Angiogenesis

The Hippo signaling pathway is increasingly recognized as a key regulator of endothelial cell (EC) proliferation, migration and vascular development. However, the roles of its upstream scaffold proteins remain poorly understood. Although WWC family proteins are widely regarded as functionally redundant activators of LATS1/2 kinases, the human genome contains a third family member, WWC3, that is absent from mice, raising the possibility of species-specific regulation of endothelial Hippo signaling. Here, we assessed the roles of WWC2 and WWC3 in human ECs using siRNA-mediated knockdown. Surprisingly, we found that WWC3 is the predominant regulator of canonical Hippo signaling, with a substantially greater effect than WWC2 on LATS1/2 phosphorylation, YAP/TAZ localization and expression of Hippo target genes. Loss of WWC3 also altered endothelial morphology and induced a partial endothelial-to-mesenchymal transition-like (EndoMT-like) phenotype. By contrast, WWC2 had a lesser effect on canonical Hippo signaling, but it was required for normal VEGF signaling dynamics. Despite these distinct molecular functions, depletion of either WWC2 or WWC3 impaired EC proliferation, migration, and cord formation in vitro. Together, our findings demonstrate that WWC family proteins perform overlapping but distinct functions in human ECs, with WWC3 acting as the predominant canonical Hippo regulator, whereas WWC2 more efficiently modulates VEGF signaling. These results reveal unexpected functional specialization among WWC proteins and suggest that regulation of Hippo signaling in human ECs differs from that inferred from mouse studies.

ANGPT2 is widely recognized as a critical regulator of pathological neovascularization. By analyzing scRNA-seq data from neonatal retinas, we demonstrate that Angpt2 transcripts are highly enriched in tip cells relative to other endothelial subtypes, where Angpt1/4 expression is absent. However, mechanisms underlying ANGPT2 function at angiogenic fronts remain inadequately understood. Here, we show that endothelial Angpt2 deletion severely disrupted retinal vascularization, characterized by neovascular tufts and micro-hemorrhage. Similar angiogenic defects also occurred in the brain, but were less evident in other tissues examined. Mechanistically, ANGPT2 insufficiency attenuated retinal tip cell invasion with aberrant mural cell coverage, compromising sprouting into non-vascularized tissues. Retinal RNA-seq analysis revealed that transcripts associated with endothelial migration and junction assembly were reduced in Angpt2 mutants compared to littermate controls, while upregulated genes were enriched in hypoxia-responsive pathways and mural cell development. Notably, abnormal mural-tip cell associations were detected within 48 hours post-Angpt2 deletion, displaying also a hypoxia-driven transcriptomic signature. These closely resemble the vascular pathologies observed in human retinopathy of prematurity. In contrast, Angpt1 insufficiency or Angpt4 deficiency primarily affected venous morphogenesis. Collectively, our findings imply that ANGPT2 is essential for driving tip cell invasion during sprouting angiogenesis, and that its insufficiency triggers hypoxia-driven vascular anomalies.

Anti-angiogenic cancer therapies attempt to withhold necessary nutrients and oxygen from growing tumors by targeting the major promoters of endothelial cell (EC) angiogenesis: vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2). Unfortunately, these treatments are often insufficient, even when coupled with chemotherapies, and fail to significantly increase survival rates. The tumor microenvironment (TME) is mechanically distinct compared to normal tissue, including increased matrix deformations or strains caused by cancer-associated fibroblasts (CAFs). In this report, we detail the specific and independent roles of two tyrosine residues, Y1054 and Y1214, on mechanical activation of VEGFR-2. Furthermore, we characterize CAF biochemical and mechanical signaling and demonstrate how ECs exhibit decreased vessel growth when co-cultured with CAFs and treated with a contractility inhibitor. Using non-phosphorylatable VEGFR-2 mutants, we reveal Y1054 and Y1214 are each necessary for EC angiogenesis, particularly in response to strain. Overall, this research highlights the need to study how mechanics in the TME promote vessel growth and thus tumor progression, which is important to consider when developing future anti-angiogenic therapies.

