Effects of Disturbed Flow on Vascular Endothelium: Pathophysiological Basis and Clinical Perspectives

See related article, pages 97–105 The arterial endothelium survives remarkably well as the interface between blood and vessel wall in an environment of constantly changing biomechanical stresses as well as acute and chronic exposure to inflammatory stimulants (eg, cytokines and hypercholesterolemia respectively).1 Cell turnover, which tends to occur in regional clusters,2 is otherwise very low in this monolayer. The endothelium also plays an important regulatory role in the pathogenesis of vascular disease. The cells readily respond to diverse stimuli through a repertoire of mechanisms to enhance their own survival even as they facilitate inflammatory, proatherogenic responses in the subendothelial tissue. The necessity to be a responsive cellular interface probably accounts for much of the endothelial phenotype heterogeneity that exists between vascular beds as well as within discrete regions of the arterial circulation.3,4 Hemodynamic characteristics that vary with blood vessel geometry predict the location of arterial sites that are susceptible to atherosclerosis.5 Curved and branching vessel geometries create sites of flow separation that contain transient flow reversals, lower average shear stresses, and occasional turbulence, (collectively, disturbed flow) and that are predictive of lesion formation. In contrast, pulsatile unidirectional laminar flow (and higher average shear stresses) is associated with regions where atherosclerosis rarely occurs, despite there being equivalent exposure to plasma risk factors such as hypercholesterolemia throughout the circulation. Although the signatures of endothelial phenotype in such regions in vivo are varied and complex, data are emerging from genomic5–7 and protein8 analyses of endothelium at such sites that identify molecular differences. Some of these are accessible for study in vitro to investigate detailed mechanisms under more controlled conditions. …

Aortic valve diseases are debilitating cardiovascular disorders associated with significant morbidity and mortality. Although there continue to be major efforts to improve the longevity of replacement valves and to improve tissue engineered substitutes,1 the underlying mechanisms that may be responsible for the initiation and development of valve pathology have received less attention than have other sclerosing cardiovascular diseases such as atherogenesis. The endothelium lining of the cardiovascular system plays an important regulatory role in vascular physiology and pathology. In similar fashion, the surfaces of valve leaflets are presumed to be generally protected (eg, anticoagulant) and regulated (eg, permeability) by the endothelium. The functional properties of endothelium or its presence/absence are associated with a variety of valve pathologies,2 and systemic endothelial dysfunction is linked to aortic valve calcification.3 However, only recently have cell and molecular studies focused on the characterization of valve endothelial phenotypes with the idea that some aspects of phenotypic change or dysfunction may contribute to valve pathologies, a situation analogous to atherogenesis. See page 1429 During the cardiac cycle, the aortic valve endothelium is subjected to complex fluid dynamics that are distinctly different on each side of the valve. As has been described for many years, arterial endothelial alignment in vivo generally follows the measured or predicted shear stress direction,4 and endothelial cells in vitro align with the dominant direction of the applied shear stress.5 The responses, which are reversible,4,6 represent endothelial structural remodeling in response to hemodynamic shear stress. It might be expected that endothelial cells isolated from aortic valves and grown in tissue culture will behave in a similar manner as arterial endothelium. However, in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Butcher et al7 demonstrate that aortic valve endothelium …

