Biomedical subjects

Gurunathan Murugesan

Publications and source records attributed to Gurunathan Murugesan.

10 recordsLinked to original sources

PubMed2006

Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation.

JAK2 V617F mutation recently was identified as a pathogenic factor in typical chronic myeloproliferative diseases (CMPD). Some forms of myelodysplastic syndromes (MDS) show a significant overlap with CMPD (classified as MDS/MPD), but the diagnostic assignment may be challenging. We studied blood or bone marrow from 270 patients with MDS, MDS/MPD, and CMPD for the presence of JAK2 V617F mutation using polymerase chain reaction, sequencing, and melting curve analysis. The detection rate of JAK2 V617F mutants for polycythemia vera, chronic idiopathic myelofibrosis, and essential thrombocythemia (n = 103) was similar to the previously reported results. In typical forms of MDS (n = 89) JAK2 V617F mutation was very rare (n = 2). However, a higher prevalence of this mutation was found in patients with MDS/MPD-U (9 of 35). Within this group, most of the patients harboring JAK2 V617F mutation showed features consistent with the provisional MDS/MPD-U entity refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T). Among 9 RARS-T patients, 6 showed the presence of JAK2 V617F mutation, and in 1 patient without mutation, aberrant, positive phospho-STAT5 staining was seen that is typically present in association with JAK2 V617F mutation. In summary, we found that RARS-T reveals a high frequency of JAK2 V617F mutation and likely constitutes another JAK2 mutation-associated form of CMPD.

Alleles↗

PubMed2006

Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis.

We developed and validated a real-time polymerase chain reaction assay using fluorescent hybridization probes and melting curve analysis to identify the JAK2 V617F mutation, which is implicated in a substantial proportion of chronic myeloproliferative disorders (CMPDs). DNA from 161 samples was isolated from peripheral blood granulocytes and formalin-fixed bone marrow clot sections in patients with CMPDs and without myeloproliferative disorders previously genotyped for the JAK2 V617F (G-->T) mutation, which included 114 wild types (GG) and 47 mutants (GT and TT). Melting curve analysis of these samples yielded 114 wild types, 42 heterozygotes, and 5 homozygotes showing 100% concordance. Analytic sensitivity of the assay for mutant DNA was 5% for the LightTyper (Roche Applied Sciences, Indianapolis, IN) and 10% for the LightCycler (Roche Applied Sciences). Consistent with earlier reports, 78% of the non-chronic myelogenous leukemia CMPD patients and 8% of non-CMPD patients displayed this mutation. This study demonstrates that clinical genotyping of the JAK2 V617F mutation can be performed by melting analysis using both freshly isolated and formalin-fixed tissues.

Bone Marrow Cells↗

PubMed2005

Creation of a large-scale genetic data bank for cardiovascular association studies.

BACKGROUND: A prerequisite for undertaking genetic association studies is the need for a genetic data bank with adequate DNA samples and a well-described clinical cohort. METHODS: We initiated a prospective single-center study enrolling 6,273 patients referred for cardiac catheterization in a genetic data bank (with eventual goal of 10,000 enrollees). Using a prescreening tool, the patients had comprehensive clinical phenotyping, including angiogram, electrocardiogram, echocardiogram, clinical history, and medication profile (Appendix A). Along with this clinical information, DNA, serum, plasma, basic metabolic panel, inflammation, and lipid panel were collected and stored in the database. RESULTS: Mean age of the patients enrolled was 64 +/- 12 years; 69% are men, 26% have diabetes, 79% have dyslipidemia, and 72% have coronary artery disease (CAD) > or = 50%. We undertook extensive quality-control measures to ensure the validity of both the clinical and DNA samples acquired into our GenBank. As part of this validation, we undertook a genetic association study to discern the effect of the apoE4 polymorphism on the risk for atherosclerosis. We are able to show that the apoE4 polymorphism is an independent risk factor for CAD. CONCLUSIONS: We have been able to create a large-scale genetic data bank as a resource to undertake genetic association studies. Key elements in implementation of this GenBank and baseline characteristics of our patient cohort are summarized. Lastly, as a "proof of concept" for the utility of this resource to discern gene variants associated with disease, we validated apoE4 polymorphism as an independent risk factor for CAD.

Aged↗

PubMed2005

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.

Animals↗

PubMed2004

Extracellular matrix-like surfactant polymers containing arginine-glycine-aspartic acid (RGD) peptides.