Uterine fibroids (leiomyomas) are highly prevalent benign tumors defined by excessive extracellular matrix (ECM) deposition, altered vascular structure, and progressive tissue stiffening, yet the cellular programs that coordinate these features remain poorly understood. Cellular senescence has been implicated in fibroid biology, but whether senescence represents a uniform state or distinct, functionally specialized cell identities within fibroids is unknown. Here, we identify the distinct heterogeneous populations of senescent cells ("senotypes") present in human fibroids and characterize their role in shaping the fibroid microenvironment. Using single-cell RNA sequencing (scRNA-seq) integrated with a senescence gene signature and protein-level validation, we identify senescent cells (SnC) distributed across fibroblast, mural, and endothelial compartments, each exhibiting distinct transcriptional programs. SnC endothelial cells (ECs) are enriched in fibroids relative to matched myometrium and activate TEAD4-associated mechanosensing, angiogenic, and immune signaling pathways, despite being associated with impaired vessel maturation in situ. In parallel, SnC fibroblast and mural populations in fibroids upregulated SRF-associated cytoskeletal and ECM programs, accompanied by increased COL6A3 expression and collagen VI deposition, consistent with tissue stiffening. Ligand-receptor and spatial analyses reveal that these SnC populations function as interconnected signaling hubs, coordinating immune cell recruitment and stromal remodeling. Importantly, analysis of human fibroids treated with collagenase demonstrated a reduction in both ECM density and SnC burden, supporting a reinforcing relationship between matrix mechanics and senescence. Together, these findings establish senescence in fibroids as a heterogeneous, mechanically reinforced, and network-driven process that links vascular dysfunction, immune signaling, and fibrosis, highlighting distinct SnC states as potential translational targets for non-surgical therapies.

Endothelial senescence is increasingly recognized as a driver of vascular pathology, while immunoglobulin G (IgG) has recently been reported to accumulate in aging tissues and induce senescence in macrophages and microglia. In cerebral cavernous malformations (CCMs), IgG accumulation has been obviously observed in CCM lesions, but the contribution of IgG to endothelial injury remains unclear. Using multi-omic profiling, endothelial models, and CCM mice, we identified IgG-secreting plasma cells enriched in lesions associated with endothelial senescence, hemorrhage, and disease severity. CCM loss-associated mTOR activation impaired lysosomal acidification and IgG processing, promoting intracellular IgG accumulation. IgG, in turn, induced NF-kB-dependent endothelial senescence. In vivo, BCMA-mediated plasma cell depletion attenuated lesion progression, whereas IgG supplementation partially restored disease severity. Anti-CD38 treatment likewise reduced IgG accumulation, endothelial senescence, hemorrhage, and lesion progression. These findings identify lysosomal dysfunction-mediated IgG as a pathogenic trigger of endothelial senescence and support targeting the plasma cell-IgG axis in CCM.

Fibroblast growth factor 2 (FGF2) is frequently induced during ischemic retinal injury and has traditionally been considered a pro-angiogenic factor based largely on studies using exogenous FGF2 administration. However, its endogenous cellular origin and physiological role remain incompletely understood. Here, we used single-cell transcriptomic analysis combined with spatial validation and rod photoreceptor-specific genetic approaches to define the endogenous role of FGF2 during oxygen-induced retinopathy (OIR). We identify rod photoreceptors as a major cellular source of ischemia-induced FGF2. Notably, Fgf2 expression remained elevated during the regression of pathological neovascularization, revealing a temporal dissociation between neuronal stress responses and vascular remodeling. Single-cell analysis further showed that Fgf2 induction occurred within a coordinated photoreceptor stress-response program involving endothelin 2 (Edn2) and B-cell lymphoma 3 (Bcl3). This transcriptional signature was independently reproduced in the N-methyl-N-nitrosourea (MNU)-induced photoreceptor degeneration model. Rod-specific deletion of Fgf2 markedly increased photoreceptor apoptosis, indicating that endogenous FGF2 contributes to photoreceptor survival under ischemic stress. In contrast, neither genetic depletion nor overexpression of FGF2 altered pathological neovascularization or vaso-obliteration. Bidirectional manipulation of FGF2 further modulated the expression of representative stress-associated genes Edn2 and Bcl3, supporting FGF2 involvement in this injury-response program. Finally, receptor expression analysis revealed relatively limited endothelial expression of Fgfr1-Fgfr4 compared with VEGF receptors, suggesting a cellular basis for the distinct effects of endogenous FGF2 and VEGF signaling. Together, these findings identify endogenous retinal FGF2 as a photoreceptor-derived survival factor that is induced during stress but is insufficient to drive pathological angiogenesis. These results support a model in which neuronal adaptation and vascular remodeling represent partially distinct responses during ischemic retinal injury.

