Angiogenesis

We investigated the genetic basis of a previously unclassified congenital vascular anomaly in a patient with multifocal cutaneous vascular tumors. Through genomic analysis, we identified a novel heterozygous de novo germline variant in GA-binding protein-alpha (GABPA), an ETS family transcription factor. Histopathologic evaluation demonstrated capillary-venous lesions lacking glucose transporter 1 expression, distinguishing this entity from common infantile hemangioma. Notably, the tumor vasculature exhibited a prominent alpha-SMA-positive perivascular cell layer, with nuclear localized GABPA detected in endothelial and perivascular cells. The GABPA c.509T>G (p.L170R) variant is absent from large population databases, including gnomAD, DeCAF, and RGC-MCPS databases, indicating it is extremely rare. The affected leucine residue is highly conserved across species and located within the Pointed (PNT) domain, a critical region mediating GABPA protein-protein interactions. AlphaMissense predicts this substitution to be highly damaging. Structure-based analysis using an AlphaFold-predicted approach, combined with in silico mutagenesis and molecular interaction analyses, revealed that the L170R substitution introduces new electrostatic interactions while disrupting native hydrophobic contacts within the PNT domain. Functional assessment in zebrafish demonstrated that mosaic stromal expression of GABPA-L170R caused abnormal vascular architecture compared with GABPA-wild-type controls, establishing a direct link between this variant and disrupted vascular development. Collectively, the genetic rarity, evolutionary conservation, predicted structural perturbation, and disruptive effects on vascular development establish GABPA-L170R as likely disease-causing variant. These findings define a new molecular mechanism underlying congenital vascular tumor formation and expand the role for ETS-family transcriptional regulation in human vascular anomalies.

ABSTRACT Endothelial cells (ECs) display marked vascular bed-specific heterogeneity, and their inflammation-induced activation is a central feature of vascular pathology. However, whether inflammatory responses are primarily shaped by intrinsic organotypic endothelial identity or by the inflammatory stimulus itself remains largely unknown. To address this question, we compared the temporal responses of brain- (hCMEC/D3), skin- (HMEC-1), and lung-derived (HuLEC-5a) human microvascular ECs to lipopolysaccharide (LPS) and interleukin-6 (IL-6/IL-6R). Across all conditions, endothelial origin was the dominant determinant of transcriptional variation, outweighing the effects of both the inflammatory stimulus and exposure time. Brain-, skin- and lung-derived ECs exhibited distinct basal inflammatory and innate immune gene expression programs and responded differently to both LPS and IL-6/IL-6R. LPS induced a rapid but largely transient response, whereas IL-6/IL-6R elicited more sustained inflammatory changes. Although transcriptional profiling identified a shared inflammatory signature across EC populations, most differentially expressed genes were vascular bed specific. These molecular differences translated into distinct functional outcomes: IL-6/IL-6R increased endothelial permeability and promoted more sustained metabolic adaptations, whereas LPS induced robust inflammatory activation without persistent barrier dysfunction. Collectively, these findings demonstrate that while inflammatory stimuli determine which signaling pathways are activated, intrinsic endothelial identity dictates the magnitude, kinetics, and functional consequences of the inflammatory response. Our results highlight the importance of incorporating vascular bed specificity into experimental models and the development of therapies targeting endothelial dysfunction and inflammation.

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.

