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Role of Pdk4 in BAPN-induecd aortic dissection in mouse models

ABSTRACT: Thoracic aortic dissection (TAD) is a life-threatening acute vascular condition with extremely high morbidity and mortality. Endothelial cells (ECs) are critical for maintaining vascular homeostasis, yet the role of Endothelial-to-Mesenchymal Transition (EndoMT)—a key cell fate process in vascular development and disease—in TAD remains poorly defined. Furthermore, the functional significance of EndoMT-gene-program activation mediated by pyruvate dehydrogenase kinase 4 (PDK4) in TAD has not been elucidated. To explore the mechanistic involvement of EndoMT in TAD, a murine TAD model was established through systemic administration of β-aminopropionitrile (BAPN). RNA sequencing was performed to profile the activation landscape of the EndoMT-gene-program in ECs for identifying key regulatory targets. Concurrently, clinical samples from TAD patients and healthy controls were analyzed to quantify mRNA and protein levels of EndoMT-related markers in aortic tissues. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) was identified as a critical regulator and validated as a downstream target of EndoMT signaling using an integrated approach combining RNA-seq, luciferase reporter assays, and immunofluorescent staining. The functional role of Pdk4 in TAD was further validated by using conditional knockout (CKO) mouse model. To facilitate better clinical translation, EC-specific Pdk4 deletion or overexpression mouse models were generated via adeno-associated virus (AAV)-mediated delivery and subjected to TAD model. Serum samples from these mice were collected, and enzyme-linked immunosorbent assay (ELISA) was used to measure levels of endothelial injury markers (sICAM1, sVCAM1, sE-Selectin) for assessing endothelial dysfunction. Additionally, the therapeutic potential of dichloroacetate (DCA), a specific PDK4 inhibitor, was evaluated by administering it to BAPN-fed mice to assess its efficacy in ameliorating TAD progression. A significant upregulation of the EndoMT-gene-program was observed in BAPN-fed murine models of TAD, aortic tissues from TAD patients, and HAECs treated with angiotensin II (Ang II). Administration of AAVEndoPdk4 directly induced TAD formation, which was exacerbated by BAPN co-treatment. Conversely, EC-specific knockdown of Pdk4 reduced TAD formation by inhibiting EndoMT signaling. The same results were also obtained in EC-specific conditional Pdk4 knockout mice (CKO, Pdk4flox/flox; Cdh5-CreERT2). PDK4 was identified as a critical factor in both TAD development and EndoMT process, with SNAI1 transcriptionally activating PDK4; vice versa, PDK4 further enhanced the activation of EndoMT signaling in both in vivo and vitro. PDK4 inhibitor, DCA, significantly slowed TAD progression in BAPN-fed mice. Mechanistically, a PDK4-EndoMT feedback loop was identified in TAD, where interruption of this self-reinforcing pathological cycle markedly attenuates the progression of TAD.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE314672 | GEO | 2026/03/27

REPOSITORIES: GEO

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Project description:Background: Protein-tyrosine-phosphatase CD45 is expressed in all nucleated cells of the hematopoietic system but has also been detected in mitral valve endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndoMT). Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony-forming cells (ECFCs) also induced expression of multiple EndoMT marker genes. We hypothesize that CD45 may contribute to atherosclerosis, a prevalent cardiovascular disease dependent on EndoMT. However, the detailed molecular mechanisms underlying how CD45 contributes to EndoMT and the impact of therapeutic manipulation of CD45 expression in atherosclerosis are entirely unknown. Method: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE-deficient (ApoE-/-/C57BL/6) background and fed them a Western diet to produce atherosclerosis. We enriched mouse aortic ECs with anti-CD31 beads to perform single-cell RNA sequencing. Cellular, biochemical, and molecular approaches were used to investigate the effect of endothelial CD45-specific deletion on EndoMT and lesion development in ApoE-/- model of atherosclerosis. Results: Endothelial ApoE-/- CD45-deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls. Single-cell RNA sequencing revealed that loss of endothelial CD45 reduces EndoMT marker expression and TGF-β signaling in atherosclerotic mice, which is associated with the reduction of lesions in the ApoE-/- mouse model. Mechanistically, the loss of CD45 results in increased FGFR2 in mouse aortic ECs and KLF2 expression in the aortic root. Consistently, endothelial CD45-deficiency inhibits EndoMT and TGF-β signaling. Conclusions: These findings demonstrate that genetic depletion of endothelial CD45 protects against EndoMT-driven atherosclerosis by promoting FGFR2 and KLF2 expression while inhibiting TGFβ signaling and EndoMT. Thus, targeting endothelial CD45 may be a novel therapeutic strategy to reduce EndoMT and treat atherosclerosis.

