Biology subjects

Orr, A. W.

Publications and source records attributed to Orr, A. W..

9 recordsLinked to original sources

bioRxiv2026

Smooth Muscle Cell Cytoglobin is a Negative Regulator of Atherosclerotic Fibrous Cap Development

Rupture of the fibrous cap is the primary cause of clinical complications from atherosclerosis. Smooth muscle cells (SMCs) are a major contributor to fibrous cap development and stability through de-differentiation to extracellular matrix-producing cells. We previously showed that the antioxidant enzyme cytoglobin (CYGB) is expressed in vascular SMCs and regulates SMC dependent vascular remodeling and gene expression. In the present study, we investigated the function of SMC-CYGB in atherosclerosis. To this end, we generated a mouse line with SMC-specific deletion of Cygb and simultaneous SMC-lineage tracing. We found that SMC specific deletion of CYGB increased fibrous cap thickness in a 17-week PCSK9-AAV8 gain of function combined with Western diet mouse model of atherosclerosis. SMC specific deletion of CYGB increased collagen deposition and SMC cellularity of the fibrous cap in the absence of changes in total plaque and necrotic core sizes. CYGB expression in SMCs was associated with transdifferentiation towards a fibroblast-like, matrix remodeling phenotype. Finally, CYGB was expressed in the fibrous cap of human coronary atherosclerotic lesions and was associated with ACTA2 positive cells. These results provide first-time evidence that SMC-CYGB reduces plaque stability by decreasing cap thickness, collagen deposition, and SMC cellularity.

physiology↗

bioRxiv2025

Macrophage EHD1 promotes inflammation and stabilizes sortilin to accelerate atherosclerosis

BackgroundMacrophages are key players in the pathogenesis of atherosclerosis. They trigger immune responses through their cell-surface receptors. However, how macrophages regulate those receptors in response to pro-inflammatory stimuli is not completely understood. Endocytic membrane trafficking involving receptor internalization, followed by endosomal transport and recycling of the internalized receptors, plays essential roles in balancing cell-surface receptors to meet cellular needs. Here, we explored the role of the endocytic regulator EHD1 in immune responses in macrophages and determined its contribution to atherosclerosis progression. MethodsEHD1 expression profiles in mouse and human plaques were determined by single-cell RNA sequencing (scRNA-seq) and immunofluorescence staining. Bone marrow transplantation (BMT) by transplanting bone marrow cells from Ehd1-/- or littermate wild-type mice to irradiated Ldlr-/- mice was performed to determine the effect of EHD1 deletion on atherosclerosis progression. In vitro mechanistic studies including inflammation signaling and endocytosis assays were performed in bone marrow-derived macrophages. ResultsEHD1 expression in macrophages is enhanced as atherosclerosis progresses in both mice and humans. Histological analysis of aortic root sections from BMT mice showed that EHD1 deletion reduces lesion size. ScRNA-seq of aortic CD45+ cells demonstrated that EHD1 deletion attenuates pro-inflammatory responses and cell-cell interactions. Mechanistic studies revealed that EHD1 accelerates the endocytic recycling of TNFR2 and activates NF-kB, leading to increased expression of inflammatory cytokines. Moreover, EHD1 interacts with retromer and stabilizes sortilin, a retrograde cargo of retromer and a risk factor for atherosclerosis. ConclusionsEHD1 promotes inflammation by enhancing TNFR2-NF-kB signaling and stabilizing sortilin, leading to accelerated atherosclerosis. Our study reveals novel roles for EHD1-mediated membrane trafficking in macrophage function and paves the way to innovative therapeutic strategies that aim to address dysregulated membrane trafficking in atherosclerosis.

cell biology↗

bioRxiv2025

Integrin-Specific Signaling Drives ER Stress-Dependent Atherogenic Endothelial Activation

