Biology subjects

Johnstone, S. R.

Publications and source records attributed to Johnstone, S. R..

8 recordsLinked to original sources

bioRxiv2026

Vascular endothelial cadherin remodeling is associated with endothelial barrier dysfunction in chronic subdural hematoma

Abstract Background: Chronic subdural hematoma (cSDH) remains a significant and increasingly prevalent cause of morbidity in older adults. Disease progression is sustained by de novo formed highly vascularized pseudomembranes and a persistent cycle of fluid accumulation and hematoma expansion. Although inflammatory and angiogenic pathways have been implicated in cSDH pathogenesis, the endothelial mechanisms underlying persistent vascular leak remain poorly understood. We investigated the cSDH vascular microenvironment in human patients and examined the role of vascular endothelial cadherin (VECAD) remodeling in cSDH endothelial dysfunction. Methods: Human cSDH membranes and fluid were evaluated by multiplex profiling, histology, immunofluorescence, immunoprecipitation, western blotting and exploratory bulk RNA sequencing. A human brain endothelial cell (HBEC) line was exposed to patient-derived cSDH fluid to assess VECAD remodeling, endothelial barrier permeability, and proliferation. Results: Multiplex profiling of patient-derived cSDH fluid detected mediators associated with endothelial cell (EC) activation, extracellular matrix remodeling, inflammation, and angiogenesis. In patient pseudomembranes, discontinuous VECAD coincided with erythrocyte extravasation. Two hyperdense (high-risk) specimens showed more extensive disruption than the hypodense (low-risk) specimen while vessel counts appeared similar. Bulk RNA sequencing identified an angiogenic environment and enrichment in cadherin regulation. A soluble VECAD (sVECAD) isoform was detected in cSDH fluid. Exposure of HBECs to patient-derived cSDH fluid increased EC proliferation, cytoplasmic VECAD (cVECAD) accumulation, and EC barrier permeability. Concurrent dexamethasone or minocycline treatment attenuated VECAD cleavage. Conclusions: VECAD disruption is a prominent feature of cSDH and is associated with EC barrier dysfunction. These findings support further evaluation of soluble VECAD as a candidate biomarker and of junction-preserving pathways as potential therapeutic targets.

pathology↗

bioRxiv2026

Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate -adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here, we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. Clonidine-sensitive sympathetic-driven hypertension was observed in all mouse lines except Y198F. Pressure myography of third-order mesenteric arteries revealed -adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A, with responses in Y198F vessels mimicking controls treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing, but found a minimal number of differentially expressed genes between phospho-dead mutants and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the 1D-adrenergic receptor as well as each phospho-dead mutant PANX1 showed that phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, basal and phenylephrine-induced S205A and Y308F currents were reduced, but ATP release resembled controls. Taken together, these findings indicate that distinct PANX1 phosphorylation determines PANX1 metabolite release versus current conducting properties and in turn, regulates physiological outcomes in the vasculature. One Sentence SummaryPANX1 Y198 phosphorylation-mediated ATP release is a major driver of -adrenergic vasoconstriction in vascular smooth muscle cells.

physiology↗

bioRxiv2025

A connexin 43 targeting peptide prevents blood vessel neointima formation

The gap junction protein connexin 43 (Cx43) is associated with human pathological vascular smooth muscle cell (SMC) proliferation and neointima formation. We previously identified mitogen-activated protein kinase (MAPK) phosphorylation of Cx43 results in binding with the cell cycle protein cyclin E, facilitating neointima formation in mice. However, the specific nature of these interactions and their relevance to human disease have not been elucidated. Using an ex vivo human saphenous vein model of neointima formation, we identified increased MAPK-phosphorylated Cx43 and cyclin E in explant tissues. We used peptide arrays to define a cyclin E-Cx43 binding region and generated CycliCx, a stearate-linked Cx43 phospho-mimetic peptide. In human coronary artery SMC, CycliCx inhibits platelet-derived growth factor-BB (PDGF-{beta})-induced changes in Cx43 trafficking and interactions with cyclin E, and stimulation of proliferation. RNAseq analysis identified CycliCx significantly inhibits PDGF-{beta}-induced proliferative pathways in SMC by limiting PDGF-{beta}-induced early G1/S phase cell cycle progression transcripts. Finally, we show CycliCx limits neointima formation in mice in vivo and in ex vivo human saphenous vein explants. Our data provide strong evidence for selective targeting of Cx43 as a viable therapeutic strategy for preventing neointimal formation in humans.