Objective: The maturation of an autologous arteriovenous fistula (AVF) depends on the coordinated hemodynamic adaptation of the inflow artery and the outflow vein. While venous remodeling has been extensively studied, the mechanisms of arterial adaptation are poorly understood. We addressed this knowledge gap with the first temporal single-cell atlas of the porcine AVF model. Methods: Six Yorkshire pigs underwent bilateral femoro-femoral AVF creation for tissue harvest at 2 and 21 days postoperatively or sham operation to collect native vessels after 2 days. Arteries and veins were analyzed by droplet-based single-cell RNA sequencing, histology, and immunohistochemistry. Results: All fistulas were patent, with flows reaching 1185.0 +/- 170.6 mL/min despite mild to moderate neointima formation in the veins. Arteries and veins demonstrated >60% endothelial denudation and increased neovascularization. Arterial remodeling was characterized by a progressive increase in venous-like transcriptional features. Arteries presented a venous-like endothelial cell (EC) composition at 21 days postop and a temporal increase in venous-associated phenotypes of myofibroblasts and fibroblasts. In both vessels, the latter populations were the main bearers of mechanosensitive signatures at 2 days. Despite the temporal convergence in phenotypes, arteries and veins retained vessel-selective characteristics. Arterial ECs expressed high levels of nitric oxide synthase, whereas venous ECs were largely inflammatory. Arterial myofibroblasts and fibroblasts had higher expression of Notch signaling transducers (HEY2, HEYL) and cellular communication network factors (CCN3, CCN5), while Wnt signaling regulators (DKK2, SFRP4) and complement genes (C3, C7, CFD) were upregulated in veins. Anti-inflammatory macrophages increased in both vessels during remodeling, likely contributing to the maturation of the fistulas. Conclusion: These analyses challenge the traditional paradigm of exclusive vein arterialization and support a complementary process of arterial venification after anastomosis. Recognition of this underappreciated phenomenon may reshape our understanding of AVF biology and guide the discovery of both vascular-wide and vessel-selective anti-stenotic therapies.

BackgroundThe progression of abdominal aortic aneurysm (AAA) is closely associated with the phenotypic switch of vascular smooth muscle cells (VSMC). While members of the Serine/Arginine-rich splicing factor (SRs) superfamily are increasingly acknowledged for their pivotal role in modulating cell phenotype, the specific contribution of these factors to AAA development has remained underexplored. MethodsThrough integrated analyses the whole transcriptome of human and murine AAA tissues derived from clinical samples and animal models, we identified serine-arginine repeat matrix 4 (SRRM4) as a candidate gene regulating AAA formation. To investigate the role of SRRM4 in AAA formation, we used global and VSMC-specific SRRM4-knockout mice to construct AAA mouse models in two different ways. The impact of SRRM4 on AAA was further investigated in in vivo and in vitro models ResultsElevated levels of SRRM4 mRNA and protein were observed in VSMC from both human and murine AAA tissues. Targeted silencing of SRRM4 in VSMC (but not in endothelial or myeloid cells) significantly attenuated AAA formation in both AngII- and CaCl2-induced mouse models. Our results identify the RE1 silencing transcription factor (REST) as a critical target gene of SRRM4 in AAA, a master regulator of cellular phenotypic stability. We show that SRRM4 modulates VSMC phenotype through alternative splicing of the REST gene, resulting in a non-functional REST4 isoform. Furthermore, our study shows that REST directly represses STAT1, a mechanism by which SRRM4 influences VSMC phenotypic transition. Regarding clinical significance, inhibition of both STAT1 expression and function was shown to be beneficial in preventing AAA formation and slowing disease progression in mouse models. ConclusionsThis investigation uncovers a previously unrecognized role of SRRM4-mediated splicing of the REST gene in the progression of AAA and identifies a novel REST/STAT1 axis critical in the phenotypic modulation of VSMC. The findings of this study may provide insights for the development of innovative therapeutic strategies for AAA. What is New?O_LIOur study demonstrates that the RNA splicing factor SRRM4 (serine/arginine repetitive matrix 4) expression is increased in human and murine abdominal aortic aneurysm (AAA) tissues and unveils a previously undescribed role of SRRM4 in regulating AAA development. C_LIO_LISRRM4 promotes AAA development and progression through inducing splicing of REST (RE1 silencing transcription factor) gene and upregulating STAT1 (signal transducer and activator of transcription 1) expression, which result in extracellular matrix degradation, inflammation, and VSMC phenotypic switching. C_LIO_LIOur study provides proof-of-concept evidence indicating the therapeutic value of STAT1 inhibition in AAA prevention and treatment. C_LI What Are the Clinical Implications?O_LIOur study defining a novel mechanism underlying the SRRM4/REST/STAT1 axis is crucial for developing new therapeutic strategies for AAA. C_LIO_LIBased on these findings, we have initiated a prospective study (REST4-AAA; ClinicalTrials.gov Identifier: NCT07056491) to evaluate REST4 expression levels in serum of AAA patients and explore its potential as a serum biomarker for AAA prediction C_LIO_LIOur findings indicate that fludarabine, a clinically available agent reported to selectively suppress STAT1, effectively attenuates AAA progression in mice, suggesting its potential for repurposing as a therapeutic strategy for AAA. C_LI