Background: Spontaneous cervical artery dissection (sCeAD) is a rare vasculopathy whose pathophysiology remains incompletely understood. Impaired vascular extracellular matrix integrity, including elastic fibers, may contribute to its development. We investigated whether serum fibrillin-1 and soluble elastin fragments (sELF) differ between patients with sCeAD and controls during the acute and chronic stages. Methods: Patients with acute sCeAD were prospectively enrolled at four German stroke centers. Blood samples were collected at baseline and after 6{+/-}1 months. Patients with a first acute ischemic stroke unrelated to sCeAD and healthy individuals served as controls. Serum fibrillin-1 and sELF concentrations were measured using enzyme-linked immunosorbent assays. Results: 61 patients with sCeAD, 53 patients with first non-CeAD ischemic stroke, and 79 healthy controls were included. After sex-matching, serum fibrillin-1 concentrations were significantly lower in patients with acute sCeAD than in healthy controls (97 [60; 192] vs. 176 [113; 269] ng/mL; p=0.009). Fibrillin-1 concentrations were also lower in both male and female patients with sCeAD than in respective healthy controls. In patients with sCeAD, fibrillin-1 concentrations increased significantly after 6 months compared with baseline (171 [130; 270] vs. 104 [67; 205] ng/mL; p=0.021). Serum fibrillin-1 concentrations were higher in men than in women across all study groups. No significant differences in sELF concentrations were observed between groups or time points. Discussion: Serum fibrillin-1 concentrations were lower during acute sCeAD and increased significantly during follow-up, whereas sELF concentrations remained unchanged. These findings support an association between circulating fibrillin-1 and acute sCeAD and warrant further investigation of its role in sCeAD pathophysiology. Pronounced sex-related differences in fibrillin-1 concentrations highlight the importance of sex-specific analyses in future.

Endothelial-to-mesenchymal transition (EndMT) has become a central mechanism in developmental biology, fibrosis, vascular disease, and cancer. We previously reported on an integrated signaling model in which TGF-{beta}2 induces EndMT through coordinated activation of Smad-dependent and Smad-independent signaling pathways converging on Snail, while GSK-3{beta} regulates Snail activity. We performed a systematic figure-by-figure reproducibility analysis of the original publication. Independent studies published between 2011 and 2026 were identified and curated according to predefined inclusion criteria. Each original experimental conclusion was evaluated for independent confirmation. In parallel, selected biochemical experiments were independently reproduced using newly acquired reagents and contemporary Western blot methodologies. Independent publications consistently reproduced each major mechanistic conclusion of the original study, including activation of Smad, ERK, PI3K/AKT, and p38 MAPK signaling, regulation of Snail expression, EndMT-associated marker switching, and GSK-3{beta}-dependent control of Snail activity. Independent laboratory experiments reproduced the principal biochemical findings using contemporary reagents and experimental workflows. The combined literature analysis and independent laboratory replication demonstrate that the mechanistic framework in our previous study has remained reproducible across multiple laboratories, endothelial cell types, disease models, and fifteen years of investigation. This work illustrates a complementary framework for assessing reproducibility that integrates direct experimental replication with cumulative independent validation.

