Atherosclerosis-prone Regions Research Articles

DLL4 promotes partial endothelial-to-mesenchymal transition at atherosclerosis-prone regions of arteries

Flowing blood regulates vascular development, homeostasis and disease by generating wall shear stress which has major effects on endothelial cell (EC) physiology. Low oscillatory shear stress (LOSS) induces a form of cell plasticity called endothelial-to-mesenchymal transition (EndMT). This process has divergent effects; in embryos LOSS-induced EndMT drives the development of atrioventricular valves, whereas in adult arteries it is associated with inflammation and atherosclerosis. The Notch ligand DLL4 is essential for LOSS-dependent valve development; here we investigated whether DLL4 is required for responses to LOSS in adult arteries. Analysis of cultured human coronary artery EC revealed that DLL4 regulates the transcriptome to induce markers of EndMT and inflammation under LOSS conditions. Consistently, genetic deletion of Dll4 from murine EC reduced SNAIL (EndMT marker) and VCAM-1 (inflammation marker) at a LOSS region of the murine aorta. We hypothesized that endothelial Dll4 is pro-atherogenic but this analysis was confounded because endothelial Dll4 negatively regulated plasma cholesterol levels in hyperlipidemic mice. We conclude that endothelial DLL4 is required for LOSS-induction of EndMT and inflammation regulators at atheroprone regions of arteries, and is also a regulator of plasma cholesterol.

Early atherogenesis: new insights from new approaches.

Purpose of reviewTo highlight recent conceptual and technological advances that have positioned the field to interrogate the cellular and molecular mechanisms contributing to the initiation of atherosclerosis, including intimal lipid accumulation, inflammation, and lesion growth.Recent findingsAdvances in the understanding of endothelial LDL transcytosis and rapid lipid uptake by intimal macrophages provide mechanistic insights into intimal LDL accumulation and the initiation of atherogenesis. Recent studies have used unbiased single-cell approaches, such as single-cell RNA sequencing and CyTOF, to characterize the cellular components of the normal intima and atherosclerotic lesions. In-vitro studies and high-resolution transcriptomic analysis of aortic intimal lipid-loaded versus lipid-poor myeloid populations in vivo suggest that lipid-loaded macrophages may not be the primary drivers of inflammation in atherosclerotic lesions.SummaryA new perspective on the complex cellular landscape of the aorta, specifically the atherosclerosis-prone regions, confirm that intimal accumulation of lipid, monocyte recruitment, and macrophage accumulation are key events in atherogenesis triggered by hypercholesterolemia. Targeting these early events may prove to be a promising strategy for the attenuation of lesion development; however, the specific details of how hypercholesterolemia acts to initiate early inflammatory events remain to be fully elucidated.

Immature Vascular Smooth Muscle Cells in Healthy Murine Arteries and Atherosclerotic Plaques: Localization and Activity.

The local development of atherosclerotic lesions may, at least partly, be associated with the specific cellular composition of atherosclerosis-prone regions. Previously, it was demonstrated that a small population of immature vascular smooth muscle cells (VSMCs) expressing both CD146 and neuron-glial antigen 2 is postnatally sustained in atherosclerosis-prone sites. We supposed that these cells may be involved in atherogenesis and can continuously respond to angiotensin II, which is an atherogenic factor. Using immunohistochemistry, flow cytometry, wound migration assay xCELLigence system, and calcium imaging, we studied the functional activities of immature VSMCs in vitro and in vivo. According to our data, these cells do not express nestin, CD105, and the leptin receptor. They are localized in atherosclerosis-prone regions, and their number increases with age, from 5.7% to 23%. Immature VSMCs do not migrate to low shear stress areas and atherosclerotic lesions. They also do not have any unique response to angiotensin II. Thus, despite the localization of immature VSMCs and the presence of the link between their number and age, our study did not support the hypothesis that immature VSMCs are directly involved in the formation of atherosclerotic lesions. Additional lineage tracing studies can clarify the fate of these cells during atherogenesis.

