disrupts the differentiation of LE, and derails uterine receptivity, resulting in implantation failure. Specifically, crucial epithelial genes, Pgr, Gata2, and Sgk1, are transcriptionally silenced through de novo deposition of H3K27me3 for LE transformation, and their sustained expression in the absence of H3K27me3 synergistically confines the nuclear translocation of FOXO1. Further functional studies identify several actin-associated genes, including Arpin, Tmod1, and Pdlim2, as novel direct targets of H3K27me3. Their aberrantly elevated expression impedes the morphological remodeling of LE, a hindrance alleviated by treatment with cytochalasin D which depolymerizes F-actin. Collectively, this study uncovers a previously unappreciated epigenetic regulatory mechanism for the transcriptional silencing of key LE genes via H3K27me3, essential for LE differentiation and thus embryo implantation.

Abstract 1129: Ddx24 In Vascular Smooth Muscle Cells Is Essential For Embryonic Vascular Development

Background: The DEAD-box family plays a crucial role in tumorigenesis and immune responses. Our previous research has identified an association between the dysfunction of DEAD-box RNA helicase 24 (DDX24) and a specific type of vascular malformation. However, the role and mechanism of DDX24 in vascular development remains unclear.Research questions: What is the role of DDX24 in regulating vascular smooth muscle cell (VSMC) function and embryonic vascular development? Aims: Our aim is to investigate the function and regulatory mechanism of DDX24 in embryonic vascular development. Methods: Cardiomyocyte/VSMC-specific Ddx24 knockout mice were generated by crossing Tagln -Cre mice with Ddx24 flox/flox transgenic mice. Stereomicroscopic photography and immunofluorescence staining were used to examine the development of blood vessels. Flow cytometry and cell proliferation assays were employed to assess the effect of DDX24 on VSMC functions. RNA sequencing and RNA immunoprecipitation were performed to uncover the downstream target of DDX24. Additionally, RNA pull-down and RNA stability experiments were conducted to elucidate the regulatory mechanism. Results: Cardiomyocyte/VSMC-specific Ddx24 knockout mice died before embryonic day 13.5 and exhibited defects in embryonic vascular formation and vascular remodeling in extraembryonic tissues. Ddx24 knockdown led to the suppression of VSMC proliferation through cell cycle arrest, likely due to increased DNA damage. DDX24 protein was found to bind to and stabilize the mRNA of FA complementation group A ( FANCA ), a gene that responds to DNA damage. Depletion of FANCA also affected cell cycle and DNA repair in VSMCs. Overexpression of FANCA successfully rescued the alterations induced by DDX24 deficiency. Conclusion: Our study illuminates the role of DDX24 in VSMC-mediated vascular development, suggesting a potential therapeutic target for diseases associated with VSMC dysfunction.

