Gurunathan Murugesan

Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein.

Endothelial cell (EC) movement is required for the development and repair of blood vessels. We have previously shown that LDL oxidized by transition metals almost completely suppressed the wound-healing migratory response of vascular EC in vitro. We now report that lysophosphatidylcholine (lysoPC), a lipid component of oxidized LDL, has an important role in the antimigratory activity of the lipoprotein. Purified 1-palmitoyl lysoPC inhibited movement with a half-maximal activity at 12-15 micrometers, and near complete inhibition at 20 micrometers; the inhibitory concentration of lysoPC was consistent with its abundance in oxidized LDL. The inhibition was not due to cytotoxicity since protein synthesis was unaffected and since EC movement was restored after removal of lysoPC. Lysophospholipid activity was dependent on lipid structure. LysoPCs containing 1-position C16 or C18 saturated fatty acids were antimigratory, but those containing C < or = 14 saturated fatty acids or polyunsaturated fatty acids were not. The activity of 1-palmitoyl lysolipids with various head groups was examined. Lysophosphatidylinositol was more antimigratory than lysophosphatidylglycerol and lysophosphatidylcholine, which were more potent than lysophosphatidylserine and lysophosphatidylethanolamine. Monoglyceride was inactive while lysophosphatidate had promigratory activity. These results are consistent with head group size rather than charge as a critical determinant of activity. To show that lysophospholipids within an intact lipoprotein were active, LDL was treated with bee venom phospholipase A2 (PLA2). The modified lipoprotein inhibited EC movement to the same extent as iron-oxidized LDL and antimigratory activity correlated with the amount of lysoPC formed. To determine antimigratory activity of lysoPC present in oxidized LDL, lipid extracts from oxidized LDL were fractionated by normal phase HPLC. The fraction comigrating with lysoPC had nearly the same activity as the total extract confirming that lysoPC (or a co-eluting lipid) was a major antimigratory molecule in oxidized LDL. These studies demonstrate that lysoPC in oxidized LDL limit EC wound healing responses in vitro, and suggest a possible role for lysolipids in limiting endothelial regeneration after a denuding injury in vivo.

Membrane microviscosity regulates endothelial cell motility

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as α-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.

Platelet CD36 surface expression levels affect functional responses to oxidized LDL and are associated with inheritance of specific genetic polymorphisms.

CD36 modulates platelet function via binding to oxidized LDL (oxLDL), cell-derived microparticles, and thrombospondin-1. We hypothesized that the level of platelet CD36 expression may be associated with inheritance of specific genetic polymorphisms and that this would determine platelet reactivity to oxLDL. Analysis of more than 500 subjects revealed that CD36 expression levels were consistent in individual donors over time but varied widely among donors (200-14,000 molecules per platelet). Platelet aggregometry and flow cytometry in a subset of subjects with various CD36 expression levels revealed a high level of correlation (r² = 0.87) between platelet activation responses to oxLDL and level of CD36 expression. A genome-wide association study of 374 white subjects from the Cleveland Clinic ASCLOGEN study showed strong associations of single nucleotide polymorphisms in CD36 with platelet surface CD36 expression. Most of these findings were replicated in a smaller subset of 25 black subjects. An innovative gene-based genome-wide scan provided further evidence that single nucleotide polymorphisms in CD36 were strongly associated with CD36 expression. These studies show that CD36 expression on platelets varies widely, correlates with functional responses to oxLDL, and is associated with inheritance of specific CD36 genetic polymorphisms, and suggest that inheritance of specific CD36 polymorphisms could affect thrombotic risk.

Endothelial cell formation of focal adhesions on hydrophilic plasma polymers

Endothelial cell (EC) formation and distribution of both actin stress fibers and focal contacts on hydrophilic plasma polymers derived from gamma-butyrolactone (GBL) and n-vinylpyrrolidone (NVP) were examined to determine their ability to support endothelial cell growth in comparison to fibronectin. One hour after seeding, cells adhered and spread moderately on fibronectin with the development of defined actin stress fibers and focal adhesions compared to NVP and GBL, on which the cells were spread with poorly developed stress fibers and a perinuclear localization of vinculin. At 3 h, cells continue to spread more on fibronectin and NVP than GBL, and the cells on fibronectin had well-defined stress fibers terminating with sharp spikes of vinculin, typical of focal adhesions. At this time point, paxillin, a signaling component of focal adhesion complex, was predominantly localized at the focal contacts for well-spread EC on fibronectin and NVP, whereas it was almost entirely concentrated in the perinuclear region of less-spread cells on GBL. However, by 24h, cells were much more spread on all three surfaces with defined stress fibers and focal contacts although EC expression of vinculin and paxillin was moderate on GBL compared to fibronectin and NVP. These results suggest that EC can form cytoskeletal structures necessary for cell survival on plasma polymers, especially on more hydrophilic NVP, which could be exploited as interface material for seeding endothelial cells.

