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Background: The modulation of vascular smooth muscle cells (VSMC) phenotypes is a highly important process in the formation of neointima and in resolution of inflammation after vascular injury. Underlying genetic mechanisms that control the phenotypic switch of VSMC in disease are not completely understood. On this basis, the phenotypic regulation of VSMC in restenosis development was examined in the mouse carotid artery injury model. Methods and Results: Wire-induced injury, an established mouse-model to study restenosis, was applied to induce vascular injury in C57BL/6 mice. Time-resolved single-cell RNA-sequencing (scRNAseq) and massive-analysis-of-cDNA-ends (MACE)-RNAseq of neointima obtained by laser capture microdissection were performed to detect genetic changes in neointima development. Single-cell RNA-sequencing of C57BL/6 mice at different time points after wire-induced injury revealed an expanding, highly proliferative activated VSMC cluster, which exhibited low expression of SMC marker genes. Analysis for differentially regulated genes (DEG) and pseudotime clustering identified a unique marker gene signature, including high expression of TIMP Metallopeptidase Inhibitor 1 (Timp1), which was identified as main ordering gene of the modulation processes′ pseudo temporal states. MACEseq confirmed upregulation of Timp1 selectively in neointima forming VSMCs. Integration with atherosclerosis scRNAseq data indicated differences in gene signatures of activated VSMC populations. Transcription factor analysis of modulated VSMC identified BACH1, a factor involved in reactive oxidative stress response and proliferation of SMCs, as potential regulatory target for re-differentiation of VSMCs after vascular injury. Conclusion: ScRNAseq identified a unique cell cluster responsible for neointima formation. Targeting the transcription factors driving the cluster (like BACH1) can be exploited to combat neointima development.

Supplemental Digital Content is available in the text., Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1−/−/NOX2−/−/NOX4−/−), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP—a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride–driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.