An Allosteric Shift in CD11c Affinity Activates a Proatherogenic State in Arrested Intermediate Monocytes.

Intermediate monocytes (iMo; CD14+CD16+) increase in number in the circulation of patients with unstable coronary artery disease (CAD), and their recruitment to inflamed arteries is implicated in events leading to mortality following MI. Monocyte recruitment to inflamed coronary arteries is initiated by high affinity ß2-integrin (CD11c/CD18) that activates ß1-integrin (VLA-4) to bind endothelial VCAM-1. How integrin binding under shear stress mechanosignals a functional shift in iMo toward an inflammatory phenotype associated with CAD progression is unknown. Whole blood samples from patients treated for symptomatic CAD including non-ST elevation MI, along with healthy age-matched subjects, were collected to assess chemokine and integrin receptor levels on monocytes. Recruitment on inflamed human aortic endothelium or rVCAM-1 under fluid shear stress was assessed using a microfluidic-based artery on a chip (A-Chip). Membrane upregulation of high affinity CD11c correlated with concomitant activation of VLA-4 within focal adhesive contacts was required for arrest and diapedesis across inflamed arterial endothelium to a greater extent in non-ST elevation MI compared with stable CAD patients. The subsequent conversion of CD11c from a high to low affinity state under fluid shear activated phospho-Syk- and ADAM17-mediated proteolytic cleavage of CD16. This marked the conversion of iMo to an inflammatory phenotype associated with nuclear translocation of NF-κB and production of IL-1ß+ We conclude that CD11c functions as a mechanoregulator that activates an inflammatory state preferentially in a majority of iMo from cardiac patients but not healthy patients.

Mechanoregulation of p38 activity enhances endoplasmic reticulum stress-mediated inflammation by arterial endothelium.

Endothelial up-regulation of VCAM-1 at susceptible sites in arteries modulates the recruitment efficiency of inflammatory monocytes that initiates atherosclerotic lesion formation. We reported that hydrodynamic shear stress (SS) mechanoregulates inflammation in human aortic endothelial cells through endoplasmic reticulum (ER) stress via activation of the transcription factor x-box binding protein 1 (XBP1). Here, a microfluidic flow channel that produces a linear gradient of SS along a continuous monolayer of endothelium was used to delve the mechanisms underlying transcriptional regulation of TNF-α-stimulated VCAM-1 expression. High-resolution immunofluorescence imaging enabled continuous detection of platelet endothelial cell adhesion molecule 1 (PECAM-1)-dependent, outside-in signaling as a function of SS magnitude. Differential expression of VCAM-1 and intercellular adhesion molecule 1 (ICAM-1) was regulated by the spatiotemporal activation of MAPKs, ER stress markers, and transcription factors, which was dependent on the mechanosensing of SS through PECAM-1 and PI3K. Inhibition of p38 specifically abrogated the rise to peak VCAM-1 at low SS (2 dyn/cm2), whereas inhibition of ERK1/2 attenuated peak ICAM-1 at high SS (12 dyn/cm2). A shear stress-regulated temporal rise in p38 phosphorylation activated the nuclear translocation of XBP1, which together with the transcription factor IFN regulatory factor 1, promoted maximum VCAM-1 expression. These data reveal a mechanism by which SS sensitizes the endothelium to a cytokine-induced ER stress response to spatially regulate inflammation promoting atherosclerosis.-Bailey, K. A., Moreno, E., Haj, F. G., Simon, S. I., Passerini, A. G. Mechanoregulation of p38 activity enhances endoplasmic reticulum stress-mediated inflammation by arterial endothelium.

Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation.

Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα.

On-chip endothelial inflammatory phenotyping.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers. These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-, lipid- and RAGE-induced inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Shear stress modulates RAGE-mediated inflammation in a model of diabetes-induced metabolic stress.

Atherosclerosis occurs preferentially at sites of disturbed blood flow despite the influence of risk factors contributing to systemic inflammation. The receptor for advanced glycation endproducts (RAGE) is a prominent mediator of inflammation in diabetes that is upregulated in atherosclerotic plaques. Our goal was to elucidate a role for arterial hemodynamics in the regulation of RAGE expression and activity. Endothelial RAGE expression was elevated at sites of flow disturbance in the aortas of healthy swine. To demonstrate a direct role for physiological shear stress (SS) in modulating RAGE expression, human aortic endothelial cells (HAEC) were exposed to high SS (HSS; 15 dyn/cm(2)), which downregulated RAGE by fourfold, or oscillatory SS (OSS; 0 ± 5 dyn/cm(2)), which upregulated RAGE by threefold, compared with static culture at 4 h. In a model of diabetes-induced metabolic stress, HAEC were chronically conditioned under high glucose (25 mM) and then simultaneously stimulated with TNF-α (0.5 ng/ml) and the RAGE ligand high mobility group box 1 (HMGB1). A 50% increase in VCAM-1 expression over TNF-α was associated with increased cytoplasmic and mitochondrial reactive oxygen species and NF-κB activity. This increase was RAGE-specific and NADPH oxidase dependent. In activated HAEC, OSS amplified HMGB1-induced VCAM-1 (3-fold) and RAGE (1.6-fold) expression and proportionally enhanced monocyte adhesion to HAEC in a RAGE-dependent manner, while HSS mitigated these increases to the level of TNF-α alone. We demonstrate that SS plays a fundamental role in regulating RAGE expression and inflammatory responses in the endothelium. These findings may provide mechanistic insight into how diabetes accelerates the nonrandom distribution of atherosclerosis in arteries.