Coronary microvascular dysfunction (CMD) is highly prevalent among patients with coronary artery disease and, as an independent risk factor, increases the risk of cardiovascular events, yet there is a lack of targeted interventions. S100a6 has been shown to regulate endothelial cell cycle progression, but its expression pattern and protective mechanism in CMD remain unclear. This study aimed to determine the endogenous protective role of S100a6 in CMD and to elucidate the molecular mechanism by which it ameliorates CMD through stabilizing the β-catenin/TCF1 complex and suppressing the activation of the PARP9/STAT1 signaling axis.

A total of 12 male ob/ob mice, aged 8 weeks and weighing 38 to 42 g, were randomly assigned into 2 groups (n=6 per group) using a random number table: the CMD group and the CMD-S100a6KD group. Mice in the CMD group received injection via tail vein of scrambled control virus, whereas those in the CMD-S100a6KD group received endothelium-specific adenovirus to knock down S100a6 expression. Additionally, six male C57BL/6J wild-type mice of the same age, weighing 18 to 22 g, were used as a healthy control group (Control group) and were fed routinely without any model construction or drug/gene intervention. Endothelial-specific knockdown of S100a6 was achieved by tail vein injection of AAV9-shS100a6 driven by an endothelium-specific Tie promoter. After 20 weeks, coronary flow reserve (CFR) and left ventricular diastolic function (E/e') were assessed by echocardiography; heart tissues were collected for immunofluorescence staining to evaluate myocardial microvessel density and related protein expression and localization. Human coronary microvascular endothelial cells (HCMECs) were cultured in vitro and treated with high glucose (25 mmol/L) combined with free fatty acids (0.5 mmol/L) for 48 h to establish a CMD cell model, followed by gene intervention using siRNA and plasmids. Experimental groups were designed according to different objectives: baseline control groups [the normal culture control group (Control group) and the CMD model group (HG group)], S100a6 loss-of-function groups (HG+siS100a6 and its siNC control), and downstream mechanism rescue groups (HG+siS100a6+siPARP9 and its siNC control; HG+siS100a6+OE-β-catenin and its empty vector control). Single-cell RNA sequencing was used to analyze the cell-subset expression of S100a6 in heart tissues, and bulk RNA sequencing was performed to identify differentially expressed genes and enriched pathways. Western blotting and qRT-PCR were used to measure the relative protein and mRNA expression levels of S100a6, β-catenin, TCF1, PARP9, STAT1, and p-STAT1. Co-immunoprecipitation was performed to validate the interaction between β-catenin and TCF1, and chromatin immunoprecipitation was used to detect the binding of TCF1 to the PARP9 promoter, thereby clarifying the molecular mechanism. Endothelial cell function was assessed by EdU proliferation assay, scratch wound healing assay, and tube formation assay.

Compared with the CMD group, mice in the CMD-S100a6KD group showed a significantly reduced CFR(P<0.05), a significantly increased E/e' ratio (P<0.05), and a significantly decreased myocardial microvessel density (P<0.05). Single-cell RNA sequencing revealed that S100a6 expression was markedly elevated overall in the heart tissues of CMD group mice, and it is also significantly upregulated in the subsets of cardiac endothelial cells (P<0.05); immunofluorescence co-localization analysis showed significantly enhanced co-localization signals of S100a6 and CD31(P<0.0001). In vitro, high glucose and high lipid stimulation significantly increased S100a6 protein expression levels in HCMECs compared with normally cultured controls (P<0.05). Knockdown of S100a6 led to a significant reduction in the number of tube branches (P<0.05), a significant decrease in scratch wound healing rate (P<0.05), and a significant decrease in EdU-positive rate (P<0.05) in HCMECs. Bulk RNA sequencing showed that PARP9 gene expression was significantly upregulated after S100a6 knockdown, and gene enrichment analysis revealed significant enrichment of the STAT1 signaling pathway. When S100a6 and PARP9 were knocked down simultaneously, STAT1 phosphorylation levels were significantly reduced compared with the S100a6 knockdown alone group (P<0.0001). Functional assays confirmed that PARP9 silencing significantly ameliorated the S100a6 knockdown-induced impairments in tube formation (P<0.05), migration capacity (P<0.05), and EdU-positive rate (P<0.05) in HCMECs. Molecular docking experiments showed that S100a6 binds within the crevice of the TCF1/β-catenin interaction interface. Knockdown of S100a6 significantly reduced β-catenin protein levels (P<0.05), significantly weakened the interaction between β-catenin and TCF1(P<0.05), and significantly decreased the binding of TCF1 to the PARP9 promoter region (P<0.05). Overexpression of β-catenin in parallel with S100a6 knockdown significantly reduced PARP9 protein expression levels (P<0.05) and reduced phosphorylated STAT1 levels (P<0.05). Functionally, β-catenin overexpression significantly rescued the S100a6 deficiency-induced reductions in tube branch number (P<0.05), scratch wound healing rate (P<0.05), and EdU-positive rate (P<0.05) in HCMECs.

S100a6 is an endogenous protective factor for coronary microcirculatory dysfunction. It regulates the activation of the PARP9/STAT1 signaling pathway through β-catenin/TCF1 complex-dependent transcriptional repression, maintains endothelial cell homeostasis, and thereby ameliorates coronary microcirculatory dysfunction.