Angiogenesis

Dysfunctional lymphangiogenesis is a component of several diseases with hypoxic microenvironments, including secondary lymphedema and solid malignancies. These vessels are ineffective at draining interstitial fluid, resulting in complications such as increased inflammation, slowed wound healing, and, for cancer patients, increased risk of metastasis. Current treatments to normalize vasculature have negative effects on healthy vessels and do not specifically target lymphatic endothelial cells (LECs). As hypoxia is known to change endothelial cell metabolism, exploiting LEC-specific metabolic pathways may provide a focused approach to restoring lymphatic function in patients. However, outside of glycolysis, changes to LEC metabolism in hypoxic conditions are understudied. To address this gap in knowledge, we examined the impact of glutamine availability on factors critical to lymphangiogenesis, including glycolysis, cell proliferation, and migration. We found that increasing glutamine availability results in increased lactate production as well as a hypoxia-specific increase in glycolytic genes HK2, GLUT1, and GLUT3. The presence of glutamine also encouraged LEC proliferation, while blocking glutamine transport reduced lactate production, HK2 expression, and slowed collective LEC migration. In a vessel formation assay, we found that glutamine increased vessel formation in normoxic conditions, but lowered vessel connectivity in hypoxic conditions, reflecting the dysfunction seen in hypoxic diseases. However, attenuating glycolysis by blocking glutamine transport caused LECs to form longer, interconnected vascular networks. This study reveals that glutamine availability can modulate LEC glycolysis, and therefore lymphangiogenesis, in a hypoxia-dependent manner. Collectively, our study identifies glutamine availability as a potential target for lymphatic vessel normalization in chronic and hypoxic diseases.

The formation of the premetastatic niche prepares distant tissues for tumor cell engraftment. Endothelial cells are critical mediators of premetastatic niche formation, orchestrating extravasation of circulating tumor cells and critical pro-tumor immune cells, such as neutrophils. In mouse models of breast cancer, we show that primary tumors upregulate the non-signaling chemokine receptor ACKR1 in the endothelium of the lung premetastatic niche. ACKR1-expressing venules were found to be preferential sites of neutrophil and tumor cell localization within lung tissue. A newly generated conditional ACKR1 allele was used to show that endothelial-specific removal of ACKR1 expression significantly reduces metastatic engraftment in the lung. When ACKR1 is activated by tumor-secreted factors, endothelial ACKR1 functions to promote neutrophil recruitment within the lung parenchyma. We conclude that ACKR1 is a critical component of the endothelial response to tumors at the metastatic site of the lung, leading to neutrophil recruitment and promotion of tumor cell metastasis. SUMMARYEndothelial cells play critical roles in breast cancer metastasis. ACKR1 is upregulated in the endothelium of the lung metastatic niche in response to primary mammary tumors. Endothelial ACKR1 expression was found to promote neutrophil infiltration into the metastatic niche and support breast tumor cell metastasis to the lung.

BackgroundAbdominal aortic aneurysm (AAA) is a disease with altered vessel wall architecture and integrity. AAA rupture is associated with high mortality. Reactive oxygen species, such as those produced by members of the NADPH oxidase (NOX) family, play a central role in several aspects of vascular physiology. In particular, the role of NOX4 appears to be highly cell and context specific. MethodsThis study analyzed the role of NOX4 in late-stage human AAA specimen and in Nox4-/- mice with experimentally induced AAA. ResultsNOX4 expression was reduced in human AAA. In a mouse model of AAA, loss of Nox4 conferred protection against AAA formation, suggesting a pathogenic role. Single cell analysis of human AAA revealed that NOX4 is primarily expressed in fibroblasts, s.mooth muscle, and endothelial cells. NOX4 mRNA expression was strongly associated with ECM synthesis and ECM remodeling pathways. Angiogenic signatures were reduced in AAA, and sub-cluster analysis of endothelial cells identified two major groups: microvascular and lymphatic endothelial cells (LEC), with very low NOX4 expression in LEC. Quantification of the vasa vasorum revealed a shift in vessel size distribution, with a reduction in the number of small vessels (<8 {micro}m) and an increase in large vessels (>26 {micro}m) correlating with increasing aortic diameter. Markers of lymphangiogenesis, including VEGFC and PROX1, were upregulated in AAA. Pseudotime trajectory analysis suggested transdifferentiation of LECs into myofibroblasts, a process associated with increased NOX4 mRNA expression. ConclusionNOX4 plays a role in the pathogenesis of AAA and is primarily expressed in fibroblasts, smooth muscle cells, and endothelial cells. Single-cell and pseudotime analyses revealed that NOX4 is associated with ECM remodeling, reduced angiogenic signatures, and the transdifferentiation of lymphatic endothelial cells into myofibroblasts. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIIn human AAA, NOX4 is associated with pro-fibrotic effects. C_LIO_LINOX4 appears to play a central role in cell differentiation processes in AAA, supporting the expansion of the fibroblast population. C_LIO_LIThe percentage of small microvessels (<8 {micro}m) is increased in human AAA, and NOX4 expression correlates positively with the proportion of small vessels. C_LIO_LIThe cell-cell communication network of endothelial cells in AAA appears to have a profile that supports fibrosis. C_LIO_LILymphatic endothelial cells and markers of lymphangiogenesis were found in AAA. C_LIO_LILymphatic endothelial cells transdifferentiate into myofibroblasts, a process accompanied by increased NOX4 expression. C_LIO_LINOX4 may serve as a mechanistic link between lymphangiogenesis and fibrosis, bridging vascular remodeling and fibrotic progression. C_LI Translational Perspective?O_LITargeting NOX4 represents a promising therapeutic strategy for mitigating fibrotic remodeling in late-stage AAA. C_LIO_LITargeting the specific receptors mediating the interaction between lymphatic endothelial cells, fibroblasts, and inflammatory cells may reveal novel therapeutic targets. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/26347161v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@688aeborg.highwire.dtl.DTLVardef@178673borg.highwire.dtl.DTLVardef@1c17f5aorg.highwire.dtl.DTLVardef@8fff06_HPS_FORMAT_FIGEXP M_FIG C_FIG

