Paul Coats

The evaluation of biological responses to polymeric scaffolds are important, given that the ideal scaffold should be biocompatible, biodegradable, promote cell adhesion and aid cell proliferation. The primary goal of this research was to measure the biological responses of cells against various polymeric and collagen electrospun scaffolds (polycaprolactone (PCL) and polylactic acid (PLA) polymers: PCL–drug, PCL–collagen–drug, PLA–drug and PLA–collagen–drug); cell proliferation was measured with a cell adhesion assay and cell viability using 5-bromo-2′-deoxyuridine (BrdU) and resazurin assays. The results demonstrated that there is a distinct lack of growth of cells against any irgasan (IRG) loaded scaffolds and far greater adhesion of cells against levofloxacin (LEVO) loaded scaffolds. Fourteen-day studies revealed a significant increase in cell growth after a 7-day period. The addition of collagen in the formulations did not promote greater cell adhesion. Cell viability studies revealed the levels of IRG used in scaffolds were toxic to cells, with the concentration used 475 times higher than the EC₅₀ value for IRG. It was concluded that the negatively charged carboxylic acid group found in LEVO is attracting positively charged fibronectin, which in turn is attracting the cell to adhere to the adsorbed proteins on the surface of the scaffold. Overall, the biological studies examined in this paper are valuable as preliminary data for potential further studies into more complex aspects of cell behaviour with polymeric scaffolds.

Pulmonary arterial hypertension (PAH) is a multi-factorial disease characterized by the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Excessive reactive oxygen species (ROS) formation resulted in alterations of the structure and function of pulmonary arterial walls, leading to right ventricular failure and death. Diabetes mellitus has not yet been implicated in pulmonary hypertension. However, recently, variable studies have shown that diabetes is correlated with pulmonary hypertension pathobiology, which could participate in the modification of pulmonary artery muscles. The metabolomic changes in PASMCs were studied in response to 25 mM of D-glucose (high glucose, or HG) in order to establish a diabetic-like condition in an in vitro setting, and compared to five mM of D-glucose (normal glucose, or LG). The effect of co-culturing these cells with an ideal blood serum concentration of cholecalciferol-D3 and tocopherol was also examined. The current study aimed to examine the role of hyperglycemia in pulmonary arterial hypertension by the quantification and detection of the metabolomic alteration of smooth muscle cells in high-glucose conditions. Untargeted metabolomics was carried out using hydrophilic interaction liquid chromatography and high-resolution mass spectrometry. Cell proliferation was assessed by cell viability and the [³H] thymidine incorporation assay, and the redox state within the cells was examined by measuring reactive oxygen species (ROS) generation. The results demonstrated that PASMCs in high glucose (HG) grew, proliferated faster, and generated higher levels of superoxide anion (O₂·⁻) and hydrogen peroxide (H₂O₂). The metabolomics of cells cultured in HG showed that the carbohydrate pathway, especially that of the upper glycolytic pathway metabolites, was influenced by the activation of the oxidation pathway: the pentose phosphate pathway (PPP). The amount of amino acids such as aspartate and glutathione reduced via HG, while glutathione disulfide, N6-Acetyl-L-lysine, glutamate, and 5-aminopentanoate increased. Lipids either as fatty acids or glycerophospholipids were downregulated in most of the metabolites, with the exception of docosatetraenoic acid and PG (16:0/16:1(9Z)). Purine and pyrimidine were influenced by hyperglycaemia following PPP oxidation. The results in addition showed that cells exposed to 25 mM of glucose were oxidatively stressed comparing to those cultured in five mM of glucose. Cholecalciferol (D3, or vitamin D) and tocopherol (vitamin E) were shown to restore the redox status of many metabolic pathways.