physiology↗

bioRxiv2025

Injury Induced Connexin 43 Expression Regulates Endothelial Wound Healing

Endothelial cell (EC) injury is a major contributing factor to vascular surgical failure. As such, understanding the mechanisms of endothelial healing is essential to the development of vascular therapeutics and procedures. Gap junctions formed by connexin 43 (Cx43) are implicated in regulating skin wound healing, but their role in endothelial healing is unknown. Secondary analysis of RNAseq data from in vivo injured mouse aortas (GEO: GSE115618), identified significant Cx43 upregulation in EC post-injury. We developed a novel in vivo model of EC injury using mouse carotid artery ligation to test the role of Cx43. We identified that EC immediately adjacent to the wound edge upregulate Cx43 protein expression, predominantly at cell-cell junctions. We show significantly delayed EC healing in a mouse model of inducible EC-specific Cx43 deletion (EC-Cx43 KO) at 24 hr post ligation. Single cell RNAseq analysis of 10,829 cells from 18 hr injured EC-WT and EC-Cx43 KO carotids revealed a Cx43-associated reduction in enrichment of EC pathways associated with migration, proliferation, and ERK/MAPK signaling pathways. Finally, the importance of Cx43 phosphorylation on EC healing was tested in mice with single-point alanine mutations (phospho-null) in known phosphorylation sites that alter Cx43 channel assembly and opening. Mice containing alanine mutations at ERK phosphorylated Cx43 serines (Cx43S255/262/279/282A) reduces healing rates similar to EC-Cx43 KO. These data suggest that EC injury-induced Cx43 upregulation, and subsequent Cx43 gap junction-mediated cell-to-cell communication are required for normal EC migration during wound healing after vascular injury. New and NoteworthyThese findings demonstrate for the first time that mechanical injury to large artery endothelium induces the expression of gap junction protein Cx43. This upregulation improves migratory and proliferative capacity of endothelial cells at the wound edge, facilitating timely wound closure. This phenomenon is dependent on appropriate gap junction function and turnover.

physiology↗

bioRxiv2023

Preserving endothelial integrity in human saphenous veins during preparation for coronary bypass surgery

ObjectiveWhile multiple factors influence coronary artery bypass graft success rates, preserving saphenous vein endothelium during surgery may improve patency. Standard methods of saphenous vein graft preparation in heparinized saline (saline) solutions result in endothelial loss and damage. Here we investigated the impact of preparing saphenous graft vessels in heparinized patient blood (blood) vs saline on cellular health and survival. MethodsSaphenous vein tissues from a total of 23 patients undergoing coronary artery bypass graft surgery were split into 2 treatment groups, 1) standard preparation using saline and 2) preparation using blood. Immediately following surgery, excess tissue was fixed for analysis. Level of endothelial coverage, oxidative stress marker 4-hydroxynonenal (4HNE), and oxidative stress protective marker nuclear factor erythroid 2-related factor 2 (NRF2) expression were evaluated. ResultsIn saline patient veins, histological analysis revealed a broken or absent luminal layer, suggesting a loss of endothelial cell (EC) coverage. Luminal cell coverage was notably preserved in blood-treated samples. Immunofluorescent staining of endothelial markers vascular endothelial cadherin (VE-cadherin) and endothelial nitric oxide (eNOS) identified a significant improvement in endothelial coverage in the blood group compared to saline. Although in both treatment groups EC expressed 4HNE indicating a similar level of oxidative stress, EC stored in blood solutions expressed higher levels of the protective transcription NRF2. ConclusionsOur data indicate that maintaining and preparing saphenous vein tissues in solutions containing heparinized blood helps preserve the endothelium and promotes vein graft health. This has the potential to improve long-term outcomes in patients. Central MessageDuring coronary artery bypass grafting, preparation of saphenous veins with heparinized saline damages the endothelium and increases oxidative stress. Heparinized blood preparation limits this endothelial loss and damage. Perspectives StatementSaphenous vein grafts are prone to failure through neointimal hyperplasia or thrombosis. Endothelial damage and loss are thought to be major contributing factors to graft failure. Here we find that preparation and preservation of saphenous vein grafts with patients own heparinized blood is sufficient to ensure endothelial preservation and protect vessels from oxidative stress compared with heparinized saline. These changes may increase long-term graft patency rates.

