Aims: The artery contains numerous cell types which contribute to multiple vascular diseases. However, the heterogeneity and cellular responses of these vascular cells during abdominal aortic aneurysm (AAA) progression have not been well characterized. Methods and results: Single-cell RNA sequencing was performed on the infrarenal abdominal aortas (IAAs) from C57BL/6J mice at Days 7 and 14 post-sham or peri-adventitial elastase-induced AAA. Unbiased clustering analysis of the transcriptional profiles from >4500 aortic cells identified 17 clusters representing nine-cell lineages, encompassing vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells, immune cells (macrophages, T cells, B cells, and dendritic cells), and two types of rare cells, including neural cells and erythrocyte cells. Seurat clustering analysis identified four smooth muscle cell (SMC) subpopulations and five monocyte/macrophage subpopulations, with distinct transcriptional profiles. During AAA progression, three major SMC subpopulations were proportionally decreased, whereas the small subpopulation was increased, accompanied with down-regulation of SMC contractile markers and up-regulation of pro-inflammatory genes. Another AAA-associated cellular response is immune cell expansion, particularly monocytes/macrophages. Elastase exposure induced significant expansion and activation of aortic resident macrophages, blood-derived monocytes and inflammatory macrophages. We also identified increased blood-derived reparative macrophages expressing anti-inflammatory cytokines suggesting that resolution of inflammation and vascular repair also persist during AAA progression. Conclusion: Our data identify AAA disease-relevant transcriptional signatures of vascular cells in the IAA. Furthermore, we characterize the heterogeneity and cellular responses of VSMCs and monocytes/macrophages during AAA progression, which provide insights into their function and the regulation of AAA onset and progression.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. BAF60c, a unique subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, is critical for cardiac and skeletal myogenesis, yet little is known about its function in the vasculature and, specifically, in AAA pathogenesis. Here, we found that BAF60c was downregulated in human and mouse AAA tissues, with primary staining to vascular smooth muscle cells (VSMCs), confirmed by single-cell RNA-sequencing. In vivo studies revealed that VSMC-specific knockout of Baf60c significantly aggravated both angiotensin II– (Ang II–) and elastase-induced AAA formation in mice, with a significant increase in elastin degradation, inflammatory cell infiltration, VSMC phenotypic switch, and apoptosis. In vitro studies showed that BAF60c knockdown in VSMCs resulted in loss of contractile phenotype, increased VSMC inflammation, and apoptosis. Mechanistically, we demonstrated that BAF60c preserved VSMC contractile phenotype by strengthening serum response factor (SRF) association with its coactivator P300 and the SWI/SNF complex and suppressing VSMC inflammation by promoting a repressive chromatin state of NF-κB target genes as well as preventing VSMC apoptosis through transcriptional activation of KLF5-dependent B cell lymphoma 2 (BCL2) expression. Our identification of the essential role of BAF60c in preserving VSMC homeostasis expands its therapeutic potential in preventing and treating AAA.

Abdominal aortic aneurysm (AAA) is a life-threatening degenerative vascular disease. Endothelial cell (EC) dysfunction is implicated in AAA. Our group recently demonstrated that Krüppel-like factor 11 (KLF11) plays an essential role in maintaining vascular homeostasis, at least partially through inhibition of EC inflammatory activation. However, the functions of endothelial KLF11 in AAA remain unknown. Here we found that endothelial KLF11 expression was reduced in the ECs from human aneurysms and was time dependently decreased in the aneurysmal endothelium from both elastase- and Pcsk9 /AngII-induced AAA mouse models. KLF11 deficiency in ECs markedly aggravated AAA formation, whereas EC-selective KLF11 overexpression markedly inhibited AAA formation. Mechanistically, KLF11 not only inhibited the EC inflammatory response but also diminished MMP9 expression and activity and reduced NADPH oxidase 2–mediated production of reactive oxygen species in ECs. In addition, KLF11-deficient ECs induced smooth muscle cell dedifferentiation and apoptosis. Overall, we established endothelial KLF11 as a potentially novel factor protecting against AAA and a potential target for intervention in aortic aneurysms.

