Reliable in vivo intravascular imaging plaque characterization: A challenge unmet.

Intravascular imaging has enabled in vivo assessment of coronary artery pathology and detection of plaque characteristics that are associated with increased vulnerability. Prospective invasive imaging studies of coronary atherosclerosis have demonstrated that invasive imaging modalities can detect lesions that are likely to progress and cause cardiovascular events and provided unique insights about atherosclerotic evolution. However, despite the undoubted value of the existing imaging techniques in clinical and research arenas, all the available modalities have significant limitations in assessing plaque characteristics when compared with histology. Hybrid/multimodality intravascular imaging appears able to overcome some of the limitations of standalone imaging; however, there are only few histology studies that examined their performance in evaluating plaque pathobiology. In this article, we review the evidence about the efficacy of standalone and multi-modality/hybrid intravascular imaging in assessing plaque morphology against histology, highlight the advantages and limitations of the existing imaging techniques and discuss the future potential of emerging imaging modalities in the study of atherosclerosis.

Interferon Regulatory Factor 5 Controls Necrotic Core Formation in Atherosclerotic Lesions by Impairing Efferocytosis.

BACKGROUND: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions. METHODS: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE-/-) mice and ApoE-/- mice with a genetic deletion of IRF5 (ApoE-/-Irf5-/-) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation. RESULTS: Both lesion and necrotic core size were significantly reduced in ApoE-/-Irf5-/- mice compared with IRF5-competent ApoE-/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c+ macrophages was evident in ApoE-/-Irf5-/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c+ macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c- macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-ß3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ. CONCLUSIONS: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c+ macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis.

Zebrafish Model for Functional Screening of Flow-Responsive Genes.

OBJECTIVE: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. APPROACH AND RESULTS: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. CONCLUSIONS: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.

Inducing Persistent Flow Disturbances Accelerates Atherogenesis and Promotes Thin Cap Fibroatheroma Development in D374Y-PCSK9 Hypercholesterolemic Minipigs.

BACKGROUND: Although disturbed flow is thought to play a central role in the development of advanced coronary atherosclerotic plaques, no causal relationship has been established. We evaluated whether inducing disturbed flow would cause the development of advanced coronary plaques, including thin cap fibroatheroma. METHODS AND RESULTS: D374Y-PCSK9 hypercholesterolemic minipigs (n=5) were instrumented with an intracoronary shear-modifying stent (SMS). Frequency-domain optical coherence tomography was obtained at baseline, immediately poststent, 19 weeks, and 34 weeks, and used to compute shear stress metrics of disturbed flow. At 34 weeks, plaque type was assessed within serially collected histological sections and coregistered to the distribution of each shear metric. The SMS caused a flow-limiting stenosis, and blood flow exiting the SMS caused regions of increased shear stress on the outer curvature and large regions of low and multidirectional shear stress on the inner curvature of the vessel. As a result, plaque burden was ≈3-fold higher downstream of the SMS than both upstream of the SMS and in the control artery (P<0.001). Advanced plaques were also primarily observed downstream of the SMS, in locations initially exposed to both low (P<0.002) and multidirectional (P<0.002) shear stress. Thin cap fibroatheroma regions demonstrated significantly lower shear stress that persisted over the duration of the study in comparison with other plaque types (P<0.005). CONCLUSIONS: These data support a causal role for lowered and multidirectional shear stress in the initiation of advanced coronary atherosclerotic plaques. Persistently lowered shear stress appears to be the principal flow disturbance needed for the formation of thin cap fibroatheroma.

Low shear stress induces M1 macrophage polarization in murine thin-cap atherosclerotic plaques.