Vasculogenic mimicry (VM) describes the ability for tumor cells to mimic endothelial cells and form tumor-lined blood vessels. Although VM is associated with poor prognosis, resistance to therapeutics and metastasis, its presence, relevance and functionality have been debated, largely due to the techniques employed to determine its presence. Dual CD31 and periodic acid-Schiff (PAS) histological staining, the method widely used to detect VM within tumor tissue, is hampered by issues with artifacts of 2D sections and does not assess active blood flow through vessels and hence cannot confirm vessel functionality. Here, we present a 3D imaging pipeline to detect functionally perfused VM and host vessels within mouse tumor models. Mice tumor models are intravenously injected with fluorescently-labelled tomato lectin which binds to the lumen of functional blood vessels. These tumors are thickly sliced, optically-cleared, stained with endothelial markers and imaged using light-sheet microscopy. We apply this imaging modality to image tumors from six breast cancer patient-derived tissue xenograft models, an orthotopic mammary cancer model and two small cell lung cancer circulating-tumor cell explant models, where VM vessels, mosaic vessels and host-VM vessel junctions can be detected. To our knowledge, this is the largest collection of 3D VM vessel images to date, providing convincing evidence of the presence and functionality of VM within tumors and defining the level of evidence necessary to detect VM in vivo.

Purpose: To understand the functional impact of macular atrophy (MA) and fibrosis in eyes with treated neovascular age-related macular degeneration (nAMD) based on deep visual sensitivity losses. Design: Cross-sectional study. Participants: One-hundred and nine eyes from 97 individuals with nAMD that had been treated for >=12 months without significant retinal fluid. Methods: All participants underwent defect-mapping microperimetry (DMP) testing - a strategy optimized to quantify the spatial extent of deep visual sensitivity losses through single 10 decibel stimuli presentations - where 208 locations were sampled in the central 8 degrees radius region. Participants also underwent color fundus photography, fundus autofluorescence and OCT imaging, which were co-registered and manually annotated for MA and fibrosis to determine their two-dimensional pointwise extent at individual test locations, and global extent in the corresponding region where DMP was performed. Main Outcome Measures: Pointwise association between the presence of MA and fibrosis with missing a stimulus on DMP and global association between the extent of MA and fibrosis with the proportion of locations missed (PLM) on DMP. Results: At a pointwise level, the presence of MA and fibrosis were independently associated with an increased likelihood of missing a stimulus on DMP (odds ratio = 42.5 and 2.5 respectively; P < 0.001 for both). However, at a global level, only an increasing extent of MA was associated with a significantly higher PLM on DMP (P < 0.001), but not fibrosis (P <= 0.227). MA extent explained a substantial proportion of the variance in the PLM (R2 = 0.97) and there was a very strong correlation between the two parameters ({rho} = 0.93). Conclusions: This study confirmed the expected functional impact of MA and fibrosis on DMP testing at a pointwise level. However, it showed at the global level that only the extent of MA, and not fibrosis, was independently associated with functional loss on DMP, and that there was a very strong structure-function correlation based on MA. These findings thus suggest that evaluating MA alone may be sufficient for capturing structural changes strongly associated with the global extent of deep visual sensitivity losses in those with treated nAMD.

Objective: Rare STAB2 loss-of-function variants emerged in population studies as being associated with elevated venous thromboembolism (VTE) risk. STAB2 encodes the cell-surface scavenger receptor stabilin-2, and reduced stabilin-2 function might increase VTE risk via elevated plasma levels of its ligands, including von Willebrand factor (VWF) and coagulation factor VIII (FVIII). Approach and Results: A 13-year-old patient with dependency on parenteral nutrition had experienced episodes of central venous catheter-related thrombosis, including the complete thrombotic occlusion of the deep pelvic veins, and pulmonary embolism. Exome sequencing for increased VTE risk identified a homozygous STAB2 splice-site variant and its effect on splicing was characterized in fibroblast-derived RNA and in a minigene splicing system. Plasma VWF, FVIII, and fibrinogen and serum hyaluronic acid (HA) levels were determined. Immunohistochemistry was performed on liver biopsy tissue. The STAB2 splice-site variant caused aberrant STAB2 mRNA splicing. The patient's healthy mother and one healthy sibling were also homozygous for the STAB2 splice-site variant but had no history of VTE. Plasma FVIII levels and VWF activity were elevated in all three, and VWF antigen was elevated in two homozygous individuals. Strikingly, approximately 1000-fold elevated HA levels were observed in all three homozygous individuals. Stabilin-2 immunohistochemistry on a liver biopsy from the proband's mother revealed no detectable signal, whereas control human liver showed strong labelling of liver sinusoidal endothelial cells. The STAB2 splice-site variant thus results in loss of detectable stabilin-2 protein. Conclusion: To current knowledge, this is the first family-based report of stabilin-2 deficiency with markedly elevated circulating HA, VWF, and FVIII levels in humans. These findings support impaired stabilin-2-dependent ligand clearance as a thrombosis susceptibility state with incomplete penetrance. The data are consistent with a significant role for stabilin-2 in circulating HA clearance in humans, but do not establish elevated HA itself as a cause of VTE.