Angiogenesis, the formation of new vessels from pre-existing ones, is essential for embryonic development and tightly regulated by VEGFA signaling. However, the contribution of additional modulators remains poorly defined. The co-receptor NRP1 is crucial for hindbrain vascularization, yet how its activity is spatiotemporally controlled is unclear. We identify the glycosylphosphatidylinositol (GPI)-anchored protease MT4-MMP as a key regulator of developmental angiogenesis. Endothelial cell-specific, inducible deletion of MT4-MMP (Mt4-mmpi{Delta}EC mice) causes an exacerbated vascular plexus in the E11.5 embryonic hindbrains. In vitro, loss of MT4-MMP in endothelial cells disrupts cell polarization and migration and enhances VEGFA-induced ERK signaling. Consistently, pERK levels are increased in hindbrain vessels from Mt4-mmpi{Delta}EC embryos, whereas they are reduced in mice with constitutive and global MT4-MMP deficiency. By combining co-expression analysis in cultured cells and embryonic hindbrains with proteomics, in silico protein modeling, and in vitro digestion assays, we identify NRP1 as a previously unrecognized MT4-MMP substrate in this context. Accordingly, inhibition of VEGFA binding to NRP1 partially rescues the aberrant angiogenic phenotype in the embryonic hindbrain of Mt4-mmpi{Delta}EC mice. Our findings reveal that MT4-MMP shapes developing brain vasculature by modulating NRP1-dependent VEGFA/ERK signaling. This newly identified MT4-MMP/NRP1 axis may have potential relevance in CNS vascular abnormalities in development and disease, as well as other pathophysiological contexts.

Vascular dysfunction and coagulopathy are hallmarks of severe COVID-19. How SARS-CoV-2 infection drives endothelial dysfunction, despite the virus not infecting or replicating in endothelial cells, remains controversial. Here, we used an in vitro co-culture model of the human pulmonary epithelial-endothelial cell barrier to investigate which inflammatory mediators drive endothelial dysfunction during SARS-CoV-2 infection. SARS-CoV-2 infection of primary human bronchial epithelial cells increased adjacent endothelial cell expression of the leukocyte adhesion marker ICAM-1, disrupted endothelial VE-cadherin junctions, promoted endothelial cell death, and promoted platelet adherence to gaps in the endothelial monolayers. Dexamethasone treatment rescued these dysregulated endothelial phenotypes in infected co-cultures, confirming that inflammatory signalling was the primary driver of SARS-CoV-2-induced endothelial dysfunction. Specifically, epithelial-derived TNF and IL-1{beta} promoted endothelial dysfunction, as inhibition of TNF or IL-1R signalling blocked SARS-CoV-2-induced endothelial dysfunction in co-cultures. SARS-CoV-2-infected wild-type mice, but not TNF, IL-1{beta}, or TNF/IL-1{beta}- deficient mice, displayed increased endothelial ICAM-1 expression, while an anti-IL-1{beta} monoclonal antibody prevented SARS-CoV-2-induced ICAM-1 expression and fibrin deposition in aged K18-ACE2 mice. Our data indicate that TNF and IL-1{beta} are the specific cytokines that drive multiple aspects of endothelial dysfunction during acute SARS-CoV-2 infection, and that inhibiting their signalling pathways may provide therapeutic benefit in preventing vascular complications of COVID-19.

