Hemodynamic disturbed flow induces differential DNA methylation of endothelial Kruppel-Like Factor 4 promoter in vitro and in vivo.

RATIONALE: Hemodynamic disturbed flow (DF) is associated with susceptibility to atherosclerosis. Endothelial Kruppel-Like Factor 4 (KLF4) is an important anti-inflammatory atheroprotective transcription factor that is suppressed in regions of DF. OBJECTIVE: The plasticity of epigenomic KLF4 transcriptional regulation by flow-mediated DNA methylation was investigated in vitro and in arterial tissue. METHODS AND RESULTS: To recapitulate dominant flow characteristics of atheroprotected and atherosusceptible arteries, human aortic endothelial cells were subjected to pulsatile undisturbed flow or oscillatory DF containing a flow-reversing phase. Differential CpG site methylation was measured by methylation-specific polymerase chain reaction, bisulfite pyrosequencing, and restriction enzyme-polymerase chain reaction. The methylation profiles of endothelium from disturbed and undisturbed flow sites of adult swine aortas were also investigated. In vitro, DF increased DNA methylation of CpG islands within the KLF4 promoter that significantly contributed to suppression of KLF4 transcription; the effects were mitigated by DNA methyltransferase (DNMT) inhibitors and knockdown of DNMT3A. Contributory mechanisms included DF-induced increase of DNMT3A protein (1.7-fold), DNMT3A enrichment (11-fold) on the KLF4 promoter, and competitive blocking of a myocyte enhancer factor-2 binding site in the KLF4 promoter near the transcription start site. DF also induced DNMT-sensitive propathological expression of downstream KLF4 transcription targets nitric oxide synthase 3, thrombomodulin, and monocyte chemoattractant protein-1. In support of the in vitro findings, swine aortic endothelium isolated from DF regions expressed significantly lower KLF4 and nitric oxide synthase 3, and bisulfite sequencing of KLF4 promoter identified a hypermethylated myocyte enhancer factor-2 binding site. CONCLUSIONS: Hemodynamics influence endothelial KLF4 expression through DNMT enrichment/myocyte enhancer factor-2 inhibition mechanisms of KLF4 promoter CpG methylation with regional consequences for atherosusceptibility.

Endothelial epigenetics in biomechanical stress: disturbed flow-mediated epigenomic plasticity in vivo and in vitro.

Arterial endothelial phenotype is regulated by local hemodynamic forces that are linked to regional susceptibility to atherogenesis. A complex hierarchy of transcriptional, translational, and post-translational mechanisms is greatly influenced by the characteristics of local arterial shear stress environments. We discuss the emerging role of localized disturbed blood flow on epigenetic mechanisms of endothelial responses to biomechanical stress, including transcriptional regulation by proximal promoter DNA methylation, and post-transcriptional and translational regulation of gene and protein expression by chromatin remodeling and noncoding RNA-based mechanisms. Dynamic responses to flow characteristics in vivo and in vitro include site-specific differentially methylated regions of swine and mouse endothelial methylomes, histone marks regulating chromatin conformation, microRNAs, and long noncoding RNAs. Flow-mediated epigenomic responses intersect with cis and trans factor regulation to maintain endothelial function in a shear-stressed environment and may contribute to localized endothelial dysfunctions that promote atherosusceptibility.

Arterial endothelial methylome: differential DNA methylation in athero-susceptible disturbed flow regions in vivo.

BACKGROUND: Atherosclerosis is a heterogeneously distributed disease of arteries in which the endothelium plays an important central role. Spatial transcriptome profiling of endothelium in pre-lesional arteries has demonstrated differential phenotypes primed for athero-susceptibility at hemodynamic sites associated with disturbed blood flow. DNA methylation is a powerful epigenetic regulator of endothelial transcription recently associated with flow characteristics. We investigated differential DNA methylation in flow region-specific aortic endothelial cells in vivo in adult domestic male and female swine. RESULTS: Genome-wide DNA methylation was profiled in endothelial cells (EC) isolated from two robust locations of differing patho-susceptibility:--an athero-susceptible site located at the inner curvature of the aortic arch (AA) and an athero-protected region in the descending thoracic (DT) aorta. Complete methylated DNA immunoprecipitation sequencing (MeDIP-seq) identified over 5500 endothelial differentially methylated regions (DMRs). DMR density was significantly enriched in exons and 5'UTR sequences of annotated genes, 60 of which are linked to cardiovascular disease. The set of DMR-associated genes was enriched in transcriptional regulation, pattern specification HOX loci, oxidative stress and the ER stress adaptive pathway, all categories linked to athero-susceptible endothelium. Examination of the relationship between DMR and mRNA in HOXA genes demonstrated a significant inverse relationship between CpG island promoter methylation and gene expression. Methylation-specific PCR (MSP) confirmed differential CpG methylation of HOXA genes, the ER stress gene ATF4, inflammatory regulator microRNA-10a and ARHGAP25 that encodes a negative regulator of Rho GTPases involved in cytoskeleton remodeling. Gender-specific DMRs associated with ciliogenesis that may be linked to defects in cilia development were also identified in AA DMRs. CONCLUSIONS: An endothelial methylome analysis identifies epigenetic DMR characteristics associated with transcriptional regulation in regions of atherosusceptibility in swine aorta in vivo. The data represent the first methylome blueprint for spatio-temporal analyses of lesion susceptibility predisposing to endothelial dysfunction in complex flow environments in vivo.