Background: Localized or segmental overgrowth is a rare condition that may co-occur with vascular malformations. These diseases generally are caused by somatic mutations in the PI3K/AKT/mTOR pathway. Most lesions are progressive and may cause symptoms including pain, functional impairment, and disfigurement. Treatment options are limited, especially for the overgrowth component. Objective: To evaluate the efficacy and safety of the mTOR inhibitor sirolimus in patients with overgrowth with or without a vascular malformation over a 6-months treatment period. Methods: We conducted a single-arm, open-label, phase II study including 18 patients (12 pediatric, six adult) with overgrowth, all of whom initiated oral sirolimus. 16/18 (88.9%) participants received oral sirolimus for 6 months, with dosing adjusted to achieve target blood trough levels of 3-8 ng/mL. Primary outcome was radiological response at six months assessed by the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Secondary outcome parameters were evaluated at three, six and nine months and included size and visual appearance of the overgrowth, quality of life (QoL), pain intensity, performance status, and safety. Results: No patient achieved complete or partial radiological response. 17 of 18 patients (94.4%) maintained stable disease at month six; one was not evaluable. QoL measured with WHOQOL-BREF improved modestly in patients aged >= 18 years (n=5, mean difference to baseline +9.3 (95%-CI: -0.9 to 19.5) in physical and +9.2 (95%-CI: 0.7 to 17.7) in psychological domains at month 6). In pediatric participants, no relevant improvement in KINDL total scores was observed over the study period. No significant changes occurred in pain scores. Performance status measured by the Lansky/Karnofsky scales showed minor improvement (mean +3.5 points (95%-CI: 1.0 to 6.1)). All patients experienced at least one adverse event, but no serious adverse events or CTCAE v5.0 grade [&ge;]3 toxicities occurred. Sirolimus was well tolerated. Six patients decided to resume sirolimus after study completion. Conclusions: Sirolimus induced minor lesion shrinkage in a subset of patients, although this did not meet the predefined radiological RECIST endpoint. However, sirolimus can lead to functional improvement in some adults, which may indicate that the primary radiological outcome did not capture patient-perceived benefit.

Background and Aims: Quantifying arterial inflammation can augment risk stratification and drug development in atherosclerosis. While perivascular adipose tissue (PVAT) density, captured as fat attenuation index (FAI) on CT angiography (CTA), is an emerging biomarker of coronary inflammation, it remains unclear whether this concept extends to carotid atherosclerosis. Methods: Using routine CTA data from hospital-based stroke cohorts, we developed a pipeline to quantify attenuation metrics of PVAT surrounding the distal common and proximal internal carotid arteries. We compared PVAT metrics between culprit and contralateral non-culprit plaques in patients with symptomatic carotid atherosclerosis and between culprit plaques and control carotid segments from patients without symptomatic atherosclerosis. In patients undergoing carotid endarterectomy, we assessed associations of CTA-derived PVAT metrics with histological and proteomic markers of plaque inflammation and stability. Results: Across 107 patients with symptomatic carotid atherosclerosis (75.5{+/-}8.7 years; 63% men), PVAT attenuation metrics did not show significant differences between culprit and contralateral non-culprit lesions (all p>0.2). We also found no evidence for higher PVAT attenuation in culprit lesions, when compared to arteries of patients without symptomatic atherosclerosis. In 82 patients undergoing endarterectomy, pre-operative CTA-derived metrics of PVAT attenuation showed no significant correlations with CD68+ macrophage areas in excised plaques (r -0.11 to 0.07, all p>0.3). Similarly, no PVAT attenuation metrics were associated with thinner fibrous cap or higher plaque levels of pro-inflammatory cytokines (IL-6, TNF). Conclusions: In contrast to coronary atherosclerosis, carotid PVAT attenuation was not associated with symptomatic culprit lesions or histological and molecular markers of plaque inflammation and vulnerability.

