SOX4 is a novel phenotypic regulator of endothelial cells in atherosclerosis revealed by single-cell analysis.

INTRODUCTION: Atherosclerotic complications represent the leading cause of cardiovascular mortality globally. Dysfunction of endothelial cells (ECs) often initiates the pathological events in atherosclerosis. OBJECTIVES: In this study, we sought to investigate the transcriptional profile of atherosclerotic aortae, identify novel regulator in dysfunctional ECs and hence provide mechanistic insights into atherosclerotic progression. METHODS: We applied single-cell RNA sequencing (scRNA-seq) on aortic cells from Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice to explore the transcriptional landscape and heterogeneity of dysfunctional ECs. In vivo validation of SOX4 upregulation in ECs were performed in atherosclerotic tissues, including mouse aortic tissues, human coronary arteries, and human renal arteries. Single-cell analysis on human aortic aneurysmal tissue was also performed. Downstream vascular abnormalities induced by EC-specific SOX4 overexpression, and upstream modulators of SOX4 were revealed by biochemical assays, immunostaining, and wire myography. Effects of shear stress on endothelial SOX4 expression was investigated by in vitro hemodynamic study. RESULTS: Among the compendium of aortic cells, mesenchymal markers in ECs were significantly enriched. Two EC subsets were subsequently distinguished, as the 'endothelial-like' and 'mesenchymal-like' subsets. Conventional assays consistently identified SOX4 as a novel atherosclerotic marker in mouse and different human arteries, additional to a cancer marker. EC-specific SOX4 overexpression promoted atherogenesis and endothelial-to-mesenchymal transition (EndoMT). Importantly, hyperlipidemia-associated cytokines and oscillatory blood flow upregulated, whereas the anti-diabetic drug metformin pharmacologically suppressed SOX4 level in ECs. CONCLUSION: Our study unravels SOX4 as a novel phenotypic regulator during endothelial dysfunction, which exacerbates atherogenesis. Our study also pinpoints hyperlipidemia-associated cytokines and oscillatory blood flow as endogenous SOX4 inducers, providing more therapeutic insights against atherosclerotic diseases.

Endothelium-targeted delivery of PPARδ by adeno-associated virus serotype 1 ameliorates vascular injury induced by hindlimb ischemia in obese mice.

Diabetic vasculopathy is a major health problem worldwide. Peripheral arterial disease (PAD), and in its severe form, critical limb ischemia is a major form of diabetic vasculopathy with limited treatment options. Existing literature suggested an important role of PPARδ in vascular homeostasis. It remains elusive for using PPARδ as a potential therapeutic target due to mostly the side effects of PPARδ agonists. To explore the roles of PPARδ in endothelial homeostasis, endothelial cell (EC) selective Ppard knockout and controlled mice were subjected to hindlimb ischemia (HLI) injury. The muscle ECs were sorted for single-cell RNA sequencing (scRNA-seq) analysis. HLI was also performed in high fat diet (HFD)-induced obese mice to examine the function of PPARδ in obese mice with delayed vascular repair. Adeno-associated virus type 1 (AAV1) carrying ICAM2 promoter to target endothelium for overexpressing PPARδ was injected into the injured muscles of normal chow- and HFD-fed obese mice before HLI surgery was performed. scRNA-seq analysis of ECs in ischemic muscles revealed a pivotal role of PPARδ in endothelial homeostasis. PPARδ expression was diminished both after HLI injury, and also in obese mice, which showed further delayed vascular repair. Endothelium-targeted delivery of PPARδ by AAV1 improved perfusion recovery, increased capillary density, restored endothelial integrity, suppressed vascular inflammation, and promoted muscle regeneration in ischemic hindlimbs of both lean and obese mice. Our study indicated the effectiveness of endothelium-targeted PPARδ overexpression for restoring functional vasculature after ischemic injury, which might be a promising option of gene therapy to treat PAD and CLI.

Author Correction: Commensal microflora-induced T cell responses mediate progressive neurodegeneration in glaucoma.

The originally published version of this Article contained an error in Figure 4. The bar chart in panel f was inadvertently replaced with a duplicate of the bar chart in panel e. This error has now corrected in both the PDF and HTML versions of the Article.

Commensal microflora-induced T cell responses mediate progressive neurodegeneration in glaucoma.

Glaucoma is the most prevalent neurodegenerative disease and a leading cause of blindness worldwide. The mechanisms causing glaucomatous neurodegeneration are not fully understood. Here we show, using mice deficient in T and/or B cells and adoptive cell transfer, that transient elevation of intraocular pressure (IOP) is sufficient to induce T-cell infiltration into the retina. This T-cell infiltration leads to a prolonged phase of retinal ganglion cell degeneration that persists after IOP returns to a normal level. Heat shock proteins (HSP) are identified as target antigens of T-cell responses in glaucomatous mice and human glaucoma patients. Furthermore, retina-infiltrating T cells cross-react with human and bacterial HSPs; mice raised in the absence of commensal microflora do not develop glaucomatous T-cell responses or the associated neurodegeneration. These results provide compelling evidence that glaucomatous neurodegeneration is mediated in part by T cells that are pre-sensitized by exposure to commensal microflora.