Targeted precision therapies are increasingly used in the treatment of individuals with vascular anomalies (VAs). This increases the need for rapid, accurate and inexpensive genetic diagnosis. Droplet digital polymerase chain reaction (ddPCR) is an alternative to next-generation sequencing (NGS), permitting rapid, highly sensitive interrogation of recurrent pathogenic mosaic variants. We examined the feasibility of ddPCR as a primary diagnostic tool in a large cohort of individuals with VAs. Lesional tissue was collected for ddPCR of up to 46 recurrent pathogenic variants across 16 genes associated with VAs. Specimens were assessed on a subset of assays for each individual based on clinical phenotype. Most individuals who had negative ddPCR results went on to high-depth gene panel or deep exome NGS, or Sanger sequencing. Here we report the phenotypic and molecular findings for 78 newly recruited and tested individuals in addition to the 60 individuals already reported from our cohort. The overall diagnostic yield for our cohort when combined with individuals previously reported was 104/138 (75%). Of 138 individuals tested, recurrent pathogenic variants were detected in 71 (51%) on ddPCR. Variants were most frequently identified in PIK3CA (n=28), TEK (n=18), GNAQ (n=12), or MAP2K1 (n=7). In a further 33 individuals, pathogenic variants were identified on NGS or Sanger sequencing. Our findings indicate that ddPCR is an efficient method achieving a high diagnostic yield in our cohort when used prior to sequencing.

Marfan syndrome (MFS) is an inherited connective tissue disorder caused by pathogenic variants in FBN1, encoding fibrillin-1, with life-threatening aortic complications arising in part from endothelial cell (EC) dysfunction. To study this in a human model, we generated hiPSC-derived ECs from three MFS patients (iMFS-ECs). We show that iMFS-ECs recapitulate known disease phenotypes, including impaired alignment in the direction of flow. Moreover, we found that iMFS-ECs do not recover from TNF--induced loss of barrier integrity, due to sustained EC contractility. iMFS-ECs exhibited TNF--induced ICAM1 upregulation and NF-{kappa}B activation comparable to healthy donor-derived hiPSC-ECs by bulk RNA-seq, while expression of genes linked to cytoskeletal arrangements, cell signaling and ECM remodeling were dysregulated. In conclusion, we show that hiPSC derived ECs can serve as a model to investigate MFS pathology. These findings establish a human iPSC platform for MFS endothelial research and suggest impaired inflammatory resolution as a novel therapeutic target.

BackgroundNeutrophil extracellular traps (NETs) contribute to immunothrombosis and arterial thrombosis. Mechanisms underlying myocardial infarction after SARS-CoV-2 vaccination remain poorly understood. ObjectivesTo investigate histopathologic and immunothrombotic features of coronary thrombi in patients with ST-elevation myocardial infarction (STEMI) after SARS-CoV-2 vaccination. MethodsWe performed a retrospective matched cohort study including patients with STEMI undergoing primary percutaneous coronary intervention between January 2021 and March 2023. Coronary thrombi obtained by aspiration were analyzed by histopathology, immunohistochemistry, and confocal microscopy for NET detection. Vaccinated and unvaccinated patients were matched by age and sex. Associations between vaccination status and thrombus characteristics were assessed after adjustment for SARS-CoV-2 serologic status. ResultsAmong 44 matched patients (23 vaccinated and 21 unvaccinated), NETs were identified in 14 vaccinated patients (61%) and 5 unvaccinated patients (24%; P = .01). Vaccination was associated with increased odds of NET-positive thrombi after adjustment for SARS-CoV-2 serology (odds ratio, 5.1; 95% CI, 1.36-19.45; P = .02). No associations were observed between vaccination and polymorphonuclear cell density, fibrin deposits, plaque fragments, or anti-platelet factor 4 staining. Among patients vaccinated within 100 days before STEMI, NET-positive thrombi were associated with shorter intervals between vaccination and myocardial infarction (median [IQR], 25 [11-64] vs 57 [40-84] days; P = .02). ConclusionsSARS-CoV-2 vaccination was associated with increased NET presence in coronary thrombi from patients with STEMI, suggesting a potential NET-mediated immunothrombotic mechanism independent of classical vaccine-induced immune thrombotic thrombocytopenia.

