There is little doubt that disturbed hemodynamic forces play a role in the development of focal atherosclerotic lesions; however, these forces are difficult to measure directly. Instead, it has been proposed that artery geometry, as the primary determinant of local hemodynamics, could be a clinically feasible surrogate “local” risk factor for atherosclerosis. To date this hypothesis has not been satisfactorily tested, owing to superficial geometric surrogates of disturbed flow, small sample sizes (effect of systemic factors) and/or confounding effects of disease on geometry. The primary objective of this thesis was to test this “geometric risk hypothesis” via direct association of definitive geometric factors and an early atherosclerosis marker (e.g. wall thickness), made possible through our access to magnetic resonance imaging and risk factor data from the Atherosclerosis Risk in Communities’ Carotid MRI sub-study. First, it was shown that the 3D geometry of the carotid bifurcation could be characterized rapidly and reliably, even for routine clinical acquisitions. Second, two novel individual geometric variables were proposed, inspired by the influence of flare and tortuosity on flow separation, which were shown to improve the prediction of disturbed flow burden compared to “conventional” shape-based geometric variables. Third, these redefined geometric factors, but not their shape-based counterparts, were shown by multiple regression to be independent predictors of wall thickness, but only after thoroughly accounting for the secondary effects of wall thickening on geometry. These findings provide strong evidence for the geometric risk hypothesis of atherosclerosis in humans group study, and provide important guidance for future investigations of geometric risk; however, the incremental value of optimized geometric risk factors is questionable relative to conventional cardiovascular risk factors, which challenges their future clinical usage as additional non-modifiable local risk factors.

There is little doubt that disturbed hemodynamic forces play a role in the development of focal atherosclerotic lesions; however, these forces are difficult to measure directly. Instead, it has been proposed that artery geometry, as the primary determinant of local hemodynamics, could be a clinically feasible surrogate “local” risk factor for atherosclerosis. To date this hypothesis has not been satisfactorily tested, owing to superficial geometric surrogates of disturbed flow, small sample sizes (effect of systemic factors) and/or confounding effects of disease on geometry. The primary objective of this thesis was to test this “geometric risk hypothesis” via direct association of definitive geometric factors and an early atherosclerosis marker (e.g. wall thickness), made possible through our access to magnetic resonance imaging and risk factor data from the Atherosclerosis Risk in Communities’ Carotid MRI sub-study. First, it was shown that the 3D geometry of the carotid bifurcation could be characterized rapidly and reliably, even for routine clinical acquisitions. Second, two novel individual geometric variables were proposed, inspired by the influence of flare and tortuosity on flow separation, which were shown to improve the prediction of disturbed flow burden compared to “conventional” shape-based geometric variables. Third, these redefined geometric factors, but not their shape-based counterparts, were shown by multiple regression to be independent predictors of wall thickness, but only after thoroughly accounting for the secondary effects of wall thickening on geometry. These findings provide strong evidence for the geometric risk hypothesis of atherosclerosis in humans group study, and provide important guidance for future investigations of geometric risk; however, the incremental value of optimized geometric risk factors is questionable relative to conventional cardiovascular risk factors, which challenges their future clinical usage as additional non-modifiable local risk factors.

Atherosclerosis is an inflammatory disease of the arterial walls and is the major cause of heart attack and stroke. Atherosclerosis is localized to curves or branches in the vasculature where disturbed blood flow alters endothelial cell (EC) gene expression and induces EC dysfunction. Epigenetics controls aberrant gene expression in many diseases, but the mechanism of flow-induced epigenetic gene regulation in ECs via DNA methylation has not been well studied until very recently. The goal of this project was to determine how the DNA methylome responds to flow, causes altered gene expression, and regulates atherosclerosis development. Here, we found that d-flow increases DNA Methyltransferase 1 (DNMT1) expression in ECs, and we hypothesized that this causes a shift in the EC methylome and transcriptome towards a pro-inflammatory, pro-atherosclerotic gene expression program, and further that this leads to atherosclerosis development. To test this hypothesis, we employed both in vitro and in vivo experimental approaches combined with genome-wide studies of the transcriptome and DNA methylome according to the following three specific aims: 1) to elucidate the role of DNA Methyltransferase 1 in EC function, 2) to uncover the DNA methylation-dependent EC gene expression response to flow, and 3) to discover and examine master regulators of EC function that are controlled by DNA methylation. The work presented here has resulted in new knowledge about the epigenetic EC shear response, details the previously unstudied EC methylome, and implicates specific loci within the genome for additional studies on their role in EC biology and atherosclerosis. This work provides a foundation for novel and more targeted therapeutic strategies for CVD.