Abstract LB079: Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4

Abstract T cell-dependent anti-tumor activity unleashed by immunotherapies such as immune checkpoint therapy (ICT) can generate durable clinical responses. However, not all patients benefit, and epigenetic and transcriptional features required by T cells for effective ICT remain to be fully established. We previously documented that the transcription factor Bhlhe40 is upregulated by ICT in tumor specific CD4 and CD8 T cells in preclinical tumor models. We validated the relevance of this by ablating Bhlhe40 in both CD4 and CD8-T cells which rendered αPD-1 or αCTLA-4 ICT ineffective in tumor models normally responsive to ICT. Our findings extended beyond preclinical models as we recently found that human tumor-specific T cells recognizing mutant neoantigens display upregulation of Bhlhe40 and CRISPR-mediated Bhlhe40 deletion in human CD8 T cells recognizing the MART-1 tumor-antigen abrogates IFNG expression. Since we previously used CD4-Cre Bhlhe40f/f (B40ΔT) mice where Bhlhe40 was deleted in both CD4 and CD8 T cells, we could not definitively delineate how Bhlhe40 was regulating CD4 versus CD8 T cells under ICT. We thus used a model where Bhlhe40 is deleted specifically in CD8 T cells (B40ΔCD8). We found that like B40ΔT mice, B40ΔCD8 mice bearing the 1956 sarcoma were insensitive to αPD-1 ICT. While B40ΔT mice were also insensitive to αCTLA-4 ICT, interestingly, B40ΔCD8 mice displayed nearly normal tumor rejection upon αCTLA-4 ICT. Thus, Bhlhe40 is required in CD8 T cells for αPD-1 response, whereas is dispensable in CD8 T cells for αCTLA-4 efficacy. Single-cell protein and transcriptomic analysis of intratumoral immune-cells from ICT-treated control B40f/f, B40ΔCD8 and B40ΔT mice revealed overlapping and distinct changes to CD4 and CD8 T cells depending on which cells had Bhlhe40 deleted and which treatment was employed. With αPD-1 or αCTLA-4, defects in expression of IFN-γ, glycolytic enzyme transcripts and cytotoxic molecules, along with increased IL-10 production in CD4 and CD8 T cells from B40ΔT mice and in B40−/− CD8 T cells in B40ΔCD8 mice was observed. Whereas αCTLA-4-treated B40ΔT mice failed to reject tumors, addition of αIL-10R led to modest tumor growth inhibition, while with αPD-1 treatment, IL-10R blockade had little effect on tumor growth. We also noted that in control B40f/f mice, αPD-1 predominately altered CD8 T cells, whereas αCTLA-4 modified CD8 and CD4 T cells, including induction of an ICOS+ IFNγ+ CD4 T cell population expressing Bhlhe40. This population was largely preserved in B40ΔCD8 mice undergoing αCTLA-4 ICT. Analysis of intratumoral macrophages revealed profound defects in ICT-induced macrophage remodeling from CX3CR1+CD206+ to iNOS+ macrophages in B40ΔT mice lacking Bhlhe40 in CD4 and CD8 T cells. Although αCTLA-4-treated B40ΔCD8 mice reject their tumors, we observed a modestly higher frequency of CX3CR1+CD206+ macrophages and lower frequency of iNOS+ macrophages as compared to αCTLA-4-treated B40f/f mice. The highest percentage of CX3CR1+CD206+ macrophages and the fewest iNOS+ macrophages was observed in αPD-1-treated B40ΔCD8 mice that completely fail to reject tumors. These results reveal distinct requirements of Bhlhe40 in CD4 and CD8 T cells that depend on which ICT is used and further highlight non-redundant mechanisms of effective αPD-1 and αCTLA-4 Citation Format: Akata Saha, Alexander S. Shavkunov, Shailbala Singh, Avery J. Salmon, Sunita Keshari, Qi Miao, Brian T. Edelson, Cassian Yee, Ken Chen, Matthew Gubin. Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB079.

GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes.

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.

Elevated levels of alcohol dehydrogenase aggravate ethanol-evoked cardiac remodeling and contractile anomalies through FKBP5-yap-mediated regulation of ferroptosis and ER stress

Alcohol intake provokes severe organ injuries including alcoholic cardiomyopathy with hallmarks of cardiac remodeling and contractile defects. This study examined the toxicity of facilitated ethanol metabolism in alcoholism-evoked changes in myocardial morphology and contractile function, insulin signaling and various cell death domains using cardiac-selective overexpression of alcohol dehydrogenase (ADH). WT and ADH mice were offered an alcohol liquid diet for 12 weeks prior to assessment of cardiac geometry, function, ER stress, apoptosis and ferroptosis. Alcohol intake provoked pronounced glucose intolerance, cardiac remodeling and contractile anomalies with apoptosis, ER stress, and ferroptosis, the effects were accentuated by ADH with the exception of global glucose intolerance. Hearts from alcohol ingesting mice displayed dampened insulin-stimulated phosphorylation of insulin receptor (tyr1146) and IRS-1 (tyrosine) along with elevated IRS-1 serine phosphorylation, the effect was augmented by ADH. Alcohol challenge dampened phosphorylation of Akt and GSK-3β, and increased phosphorylation of c-Jun and JNK, the effects were accentuated by ADH. Alcohol challenge promoted ER stress, FK506 binding protein 5 (FKBP5), YAP, apoptosis and ferroptosis, the effects were exaggerated by ADH. Using a short-term ethanol challenge model (3 g/kg, i.p., twice in three days), we found that inhibition of FKBP5-YAP signaling or facilitated ethanol detoxification by Alda-1 alleviated ethanol cardiotoxicity. In vitro study revealed that the ethanol metabolite acetaldehyde evoked cardiac contractile anomalies, lipid peroxidation, and apoptosis, the effects of which were mitigated by Alda-1, inhibition of ER stress, FKBP5 and YAP. These data suggest that facilitated ethanol metabolism via ADH exacerbates alcohol-evoked myocardial remodeling, functional defects, and insulin insensitivity possibly through a FKBP5-YAP-associated regulation of ER stress and ferroptosis.