IntroductionAnti-vascular endothelial growth factor (anti-VEGF) therapies are standards of care for vision-threatening retinal diseases. This retrospective observational study describes demographics, utilization, best recorded visual acuity (BRVA), and safety among eyes with neovascular age-related macular degeneration (nAMD), diabetic retinopathy (DR), diabetic macular edema (DME), or retinal vein occlusion (RVO) treated with the biosimilar aflibercept-ayyh (PAVBLU(R)) in routine clinical practice. MethodsElectronic medical records from the Retina Consultants of America database of patients receiving aflibercept-ayyh (12/1/2024-10/31/2025) were analyzed, focusing on eyes with [&ge;]84 days of follow-up. The index date was the first documented aflibercept-ayyh injection. Postindex data were used to assess treatment patterns, BRVA (Wilcoxon signed rank test), and adverse events of special interest (AESIs). ResultsA total of 1,000 consecutive eyes from 989 patients received 3,730 injections of aflibercept-ayyh; most (91%) switched from prior anti-VEGF therapy and 9% were anti-VEGF treatment-naive. Disease distribution was 58% nAMD, 19% RVO, 16% DME, and 7% DR. Among switchers, median (IQR) number of prior injections was 21 (8-46). Median (IQR) follow-up was 6.0 months (4.6-7.1). Median (IQR) number of aflibercept-ayyh injections per eye was 4 (3-5). Among eyes with [&ge;]84 days of follow-up (n=889), mean BRVA expressed as logarithm of minimum angle of resolution (logMAR) remained stable for switchers (0.4 to 0.4; P=0.96) and improved from baseline in anti-VEGF-naive eyes (0.5 to 0.4; P<0.01). Confirmed AESIs included iritis (n=2; 0.05% of injections), with no events of vitreous cells, endophthalmitis, retinal detachment, retinal vasculitis, or vitreous hemorrhage. ConclusionIn this descriptive real-world analysis, aflibercept-ayyh was associated with stable visual acuity in previously treated eyes and vision improvement in treatment-naive eyes, with no new or unexpected safety findings, consistent with expectations for aflibercept. These findings add real-world experience to preexisting evidence demonstrating no clinically meaningful differences between aflibercept-ayyh (PAVBLU(R)) and reference aflibercept (EYLEA(R)). KEY SUMMARY POINTSO_ST_ABSWhy carry out this study?C_ST_ABSO_LIThe anti-vascular endothelial growth factor (VEGF) drug aflibercept, approved in 2011 and marketed in the United States as EYLEA(R),* has demonstrated efficacy in treating retinal diseases such as neovascular age-related macular degeneration (nAMD), diabetic retinopathy (DR), diabetic macular edema (DME), or retinal vein occlusion (RVO) and is a standard of care for these disorders. C_LIO_LIAflibercept-ayyh is a biosimilar to aflibercept that has demonstrated comparable efficacy and safety in the treatment of nAMD in a randomized controlled clinical trial. C_LIO_LIThis study describes the real-world use patterns, vision outcomes, and safety of aflibercept-ayyh in clinical settings in the United States for the treatment of nAMD, DR, DME, and RVO. C_LI What was learned from the study?O_LIIn this real-world study of 1,000 consecutive eyes treated with the biosimilar aflibercept-ayyh in patients with retinal diseases, we observed no new safety concerns and that aflibercept-ayyh maintained visual acuity in eyes switching anti-VEGF agents and improved vision in anti-VEGF-naive eyes, consistent with expected responses to aflibercept. C_LIO_LIThese findings support aflibercept-ayyh as a suitable treatment option when anti-VEGF therapy is indicated. *EYLEA(R) is a registered trademark of Regeneron Pharmaceuticals, Inc. PAVBLU(R) is a registered trademark of Amgen Inc. C_LI

Tumor cells commonly exhibit aerobic glycolysis and produce lactate despite oxygen availability. Lactate dehydrogenase (LDH) catalyzes pyruvate-lactate interconversion and regulates intracellular lactate levels. Endothelial cells also depend on glycolysis for ATP production, which prompted us to investigate LDH in canine hemangiosarcoma (HSA), a malignant endothelial tumor. We inhibited LDH with (R)-GNE-140 or sodium oxamate in two canine HSA cell lines (HU-HSA-2 and HU-HSA-3) and generated HU-HSA-3 clones with knockout of LDHA or LDHB to evaluate the effects of LDH perturbation. (R)-GNE-140 and sodium oxamate suppressed proliferation and reduced global histone lactylation levels in both cell lines. mRNA-sequencing (mRNA-seq) of (R)-GNE-140-treated HU-HSA-2 cells identified cholesterol/lipid metabolism-related gene sets among the top negatively enriched pathways. Representative cholesterol/lipid metabolism genes responded differently depending on cell lines and inhibitors. (R)-GNE-140 decreased these genes in HU-HSA-2 but not HU-HSA-3, whereas sodium oxamate decreased them in HU-HSA-3 with limited effects in HU-HSA-2. In HU-HSA-3, LDHA and LDHB knockout clones decreased SREBP2 expression and reduced the number of lipid droplets. Fluvastatin, a cholesterol metabolism inhibitor, inhibited HSA cell growth in vitro but did not significantly suppress tumor growth in two HSA patient-derived xenograft (PDX) models. In contrast, combined fluvastatin and dipyridamole treatment inhibited proliferation in vitro and tumor growth in PDX models. Collectively, these results suggest a context-dependent association between LDH and cholesterol/lipid metabolism in canine HSA cell lines and provide a rationale for further evaluation of combined cholesterol pathway inhibition.