Macrophages, a significant component of atherosclerotic plaques vulnerable to acute complications, can be pro-inflammatory (designated M1), regulatory (M2), lipid- (Mox) or Heme-induced (Mhem). We showed previously that low (LSS) and oscillatory (OSS) shear stress cause thin-cap fibroatheroma and stable smooth muscle cell-rich plaque formation respectively in ApoE-knockout (ApoE(-/-)) mice. Here we investigated whether different shear stress conditions relate to specific changes in macrophage polarization and plaque morphology by applying a shear stress-altering cast to the carotid arteries of high fat-fed ApoE(-/-) mice. The M1 markers iNOS and IRF5 were highly expressed in macrophage-rich areas of LSS lesions compared to OSS lesions 6weeks after cast placement, while the M2 marker Arginase-1, and Mox/Mhem markers HO-1 and CD163 were elevated in OSS lesions. Our data indicates shear stress could be an important determinant of macrophage polarization in atherosclerosis, with low shear promoting M1 programming.

Thin-cap fibroatheroma rupture is associated with a fine interplay of shear and wall stress.

In this review, we summarized the effect of mechanical factors (shear and wall stress) on thin-cap fibroatheroma formation and rupture. To make this review understandable for a biology-oriented audience, we start with detailed definitions of relevant mechanical metrics. We then describe how biomechanics has supported histopathologic efforts to understand the basis of plaque rupture. In addition to plaque rupture, biomechanics also contributes toward the progression of thin-cap fibroatheroma through a multitude of reported mechanobiological mechanisms. We thus propose a new mechanism whereby both shear stress and wall stress interact to create thin-cap fibroatheromas. Specifically, when regions of certain blood flow and wall mechanical stimuli coincide, they synergistically create inflammation within the cellular environment that can lead to thin-cap fibroatheroma rupture. A consequence of this postulate is that local shear stress is not sufficient to cause rupture, but it must coincide with regions of local tissue stiffening and stress concentrations that can occur during plaque progression. Because such changes to the wall mechanics occur over a micrometer scale, high spatial resolution imaging techniques will be necessary to evaluate this hypothesis and ultimately predict plaque rupture in a clinical environment.

Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors.

In order to fully understand the dynamics of processes within biological lipid membranes, it is necessary to possess an intimate knowledge of the physical state and ordering of lipids within the membrane. Here we report the use of three molecular rotors based on meso-substituted boron-dipyrrin (BODIPY) in combination with fluorescence lifetime spectroscopy to investigate the viscosity and phase behaviour of model lipid bilayers. In phase-separated giant unilamellar vesicles, we visualise both liquid-ordered (Lo) and liquid-disordered (Ld) phases using fluorescence lifetime imaging microscopy (FLIM), determining their associated viscosity values, and investigate the effect of composition on the viscosity of these phases. Additionally, we use molecular dynamics simulations to investigate the orientation of the BODIPY probes within the bilayer, as well as using molecular dynamics simulations and fluorescence correlation spectroscopy (FCS) to compare diffusion coefficients with those predicted from the fluorescence lifetimes of the probes.

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.

In vivo mapping of vascular inflammation using the translocator protein tracer 18F-FEDAA1106.

Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro-2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5-methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18F-FEDAA1106 and 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG, a marker of glucose metabolism) for positron emission tomographic (PET) imaging of vascular inflammation. This was tested using a murine model in which focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18F-FEDAA1106 or 18F-FDG was registered to anatomic data generated by computed tomographic (CT)/CT angiography. Standardized uptake values were significantly increased at cuffed compared to contralateral arteries using either 18F-FEDAA1106 (p < .01) or FDG (p < .05). However, the 18F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the noninflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.

Fibromodulin deficiency reduces low-density lipoprotein accumulation in atherosclerotic plaques in apolipoprotein E-null mice.

OBJECTIVE: The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques. METHODS AND RESULTS: Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development. CONCLUSIONS: The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability.

Assessment of the nano-mechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy.