Aging is the foremost risk factor for metabolic syndrome and atherosclerosis, which is a principal cause of cardiovascular diseases (CVDs). Vascular endothelial cells (ECs), which line the vascular intima, play a central role in maintaining vascular homeostasis. Their dysfunction, marked by impaired barrier function, inflammation, and metabolic dysregulation, constitutes an early and pivotal event in atherogenesis. As key sensors of hemodynamic forces, ECs are constantly exposed to blood flow-induced shear stress, which exert divergent effects on metabolism depending on the flow pattern. Laminar flow with relatively high shear stress (LS), as a critical atheroprotective factor, maintains EC quiescence and promotes anti-inflammatory responses and antioxidant defense, whereas disturbed flow with low and oscillatory shear stress (OS), induces the athero-susceptible signaling network to activate glycolysis and inflammation in ECs. While genetic, epigenetic, and molecular signaling mechanisms in EC physiology and pathophysiology have been extensively explored, the crucial role of EC metabolism in EC dysfunction and atherogenesis remains largely understudied. By serving as precursors, intermediates, and end products of cellular processes, metabolites offer a dynamic snapshot of endothelial metabolic states under both physiological and pathophysiological conditions. With aging, ECs undergo profound metabolic reprogramming, including disrupted glycolysis, mitochondrial dysfunction, and altered redox homeostasis. In healthy vasculature, ECs maintain quiescence and metabolic homeostasis, primarily relying on glycolysis for energy. With aging, the gradual accumulation of atherosclerotic risk factors, including oxidative stress, inflammation, dyslipidemia, and hyperglycemia, drives metabolic reprogramming in ECs, particularly in regions exposed to disturbed flow with OS, ultimately leading to EC dysfunction and atherosclerosis. This review summarizes recent advances in age-related metabolic reprogramming in ECs and its contribution to atherosclerosis, particularly focusing on the dysregulation of glycolysis, fatty acid metabolism, amino acid metabolism, and mitochondrial respiration induced by age and fluid shear stress. This review also outlines recent methodologies for profiling EC metabolism, and discusses potential therapeutic applications of targeting EC metabolism to prevent or delay the development of atherosclerosis.

Endothelial cells respond to forces generated by laminar blood flow with changes in vasodilation, anticoagulant, fibrinolytic, or anti-inflammatory functions which preserve vessel patency. These responses to flow shear stress are primarily mediated by the modulation of the following transcription factors: Krüppel-like factors 2 and 4 (KLF2 and KLF4). Notably, disturbed flow patterns, which are found in vascular areas predisposed to atherosclerosis, significantly reduce the endothelial expression of KLF2 and KLF4, resulting in changes in the transcriptome that exacerbate inflammation and thrombosis. The endothelial CCM (Cerebral Cavernous Malformation) complex, comprising KRIT1 (Krev1 interaction trapped gene 1), CCM2 (Malcavernin), and CCM3 (Programmed cell death protein 10), suppresses the expression of KLF2 and KLF4. Loss of function of the CCM complex has recently been suggested to protect from coronary atherosclerosis in humans. We thus hypothesized that the silencing of KRIT1, the central scaffold of the CCM complex, can normalize the atherogenic effects of disturbed flow on the human endothelial transcriptome. Bulk RNA sequencing (RNA-seq) was conducted on human umbilical vein endothelial cells (HUVECs) after the expression of KRIT1 was silenced using specific small interfering RNA (siRNA). The endothelial cells were exposed to three different conditions for 24 h, as follows: pulsatile shear stress (laminar flow), oscillatory shear stress (disturbed flow), and static conditions (no flow). We found that silencing the KRIT1 expression in HUVECs restored the expression of the transcription factors KLF2 and KLF4 under oscillatory shear stress. This treatment resulted in a transcriptomic profile similar to that of endothelial cells under pulsatile shear stress. These findings suggest that inhibition of the CCM complex in endothelium plays a vasoprotective role by reactivating a protective gene program to help endothelial cells resist disturbed blood flow. Targeting CCM genes can activate well-known vasoprotective gene programs that enhance endothelial resilience to inflammation, hypoxia, and angiogenesis under disturbed flow conditions, providing a novel pathway for preventing atherothrombosis.

Endothelial cell (EC)-specific Ctgf/Ccn2 expression increases EC reprogramming and atherosclerosis.

Endothelial cells respond to forces generated by laminar blood flow with changes in vasodilation, anticoagulant, fibrinolytic, or anti-inflammatory functions which preserve vessel patency. These responses to flow sheer stress are primarily mediated by the modulation of transcription factors Krüppel-like factors 2 and 4 (KLF2 and KLF4). Notably, disturbed flow patterns, which are found in vascular areas predisposed to atherosclerosis, significantly reduce the endothelial expression of KLF2 and KLF4, resulting in changes in the transcriptome that exacerbate inflammation and thrombosis. The endothelial CCM complex, comprising KRIT1, CCM2, and CCM3, suppresses the expression of KLF2 and KLF4. Loss of function of the CCM complex has recently been suggested to protect from coronary atherosclerosis in humans. We thus hypothesized that silencing of KRIT1, the central scaffold of the CCM complex, can normalize the atherogenic effects of disturbed flow on the human endothelial transcriptome. Bulk RNA sequencing (RNA-seq) was conducted on human umbilical vein endothelial cells (HUVECs) after the expression of KRIT1 was silenced using specific siRNAs. The endothelial cells were exposed to three different conditions for 24 hours: pulsatile shear stress (laminar flow), oscillatory shear stress (disturbed flow), and static conditions (no flow). We found that silencing KRIT1 expression in HUVECs restored the expression of the transcription factors KLF2 and KLF4 under oscillatory shear stress. This treatment resulted in a transcriptomic profile similar to that of endothelial cells under pulsatile shear stress. These findings suggest that inhibition of the CCM complex in endothelium plays a vasoprotective role by reactivating a protective gene program to help endothelial cells resist disturbed blood flow. Targeting CCM genes can activate well-known vasoprotective gene programs that enhance endothelial resilience to inflammation, hypoxia, and angiogenesis under disturbed flow conditions, providing a novel pathway for preventing atherosclerosis.

