Introduction Following a cerebrovascular event, the associated risks for further major adverse cerebro- and cardiovascular events and death (MACE) and important aspects of cognitive, mental and patient-reported outcomes are currently not understood, particularly long-term. Here, we present the study design of the ongoing Berlin Long-term Observation of Vascular Events (BeLOVE) stroke stratum and report data of the first study phase.Methods and analysis BeLOVE is a prospective, longitudinal, observational, hospital-based cohort study. Its stroke stratum enrols adult patients with acute ischaemic stroke, transient ischaemic attack (TIA) or non-traumatic intracerebral haemorrhage. Patients undergo deep phenotyping including cerebral and cardiac MRI, ECG, echocardiography and bio-sampling including multi-omics analyses. Regular, standardised follow-ups take place annually over a period of up to 10 years and record the frequency of MACE as the primary outcome.Secondary outcomes include the frequency, progression and interactions of functional impairments, namely post-stroke cognition, pain, depression, seizures and their relationship to quality of life.The first study phase included 758 patients (median 69 years, 37% female). At 2-year follow-up, the cumulative incidence (95% CI) of the composite primary endpoint MACE was 0.107 (0.085 to 0.132) and that of first ischaemic stroke, first myocardial infarction and death were 0.066 (0.049 to 0.086), 0.015 (0.008 to 0.026) and 0.040 (0.027 to 0.056), respectively.Ethics and dissemination Each participant will provide informed written consent during the acute in-hospital phase. Data will be available for research purposes via a written request to the data use and access committee.Trial registration number German Clinical Trials Register: http://www.drks.de/DRKS00023323 on 4 November 2020.

Engineering vascularized human liver tissue for in vitro and in vivo applications remains a major challenge. Here, we describe a scalable approach to generate human liver spheroids with self-organized, lumen-containing vascular networks and demonstrate their use as building blocks for fabricating vascularized single and multilayer tissue constructs. Spheroids were formed from HepaRG liver cells, human umbilical vein endothelial cells, and adipose tissue-derived mesenchymal stem cells. Including the latter in specific ratios prevented a spatial segregation of hepatic and endothelial compartments, enabling endothelial network formation. We present two media for culturing these spheroids: a serum-reduced medium and a defined serum-free medium containing Gibco KnockOut Serum Replacement. These media supported the long-term maintenance of hepatocytes in a metabolically active, relatively mature state, as well as the persistence of endothelial networks. Spheroid-derived endothelial cells established anastomoses with external endothelial channels in microfluidic devices, and upon grafting into mouse liver tissue extended into the host parenchyma. Moreover, endothelial sprouts emerging from the spheroids formed inter-spheroid connections within permissive hydrogels, a process that depended on the inter-spheroid distance. Finally, we demonstrate the fabrication of planar tissue layers with vascularly interconnected spheroids and the creation of a macroscale tissue construct from several such layers.

Abstract Perfusable vascular microphysiological systems are increasingly used to model angiogenesis, tissue crosstalk, and disease. However, many platforms still rely on oscillatory, discontinuous, or poorly controlled perfusion regimes, limiting the study of sustained flow-dependent vascular remodeling. Here, we establish a tunable, unidirectional laminar flow workflow for long-term perfusion of angiogenic vasculature-on-chip cultures and use it to investigate endothelial, perivascular, and immune cell responses to sustained flow. Using an AIM Biotech microfluidic platform containing 14-day-old human umbilical vein endothelial cell-derived angiogenic sprouts and pericytes, continuous perfusion enabled intraluminal transport of 1 μm tracer beads through vessels, demonstrating stable flow across the vascular bed. Sustained laminar flow induced endothelial remodeling at both the mother vessel and sprout levels, with cellular alignment evident in both compartments. Quantitative analysis of the mother vessel further revealed Golgi polarization against the direction of flow. Sustained perfusion also increased pericyte recruitment to angiogenic sprouts and reduced endothelial proliferation within the mother vessel, consistent with flow-driven vascular maturation and quiescence. Live-cell imaging further captured directional endothelial migration against the flow, lumen remodeling, and dynamic pericyte behavior under continuous perfusion. In immune-cell assays performed under continuous-flow conditions, interactions with untreated endothelium were limited, whereas inflammatory activation increased immune-cell adhesion and crawling. These observations suggest that sustained flow supports a quiescent endothelial phenotype and demonstrate the suitability of the platform for studying inflammatory activation and immune-vascular communication under controlled hemodynamic conditions. Beyond its biological relevance, the workflow provides practical advantages for live-cell imaging, low medium consumption, and downstream perturbation studies. Moreover, the modular design of the platform makes it well suited for vascular-organ crosstalk applications. Collectively, these results establish laminar flow angiogenic vasculature-on-chip as an experimentally tractable model for studying vascular mechanobiology, vascular maturation, and dynamic cell interactions under defined hemodynamic conditions.