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Using a modular approach, a vascularized tissue construct is created by embedding functional cells within submillimeter-sized collagen cylinders (modules) while the outside surfaces are seeded with endothelial cells (EC). The void spaces created by randomly packing modules into a container form EC-lined perfusion channels. Upon implantation, the tissues are remodeled by and integrated into the host and experience, to some degree, immune and inflammatory responses. This work utilized microfluidic techniques to study and model the tissue remodeling in vitro in the absence of the host response. When the construct’s tortuous perfusion channels were reproduced in poly(dimethylsiloxane) microfluidic devices and lined with EC, perfusion at higher flow rates reduced EC activation and maintained the desired quiescent EC phenotype. When applying these results to collagen constructs, higher flow rates were not achievable due to the weak mechanical properties of collagen. To increase the collagen’s mechanical strength, a semi-synthetic collagen/poloxamine-methacrylate hydrogel was examined but due to its heterogeneous surface composition, there was inadequate EC attachment and the material was deemed unsuitable for this application. Proceeding with lower flow rates, tissues assembled within microfluidic perfusion chambers from EC-seeded collagen modules showed that over the course of 24 hours, perfusion did not significantly increase activation but instead increased KLF2 expression, a transcription factor involved in the establishment of EC quiescence, and disrupted VE-cadherin bonds between adjacent EC. However, after 1 week of perfusion, the majority of EC were lost. To ameliorate this loss, mesenchymal stromal cells (MSC) were embedded within the modules in order to take advantage of their anti-apoptotic and immunomodulation effects. The MSC temporarily mitigated the loss of the EC but did not prevent it. They did, however, take on a phenotype similar to smooth muscle cells and migrated towards the EC. Perhaps this indicates that the combination of EC, MSC and perfusion drives the creation and assembly of pseudo vessels. Together, the microfluidic techniques used in this study to assemble and perfuse modular tissues revealed new insights into the remodeling process and exposed critical issues surrounding the adaptation of the EC to the combination of perfusion, remodeling and changing flow fields.

Using a modular approach, a vascularized tissue construct is created by embedding functional cells within submillimeter-sized collagen cylinders (modules) while the outside surfaces are seeded with endothelial cells (EC). The void spaces created by randomly packing modules into a container form EC-lined perfusion channels. Upon implantation, the tissues are remodeled by and integrated into the host and experience, to some degree, immune and inflammatory responses. This work utilized microfluidic techniques to study and model the tissue remodeling in vitro in the absence of the host response. When the construct’s tortuous perfusion channels were reproduced in poly(dimethylsiloxane) microfluidic devices and lined with EC, perfusion at higher flow rates reduced EC activation and maintained the desired quiescent EC phenotype. When applying these results to collagen constructs, higher flow rates were not achievable due to the weak mechanical properties of collagen. To increase the collagen’s mechanical strength, a semi-synthetic collagen/poloxamine-methacrylate hydrogel was examined but due to its heterogeneous surface composition, there was inadequate EC attachment and the material was deemed unsuitable for this application. Proceeding with lower flow rates, tissues assembled within microfluidic perfusion chambers from EC-seeded collagen modules showed that over the course of 24 hours, perfusion did not significantly increase activation but instead increased KLF2 expression, a transcription factor involved in the establishment of EC quiescence, and disrupted VE-cadherin bonds between adjacent EC. However, after 1 week of perfusion, the majority of EC were lost. To ameliorate this loss, mesenchymal stromal cells (MSC) were embedded within the modules in order to take advantage of their anti-apoptotic and immunomodulation effects. The MSC temporarily mitigated the loss of the EC but did not prevent it. They did, however, take on a phenotype similar to smooth muscle cells and migrated towards the EC. Perhaps this indicates that the combination of EC, MSC and perfusion drives the creation and assembly of pseudo vessels. Together, the microfluidic techniques used in this study to assemble and perfuse modular tissues revealed new insights into the remodeling process and exposed critical issues surrounding the adaptation of the EC to the combination of perfusion, remodeling and changing flow fields.

Peripheral vascular grafts are used for the treatment of peripheral arterial disease and arteriovenous grafts for vascular access in end stage renal disease. The development of neo-intimal hyperplasia and thrombosis in the distal anastomosis remains the main reason for occlusion in that region. The local haemodynamics produced by a graft in the host vessel is believed to significantly affect endothelial function. Single spiral flow is a normal feature in medium and large sized vessels and it is induced by the anatomical structure and physiological function of the cardiovascular system. Grafts designed to generate a single spiral flow in the distal anastomosis have been introduced in clinical practice and are known as spiral grafts. In this work, spiral peripheral vascular and arteriovenous grafts were compared with conventional grafts using ultrasound and computational methods to identify their haemodynamic differences. Vascular-graft flow phantoms were developed to house the grafts in different surgical configurations. Mimicking components, with appropriate acoustic properties, were chosen to minimise ultrasound beam refraction and distortion. A dual-beam two-dimensional vector Doppler technique was developed to visualise and quantify vortical structures downstream of each graft outflow in the cross-flow direction. Vorticity mapping and measurements of circulation were acquired based on the vector Doppler data. The flow within the vascular-graft models was simulated with computed tomography based image-guided modelling for further understanding of secondary flow motions and comparison with the experimental results. The computational assessments provided a three-dimensional velocity field in the lumen of the models allowing a range of fluid dynamic parameters to be predicted. Single- or double-spiral flow patterns consisting of a dominant and a smaller vortex were detected in the outflow of the spiral grafts. A double- triple- or tetra-spiral flow pattern was found in the outflow of the conventional graft, depending on model configuration and Reynolds number. These multiple-spiral patterns were associated with increased flow stagnation, separation and instability, which are known to be detrimental for endothelial behaviour. Increased in-plane mixing and wall shear stress, which are considered atheroprotective in normal vessels, were found in the outflow of the spiral devices. The results from the experimental approach were in agreement with those from the computational approach. This study applied ultrasound and computational methods to vascular-graft phantoms in order to characterise the flow field induced by spiral and conventional peripheral vascular and arteriovenous grafts. The results suggest that spiral grafts are associated with advanced local haemodynamics that may protect endothelial function and thereby may prevent their outflow anastomosis from neo-intimal hyperplasia and thrombosis. Consequently this work supports the hypothesis that spiral grafts may decrease outflow stenosis and hence improve patency rates in patients.