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy - a form of autophagy that degrades ferritin to release labile ferrous iron - and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. MethodsEndothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ResultsSurgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. ConclusionsOur results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

Syndecans are transmembrane proteoglycans that regulate angiogenesis through both their glycosaminoglycan chains and core proteins. While roles for all four mammalian syndecans in new blood vessel formation are well established, it has more recently emerged that their extracellular core proteins contain discrete bioactive regulatory sequences capable of influencing cellular processes, including angiogenesis. We previously demonstrated that the syndecan-3 (SDC3) ectodomain possesses anti-angiogenic activity independent of its heparan sulphate chains. Here, we identified and characterised a novel anti-angiogenic sequence within the SDC3 ectodomain. Using recombinant truncation mutants, endothelial migration assays and peptide mapping, we localised activity to a discrete region of the extracellular domain and subsequently defined a conserved minimal nine amino acid peptide, QM111, that retained full biological activity. QM111 inhibited endothelial cell migration and angiogenic sprouting in both rat aortic ring and mouse choroidal explant models. Intrinsic disorder analysis revealed that QM111 resides within a region of comparatively reduced disorder, consistent with other syndecan regulatory sequences. This supports the concept that syndecan ectodomains contain conserved functional modules embedded within intrinsically disordered extracellular domains. QM111 did not induce inflammatory chemokine production, exhibited no detectable cytotoxicity, and retained substantial stability in human serum and vitreous humour. Finally, QM111 displayed anti-angiogenic activity comparable to the previously described syndecan-2-derived peptide QM107, with combination treatment producing more robust inhibition of angiogenesis. These findings identify QM111 as a novel endogenous anti-angiogenic peptide and support the concept that syndecan ectodomains are reservoirs of biologically active regulatory sequences with therapeutic potential. The work further establishes syndecan-derived peptides as a promising platform for the development of next-generation anti-angiogenic therapies.

Purpose: To validate an automated pipeline to detect and quantify focal retinal and choroidal pulsation areas that are synchronous with the cardiac cycle in video indocyanine green angiography (ICGA). Design: Retrospective, observational, hypothesis-generating validation study Subjects, Participants: Consecutive patients with a diagnosis of central serous chorioretinopathy (CSCR) in one or both eyes. Methods: Videos were acquired on Heidelberg HRA+OCT. The pipeline consisted of three steps: signal extraction, foci detection, and quantification. After registration of the constituent frames, each pixel's intensity signal was analyzed at the presumed cardiac frequency (tested from a sample of three detectable frequencies). A synchrony score combining local phase coherence with oscillation amplitude was then derived and computed using a standard deviation ({sigma}) above each video's background oscillation value. Two masked graders marked the retinal and choroidal pulsation areas twice. We compared detection of the pulsation areas against grader consensus using a receiver operating characteristic curve (using multiple grid sizes to divide the scan area) and, separately, using a signal-based area-reduction method to obtain an optimum {sigma} value. Main Outcome Measures: Agreement between the automated algorithm and human graders in detection of pulsation foci, and the optimum threshold multiplier ({sigma}). Results: We studied 20 ICGA videos from 20 eyes. At the 16-pixel grid size, the pipeline achieved a mean area under the curve (AUC) of 0.914, sensitivity of 0.86, and specificity of 0.80. Grader agreement improved with larger grid size, reaching substantial-to-strong levels for choroidal annotations. The two independent validation methods demonstrated similar {sigma} values that differed by 0.62{sigma}, supporting {sigma}=4.0 as the optimum value. Conclusions: We report the first automated method to quantify retinal and choroidal vascular pulsation on video ICGA. It measures pixels that oscillate over time with the presumed cardiac cycle and works reliably at the spatial scale (grid level) where experts agree. Pulsatile hemodynamics may add a new vascular biomarker for glaucoma, diabetes, hypertension, and pachychoroid diseases.