Abstract Heart failure with preserved ejection fraction (HFpEF) is widely linked to endothelial dysfunction, yet the molecular pathways translating cardiometabolic stress into microvascular remodeling remain poorly defined. Here, we identify endothelial YAP/TAZ signaling as a mechanistic regulator of sex-divergent vascular responses in HFpEF. Plasma proteomics from the UK Biobank revealed elevated circulating YAP1 levels associated with heart failure and increased mortality, particularly in male patients, where YAP1 coincided with increased levels of the endothelial activation marker ESM1. In a hypertensive cardiorenal mouse model, endothelial YAP/TAZ deletion preserved cardiac function, whereas endothelial TAZ gain-of-function aggravated disease. Under cardiometabolic stress (TNFα and high glucose), endothelial cells exhibited sex-specific rewiring of YAP/TAZ-dependent transcriptional programs. Male endothelial cells showed increased extracellular YAP1 release, angiogenic instability with impaired extracellular matrix remodeling, whereas female cells adopted an immune-primed, stress-adaptive phenotype. Mechanistically, cardiometabolic stress uncoupled canonical YAP-TEAD transcription and engaged alternative cofactors, including VGLL3 and VGLL4, thereby reshaping the endothelial secretome and propagating sex-divergent microvascular remodeling. These findings identify endothelial YAP/TAZ rewiring as a molecular switch that converts cardiometabolic stress into sex-divergent microvascular remodeling in HFpEF and connect this process to circulating YAP1 and ESM1 in patients.

BACKGROUND: Hereditary hemorrhagic telangiectasia is a genetic disorder caused by loss-of-function mutations in components of the bone morphogenetic protein signaling pathway, leading to arteriovenous malformations. Most prior work has treated BMP (bone morphogenetic protein) component depletion as mechanistically interchangeable, yet whether distinct genes converge on a shared mechanism remains unclear. We aimed to understand the molecular relationship between BMP signaling and endothelial flow response that leads to arteriovenous malformation formation. METHODS: We expose human endothelial monolayers treated with small interfering RNA against SMAD4 or ALK1 to laminar flow and analyze flow-responsive transcriptomics, flow-responsive BMP signaling activation dynamics, cell polarity, and morphology. We analyze the cell-autonomous and noncell-autonomous migration dynamics of endothelial cells treated with siSMAD4 or siALK1 . Using the postnatal mouse retina model, we study endothelial cell distribution changes over time in mosaic settings, and assess the remodeling capabilities of Smad4 iECKO or Alk1 iECKO , relative to littermate controls. RESULTS: This study shows that depletion of SMAD4 or ALK1 leads to fundamentally distinct mechanisms of vascular malformation. SMAD4 deficiency enhances endothelial responses to blood flow, including transcriptional activation and migration against flow, causing excessive capillary pruning and the development of single large shunts. In contrast, ALK1 deficiency disrupts flow sensing, impairs cell polarization and migration, and promotes a persistent angiogenic state, resulting in dense, hypervascularized networks. RNA sequencing across static and flow conditions identifies both flow-dependent and flow-independent transcriptional changes, suggesting early defects in endothelial fate specification. Mosaic in vitro models show that mutant cells co-opt neighboring wild-type cells, while in vivo tracking confirms mutation-specific migration behavior. CONCLUSIONS: These findings reveal divergent cellular programs driving arteriovenous malformations and underscore the need for gene-specific diagnostic and therapeutic strategies.

