Endothelial reprogramming by disturbed flow revealed by single-cell RNA and chromatin accessibility study

Andueza, Aitor; Kumar, Sandeep; Kim, Ju‐Young; Kang, Dong Won; Mumme, Hope L; Perez, Julian I.; Jo, Hanjoong

doi:10.1101/2020.07.15.205427

Cited by 19 publications

(35 citation statements)

References 72 publications

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“…KLK10 mRNA (Figure 1g), KLK10 protein in cell lysates (Figure 1h,i), and secreted protein in the conditioned media (Figure 1j) were decreased by OS and increased by LS, confirming the role of KLK10 as a flow-sensitive gene and protein in vivo and in vitro. We further confirmed the flow-dependent expression of Klk10 by re-analyzing the single-cell RNA sequencing (scRNAseq) and ATAC sequencing (scATACseq) data sets that we recently published using the PCL model 38 . For the scRNAseq and scATACseq study, single-cells and -nuclei obtained from the LCAs and RCAs, respectively, at 2 days or 2 weeks after the PCL were used.…”

Section: Klk10 Expression Is Increased By S-flow and Decreased By D-flow In Ecs In Vitro And In Vivosupporting

confidence: 62%

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

Williams

Mahmoud

Liu

et al. 2022

eLife

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Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo& mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs (HAECs), as determined by NFkB activation, expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), and monocyte adhesion. Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

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Section: Klk10 Expression Is Increased By S-flow and Decreased By D-flow In Ecs In Vitro And In Vivosupporting

confidence: 62%

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

Williams

Mahmoud

Liu

et al. 2022

eLife

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“…We further confirmed the flow-dependent expression of KLK10 by re-analyzing the single-cell RNA sequencing (scRNAseq) and ATAC sequencing (scATACseq) data sets that we recently published using the PCL model 38 . For the scRNAseq and scATACseq study, single-cells and -nuclei obtained from the LCAs and RCAs, respectively, at 2 days or 2 weeks after the PCL were used.…”

Section: Introductionsupporting

confidence: 61%

“…Interestingly, we found that HTRA1 was upregulated by OS in HAECs (Figure 7e,g), indicating HTRA1 itself is shear sensitive. This finding was further validated by qPCR in HAECs (Figure S12a) and by scRNAseq (Figure S13) and scATACseq analyses (Figure S14) in the mouse PCL study 38 . These results show KLK10 and HTRA1 expression are inversely regulated by flow.…”

Section: Htra1 Binds Klk10 Regulating Its Anti-inflammatory and Barrier Protective Functionmentioning

confidence: 59%

“…For the scRNAseq and scATACseq study, single-cells and -nuclei obtained from the LCAs and RCAs, respectively, at 2 days or 2 weeks after the PCL were used. As described previously, the carotid artery wall cells were identified as EC clusters (E1-E8), smooth muscle cells (SMC), fibroblasts (Fibro), monocytes/macrophages (Mo1-4), dendritic cells (DC) and T-cells 38 (Figure 1K and I). E2 and E3 represent athero-protected endothelial populations exposed to s-flow in the RCA, whereas E5 and E7 represent ECs exposed to acute d-flow (for 2 days).…”

Section: Introductionmentioning

confidence: 60%

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Stable Flow-induced Expression of KLK10 Inhibits Endothelial Inflammation and Atherosclerosis

Williams

Mahmoud

Liu

et al. 2021

Preprint

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Introduction: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (dflow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10, a secreted serine protease) as a flow-sensitive gene in arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Methods and Results: Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated KLK10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced inflammation and permeability dysfunction in human artery ECs (HAECs). Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of KLK10- expressing plasmids inhibited atherosclerosis in ApoE-/- mice. Studies using pharmacological inhibitors and siRNAs revealed that the anti-inflammatory effects of KLK10 were mediated by a Protease Activated Receptors (PAR1/2)-dependent manner. However, unexpectedly, KLK10 did not cleave the PARs. Through a proteomics study, we identified HTRA1 (High-temperature requirement A serine peptidase 1), which bound and cleaved KLK10. Further, siRNA knockdown of HTRA1 prevented KLK10's anti-inflammatory and barrier protective function in HAECs, suggesting that HTRA1 regulates KLK10 function. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. Conclusion: KLK10 is a flow-sensitive endothelial protein and, in collaboration with HTRA1, serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

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“…A recent study that used scRNA‐seq and scATAC‐seq to assess the molecular response of BECs to carotid artery ligation revealed reprogramming of enhancer accessibility that was linked to changes in gene expression and cell function. [ 154 ] It will be interesting to determine if lineage‐dependent differences in local chromatin composition impact endothelial function during development and in disease. This functional specialization may include differential control of metabolism, vasorelaxation, angiocrine secretion, disease progression, and organ regeneration.…”

Section: Discussionmentioning

confidence: 99%

Endothelial ontogeny and the establishment of vascular heterogeneity

et al. 2021

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The establishment of distinct cellular identities was pivotal during the evolution of Metazoa, enabling the emergence of an array of specialized tissues with different functions. In most animals including vertebrates, cell specialization occurs in response to a combination of intrinsic (e.g., cellular ontogeny) and extrinsic (e.g., local environment) factors that drive the acquisition of unique characteristics at the single‐cell level. The first functional organ system to form in vertebrates is the cardiovascular system, which is lined by a network of endothelial cells whose organ‐specific characteristics have long been recognized. Recent genetic analyses at the single‐cell level have revealed that heterogeneity exists not only at the organ level but also between neighboring endothelial cells. Thus, how endothelial heterogeneity is established has become a key question in vascular biology. Drawing upon evidence from multiple organ systems, here we will discuss the role that lineage history may play in establishing endothelial heterogeneity.

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Endothelial reprogramming by disturbed flow revealed by single-cell RNA and chromatin accessibility study

Cited by 19 publications

References 72 publications

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

Stable Flow-induced Expression of KLK10 Inhibits Endothelial Inflammation and Atherosclerosis

Endothelial ontogeny and the establishment of vascular heterogeneity

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