CDC42BPA::BRAF represents a novel fusion in desmoplastic infantile ganglioglioma/desmoplastic infantile astrocytoma.

Summary Desmoplastic infantile ganglioglioma/desmoplastic infantile astrocytoma (DIG/DIA) are low-grade glial/glioneuronal tumors occurring predominantly in the cerebral hemispheres of infants. DIG/DIA exhibit BRAF or RAF1 alterations leading to oncogenic mitogen-activated protein kinase (MAPK) pathway activation. Here, we report the discovery of the novel CDC42BPA::BRAF fusion in a three-month-old patient with left frontotemporal DIA using DNA sequencing. Independent validation was performed through RNA sequencing. This fusion joins the kinase domains of BRAF and CDC42BPA, potentially constitutively activating both. It marks the first report of a fusion involving the actomyosin regulatory kinase CDC42BPA/MRCKα in brain tumors, suggesting potential involvement of actin remodeling defects in DIG/DIA. Surgical excision is curative for DIG/DIA, but incomplete resection, recurrence, malignant transformation, or metastases may necessitate adjuvant chemotherapy, posing risks. Identifying and excluding molecular alterations is crucial for selecting targeted therapies, such as BRAF and MEK inhibitors. These options present potential treatments with lower toxicity compared to conventional chemotherapy.

Hereditary conditions associated with elevated cancer risk in childhood

The widespread use of next-generation sequencing (NGS) technologies revealed that a significant percentage of tumors in children develop as a part of monogenic hereditary diseases. Predisposition to the development of pediatric neoplasms is characteristic of a wide range of conditions including hereditary tumor syndromes, primary immunodeficiencies, rasopathies, and phakomatosis. The mechanisms of tumor molecular pathogenesis are diverse and include disturbances in signaling cascades, defects in DNA repair, chromatin remodeling, and microRNA processing. Timely diagnosis of tumor-associated syndromes is important for the proper choice of cancer treatment, genetic counseling of families, and the development of surveillance programs. The review describes the spectrum of neoplasms characteristic of the most common syndromes and the molecular pathogenesis of these diseases.

Hippo Signaling Mediates TGFβ-Dependent Transcriptional Inputs in Cardiac Cushion Mesenchymal Cells to Regulate Extracellular Matrix Remodeling.