BACKGROUND: The chromosome 9p21.3 locus is among the strongest genetic risk loci for cardiovascular disease (CVD), yet the regulatory mechanisms underlying this association remain poorly understood. Previous vascular disease studies focused predominantly on the role of this locus in smooth muscle cell (SMC) dysfunction, whereas its potential role in endothelial cells (ECs) remains less well characterized. Here, we sought to identify candidate regulatory variants at 9p21.3, their distal target genes, and effector transcripts that contribute to CVD-related phenotypes in ECs. METHODS: Promoter capture Hi-C was used to map chromatin interactions at 9p21.3 in primary human ascending aortic SMCs (n=6), supplementing the previously obtained ECs (n=16) dataset. To support the regulatory relevance of these interactions, expression quantitative trait locus analysis was performed in 136 vascular biopsies. Variant effects were prioritized in silico using AlphaGenome. The regulatory activity of the prioritized region was validated using CRISPR activation. RESULTS: Chromatin interaction mapping identified a complex regulatory architecture at the 9p21.3 locus involving 19 putative regulatory elements and 15 genes across ECs and SMCs. MIR31HG showed the most recurrent distal promoter connectivity with the 9p21.3 region. Consistent with the chromatin interaction map, expression of MIR31HG transcripts was associated with genetic variation at the 9p21.3 locus. In ECs, the lncRNA MIR31HG, but not miR-31, was responsive to hemodynamic flow. Depletion of MIR31HG in ECs was associated with endothelial activation. Epigenetic tracks and deep learning predictions highlighted an active enhancer element at 9p21.3, which was subsequently validated by CRISPR activation and increased MIR31HG expression, confirming its capacity to regulate MIR31HG over a large genomic distance of approximately 538 kb. CONCLUSIONS: Altogether, these data implicate lncRNA MIR31HG as a likely effector of the 9p21.3 CVD locus, linking the cis-regulatory element and heritable genetic variation in the respective region to endothelial activation through long-range enhancer regulation.

Purpose: Post-marketing reports have associated faricimab, the first intravitreal bispecific antibody, with intraocular inflammation (IOI). Its inflammatory phenotype, temporal pattern and systemic safety relative to established anti-VEGF agents remain unclear. We compared faricimab, aflibercept and ranibizumab across three independent pharmacovigilance databases. Methods: We analysed FAERS reports from Q1 2022 to Q2 2026, with external validation in JADER (PMDA, Japan) and EudraVigilance. Reports were de-duplicated, restricted to primary-suspect reports, and a pre-specified list of 36 non-adverse-event preferred terms (PTs) was excluded. We computed reporting odds ratios (ROR), proportional reporting ratios and Bayesian information components, with Benjamini-Hochberg false discovery rate control. Head-to-head RORs were adjusted for demographic confounders by logistic regression. Time to onset (TTO) was modelled with Weibull distributions. Sensitivity analyses addressed masking and differential market duration. Signals were graded with a pre-specified bidirectional cross-database evidence framework (Confirmed, Consistent, Unconfirmed, Discordant). Results: Faricimab was reported more often for anterior-chamber and vitreal inflammation. Adjusted RORs (comparator versus faricimab, values <1 indicating faricimab excess) were 0.27 (95% CI 0.18-0.40) for iridocyclitis and 0.45 (0.35-0.58) for uveitis versus aflibercept, and 0.13 (0.06-0.24) and 0.12 (0.06-0.20) versus ranibizumab. All four PTs were Confirmed in JADER against both comparators; for iridocyclitis, RORs were 0.048 versus aflibercept and 0.109 versus ranibizumab. Faricimab IOI arose late (median TTO 100 days, IQR 31-182, versus 7 days for both comparators; P = 2.6E-14) and its Weibull onset distribution was markedly flatter (shape 0.81 versus {approx}0.42). In contrast, aflibercept predominated for endophthalmitis-spectrum events and intraocular pressure elevation, and ranibizumab for vitreous haemorrhage, the latter confirmed in JADER. Faricimab showed lower reporting across cardiac, nervous system, vascular and renal system organ classes, confirmed in EudraVigilance. An unadjusted FAERS death signal (ROR 3.16, aflibercept versus faricimab) attenuated after adjustment (aROR {approx}1.8) and was absent in outcome-based JADER analyses. Conclusions: Faricimab exhibits a reproducible, delayed-onset, anterior-predominant IOI phenotype alongside a comparatively favourable systemic safety profile. Clinicians should be aware that faricimab-associated IOI may arise weeks to months after injection. Prospective studies with exposure denominators are needed to quantify absolute risk.