Endothelial cell expression of monocyte chemotactic protein-1, tissue factor, and thrombomodulin on hydrophilic plasma polymers

Endothelial cells (EC) from human aortas, microvessels, and pulmonary arteries were examined for their expression and activity of monocyte chemotactic protein-1 (MCP-1), tissue factor, and thrombomodulin in response to tumor necrosis factor-alpha (TNFalpha) on the hydrophilic plasma polymers gamma-butyrolactone (GBL) and N-vinyl-2-pyrrolidone (NVP), along with a fibronectin (FN) control. RNAs isolated from EC grown on these substrates were subjected to reverse transcription-polymerase chain reaction (RT-PCR) and dot-blot analysis. EC expression of MCP-1 and tissue factor was very low in the absence of TNFalpha but high for constitutively expressed thrombomodulin. TNFalpha induced EC expression and activity of MCP-1 and tissue factor and suppressed that of thrombomodulin on all substrates. Greater differences were seen with regard to cell origin, but little difference was seen among substrates. Basal secretion of MCP-1 was very low in aortic and pulmonary artery EC and even less in microvascular EC. TNFalpha increased MCP-1 secretion significantly in aortic and pulmonary artery EC but to a lesser extent in microvascular EC. In contrast, tissue factor expression was greater in pulmonary artery EC compared to microvascular and aortic EC. Basal expression of thrombomodulin was largely comparable for all three cell types grown on different surfaces, but TNFalpha suppressed thrombomodulin to different extents depending on the origin of the EC. The activity of tissue factor and thrombomodulin and the secretion of MCP-1 by EC were largely correlated with the expression of these genes. We conclude that EC origin may be an important determinant of cellular function on hydrophilic plasma polymer substrates. However, the differences in cellular function due to variations in substrate surface hydrophilicity could have been masked by the extracellular matrix remodeling that presumably occurred during EC growth to confluence.

Integrin-Dependent Interaction of Human Vascular Endothelial Cells on Biomimetic Peptide Surfactant Polymers

Biomimetic surfactant polymers designed by molecular grafting of pendant RGD peptides (Pep) and dextran oligosaccharides (Dex) in different ratios onto the backbone of poly(vinyl amine) (PVAm) were examined for their ability to promote endothelial cell (EC) growth. Adhesion, formation of focal contacts, and expression of integrin receptors were examined in EC seeded onto a series of novel surfactants containing 100% dextran (PVAm[Pep (0%)]) to 100% peptide (PVAm[Pep (100%)]) compared to fibronectin control. Interaction of EC on polymer was specific, as soluble GRGDSP, but not GRGESP, was able to inhibit both adhesion and spreading of EC. At three hours, EC attachment and spreading were rapid and comparable on fibronectin and PVAm[Pep (100%)], rounded on PVAm[Pep (0%)], and intermediate on PVAm[Pep (25%)], (PVAm[Pep (50%)], and PVAm[Pep (75%)], with increasing peptide ratio favoring more spreading, although all the substrates had similar hydrophilicity. Cells that spread well on fibronectin and PVAm[Pep (100%)] had sharp spikes of vinculin localized at the termination point of actin stress fibers. Formation of stress fibers and focal adhesions on other substrates were correlated with spreading pattern of EC and the peptide content. EC seeded on fibronectin expressed f 5 g 1 integrins all along the stress fibers and throughout the entire cytoskeleton, but this distribution pattern was less prominent on PVAm[Pep (100%)]. However, expression and distribution of vitronectin receptors ( f v g 3 ) were similar on both fibronectin and PVAm[Pep (100%)], suggesting a strong cell adhesion on PVAm[Pep (100%)]. Viability of EC was also comparable on both fibronectin and PVAm[Pep (100%)] at 24 h. Substrates with high proportion of dextran limited cell adhesion, probably by decreasing protein adsorption. These results suggest that it may be possible to engineer substrates that promote cell adhesion in a receptor-dependent manner while blocking nonspecific protein adsorption, which may have potential as interface materials for prostheses used in cardiovascular system.