Lymphatic vessels are formed during embryonic and postnatal development to facilitate interstitial fluid clearance and immune regulation after birth. Their organ-specific heterogeneity in organisation and function is preceded by heterogenous origins of lymphatic endothelial cells (LECs), the main building blocks of lymphatic vessels. In the dermis, a subset of LECs was reported to arise from blood capillaries, which themselves differentiate, in part, from paraxial mesoderm. However, it is not known whether additional cell lineages contribute to the dermal LEC population. Here, we have combined transcriptomic analyses with genetic lineage tracing and wholemount immunostaining to show that 60% of LECs in the embryonic day (E) 13.5 and E15.5 dermis are derived from a cell lineage that expresses Csf1r, a marker of myeloid cells and their progeny. Csf1r lineage LECs persist in adult dermal lymphatic vasculature and are indispensable for normal lymphatic development, because Prox1 deletion within the Csf1r lineage causes dermal oedema and blood-filled lymphatic vessels. As Csf1r lineage dermal LECs do not themselves express Csf1r and also do not arise from Csf1r-expressing differentiated myeloid cells, our findings imply the existence of a Csf1r-expressing non-LEC precursor population for the majority of dermal LECs and will prompt further work to identify this cell population.

AbstractHereditary Hemorrhagic Telangiectasia (HHT) is a genetic vascular disorder characterized by distinct vascular malformations, including deep organ arteriovenous malformations (AVMs) and mucocutaneous telangiectasias. People with HHT inherit monoallelic pathogenic variants in members of the TGF{beta} signaling cascade (ACVRL1, ENG and SMAD4), resulting in a loss of gene function and dysangiogenesis. While these heterozygous inactivating mutations are present in all cells, malformations develop locally, indicating a focal trigger of onset. Indeed, recent human sequencing studies revealed that second-hit somatic mutations, resulting in complete bi-allelic loss of gene function, are linked to lesion formation in the three major types of HHT (HHT1, HHT2, JP/HHT). To model the loss of heterozygosity (LOH) associated with HHT patients, we generated new Eng and Smad4 HHT mouse models whereby endothelial cell-specific, somatic LOH mutations are induced within a heterozygous loss of function background (HHT-iEC-LOH). The HHT-iEC-LOH models recapitulate the mosaic makeup of patient malformations and indicate that multiple, distinct secondary somatic mutations can contribute to AVM onset. Utilizing immunofluorescent staining, blue latex vasculature casting, weighted tracer perfusions, and lineage tracing studies, HHT-iEC-LOH models were phenotypically assessed and compared to traditional inducible endothelial cell knockout (HHT-iECKO) HHT models. Overall, HHT-iEC-LOH mice exhibit increased malformation frequency and vascular phenotypes that are comparable or exceed the severity of iECKO models. Significantly, HHT-iEC-LOH mice can be induced early in development and live into adulthood, displaying persistent cerebrovascular phenotypes. The heightened patient representation offered by these newly developed models enables the study of long-term disease progression and testing of therapeutic interventions.

ABSTRACT/SUMMARYVascular remodeling within the developing fetus and placenta is essential for supporting the growth and function of emerging tissues and organs. Pericytes (PCs) play a central role in stabilizing and maturing microvascular networks by extending along endothelial cells (ECs) and reinforcing vessel integrity. In the placenta, as in other organs, PC-EC communication is mediated in part by platelet-derived growth factor-BB (PDGF-BB) signaling, which governs PC differentiation, proliferation, migration, and survival, ultimately enabling their recruitment and retention along capillaries. In this study, we identified progressive PC investment along feto-placental capillaries in both murine and human tissues across gestation, supported by morphological and molecular evidence. Placental PCs displayed phenotypic heterogeneity comparable to that observed in the brain and heart, suggesting conserved diversity across organ systems. In addition to characterizing PC dynamics, we examined the expression of recently identified soluble PDGF Receptor-{beta} (sPDGFR{beta}) isoforms. These variants were detected at the protein and transcript levels in mouse and human placentas, as well as in a murine trophoblast-embryonic stem cell (TESC) differentiation model that recapitulates aspects of early placental vascular development. Within this model, sPDGFR{beta} expression was independent of ADAM10 activity and exogenous growth factors during early vessel formation but was markedly upregulated during hypoxia. To assess how elevated sPDGFR{beta} might influence PDGF-BB signaling, we exposed TESCl-derived vascular networks to excess PDGF-BB with or without a sPDGFR{beta} mimetic. PDGF-BB alone reduced full-length PDGFR{beta} levels while increasing receptor phosphorylation, consistent with known ligand-induced regulatory mechanisms. Inclusion of the sPDGFR{beta} mimetic shifted these responses toward baseline, suggesting a potential modulatory or feedback role for soluble receptor variants. Together, these findings demonstrate that PCs are progressively recruited to placental capillaries and exhibit diverse phenotypes during development, and that soluble PDGFR{beta} isoforms may modulate PDGF-BB signaling in a manner sensitive to oxygen tension. Understanding these mechanisms provides insight into the regulation of placental vascular maturation and may inform strategies to improve human health by targeting disorders rooted in impaired placental development.