Retinal vascular features are proposed biomarkers of systemic health, yet their longitudinal behaviour and relationship to systemic health remain poorly understood. We quantified retinal vascular morphology from serial colour fundus photographs in the Canadian Longitudinal Study on Aging, including 21,562 participants at baseline with good quality images. On average, vascular complexity and density decreased between baseline visit and follow-up 2 (mean interval 5.7 years), whereas tortuosity, arteriolar widths, and venular widths increased. Baseline vascular features were associated with demographic, cardiovascular, metabolic and behavioural traits, whereas altered longitudinal trajectories were associated principally with older age and higher low-density lipoprotein cholesterol. Within-person increases in mean arterial pressure covaried with lower vascular complexity, density and vessel widths. In survival analyses, independently of baseline vascular features, increases in fractal dimension were associated with lower hazard of subsequent all-cause mortality (hazard ratio = 0.92 per standard deviation, 95% confidence interval (CI) = 0.86 - 0.98), whereas arteriolar widening was associated with higher mortality (hazard ratio = 1.09, 95% CI = 1.02 - 1.17). No retinal change metric was associated with major adverse cardiovascular events after false-discovery-rate correction. These findings identify retinal vascular remodelling as a dynamic phenotype associated with systemic health and subsequent mortality, and motivate evaluation of multi-time-point retinal imaging in oculomics.

Background: Platelets are critical drivers of thrombo-inflammatory responses in different cardiovascular diseases. Abdominal aortic aneurysm (AAA) is a progressive, life-threatening vascular disorder mainly characterised by chronic inflammation, extracellular matrix degradation, and the formation of a platelet-rich intraluminal thrombus (ILT). Experimental and clinical evidence identified platelets as main players in AAA pathology as evidenced by elevated platelet activation and procoagulant activity that critically contribute to AAA progression. Methods: The present study investigated the contribution of glycoprotein (GP)Ib alpha, the von Willebrand factor (VWF)-binding subunit of the platelet GPIb-IX-V complex, to AAA initiation and progression in experimental AAA using the ePPE mouse model and in patients. Results: Genetic ablation of platelet GPIb alpha significantly attenuated early aneurysm expansion in experimental AAA, indicating a critical role for GPIb alpha during the initial stages of aneurysm development. This initial effect was compensated at later time points showing no differences in aneurysm progression between groups. Notably, genetic deletion of GPIb alpha induced a constitutively hyperactive platelet phenotype already in naive mice that was further amplified during experimental AAA. This elevated platelet hyperactivity was mainly due to increased GPVI activation of platelets 28 days post-surgery. To assess the clinical relevance, spatial profiles of human ILT specimens from patients with AAA were analysed. In the ILT, we detected a highly compartmentalised distribution of GPIb alpha and VWF with pronounced enrichment within the luminal layer. In parallel, circulating VWF activity as well as platelet surface expression of GPIb alpha were significantly increased in patients with AAA. Conclusion: Collectively, these findings identify a dysregulated GPIb alpha-VWF axis in human AAA pathology, mainly characterised by enhanced platelet GPIb alpha surface expression and increased activity of circulating VWF.