Biomechanical factors in atherosclerosis: mechanisms and clinical implications.

Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of atherosclerotic lesions, which develop at regions of arteries that are exposed to complex blood flow. In addition, plaque progression and eventually plaque rupture is influenced by a complex interaction between biological and mechanical factors-mechanical forces regulate the cellular and molecular composition of plaques and, conversely, the composition of plaques determines their ability to withstand mechanical load. A deeper understanding of these interactions is essential for designing new therapeutic strategies to prevent lesion development and promote plaque stabilization. Moreover, integrating clinical imaging techniques with finite element modelling techniques allows for detailed examination of local morphological and biomechanical characteristics of atherosclerotic lesions that may be of help in prediction of future events. In this ESC Position Paper on biomechanical factors in atherosclerosis, we summarize the current 'state of the art' on the interface between mechanical forces and atherosclerotic plaque biology and identify potential clinical applications and key questions for future research.

Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium.

OBJECTIVE: Endothelial transcription factors Krüppel-like factor 4 (KLF4) and KLF2 are implicated in protection against atherogenesis. Steady-state microRNA (miR) regulation of KLFs in vivo is accessible by screening region-specific endothelial miRs and their targets. METHODS AND RESULTS: A subset of differentially expressed endothelial miRs was identified in atherosusceptible versus protected regions of normal swine aorta. In silico analyses predicted highly conserved binding sites in the 3'-untranslated region (3'UTR) of KLF4 for 5 miRs of the subset (miR-26a, -26b, -29a, -92a, and -103) and a single binding site for a miR-92a complex in the 3'UTR of KLF2. Of these, only miR-92a knockdown and knock-in resulted in responses of KLF4 and KLF2 expression in human arterial endothelial cells. Dual luciferase reporter assays demonstrated functional interactions of miR-92a with full-length 3'UTR sequences of both KLFs and with the specific binding elements therein. Two evolutionarily conserved miR-92a sites in KLF4 3'UTR and 1 site in KLF2 3'UTR were functionally validated. Knockdown of miR-92a in vitro resulted in partial rescue from cytokine-induced proinflammatory marker expression (monocyte chemotactic protein 1, vascular cell adhesion molecule-1, E-selectin, and endothelial nitric oxide synthase) that was attributable to enhanced KLF4 expression. Leukocyte-human arterial endothelial cell adhesion experiments supported this conclusion. In swine aortic arch endothelium, a site of atherosusceptibility where miR-92a expression was elevated, both KLFs were expressed at low levels relative to protected thoracic aorta. CONCLUSIONS: miR-92a coregulates KLF4 and KLF2 expression in arterial endothelium and contributes to phenotype heterogeneity associated with regional atherosusceptibility and protection in vivo.

MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro.