KRIT1 Deficiency Promotes Aortic Endothelial Dysfunction.

Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/− mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.

SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis

SUMMARYAtherosclerosis, which underlies life-threatening cardiovascular disorders including myocardial infarction and stroke1, is initiated by low density lipoprotein cholesterol (LDL) passage into the artery wall and engulfment by macrophages, leading to foam cell formation and lesion development2, 2, 3, 3. How circulating LDL enters the artery wall to instigate atherosclerosis is unknown. Here we show in mice that scavenger receptor, class B type 1 (SR-B1) in endothelial cells mediates LDL delivery into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and LDL transcytosis across endothelial monolayers requires its direct binding to SR-B1 and an 8 amino acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4)4. DOCK4 promotes SR-B1 internalization and LDL transport by coupling LDL binding to SR-B1 with Rac1 activation. SR-B1 and DOCK4 expression are increased in atherosclerosis-prone regions of the mouse aorta prior to lesion formation, and in human atherosclerotic versus normal arteries. These findings challenge the long-held concept that atherogenesis involves passive LDL movement across a compromised endothelial barrier. Interventions inhibiting endothelial delivery of LDL into the artery wall may represent a new therapeutic category in the battle against cardiovascular disease.

Effect of angulation and Reynolds number on recirculation at the abdominal aorta-renal artery junction

We investigate the effect of renal artery angle variation and Reynolds number on the probability of formation of atherosclerosis-prone regions at the abdominal aorta-renal artery junction. The rheologically accurate shear-thinning Yeleswarapu model for blood is used to simulate flow in a 3-Dimensional T-junction whose dimensions are those of the human abdominal aorta-renal artery junction. The recirculation length and wall shear stresses are evaluated at the junction for steady, laminar flow using ANSYS FLUENT v14.5. The recirculation length and wall shear stresses are calculated for Reynolds number of 500–1500, and the angle of renal artery (with the horizontal axis) of −20° to +20°. Computational fluid dynamics analysis for the flow conditions used in present study show that the flow recirculation and low wall shear stress regions overlap at the top surface of renal artery downstream of the entrance and at the curved surface of abdominal aorta downstream of the junction, making these regions susceptible to atherosclerosis. Further investigations show that positive angles of renal artery have larger area of recirculation and larger low wall shear stress regions. The recirculation length increases with Reynolds number, and it is maximum when θ = +20°, and minimum when θ = −20°.

Shear Stress Counteracts Endothelial CX3CL1 Induction and Monocytic Cell Adhesion.

Flow conditions critically regulate endothelial cell functions in the vasculature. Reduced shear stress resulting from disturbed blood flow can drive the development of vascular inflammatory lesions. On endothelial cells, the transmembrane chemokine CX3CL1/fractalkine promotes vascular inflammation by functioning as a surface-expressed adhesion molecule and by becoming released as soluble chemoattractant for monocytic cells expressing the receptor CX3CR1. Here, we report that endothelial cells from human artery, vein, or microvasculature constitutively express CX3CL1 when cultured under static conditions. Stimulation with TNFα under static or very low shear stress conditions strongly upregulates CX3CL1 expression. By contrast, CX3CL1 induction is profoundly reduced when cells are exposed to higher shear stress. When endothelial cells were grown and subsequently stimulated with TNFα under low shear stress, strong adhesion of monocytic THP-1 cells to endothelial cells was observed. This adhesion was in part mediated by transmembrane CX3CL1 as demonstrated with a neutralizing antibody. By contrast, no CX3CL1-dependent adhesion to stimulated endothelium was observed at high shear stress. Thus, during early stages of vascular inflammation, low shear stress typically seen at atherosclerosis-prone regions promotes the induction of endothelial CX3CL1 and monocytic cell recruitment, whereas physiological shear stress counteracts this inflammatory activation of endothelial cells.

