Biology subjects

Stout, R.

Publications and source records attributed to Stout, R..

4 recordsLinked to original sources

bioRxiv2026

Endothelial AMBRA1 loss contributes to vascular dysregulation facilitating metastatic progression in early-stage melanoma

Validated biomarkers for identifying patients with early-stage melanomas at high risk of metastasis are limited. We have previously shown that loss of Activating Molecule in Beclin-1-Regulated Autophagy (AMBRA1) and loricrin in the epidermal microenvironment is associated with tumour recurrence. Here we show that endothelial AMBRA1 deficiency contributes to vascular dysfunction and melanoma progression. Melanoma-conditioned media or exposure to TGF{beta}1-3 ligands induced post-transcriptional loss of AMBRA1 in endothelial cells. Transcriptomic profiling in both in vitro and human melanoma single-cell RNA sequencing datasets showed activation of cell-cycle and metabolic programmes alongside suppression of endothelial junction, adhesion and immune-supportive pathways in AMBRA1-deficient endothelial cells. Functionally, AMBRA1 loss increased endothelial cell proliferation, accelerated early tubulogenesis, impaired three-dimensional spheroid adhesion, stabilised endothelial-to-mesenchymal transition-associated transcription factors Slug and Snail, and reduced Claudin-5, VE-cadherin and N-cadherin, indicative of endothelial plasticity and dysfunction. Furthermore, loss of AMBRA1 in a subset of intratumoural and peritumoural blood and lymphatic vessels correlated significantly with metastatic progression in a cohort of 121 non-ulcerated primary AJCC stage I/II melanomas classified as AMBLor at-risk. Collectively, these findings position endothelial AMBRA1 as a regulator of vascular plasticity, dysfunction, and immune-supportive endothelial function in early-stage melanomas, and a marker of a permissive microenvironment associated with metastasis.

cancer biology↗

bioRxiv2025

Thyroid hormone promotes fetal neurogenesis

Maternal low thyroxine (T4) serum levels during the first trimester of pregnancy correlate with cerebral cortex volume and mental development of the progeny, but why neural cells during early fetal brain development are vulnerable to maternal T4 levels remains unknown. In this study, using iPSCs obtained from a boy with a loss-of-function mutation in MCT8--a transporter previously identified as critical for thyroid hormone uptake and action in neural cells--we demonstrate that thyroid hormones induce transcriptional changes that promote the progression of human neural precursor cells along the dorsal projection trajectory. Consistent with these findings, single-cell, spatial, and bulk transcriptomics from MCT8-deficient cerebral organoids and cultures of human neural precursor cells underscore the necessity for optimal thyroid hormone levels for these cells to differentiate into neurons. The controlled intracellular activation of T4 signaling occurs through the transient expression of the enzyme type 2 deiodinase, which converts T4 into its active form, T3, alongside the coordinated expression of thyroid hormone nuclear receptors. The intracellular activation of T4 in NPCs results in transcriptional changes important for their division mode and cell cycle progression. Thus, T4 is essential for fetal neurogenesis, highlighting the importance of adequate treatment for mothers with hypothyroidism.

neuroscience↗

bioRxiv2025

Orally Delivered Milk-Derived Nanovesicles Loaded with Connexin 43 Peptides forTargeted Cardiac Ischemia-Reperfusion Therapy

Extracellular vesicles have emerged as promising nanocarriers for targeted drug delivery, but their therapeutic potential is limited by challenges related to administration route, loading, targeted delivery and production at scale. Here, we report an innovative approach for targeted delivery of therapeutic peptides to injured tissues using milk-derived small extracellular vesicles (mEVs) as an abundant, safe, orally administrable nanoplatform. We demonstrate that a sub-population of mEVs naturally contain Connexin 43 (Cx43) and its Carboxyl-Terminal (CT) polypeptides, which have been shown to play crucial roles in wound healing and tissue repair. Leveraging this intrinsic property, we developed an esterification method to efficiently and uniformly load mEVs with enhanced levels of an exogenous Cx43 CT peptide (CT11 - RPRPDDLEI), as assessed by flow cytometry-based vesicle quantification and mass spectrometry. These engineered mEVs exhibited remarkable injury targeting capabilities, with > 30-fold increases in uptake by injured cells compared to non-wounded cells in vitro and preferential accumulation in wounded tissues in vivo. Notably, CT11-loaded mEVs orally administered after myocardial infarction reduced infarct size by >60% and preserved heart function in a mouse model of ischemia-reperfusion injury. This study represents a significant advance in nanomedicine, demonstrating the utilization of naturally occurring milk-derived extracellular vesicles as an oral delivery system for therapeutic peptides, achieving unprecedented targeting efficiency and efficacy in the treatment of myocardial ischemia-reperfusion injury.

bioengineering↗

bioRxiv2021

Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if blood-brain-barrier integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

neuroscience↗