Disturbed flow is one of the pathological initiators of endothelial dysfunction in intimal hyperplasia (IH) which is commonly seen in vascular bypass grafts, and arteriovenous fistulas. Various in vitro disease models have been designed to simulate the hemodynamic conditions found in the vasculature. Nonetheless, prior investigations have encountered challenges in establishing a robust disturbed flow model, primarily attributed to the complex bifurcated geometries and distinctive flow dynamics. In the present study, we aim to address this gap by introducing an in vitro bypass flow model capable of inducing disturbed flow and other hemodynamics patterns through a pulsatile flow in the same model. To assess the model's validity, we employed computational fluid dynamics (CFD) to simulate hemodynamics and compared the morphology and functions of human umbilical venous endothelial cells (HUVECs) under disturbed flow conditions to those in physiological flow or stagnant conditions. CFD analysis revealed the generation of disturbed flow within the model, pinpointing the specific location in the channel where the effects of disturbed flow were observed.High-content screening, a single-cell morphological profile assessment, demonstrated that HUVECs in the disturbed flow area exhibited random orientation, and morphological features were significantly distinct compared to cells in the physiological flow or stagnant condition after a two days of flow exposure. Furthermore, HUVECs exposed to disturbed flow underwent extensive remodeling of the adherens junctions and expressed higher levels of endothelial cell activation markers compared to other hemodynamic conditions.In conclusion, our in vitro bypass flow model provides a robust platform for investigating the associations between disturbed flow pattern and vascular diseases.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a complex adhesion and signaling molecule expressed by endothelial cells, platelets, and leukocytes.1,2 On endothelial cells this transmembrane glycoprotein member of the immunoglobulin gene superfamily is concentrated at intercellular junctions and cycles through vesicle-like structures contiguous with the lateral plasma membrane, termed the lateral border recycling compartment.3 Homophilic adhesive interactions between PECAM-1 on leukocytes and endothelial cells mediate leukocyte migration through endothelial cell monolayers (diapedesis) in vitro and in vivo and through the perivascular basement membrane.2,4 As a signaling molecule, PECAM-1 transduces signals required for proinflammatory adhesion molecule expression by endothelial cells. However, PECAM-1 can also inhibit inflammatory and immune responses.2 Thus, PECAM-1 has the potential to influence atherogenesis in more than one way. See accompanying articles on pages 1996 and 2003 Usually the deficiency of a molecule leads to an overall increase, decrease, or no change in murine atherosclerotic lesion burden, but the distribution of lesions in the arterial tree remains unchanged, with the majority of lesions occurring in the aortic root, the lesser (inner) curvature of the ascending aorta and near ostia of arterial branches in the descending aorta.5 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , two articles detail independent obserations that deficiency of PECAM-1 results in an altered distribution of atherosclerotic lesions. Goel et al6 evaluated atherosclerotic lesion development in LDL receptor deficient ( ldlr −/−) mice by measuring lipid accumulation using oil red O staining in en face preparations of the aorta and cross-sections of the aortic root, as well as micro computed tomography of osmium tetroxide stained proximal aorta and …

Atherosclerosis is an inflammatory disease that develops preferentially at bends and branches of the vasculature exposed to disturbed flow and low shear stress (LSS). These mechanical conditions modify endothelial cell...

