Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm's canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell-likely accounting for increased outflow resistance-that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness-and its biomechanical effects on pore formation-as a therapeutic target in glaucoma.cell mechanics | primary open-angle glaucoma | modulus | cytoskeleton

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 2/2 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 20.67 + 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.

The bulk of aqueous humor passing through the conventional outflow pathway must cross the inner wall endothelium of Schlemm’s canal (SC), likely through micron-sized transendothelial pores. SC pore density is reduced in glaucoma, possibly contributing to obstructed aqueous humor outflow and elevated intraocular pressure (IOP). Little is known about the mechanisms of pore formation; however, pores are often observed near dome-like cellular outpouchings known as giant vacuoles (GVs) where significant biomechanical strain acts on SC cells. We hypothesize that biomechanical strain triggers pore formation in SC cells. To test this hypothesis, primary human SC cells were isolated from three non-glaucomatous donors (aged 34, 44 and 68), and seeded on collagen-coated elastic membranes held within a membrane stretching device. Membranes were then exposed to 0%, 10% or 20% equibiaxial strain, and the cells were aldehyde-fixed 5 minutes after the onset of strain. Each membrane contained 3–4 separate monolayers of SC cells as replicates (N = 34 total monolayers), and pores were assessed by scanning electron microscopy in 12 randomly selected regions (~65,000 μm2 per monolayer). Pores were identified and counted by four independent masked observers. Pore density increased with strain in all three cell lines (p < 0.010), increasing from 87±37 pores/mm2 at 0% strain to 342±71 at 10% strain; two of the three cell lines showed no additional increase in pore density beyond 10% strain. Transcellular “I-pores” and paracellular “B-pores” both increased with strain (p < 0.038), however B-pores represented the majority (76%) of pores. Pore diameter, in contrast, appeared unaffected by strain (p = 0.25), having a mean diameter of 0.40 μm for I-pores (N = 79 pores) and 0.67 μm for B-pores (N = 350 pores). Pore formation appears to be a mechanosensitive process that is triggered by biomechanical strain, suggesting that SC cells have the ability to modulate local pore density and filtration characteristics of the inner wall endothelium based on local biomechanical cues. The molecular mechanisms of pore formation and how they become altered in glaucoma may be studied in vitro using stretched SC cells.

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.

C oronary heart disease is projected to remain the worldwide leading cause of death until 2030.1 Coronary heart disease is a major cause of morbidity and reduced quality of life with enormous economic consequences.2,3 Atherosclerosis, a multifocal lipid-driven inflammatory process, is the principal underlying pathology in patients with coronary heart disease, which commonly presents clinically with symptoms secondary to luminal narrowing of an epicardial coronary artery or 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. an acute coronary syndrome. The latter is a major cause of coronary heart disease death and most commonly results from rupture at the site of a thin cap fibroatheroma (TCFA) leading to coronary thrombosis. 4The precise environmental cues that lead plaques toward an advanced and high-risk phenotype are not yet fully elucidated, but disturbed blood flow is thought to play a central role in both lesion initiation and progression.5 Disturbed flow is most frequently quantified by metrics of shear stress, which is the frictional force imposed by blood flowing over the endothelial surface, and association between these metrics and coronary atherosclerotic lesion stage have been demonstrated in vivo in both animal models 6-9 and patients. 10,11 However, few studies have investigated the impact of prevalent shear co...