The transforming growth factor beta (TGFβ) and Hippo signaling pathways are evolutionarily conserved pathways that play a critical role in cardiac fibroblasts during embryonic development, tissue repair, and fibrosis. TGFβ signaling and Hippo signaling are also important for cardiac cushion remodeling and septation during embryonic development. Loss of TGFβ2 in mice causes cardiac cushion remodeling defects resulting in congenital heart disease. In this study, we used in vitro molecular and pharmacologic approaches in the cushion mesenchymal cell line (tsA58-AVM) and investigated if the Hippo pathway acts as a mediator of TGFβ2 signaling. Immunofluorescence staining showed that TGFβ2 induced nuclear translocation of activated SMAD3 in the cushion mesenchymal cells. In addition, the results indicate increased nuclear localization of Yes-associated protein 1 (YAP1) following a similar treatment of TGFβ2. In collagen lattice formation assays, the TGFβ2 treatment of cushion cells resulted in an enhanced collagen contraction compared to the untreated cushion cells. Interestingly, verteporfin, a YAP1 inhibitor, significantly blocked the ability of cushion cells to contract collagen gel in the absence or presence of exogenously added TGFβ2. To confirm the molecular mechanisms of the verteporfin-induced inhibition of TGFβ2-dependent extracellular matrix (ECM) reorganization, we performed a gene expression analysis of key mesenchymal genes involved in ECM remodeling in heart development and disease. Our results confirm that verteporfin significantly decreased the expression of α-smooth muscle actin (Acta2), collagen 1a1 (Col1a1), Ccn1 (i.e., Cyr61), and Ccn2 (i.e., Ctgf). Western blot analysis indicated that verteporfin treatment significantly blocked the TGFβ2-induced activation of SMAD2/3 in cushion mesenchymal cells. Collectively, these results indicate that TGFβ2 regulation of cushion mesenchymal cell behavior and ECM remodeling is mediated by YAP1. Thus, the TGFβ2 and Hippo pathway integration represents an important step in understanding the etiology of congenital heart disease.

Cardiomyocyte-specific deletion of endothelin receptor A (ETA) obliterates cardiac aging through regulation of mitophagy and ferroptosis

Advanced aging evokes unfavorable changes in the heart including cardiac remodeling and contractile dysfunction although the underlying mechanism remains elusive. This study was conducted to evaluate the role of endothelin-1 (ET-1) in the pathogenesis of cardiac aging and mechanism involved. Echocardiographic and cardiomyocyte mechanical properties were determined in young (5–6 mo) and aged (26–28 mo) wild-type (WT) and cardiomyocyte-specific ETA receptor knockout (ETAKO) mice. GSEA enrichment identified differentially expressed genes associated with mitochondrial respiration, mitochondrial protein processing and mitochondrial depolarization in cardiac aging. Aging elevated plasma levels of ET-1, Ang II and suppressed serum Fe2+, evoked cardiac remodeling (hypertrophy and interstitial fibrosis), contractile defects (fractional shortening, ejection fraction, cardiomyocyte peak shortening, maximal velocity of shortening/relengthening and prolonged relengthening) and intracellular Ca2+ mishandling (dampened intracellular Ca2+ release and prolonged decay), the effects with the exception of plasma AngII, ET-1 and Fe2+ were mitigated by ETAKO. Advanced age facilitated O2− production, carbonyl protein damage, cardiac hypertrophy (GATA4, ANP, NFATc3), ER stress, ferroptosis, compromised autophagy (LC3B, Beclin-1, Atg7, Atg5 and p62) and mitophagy (parkin and FUNDC1), and deranged intracellular Ca2+ proteins (SERCA2a and phospholamban), the effects of which were reversed by ETA ablation. ET-1 provoked ferroptosis in vitro, the response was nullified by the ETA receptor antagonist BQ123 and mitophagy inducer CsA. ETA but not ETB receptor antagonism reconciled cardiac aging, which was abrogated by inhibition of mitophagy and ferroptosis. These findings collectively denote promises of targeting ETA, mitophagy and ferroptosis in the management of aging-associated cardiac remodeling and contractile defect.

The role of Semaphorin 3A in oral diseases.

Semaphorin 3A (SEMA3A), also referred to as H-Sema III, is a molecule with significant biological importance in regulating physiological and pathological processes. However, its role in oral diseases, particularly its association with inflammatory immunity and alveolar bone remodeling defects, remains poorly understood. This comprehensive review article aims to elucidate the recent advances in understanding SEMA3A in the oral system, encompassing nerve formation, periodontitis, pulpitis, apical periodontitis, and oral squamous cell carcinoma. Notably, we explore its novel function in inflammatory immunomodulation and alveolar bone formation during oral infectious diseases. By doing so, this review enhances our comprehension of SEMA3A's role in oral biology and opens up possibilities for modulatory approaches and potential treatments in oral diseases.