Hemodynamics significantly impact the biology of endothelial cells (ECs) lining the blood vessels. ECs are exposed to various hemodynamic forces, particularly frictional shear stress from flowing blood. While physiological flows are critical for the normal functioning of ECs, abnormal flow dynamics, known as disturbed flows, may trigger endothelial dysfunction leading to atherosclerosis and other vascular conditions. Such flows can occur due to sudden geometrical variations and vascular abnormalities in the cardiovascular system. In the current study, a microfluidic system was used to investigate the impact of different flow conditions (i.e, normal vs. disturbed) on ECs in vitro. We particularly explored the relationship between specific flow patterns and cellular pathways linked to oxidative stress and inflammation related to atherosclerosis. Here, we utilized a 2D cell culture perfusion system featuring an immortalized human vascular endothelial cell line (EA.hy926) connected to a modified peristaltic pump system to generate either steady laminar flows, representing healthy conditions, or disturbed oscillatory flows, representing diseased conditions. EA.hy926 were exposed to an oscillatory flow shear stress of 0.5 dynes/cm2 or a laminar flow shear stress of 2 dynes/cm2 up to 24 h. Following flow exposure, cells were harvested from the perfusion chamber for quantitative PCR analysis of gene expression. Reactive oxygen species (ROS) generation under various shear stress conditions was also measured using DCFDA/H2DCFDA fluorescent assays. Under oscillatory shear stress flow conditions (0.5 dynes/cm2), EA.hy926 ECs showed a 3.5-fold increase in the transcription factor nuclear factor (NFκ-B) and a remarkable 28.6-fold increase in cyclooxygenase-2 (COX-2) mRNA expression, which are both proinflammatory markers, compared to static culture. Transforming growth factor-beta (TGFβ) mRNA expression was downregulated in oscillatory and laminar flow conditions compared to the static culture. Apoptosis marker transcription factor Jun (C-Jun) mRNA expression increased in both flow conditions. Apoptosis marker C/EBP homologous protein (CHOP) mRNA levels increased significantly in oscillatory flow, with no difference in laminar flow. Endothelial nitric oxide synthase (eNOS) mRNA expression was significantly decreased in cells exposed to oscillatory flow, whereas there was no change in laminar flow. Endothelin-1 (ET-1) mRNA expression levels dropped significantly by 0.5- and 0.8-fold in cells exposed to oscillatory and laminar flow, respectively. ECs subjected to oscillatory flow exhibited a significant increase in ROS at both 4 and 24 h compared to the control and laminar flow. Laminar flow-treated cells exhibited a ROS generation pattern similar to that of static culture, but at a significantly lower level. Overall, by exposing ECs to disturbed and normal flows with varying shear stresses, significant changes in gene expression related to inflammation, endothelial function, and oxidative stress were observed. In this study, we present a practical, optimized system as an in vitro model that can be employed to investigate flow-associated diseases, such as atherosclerosis and aortic aneurysm, thereby supporting the understanding of the underlying molecular mechanisms.

Introduction Atherosclerosis, an underlying cause of cardiovascular disease, is a focal disease of arteries. Haemodynamic shear stress exerted on the endothelium by the flowing blood plays an important role in the pathogenesis of atherosclerosis and spatial distribution of atherosclerotic lesions. Low, oscillatory shear stress promotes atherosclerosis by regulating endothelial cell (EC) viability and function, while high shear is athero-protective. To elucidate the molecular mechanisms underlying the effects of shear stress on EC physiology, we used microarray technology coupled to computational fluid dynamics to define the EC transcriptome at low and high shear regions of the porcine aorta. The study identified 60 putative regulators of apoptosis that were differentially expressed at the two aortic regions. We hypothesised that this gene set includes key regulators of EC survival in response to haemodynamic forces. Methods To assess the function of specific genes in ECs and their potential role in the response to haemodynamic force, we have used zebrafish, which is an emerging vertebrate model in vascular biology. We selected five putative regulators of EC apoptosis that were differentially expressed between high and low shear stress region of the porcine aorta. Their expression was transiently knocked down in zebrafish embryos with gene-specific morpholino antisense oligonucleotides, while blood flow was modulated using the silent heart model (cardiac troponin T deficiency) or tricaine treatment. The effect of gene silencing on EC apoptosis was examined by immunofluorescent staining using antibodies that recognise active caspase 3 and by TUNEL assay. Results Our study reveals that cessation of blood flow promotes EC apoptosis in zebrafish embryos. Morpholino-mediated knockdown of two candidate genes from the microarray analysis, TP53 apoptosis effector ( PERP ) or programmed cell death protein 2-like ( PDCD2L) , reduced apoptosis in ECs exposed to flow cessation by approximately 30% (p PERP or PDCD2L ). These data indicate that PERP and PDCD2L have a role in promoting EC apoptosis in the absence of haemodynamic forces. Conclusions We have established a platform for functional screening of flow-regulated genes in zebrafish. Initial screening identified two novel regulators of EC dysfunction, PERP which is a target of the pro-apoptotic transcription factor p53, and PDCD2L which has sequence similarity with the apoptosis effector RP8. Future work will be focused on defining the mechanism by which PERP and PDCD2L induce apoptosis in ECs.