Background: Spontaneous coronary artery dissection (SCAD) causes acute coronary syndrome that predominantly affects women. It is not known why SCAD occurs in specific coronary artery segments. We aimed to identify anatomical and hemodynamic factors that lead to SCAD. Methods: We studied 36 women with angiographically-confirmed SCAD from more than 20 hospital sites and 75 sex- and ethnicity-matched control participants with normal coronary anatomy. Coronary arteries were reconstructed from computed tomography coronary angiography (CTCA) to quantify vessel geometry (curvature, diameter, torsion) and flow-derived metrics (time-averaged endothelial shear stress [TAESS], topological shear variation index [TSVI], oscillatory shear index [OSI], and relative residence time [RRT]) at the tree (left/right), territory (LAD, LCx, RCA), and lesion levels. Results: Compared with controls, SCAD-affected coronary arteries had greater curvature and higher TAESS and TSVI at the whole-tree level (all p?0.007). At the vessel (territory) level, SCAD-affected arteries were smaller in average diameter and showed higher curvature, TAESS, and TSVI than matched control vessels (all p?0.047). Within the same patient, SCAD lesion segments were characterized by smaller diameter, lower torsion, and higher TAESS and TSVI than non-affected segments from the same coronary tree (all p?0.001; curvature borderline). A model combining curvature, TAESS, and TSVI discriminated SCAD from controls with AUC 0.95 (left tree) and 0.97 (right tree); adding diameter yielded AUCs >0.91 at the territory level. Conclusions: SCAD was associated with a reproducible multi-scale signature of smaller vessel caliber and higher, more variable endothelial shear stress supporting a hemodynamic contribution to SCAD clustering in specific coronary arteries and segments.

Purpose: To evaluate whether initiation of GLP-1 receptor agonists (GLP-1RAs) is associated with anti-VEGF treatment burden in type 2 diabetes patients with diabetic macular edema (DME) in the IRIS(R) Registry (Intelligent Research in Sight). Methods: Incident GLP-1RA initiators were matched 1:1 with controls via Mahalanobis distance matching (9,896 pairs; N=19,792) on sociodemographics, DME risk factors, and factors influencing GLP-1RA prescription including hypertension, obesity, chronic kidney disease. A longitudinal mixed-effects event-study model evaluated monthly anti-VEGF injection frequency over a 36-month window (12 months before through 24 months after initiation), adjusting for DME duration. Visual acuity (VA) and central subfield thickness (CST) were secondary outcomes. Results: Following GLP-1RA initiation, anti-VEGF injection trajectories did not significantly differ between the matched GLP-1RA and control cohorts (interaction coefficients -0.18 to 1.59, P>0.05). Likewise, no differences in VA were observed between cohorts (-0.05 to 0.04 logMAR, P>0.05) or CST (-14.12 to 33.58 {micro}m, P>0.05). Conclusion: In these matched cohorts, GLP-1RA initiation was not associated with the trajectory of anti-VEGF use or changes in VA or CST. Precis We used the American Academy of Ophthalmology IRIS(R) Registry (Intelligent Research in Sight) to identify patients with DME. In 19,792 matched patients, there was no significant reduction in injection frequency post GLP1-RA initiation and no significant change in VA or CST.

