Influence of shear stress magnitude and direction on atherosclerotic plaque composition.

The precise flow characteristics that promote different atherosclerotic plaque types remain unclear. We previously developed a blood flow-modifying cuff for ApoE-/- mice that induces the development of advanced plaques with vulnerable and stable features upstream and downstream of the cuff, respectively. Herein, we sought to test the hypothesis that changes in flow magnitude promote formation of the upstream (vulnerable) plaque, whereas altered flow direction is important for development of the downstream (stable) plaque. We instrumented ApoE-/- mice (n = 7) with a cuff around the left carotid artery and imaged them with micro-CT (39.6 µm resolution) eight to nine weeks after cuff placement. Computational fluid dynamics was then performed to compute six metrics that describe different aspects of atherogenic flow in terms of wall shear stress magnitude and/or direction. In a subset of four imaged animals, we performed histology to confirm the presence of advanced plaques and measure plaque length in each segment. Relative to the control artery, the region upstream of the cuff exhibited changes in shear stress magnitude only (p < 0.05), whereas the region downstream of the cuff exhibited changes in shear stress magnitude and direction (p < 0.05). These data suggest that shear stress magnitude contributes to the formation of advanced plaques with a vulnerable phenotype, whereas variations in both magnitude and direction promote the formation of plaques with stable features.

Quantification of plaque stiffness by Brillouin microscopy in experimental thin cap fibroatheroma.

Plaques vulnerable to rupture are characterized by a thin and stiff fibrous cap overlaying a soft lipid-rich necrotic core. The ability to measure local plaque stiffness directly to quantify plaque stress and predict rupture potential would be very attractive, but no current technology does so. This study seeks to validate the use of Brillouin microscopy to measure the Brillouin frequency shift, which is related to stiffness, within vulnerable plaques. The left carotid artery of an ApoE(-/-)mouse was instrumented with a cuff that induced vulnerable plaque development in nine weeks. Adjacent histological sections from the instrumented and control arteries were stained for either lipids or collagen content, or imaged with confocal Brillouin microscopy. Mean Brillouin frequency shift was 15.79 ± 0.09 GHz in the plaque compared with 16.24 ± 0.15 (p < 0.002) and 17.16 ± 0.56 GHz (p < 0.002) in the media of the diseased and control vessel sections, respectively. In addition, frequency shift exhibited a strong inverse correlation with lipid area of -0.67 ± 0.06 (p < 0.01) and strong direct correlation with collagen area of 0.71 ± 0.15 (p < 0.05). This is the first study, to the best of our knowledge, to apply Brillouin spectroscopy to quantify atherosclerotic plaque stiffness, which motivates combining this technology with intravascular imaging to improve detection of vulnerable plaques in patients.

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.

The effect of varying glucose levels on the ex vivo crystalline lens: implications for hyperglycaemia-induced refractive changes.

PURPOSE: Refractive changes in diabetic eyes have long been reported but with equivocal results. The lens has been a more recent focus as the source of any change but it is possible that multiple sources of variation have made it difficult to demonstrate a systematic change clinically. The aim of this study was therefore to use a bovine lens model to investigate the optical changes in hyperglycaemia and when lenses are returned to normal glucose levels as would occur following commencement of treatment. METHOD: Bovine eyes were obtained and their lenses excised under sterile conditions before placing them in culture medium within an incubator using standard tissue culture techniques. In the first experiment, lenses were transferred into culture medium containing 5 mm (n = 12), 15 mm (n = 12) and 30 mm (n = 12) glucose. Measurements were made of the change in back vertex focusing distance with equatorial lens diameter using the ScanTox(™) measurement system. From these measurements, the back vertex focal length and primary longitudinal spherical aberration were derived. In a second experiment, lenses maintained at 30 mm glucose (n = 7) were stepped down to 5 mm glucose to simulate starting diabetic therapy and measured in the same way. Changes over time were assessed with a linear regression model. RESULTS: A trend towards myopia was observed with increasing hyperglycaemia, this was not statistically significant. When lenses were stepped-down from hyperglycaemia to normal physiological levels of glucose, a hyperopic shift was observed in line with published clinical studies that again failed to reach statistical significance. High variability in the measurement on longitudinal spherical aberration prevented any significant trends being measured. CONCLUSIONS: Our results suggest that there are no consistent crystalline lens-induced refractive changes following exposure to hyperglycaemia for time-periods up to 5 days used in the current study. It is possible that bovine lenses are able to offset the raised osmotic pressure from high glucose levels in the short-term by a process of osmoregulation and that repeated osmotic stress or longer term exposure may be required to induce the changes in refraction that are seen clinically.

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.