Modeling the Effect of Red Blood Cells Deformability on Blood Flow Conditions in Human Carotid Artery Bifurcation.

The purpose of this work is to predict the effect of impaired red blood cells (RBCs) deformability on blood flow conditions in human carotid artery bifurcation. First, a blood viscosity model is developed that predicts the steady-state blood viscosity as a function of shear rate, plasma viscosity, and mechanical (and geometrical) properties of RBC's. Viscosity model is developed by modifying the well-known Krieger and Dougherty equation for monodisperse suspensions by using the dimensional analysis approach. With the approach, we manage to account for the microscopic properties of RBC's, such as their deformability, in the macroscopic behavior of blood via blood viscosity. In the second part of the paper, the deduced viscosity model is used to numerically predict blood flow conditions in human carotid artery bifurcation. Simulations are performed for different values of RBC's deformability and analyzed by investigating parameters, such as the temporal mean wall shear stress (WSS), oscillatory shear index (OSI), and mean temporal gradient of WSS. The analyses show that the decrease of RBC's deformability decrease the regions of low WSS (i.e., sites known to be prevalent at atherosclerosis-prone regions); increase, in average, the value of WSS along the artery; and decrease the areas of high OSI. These observations provide an insight into the influence of blood's microscopic properties, such as the deformability of RBC's, on hemodynamics in larger arteries and their influence on parameters that are known to play a role in the initiation and progression of atherosclerosis.

Assessment of shear stress related parameters in the carotid bifurcation using mouse-specific FSI simulations

The ApoE−/− mouse is a common small animal model to study atherosclerosis, an inflammatory disease of the large and medium sized arteries such as the carotid artery. It is generally accepted that the wall shear stress, induced by the blood flow, plays a key role in the onset of this disease. Wall shear stress, however, is difficult to derive from direct in vivo measurements, particularly in mice. In this study, we integrated in vivo imaging (micro-Computed Tomography-µCT and ultrasound) and fluid–structure interaction (FSI) modeling for the mouse-specific assessment of carotid hemodynamics and wall shear stress. Results were provided for 8 carotid bifurcations of 4 ApoE−/− mice. We demonstrated that accounting for the carotid elasticity leads to more realistic flow waveforms over the complete domain of the model due to volume buffering capacity in systole. The 8 simulated cases showed fairly consistent spatial distribution maps of time-averaged wall shear stress (TAWSS) and relative residence time (RRT). Zones with reduced TAWSS and elevated RRT, potential indicators of atherosclerosis-prone regions, were located mainly at the outer sinus of the external carotid artery. In contrast to human carotid hemodynamics, no flow recirculation could be observed in the carotid bifurcation region.

MiR-92a

During life, our arteries with their 3 layers, numerous curvatures, branch points, and bifurcations, are continuously exposed to different flow and shear stress conditions. At branch points and bifurcations, especially oscillatory shear stress activates the endothelium, making these arterial sites prone to develop atherosclerosis.1,2 The subendothelial deposition and subsequent modification of lipids, such as low-density lipoprotein (LDL), further enhances this vascular inflammation. Along with endothelial activation, both the innate and adaptive immune system come into play, resulting in further recruitment of leukocytes and formation of atherosclerotic plaques.1,2 Article, see p 434 To keep the development of atherosclerosis under control, our arteries are subjected to a tight regulation to maintain their homeostasis. Regulatory RNAs, including micro-RNAs, are powerful post-transcriptional mediators that control vascular homeostasis, and their deregulation can result in aggravation of endothelial dysfunction and aggravation of vascular disease. Micro-RNAs can therefore be considered as potential novel therapeutics to treat cardiovascular disease.3 In this issue of Circulation Research , Loyer et al4 performed an elegant screening to identify micro-RNAs affecting atherosclerosis, the so-called atheromiRs, in endothelial cells. The authors selected the atheromiRs in in vitro settings using human umbilical vein endothelial cells based on 2 criteria that are highly relevant for the pathogenesis of atherosclerosis: (1) miRs that exhibit a change in expression by exposure to oxidized LDL (oxLDL) under low shear stress and (2) miRs that are not affected by oxLDL exposure under high shear stress conditions. The miR that was most dysregulated and fulfilled these criteria was miR-92a, which was highly expressed in atherosclerosis-prone regions in normocholesterolemic ldlr −/− mice compared with atherosclerosis-resistant regions in these mice. This difference in miR-92a expression was …

