Modelling the Impact of Cell Seeding Strategies on Cell Survival and Vascularisation in Engineered Tissue

Currently, the design of tissue engineered constructs for peripheral nerve repair is informed predominantly by experiments. However, translation to the clinical setting is slow, and engineered tissues have not surpassed the outcomes achieved by nerve grafts. Therapeutic cell survival and vascularisation are important for the assimilation of engineered tissue, and vascularisation provides vital directional cues for regenerating nerves. In this thesis, mathematical modelling informed by experimental data is used to investigate the impact of different therapeutic cell seeding strategies on cell survival and vascularisation in engineered tissue nerve repair constructs. A mathematical model of interactions between cells, oxygen and vascular endothelial growth factor (VEGF), consisting of three partial differential equations, is developed and parameterised against in vitro data. Key cell type-specific parameter values are derived, and the model is then used to simulate cell-solute interactions in a nerve repair construct over the first five days post-implantation in vivo. Simulations using uniform seeding cell densities of 88 and 13 × 10⁶ cells/ml result in the highest mean viable cell densities across the construct after 1 and 5 days respectively. However, simulations using seeding densities in the range of 200 – 300 ×10⁶ cells/ml result in steeper VEGF gradients and higher total VEGF concentrations across the construct, which could be beneficial for vascularisation. Simulations incorporating a porous construct sheath result in higher viable cell density predictions, but also lower total VEGF concentrations, than those run using an impermeable sheath. Subsequently, the cell-solute model is combined with a discrete model of angiogenesis that simulates vascular growth in response to gradients of VEGF. Simulation results suggest that different cell seeding strategies could influence the density, rate and morphology of vascularisation. The predictions generated in this work demonstrate how mathematical modelling as part of a wider multidisciplinary approach can provide direction for future experimental work

Hypertension:A problem of organ blood flow supply-demand mismatch

This review introduces a new hypothesis that sympathetically mediated hypertensive diseases are caused, in the most part, by the activation of visceral afferent systems that are connected to neural circuits generating sympathetic activity. We consider how organ hypoperfusion and blood flow supply–demand mismatch might lead to both sensory hyper-reflexia and aberrant afferent tonicity. We discuss how this may drive sympatho-excitatory-positive feedback and extend across multiple organs initiating, or at least amplifying, sympathetic hyperactivity. The latter, in turn, compounds the challenge to sufficient organ blood flow through heightened vasoconstriction that both maintains and exacerbates hypertension

The effect of cholinergic loss on the structure and function of the neurovascular unit: implications for cerebral amyloid angiopathy

Innervation of cerebral blood vessels is important for the regulation of vascular tone and adequate cerebral perfusion. Alzheimer’s disease (AD) is characterised by a loss of cholinergic innervation of the neurovascular unit (NVU). This loss may contribute not only to inefficient cerebrovascular perfusion, but also to the failure of removal of amyloid-β (Aβ), leading to its accumulation as cerebral amyloid angiopathy (CAA). This hypothesis was tested by mimicking loss of cholinergic innervation of the cortex and hippocampus in adult male C57BL/6 mice using the immunospecific toxin mu-p75-saporin. Using quadruple labelling immunohistochemistry and 3D reconstructions of the NVU, loss of perivascular cholinergic innervation was observed at the smooth muscle cells and basement membrane in the cortex and hippocampus, with additional perivascular loss at the astrocyte endfeet in the cortex. Arterial spin labelling fMRI revealed no differences in resting cerebral blood flow between control and saporin-treated mice in either the cortex or hippocampus. However, denervated vessels in the cortex, but not the hippocampus, failed to respond to pharmacological stimulation of endothelial nitric oxide synthase (eNOS). Further studies revealed a decrease in eNOS expression in the cortex, but an increase in eNOS expression and activity in the hippocampus following loss of cholinergic input. No differences were noted between control and saporin-treated mice in the efficiency of removal of Aβ along perivascular basement membranes in either the cortex or hippocampus. Treatment of TetO-APPSweInd mice with saporin resulted in a trend towards higher CAA load. These data suggest that there are innate differences between NVUs of the cortex and the hippocampus and a difference in their functional response to loss of cholinergic input. Loss of cholinergic input at the NVU may contribute to accumulation of Aβ; however, this likely requires additional pathological factors for CAA to develop

Investigating the expression of P2 purinergic receptors in sympathetic ganglia and perivascular nerves of arteries

Hypertension, also known as high blood pressure, is a serious medical condition that can increase the risk of several diseases including heart failure, stroke, and chronic kidney disease. It is a major cause of premature death worldwide, affecting up to one in every four men and one in every five women. One of the factors that play a role in hypertension progression is blood vessel innervation via perivascular sympathetic nerves. The importance of ATP released from sympathetic nerves as an extracellular signalling molecule is now well known and evidence is accumulating that ATP, and other nucleotides (ADP, UTP and UDP) play key roles in cardiovascular physiology and pathology via P2X (ion channel) and P2Y (G protein-coupled) receptors. Pre-junctional nerve terminals are equipped with a number of auto- and/or heteroreceptors, including ionotropic P2X and metabotropic P2Y receptors. Pre-junctional purinergic receptors serve as modulation sites of neurotransmitter release via ATP and other nucleotides released by neuronal activity and pathological signals. However, very little is currently known about the expression of P2 receptors in sympathetic post-ganglionic nerves that innervate blood tissues, where they have a potential pre-junctional role in regulating neurotransmission. This project aimed to characterise the P2 receptor profiles in mouse superior cervical ganglion and perivascular nerve terminals of the superior mesenteric artery and carotid artery. In addition, we aimed to investigate the expression and function of P2 receptors in human SH-SY5Y cells and in vitro differentiated SH-SY5Y cells as a model of adrenergic sympathetic neurons. RT-PCR revealed the presence of mRNA in all P2 receptors was expressed in superior cervical ganglion. However, some receptors (P2X2, P2X3, P2X5, P2X6, P2Y1, P2Y4 and P2Y12) were expressed in the superior cervical ganglion, but not expressed in the superior mesenteric artery and carotid artery. We suggest that receptor subtypes detected in sympathetic ganglia but not arteries could be expressed by sympathetic post-ganglionic nerves that innervate arteries and could serve as auto- and/or heteroreceptors to modulate neurotransmitters release. We confirmed the expression of all P2X (with the exception of P2X5 and P2X6) and P2Y1 at the protein level by immunohistochemistry, and we found these receptors colocalised with the general marker of neurons (PGP9.5), the adrenergic sympathetic marker tyrosine hydroxylase in superior cervical ganglion and perivascular nerves within the adventitia of the arteries. Moreover, it was determined in this project that the P2X agonist (BzATP) promoted intracellular calcium responses in differentiated SH-SY5Y cells are principally mediated by P2X7 receptors. The data outlined that P2X7 receptors are functionally active in differentiated SH-SY5Y cells and may be a novel and interesting drug target for modulating neurotransmission and thus may have potential therapeutic applications in vascular diseases including hypertension. Further research is required to fully investigate the physiological roles of all of the P2 receptor subtypes expressed in sympathetic ganglia and pre-junctional sympathetic nerve terminals of arteries