GPX4-dependent ferroptosis has emerged as a therapeutic strategy for cancer treatment. Here, we demonstrated that protein kinase A (PKA) participates in the regulation of ferroptosis by controlling the m6A modification of GPX4 in an ALKBH5-dependent manner. Notably, we identified ALKBH5, an m6A demethylase, as a novel target of PKA, which drives phosphorylation-dependent degradation of ALKBH5 protein. Moreover, the deletion of ALKBH5 represses ferroptotic cell death by maintaining GPX4 m6A modification and stability. Thus, by regulating ALKBH5-dependent GPX4 stability, PKA acts as a key regulator of ferroptosis. Our study unveils the involvement of PKA in m6A modification, which could control GPX4-dependent ferroptosis and tumor progression.

Cell polarity involves the asymmetric distribution of cellular components such as signalling molecules and organelles within a cell, alterations in cell morphology and cell-cell contacts. Advances in fluorescence microscopy and deep learning algorithms open up a wealth of unprecedented opportunities to characterise various aspects of cell polarity, but also create new challenges for comprehensible and interpretable image data analysis workflows to fully exploit these new opportunities. Here we present Polarity-JaM, an open source package for reproducible exploratory image analysis that provides versatile methods for single cell segmentation, feature extraction and statistical analysis. We demonstrate our analysis using fluorescence image data of endothelial cells and their collective behaviour, which has been shown to be essential for vascular development and disease. The general architecture of the software allows its application to other cell types and imaging modalities, as well as seamless integration into common image analysis workflows, see https://polarityjam.readthedocs.io . We also provide a web application for circular statistics and data visualisation, available at www.polarityjam.com , and a Napari plug-in, each with a graphical user interface to facilitate exploratory analysis. We propose a holistic image analysis workflow that is accessible to the end user in bench science, enabling comprehensive analysis of image data.

Coronary arteries develop under constant mechanical stress. However, the role of mechanosensitive ion channels in this process remains poorly understood. Here we show that the ion channel PIEZO2, which responds to mechanical stimuli, is expressed in specific coronary endothelial cell populations during a critical phase of coronary vasculature remodeling. These Piezo2+ coronary endothelial cells show distinct transcriptional profiles and have mechanically activated ionic currents. Strikingly, PIEZO2 loss-of-function mouse embryos and mice with human pathogenic variants of PIEZO2 show abnormal coronary vessel development and cardiac left ventricular hyperplasia. We conclude that an optimal balance of PIEZO2 channel function contributes to proper coronary vessel formation, structural integrity and remodeling, and is likely to support normal cardiac function. Our study highlights the importance of mechanical cues in cardiovascular development and suggests that defects in this mechanosensing pathway may contribute to congenital heart conditions.

Background Ischemic stroke remains a leading cause of morbidity and mortality worldwide, with limited treatment options available. Vascular dysfunction is a key pathomechanism, and brain endothelial cells (bECs) play a critical role in determining stroke outcomes. This study investigates the specific roles of YAP (yes‐associated protein 1) and TAZ (WW domain containing transcription regulator 1) in regulating bEC functions during stroke. Methods Mice underwent 30‐minute middle cerebral artery occlusion (MCAo) followed by reperfusion to model ischemic stroke. TAZ reporter mice were used to track stroke‐induced subcellular changes in TAZ expression. Tamoxifen‐inducible endothelial‐specific Y ap / T az knockout and control mice were used to study YAP/TAZ’s role in bEC function post‐stroke. Stroke outcomes were measured by magnetic resonance imaging and NeuN (neuronal nuclei)‐associated lesion analysis. Properties of bECs were assessed via immunohistochemistry and RNA sequencing. Inflammatory parameters were analyzed by flow cytometry of brain immune cells and quantitative polymerase chain reaction. Results Middle cerebral artery occlusion/reperfusion regulated Yap , Taz , and YAP/TAZ target gene expression in the brain. TAZ reporter mice confirmed stroke‐induced endothelial YAP/TAZ activation. Endothelial‐specific loss of YAP/TAZ reduced infarct volumes at 4 weeks after MCAo without impairing stroke‐induced angiogenesis, revealing an unexpected neuroprotective role for endothelial YAP/TAZ depletion. YAP/TAZ deficiency modulated cGAS−STING (cyclic GMP‐AMP synthase−stimulator of interferon genes) and Wnt (wingless‐related integration site) signaling genes in bECs and promoted myeloid cell recruitment and an anti‐inflammatory vascular environment during the subacute phase of stroke. Conclusions Our data suggest that endothelial YAP/TAZ affects the inflammatory milieu subacutely after ischemia and thereby influences the chronic course of stroke. Modulation of YAP/TAZ activity in ECs may be a promising therapeutic target to promote neuroprotection after stroke.