Vascular matrix biology and bioengineering workshop IV and biology of signaling in the cardiovascular system workshop III October 20–24, 2013

Previous work has shown that ECM remodeling is a key component of the inflammatory activation of the endothelium in atherosclerosis, which occurs in regions of arteries subject to disturbed flow. Fibronectin is deposited in these atherosclerosis-prone regions of arteries even in WT mice. Fluid shear-dependent inflammatory signaling mediated by NF-jB or JNK is selectively induced in endothelial cells on fibronectin whereas it is suppressed on basement membrane proteins. Investigation of the mechanism behind these effects showed that PKA is selectively activated in cells on basement membrane and suppresses inflammatory signaling. We therefore investigated the integrin receptors that mediate ECM binding. The major fibronectin receptor, a5b1, and the major collagen/laminin receptor, a2b1, share the common b1 subunit. We therefore analyzed the cytoplasmic domains of the unique integrin alpha subunits. A chimera in which the integrin a5 cytosolic tail was replaced by the a2 cytosolic tail was examined. Endothelial cells expressing the integrin a5/2 chimera adhered and spread normally on fibronectin. However, shear stress activated PKA and inhibited NF-jB, similar to cells on basement membranes. Pharmacological inhibition of PDE4 activity abolished fibronectin-specific NF-jB activation, indicating that cAMP degradation is required for the pro-inflammatory effects of fibronectin. PDE4D5 directly and specifically associates with the integrin a5 cytosolic tail. Knock-down of PDE4D impaired flow-dependent NFjB activation in the cells on fibronectin. These data suggest that the interaction of the integrin a5 cytoplasmic domain with PDE4D mediates the pro-inflammatory effects of fibronectin by inhibiting the anti-inflammatory cAMP/PKA pathway.

Endothelial Cell Sensing of Flow Direction

Atherosclerosis-prone regions of arteries are characterized by complex flow patterns where the magnitude of shear stress is low and direction rapidly changes, termed disturbed flow. How endothelial cells sense flow direction and how it impacts inflammatory effects of disturbed flow are unknown. We therefore aimed to understand how endothelial cells respond to changes in flow direction. Using a recently developed flow system capable of changing flow direction to any angle, we show that responses of aligned endothelial cells are determined by flow direction relative to their morphological and cytoskeletal axis. Activation of the atheroprotective endothelial nitric oxide synthase pathway is maximal at 180° and undetectable at 90°, whereas activation of proinflammatory nuclear factor-κB is maximal at 90° and undetectable at 180°. Similar effects were observed in randomly oriented cells in naive monolayers subjected to onset of shear. Cells aligned on micropatterned substrates subjected to oscillatory flow were also examined. In this system, parallel flow preferentially activated endothelial nitric oxide synthase and production of nitric oxide, whereas perpendicular flow preferentially activated reactive oxygen production and nuclear factor-κB. These data show that the angle between flow and the cell axis defined by their shape and cytoskeleton determines endothelial cell responses. The data also strongly suggest that the inability of cells to align in low and oscillatory flow is a key determinant of the resultant inflammatory activation.