B-170 Identifying Metabolic Phenotyping Variability in a Cellular Model of Barth Syndrome

Abstract Introduction Barth Syndrome is an X-linked multisystem disease caused by the mutation in the TAZ gene (TAZ, G 4.5, OMIM 300394) that encodes for acyltransferase taffazin. Taffazin is a mitochondrial protein highly expressed in heart and skeletal muscle and has a central role in cardiolipin remodeling process. TAZ is the only known single gene defect in cardiolipin remodeling, with more than a hundred TAZ mutations identified. However, no systematic correlation has been established between TAZ genotypes and clinical or biochemical phenotypes in Barth syndrome. There is a large phenotypic heterogeneity among affected individuals sometimes even within the same family. This raises a possibility of genetic and metabolic modifiers involved in TAZ clinical and metabolic phenotypes. The aim of this study is to apply LC-MS/MS untargeted and targeted metabolic analyses to explore possible correlations between two TAZ genotypes and metabolome in a cellular model of Barth Syndrome. Methods Mutant TAZ 3(c.647G>T), TAZ 4(c.778-24_778-7delinsA) fibroblast and matching by donors’ age and gender control fibroblast lines (healthy#1, healthy #2) were cultured under identical conditions. Cells (n = 6 for each genotype) were harvested and extracted followed Bligh and Dyer protocol. Extracted metabolites were analyzed by Agilent 6545 Q-TOF coupled with 1290 Infinity II HPLC. Chromatographic separation was achieved by using a Water Premier BEH Z-HILIC Column (2.5 µM, 2.1 × 150 mm) and Waters X-select HSS T3 (2.5 µM, 2.1 × 100 mm) columns for polar and non-polar metabolites respectively. Raw data was normalized with internal standard and total cellular protein amounts. Statistical and pathway enrichment analyses were performed with a chemometric platform Mass Profiler Professional (Agilent) and Metaboanalyst webtool. Results Data was treated first as for two separate genotypes (healthy vs TAZ) followed and compared to the analysis with four separate genotypes (healthy#1, healthy #2 TAZ c.647G>T, TAZ c.778-24_778-7delinsA). Metabolites contributing to discrimination of the genotypes based on metabolic profiles were identified by using a univariate analysis (P < 0.05) and a supervised PLSDA (VIP>1 for small polar metabolites and VIP > 3 for non-polar molecules). The differentially expressed metabolites included amino acids, acylcarnitine, Krebs cycle intermediates, ATP, 3-methylglutaconic acid, fatty acids and a variety of phospholipids. Pathway enrichment analysis revealed that several metabolic pathways were involved. Conclusions Our analyses reveal that genetic variations in TAZ gene cause the differences in the abundance of certain metabolites and affected pathways. This leads us to the conclusion that from the point of view of metabolic pathophysiology, TAZ should be considered beyond a single-gene defect and a more personalized approach is needed in search for potential therapies.

DDX24 Is Essential for Cell Cycle Regulation in Vascular Smooth Muscle Cells During Vascular Development via Binding to FANCA mRNA.

The DEAD-box family is essential for tumorigenesis and embryogenesis. Previously, we linked the malfunction of DDX (DEAD-box RNA helicase)-24 to a special type of vascular malformation. Here, we aim to investigate the function of DDX24 in vascular smooth muscle cells (VSMCs) and embryonic vascular development. Cardiomyocyte (CMC) and VSMC-specific Ddx24 knockout mice were generated by crossing Tagln-Cre mice with Ddx24flox/flox transgenic mice. The development of blood vessels was explored by stereomicroscope photography and immunofluorescence staining. Flow cytometry and cell proliferation assays were used to verify the regulation of DDX24 on the function of VSMCs. RNA sequencing and RNA immunoprecipitation coupled with quantitative real-time polymerase chain reaction were combined to investigate DDX24 downstream regulatory molecules. RNA pull-down and RNA stability experiments were performed to explore the regulation mechanism of DDX24. CMC/VSMC-specific Ddx24 knockout mice died before embryonic day 13.5 with defects in vessel formation and abnormal vascular remodeling in extraembryonic tissues. Ddx24 knockdown suppressed VSMC proliferation via cell cycle arrest, likely due to increased DNA damage. DDX24 protein bound to and stabilized the mRNA of FANCA (FA complementation group A) that responded to DNA damage. Consistent with the function of DDX24, depletion of FANCA also impacted cell cycle and DNA repair of VSMCs. Overexpression of FANCA was able to rescue the alterations caused by DDX24 deficiency. Our study unveiled a critical role of DDX24 in VSMC-mediated vascular development, highlighting a potential therapeutic target for VSMC-related pathological conditions.