Pathogenic variants of COL4A1 and COL4A2, which encode the 1 and 2 chains of type IV collagen in vascular basement membranes, cause cerebral small vessel disease and intracerebral hemorrhage, but the signaling mechanisms that precede vascular damage remain incompletely defined. Here, we investigated cerebral arterial function in mice heterozygous for the Col4a2+/G646D mutation. Cerebral pial arteries from aged (12-month-old), but not young (3-month-old), Col4a2+/G646D mice exhibited impaired pressure-induced myogenic tone, whereas downstream parenchymal arterioles retained normal myogenic responsiveness, revealing a segment-selective vascular defect. The frequency of subcellular Ca2+ signals (Ca2+ sparks) generated by the release of Ca2+ through ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) was increased, and large-conductance Ca2+-activated K+ (BK) channel activity was elevated, in cerebral pial artery smooth muscle cells (SMCs) from aged Col4a2+/G646D mice. SR Ca2+ store load and Ryr2 transcript abundance were unchanged in these cells, but Stimulated Emission Depletion super-resolution microscopy revealed that RyR2 protein clusters were enlarged compared with controls. Protein kinase A (PKA) activation reproduced RyR2 cluster enlargement, whereas PKA inhibition normalized RyR2 cluster organization and BK channel activity in SMCs from aged Col4a2+/G646D mice. These findings identify age-associated, PKA-dependent, nanoscale remodeling of RyR2 clusters as a previously unknown mechanism underlying cerebral arterial dysfunction in COL4A2-related disease.

MYC amplification is the most common genetic alteration in radiation-associated angiosarcoma and is also present in a subset of de novo primary angiosarcoma, yet its oncogenic role in this vascular malignancy remains unclear. Here, we report that inducible, endothelial-specific MYC overexpression alone was sufficient to drive angiosarcomagenesis in mice. The resulting pulmonary angiosarcoma tumors exhibited impaired endothelial differentiation, indicative of MYC-driven lineage plasticity. Notably, MYC inactivation in dedifferentiated allografts induced growth arrest and restored endothelial gene expression, demonstrating MYC-dependent reversible cellular plasticity. Integrative analyses of MYC ChIP-seq, ATAC-seq, and RNA-seq revealed that overexpressed MYC preferentially occupied highly accessible promoters and activated genes involved in cell growth. Consistent with the murine findings, MYC-amplified human angiosarcomas were enriched for MYC target and cell-cycle programs and depleted of endothelial lineage signatures. MYC overexpression in human endothelial and angiosarcoma cells recapitulated these transcriptional changes, enhanced proliferation, and impaired endothelial tube formation. Together, our results establish MYC as an oncoprotein that promotes endothelial lineage plasticity to drive angiosarcoma initiation and progression.

Objective: Dendritic cells (DCs) are priming local immune responses, and their tissue-associated activity increases in inflammatory conditions. In patients with coronary artery disease (CAD), circulating dendritic cell precursors (DCPs) are reduced. This study investigates circulating DCPs and tissue densities of migrated DCs in CAD patients undergoing bypass surgery, focussing on patients with chronic kidney disease (CKD) as a risk factor for CAD and a condition of chronic vascular inflammation. Approach and Results: Circulating DCPs were measured in the peripheral blood (before surgery) using fluorescence-activated cell sorting (FACS) in n=59 patients and results were compared with an age-matched healthy control group (n=45). DCs were stained (Fascin) in circular sections of the internal thoracic artery taken during bypass surgery and mature (CD83+) and immature (CD209+) DCs were differentiated. Cell densities were obtained digitally. Descriptive statistics and correlation analysis generated hypotheses. Circulating myeloid DCPs were reduced (p<0.001), concomitant CKD added no further reduction. Immature DCs were predominantly detected in the adventitia, mature DCs dominated in the intima (p<0.001). In CKD, reduced circulating myeloid DCPs correlated with immature DCs in the vessel wall (r=0.87; p=0.002) and rather mature DCs were detectable; in patients without CKD immature DCs were dominating. C-reactive protein correlated with mature DCs (r=0.38; p=0.026). Conclusions: Quantities of DCPs and mature and immature DCs vary between the vessel wall layers. The dominance of immature myeloid DCs in the adventitia suggests activation of the vascular-associated lymphoid tissue (VALT). Intensified inflammation associated with CKD may have promoted the maturation of myeloic DCs.

