Blood flow and vascular shear stress patterns play a significant role in inducing and modulating physiological responses of endothelial cells (ECs). Pulsatile shear (PS) is associated with an atheroprotective endothelial phenotype, while oscillatory shear (OS) is associated with an atheroprone endothelial phenotype. Although mechanisms of endothelial shear response have been extensively studied, most studies focus on characterization of single molecular pathways, mainly at fixed time points after stress application. Here, we carried out a longitudinal time-series study to measure the transcriptome after the application of PS and OS. We performed systems analyses of transcriptional data of cultured human vascular ECs to elucidate the dynamics of endothelial responses in several functional pathways such as cell cycle, oxidative stress, and inflammation. By combining the temporal data on differentially expressed transcription factors and their targets with existing knowledge on relevant functional pathways, we infer the causal relationships between disparate endothelial functions through common transcriptional regulation mechanisms. Our study presents a comprehensive temporally longitudinal experimental study and mechanistic model of shear stress response. By comparing the relative endothelial expressions of genes between OS and PS, we provide insights and an integrated perspective into EC function in response to differential shear. This study has significant implications for the pathogenesis of vascular diseases.

The two main blood flow patterns, namely, pulsatile shear (PS) prevalent in straight segments of arteries and oscillatory shear (OS) observed at branch points, are associated with atheroprotective (healthy) and atheroprone (unhealthy) vascular phenotypes, respectively. The effects of blood flow-induced shear stress on endothelial cells (ECs) and vascular health have generally been studied using human umbilical vein endothelial cells (HUVECs). While there are a few studies comparing the differential roles of PS and OS across different types of ECs at a single time point, there is a paucity of studies comparing the temporal responses between different EC types. In the current study, we measured OS and PS transcriptomic responses in human aortic endothelial cells (HAECs) over 24 h and compared these temporal responses of HAECs with our previous findings on HUVECs. The measurements were made at 1, 4, and 24 h in order to capture the responses at early, mid, and late time points after shearing. The results indicate that the responses of HAECs and HUVECs are qualitatively similar for endothelial function-relevant genes and several important pathways with a few exceptions, thus demonstrating that HUVECs can be used as a model to investigate the effects of shear on arterial ECs, with consideration of the differences. Our findings show that HAECs exhibit an earlier response or faster kinetics as compared to HUVECs. The comparative analysis of HAECs and HUVECs presented here offers insights into the mechanisms of common and disparate shear stress responses across these two major endothelial cell types.

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -β, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor β2 (TGFβ2) secretion and cleavage. TGFβ2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFβ2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.