Background: Phenotyping of atherosclerotic plaque vulnerability has largely relied on histopathology that captures structural features, but does not fully account for clinical presentation. Proteomic profiling could uncover molecular readouts of vulnerability that refine plaque phenotyping and provide mechanistic insights. Yet, the proteomic signatures associated with plaque rupture and symptomatic presentation are poorly characterized. Methods: We profiled paired carotid plaque tissue and preoperative plasma from 88 patients undergoing carotid endarterectomy (51 symptomatic, 37 asymptomatic) using the Olink Explore 3072 platform. We related plaque protein abundance to symptomatic presentation and quantitative histopathological features, and compared the performance of histopathology- vs. proteomics-based models for discriminating symptomatic disease. Next, we developed proteomic signatures of cellular abundance and explored their associations with plaque phenotypes by using plaque single-cell RNA-sequencing (scRNA-seq) data. Finally, we assessed plaque-plasma concordance across 2,837 shared proteins. Results: Across 2,837 plaque proteins, 19 were differentially expressed in symptomatic plaques related to distinct clinical events, highlighting pathways related to neutrophil degranulation and innate immune system. FGFBP1 showed the strongest association with symptomatic presentation (log2 fold change = 1.14; P = 1.82 x 10^-6). Proteins associated with a composite vulnerability index based on histopathology were enriched for inflammatory pathways, including TNF signaling through NF{kappa}B, complement activation, and IL6-JAK-STAT3 signaling. Individual proteins also mapped to specific histopathological features, including CXCL8 associated with macrophage burden and lipid core size, and EPHB4 and PKN3 with neovascularization. A proteomics-based model discriminated symptomatic from asymptomatic plaques substantially better than a histopathology-based model (AUC 0.83 vs. 0.66; P = 0.026). Integration with scRNA-seq data enabled the development of cell-class signatures that correlated with histopathology readouts, including macrophage burden, smooth muscle cell content, and neovascularization. Plaque and plasma protein levels showed limited overall correspondence (median {rho}=0.11), although selected proteins, including FGFBP1, demonstrated concordant associations in plasma. Conclusions: Deep proteomic profiling of human carotid plaques identifies molecular signatures of symptomatic atherosclerosis that extend beyond conventional histopathology. These signatures implicate neutrophil activation and inflammatory signaling pathways as key determinants of plaque vulnerability. Although plaque and plasma proteomes are largely distinct, selected proteins may represent promising circulating biomarkers for future risk stratification.

Individuals with inherited retinal dystrophies (IRDs) undergo a slow, genetically heterogeneous loss of vision, yet how the visual cortex and non-visual sensory, motor, and psychological systems adapt to this deprivation remains poorly characterized. Existing evidence comes mainly from single-modality, cross-sectional studies that rarely account for genetic heterogeneity, making it hard to distinguish adaptive change from a direct, non-retinal mutation effect, since several IRD genes are not retina-specific. To address this gap, we designed an observational, longitudinal, multimodal protocol that combines ophthalmological, genetic, and in silico characterization with electrophysiological (steady-state visual evoked potentials and TMS-EEG), chemosensory, sensorimotor, and psycho-personological assessments. Patients aged 18 to 75 years with rod-cone (retinitis pigmentosa, Usher syndrome) or cone and cone-rod dystrophies will be assessed at baseline (T0) and at an 18-month follow-up (T1); sighted controls, matched for age, sex, and handedness, will complete the same battery once. Importantly, pairing genotypic with phenotypic data allows changes in non-visual domains to be interpreted against, rather than independently of, each patient's molecular background. We expect individuals with IRDs to differ from controls in visual cortical responsiveness and in selected non-visual sensory and sensorimotor measures, with genotype-related differences explored where sample size permits. Given the rarity of IRDs, the design is exploratory and emphasizes effect sizes and individual variability over large-sample inference. The protocol was approved by the Ethics Committee of the University of Verona (CARP 08.R1/2024) and follows the Declaration of Helsinki and the GDPR; findings will be disseminated through peer-reviewed publications and shared with patients and IRD patient associations.