physiology↗

bioRxiv2023

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting [~]3% of the US population. It is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). The mechanism underlying AF is not well understood and current treatments are limited to managing this progressive disease, rather than arresting the underlying pathology. We previously identified edema-induced disruption of sodium channel (NaV1.5) -rich intercalated disk (ID) nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. We identified two molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. AF incidence was increased in untreated mice exposed to VEGF relative to vehicle controls. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions (GJs), measured by a distance transformation-based spatial analysis of 3D confocal images of IDs. Similar effects were observed with NaV1.5 localized near mechanical junctions (MJs) composed of N-cad. Blocking connexin43 hemichannels (CT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared to VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from GJs to control levels and reduced MJ-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small molecule inhibitors of Cx43 and Panx1.

physiology↗

bioRxiv2023

A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and is Differentially Regulated during Hypoxia and Aging

ABSTRACT/SUMMARYThe platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFR{beta}) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFR{beta} (sPDGFR{beta}) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFR{beta} variants, and specifically during tissue homeostasis. Here, we found sPDGFR{beta} protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFR{beta} isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFR{beta} by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFR{beta} transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFR{beta} protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFR{beta} variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFR{beta} variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFR{beta} likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFR{beta} in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion - critical processes underlying neuronal health and function, and in turn memory and cognition.

cell biology↗

bioRxiv2019

Endothelial pannexin 1 channels control inflammation by regulating intracellular calcium

In BriefInterleukine-1 beta (IL-1{beta}) has been identified as a critical factor that contributes to the inflammatory response in cardiovascular disease (e.g., atherosclerosis). Pannexin 1 (Panx1) channel activity in endothelial cells regulates localized inflammatory cell recruitment. In response to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular calcium to control the production and release of cytokines including IL-1{beta}.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC=\"FIGDIR/small/750323v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@59e72borg.highwire.dtl.DTLVardef@57bd5corg.highwire.dtl.DTLVardef@50ed73org.highwire.dtl.DTLVardef@1151f56_HPS_FORMAT_FIGEXP M_FIG C_FIG AbstractThe proinflammatory cytokine IL-1{beta} is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1{beta} expression and release are tightly controlled by changes in intracellular Ca2+. In addition, purinergic signaling through ATP release has also been reported to promote IL-1{beta} production. Despite this, the mechanisms that control IL-1{beta} synthesis and expression have not been identified. The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, especially during inflammation. However, resolution of purinergic signaling occurs quickly due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that TNF signaling induced NF{kappa}{beta}-associated Panx1 transcription. Genetic inhibition of Panx1 reduced the expression and secretion of IL-1{beta}. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL1-{beta} expression was not altered after direct ATP stimulation, following degradation of ATP by apyrase, or after pharmacological blockade of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, control IL-1{beta} production. Because Panx1 forms a large pore channel, we hypothesized it may act to passively diffuse Ca2+ into the cell upon opening to regulate IL-1{beta}. High-throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated intracellular Ca2+. Genetic or pharmacological inhibition of Panx1 reduced TNF-associated increases in intracellular Ca2+, and IL-1{beta} transcription. Furthermore, we found that the Ca2+-sensitive NF{kappa}{beta}-p65 protein failed to localize to the nucleus after genetic or pharmacological block of Panx1. Taken together, our study provides the first evidence that intracellular Ca2+ regulation via the Panx1 channel induces a feed-forward effect on NF{kappa}{beta} to regulate IL-1{beta} synthesis and release in endothelium during inflammation.

cell biology↗