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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session C

Poster H15 - Histological Confirmation of Disturbed Flow-Induced Protein Expression in Carotid Arteries of Mice with Sickle Cell Disease

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Sickle cell disease (SCD) is characterized by mutation of the beta globin gene, causing sickling of red blood cells. Children with SCD are at an elevated risk of stroke, with up to 37% experiencing a silent stroke by the age of 14. The cause of this risk is unclear, but the suggested reason is accumulated arterial damage, indicated by increased elastic lamina fragmentation. Previously, we showed that cathepsin K, a powerful elastase and collagenase, is upregulated in the arteries of mice with SCD. This expression is mediated biochemically by increased levels of proinflammatory cytokine TNFα, and biomechanically by low and oscillatory shear stress on endothelial cells caused by disturbed flow. Cystatin C, the endogenous inhibitor of cathepsins, is inversely regulated, being elevated by high, unidirectional shear stress. Wall shear stress (WSS), the frictional force of blood flow on the vessel wall, can vary throughout the vessel due to asymmetrical flow profiles. Flow disturbances are generally known to occur at sharp curvatures or bifurcations. SCD is unique in that transient adhesions and red blood cells aggregations can disturb flow in straight regions of arteries.
Computational fluid dynamics (CFD) is a powerful tool to simulate arterial blood flow. To visualize localized differences in protein expression induced by flow disturbances, we are matching CFD-derived WSS profiles to histological analysis of artery sections. We hypothesize that there will be lower levels of cystatin C expression and increased elastic lamina degradation at sites of disturbed flow in the carotid arteries of mice with SCD.

Materials and

Methods:  Magnetic resonance angiography (MRA) was used to image Townes humanized sickle cell transgenic mice. Each model consisted of mice with wild-type (AA), sickle-trait (AS), and sickle cell (SS) genotypes. We isolated the common carotid arteries by segmenting the MRA images in Materialise Mimics, before creating 3-dimensional finite element models of each vessel. For each model, we ran blood flow simulations in SimVascular, using a steady-state, no-slip boundary condition. Using ParaView, we visualized metrics from the results of our simulations, such as blood velocity and wall shear stress. Mice were euthanized at 5 or 7 months of age , perfusion fixed, and their carotid arteries were removed, fixed, and embedded in paraffin. We then sectioned from the base of the vessel to the carotid bifurcation using a Leica Biosystems HistoCore MULTICUT, with attention to identified stenotic regions. We performed Verhoeff Van Gieson elastin staining, Masson’s trichrome staining, and immunohistochemistry staining for cystatin C and cathepsin K. Stained slides were imaged using a Zeiss confocal microscope and a Hamamatsu NanoZoomer before analysis in ImageJ. Statistical results were compiled using Prism.

Results, Conclusions, and Discussions: From the CFD performed on reconstructed mouse carotid arteries, SS mice showed an increase in the number and area of regions of low and oscillatory WSS compared to AA mice. Furthermore, we observed localization of low WSS in SS mice to a particular side of the vessel compared to a more uniform distribution in AA mice. This was shown through cross-sectional WSS profiles taken from the segment of the common carotid artery before the bifurcation, depicting the distribution of shear stresses throughout the circumference of the artery wall. Stained histological sections corresponding to location of the WSS profiles along the length of the computationally reconstructed arterial geometries were orientated to match the WSS profiles (Figure 1). CFD results were confirmed by localized damage in the artery wall, indicated by an increased number of elastic lamina breaks in areas of low shear stress. In areas with increased elastic lamina degradation, we observed lower levels of immunostaining for cystatin C. Conversely, we observed higher levels of cystatin C expression in areas with intact elastin. This suggests that disturbed flow causes lower expression of cystatin C, allowing cathepsins to be active and degrade their target substrate, elastin. This indicates the asymmetry around the circumference of the artery that was identified by the CFD analysis and confirmed by immunohistochemical staining of the shear stress-regulated inhibitor of cathepsins, cystatin C. Comparing the results of CFD to histological analysis allows us to understand how disturbed flow in SCD leads to differential protein expression and the subsequent arterial remodeling that is thought to cause stroke.

Acknowledgements (Optional):  NIH-BESIP, NIBIB, BETA Center, and the MATRICES Lab