"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Purification and Analysis of Circulating Lipid Particles.

Lipid particles found in circulating extracellular fluids such as blood or lymph are essential for cellular homeostasis, metabolism and survival. Such particles provide essential lipids and fats which enable cells to synthesize new membranes and regulate different biochemical pathways. Imbalance in lipid particle metabolism can cause pathological states such as atherosclerosis. Here, elevated low-density lipoprotein (LDL) accumulation leads to fat-filled lesions or plaques in arterial walls. In this chapter, we provide a detailed set of protocols for the rapid and safe purification of lipid particles from human blood using high-speed ultracentrifugation. We provide a detailed set of assays for further analysis of the biochemical and cellular properties of these lipid particles. By combining these assays, we can better understand the complex roles of different lipid particles in normal physiology and disease pathology.

Structural Basis for Vascular Endothelial Growth Factor Receptor Activation and Implications for Disease Therapy.

Vascular endothelial growth factors (VEGFs) bind to membrane receptors on a wide variety of cells to regulate diverse biological responses. The VEGF-A family member promotes vasculogenesis and angiogenesis, processes which are essential for vascular development and physiology. As angiogenesis can be subverted in many disease states, including tumour development and progression, there is much interest in understanding the mechanistic basis for how VEGF-A regulates cell and tissue function. VEGF-A binds with high affinity to two VEGF receptor tyrosine kinases (VEGFR1, VEGFR2) and with lower affinity to co-receptors called neuropilin-1 and neuropilin-2 (NRP1, NRP2). Here, we use a structural viewpoint to summarise our current knowledge of VEGF-VEGFR activation and signal transduction. As targeting VEGF-VEGFR activation holds much therapeutic promise, we examine the structural basis for anti-angiogenic therapy using small-molecule compounds such as tyrosine kinase inhibitors that block VEGFR activation and downstream signalling. This review provides a rational basis towards reconciling VEGF and VEGFR structure and function in developing new therapeutics for a diverse range of ailments.

Scavenger Receptors as Biomarkers and Therapeutic Targets in Cardiovascular Disease.

The process of atherosclerosis leads to the formation of plaques in the arterial wall, resulting in a decreased blood supply to tissues and organs and its sequelae: morbidity and mortality. A class of membrane-bound proteins termed scavenger receptors (SRs) are closely linked to the initiation and progression of atherosclerosis. Increasing interest in understanding SR structure and function has led to the idea that these proteins could provide new routes for cardiovascular disease diagnosis, management, and treatment. In this review, we consider the main classes of SRs that are implicated in arterial disease. We consider how our understanding of SR-mediated recognition of diverse ligands, including modified lipid particles, lipids, and carbohydrates, has enabled us to better target SR-linked functionality in disease. We also link clinical studies on vascular disease to our current understanding of SR biology and highlight potential areas that are relevant to cardiovascular disease management and therapy.

Should dialysis-dependent patients with upper limb arterio-venous fistulae undergoing coronary artery bypass grafting avoid having ipsilateral in situ mammary artery grafts?

A best evidence topic in cardiac surgery was written according to a structured protocol. The question addressed was whether dialysis-dependent patients with upper limb arterio-venous fistulae (AVFs) undergoing coronary artery bypass grafting should avoid having ipsilateral in situ internal mammary artery (IMA) grafts. A literature search performed yielded 28 peer reviewed articles, of which 21 represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. The papers identified included 478 patients, of whom 219 had in situ IMA grafts with ipsilateral upper limb arterio-venous fistulae. There was a substantial variation between the papers, from single case reports to small retrospective cohort studies, but no randomized, controlled trials. The largest retrospective study included 155 patients and followed up for up to 5 years. Methods used to determine coronary steal included clinical assessment, electrocardiogram or echocardiographic changes, Doppler ultrasound of mammary arteries and angiography. The aggregate evidence suggested that 61 of the 219 patients with ipsilateral IMA grafts developed some clinical or physiological evidence of malperfusion during the use of the AVFs for dialysis. Comparisons with the contralateral IMA suggested that 27 of the 61 patients suffered similar problems when dialysis was applied. A number of studies used controls, including in situ right internal mammary artery (RIMA) flow and patients not on dialysis. In total, 32 patients had their in situ RIMA flow measurements studied, of which none showed any statistically significant flow alteration. While further strong evidence to demonstrate long-term outcomes is required, we recommend the avoidance, where possible, of ipsilateral in situ IMA grafts in patients with an upper limb AVF. There is sufficient experimental and anecdotal evidence to suggest that steal occurs and that in some patients, this has clinical implications on both morbidity and mortality. In this scenario, the use of the contralateral mammary is strongly advocated to maximize the patency of grafts in an already high-risk population.