Abstract P2171: Lentivirus TAZ Gene Therapy Restores Metabolic Functions In Taz Ko Mice Model Of Barth Syndrome

Introduction: Barth Syndrome is an X-linked disorder caused by mutation in thegene encoding tafazzin (or TAZ ), characterized by dilated cardiomyopathy, muscularhypotonia and cyclic neutropenia. TAZ is a mitochondrial acyltransferase that generatesmature cardiolipin (CL) from monolysocardiolipin (MLCL) and is essential formitochondrial morphology and function. Patients with BTHS exhibit a characteristicreduction in the levels of CL and an accumulation of MLCL. Currently, there is no targetedtreatment exists for these patients. We characterized a global TAZ knockout mousemodel of BTHS and tested the efficacy of Lentivirus-mediated TAZ gene therapy. Methods: We generated third generation lentiviral constructs without an insert, withGreen Fluorescent Protein, with recombinant tafazzin and with recombinant tafazzinmodified to contain a cellular penetrating peptide (hTAZ-CPP) that promotes uptake intotafazzin-deficient cells. We tested the efficacy of these recombinant lentiviruses, first inmouse embryo fibroblasts (MEF) isolated from tafazzin knockout mice and then intafazzin knockout mice after systemic administration. Results: We have found that treatment with hTAZ-CPPs reduces the MLCL/CL ratio tonear wild type levels, thereby rescuing the defect in cardiolipin remodeling in TAZ KOMEFs. Serial echocardiograms revealed significant increase in septal wall thickness andsignificant decrease in ejection fraction, fractional shortening and global longitudinal strainin TAZ KO mice. The TAZ KO mice were treated systemic lentivirus at 5 weeks of ageand then echocardiograms were performed at every 4 weeks until 16 weeks. After thetreatment septal wall thickness continued to increase and ejection fraction, fractionalshortening and global longitudinal strain continued to decrease in the empty virus or GFPtreated mice, whereas they are remained stable in those treated with hTAZ-CPPs. Conclusion: These findings suggest that Lentivirus-mediated TAZ gene replacementtherapy enhanced with cell penetrating peptides has potential as a future treatment forBTHS.

Mathematical models of developmental vascular remodelling: A review.

Over the past 40 years, there has been a strong focus on the development of mathematical models of angiogenesis, while developmental remodelling has received little such attention from the mathematical community. Sprouting angiogenesis can be seen as a very crude way of laying out a primitive vessel network (the raw material), while remodelling (understood as pruning of redundant vessels, diameter control, and the establishment of vessel identity and hierarchy) is the key to turning that primitive network into a functional network. This multiscale problem is of prime importance in the development of a functional vasculature. In addition, defective remodelling (either during developmental remodelling or due to a reactivation of the remodelling programme caused by an injury) is associated with a significant number of diseases. In this review, we discuss existing mathematical models of developmental remodelling and explore the important contributions that these models have made to the field of vascular development. These mathematical models are effectively used to investigate and predict vascular development and are able to reproduce experimentally observable results. Moreover, these models provide a useful means of hypothesis generation and can explain the underlying mechanisms driving the observed structural and functional network development. However, developmental vascular remodelling is still a relatively new area in mathematical biology, and many biological questions remain unanswered. In this review, we present the existing modelling paradigms and define the key challenges for the field.