Anti-Inflammatory and Antiatherogenic Role of BMP Receptor II in Endothelial Cells

Atherosclerosis is an inflammatory disease with multiple underlying metabolic and physical risk factors. Bone morphogenic protein 4 (BMP4) expression is increased in endothelium in atherosclerosis-prone regions and is known to induce endothelial inflammation, endothelial dysfunction, and hypertension. BMP actions are mediated by 2 different types of BMP receptors (BMPRI and BMPRII). Here, we show a surprising finding that loss of BMPRII expression causes endothelial inflammation and atherosclerosis. Using BMPRII siRNA and BMPRII(+/-) mice, we found that specific knockdown of BMPRII, but not other BMP receptors (Alk1, Alk2, Alk3, Alk6, ActRIIa, and ActRIIb), induced endothelial inflammation in a ligand-independent manner by mechanisms mediated by reactive oxygen species, nuclear factor-KappaB, and reduced nicotinamide adenine dinucleotide phosphate oxidases. Further, BMPRII(+/-)ApoE(-/-) mice developed accelerated atherosclerosis compared with BMPRII(+/+)ApoE(-/-) mice. Interestingly, we found that multiple proatherogenic stimuli, such as hypercholesterolemia, disturbed flow, prohypertensive angiotensin II, and the proinflammatory cytokine (tumor necrosis factor-α), downregulated BMPRII expression in endothelium, whereas antiatherogenic stimuli, such as stable flow and statin treatment, upregulated its expression in vivo and in vitro. Moreover, BMPRII expression was significantly diminished in human coronary advanced atherosclerotic lesions. Also, we were able to rescue the endothelial inflammation induced by BMPRII knockdown by overexpressing the BMPRII wild type, but not by the BMPRII short form lacking the carboxyl-terminal tail region. These results suggest that BMPRII is a critical, anti-inflammatory, and antiatherogenic protein that is commonly targeted by multiple pro- and antiatherogenic factors. BMPRII may be used as a novel diagnostic and therapeutic target in atherosclerosis.

Which diameter and angle rule provides optimal flow patterns in a coronary bifurcation?

The branching angle and diameter ratio in epicardial coronary artery bifurcations are two important determinants of atherogenesis. Murray's cubed diameter law and bifurcation angle have been assumed to yield optimal flows through a bifurcation. In contrast, we have recently shown a 7/3 diameter law (HK diameter model), based on minimum energy hypothesis in an entire tree structure. Here, we derive a bifurcation angle rule corresponding to the HK diameter model and critically evaluate the streamline flow through HK and Murray-type bifurcations. The bifurcations from coronary casts were found to obey the HK diameter model and angle rule much more than Murray's model. A finite element model was used to investigate flow patterns for coronary artery bifurcations of various types. The inlet velocity and pressure boundary conditions were measured by ComboWire. Y-bifurcation of Murray type decreased wall shear stress-WSS (10%–40%) and created an increased oscillatory shear index-OSI in atherosclerosis-prone regions as compared with HK-type bifurcations. The HK-type bifurcations were found to have more optimal flow patterns (i.e., higher WSS and lower OSI) than Murray-type bifurcations which have been traditionally believed to be optimized. This study has implications for changes in bifurcation angles and diameters in percutaneous coronary intervention.

VEGFR2 signalling contributes to increased endothelial susceptibility to TNF-α under chronic non-uniform shear stress