Vascular disease often begins as a failure to read mechanical force. Cerebral cavernous malformations (CCMs) are leaky, brain-invading capillary clusters that cause stroke and seizures and arise from endothelial loss of the CCM complex. They form exclusively in low-flow vessels but existing models cannot explain this selectivity. We previously established that CCM-mutant endothelium inverts its adhesive balance, weakening cell-cell junctions while building matrix-degrading adhesions. The mechanosensors imposing this switch are unknown. Here we show that the mechanosensitive Ca2+ channels PIEZO1, TRPV2 and TRPV4 are overexpressed in the endothelium of CCM lesions in a 42-patient cohort, and that lesion volume scales with their expression level. Mechanistically, PIEZO1 partners with TRPV4 to destabilise cell-cell junctions and with TRPV2 to reinforce cell-matrix adhesions; silencing TRPV4 restores junctional integrity and abolishes KLF4 induction, whereas silencing TRPV2 or inhibition with tranilast abolishes matrix degradation and invasion. In vivo, trpv4 knockdown or inhibition with GSK2798745 restores atrioventricular canal morphogenesis in ccm2-mutant zebrafish. Gain of mechanosensory channel function is thus a causal step in CCM pathogenesis, upstream of the KLF program. Combined therapeutic targeting of TRPV2 and TRPV4 with inhibitors validated in humans offers a route to therapy.

Purpose To evaluate the safety, tolerability, and preliminary bioactivity of a single suprachoroidal injection of AXT107 (Gersizangitide), a multimodal integrin-disrupting peptide that acts on both the VEGFA pathway and angiopoietin-Tie2 pathway, in eyes with neovascular age-related macular degeneration (nAMD). Design Phase 1/2a, open-label, dose-escalation clinical trial. Participants Fifteen subjects with active nAMD, including 11 treatment-experienced partial responders to intravitreal anti-vascular endothelial growth factor (VEGF) therapy and 4 treatment-naive subjects; 14 eyes met the protocol-specified baseline imaging criterion and were included in efficacy analyses. Methods Study eyes received a single suprachoroidal injection of AXT107 microparticles at doses of 0.125, 0.25, or 0.5 mg and were followed for 40 weeks. Protocol-specified rescue intravitreal anti-VEGF treatment was permitted from week 12 based on protocol-specified best-corrected visual acuity (BCVA) or central subfield thickness (CST) criteria; anti-VEGF treatment before Week 12 was investigator-initiated. Primary outcomes were ocular and systemic safety through week 40; secondary outcomes included change in CST, change in BCVA, and time to first protocol-specified rescue injection. Main Outcome Measures Incidence of ocular and systemic adverse events, intraocular pressure (IOP), CST, BCVA, and time to first protocol-specified rescue injection. Results Twenty subjects were screened and 15 enrolled across 4 U.S. sites; 3 eyes received 0.125 mg, 3 received 0.25 mg, and 9 received 0.5 mg of AXT107. No serious adverse events related to AXT107 occurred, and no cases of endophthalmitis, retinal detachment, suprachoroidal hemorrhage, or clinically significant intraocular inflammation were observed through week 40. No clinically meaningful intraocular pressure (IOP) elevations occurred at any dose, and no subject required chronic IOP-lowering therapy. Among the 14 eligible eyes included in efficacy analyses, 2 eyes (01-04 and 01-05) completed 40 weeks without protocol-specified anti-VEGF rescue, and 1 eye (02-04) first received protocol-specified rescue at week 36. At the last on-treatment visit, BCVA changes ranged from +15 to -31 ETDRS letters; 3 eyes gained[&ge;]5 letters, 3 gained[&ge;]10 letters, and 1 gained[&ge;]15 letters. Among the 14 eligible eyes, anatomical improvements in CST were observed in at least 1 eye in each dose cohort. The most durable responses were seen in treatment-experienced eyes receiving 0.5 mg (median durability of 36 weeks), whereas treatment-naive 0.5-mg eyes showed more variable courses and generally earlier protocol-specified rescue in this small cohort. Conclusions In this first-in-human study, a single suprachoroidal injection of AXT107 up to 0.5 mg was well tolerated through Week 40, with no drug-related serious adverse events, no clinically significant intraocular inflammation, and no sustained IOP elevations. After a single suprachoroidal AXT107 injection, 2 eyes completed 40 weeks without protocol-specified rescue anti-VEGF treatment, and 1 additional eye received its first protocol-specified rescue treatment at Week 36. The exploratory observation that durability appeared greatest in treatment-experienced eyes supports further controlled study of AXT107, particularly in treatment-experienced nAMD populations.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.