Natriuretic Peptide Signaling in Uterine Biology and Preeclampsia.

Endometrial decidualization is a uterine process essential for spiral artery remodeling, embryo implantation, and trophoblast invasion. Defects in endometrial decidualization and spiral artery remodeling are important contributing factors in preeclampsia, a major disorder in pregnancy. Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood volume and pressure. ANP is also generated in non-cardiac tissues, such as the uterus and placenta. In recent human genome-wide association studies, multiple loci with genes involved in natriuretic peptide signaling are associated with gestational hypertension and preeclampsia. In cellular experiments and mouse models, uterine ANP has been shown to stimulate endometrial decidualization, increase TNF-related apoptosis-inducing ligand expression and secretion, and enhance apoptosis in arterial smooth muscle cells and endothelial cells. In placental trophoblasts, ANP stimulates adenosine 5'-monophosphate-activated protein kinase and the mammalian target of rapamycin complex 1 signaling, leading to autophagy inhibition and protein kinase N3 upregulation, thereby increasing trophoblast invasiveness. ANP deficiency impairs endometrial decidualization and spiral artery remodeling, causing a preeclampsia-like phenotype in mice. These findings indicate the importance of natriuretic peptide signaling in pregnancy. This review discusses the role of ANP in uterine biology and potential implications of impaired ANP signaling in preeclampsia.

Runx1 regulates critical factors that control uterine angiogenesis and trophoblast differentiation during placental development.

During early pregnancy in humans and rodents, uterine stromal cells undergo a remarkable differentiation to form the decidua, a transient maternal tissue that supports the growing fetus. It is important to understand the key decidual pathways that orchestrate the proper development of the placenta, a key structure at the maternal-fetal interface. We discovered that ablation of expression of the transcription factor Runx1 in decidual stromal cells in a conditional Runx1-null mouse model (Runx1d/d) causes fetal lethality during placentation. Further phenotypic analysis revealed that uteri of pregnant Runx1d/d mice exhibited severely compromised decidual angiogenesis and a lack of trophoblast differentiation and migration, resulting in impaired spiral artery remodeling. Gene expression profiling using uteri from Runx1d/d and control mice revealed that Runx1 directly controls the decidual expression of the gap junction protein connexin 43 (also known as GJA1), which was previously shown to be essential for decidual angiogenesis. Our study also revealed that Runx1 controls the expression of insulin-like growth factor (IGF) 2 and IGF-binding protein 4 (IGFBP4) during early pregnancy. While Runx1 deficiency drastically reduced the production of IGF2 by the decidual cells, we observed concurrent elevated expression of the IGFBP4, which regulates the bioavailability of IGFs, thereby controlling trophoblast differentiation. We posit that dysregulated expression of GJA1, IGF2, and IGFBP4 in Runx1d/d decidua contributes to the observed defects in uterine angiogenesis, trophoblast differentiation, and vascular remodeling. This study therefore provides unique insights into key maternal pathways that control the early phases of maternal-fetal interactions within a critical window during placental development.

