Piezo1-mediated regulation of smooth muscle cell volume in response to enhanced extracellular matrix rigidity

Background and Purpose: Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined. Experimental Approach: Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation. Key Results: On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were upregulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation. Conclusions and Implications: In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes

Nano- and micro-structured temperature-sensitive hydrogels for rapidly responsive devices

This thesis aims to extend the understanding and explore the application of temperature-responsive hydrogel systems by integrating microelectromechanical systems (MEMS). Stimuli-responsive hydrogel systems are immensely investigated and applied in numerous fields, and interfacing with micro- and nano-fabrication techniques will open up more possibilities. In Chapter 2, the first biologically relevant, in vitro cell stretching device based on hydrogel surface instability was developed. This dynamic platform is constructed by embedding micro-heater devices under temperature-responsive surface-attached hydrogels. The fast and regional temperature change actuates the stretching and relaxation of the seeded human artery smooth muscle cell (HASMC) via controllable surface creasing instability. This device is engineered to mimic the in vivo environment of HASMCs, with independent control over substrate stiffness, mechanical cues and peptide attachment chemistry, and the response of HASMCs is inspected by the differentiation marker expression change. In Chapter 3, the swelling and deswelling kinetics of hydrogel sheets with high polymer content is inspected, with micro-heaters providing abrupt local temperature change. Poly(N-isopropylacrylamide) (PNIPAM) molecules can form hydrogen bonds with both water molecules and polymer chains, while poly(N,N-diethylacrylamide) (PDEAM) molecules can only form hydrogen bonds with water. The kinetics of the two hydrogel systems are systematically compared, revealing that while PDEAM shows one-step mass transport-limited kinetics, PNIPAM shows two-step kinetics behavior, presumably reflecting the strong influence of inter-molecular hydrogen bonding. The following two chapters document the attempts to further investigate into the hydrogel/MEMS interface. In Chapter 4, photo-patterning technique assists the study of regional modulus contrast influencing the formation of creases on the soft hydrogel surface, and it is demonstrated that the dimensions of the stiff patterns are relevant in directing the creasing direction. In Chapter 5, a photo-patternable sacrificial layer is designed based on crosslinking chemistry and gelation physics to potentially enable the construction of more complex MEMS devices

Assessment of the nanomechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy

Coronary atherosclerosis is a major cause of mortality and morbidity worldwide. Despite its systemic nature, atherosclerotic plaques form and develop at “predilection” sites often associated with disturbed biomechanical forces. Therefore, computational approaches that analyse the biomechanics (blood flow and tissue mechanics) of atherosclerotic plaques have come to the forefront over the last 20 years. Assignment of appropriate material properties is an integral part of the simulation process. Current approaches for derivation of material properties rely on macro-mechanical testing and are agnostic to local variations of plaque stiffness to which collagen microstructure plays an important role. In this work we used Atomic Force Microscopy to measure the stiffness of healthy and atherosclerotic coronary arteries and we hypothesised that are those are contingent on the local microstructure. Given that the optimal method for studying mechanics of arterial tissue with this method has not been comprehensively established, an indentation protocol was firstly developed and optimised for frozen tissue sections as well as a co-registration framework with the local collagen microstructure utilising the same tissue section for mechanical testing and histological staining for collagen. Overall, the mechanical properties (Young’s Modulus) of the healthy vessel wall (median = 11.0 kPa, n=1379 force curves) were found to be significantly stiffer (p=1.3410-10) than plaque tissue (median=4.3 kPa, n=1898 force curves). Within plaques, lipid-rich areas (median=2.2 kPa, n=392 force curves) were found significantly softer (p=1.4710-4) than areas rich in collagen, such as the fibrous cap (median=4.9 kPa, n=1506 force curves). No statistical difference (p=0.89) was found between measurements in the middle of the fibrous cap (median=4.8 kPa, n=868 force curves) and the cap shoulder (median=5.1 kPa, n=638 force curves). Macro-mechanical testing methods dominate the entire landscape of material testing techniques. Plaques are very heterogenous in composition and macro-mechanical methods are agnostic to microscale variations in plaque stiffness. Mechanical testing by indentation may be better suited to quantify local variations in plaque stiffness, that are potent drivers of plaque rupture.Open Acces

The use of Fluid Haemodynamics in the Diagnosis of Cardiovascular Disease

Currently the diagnostic methods used to detect cardiovascular disease largely rely on the inference of the presence of arterial stenosis. There is a clinical interest in the development of a diagnostic screening technique which can indicate the risk of developing cardiovascular disease at an early stage so that non-surgical treatments can be applied. The goal of this work was to develop and validate a diagnostic screening technique for cardiovascular disease using the mechanical biomarker wall shear stress. Improvements in wall shear stress measurements were made by using a 2D Fourier transform to extract additional spectral information from the ultrasound pulse and decrease the spectral variance by integrating across the bandwidth of transmitted frequencies. This technique was validated for a series of anatomically realistic flow phantoms which precisely mimicked the progression of wall stiffening that characterises cardiovascular disease. The newly developed spectral analysis technique demonstrated a higher diagnostic performance than the other techniques tested, both in terms of a greater degree of significance in detecting differences in vessel wall stiffness and in terms of the sensitivity and specificity of the technique. The technique could not be tested in pulsatile flow due to hardware limitations, but preliminary testing indicated that the increased performance of the technique would likely transfer to a physiological flow regime. The results of this work indicated that the algorithm had the potential to rival the diagnostic power of the current gold standard while being applicable at an earlier stage of cardiovascular disease