A chronic proinflammatory state precedes pathological change in arterial endothelial cells located within regions of susceptibility to atherosclerosis. The potential contributions of regulatory microRNAs to this disequilibrium were investigated by artery site-specific profiling in normal adult swine. Expression of endothelial microRNA10a (miR-10a) was lower in the athero-susceptible regions of the inner aortic arch and aorto-renal branches than elsewhere. Expression of Homeobox A1 (HOXA1), a known miR-10a target, was up-regulated in the same locations. Endothelial transcriptome microarray analysis of miR-10a knockdown in cultured human aortic endothelial cells (HAEC) identified IkappaB/NF-kappaB-mediated inflammation as the top category of up-regulated biological processes. Phosphorylation of IkappaBalpha, a prerequisite for IkappaBalpha proteolysis and NF-kappaB activation, was significantly up-regulated in miR-10a knockdown HAEC and was accompanied by increased nuclear expression of NF-kappaB p65. The inflammatory biomarkers monocyte chemotactic protein 1 (MCP-1), IL-6, IL-8, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin were elevated following miR-10a knockdown. Conversely, knockin of miR-10a (a conservative 25-fold increase) inhibited the basal expression of VCAM-1 and E-selectin in HAEC. Two key regulators of IkappaBalpha degradation--mitogen-activated kinase kinase kinase 7 (MAP3K7; TAK1) and beta-transducin repeat-containing gene (betaTRC)--contain a highly conserved miR-10a binding site in the 3' UTR. Both molecules were up-regulated by miR-10a knockdown and suppressed by miR-10a knockin, and evidence of direct miR-10a binding to the 3' UTR was demonstrated by luciferase assay. Comparative expression studies of endothelium located in athero-susceptible aortic arch and athero-protected descending thoracic aorta identified significantly up-regulated MAP3K7, betaTRC, phopho-IkappaBalpha, and nuclear p65 expression suggesting that the differential expression of miR-10a contributes to the regulation of proinflammatory endothelial phenotypes in athero-susceptible regions in vivo.

Chronic endoplasmic reticulum stress activates unfolded protein response in arterial endothelium in regions of susceptibility to atherosclerosis.

RATIONALE: Endothelial function and dysfunction are central to the focal origin and regional development of atherosclerosis; however, an in vivo endothelial phenotypic footprint of susceptibility to atherosclerosis preceding pathological change remains elusive. OBJECTIVE: To conduct a comparative multi-site genomics study of arterial endothelial phenotype in atherosusceptible and atheroprotected regions. METHODS AND RESULTS: Transcript profiles of freshly isolated endothelial cells from 7 discrete arterial regions in normal swine were analyzed to determine the steady state in vivo endothelial phenotypes in regions of varying susceptibilities to atherosclerosis. The most abundant common feature of the endothelium of all atherosusceptible regions was the upregulation of genes associated with endoplasmic reticulum (ER) stress. The unfolded protein response pathway, induced by ER stress, was therefore investigated in detail in endothelium of the atherosusceptible aortic arch and was found to be partially activated. ER transmembrane signal transducers IRE1alpha and ATF6alpha and their downstream effectors, but not PERK, were activated concomitant with a higher transcript expression of protein folding enzymes and chaperones, indicative of ER stress in vivo. CONCLUSIONS: The findings demonstrate the prevalence of chronic endothelial ER stress and activated unfolded protein response in vivo at atherosusceptible arterial sites. We propose that chronic localized biological stress is linked to spatial susceptibility of the endothelium to the initiation of atherosclerosis.

Hypercholesterolemia induces side-specific phenotypic changes and peroxisome proliferator-activated receptor-gamma pathway activation in swine aortic valve endothelium.

UNLABELLED: Background- The endothelium of healthy aortic valves expresses different phenotypes on the aortic and ventricular sides. On the aortic side, which is susceptible to aortic valve sclerosis, there is a balanced coexpression of both propathological and protective pathways. Side-specific global gene expression can address endothelial phenotype balance in early aortic valve sclerosis. METHODS AND RESULTS: Adult male swine were fed a hypercholesterolemic or an isocaloric normal diet for 2-week and 6-month periods. Hypercholesterolemia induced localized lipid insudation confined to the aortic side of the leaflet. Transcript profiling of valve endothelial populations showed that the susceptible aortic side was more sensitive to 2-week hypercholesterolemia than the ventricular side (1,325 vs 87 genes were differentially expressed). However, greater sensitivity was not evidence of a dysfunctional phenotype. Instead, pathway analyses identified differential expression of caspase 3-, peroxisome proliferator-activated receptor gamma-, TNF-alpha-, and nuclear factor-kappaB-related pathways that were consistent with a protective endothelial phenotype. This was confirmed at the protein level at 2 weeks and persisted at 6 months. CONCLUSIONS: In a large animal model at high spatial resolution, endothelium on the pathosusceptible side of the aortic valve leaflet is responsive to hypercholesterolemia. Transcript profiles indicative of a protective phenotype were induced and persisted on the side prone to aortic valve sclerosis.