1529-P: Hypothalamic FGF1 Regulates Systemic Glucose and Energy Homeostasis

Systemic glucose and energy homeostasis is maintained by interorgan communication networks and the hypothalamus plays a key part in this process. Many research are being conducted to investigate the hypothalamus as a therapeutic target for metabolic diseases such as diabetes and obesity. Recent studies demonstrate that a single intracerebroventricular (icv) injection of FGF1 causes sustained glucose remission in diabetic rodent models (Scarlett et al.), and further study is being conducted. However, there are limited findings about how metabolic status affects endogenous brain FGF1 expression and how brain FGF1 plays in energy homeostasis. A study published in 2012 (Jonker et al.) found severe insulin resistance and a defect in adipose tissue remodeling in HFD-fed FGF1 null-mice. However, these investigations have a limitation in that it is not obvious which tissue/organ mediates insulin resistance due to FGF1 depletion. We found that CNS-deletion of FGF1 (BKO) shows impaired glucose sensing and decreased ERK/STAT3 signaling in ventromedial hypothalamus (VMH) and arcuate nucleus (ARC). This decreased leptin sensitivity leads to decreased muscle glucose uptake following glucose intolerance. We evaluated FGF1 expression in hypothalamus in several metabolic states such as fasting-feeding or other postprandial stimuli to discover which hypothalamic area mediates endogenous FGF1 activity. Interestingly, FGF1 expression in the VMH, but not other brain regions, are increased upon feeding or glucose administration and this induction of FGF1 does not require leptin or insulin. Acute FGF1 deletion in adult VMH causes glucose intolerance as well as increased body weight gain due to reduced leptin sensitivity and brown fat dysfunction, which leads to decreased energy expenditure. In keeping with this finding, germ-line ablation of FGF1 in VMH neurons results in increased adiposity. We propose that endogenous brain FGF1 regulates glucose and energy homeostasis through modulating VMH leptin sensitivity. Disclosure H.Shin: None. J.Suh: None.

Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells.

Polydom is an extracellular matrix protein involved in lymphatic vessel development. Polydom-deficient mice die immediately after birth due to defects in lymphatic vessel remodeling, but the mechanism involved is poorly understood. Here, we report that Polydom directly binds to Tie1, an orphan receptor in the Angiopoietin-Tie axis, and facilitates migration of lymphatic endothelial cells (LECs) in a Tie1-dependent manner. Polydom-induced LEC migration is diminished by PI3K inhibitors but not by an ERK inhibitor, suggesting that the PI3K/Akt signaling pathway is involved in Polydom-induced LEC migration. In line with this possibility, Akt phosphorylation in LECs is enhanced by Polydom although no significant Tie1 phosphorylation is induced by Polydom. LECs also exhibited nuclear exclusion of Foxo1, a signaling event downstream of Akt activation, which was impaired in Polydom-deficient mice. These findings indicate that Polydom is a physiological ligand for Tie1 and participates in lymphatic vessel development through activation of the PI3K/Akt pathway.

In organello real-time NMR metabolomics of mitochondria: An S. cerevisiae model of Barth syndrome displays perturbed metabolism.

Mitochondria are central to cellular metabolism, generating more than 90% of the ATP energy currency of the cell. Dysfunction of mitochondria occurs in a number of diseases including cancer, cardiopathy, and neurodegeneration, as well as heritable mitochondrial diseases such as Barth syndrome (BTHS). BTHS is caused by faulty cardiolipin (CL) remodeling, wherein mutations of the transacylase Tafazzin result in a defective enzyme and a build-up of the precursor, monolysocardiolipin. The aim of this study is to investigate how defects in CL remodeling affect the tricarboxylic acid (TCA) cycle. The rationale, is that CL – the hallmark lipid of mitochondria, is crucial for proper cristae morphology and mitochondrial bioenergetic function. Since enzymes in the TCA cycle are membrane-bound, altered lipid content could perturb mitochondrial membranes, and hence the activities of enzymes involved in metabolism. We used real-time 1H-13C HSQC NMR to characterize the fate of 13C-pyruvate in mitochondria isolated from S. cerevisiae. We compared wild-type (WT) mitochondria with ∆taz1 - a knockout that models BTHS, and ∆crd1 – a CL remodeling mutant that lacks CL completely. Following uptake of 13C-pyruvate, fifteen new 13C-labeld metabolites could be identified in the mitochondria over the course of ∼2 h. While many metabolites showed similar kinetics between the three strains, levels of mevalonate - a precursor in cholesterol synthesis, appears to be elevated in ∆taz1. Synthesis of glutamine showed large differences between all three strains. Overall, the turnover of pyruvate, appeared more sluggish in the CL remodeling mutants compared to WT. The studies demonstrate that proper CL remodeling is required for optimal TCA cycle activity, and may shed light on the etiology of BTHS, as well as point the way to therapeutic interventions.