Biology subjects

Cockerham, E. D.

Publications and source records attributed to Cockerham, E. D..

3 recordsLinked to original sources

bioRxiv2022

Spatially Resolved Metabolites in Stable and Vulnerable Human Atherosclerotic Plaques Identified by Mass Spectrometry Imaging

Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging (MSI) on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and HMDB, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheroma. We then integrated these results with an RNA-sequencing dataset comparing stable and unstable human atherosclerosis. Upon integrating our MSI results with the RNA-seq dataset, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. Acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindole-acetic acid was enriched in the fibrous cap. Our work herein represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/494038v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@111368org.highwire.dtl.DTLVardef@1112d99org.highwire.dtl.DTLVardef@c8cdb2org.highwire.dtl.DTLVardef@13895d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2019

The Differential Roles of the Adaptor Proteins Nck1 and Nck2 in Shear Stress-Induced Endothelial Activation.

While CANTOS established the role of treating inflammation in atherosclerosis, our understanding of endothelial activation at atherosclerosis-prone sites remains limited. Disturbed flow at atheroprone regions primes plaque inflammation by enhancing endothelial NF-{kappa}B signaling. Herein, we demonstrate a novel role for the Nck adaptor proteins in disturbed flow-induced endothelial activation. Although highly similar, only Nck1 deletion, but not Nck2 deletion, limits flow-induced NF-{kappa}B activation and proinflammatory gene expression. Nck1 knockout mice show reduced endothelial activation and inflammation in both models of disturbed flow and high fat diet-induced atherosclerosis. Bone marrow chimeras confirm that vascular Nck1, but not hematopoietic Nck1, mediates this effect. In contrast, endothelial Nck2 depletion does not affect endothelial activation or atherosclerosis. Domain swap experiments and point mutations identify the Nck1 SH2 domain and the first SH3 domain as critical for flow-induced endothelial activation. We further characterize Nck1s proinflammatory role by identifying interleukin-1 type I receptor kinase-1 (IRAK-1) as a Nck1-selective binding partner, demonstrating IRAK-1 activation by disturbed flow requires Nck1 in vitro and in vivo, showing endothelial Nck1 and IRAK-1 staining in early human atherosclerosis, and demonstrating that disturbed flow-induced endothelial activation requires IRAK-1. Taken together, our data reveal a hitherto unknown link between Nck1 and IRAK-1 in atherogenic inflammation.

pathology↗

bioRxiv2019

The Adaptor Protein Nck1, but not Nck2, Mediates Shear Stress-Induced Endothelial Permeability.

Alteration in hemodynamic shear stress at atheroprone sites promotes endothelial paracellular pore formation and permeability. Previously, we have reported that a peptide inhibitor to Nck prevented shear stress-induced p21 activated kinase (PAK) activation and endothelial permeability. However, the specificity of this peptide is unclear, and the role of individual Nck isoforms remain unknown. Here, we show that genetic deletion of Nck1/2 adaptor proteins significantly ameliorates shear stress induced permeability, and selective isoform depletion suggests distinct signaling mechanisms. Only Nck1 deletion significantly reduces flow-induced paracellular pore formation and permeability, whereas Nck2 depletion has no significant effects. Additionally, Nck1 reexpression, but not Nck2, restores shear stress-induced permeability in Nck1/2 knockout cells, confirming the non-compensating roles. In vivo, using the partial carotid ligation model of disturbed flow, Nck1 knockout prevented the increase in vascular permeability, as assessed by both Evans blue extravasation and leakage of plasma fibrinogen into the vessel wall. Domain swap experiments mixing SH2 (phosphotyrosine binding) and SH3 (proline rich binding) domains between Nck1 and Nck2 showed a dispensable role for SH2 domains but a critical role for the Nck1 SH3 domains in rescuing shear stress-induced endothelial permeability. Consistent with this, both Nck1 and Nck2 bind to PECAM-1 (SH2 dependent) in response to shear stress, but only Nck1 ablation interferes with shear stress-induced PAK2 activation (SH3 dependent). This work provides the first evidence that Nck1 and Nck2 play distinct roles in flow-induced vascular permeability.\n\nNew and NoteworthyThe present study shows a specific role for Nck1 in endothelial permeability in response to shear stress. Using in vitro and in vivo models, we demonstrate improvement in endothelial barrier integrity in cells subjected to disturbed flow only following Nck1 but not Nck2 deletion. Selective Nck1 inhibition may limit endothelial permeability at sites of disturbed flow to reduce atherosclerosis without affecting angiogenesis, which requires both Nck1 and Nck2 inhibition.

molecular biology↗