Aims Heart failure remains a leading cause of morbidity and mortality worldwide. Suitable in vitro models to accurately replicate the pathological environment in heart failure with reduced and preserved ejection fraction (HFrEF/HFpEF) are limited, hampering mechanistic studies and drug screening. In particular, these models rarely incorporate immune cells, which play a critical role in heart failure. To address these limitations, we developed an isogenic 3D induced pluripotent stem cell (iPSC)-derived cardiac spheroid model incorporating monocytes. Methods and results Cardiac spheroids were assembled from three healthy female iPSC lines: three-cell-type (3CT) spheroids consisting of iPSC-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells, and four-cell-type (4CT) spheroids additionally containing monocytes. After six days of culture, established spheroids were treated for 24 h with different known heart failure-associated triggers (glucose & tumour necrosis factor alpha (TNFα) or ischaemia with/without reoxygenation). Differences between treated and control 3CT and 4CT spheroids were investigated at the cellular, molecular, and functional levels using confocal microscopy, RNA expression (qPCR and RNA sequencing), protein secretion using proximity extension assay technology (Olink), and functional analyses of beating rate, contraction, and relaxation. The results confirmed successful monocyte integration in 4CT spheroids, and only spheroids with monocytes (4CTs) exhibited changes in beating rate and relaxation duration upon stimulation, highlighting the necessity of incorporating immune cells to successfully mimic heart failure-associated functional changes. Along with a more pronounced global transcriptomic treatment response and inflammatory changes, additional transcriptomic alterations previously linked to heart failure in patients, as well as changes in metabolism, ion channels, and extracellular matrix pathways, were observed in 4CT compared with 3CT spheroids. Conclusion We showed that immune cell incorporation enhances the functional and transcriptional responses of engineered cardiac tissue to relevant heart failure triggers in vitro and is essential for future studies to elucidate the cellular crosstalk and pathomechanisms. Translational perspective Heart failure continues to be a predominant cause of morbidity and mortality, necessitating the development of innovative therapeutic strategies, particularly in light of the rising prevalence of obesity and diabetes mellitus. We introduced an isogenic in vitro spheroid model comprising iPSC-derived cardiomyocytes, cardiac fibroblasts, endothelial cells, and monocytes to examine the effects of heart failure-associated triggers on cardiac tissue. Our findings indicate that spheroids incorporating monocytes exhibit a more pronounced response to heart failure-associated triggers and demonstrate greater differential transcriptional and functional responses than spheroids lacking immune cells. This model ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, SFB-1470-A03, SFB-1470–A02, SFB-1470–Z01 European Research Council, https://ror.org/0472cxd90, 725229, 101078307 European Commission, 874827, 801540 Fondation Leducq, https://ror.org/01czwga19, Transatlantic Networks of Excellence: AtheroGEN German Centre for Cardiovascular Research, https://ror.org/031t5w623, 81X3100210, 81X2100282 Dutch Research Council, 024.003.013