Side-specific expression of activated leukocyte adhesion molecule (ALCAM; CD166) in pathosusceptible regions of swine aortic valve endothelium.

Aortic valve sclerosis (AVS), an early form of aortic valve disease, develops preferentially on the aortic side of valve leaflets, a predilection that is reflected in an heterogeneous side-specific gene expression profile. It has been ascertained that hypercholesterolemia is sufficient to initiate the endothelial expression of activated leukocyte adhesion molecule (ALCAM; CD166), restricted to the aortic side of the leaflet. Intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1)--both of which are more typically associated with early arterial inflammation--are not differentially expressed. ALCAM up-regulation by hypercholesterolemia suggests a side-specific spatial role in the recruitment of leukocytes to AVS sites.

Prelesional arterial endothelial phenotypes in hypercholesterolemia: universal ABCA1 upregulation contrasts with region-specific gene expression in vivo.

Atherosclerosis originates as focal arterial lesions having a predictable distribution to regions of bifurcations, branches, and inner curvatures where blood flow characteristics are complex. Distinct endothelial phenotypes correlate with regional hemodynamics. We propose that systemic risk factors modify regional endothelial phenotype to influence focal susceptibility to atherosclerosis. Transcript profiles of freshly isolated endothelial cells from three atherosusceptible and three atheroprotected arterial regions in adult swine were analyzed to determine the initial prelesional effects of hypercholesterolemia on endothelial phenotypes in vivo. Cholesterol efflux transporter ATP-binding cassette transporter A1 (ABCA1) was upregulated at all sites in response to short-term high-fat diet. Proinflammatory and antioxidative endothelial gene expression profiles were induced in atherosusceptible and atheroprotected regions, respectively. However, markers for endoplasmic reticulum stress, a signature of susceptible endothelial phenotype, were not further enhanced by brief hypercholesterolemia. Both region-specific and ubiquitous (ABCA1) phenotype changes were identified as early prelesional responses of the endothelium to hypercholesterolemia.

Endothelial heterogeneity associated with regional athero-susceptibility and adaptation to disturbed blood flow in vivo.

Endothelial phenotypic heterogeneity plays an important role in the susceptibility of the cardiovascular system to disease. Arteries and heart valves are susceptible to chronic inflammatory disease in regions of blood flow disturbance that implicates hemodynamic forces and transport characteristics as prominent influences on endothelial phenotype. By combining in vivo high-throughput genomics (discovery science) and in vitro mechanistic approaches (reductionist science), we present endothelial patho-susceptibility as an imbalance of multiple interrelated pathways that sensitize the cells to pathological change. The recently identified association of endoplasmic reticulum stress with endothelium in regions of flow disturbance is outlined as an important example of susceptible phenotype linked to proinflammatory and oxidative stress pathways.

Hypercholesterolemia suppresses inwardly rectifying K+ channels in aortic endothelium in vitro and in vivo.

Inwardly rectifying K+ (Kir) channels are responsible for maintaining endothelial membrane potential and play a key role in endothelium-dependent vasorelaxation. In this study, we show that endothelial Kir channels are suppressed by hypercholesterolemic levels of lipoproteins in vitro and by serum hypercholesterolemia in vivo. Specifically, exposing human aortic endothelial cells to acetylated low-density lipoprotein or very low density lipoprotein resulted in a time- and concentration-dependent decrease in Kir current that correlated with the degree of cholesterol loading. The suppression was fully reversible by cholesterol depletion. Furthermore, a decrease in Kir current resulted in depolarization of endothelial membrane potential. Most important, the flow sensitivity of Kir currents was also impaired by cholesterol loading. Specifically, flow-induced increase in Kir current was suppressed by 70%, and flow-induced hyperpolarization was almost completely abrogated. Furthermore, we show that hypercholesterolemia in vivo also strongly suppresses endothelial Kir currents and causes a shift in endothelial membrane potential, as determined by comparing the currents in aortic endothelial cells freshly isolated from healthy or hypercholesterolemic pigs. Therefore, we suggest that suppression of Kir current is one of the important factors in hypercholesterolemia-induced endothelial dysfunction.

Exploring human/animal intersections: converging lines of evidence in comparative models of aging.

At a symposium convened on March 8, 2007 by the Institute on Aging at the University of Pennsylvania, researchers from the University's Schools of Medicine and Veterinary Medicine explored the convergence of aging research emerging from the two schools. Studies in human patients, animal models, and companion animals have revealed different but complementary aspects of the aging process, ranging from fundamental biologic aspects of aging to the treatment of age-related diseases, both experimentally and in clinical practice. Participants concluded that neither animal nor human research alone will provide answers to most questions about the aging process. Instead, an optimal translational research model supports a bidirectional flow of information from animal models to clinical research.