Atherogenic endothelial activation arises from both the local arterial microenvironment--characterized by altered extracellular matrix composition and disturbed blood flow--and soluble proinflammatory stimuli such as oxidized low-density lipoprotein (oxLDL). Fibronectin, a provisional extracellular matrix protein enriched at atheroprone sites, enhances endothelial activation and inflammation triggered by oxLDL and disturbed flow. Although endoplasmic reticulum (ER) stress contributes to vascular dysfunction, the role of matrix composition in regulating ER stress remains unknown. We show that oxLDL and disturbed flow induce ER stress selectively in endothelial cells adhered to fibronectin, whereas both stimuli fail to induce ER stress in cells on basement membrane proteins. This matrix-specific ER stress response requires integrin activation, as endothelial cells deficient for integrin activation (talin1 L325R mutation) fail to activate ER stress in response to disturbed flow and oxLDL and direct stimulation of integrin activation using CHAMP peptides is sufficient to trigger ER stress. Blunting endothelial expression of fibronectin-binding integrins (5, v) using siRNA prevents ER stress in response to atherogenic stimuli in vitro, whereas endothelial 5 and v deletion reduces ER stress at atheroprone sites in vivo. The mechanisms driving integrin-dependent ER stress remain unclear, since matrix composition does not affect protein translation, unfolded protein accumulation, or superoxide production, and scavenging superoxide (TEMPOL) does not reduce integrin-dependent ER stress. Inhibiting ER stress with TUDCA reduces proinflammatory and metabolic gene expression (bulk RNAseq) but does not prevent NF-{kappa}B activation, a classic proinflammatory transcription factor. Rather, TUDCA prevents activation of c-jun N-terminal kinase (JNK) and c-jun activation, and blocking JNK (SP600126) or c-Jun activity (TAM67) prevents proinflammatory gene expression following both stimuli. Together, these findings offer new insight into how the arterial microenvironment contributes to atherogenesis, with fibronectin-binding integrin signaling promotes ER stress in response to mechanical and metabolic stressors, thereby amplying proinflammatory endothelial activation through JNK-c-Jun signaling. HighlightsO_LIFibronectin promotes endothelial ER stress in response to oxLDL and disturbed flow via integrin 5{beta}1 and v{beta}3 signaling. C_LIO_LIMatrix-specific ER stress occurs independently of oxidative stress or misfolded protein accumulation, indicating a non-canonical UPR activation. C_LIO_LIER stress amplifies proinflammatory gene expression through JNK-c-Jun signaling, without activating NF-{kappa}B. C_LIO_LITargeting the integrin-ER stress-JNK axis may offer new therapeutic strategies for early atherosclerosis. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/654582v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@ed7ca3org.highwire.dtl.DTLVardef@191c238org.highwire.dtl.DTLVardef@660f3dorg.highwire.dtl.DTLVardef@7287a4_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

pathology↗

bioRxiv2024

Polycomb Repressive Complex 2 promotes atherosclerotic plaque vulnerability

Key findings1. PRC2 regulates EC shear stress responses. 2. PRC2 governs Klf2/4 suppression downstream of Pcdhg. 3. High PRC2 in ASCVD-prone arterial regions suppresses Klf2/4 to promote ASCVD. 4. Athero-protective Klf2/4 induction upon PRC2 inhibition requires Notch signaling. 5. Tazemetostat, an FDA approved PRC2 inhibitor, slows ASCVD progression and improves markers of plaque stability. Atherosclerotic cardiovascular disease (ASCVD), the leading cause of mortality worldwide, is driven by endothelial cell inflammatory activation and counter-balanced by anti-inflammatory transcription factors Klf2 and Klf4 (Klf2/4). Understanding vascular endothelial inflammation to develop effective treatments is thus essential. Here, we identify, Polycomb Repressive Complex (PRC) 2, which blocks gene transcription by trimethylating histone3 Lysine27 in gene promoter/enhancers, as a potent, therapeutically targetable determinant of vascular inflammation and ASCVD progression. Bioinformatics identified PRC2 as a direct suppressor of Klf2/4 transcription. Klf2/4 transcription requires Notch signaling, which reverses PRC2 modification of Klf2/4 promoter/enhancers. PRC2 activity is elevated in human ASCVD endothelium. Treating mice with established ASCVD with tazemetostat, an FDA approved pharmacological inhibitor of PRC2, slowed plaque progression by 50% and drastically improved markers of plaque stability. This study elucidates a fundamental mechanism of vascular inflammation, thus identifying a potential method for treating ASCVD and possibly other vascular inflammatory diseases.