Intracerebral haemorrhage (ICH) is a severe form of stroke with high morbidity and mortality rates. For survivors, acute haematoma expansion strongly determines neurological outcome. Although blood pressure reduction is widely investigated as a strategy to limit haematoma growth, the haemodynamic mechanisms regulating haemorrhage development remain poorly understood. Zebrafish provide a tractable in vivo model to study cerebrovascular biology and spontaneous ICH, yet the contribution of vascular regulation to haemorrhage onset and expansion has not been explored in this species. Here, we investigated whether pharmacological modulation of vascular dilation influences ICH development in zebrafish larvae. We first characterised vascular changes during the developmental window in which spontaneous ICH occurs and observed increased heart rate and progressive reductions in arterial diameter between 2 and 3 days post-fertilisation, suggesting increased vascular resistance. We then tested whether vasoconstriction promotes haemorrhage using angiotensin II, which induced systemic and cerebrovascular vasoconstriction but did not increase ICH incidence or haematoma size in two independent ICH models. In contrast, pharmacological vasodilation using sodium nitroprusside or isoproterenol significantly reduced haematoma size in a high-incidence model of atorvastatin-induced ICH. Live imaging of cerebral blood flow revealed that vasodilation was associated with the confinement of red blood cells around affected vessels rather than dispersing into the brain ventricles. Together, these findings indicate that vascular dilation modulates haemorrhage progression in zebrafish ICH and establish this model as a platform to investigate haemodynamic mechanisms regulating haematoma expansion.

BackgroundThe hyperactivating p.L914F mutation in TIE2, a receptor tyrosine kinase that is essential for vascular development and function, has been found to drive sporadic venous malformation (VM). While germline or early developmental expression of the mutation is thought to be lethal, mosaic or somatic expression is expected to result in VM disease. However, this has never been shown experimentally. Therefore, we utilized a genetic murine model of TIE2 p.L914F to examine the effects of the mutation in the mosaic condition. ResultsUsing an mTmG reporter mouse, we show that the CMV-Cre mouse line drives mosaic Cre recombination during early embryonic development. We then crossed B6-Tg(Rosa26-TIE2L914F)EBos (TIE2L914F) mice to CMV-Cre mice, to drive mosaic expression of TIE2 p.L914F during development. The offspring of these mice did not have the expected Mendelian ratio of mutant to control animals, indicating that mutant mice experienced partial lethality during development. Furthermore, surviving CMV-Cre;TIE2L914F mutant offspring developed a VM phenotype, with the formation of massively enlarged venous/capillary vessels in various tissues. ConclusionsIn this study, we show that mosaic embryonic expression of the VM-causative mutation TIE2 p.L914F causes partial embryonic lethality and the formation of a VM phenotype. This a novel in vivo model of mutant TIE2-driven VM disease and it illustrates that the extent of the mutational event during development will contribute to varying levels of severity in the VM phenotype.