Specific and efficient smooth muscle cell–targeted (SMC-targeted) gene deletion is typically achieved by pairing SMMHC-CreER ^T2 -Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreER ^T2 , is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreER ^T2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing bacterial artificial chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreER ^T2 -Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11 -driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the CreNLS ^P2A or CreER ^T2–P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre-knockin mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive ( Myh11 -CreNLS ^P2A ) and inducible ( Myh11 -CreER ^T2–P2A ) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11 -CreER ^T2 -RAD mice and the Itga8 -CreER ^T2 mouse models, our models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.

The pathophysiology of chronic thromboembolic pulmonary hypertension (CTEPH) is largely unknown. Although pulmonary endarterectomy (PEA) is potentially curative, inoperable patients and persistent pulmonary hypertension (PH) following surgery remain a significant problem. In this study, we aim to describe the histopathological characteristics of CTEPH and explore the potential relationship between pulmonary arterial lesions, radiological parameters, and clinical manifestations. Endarterectomized tissues from 81 consecutive patients of CTEPH were carefully collected, sectioned, and examined by experienced pathologists. Pertinent clinical and radiological data were obtained from medical records and operative reports. Neointima, fresh/organized thrombi, recanalized regions, and atherosclerotic lesions were microscopically examined as previously described. Thrombi and atherosclerosis were dominant in UCSD classification level I PEA materials, while recanalized neo-vessels were more frequently observed in UCSD classification level III cases. Degenerative changes of the extracellular matrix were also noticed in the vascular bed. Atherosclerotic lesions were more frequently observed in cases with higher ratio of the pulmonary artery diameter to ascending aorta diameter (PA/AA) reflected by computed tomographic pulmonary arterial scanning. Furthermore, the removal of pulmonary artery complex lesions (with the combination of three to four types of lesions) by PEA was associated with lower postoperative mean pulmonary arterial pressure (mPAP) and decreased incidences of persistent PH. Our study demonstrates that the histopathological features of CTEPH are strongly linked with clinical manifestations and the postoperative outcome after PEA. These data may provide possible evidence for further studies in searching for appropriate causal factors underlying this disease.

BACKGROUND: Chronic thromboembolic pulmonary hypertension is characterized by proximal pulmonary artery obstruction and distal microvasculopathy. However, the mechanisms driving this dual-compartment pulmonary vascular remodeling remain unclear. METHODS: Male Sprague-Dawley rats were injected with gelatin sponge combined with SU5416 as a secondary insult. Hemodynamics, echocardiography, and pulmonary vascular remodeling were evaluated to investigate the development of chronic thromboembolic pulmonary hypertension. Single-cell RNA sequencing of rat lung tissue was conducted to elucidate the molecular mechanisms underlying pulmonary vascular remodeling. The results were validated by immunofluorescence and cell-based experiments. RESULTS: The optimal size range of gelatin sponge for large pulmonary artery obstruction was 710 to 1000 µm, which synergized with a low dose of SU5416 (10 mg/kg) to induce significant increases in right ventricular systolic pressure and right ventricular hypertrophy at 5 weeks. The model exhibited persistent elastic pulmonary artery obstruction and remodeling, along with significant wall thickening and muscularization of pulmonary microvessels. Single-cell transcriptomic analysis revealed a significant reduction in microvascular endothelial cells and an increase in smooth muscle cells in the chronic thromboembolic pulmonary hypertension rats. STAT3 , EGR1 , and HIF1A were identified as key genes driving aberrant smooth muscle cell proliferation. The Sell (encoding L-selectin)- Podxl (encoding PODXL, podocalyxin) ligand-receptor pair was found specifically in diseased rats and mediated immune cell–endothelial cell interactions. L-selectin promoted neutrophil adhesion and dysfunction in pulmonary arterial and microvascular endothelial cells, both of which were reversed by PODXL knockdown. CONCLUSIONS: Our new model recapitulates human chronic thromboembolic pulmonary hypertension pathophysiology and is useful for understanding pulmonary microvasculopathy. Sell-Podxl is a previously unrecognized link between inflammation and vascular remodeling, offering a potential therapeutic target.