Endothelial protein kinase C isoform identity and differential activity of PKCzeta in an athero-susceptible region of porcine aorta.

Endothelial protein kinase C (PKC) signaling was investigated in different regions of normal porcine aorta. The locations map to differential atherosclerotic susceptibility and correlate with sites of disturbed (DF) or undisturbed (UF) local flow profiles. Endothelial lysates were isolated from the inner curvature of the aortic arch (DF; athero-susceptible) and a nearby UF region of the descending thoracic aorta (UF; athero-protected), and in some experiments a distant athero-protected UF site, the common carotid artery. Total endothelial PKC activity in the DF regions was 145% to 240% of that in both UF locations (P<0.05), whereas the UF regions were not significantly different from each other. PKC protein isoforms alpha, beta, epsilon, iota, lambda, and zeta were expressed in similar proportions in both aortic regions, suggesting that differences of kinase activity were not directly attributable to expression levels. Inhibition of members of the "conventional" and "novel" PKC families had no differential effect on regional kinase activity. However, inhibition of PKCzeta, a member of the "atypical" PKC family, reduced the DF lysate kinase activity to that of UF levels (NS P=0.35). Differential phosphorylation of PKCzeta Thr410 and Thr560, along with increased levels of PKCzeta degradation products in UF endothelial lysates, suggested posttranslational modification of PKCzeta as the basis for site-specific differences in vivo. Steady-state regional heterogeneity of an important family of regulatory proteins in intact arterial endothelium in vivo may link localized athero-susceptibility and the associated hemodynamic environment.

Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.

Calcific aortic valve sclerosis involves inflammatory processes and occurs preferentially on the aortic side of endothelialized valve leaflets. Although the endothelium is recognized to play critical roles in focal vascular sclerosis, the contributions of valvular endothelial phenotypes to aortic valve sclerosis and side-specific susceptibility to calcification are poorly understood. Using RNA amplification and cDNA microarrays, we identified 584 genes as differentially expressed in situ by the endothelium on the aortic side versus ventricular side of normal adult pig aortic valves. These differential transcriptional profiles, representative of the steady state in vivo, identify globally distinct endothelial phenotypes on opposite sides of the aortic valve. Several over-represented biological classifications with putative relevance to endothelial regulation of valvular homeostasis and aortic-side vulnerability to calcification were identified among the differentially expressed genes. Of note, multiple inhibitors of cardiovascular calcification were significantly less expressed by endothelium on the disease-prone aortic side of the valve, suggesting side-specific permissiveness to calcification. However, coexisting putative protective mechanisms were also expressed. Specifically, enhanced antioxidative gene expression and the lack of differential expression of proinflammatory molecules on the aortic side may protect against inflammation and lesion initiation in the normal valve. These data implicate the endothelium in regulating valvular calcification and suggest that spatial heterogeneity of valvular endothelial phenotypes may contribute to the focal susceptibility for lesion development.

Biofluids, cell mechanics and epigenetics: Flow-induced epigenetic mechanisms of endothelial gene expression.

Epigenetics is the regulation of gene expression (transcription) in response to changes in the cell environment through genomic modifications that largely involve the non-coding fraction of the human genome and that cannot be attributed to modification of the primary DNA sequence. Epigenetics is dominant in establishing cell fate and positioning during programmed embryonic development. However the same pathways are used by mature postnatal and adult mammalian cells during normal physiology and are implicated in disease mechanisms. Recent research demonstrates that blood flow and pressure are cell environments that can influence transcription via epigenetic pathways. The principal epigenetic pathways are chemical modification of cytosine residues of DNA (DNA methylation) and of the amino tails of histone proteins associated with DNA in nucleosomes. They also encompass the post-transcriptional degradation of mRNA transcripts by non-coding RNAs (ncRNA). In vascular endothelium, epigenetic pathways respond to temporal and spatial variations of flow and pressure, particularly hemodynamic disturbed blood flow, with important consequences for gene expression. The biofluid environment is linked by mechanotransduction and solute transport to cardiovascular cell phenotypes via signaling pathways and epigenetic regulation for which there is an adequate interdisciplinary infrastructure with robust tools and methods available. Epigenetic mechanisms may be less familiar than acute genomic signaling to Investigators at the interface of biofluids, biomechanics and cardiovascular biology. Here we introduce a biofluids / cellular biomechanics readership to the principal epigenetic pathways and provide a contextual overview of endothelial epigenetic plasticity in the regulation of flow-responsive transcription.