Project description:Background: Biological-sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (EC) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC. Methods: We used aortic EC that was isolated from 13-week-old female and male mice as well as EC-lineage tracing (Cdh5-CreERT2 Rosa-YFP) atherosclerotic Apoe‒/‒ mice to investigate the biological sexual dimorphism of the EC functions in vitro and in vivo. RNA sequencing (RNAseq) analyses were performed on male and female mouse aortic EC and human lung EC. Results: In vitro, female mouse aortic EC (F_MAEC) showed more apoptosis and higher cellular ROS levels, lower mitochondrial membrane potential (ΔΨm), and lower levels of mitochondrial transcription factor A (TFAM) than male aortic EC (M_MAEC). Additionally, F_MAEC had decreased angiogenic potential (tube formation, cell viability, and proliferation) compared to M_MAEC. In vivo, female mice had significantly higher lipid accumulation within the aortas, less efficient outward vessel remodeling, and lower endothelial-mediated vasorelaxation than males. Using the EC-lineage tracing approach, we found that female lesions had significantly lower rates of intraplaque neovascularization and Endothelial-to-Mesenchymal (EndoMT) transition within advanced atherosclerotic lesions but higher incidents of missing EC lumen coverage and higher levels of oxidative products and apoptosis. RNAseq analyses revealed that both mouse and human female EC had higher expression of genes associated with inflammation and apoptosis and lower expression of genes related to angiogenesis and oxidative phosphorylation than male EC. Conclusions: Our study delineates critical sex-specific differences in EC relevant to pro-inflammatory, pro-oxidant, and angiogenic characteristics, which are entirely consistent with a vulnerable phenotype in females. Our results provide a biologic basis for sex-specific pro-atherosclerotic mechanisms.

Project description:Vascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive an endothelial-to-mesenchymal transition (EndoMT), where endothelial cells undergo a phenotypic switch to mesenchymal-like cells that are pro-inflammatory and pro-fibrotic. In this study, we sought to determine whether obesity can promote EndoMT in adipose tissue. Mice in which endothelial cells are lineage-traced with eYFP were fed a high-fat/high-sucrose (HF/HS) or Control diet for 13, 26, and 52 weeks, and EndoMT was assessed in adipose tissue depots as percentage of CD45-CD31-Acta2+ mesenchymal-like cells that were eYFP+. EndoMT was also assessed in human adipose endothelial cells through cell culture assays and by the analysis of single cell RNA sequencing datasets obtained from the visceral adipose tissues of obese individuals. Quantification by flow cytometry showed that mice fed a HF/HS diet display a time-dependent increase in EndoMT over Control diet in subcutaneous adipose tissue (+3.0%, +2.6-fold at 13 weeks; +10.6%, +3.2-fold at 26 weeks; +11.8%, +2.9-fold at 52 weeks) and visceral adipose tissue (+5.5%, +2.3-fold at 13 weeks; +20.7%, +4.3-fold at 26 weeks; +25.7%, +4.8-fold at 52 weeks). Transcriptomic analysis revealed that EndoMT cells in visceral adipose tissue have enriched expression of genes associated with inflammatory and TGFb signaling pathways. Human adipose-derived microvascular endothelial cells cultured with TGF-β1, IFN-γ, and TNF-⍺ exhibited a similar upregulation of EndoMT markers and induction of inflammatory response pathways. Analysis of single cell RNA sequencing datasets from visceral adipose tissue of obese patients revealed a nascent EndoMT sub-cluster of endothelial cells with reduced PECAM1 and increased ACTA2 expression, which was also enriched for inflammatory signaling genes and other genes associated with EndoMT. These experimental and clinical findings show that chronic obesity can accelerate EndoMT in adipose tissue. We speculate that EndoMT is a feature of adipose tissue dysfunction that contributes to local inflammation and the systemic metabolic effects of obesity.

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Role of Pdk4 in BAPN-induecd aortic dissection in mouse models

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