We report on a novel series of biomimetic polymers exhibiting interfacial properties similar to the extracellular matrix. A series of well-defined surfactant polymers were synthesized by simultaneously incorporating arginine-glycine-aspartic acid (RGD) peptide, dextran oligosaccharide, and hexyl ligands with controlled feed ratios onto a poly(vinyl amine) (PVAm) backbone. The peptide sequence was H-GSSSGRGDSPA-NH(2) (Pep) having a hydrophilic extender at the amino terminus and capped carboxy terminus. The peptide-to-dextran (Pep:Dex) ratios were varied to create surfactants having 0, 25, 50, 75, and 100 mol-% peptide relative to dextran. The surfactants were characterized by IR, NMR and atomic force microscopy (AFM) for composition and surface active properties. AFM confirmed full surface coverage of PVAm(Pep)(100%) on graphite, and supported the mechanism of interdigitation of hexyl ligands between surfactant molecules within a specified range of hexyl chain densities. the attachment and growth of human pulmonary artery endothelial cells on the PVAm(Pep)(100%) surface was identical to the fibronectin positive control. Cell adhesion decreased dramatically with decreasing peptide density on the surfactant polymers. Molecular model of a peptide surfactant polymer, consisting of poly(vinyl amine) backbone with peptide, dextran oligosaccharide and hexyl branches coupled to the polymer chain.

Absorption↗

PubMed2003

Lysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines.

The role of lysophosphatidylcholine (LPC) in the induction of MCP-1, IL-8 and RANTES, which are chemotactic factors to monocytes, neutrophils and lymphocytes, respectively, by human vascular endothelial cells (EC), was examined. LPC induced the expression of MCP-1 and IL-8 in a concentration- and time-dependent manner in microvascular EC (MVEC) and in large vessel EC from aorta, pulmonary artery and umbilical vein. LPC also induced RANTES in MVEC but not in large vessel EC. Signaling pathways responsible for LPC induction of chemokines were examined in MVEC. LPC and TNFalpha, a cytokine secreted in sites of inflammation, additively stimulated RANTES expression. LPC did not augment TNFalpha induction of MCP-1 or IL-8. A platelet-activating factor receptor antagonist (BN52021) failed to block LPC induction of MVEC chemokines, but the G(i)-protein inhibitor pertussis toxin partially blocked LPC induction of RANTES and IL-8. LPC activated multiple kinases in MVEC; it increased the phosphorylation of ERK1/2, AKT and p38 MAP kinase in a time-dependent manner. An inhibitor of the MAPK/ERK pathway, PD98059, blocked the phosphorylation of ERK1/2 and RANTES induction by LPC, but augmented IL-8 induction. LY294002, a specific inhibitor of phosphoinositide 3 kinase (PI3 kinase), blunted the phosphorylation of AKT and inhibited LPC induction of RANTES more strongly than IL-8. Inhibition of p38 MAP kinase pathway by SB202190 also blocked LPC-induced expression of IL-8 and RANTES. Our results suggest that LPC induction of chemokines in MVEC is distinct from that in large vessel EC, and required the activities of MAP kinases and PI3 kinase for the induction of RANTES and IL-8. We speculate that the presence of LPC, a bioactive lipid product of phospholipase A(2) (PLA(2)) and a constituent of oxidized low-density lipoprotein, can differentially influence the chemotaxis of particular leukocyte subpopulations during inflammation.

Aorta↗

PubMed2002

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.

4-Butyrolactone↗

PubMed2002

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 alpha-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.

Animals↗

PubMedDate not supplied

Human microvascular endothelial cell growth and migration on biomimetic surfactant polymers.

Successful engineering of a tissue-incorporated vascular prosthesis requires cells to proliferate and migrate on the scaffold. Here, we report on a series of "ECM-like" biomimetic surfactant polymers that exhibit quantitative control over the proliferation and migrational properties of human microvascular endothelial cells (HMVEC). The biomimetic polymers consist of a poly(vinyl amine) (PVAm) backbone with hexanal branches and varying ratios of cell binding peptide (RGD) to carbohydrate (maltose). Proliferation and migration behavior of HMVEC was investigated using polymers containing RGD: maltose ratios of 100:0, 75:25 and 50:50, and compared with fibronectin (FN) coated glass (1 microg/cm2). A radial Teflon fence migration assay was used to examine HMVEC migration at 12 h intervals over a 48 h period. Migration was quantified using an inverted optical microscope, and HMVEC were examined by confocal microscopy for actin and focal adhesion organization/ arrangement. Over the range of RGD ligand density studied (approximately 0.19-0.6 peptides/nm2), our results show HMVEC migration decreases with increasing RGD density in the polymer. HMVEC were least motile on the 100% RGD polymer (approximately 0.38-0.6 peptides/nm2) with an average migration of 0.20 mm2/h in area covered, whereas HMVEC showed the fastest migration of 0.48+/-0.06 mm2/h on the 50% RGD surface ( approximately 0.19-0.30 peptides/nm2). In contrast, cell proliferation increased with increasing surface peptide density; proliferation on the 50% RGD surface was 1.5%+/-0.06/h compared with 2.2%+/-0.07/h on the 100% RGD surface. Our results show that surface peptide density affects cellular functions such as growth and migration, with the highest peptide density supporting the most proliferation but the slowest migration.

Biomimetic Materials↗

PubMedDate not supplied

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 alpha5beta1 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 (alpha(v)beta3) 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.

Biomimetic Materials↗