Guidance varies regarding the optimal timing of arteriovenous fistula (AVF) creation. The aim of this study was to evaluate the association between uraemia, haemodialysis and early AVF failure. Immunoblotting and cell proliferation assays were performed on vascular smooth muscle cells (VSM) cells isolated from long saphenous vein samples to evaluate the cells’ ability to proliferate when stimulated with uraemic (post-dialysis) and hyperuraemic (pre-dialysis) serum. Clinical data was collected prospectively for 569 consecutive radiocephalic (RCF) and brachiocephalic (BCF) fistulae. The primary outcome was AVF failure at 6 weeks. Dialysis status (haemodialysis (HD); pre-dialysis (Pre-D)), eGFR and serum urea were evaluated to determine if they affected early AVF failure. Human VSM cells demonstrated increased capacity to proliferate when stimulated with hyperuraemic serum. There was no significant difference in early failure rate of either RCF or BCF depending on dialysis status (pre-D RCF 31.4% (n = 188); pre-D BCF 22.4% (n = 165); HD RCF 29.3% (n = 99); HD BCF 25.9% (n = 116); p = 0.34). There was no difference in mean eGFR between those patients with early AVF failure and those without (11.2+/-0.2 ml/min/1.73 m2 vs. 11.6+/-0.4 ml/min/1.73 m2; p = 0.47). Uraemia was associated with early AVF failure (serum urea: 35.0+/-0.7 mg/dl vs. 26.6+/-0.3 mg/dl (p < 0.001)). We present the first in vivo evidence of an association between adverse early AVF outcomes and uraemia. This is supported mechanistically by in vitro work demonstrating a pro-mitogenic effect of hyperuraemic serum. We hypothesise that uraemia-driven upregulation of VSM cell proliferation at the site of surgical insult in contributes to higher early AVF failure rates.

Peripheral vascular disease (PVD) is associated with numerous pathophysiological adaptations of the microvasculature. Considering this, active and passive pressure-dependent and pressure-independent mechanisms of vascular control were studied in small resistance arteries isolated from patients with PVD. Using pressure myography and confocal microscopy, human skeletal muscle arteriolar structure and function were compared between paired arteries; one isolated from the healthy non-diseased proximal skeletal muscle vascular bed (PSM, internal control) and the other from the diseased ischaemic part of the leg [distal skeletal muscle (DSM)]. Structurally, arteries isolated from the diseased part of the leg displayed significant atrophy compared with the non-diseased arteries. Functionally, no differences were observed in the fundamental ability small resistance arteries to contract or relax. However, active pressure-dependent myogenic contraction was significantly reduced in DSM arteries compared with PSM arteries. DSM versus PSM; 3 +/- 1% versus 22 +/- 4% and 3.4% +/- 1.1% versus 25 +/- 4% at 80 and 120 mmHg, respectively. Furthermore, structural remodelling in DSM arteries could also be correlated with significant changes in vascular wall mechanics. DSM arteries displayed significantly greater incremental dispensability, wall stress and wall strain compared with PSM arteries as a product of pressure-dependent distension. These alterations in pressure-dependent active myogenic tone and passive mechanical properties goes some way to explain uncontrolled orthostatic-dependent changes in leg fluid volume and oedema formation experienced by these patients.

In critical limb ischaemia (CLI), the ability to regulate regional blood flow in the diseased portion of the leg would appear to be severely compromised. Considering this, pressure-dependent myogenic and mechanical properties of resistance arterioles isolated from control subjects and from patients with CLI were studied. Using confocal microscopy and pressure myography, subcutaneous resistance arteriole structure and function were compared between subcutaneous arterioles isolated from healthy volunteers [control subcutaneous (CS)] and non-diseased proximal subcutaneous (PS; internal control) and distal subcutaneous (DS) arterioles from the diseased ischaemic part of the limb from patients with CLI. Significant wall atrophy was observed in DS arterioles compared with PS and CS arterioles. Passive pressure-dependent mechanical properties were significantly altered in the diseased arterioles compared with PS and CS arterioles. Active pressure-dependent myogenic tone was completely absent in DS arterioles. The atrophic structural remodelling in DS arterioles were correlated with the changes in vascular mechanics, but not with the ability of these arterioles to contract in response to chemical stimuli. However, active pressure-dependent myogenic tone was absent in the DS arterioles. The combination of altered pressure-dependent passive mechanical and active myogenic tone goes some way in explaining CLI sequelae and poor outcome following surgical revascularization experienced by these patients.