Arteriovenous malformations (AVMs) are a hallmark of hereditary haemorrhagic telangiectasia (HHT) and arise from abnormal vascular remodelling. Although AVM formation has been associated with disruptions in the BMP9/10 signalling pathway, the distinct contributions of its components remain unclear. Here, we combine in vitro mosaic endothelial cell (EC) cultures with agent-based modelling (ABM) to investigate how knockdown of the BMP9/10 pathway components ALK1 and SMAD4 alters cell-cell interactions and collective vascular organisation. Using cell tracking data, we inferred the mechanical interactions between neighbouring ECs in 2D monolayers by applying Approximate Bayesian Computation to an ABM of cell migration. SMAD4 knockdown increased the motile forces generated by ECs, while ALK1 knockdown weakened the ability of cells to push apart and rearrange with their neighbours, both resulting in greater mixing and fluidity within the cell layer. When these altered interaction profiles were incorporated into an ABM of collective EC dynamics during vascular remodelling, they gave rise to distinct AVM-prone phenotypes. SMAD4-deficient populations exhibited local blockages as highly polarised cells migrated excessively, whereas ALK1-deficient populations disrupted vascular patterning by impairing coordinated polarity and neighbour separation, favouring flow reversal. Our findings suggest that canonical BMP9/10 pathway defects destabilise the biomechanical balance of endothelial interactions, leading to emergent collective behaviours that predispose vessels to AVM formation. Significance Statement Arteriovenous malformations (AVMs) are abnormal connections between arteries and veins that disrupt blood flow and can cause stroke or life-threatening bleeding. They are a hallmark of hereditary haemorrhagic telangiectasia (HHT), a genetic vascular disorder, but the cellular events that initiate AVMs remain unclear. Using a combination of endothelial cell cultures and computational modelling, we show that loss of two key HHT-linked genes, ALK1 and SMAD4, disrupts the coordinated movement and interactions of endothelial cells. Although both deficiencies lead to AVM-like behaviours, they do so through distinct mechanisms. SMAD4 loss drives excessive collective migration, whereas ALK1 loss impairs polarity and neighbour separation. These findings provide a mechanistic framework for how genetic defects could contribute to cell-cell dynamics observed in vascular malformations. ### Competing Interest Statement The authors have declared no competing interest. Fondation Leducq (Leducq Foundation), 17 CVD 03 UKRI | Engineering and Physical Sciences Research Council (EPSRC), EP/X025705/1 Deutsches Zentrum für Herz-Kreislaufforschung (DZHK), 329389797, CRC1444, CRC1470