Nano-indentation techniques might be better equipped to assess the heterogeneous material properties of plaques than macroscopic methods but there are no bespoke protocols for this kind of material testing for coronary arteries. Therefore, we developed a measurement protocol to extract mechanical properties from healthy and atherosclerotic coronary artery tissue sections. Young's modulus was derived from force-indentation data. Metrics of collagen fibre density were extracted from the same tissue, and the local material properties were co-registered to the local collagen microstructure with a robust framework. The locations of the indentation were retrospectively classified by histological category (healthy, plaque, lipid-rich, fibrous cap) according to Picrosirius Red stain and adjacent Hematoxylin & Eosin and Oil-Red-O stains. Plaque tissue was softer (p < 0.001) than the healthy coronary wall. Areas rich in collagen within the plaque (fibrous cap) were significantly (p < 0.001) stiffer than areas poor in collagen/lipid-rich, but less than half as stiff as the healthy coronary media. Young's moduli correlated (Pearson's ρ = 0.53, p < 0.05) with collagen content. Atomic force microscopy (AFM) is capable of detecting tissue stiffness changes related to collagen density in healthy and diseased cardiovascular tissue. Mechanical characterization of atherosclerotic plaques with nano-indentation techniques could refine constitutive models for computational modelling.

Disturbed blood flow induces RelA expression via c-Jun N-terminal kinase 1: a novel mode of NF-κB regulation that promotes arterial inflammation.

RATIONALE: The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB transcription factors are uncertain. OBJECTIVE: We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation. METHODS AND RESULTS: Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-κB expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-κB may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-κB expression and inflammation in murine carotid arteries via JNK1. CONCLUSIONS: We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-κB expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK-NF-κB crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.

Considering the Influence of Coronary Motion on Artery-Specific Biomechanics Using Fluid-Structure Interaction Simulation.

The endothelium in the coronary arteries is subject to wall shear stress and vessel wall strain, which influences the biology of the arterial wall. This study presents vessel-specific fluid-structure interaction (FSI) models of three coronary arteries, using directly measured experimental geometries and boundary conditions. FSI models are used to provide a more physiologically complete representation of vessel biomechanics, and have been extended to include coronary bending to investigate its effect on shear and strain. FSI both without- and with-bending resulted in significant changes in all computed shear stress metrics compared to CFD (p = 0.0001). Inclusion of bending within the FSI model produced highly significant changes in Time Averaged Wall Shear Stress (TAWSS) + 9.8% LAD, + 8.8% LCx, - 2.0% RCA; Oscillatory Shear Index (OSI) + 208% LAD, 0% LCx, + 2600% RCA; and transverse wall Shear Stress (tSS) + 180% LAD, + 150% LCx and + 200% RCA (all p < 0.0001). Vessel wall strain was homogenous in all directions without-bending but became highly anisotropic under bending. Changes in median cyclic strain magnitude were seen for all three vessels in every direction. Changes shown in the magnitude and distribution of shear stress and wall strain suggest that bending should be considered on a vessel-specific basis in analyses of coronary artery biomechanics.

A physics-based machine learning technique rapidly reconstructs the wall-shear stress and pressure fields in coronary arteries.

With the global rise of cardiovascular disease including atherosclerosis, there is a high demand for accurate diagnostic tools that can be used during a short consultation. In view of pathology, abnormal blood flow patterns have been demonstrated to be strong predictors of atherosclerotic lesion incidence, location, progression, and rupture. Prediction of patient-specific blood flow patterns can hence enable fast clinical diagnosis. However, the current state of art for the technique is by employing 3D-imaging-based Computational Fluid Dynamics (CFD). The high computational cost renders these methods impractical. In this work, we present a novel method to expedite the reconstruction of 3D pressure and shear stress fields using a combination of a reduced-order CFD modelling technique together with non-linear regression tools from the Machine Learning (ML) paradigm. Specifically, we develop a proof-of-concept automated pipeline that uses randomised perturbations of an atherosclerotic pig coronary artery to produce a large dataset of unique mesh geometries with variable blood flow. A total of 1,407 geometries were generated from seven reference arteries and were used to simulate blood flow using the CFD solver Abaqus. This CFD dataset was then post-processed using the mesh-domain common-base Proper Orthogonal Decomposition (cPOD) method to obtain Eigen functions and principal coefficients, the latter of which is a product of the individual mesh flow solutions with the POD Eigenvectors. Being a data-reduction method, the POD enables the data to be represented using only the ten most significant modes, which captures cumulatively greater than 95% of variance of flow features due to mesh variations. Next, the node coordinate data of the meshes were embedded in a two-dimensional coordinate system using the t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm. The reduced dataset for t-SNE coordinates and corresponding vector of POD coefficients were then used to train a Random Forest Regressor (RFR) model. The same methodology was applied to both the volumetric pressure solution and the wall shear stress. The predicted pattern of blood pressure, and shear stress in unseen arterial geometries were compared with the ground truth CFD solutions on "unseen" meshes. The new method was able to reliably reproduce the 3D coronary artery haemodynamics in less than 10 s.

Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells.

High laminar shear stress (HLSS) is vasculoprotective partly through induction of Kruppel-like factor 2 (KLF2). Connexin37 (Cx37) is highly expressed in endothelial cells (ECs) of healthy arteries, but not in ECs overlying atherosclerotic lesions. Moreover, Cx37 deletion in apolipoprotein E-deficient (ApoE(-/-)) mice increases susceptibility to atherosclerosis. We hypothesized that shear stress, through KLF2 modulation, may affect Cx37 expression in ECs. Cx37 expression and gap-junctional intercellular (GJIC) dye transfer are prominent in the straight portion of carotid arteries of ApoE(-/-) mice, but are reduced at the carotid bifurcation, a region subjected to oscillatory flow. Shear stress-modifying vascular casts were placed around the common carotid artery of ApoE(-/-) mice. Whereas Cx37 expression was conserved in HLSS regions, it was downregulated to ~50% in low laminar or oscillatory flow regions. To study the mechanisms involved, HUVECs or bEnd.3 cells were exposed to flow in vitro. Cx37 and KLF2 expression were increased after 24h of HLSS. Interestingly, shear-dependent Cx37 expression was significantly reduced after silencing of KLF2. Moreover after exposure to simvastatin, a well-known KLF2 inducer, KLF2 binds to the Cx37 promoter region as shown by ChIP. Finally, GJIC dye transfer was highly reduced after KLF2 silencing and was increased after exposure to simvastatin. HLSS upregulates the expression of Cx37 in ECs by inducing its transcription factor KLF2, which increases intercellular communication. Therefore, this effect of shear stress on Cx37 expression may contribute to the synchronization of ECs and participate in the protective effect of HLSS.

Dexamethasone arterializes venous endothelial cells by inducing mitogen-activated protein kinase phosphatase-1: a novel antiinflammatory treatment for vein grafts?

BACKGROUND: Vein grafting in coronary artery surgery is complicated by a high restenosis rate resulting from the development of vascular inflammation, intimal hyperplasia, and accelerated atherosclerosis. In contrast, arterial grafts are relatively resistant to these processes. Vascular inflammation is regulated by signaling intermediaries, including p38 mitogen-activated protein (MAP) kinase, that trigger endothelial cell (EC) expression of chemokines (eg, interleukin-8, monocyte chemotactic protein-1) and other proinflammatory molecules. Here, we have tested the hypothesis that p38 MAP kinase activation in response to arterial shear stress (flow) may occur more readily in venous ECs, leading to greater proinflammatory activation. METHODS AND RESULTS: Comparative reverse-transcriptase polymerase chain reaction and Western blotting revealed that arterial shear stress induced p38-dependent expression of monocyte chemotactic protein-1 and interleukin-8 in porcine jugular vein ECs. In contrast, porcine aortic ECs were protected from shear stress-induced expression of p38-dependent chemokines as a result of rapid induction of MAP kinase phosphatase-1. However, we observed with both cultured porcine jugular vein ECs and perfused veins that venous ECs can be protected by brief treatment with dexamethasone, which induced MAP kinase phosphatase-1 to suppress proinflammatory activation. CONCLUSIONS: Arterial but not venous ECs are protected from proinflammatory activation in response to short-term exposure to high shear stress by the induction of MAP kinase phosphatase-1. Dexamethasone pretreatment arterializes venous ECs by inducing MAP kinase phosphatase-1 and may protect veins from inflammation.