BackgroundPost-viral diseases, including post-COVID-19 syndrome (PCS) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), cause substantial long-term morbidity. Persistent cardiovascular (CV) risk after acute infection highlights the need for accessible tools to quantify microvascular health. MethodsAll Eyes on PCS is a prospective, observational study investigating the retinal microcirculation using retinal vessel analysis (RVA). We compared RVA parameters in 102 PCS patients with 204 age- and sex-matched healthy controls (HC, matched from n = 303). Secondary matched analyses included never infected controls (NI, n = 96), recovered individuals (n = 102), PCS patients, and ME/CFS patients (n = 62). Laboratory variables, circulating markers of endothelial dysfunction (ED) and inflammation were compared between cohorts and their associations with RVA parameters were examined. ResultsCompared with HC, PCS patients showed reduced venular flicker-induced dilation (3.7 {+/-} 2.2% vs. 4.5 {+/-} 2.7%, p = 0.005), narrow retinal arterioles (CRAE, 178.3 {+/-} 15.5 {micro}m vs. 183.3 {+/-} 15.9 {micro}m, p = 0.009), and lower arteriolar-to-venular ratio (0.83 {+/-} 0.06 vs. 0.86 {+/-} 0.07, p = 0.004). Findings persisted after adjustment for CV factors and remained evident in an extended secondary matched analysis across NI, recovered, and PCS patients. ME/CFS patients showed the most pronounced alterations. PCS severity correlated with lower AVR (r = -0.21, p = 0.037) and reduced arteriolar FID (r = -0.21, p = 0.039), particularly for neurocognitive symptoms. IL-6, ICAM-1 and VCAM-1 were elevated in PCS and ME/CFS and lower AVR correlated with inflammatory and iron-related markers (all adjusted p < 0.01). A combined model discriminated ME/CFS patients with good accuracy (AUC = 0.80). ConclusionsPCS is associated with persistent ED, most pronounced in ME/CFS patients and linked to symptom severity and ongoing inflammation. RVA may provide a noninvasive, readout of ED in post-viral syndromes. Trial RegistrationThe All Eyes on PCS Study has previously been registered at ClinicalTrials.gov (NCT05635552). Novelty and SignificanceO_ST_ABSWhat is known?C_ST_ABS- PCS and ME/CFS are associated with persistent endothelial dysfunction and increased long-term cardiovascular risk. - Neurocognitive symptoms in post-viral syndromes have been linked to impaired neurovascular coupling. - Retinal vessel analysis provides a validated, non-invasive readout of systemic and cerebral microvascular health. What new information does this article contribute?- PCS is characterized by persistent functional and structural retinal microvascular dysfunction - Retinal endothelial dysfunction scales continuously with post-viral disease severity and is most pronounced in patients fulfilling ME/CFS criteria. - Retinal microvascular alterations are linked to inflammatory-endothelial activation and iron dysregulation, identifying a biologically coherent vascular phenotype. This study provides the first comprehensive human in vivo assessment of retinal microvascular structure and function across the full post-COVID-19 spectrum, from never infected controls to recovered individuals, PCS patients, and those fulfilling ME/CFS criteria. Using retinal vessel analysis as a surrogate of neurovascular and endothelial function, we demonstrate that endothelial dysfunction persists in patients with ongoing post-viral symptomatology. Retinal venular flicker-induced dilation, arteriolar caliber, and autoregulatory capacity decline progressively with increasing clinical severity, indicating a dose-response relationship between microvascular injury and post-infectious disease burden. Importantly, these vascular alterations are linked to sustained inflammatory and endothelial activation and to disturbances in iron homeostasis, indicating an inflammatory-endothelial axis rather than isolated cardiovascular risk. By integrating microvascular phenotyping with symptom profiles and circulating biomarkers, this work identifies retinal endothelial dysfunction as a mechanistically informative and clinically accessible marker of post-viral disease severity. These findings advance understanding of post-infectious vascular pathology and provide a translational framework for biological stratification and risk assessment in PCS and ME/CFS.

PurposeAnti-vascular endothelial growth factor (anti-VEGF) therapy is the standard of care for neovascular age-related macular degeneration (AMD), yet many patients exhibit persistent retinal degeneration, fibrosis, and incomplete therapeutic response. The molecular pathways underlying this incomplete response remain poorly understood. We sought to identify VEGF-independent signaling pathways active in the vitreous of anti-VEGF-treated AMD patients. MethodsWe performed multiplex antibody-based proteomic profiling of 1,000 human proteins in vitreous samples from patients with neovascular AMD receiving anti-VEGF therapy (n=8) and comparative controls (n=6). Differential protein expression was assessed using one-way ANOVA, followed by gene ontology and pathway enrichment analyses. Drug-target relationships were evaluated to identify potential opportunities for therapeutic repositioning. ResultsWe identified 107 differentially expressed proteins (p<0.05), including key regulators of immune signaling, angiogenesis, and metabolism. Notably, multiple components of cytotoxic lymphocyte pathways were dysregulated, including IL-21R, SIGLEC-7, CTLA4, and IL-2-associated signaling. Enrichment analyses revealed significant activation of pathways related to T-cell activation, interleukin signaling, and leukocyte-mediated cytotoxicity. These immune signatures persisted despite suppression of VEGF signaling. Several clinically available immunomodulatory agents--including abatacept, sirolimus, and dupilumab--targeted pathways identified in this dataset. ConclusionsAnti-VEGF-treated neovascular AMD exhibits persistent cytotoxic immune signaling in the vitreous, suggesting that VEGF-independent immune mechanisms may contribute to ongoing retinal damage and incomplete therapeutic response. These findings provide a rationale for combination therapeutic strategies targeting both angiogenic and immune pathways in AMD.