Background: Acute heart failure (AHF) patients face poor outcomes, particularly within the high-risk 90-day post-discharge phase. Additional biomarkers reflecting the multifaced pathophysiology of AHF are necessary to improve outcome prediction. Objective: This study aimed to identify novel plasma protein biomarkers and biological pathways related to survival and recovery following an AHF event. Methods: Plasma samples were obtained from patients enrolled in the BeLOVE (Berlin Long-Term Observation of Vascular Events) cohort during an AHF event, at 90-day follow-up, and from a reference group (patients with cardiovascular risk but no recent AHF). Proteomics analysis was performed using both Mass Spectrometry (MS) and Olink Explore (Uppsala, Sweden). Cox proportional-hazards regression identified plasma proteins linked to 90-day all-cause mortality post-AHF. Results: Out of 2,324 proteins analyzed from Olink and 533 from MS, 67 were significantly associated with 90-day mortality (|lnHR|>0.4, FDR<0.05). Inflammation, apoptosis, and extracellular matrix remodeling were key mortality predictors, while metabolic proteins, coagulation control, and complement system associated with survival. TNF receptor family members showed promise for risk stratification beyond natriuretic peptides. Comparison of plasma from survivors at acute setting and 90-day follow-up identified 591 significantly altered proteins via Olink and 131 via MS (logFC>75th percentile, FDR<0.05). CA125 (logFC = -1.8 [-2.2,-1.4], FDR ≈ 2e-8 ) emerged as a biomarker for clinical recovery, declining more sharply than NT-proBNP. Conclusions: Combining targeted and untargeted proteomic approaches identifies novel pathways and biomarkers to refine risk stratification in AHF. Profiling of proteomic changes post-AHF provides critical insights into the molecular processes underlying disease progression and recovery. ### Competing Interest Statement FE reports grants from German Research Foundation (DFG), grants from German Ministry of Education and Research, grants from the German Heart Foundation; during the conduct of the study; personal fees and non-financial support from Novartis, grants and personal fees from Boehringer Ingelheim, personal fees from CVRx, Pfizer, Medtronic, grants and personal fees from Servier, personal fees from MSD, personal fees from Merck & Co., grants from AstraZeneca, personal fees from Bayer, personal fees from Resmed, personal fees from Berlin Chemie, grants from Thermo Fischer, personal fees from Vifor Pharma, personal fees from PharmaCosmos outside the submitted work. CHN reports Grants from German Center for cardiovascular research (DZHK) and German Center for neurodegenerative disease (DZNE). Honoraria for lectures from Alexion, Astra Zeneca, Bayer, BMS, Novartis and Pfizer. ME reports grants from Bayer and fees paid to the Charité from Abbot, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, BMS, Daiichi Sankyo, Amgen, Sanofi, Novartis, Pfizer, all outside the submitted work. ME received funding from DFG under Germanys Excellence Strategy - EXC-2049 - 390688087, Collaborative Research Center ReTune TRR 295- 424778381, BMBF, DZNE, DZHK, EU, Corona Foundation, and Fondation Leducq. HG reports grants from the DFG, the Leducq Foundation, the Federal Ministry of Education and Research (BMBF) and the DZHK during the conduct of the study, outside of the submitted work. UL reports research funding from DZHK; Fondation Leducq; research grants from Novartis, Bayer and Amgen. KM declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. DNM received funding for research from Bayer Healthcare, Deutsche Forschungsgemeinschaft and from BMBF. BP reports personal fees and other from Bayer Healthcare, personal fees and other from MSD, personal fees and other from Novartis, personal fees from Astrazeneca, grants and personal fees from Servier, personal fees from Medscape, outside the submitted work. No other relationships or activities that could appear to have influenced the submitted work have exist beyond those listed. TP received grants from the BMBF, the Federal Ministry of Food and Agriculture (BMEL), the Federal Ministry for Economic Affairs and Energy (BMWi), the DFG, Deutsche Herzstiftung, German Academic Exchange Service (DAAD). JSM reports grants from Bayer Healthcare, non-financial support from Siemens healthineers, non-financial support from Circle cardiovascular, non-financial support from Medis, outside the submitted work, and Bayer Healthcare, Advisor. Furthermore, funding for research from the EU, DZHK, Deutsche Herzstiftung. UK received research grants and honoraria (speaker/ advisory board) from BAYER AG, and received honoraria (speaker/ advisory board) from Amarin, Apontis Pharma, Berlin Chemie, Novartis, Oviva, Sanofi, and Servier. UK is supported the DFG (SFB-1470-A09) and by the DZHK, BER 5.4 PR. ### Funding Statement This work was supported by the following grant: the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 437531118 - SFB- 1470-B05; SFB-1470-Z02) and the DZHK (German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK)) site project ″Multiscale mechanistic phenotyping in Heart Failure with reduced ejection fraction″ (TYPE-HF II, project number 81Z0100204). G.G.S. is supported by grant from the DZHK (German Centre for Cardiovascular Research - 81X3100210; 81X2100282); the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation - SFB-1470-A02; SFB-1470-Z01) and the European Research Council - ERC StG 101078307. The Berlin Institute of Health (BIH) funded the basic infrastructure, recruitment as well as deep phenotyping after 90 days. There is no financial remuneration for study participation, except for reimbursement of the transportation cost that patients had related to the BTU visits. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Charité-Universitätsmedizin Berlin gave ethical approval of this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Bone has the unique ability to regenerate without scarring, yet the cellular dynamics and directional organization underlying this process, and how they are linked to marrow reconstitution, remain incompletely understood. To investigate these mechanisms, we developed a novel double osteotomy model in mice, involving transplantation of a 2-mm bone graft between genetically distinct fluorescent reporter lines (yellow fluorescent protein and red fluorescent protein). This approach enabled precise tracking of cellular migration and vascularization over 3, 7, and 14 d post-transplantation. Our findings revealed directed migration of host-derived proximal bone marrow cells into the graft, starting at day 3 and leading to complete host cell infiltration by day 14. CD146-positive blood vessels, mainly originating from the proximal host marrow, invaded the graft coinciding with graft marrow remodeling. Marrow disintegration within the graft occurred prior to vascular and cellular invasion, with subsequent reconstitution progressing from the proximal side. Flushing the graft marrow cavity prior to transplantation resulted in more extensive marrow niche formation by day 14 suggesting that marrow reconstitution can proceed more rapidly without the need for prior remodeling. This study introduces a new model to dissect the spatial and temporal coordination of cellular migration and vessel invasion during bone regeneration. Our results uncover the directional nature of healing and underscore the critical role of marrow reconstitution in guiding regenerative processes-insights that may inform surgical and biomaterial strategies to enhance bone repair.