ObjectivesVascular endothelial growth factor receptor 2 (VEGFR2), a tyrosine kinase receptor activated by VEGF and shear stress, is critically involved in endothelial mechanotransduction. We investigated the role of VEGFR2 in non-uniform shear stress-induced endothelial susceptibility to inflammatory stimuli. MethodsEndothelial cells (ECs) were exposed to non-uniform shear stress, followed by stimulation with TNF-α. ECs were transfected with siRNAs against VEGFR2. Alternatively, ECs were treated with blocking antibody against VEGFR2, or with inhibitors of VEGFR2 (ZM 323881), PI3K (LY 294002), or Src-kinase (PP2). THP-1 monocytes were used for dynamic adhesion assays. Endothelial protein expression was determined by immunofluorescence. ResultssiRNA against VEGFR2 decreased VEGFR2 protein expression by 40% as determined by Western blotting. In endothelial cells exposed to non-uniform shear stress, VEGFR2 knockdown inhibited TNF-α-induced NF-κB translocation to the nucleus, and the upregulation of VCAM-1 and E-selectin. Consequently, monocytic cell recruitment to endothelium under non-uniform shear stress conditions was reduced. Similar effects were observed by blocking VEGFR2 activity using a specific inhibitor ZM 323881, or an antibody against VEGFR2 before TNF-α stimulation. Inhibition of PI3K with LY 294002 significantly reduced non-uniform shear stress-induced endothelial susceptibility to TNF-α, whereas blocking Src-kinase with PP2 was ineffective. ConclusionVEGFR2 is critically involved in adhesion molecule induction and monocytic cell recruitment to endothelium in response to non-uniform shear stress and TNF-α. Targeting the mechanosensory cascade can prevent endothelial activation in atherosclerosis-prone regions.

The effects of stenosis severity on the hemodynamic parameters—assessment of the correlation between stress phase angle and wall shear stress

To study the effects of increase in the degree of stenosis severity and subsequent complexity of hemodynamic patterns on hemodynamic parameters, experimental investigations and numerical simulations were performed. The correlations between the large negative Stress Phase Angle (SPA), the low mean Wall Shear Stress (WSS) and high Oscillatory Shear Index (OSI) were investigated at the distal shoulder and post-stenotic regions as the outcomes of elevated stenosis severity. Models included non-Newtonian fluid flow in stenotic arteries with 30–80% symmetrical stenoses. To study the interactions between pulsatile WSS and pulsatile wall circumferential stress (WCS) acting on endothelial cells, SPA as the phase difference between WSS and WCS waves was used. Moreover, the distribution of SPA on the lumen axis was compared to the distributions of the mean WSS and OSI that have been regarded until now as the determinants of atherosclerosis-prone regions. Results indicate that an increase in stenosis severity, not only affects the mean WSS, mean WCS and pulse amplitudes, but also influences the phase difference between them. The SPA is large negative on the distal shoulder and post-stenotic areas where atherosclerotic plaque develops. The increasing stenosis severity and the subsequent increasing complexity of hemodynamic patterns affect the correlation between any of the low mean WSS and high OSI with large negative SPA, such that it not only leads to create and develop some regions where the correlation between any of the low mean WSS and high OSI with large negative SPA is well but also leads to create and develop other regions where such correlations fail.

Attenuated atherogenesis in apolipoprotein E-deficient mice lacking amyloid precursor protein

ObjectiveRecent evidence suggests that amyloid precursor protein (APP) is overexpressed in atherosclerosis-prone regions of mouse aorta. We therefore investigated in the present study whether APP has a role in the progression and composition of atherosclerotic plaques. Methods and resultsApolipoprotein E-deficient (apoE−/−) mice were crossbred with animals lacking APP (APP−/−). After 16 weeks on a Western-type diet, apoE−/− and APP−/−/apoE−/− mice showed similar cholesterol levels. However, atherosclerotic plaque size was significantly reduced in the distal thoracic aorta (90% reduction) and abdominal aorta (75% reduction) of APP−/−/apoE−/− mice as compared to apoE−/−. Plaques at the level of the aortic valves were not different in size, but showed a more stable phenotype in APP−/−/apoE−/− mice, as indicated by a reduced macrophage content, an increased amount of collagen and a thicker fibrous cap. ConclusionOur findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability.