BackgroundClassical myeloproliferative neoplasms (MPN)--essential thrombocythemia, polycythemia vera, and primary myelofibrosis--are characterized by clonal hematopoiesis, overproduction of mature blood cells, and a high burden of thromboembolic events. Although thrombosis is the leading cause of morbidity and mortality in MPN, the contribution of the vascular endothelium remains incompletely defined. We investigated patient-derived endothelial colony-forming cells (ECFCs) as a surrogate for vascular endothelium in individuals with JAK2 V617F-mutated MPN. MethodsECFCs were cultured from peripheral blood of patients with MPN and healthy controls, phenotyped for thrombo-inflammatory and adhesive markers, tested for JAK2 V617F, and profiled by bulk RNA sequencing. Functional assays assessed endothelial-dependent factor Xa generation. Transcriptomes were benchmarked against public HUVEC reference datasets processed through an identical quantification pipeline. ResultsECFCs were obtained more frequently and in greater numbers from patients with MPN than from controls, indicating enhanced endothelial regenerative or activation potential. MPN ECFCs exhibited increased von Willebrand factor and P-selectin expression and release, along with elevated endothelial cell-dependent factor Xa generation, consistent with a thrombo-inflammatory, procoagulant phenotype. JAK2 V617F was not detected in any ECFC colonies, supporting a non-clonal origin of these endothelial abnormalities. Transcriptomic analysis identified 289 differentially expressed genes in MPN versus control ECFCs, with pathway enrichment revealing coordinated dysregulation of blood coagulation, platelet activation, plasminogen regulation, vascular permeability, extracellular matrix organization, and angiogenesis. Benchmarking against HUVEC datasets confirmed strong endothelial identity of ECFC-derived cells, with MPN-associated changes reflecting endothelial activation rather than loss of endothelialness. ConclusionsECFCs from patients with JAK2-mutated MPN display functional and transcriptomic signatures of endothelial dysfunction in the absence of detectable driver mutations. These findings support a model in which a primed, thrombo-inflammatory endothelium cooperates with clonal hematopoiesis to promote the heightened thrombotic risk characteristic of MPN.

Understanding how specific VWF variants disrupt endothelial processing and function is central to elucidating von Willebrand disease (VWD) pathophysiology. However, current in vitro systems lack either the endothelial specificity or the genetic flexibility required for systematic variant characterization. Here, we present a genetically defined VWF-knockout cord-blood-derived endothelial colony-forming cell (VWF-KO cbECFC) model that enables controlled reintroduction of VWF variants in a physiologically relevant endothelial context. Using a patient with type 3 VWD carrying the homozygous pathogenic variant p.M771V and a second homozygous variant of uncertain significance p.R2663P as a reference, we demonstrate that expression of p.M771V in VWF-KO cbECFCs reproduces the patients intracellular processing defect and loss of high-molecular-weight multimers, whereas p.R2663P behaves as a benign allele. These findings establish the models ability to accurately distinguish pathogenic from non-pathogenic variants. Comparative analyses with HEK293 cells show that VWF-KO cbECFCs provide superior subcellular resolution, reliably forming authentic Weibel-Palade bodies (WPBs) and faithfully revealing ER retention phenotypes that remain ambiguous in non-endothelial systems. The proliferative capacity of cbECFCs further enables scalable and reproducible experimentation, overcoming major limitations associated with patient-derived ECFCs. Looking ahead, the VWF-KO cbECFC platform offers broad potential for VWF and VWD research. Its endothelial identity and genetic flexibility make it suitable for investigating VWF biosynthesis and trafficking, secretion dynamics, WPB biology, angiogenic processes, and shear-dependent VWF function. This system therefore provides a versatile foundation for mechanistic studies, systematic variant assessment, and future translational applications.

Pericytes are mural cells that provide support to the endothelium of small blood vessels. Pericyte soma are regularly spaced along vessels, and their processes overlap only slightly. Given that vessel patterning is imprecise, we explore the interplay between vessel growth and pericyte recruitment that leads to even pericyte spacing. After recruitment to the zebrafish brain central arteries (CtAs), pericytes undergo rapid expansion, followed by morphological differentiation. Blocking angiogenesis by reducing Gpr124 (Wnt) or Vegf signaling reduces the length of the vessel network and the number of pericytes, preserving spacing, suggesting proportional recruitment of pericytes to cover the network and the territorial nature of pericytes. However, these initial brain pericytes have low proliferation rates. We demonstrate that additional pericytes are recruited firstly through migration of col5a1- and later col1a2-expressing fibroblasts into the brain. These second-wave pericytes retain some fibroblast properties and show elevated col1a2 levels in a model of pericyte loss (notch3 mutants). Our data provide new insights into the developmental timing, expansion, and novel origins of late-arriving brain pericytes during embryogenesis. SUMMARY STATEMENTThis article demonstrates that brain pericytes originate from multiple sources, including fibroblast-derived populations, and how pericyte numbers are adjusted in proportion to vessel development.