Background. Cardiovascular disease is a leading cause of death among women with breast cancer, and the 2026 ACC/AHA dyslipidemia guideline endorses coronary artery calcium (CAC) scoring to guide statin therapy before cardiotoxic treatment. Breast cancer patients routinely undergo staging 18F-fluorodeoxyglucose PET/CT, whose low-dose CT visualizes the coronary arteries, thus enabling CAC quantification at no additional cost or radiation. Methods. In this single-center retrospective study, consecutive women with newly diagnosed breast cancer undergoing staging 18F-FDG PET/CT (2009?2021) had semi-automated Agatston CAC scoring performed on the low-dose CT and were stratified by CAC presence (CAC-P) versus absence (CAC-A). We assessed a composite of cardiac diagnostic testing (stress testing, coronary CT angiography, invasive angiography), clinical events, and reclassification of statin eligibility per ACC/AHA guideline thresholds in a prevention-eligible subgroup. Results. Among 276 women (mean age 55.5 years; median follow-up 7.1 years), CAC was present in 68 (25%) but was clinically reported in only 5.4%. CAC-P was associated with more cardiac testing (34% vs 12%; age-adjusted hazard ratio 2.75, 95% CI 1.43?5.28) and, though underpowered, with more atherosclerotic events (7.4% vs 1.4%), but not with the all-cause composite. In the prevention-eligible subgroup (n=39), CAC scoring would have changed statin eligibility in 64%, initiating therapy in 62% of CAC-P women and supporting de-prescribing in 67% of CAC-A women. Conclusions. CAC can be feasibly quantified from staging PET/CT in women with breast cancer and would frequently reclassify statin eligibility at no additional cost or radiation, yet is rarely reported.

Background: APOE4, the variant of apolipoprotein E carried by 25% of individuals, is a common genetic risk factor for cardiovascular disease (CVD). Although ApoE classically participates in lipid transport, APOE4-associated risk goes beyond impact on circulating lipids. Life-threatening CVD events including myocardial infarction and stroke are driven by atherogenesis and thrombosis. In mice ApoE4 increases atherosclerosis severity, but whether other major drivers of CVD events are influenced by ApoE4 is unknown. Methods: GWAS data for venous thromboembolism (VTE) were analyzed. In humanized APOE3 (hE3) and APOE4 (hE4) mice, thrombosis was assessed by intravital microscopy (IVM) in the mesenteric microcirculation and by inferior vena cava (IVC) partial ligation. Actions of ApoE3 versus ApoE4 on endothelial cells (EC) and their underpinnings were studied in cultured human and mouse aortic EC, interrogating interactomes with immunoprecipitation-mass spectrometry and quantifying the secretion of Von Willebrand Factor (vWF), a critical initiator of thrombosis. Single cell transcriptomics datasets were queried do localize endothelial cell gene expression. Results: GWAS showed that APOE4 is associated with increased VTE risk, and whereas plasma lipids were similar, both microvascular and venous thrombosis were markedly increased in hE4 compared to hE3 mice. In cultured EC, whereas ApoE3 attenuated vWF secretion, it was enhanced by ApoE4, and both processes were mediated by ApoE receptor 2 (ApoER2). ApoE4, but not ApoE3, suppressed VEGF eNOS activation and NO production by causing the recruitment of the protein phosphatase 2A (PP2A) catalytic subunit to ApoER2 and the activation of PP2A. PP2A deletion prevented ApoE4-induced eNOS antagonism and vWF secretion by preserving Akt activation, and the NO donor spermine NONOate negated apoE4 stimulation of vWF secretion. PP2A activity was increased in hE4 aortas and IVC, and EC ApoER2 deletion or pharmacologic PP2A inhibition fully prevented exaggerated thrombosis in hE4 mice. In human great saphenous vein ApoER2 is primarily expressed in valvular endothelium. Conclusions: APOE4 is a risk allele for thrombosis, and ApoE4 is prothrombotic in microvasculature and veins in mice. Mechanistically, the ApoE4-EC ApoER2 tandem enhances vWF secretion by recruiting and activating PP2A and antagonizing eNOS, resulting in exaggerated thrombosis. In human veins ApoER2 is expressed in valvular endothelium, which is the most common site of initiation of venous thrombosis. Targeting these processes may afford protection from both primary thrombotic disorders like VTE and acute CVD events such as myocardial infarction and stroke in 25% of the population.