See related article, pages 200–208 The endothelium is an expansive spatially distributed organ.1 Endothelial cells participate in a large number of physiological processes including the control of vasomotor tone, the trafficking of cells and nutrients, the regulation of permeability, and the maintenance of blood fluidity. In addition, the endothelium mediates new blood vessel formation, contributes to the balance of pro- and antiinflammatory mediators, and may play a role in antigen presentation. In accomplishing these tasks, the endothelium exhibits a remarkable “division of labor”. For example, arteriolar endothelium is primarily responsible for mediating vasomotor tone; endothelium in postcapillary venules regulates leukocyte trafficking; capillary endothelial cells display organ-specific barrier properties (eg, blood brain barrier versus fenestrated, discontinuous endothelium in hepatic sinusoids); and endothelial cells from different vascular beds balance local hemostasis via the expression of site-specific patterns of anticoagulants and procoagulants.2 In recent years, in vivo phage display and direct proteome mapping of the intact vasculature have revealed a rich diversity in endothelial cell surface markers.3,4 Any consideration of the mechanisms underlying endothelial heterogeneity is best framed around the time-honored debate of nature versus nurture (which will be addressed here in reverse order) (Figure). Mechanisms of endothelial cell heterogeneity. Relative importance of epigenetics and microenvironment in mediating site-specific phenotypes is indicated by +. The table is designed to provide a conceptual framework; the scores are largely speculative and will require ongoing experimental validation. ### Nurture Site-specific endothelial cell phenotypes may be initiated and maintained by signals residing in the extracellular environment. The endothelium is analogous to a barcode reader, constantly taking inventory of its surrounding extracellular environment on the luminal side (circulating blood and its constituents), the abluminal side, and at the endothelial junctions. Environmental cues may be classified into biomechanical or biochemical. Biochemical forces include shear stress and strain. …

Atherosclerotic lesions occur non-randomly at vascular niches in bends and bifurcations where fluid flow can be characterized as "disturbed" (low shear stress with both forward and retrograde flow). Endothelial cells (ECs) at these locations experience significantly lower average shear stress without change in the levels of pressure or strain, which affects the local balance in mechanical stresses. Common in vitro models of atherosclerosis focus primarily on shear stress without accounting for pressure and strain loading. To overcome this limitation, we used our microfluidic endothelial cell culture model (ECCM) to achieve accurate replication of pressure, strain, and shear stress waveforms associated with both normal flow seen in straight sections of arteries and disturbed flow seen in the abdominal aorta in the infrarenal segment at the wall distal to the inferior mesenteric artery (IMA), which is associated with high incidence of atherosclerotic lesion formation. Human aortic endothelial cells (HAECs) were cultured within the ECCM under both normal and disturbed flow and evaluated for cell shape, cytoskeletal alignment, endothelial barrier function, and inflammation using immunofluorescence microscopy and flow cytometry. Results clearly demonstrate quantifiable differences between cells cultured under disturbed flow conditions, which are cuboidal with short and randomly oriented actin microfilaments and show intermittent expression of β-Catenin and cells cultured under normal flow. However, in the absence of pro-inflammatory stimulation, the levels of expression of activation markers: intra cellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular endothelial cell growth factor - receptor 2 (VEGF-R2) known to be involved in the initiation of plaque formation were only slightly higher in HAECs cultured under disturbed flow in comparison to cells cultured under normal flow.

IntroductionAtherosclerosis develops at branches and bends of arteries exposed to disturbed blood flow, whereas regions exposed to uniform flow are protected. Disturbed flow generates low, oscillatory wall shear stress (WSS)...

eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Wild type mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.