Vascular endothelial (VE)-cadherin is essential for maintaining endothelial junctional barrier integrity. The Angiopoietin-1 (Ang-1)/Tie2 axis induced Akt1 activation is crucial for maintaining endothelial junctional barrier by inhibiting FoxO1 and suppressing expression of Angiopoietin-2 (Ang-2), a Tie2 antagonist. Systemic inflammatory conditions such as sepsis, Akt1 expression is reduced, whereas FoxO1-dependent Ang-2 expression is increased, resulting in endothelial barrier dysfunction. We previously showed that the TLR4/FoxO1 axis induces the ubiquitin E3 ligase CHFR, which promotes endothelial barrier disruption by targeting VE-cadherin for ubiquitylation and degradation. However, little is known about Akt1 expression during vascular inflammation. Here, we identified FoxO1-dependent CHFR expression as a key mechanism driving K48-linked polyubiquitylation and proteasomal degradation of Akt1 in endothelial cells (EC). LPS-induced K48-linked ubiquitylation of Akt1 was prevented in CHFR-depleted human EC and in endothelial-specific Chfr knockout (Chfr{Delta}EC) mice. Accordingly, CHFR depletion increased Akt1 and VE-cadherin expression in both human lung EC and Chfr{Delta}EC mice. Chfr{Delta}EC mouse lungs also exhibited elevated Ang-1 and Tie2 expression, and Ang-1 stimulation induced sustained Akt1 phosphorylation in CHFR-deficient EC. Moreover, CHFR depletion prevented LPS-induced expression of FoxO1 and Ang-2 in EC. Mechanistically, CHFR interacted with phosphorylated Akt1 and mediated its ubiquitylation at lysine residues K30, K39, K154, and K268. Expression of a ubiquitylation-deficient Akt1 mutant prevented LPS-induced VE-cadherin degradation and vascular injury. Collectively, these findings identify CHFR as a critical regulator of endothelial inflammatory responses by controlling Akt1 stability and VE-cadherin expression during inflammation.

BACKGROUNDAbdominal aortic aneurysm (AAA) is a life-threatening condition with >80% mortality upon rupture and no effective pharmacotherapy available. Despite epidemiological evidence linking metformin use to reduced AAA progression, its mechanism remains elusive. Notably, peroxisome proliferator-activated receptor {gamma} coactivator 1 (PGC-1, encoded by Ppargc1a) is downregulated in human AAA, yet its functional role in metformins protection is unknown. METHODSWe employed porcine pancreatic elastase (PPE)-induced murine AAA, VSMC-specific Ppargc1a knockout (Ppargc1aVSMC-KO), primary VSMC senescence models, and pharmacological inhibition (Compound C for AMPK; Ex-527 for SIRT1) to define the AMPK-SIRT1-PGC-1 axis. RESULTSMetformin significantly inhibited AAA expansion, suppressed VSMC senescence (p53/p21{downarrow}, SA-{beta}-gal{downarrow}), and preserved contractile phenotype (SMTN{uparrow}, IL-6/TNF-{downarrow}). Crucially, all benefits were abrogated in Ppargc1aVSMC-KO mice, which exhibited accelerated aneurysm growth, mitochondrial fragmentation, ATP depletion, and ROS accumulation. Mechanistically, metformin activated AMPK/SIRT1 to upregulate PGC-1; AMPK or SIRT1 inhibition blocked this cascade and reversed protection. CONCLUSIONMetformin restrains AAA by restoring VSMC mitochondrial homeostasis via the AMPK/SIRT1[-&gt;]PGC-1 axis, positioning PGC-1 as a non-redundant, cell-autonomous guardian against vascular degeneration. These findings provide a mechanistic foundation for repurposing metformin and developing PGC-1-targeted therapies in AAA.

PurposeThe short lifespan of primary normal choroidal melanocytes (NCMs) in vitro represents a major barrier to mechanistic, functional, and translational studies of choroid biology and uveal melanoma (UM). This study aimed to establish and characterize immortalized human NCM lines that retain melanocytic function, maintain a non-cancerous profile, and are amenable to gene editing. MethodsNCMs from four donors were immortalized by lentiviral transduction of Cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and human Telomerase reverse transcriptase (hTERT), establishing NCM-K4DT lines. Their morphology, melanocytic marker expression, proliferation and functional properties (melanin synthesis, tyrosinase activity) were evaluated. Genomic stability was assessed by targeted mutation profiling, karyotyping, and copy number variation analysis. The tumorigenicity was tested in immunodeficient mice. Plasmid-based CRISPR/Cas9 editing was performed to determine their suitability for gene editing. ResultsNCM-K4DT lines retained dendritic-shaped morphology, pigmentation, and expression of PMEL, TYRP1, Melan-A, and SOX10. Cells exhibited enhanced proliferative capacity with preserved cell cycle regulation. Melanin production and tyrosinase activity were comparable to primary NCMs. Genomic profiling confirmed the absence of UM-associated driver mutations and chromosomal abnormalities. In vivo growth assays demonstrated no tumorigenic potential. Notably, NCM-K4DT cells were efficiently edited by CRISPR/Cas9. ConclusionsNCM-K4DT lines represent stable, non-cancerous, and genetically tractable models for studying choroidal melanocyte biology, modeling UM-associated mechanisms, and advancing therapeutic development in ocular research.