cell biology↗

bioRxiv2024

Gamma protocadherins in vascular endothelial cells inhibit Klf2/4 to promote atherosclerosis

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide1. Laminar shear stress (LSS) from blood flow in straight regions of arteries protects against ASCVD by upregulating the Klf2/4 anti-inflammatory program in endothelial cells (ECs)2-8. Conversely, disturbed shear stress (DSS) at curves or branches predisposes these regions to plaque formation9,10. We previously reported a whole genome CRISPR knockout screen11 that identified novel inducers of Klf2/4. Here we report suppressors of Klf2/4 and characterize one candidate, protocadherin gamma A9 (Pcdhga9), a member of the clustered protocadherin gene family12. Pcdhg deletion increases Klf2/4 levels in vitro and in vivo and suppresses inflammatory activation of ECs. Pcdhg suppresses Klf2/4 by inhibiting the Notch pathway via physical interaction of cleaved Notch1 intracellular domain (NICD Val1744) with nuclear Pcdhg C-terminal constant domain (CCD). Pcdhg inhibition by EC knockout (KO) or blocking antibody protects from atherosclerosis. Pcdhg is elevated in the arteries of human atherosclerosis. This study identifies a novel fundamental mechanism of EC resilience and therapeutic target for treating inflammatory vascular disease.

cell biology↗

bioRxiv2024

CD14 Blockade Does Not Improve Outcomes of Deep Vein Thrombosis Following Inferior Vena Cava Stenosis in Mice

BackgroundNeutrophil-mediated persistent inflammation and neutrophil extracellular trap formation (NETosis) promote deep vein thrombosis (DVT). CD14, a co-receptor for toll-like receptor 4 (TLR4), is actively synthesized by neutrophils, and the CD14/TLR4 signaling pathway has been implicated in proinflammatory cytokine overproduction and several aspects of thromboinflammation. The role of CD14 in the pathogenesis of DVT remains unclear. ObjectiveTo determine whether CD14 blockade improves DVT outcomes. MethodsBulk RNA sequencing and proteomic analyses were performed using isolated neutrophils following inferior vena cava (IVC) stenosis in mice. DVT outcomes (IVC thrombus weight and length, thrombosis incidence, neutrophil recruitment, and NETosis) were evaluated following IVC stenosis in mice treated with a specific anti-CD14 antibody, 4C1, or control antibody. ResultsMice with IVC stenosis exhibited increased plasma levels of granulocyte colony-stimulating factor (G-CSF) along with a higher neutrophil-to-lymphocyte ratio and increased plasma levels of cell-free DNA, elastase, and myeloperoxidase. Quantitative measurement of total neutrophil mRNA and protein expression revealed distinct profiles in mice with IVC stenosis compared to mice with sham surgery. Neutrophils of mice with IVC stenosis exhibited increased inflammatory transcriptional and proteomic responses, along with increased expression of CD14. Treatment with a specific anti-CD14 antibody, 4C1, did not result in any significant changes in the IVC thrombus weight, thrombosis incidence, or neutrophil recruitment to the thrombus. ConclusionThe results of the current study are important for understanding the role of CD14 in the regulation of DVT and suggest that CD14 lacks an essential role in the pathogenesis of DVT following IVC stenosis.

pathology↗

bioRxiv2020

Integrin Affinity Modulation Critically Regulates Atherogenic Endothelial Activation in vitro and in vivo

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of 5{beta}1 integrins in endothelial cells in vitro, and 5{beta}1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of 5{beta}1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread, but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity 5{beta}1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of 5{beta}1 integrins in talin1 L325R cells partially restores fibronectin deposition, whereas NF-{kappa}B activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.

cell biology↗

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↗