Molecular Subtype Classification of Formalin-Fixed, Paraffin-Embedded Diffuse Large B-Cell Lymphoma Samples on the ICEPlex® System

Microarray gene expression profiling (GEP) has been used to identify molecular subtypes of diffuse large B-cell lymphoma (DLBCL) based on the similarity of GEP to a putative “cell of origin” (COO) and defines two molecular subtypes: activated B-cell-like (ABC) DLBCL and germinal centre B-cell-like (GCB) DLBCL (Alizadeh, et al 2000). This dichotomization has prognostic and biological significance, with the ABC subtype having a worse outcome and distinct pathobiology that includes activation of the B-cell receptor and nuclear factor (NF)-κB pathways (Lenz, et al 2008). Attempts to reduce this subclassification to practice using immunohistochemistry are fraught with technical and interpretive issues such that a need for practical quantitative molecular assays exists (Coutinho, et al 2013, de Jong, et al 2007, de Jong, et al 2009, Salles, et al 2011). Indeed, studies have refined this COO concept using limited gene sets, including a 14-gene model to assign ABC and GCB DLBCL subtype developed by Wright et al (2003) as well as a recently-described DLBCL subtyping assay based on parsimonious digital gene expression (Nanostring) technology (Scott, et al 2014). In order to support clinical trials for therapies targeting populations enriched in ABC DLBCL and to offer prognostic information for DLBCL patients as part of our clinical service, we developed a novel multiplex, single-tube, gene expression assay on the ICEPlex® system (PrimeraDx, Mansfield, MA), which allows differentiation between GCB and ABC DLBCL subtypes in formalin-fixed paraffin-embedded (FFPE) specimens using a Food and Drug Administration-cleared platform.

P2RY1 and P2RY12 polymorphisms and on‐aspirin platelet reactivity in patients with coronary artery disease

Introduction:  Association of P2RY1 and P2RY12 polymorphisms with on‐aspirin platelet reactivity was investigated.

Pilot Candidate Gene Analysis of Patients ≥60 Years Old With Aortic Stenosis Involving a Tricuspid Aortic Valve

The potential genetic basis of aortic stenosis in older people is poorly understood. A total of 265 patients with aortic stenosis involving tricuspid aortic valves and 961 controls were genotyped for ≤660 candidate single nucleotide polymorphisms (SNPs). After dividing the patients and controls into training and validation sets, we tested the correlation of the SNPs with the age-adjusted aortic valve area, determined by echocardiography or cardiac catheterization. A bootstrapped global p value of ≤0.005 was considered evidence of a possible significant correlation. The cases were aged 73 ± 7 years, and 72.7% were men. The median aortic valve area was 1.0 cm(2) (interquartile range 0.7 to 1.5). The controls were aged 69 ± 6 years, and 69.8% were men. The minor allele frequency was 21% ± 15% (37% <0.20). Three SNPs met the criteria for significant correlation (rs2276288 [MYO7A], p = 0.001; rs5194 [AGTR1], p = 0.004; rs207 307 [ELN], p = 0.005). Another 2 SNPs reached borderline significance (p ≤0.008). In conclusion, we report 3 SNPs to be associated with aortic stenosis involving tricuspid aortic valves in older subjects. Given the concerns regarding the problem of multiple statistical testing, validation studies are required to further assess these correlations.

LightTyper™ platform for high-throughput clinical genotyping

DNA sequence variations due to single nucleotide changes or polymorphisms (SNPs) have demonstrated an association with certain diseases as causative agents or surrogate biomarkers. Identification and genotyping of SNPs requires reliable and robust technologies. Multiple genotyping platforms are available to detect SNPs. Although many of these platforms meet the requirements of the research environment, technologies have also emerged for high-throughput clinical genotyping as well. The LightTyper™ is one such platform, providing SNP identification by employing melting curve analysis of fluorescently labeled probes. The LightTyper has been used to identify SNPs associated with myocardial infarction, developing and validating assays for approximately 100 SNPs in 30 candidate genes. The LightTyper is also amenable to the use of assays already developed for the LightCycler™, which is widely used in clinical laboratories. The initial experience presented here suggests the potential use of the LightTyper for high-throughput clinical genotyping.