Despite considerable progress, the underlying mechanisms that enable scar-free regeneration in bone after injury are still not well understood. Here, we compared the spatiotemporal distribution of SOX9-positive chondrocytes, SPARC-positive hypertrophic chondrocytes and osteoblasts, versus Osterix-positive osteoblasts, i.e. key cell types in cartilage and bone formation, in the fracture gap of a mouse osteotomy model with the orderly sequence of events observed in the growth plate. We show that external mechanical stability determines the spatial distribution of osteoblastic and chondrogenic cell populations at day 7, thus defining the site of chondrogenesis initiation. At day 14, only rigid, but not semi-rigid fixation promoted the formation of avascular regions within previously vascularized areas. We thus propose a model how mechanical stabilization promotes bone healing: Blood vessel growth into the hematoma is followed by localized vascular degradation as chondrogenesis progresses, ultimately leading to vascular regrowth via endochondral ossification initiated at the tips of the distal bones. Deepening our understanding of these processes and how they ultimately relate to scar-free bone regeneration is of significant medical relevance as they can provide instructions how to promote fracture healing.

Caspases are known for their roles in cell death and inflammation. However, emerging evidence suggests they also mediate non-lethal processes, governed by a finely tuned balance of localization, activity, kinetics, and substrate availability. Given that many caspase substrates are implicated in mechanoadaptive processes, we investigated if caspases contribute to morphological adaptation of human pulmonary artery endothelial cells to fluid shear stress and other morphology-altering stimuli in vitro. Using selective inhibitors, we screened all major caspases for a role in endothelial cell adaptation to unidirectional laminar shear stress (15 dyn/cm2, 72 h). Selective inhibition of caspase-6, but not other caspases, impaired morphological shear adaptation. Only 5.5% of caspase-6-inhibited cells shear-adapted vs. 75.2% of vector controls. Live-cell FRET imaging revealed progressive caspase-6 activation starting at 18 h of shear stress, coinciding with the onset of morphological remodeling. The active caspase-6 localized predominantly perinuclearly, while caspase-3 remained inactive throughout shear exposure. Caspase-6 inhibition did not affect elongation in response to alternative biomechanical or biochemical stimuli, including uniaxial cyclic stretch (5%, 1 Hz), spatial confinement on narrow micropatterned RGD-lines, or TNF-α stimulation, nor did it impair cell adhesion, directed migration, wound healing, or barrier recovery after wounding. Our study uncovers a previously unidentified role of caspase-6 as a non-apoptotic, mechanosensitive effector specifically required for shear-induced morphological adaptation of pulmonary artery endothelial cells, highlighting a novel regulatory axis in vascular mechanoadaptation.

Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder caused by mutations in the bone morphogenetic protein signaling pathway, leading to arteriovenous malformations. While previously thought to share molecular and cellular dysregulation, this study reveals highly distinct mechanisms depending on whether mutations occur in Alk1 or SMAD4. Loss of SMAD4 enhances endothelial cell responses to flow, including flow-regulated transcription and cell migration against blood flow, causing excessive pruning of capillaries and the formation of single large shunts. Conversely, Alk1 deficiency disrupts endothelial flow responses, including cell polarization and directional migration, leading to a dense vascular network and the persistence of a malformation nidus. In vivo cell population tracking of mutant cells validates unique endothelial cell migration defects. Mosaic cell culture models further illustrate that mutant cells co-opt wild-type cells driving distinct Alk1 or SMAD4 mutant-like behavioral defects. These findings demonstrate that arteriovenous malformations develop through fundamentally different cellular mechanisms based on the specific genetic mutation emphasizing the need for tailored diagnostic and therapeutic strategies.

