Derek T. Warren

Cardiovascular disease involves coordinated remodelling across multiple biological processes. Gene-level signatures vary substantially between studies because different combinations of genes can support similar biological processes. Pathway-level analyses can provide more stable representations of these processes but typically consider pathways independently. We hypothesised that grouping related pathways into conserved biological functions and quantifying their relative weighting would reveal a higher-order, transferable property of vascular tissue that we term functional organisation. We quantified the relative weighting of six conserved biological functions across independent transcriptomic datasets spanning human vascular disease, experimental models and therapeutic interventions. Vascular tissues exhibited a reproducible functional organisation defined by the balance of these functions. A dominant remodelling trajectory captured coordinated, nonlinear rebalancing of structural, immune, signalling and metabolic programmes, while a second dimension distinguished contractile/ECM organisation from immune activity. This organisation was preserved across independently reconstructed reference cohorts and remained robust to analytical sensitivity testing. When independent datasets were projected into this fixed framework, biological and clinical phenotypes occupied coherent positions along the remodelling landscape. Plaque-derived vascular, stromal and immune cell populations also occupied ordered functional positions, linking cellular heterogeneity to tissue-level organisation. The same organisational structure generalised across atherosclerosis, peripheral vascular disease and abdominal aortic aneurysm and revealed continuous biological heterogeneity within conventional clinical classifications. Genetic, pharmacological and dietary perturbations reproducibly shifted functional organisation, demonstrating that organisational state is dynamic and biologically responsive. Together, these findings identify functional organisation as a reproducible tissue-level property of vascular remodelling and provide a framework for understanding cardiovascular disease as coordinated rebalancing of biological functions rather than alteration of individual pathways in isolation.

Tunneling nanotubes (TNTs) are actin-based cytoplasmic connections that can mediate intercellular transfer of various cellular cargo and have been implicated in cancer progression and chemoresistance. However, the signalling mechanisms driving their formation remain poorly understood. Given the frequent dysregulation of EGFR and c-Met signalling in non-small cell lung cancer (NSCLC), and prior evidence of TNTs in lung adenocarcinoma patient samples, we investigated the role of EGFR and c-Met receptor signalling crosstalk in TNT induction in A549 lung adenocarcinoma cells. Stimulation with EGF, HGF, or in combination induced a concentration dependent increase in the formation of TNTs. TNTs exhibited typical characteristics, including F-actin expression, non-adherence to the substratum and facilitated intercellular trafficking of lysosomes, mitochondria, and lipid vesicles. EGFR was identified as a novel component of TNTs, but had little co-localisation with the c-Met receptor. Co-stimulation with HGF and EGF did not produce consistent additive or synergistic effects on TNT formation, suggesting shared downstream signalling. Furthermore, although EGFR and c-Met inhibition blocked EGF- and HGF-induced TNTs respectively, inhibition of both receptors was required to suppress TNTs following dual HGF/EGF treatment. Interestingly, blocking the EGF receptor alongside c-Met resulted in a more potent inhibition of HGF-induced TNTs, indicating crosstalk. Furthermore, inhibition of downstream MEK and PI3K pathways reduced HGF- or EGF- induced TNT formation, but dual inhibition was required to completely block TNT formation in HGF+EGF co-stimulated cells. These findings reveal a novel convergence of EGFR and c-Met and their downstream MAPK/PI3K pathways in TNT regulation, which can have important clinical implications in combinatorial receptor and cell signalling pathway targeting in NSCLC.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, can invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis. Here, we show that FN stimulates human VSMCs to secrete small extracellular vesicles (sEVs) by activating the β1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery preventing cellular spreading and adhesion. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI on the sEV surface. In vitro assays identified collagen VI as playing a key role in cell adhesion and invasion directionality. Taken together, our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collagen VI enriched sEVs by VSMCs. These sEVs stimulate directional invasion, most likely by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction force to enable VSMC movement within the complex vascular ECM network.

Display technologies are used extensively in the discovery of peptides and antibodies towards the development of new medicines and diagnostic tools. Phage display technology enables the filtering of 10 random peptides/antibodies down to and enriched pool of candidates with favourable binding affinities. In recent years, next-generation DNA sequencing technologies have increased the precision and accuracy with which the peptide sequences of phage display library clones are identified. Inaccuracies in DNA sequencing such as substitutions, insertions and deletions in the library oligonucleotide region have the potential to result in the identification of erroneous candidate sequences. Here, we describe a P ython P ipeline for P hage A nalysis through a N ormative U nified T oolset (P3ANUT) which employs Levenshtein distance, k-mer approaches and a novel encoding scheme on paired-end sequencing outputs to correct sequencing errors from next-generation sequencing outputs of display library screens. We introduce an easy-to-use and highly customisable computational tool with graphical user- and command line interfaces to process entire datasets within a single input, as well as visualisation tools for candidate analysis and data generation. P3ANUT shows significant improvements in read recovery, overall read quality, and runtime compared to a previously published pipeline. GRAPHICAL