Biomechanical characterization of the remodeling of atherosclerotic arteries and plaque rupture

Empirical thesis.Bibliography: pages 140-152.Chapter 1. Introduction -- Chapter 2. Literature reviews -- Chapter 3. Mechanical characterization of the lamellar structure of human abdominal aorta in the development of atherosclerosis : an atomic force microscopy study -- Chapter 4. Progressive changes of elastic moduli of arterial wall and atherosclerotic plaque components during plaque development in human coronary arteries -- Chapter 5. Arterial wall remodeling in the development of atherosclerotic plaques: mechanical stress analys -- Chapter 6. Stress analysis of fracture of atherosclerotic plaques : crack propagation modeling -- Chapter 7. Conclusions, limitations and future directions -- Appendices -- References.Myocardial infarction is one of the leading causes of death in the world, resulting mostly from the sudden rupture of atherosclerotic plaques. Atherosclerotic plaques often form in specific regions within the arterial tree characterized by complex blood flow patterns. The progression of initial plaques depends on arterial wall remodeling defined as any persistent changes within the composition and size of the artery allowing adaptation to new circumstance. Plaques that are prone to rupture may often be clinically silent until the time of rupture; hence, the detection of vulnerable plaques is of great importance. It has been hypothesized that mechanical fatigue caused by pulsatile blood pressure is the main mechanism underlying atherosclerotic plaque rupture. This thesis aimed to characterise the remodeling and rupture of atherosclerotic plaques from a mechanical perspective.For the characterization of remodeling of atherosclerotic arteries, the alteration of composition and geometry of atherosclerotic arteries were examined separately. To analyse the modification of mechanical properties of atherosclerotic lesion components with plaque development, 20 human abdominal aortas, and 20 human coronary arteries were extracted at autopsy from subjects who died mostly due to post-accident complications. The force-spectroscopy mode of the atomic force microscopy (AFM) and histological examination were used to determine the elastic moduli of specified locations within samples. To investigate the leading causes of expansive remodeling of atherosclerotic arteries, as well as its consequences, many idealised models mimicking different stages of plaque development were designed. Using fluid-solid interaction analysis, the distribution of mechanical stresses among different models was estimated and the results were compared. For the mechanical characterization of plaque rupture, the geometry of atherosclerotic coronary plaques was reconstructed from histological images. Pulsatile blood pressure was considered as the external load and stress distribution within each model was estimated using finite element method. The process of mechanical fatigue failure within atherosclerotic plaques was simulated based on fracture mechanics roles. Then, the effect of plaque morphology, mean and pulse blood pressure and lipid pool stiffness on the number of fatigue cycles required for the fracture of atherosclerotic plaques was investigated.The outcomes of the AFM test on the atherosclerotic abdominal aorta and coronary arteries indicated the high variability of Young's modulus at different locations of plaque. Fibrous cap showed a lower stiffness than the fibrous tissue beneath the lipid pool. Calcification zones and lipid pools were the stiffest and softest components of atherosclerotic lesions respectively. With atherosclerotic plaque development, reduction of elastin lamellae stiffness, as well as stiffening of inter-lamellar zones, were detected in the medial layer of the diseased portion of the abdominal aortic wall. Moreover, the increase of media stiffness due to the build-up of fibrosis tissue and reduction of the elastic modulus of internal elastic lamina was observed in coronary arteries. Significant differences were observed between the stiffness of the medial layer in diseased parts and free-plaque segments in incomplete plaques. The results of computational modeling on the remodeling of atherosclerotic arteries showed that in atherosclerotic plaques with expansive remodeling, the level of endothelial shear stress, as well as the level wall circumferential stress in the diseased-free wall of the artery, remain approximately in the physiological range. However, higher levels of stress are induced at the shoulder and cap of plaques with expansive remodeling compared to atherosclerotic plaques which do not exhibit remodeling. With the numerical simulation of fatigue failure of atherosclerotic plaques, it was found that the required time for the plaque rupture decreased with increase of mean and pulse pressure and with reduction of lipid pool stiffness.Development of atherosclerotic plaques leads to alteration of micromechanical properties of the arterial wall in both elastic and muscular arteries. Findings suggest that most atherosclerotic arteries exhibit expansive remodeling to preserve the normal level of mechanical stresses sensed by endothelial and smooth muscles cells. The increase of pulse and mean blood pressure, intensification of stiffness mismatch between plaque components, as well as the expansive remodeling of atherosclerotic arteries, can increase the risk of plaque rupture.Mode of access: World wide web1 online resource (xxi, 152 pages) illustrations (some colour