biostudies-arrayexpressShailaja SeetharamanHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16095Our goal was to identify differentially expressed genes in control endothelial cells subjected to varying mechanical forces (flow).biostudies-arrayexpressNucleic Acid Extraction - TeloHAECs subjected to UF and DF were lysed, and total RNA was collected using a NucleoSpin RNA kit (Macherey-Nagel, #740955).Sequencing - Libraries were then sequenced using Illumina NovaSEQ6000 (illumina provided reagents and protocols) with ~60M PE reads/sample.Growth Protocol - TeloHAECs are grown in EGM-2 growth media (Lonza).Sample Collection - Cells were cultured in 6-well plates with EGM-2 medium supplemented with 4% dextran (Sigma-Aldrich, St. Louis, MO, 31392). Hemodynamic flows were applied to using a 1o tapered stainless steel motorised cone (UMD-17 (Arcus20 Technology, Livermore CA). The rotation of the cone captures healthy unidirectional flow (UF) seen in athero-protective distal internal carotid artery, while the disturbed flow (DF) mimics athero-susceptible human carotid artery. During flow application to cells, the flow device was placed at 37C with 5%CO2. TeloHAECs subjected to UF and DF were lysed and RNA was extracted as mentioned below. Two technical replicates for each condition were pooled together to obtain an experimental replicate.Library Construction - Using a TruSEQ mRNA RNA-SEQ library preparation protocol (Illumina provided), strand-specific RNA-SEQ libraries were prepared.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - The reads were aligned to the Homo Sapiens genome hg19/GCh37 reference by psuedoalignment using Kallisto 0.46.1.Data Transformation - DESeq2 (version 1.40.2) in R software (version 4.3.1) was used to normalize read counts per transcript and perform differential gene expression analysis.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Endothelial tissues are essential mechanosensors in the vasculature and facilitate adaptation to various blood flow-induced mechanical cues. Defects in endothelial mechanoresponses can perturb tissue remodelling and functions leading to cardiovascular disease progression. In this context, the precise mechanisms of endothelial mechanoresponses contributing to normal and diseased tissue functioning remain elusive. Here, we sought to uncover how flow-mediated transcriptional regulation drives endothelial mechanoresponses in healthy and atherosclerotic-prone tissues. Using bulk RNA sequencing, we identify novel mechanosensitive genes in response to healthy unidirectional flow (UF) and athero-prone disturbed flow (DF). We find that the transcription as well as protein expression of Four-and-a-half LIM protein 2 (FHL2) are enriched in athero-prone DF both <i>in vitro</i> and <i>in vivo</i>. We then demonstrate that the exogenous expression of FHL2 is necessary and sufficient to drive discontinuous adherens junction morphology and increased tissue permeability. This athero-prone phenotype requires the force-sensitive binding of FHL2 to actin. In turn, the force-dependent localisation of FHL2 to stress fibres promotes microtubule dynamics to release the RhoGEF, GEF-H1, and activate the Rho-ROCK pathway. Thus, we unravelled a novel mechanochemical feedback wherein force-dependent FHL2 localisation promotes hypercontractility. This misregulated mechanoresponse creates highly permeable tissues, depicting classic hallmarks of atherosclerosis progression. Overall, we highlight crucial functions for the FHL2 force-sensitivity in tuning multi-scale endothelial mechanoresponses.RNA-seq of coding RNAHomo sapiensMechanosensitive FHL2 tunes endothelial functionShailaja SeetharamanfalseRNAseq of human aortic endothelial cells (TeloHAECs) under unidirectional flow (UF) and disturbed flow (DF).Our goal was to identify differentially expressed genes in control endothelial cells subjected to varying mechanical forces (flow).2026-04-10T00:00:00Z2026-04-10T01:02:14.155Z2025-11-13T15:36:58.04ZE-MTAB-1609538948838ERP183896EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_000418410.1101/2024.06.16.599227