JAG1-NOTCH4 mechanosensing drives atherosclerosis.

Endothelial cell (EC) sensing of disturbed blood flow triggers atherosclerosis, a disease of arteries that causes heart attack and stroke, through poorly defined mechanisms. The Notch pathway plays a central role in blood vessel growth and homeostasis, but its potential role in sensing of disturbed flow has not been previously studied. Here, we show using porcine and murine arteries and cultured human coronary artery EC that disturbed flow activates the JAG1-NOTCH4 signaling pathway. Light-sheet imaging revealed enrichment of JAG1 and NOTCH4 in EC of atherosclerotic plaques, and EC-specific genetic deletion of Jag1 (Jag1ECKO) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1ECKO mice demonstrated that Jag1 suppresses subsets of ECs that proliferate and migrate. We conclude that JAG1-NOTCH4 sensing of disturbed flow enhances atherosclerosis susceptibility by regulating EC heterogeneity and that therapeutic targeting of this pathway may treat atherosclerosis.

c-Jun N-terminal kinase primes endothelial cells at atheroprone sites for apoptosis.

OBJECTIVE: Atherosclerosis is a focal disease that occurs predominantly at branches and bends of the arterial tree. Endothelial cells (EC) at atherosusceptible sites are prone to injury, which can contribute to lesion formation, whereas EC at atheroprotected sites are resistant. The c-Jun N-terminal kinase (JNK) is activated constitutively in EC at atherosusceptible sites but is inactivated at atheroprotected sites by mitogen-activated protein kinase phosphatase-1 (MKP-1). Here, we examined the effects of JNK activation on EC physiology at atherosusceptible sites. METHODS AND RESULTS: We identified transcriptional programs regulated by JNK by applying a specific pharmacological inhibitor to cultured EC and assessing the transcriptome using microarrays. This approach and subsequent validation by gene silencing revealed that JNK positively regulates the expression of numerous proapoptotic molecules. Analysis of aortae of wild-type, JNK1(-/-), and MKP-1(-/-) mice revealed that EC at an atherosusceptible site express proapoptotic proteins and are primed for apoptosis and proliferation in response to lipopolysaccharide through a JNK1-dependent mechanism, whereas EC at a protected site expressed lower levels of proapoptotic molecules and were protected from injury by MKP-1. CONCLUSIONS: Spatial variation of JNK1 activity delineates the spatial distribution of apoptosis and turnover of EC in arteries.

Mechanosensitive pathways are regulated by mechanosensitive miRNA clusters in endothelial cells.

Shear stress is known to affect many processes in (patho-) physiology through a complex, multi-molecular mechanism, termed mechanotransduction. The sheer complexity of the process has raised questions how mechanotransduction is regulated. Here, we comprehensively evaluate the literature about the role of small non-coding miRNA in the regulation of mechanotransduction. Regulation of mRNA by miRNA is rather complex, depending not only on the concentration of mRNA to miRNA, but also on the amount of mRNA competing for a single mRNA. The only mechanism to counteract the latter factor is through overarching structures of miRNA. Indeed, two overarching structures are present miRNA families and miRNA clusters, and both will be discussed in details, regarding the latest literature and a previous conducted study focussed on mechanotransduction. Both the literature and our own data support a new hypothesis that miRNA-clusters predominantly regulate mechanotransduction, affecting 65% of signalling pathways. In conclusion, a new and important mode of regulation of mechanotransduction is proposed, based on miRNA clusters. This finding implicates new avenues for treatment of mechanotransduction and atherosclerosis.