Cardiovascular diseases including atherosclerosis and heart failure, arise from the intricate interplay of metabolic, immune, and neural dysregulation within vascular and cardiac tissues: This review focuses on integrating recent advances in metabolic and immune crosstalk of the cardiac vasculature that affects cardiometabolic health and disease progression. Coronary and lymphatic endothelial cells regulate cardiac metabolism, and their dysfunction is linked to cardiovascular diseases. Lymphatics maintain tissue homeostasis, including clearing metabolic waste, lipids, and immune cells, and their maladaptation in metabolic diseases worsens outcomes. Altered vascular endothelial metabolism in heart failure drives immune-mediated inflammation, fibrosis, and adverse cardiac remodeling. Concurrently, artery tertiary lymphoid organs formed in the adventitia of advanced atherosclerotic arteries, serve as pivotal neuroimmune hubs, coordinating local immunity through T and B cell activation and neurovascular signaling via artery-brain circuits. T cells within plaques and artery tertiary lymphoid organs undergo clonal expansion as a result of peripheral tolerance breakdown, with proinflammatory CD4+ and CD8+ subsets amplifying atherosclerosis, effects further shaped by systemic immune activation. Therapeutic strategies targeting endothelial cell metabolism, lymphatic dysfunction, neuroimmune crosstalk, and T cell plasticity hold promise for integrated cardiovascular disease management.

Collaterals are unique blood vessels present in many healthy tissues that cross-connect distal-end arterioles of adjacent arterial trees, thus providing alternate routes of perfusion. Stroke patients with superior pial collateral flow respond better to treatments and present with an overall improved prognostic outcome. However, how pial collaterals develop in the embryo and how they reactivate upon stroke remains unclear. Here, using lineage tracing in combination with three-dimensional imaging, we demonstrate that mouse embryos employ a novel mechanism to build pial collaterals, distinct from their outward remodeling following stroke. Endothelial cells (ECs) of arterial and microvascular origin invade already existing pre-collateral vascular structures in a process which we termed mosaic colonization. Arterialization of these pre-collateral vascular segments happens concurrently with mosaic colonization. Despite having a smaller proliferative capacity, embryonic arterial cells represent the majority of cells that migrate to form nascent collaterals; embryonic microvascular cells, despite their higher proliferative potential, form only about a quarter of collateral endothelial cells. Moreover, postnatal collateral growth relies much more on self-replenishment of arterial cells than on microvascular contribution. Following ischemic injury, pial collateral outward remodeling relies on local cell proliferation rather than recruitment of non-arterial cells. Together, these findings establish distinct cellular mechanisms underlying pial collateral development and ischemic remodeling, raising the prospect for future research to identify novel, collateral-specific therapeutic strategies for ischemic stroke.

Mechanical stimuli, particularly laminar blood flow, play a crucial role in shaping the vascular system. Changes in the rate of blood flow manifest in altered shear stress, which activates signaling cascades that drive vascular remodeling. Consistently, dysregulation of the endothelial response to fluid shear forces and aberrant flow patterns both lead to pathological conditions, including impaired blood vessel development and atherosclerosis. Despite its importance, the mechanisms driving the coordinated cell behavior underlying vascular remodeling are not fully understood. Combining classical cell biological approaches with advanced image analysis, mathematical modeling, biomimetic strategies, and in vivo studies, we identify the planar cell polarity (PCP) protein Vangl1 as an enforcer of flow-dependent cell dynamics in the vascular system. We demonstrate that shear stress triggers the relocation of Vangl1 from an internal reservoir to the plasma membrane at the initiation of cell remodeling. Membrane enrichment of Vangl1 is mediated by a Coronin1C-dependent shift in the equilibrium between endo- and exocytosis and results in the spatial reorganization of another essential PCP protein, Frizzled6 (Fzd6). The resulting mutual exclusion of the core PCP proteins Fzd6 and Vangl1 augments differential junctional and cytoskeletal dynamics along the flow axis. Loss of Vangl1 limits the ability of endothelial cells to respond to shear forces in a coordinated fashion, resulting in irregular cell alignment along the flow direction and erroneous vessel sprouting. Together, these studies introduce core PCP signaling as a determinant of collective cell dynamics and organization of the vascular system. ### Competing Interest Statement The authors have declared no competing interest. The data that support the findings of this study are available from the corresponding authors upon reasonable request. No restrictions apply.