A library of ferrocenyl β‐diketonate compounds with varying degrees of aromatic functionality have been synthesized and fully characterized. This includes cyclic voltammetry and the analysis of four new structures by single crystal X‐ray diffraction. The compounds cytotoxic potential has been determined by MTT screening against pancreatic carcinoma (MIA PaCa‐2), ovarian adenocarcinoma (A2780), breast adenocarcinomas (MDA‐MB‐231 and MCF‐7) and normal epithelial retinal (ARPE‐19). The compounds show a general trend, where increasing the number of aromatic rings in the molecule yields an increase in cytotoxicity and follows the trend anthracenyl>naphthyl>phenyl>methyl. The compounds are particularly sensitive to the triple negative cancer cell line MDA‐MB‐231, and the potential modes of action have been studied by production of reactive oxygen species using fluorescence microscopy and cell morphology using Scanning Electron Microscopy. All assays highlight the ferrocenyl β‐diketonate with an anthracenyl substituent to be the lead compound in this library. The decomposition of this compound was also observed within cells, yielding a cytotoxic fluorescent molecule, which has been visualized by confocal microscopy.

During ageing, the extracellular matrix of the aortic wall becomes more rigid. In response, vascular smooth muscle cells (VSMCs) generate enhanced contractile forces. Our previous findings demonstrate that VSMC volume is enhanced in response to increased matrix rigidity, but our understanding of the mechanisms regulating this process remain incomplete. In this study, we show that microtubule stability in VSMCs is reduced in response to enhanced matrix rigidity via Piezo1-mediated Ca2+ influx. Moreover, VSMC volume and Ca2+ flux is regulated by microtubule dynamics; microtubule-stabilising agents reduced both VSMC volume and Ca2+ flux on rigid hydrogels, whereas microtubule-destabilising agents increased VSMC volume and Ca2+ flux on pliable hydrogels. Finally, we show that disruption of the microtubule deacetylase HDAC6 uncoupled these processes and increased α-tubulin acetylation on K40, VSMC volume and Ca2+ flux on pliable hydrogels, but did not alter VSMC microtubule stability. These findings uncover a microtubule stability switch that controls VSMC volume by regulating Ca2+ flux. Taken together, these data demonstrate that manipulation of microtubule stability can modify VSMC response to matrix stiffness.

Background and Purpose: Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined.Experimental Approach: Human aortic‐VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation.Key Results: On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic‐like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin‐1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin‐1 gene expression were up‐regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation.Conclusions and Implications: In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin‐1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin‐1 gene expression is observed in disease relevant VSMC phenotypes.

The skin is a multifunctional organ, forming a barrier between the external and internal environment, thereby functioning as an initial safeguard against foreign and harmful environmental and biological factors. The barrier function is governed by the epidermis, a stratified epithelium composed of an outermost cornified envelope, several differentiated layers, and an innermost basal layer, anchored to the basement membrane (BM). Below the BM lies the dermis, which is composed of an extracellular matrix (ECM), formed by the dermal fibroblasts and dermal adipose tissue. The skin is subject to constant cellular turnover (terminal differentiation) that maintains the homeostatic state of the skin. Recently, autophagy has been implicated in epidermal differentiation and in preserving homeostasis in the skin. The AKT/mTORC1 pathway is a key regulator for epidermal development and differentiation, and conditions (e.g., psoriasis) are linked to the hyperactivation of these key autophagy regulators. A further process known as LC3-associated phagocytosis (LAP) uses some but not all components of autophagy. The Atg16l1 E230 mouse model (E230), deficient for LAP, has been widely used to study the effects of LAP-deficiency and autophagy in tissue homeostasis. Here, the E230 model was used to study the relationship between skin homeostasis and LAP and to determine whether LAP-deficient mice (WD) display a cutaneous skin phenotype. Histological analysis of male 1-year-old Wild Type (WT) and WD mice skin revealed morphological differences in dorsal and tail skin. qPCR analysis of key keratins showed no differences (p > 0.05) when compared between genotypes. This was confirmed by western blotting studies. In addition, the expression of general proliferation markers (Akt & ERK2) showed a small reduction (p < 0.05) in the WD mice skin. General skin barrier formation was assessed by dye permeation assays, which demonstrated full and proper formation of the skin barrier at E18.5 in both WT and WD. Notable dermal thinning in the WD mice skin (p < 0.0001), led us to examine the biomechanical properties to dismiss any abnormalities relating to biomechanical functionality. Results indicate that WT and WD mouse skin show identical biomechanical properties at these ages (p < 0.05 and p < 0.01, respectively). In summary, the noted differences in the dermal and epidermal histology of WD skin were shown to be functionally insignificant in terms of epidermal permeability and dermal biomechanics.