Shear stress preconditioning modulates endothelial susceptibility to circulating TNF-α and monocytic cell recruitment in a simplified model of arterial bifurcations

Objective Atherosclerotic plaque formation results from a combination of local shear stress patterns and inflammatory processes. This study investigated the endothelial response to shear stress in combination with the inflammatory cytokine TNF-α in a simplified model of arterial bifurcation. Methods Human umbilical vein endothelial cells (ECs) were exposed to laminar or non-uniform shear stress in bifurcating flow-through slides, followed by stimulation with TNF-α. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells. Endothelial protein expression was determined by immunofluorescence. Results Adhesion of monocytic cells to unstimulated ECs was nearly undetectable under laminar shear stress and was slightly increased under non-uniform shear stress. Exposure of ECs to non-uniform shear stress in combination with TNF-α induced a 12-fold increase in monocytic cell recruitment and a significant induction of endothelial E-selectin and VCAM-1 expression. Both these effects were prevented in ECs exposed to laminar shear stress. The significant differences in TNF-α-induced monocytic cell recruitment and adhesion molecule expression between laminar and non-uniform shear stress regions were abolished in the absence of shear stress preconditioning. Simvastatin (1 μmol/L) suppressed the non-uniform shear stress- and TNF-α-induced increase in monocytic cell adhesion by about 30% via inhibition of VCAM-1 expression. Resveratrol, the active component of red wine, inhibited the expression of both VCAM-1 and E-selectin, and reduced monocytic cell recruitment by 50% at 20 μmol/L. Conclusions Non-uniform shear stress induces endothelial susceptibility to circulating TNF-α and adhesion of monocytic cells. Interference with this process may inhibit inflammatory response in atherosclerosis-prone regions.

Shc coordinates signals from intercellular junctions and integrins to regulate flow-induced inflammation

Atherosclerotic plaques develop in regions of the vasculature associated with chronic inflammation due to disturbed flow patterns. Endothelial phenotype modulation by flow requires the integration of numerous mechanotransduction pathways, but how this is achieved is not well understood. We show here that, in response to flow, the adaptor protein Shc is activated and associates with cell–cell and cell–matrix adhesions. Shc activation requires the tyrosine kinases vascular endothelial growth factor receptor 2 and Src. Shc activation and its vascular endothelial cadherin (VE-cadherin) association are matrix independent. In contrast, Shc binding to integrins requires VE-cadherin but occurs only on specific matrices. Silencing Shc results in reduction in both matrix-independent and matrix-dependent signals. Furthermore, Shc regulates flow-induced inflammatory signaling by activating nuclear factor κB–dependent signals that lead to atherogenesis. In vivo, Shc is activated in atherosclerosis-prone regions of arteries, and its activation correlates with areas of atherosclerosis. Our results support a model in which Shc orchestrates signals from cell–cell and cell–matrix adhesions to elicit flow-induced inflammatory signaling.

ABCA1 is upregulated in athero‐protected regions of arteries in response to brief hypercholesterolemia treatment in vivo

Endothelial cells (EC) in atherosclerosis‐prone regions of arteries display distinct gene expression profiles compared to athero‐protected regions. We investigated the differential alteration of EC phenotype at these sites during brief hypercholesterolemia. Eleven adult male swine were fed a normal chow diet (n=6) or an isocaloric high fat diet (n=5) for a period of 2 weeks. EC RNA was isolated from 3 protected (carotid, descending thoracic, renal arteries) and 3 susceptible (aortic arch, renal branch, abdominal aorta) arterial regions, linearly amplified and hybridized to porcine microarrays. Fifty eight lipid metabolism related genes were differentially expressed in ECs from all regions in response to hypercholesterolemia. However, ATP Binding Cassette A1 (ABCA1), an endothelial transmembrane protein that regulates phospholipids and cholesterol homeostasis inside the cell in response to changing levels of cholesterol in the blood, was specifically upregulated in athero‐protected regions. This response was also validated at the protein level by immunohistochemistry. Region‐specific upregulation of ABCA1 transcript and protein in ECs demonstrates a site‐specific differential EC phenotypic response to hypercholesterolemia. Supported by grants from NHLBI.