Circulation research最新文献

Background: Genome-wide association studies implicate common genetic variations in the LRP1 (low-density lipoprotein receptor-related protein 1 gene) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown.

Methods: Fine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells were genome-edited using CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) to delete or modify candidate enhancer regions and generate LRP1 knockout cell lines. Cells were differentiated into smooth muscle cells through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown, and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA sequencing, and mass spectrometry.

Results: Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure, and spontaneous coronary artery dissection. We found the rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL (Zinc Finger Protein SNAI1) to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 knockout decreased induced pluripotent stem cell-derived smooth muscle cell proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix and connective tissue development. LRP1 knockout and deletion of rs11172113 enhancer showed potentiated canonical TGF-β (transforming growth factor beta) signaling through enhanced phosphorylation of SMAD2/3 (Mothers against decapentaplegic homolog 2/3). Analyses of the protein content of decellularized extracts indicated partial extracellular matrix remodeling involving enhanced secretion of CYR61 (cystein rich angiogenic protein 61), a known LRP1 ligand involved in vascular integrity and TIMP3 (Metalloproteinase inhibitor 3), implicated in extracellular matrix maintenance and also known to interact with LRP1.

Conclusions: Our findings support allele-specific LRP1 expression repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in smooth muscle cells to remodel the extracellular matrix enhanced by TGF-β as a potential mechanism of this pleiotropic locus for vascular diseases.

Background: Disturbed metabolism and transport of citrate play significant roles in various pathologies. However, vascular citrate regulation and its potential role in aortic aneurysm (AA) development remain poorly understood.

Methods: Untargeted metabolomics by mass spectrometry was applied to identify upregulated metabolites of the tricarboxylic acid cycle in AA tissues of mice. To investigate the role of citrate and its transporter ANK (progressive ankylosis protein) in AA development, vascular smooth muscle cell (VSMC)-specific Ank-knockout mice were used in both Ang II (angiotensin II)- and CaPO₄-induced AA models.

Results: Citrate was abnormally increased in both human and murine aneurysmal tissues, which was associated with downregulation of ANK, a citrate membrane transporter, in VSMCs. The knockout of Ank in VSMCs promoted AA formation in both Ang II- and CaPO₄-induced AA models, while its overexpression inhibited the development of aneurysms. Mechanistically, ANK deficiency in VSMCs caused abnormal cytosolic accumulation of citrate, which was cleaved into acetyl coenzyme A and thus intensified histone acetylation at H3K23, H3K27, and H4K5. Cleavage under target and tagmentation analysis further identified that ANK deficiency-induced histone acetylation activated the transcription of inflammatory genes in VSMCs and thus promoted a citrate-related proinflammatory VSMC phenotype during aneurysm diseases. Accordingly, suppressing citrate cleavage to acetyl coenzyme A downregulated inflammatory gene expression in VSMCs and restricted ANK deficiency-aggravated AA formation.

Conclusions: Our studies define the pathogenic role of ANK deficiency-induced cytosolic citrate accumulation in AA pathogenesis and an undescribed citrate-related proinflammatory VSMC phenotype. Targeting ANK-mediated citrate transport may emerge as a novel diagnostic and therapeutic strategy in AA.

Background: Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability.

Methods: Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2^iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm²) or oscillatory shear (0.5±4 dynes/cm²) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and optoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function.

Results: We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2^iΔEC mice showed heightened vascular permeability. Kindlin-2^iΔEC were bred onto ApoE^-/- mice to generate Kindlin-2^iΔEC; ApoE^-/- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation.

Conclusions: Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis.

Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.

Background: Given the growing acknowledgment of the detrimental effects of excessive myocardial fibrosis on pathological remodeling after myocardial ischemia-reperfusion injury (I/R), targeting the modulation of myocardial fibrosis may offer protective and therapeutic advantages. However, effective clinical interventions and therapies that target myocardial fibrosis remain limited. As a promising chimeric antigen receptor (CAR) cell therapy, whether CAR macrophages (CAR-Ms) can be used to treat I/R remains unclear.

Methods: The expression of FAP (fibroblast activation protein) was studied in mouse hearts after I/R. FAP CAR-Ms were generated to target FAP-expressing cardiac fibroblasts in mouse hearts after I/R. The phagocytosis activity of FAP CAR-Ms was tested in vitro. The efficacy and safety of FAP CAR-Ms in treating I/R were evaluated in vivo.

Results: FAP was significantly upregulated in activated cardiac fibroblasts as early as 3 days after I/R. Upon demonstrating their ability to engulf FAP-overexpressing fibroblasts, we intravenously administered FAP CAR-Ms to mice at 3 days after I/R and found that FAP CAR-Ms significantly improved cardiac function and reduced myocardial fibrosis in mice after I/R. No toxicities associated with FAP CAR-Ms were detected in the heart or other organs at 2 weeks after I/R. Finally, we found that FAP CAR-Ms conferred long-term cardioprotection against I/R.

Conclusions: Our proof-of-concept study demonstrates the therapeutic potential of FAP CAR-Ms in alleviating myocardial I/R and potentially opens new avenues for the treatment of a range of heart diseases that include a fibrotic phenotype.