Spatial phenotyping of the endocardial endothelium as a function of intracardiac hemodynamic shear stress.

Despite substantial evidence for the central role of hemodynamic shear stress in the functional integrity of vascular endothelial cells, hemodynamic and molecular regulation of the endocardial endothelium lining the heart chambers remains understudied. We propose that regional differences in intracardiac hemodynamics influence differential endocardial gene expression leading to phenotypic heterogeneity of this cell layer. Measurement of intracardiac hemodynamics was performed using 4-dimensional flow MRI in healthy humans (n=8) and pigs (n=5). Local wall shear stress (WSS) and oscillatory shear indices (OSI) were calculated in three distinct regions of the LV - base, mid-ventricle (midV), and apex. In both the humans and pigs, WSS values were significantly lower in the apex and midV relative to the base. Additionally, both the apex and midV had greater oscillatory shear indices (OSI) than the base. To investigate regional phenotype, endocardial endothelial cells (EEC) were isolated from an additional 8 pigs and RNA sequencing was performed. A false discovery rate of 0.10 identified 1051 differentially expressed genes between the base and apex, and 321 between base and midV. Pathway analyses revealed apical upregulation of genes associated with translation initiation. Furthermore, tissue factor pathway inhibitor (TFPI; mean 50-fold) and prostacyclin synthase (PTGIS; 5-fold), genes prominently associated with antithrombotic protection, were consistently upregulated in LV apex. These spatio-temporal WSS values in defined regions of the left ventricle link local hemodynamics to regional heterogeneity in endocardial gene expression.

Spatial microstimuli in endothelial mechanosignaling.

Descriptive and quantitative analyses of microstimuli in living endothelial cells strongly support an integrated mechanism of mechanotransduction regulated by the spatial organization of multiple structural and signaling networks. Endothelial responses to blood flow are regulated at multiple levels of organization extending over scales from vascular beds to single cells, subcellular structures, and individual molecules. Microstimuli at the cellular and subcellular levels exhibit temporal and spatial complexities that are increasingly accessible to measurement. We address the cell and subcellular physical interface between flow-related forces and biomechanical responses of the endothelial cell. Live cell imaging and computational analyses of structural dynamics, two important approaches to microstimulation at this scale, are briefly reviewed.

Integrated Regional Cardiac Hemodynamic Imaging and RNA Sequencing Reveal Corresponding Heterogeneity of Ventricular Wall Shear Stress and Endocardial Transcriptome.

BACKGROUND: Unlike arteries, in which regionally distinct hemodynamics are associated with phenotypic heterogeneity, the relationships between endocardial endothelial cell phenotype and intraventricular flow remain largely unexplored. We investigated regional differences in left ventricular wall shear stress and their association with endocardial endothelial cell gene expression. METHODS AND RESULTS: Local wall shear stress was calculated from 4-dimensional flow magnetic resonance imaging in 3 distinct regions of human (n=8) and pig (n=5) left ventricle: base, adjacent to the outflow tract; midventricle; and apex. In both species, wall shear stress values were significantly lower in the apex and midventricle relative to the base; oscillatory shear index was elevated in the apex. RNA sequencing of the endocardial endothelial cell transcriptome in pig left ventricle (n=8) at a false discovery rate ≤10% identified 1051 genes differentially expressed between the base and the apex and 327 between the base and the midventricle; no differentially expressed genes were detected at this false discovery rate between the apex and the midventricle. Enrichment analyses identified apical upregulation of genes associated with translation initiation including mammalian target of rapamycin, and eukaryotic initiation factor 2 signaling. Genes of mitochondrial dysfunction and oxidative phosphorylation were also consistently upregulated in the left ventricular apex, as were tissue factor pathway inhibitor (mean 50-fold) and prostacyclin synthase (5-fold)-genes prominently associated with antithrombotic protection. CONCLUSIONS: We report the first spatiotemporal measurements of wall shear stress within the left ventricle and linked regional hemodynamics to heterogeneity in ventricular endothelial gene expression, most notably to translation initiation and anticoagulation properties in the left ventricular apex, in which oscillatory shear index is increased and wall shear stress is decreased.