Xiaohong Wang

Endothelial cell (EC) mechanosensing is essential for vascular homeostasis and atherosclerosis development, though the influence of blood flow patterns on EC signaling is not fully understood. Discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, acts as a mechanosensor linking shear force to endothelial responses. Atheroprotective laminar shear flow induces rapid, transient DDR1 activation and condensation through liquid-liquid phase separation without harmful effects, while atherogenic shear causes delayed, sustained condensation, leading to Yes-associated protein (YAP) activation and downstream pathological signaling. We show that heat shock protein 27 (HSP27) regulates shear-dependent DDR1 condensation and phase transition. Specifically, the interaction between DDR1 and HSP27, mediated by their respective domains, is crucial for DDR1 condensate disassembly. Atherogenic shear, unlike atheroprotective shear, triggers prolonged DDR1-mediated HSP27 phosphorylation, promoting DDR1 gel transition and activating YAP signaling. The DDR1-HSP27 axis is key in endothelial YAP activation and atherogenesis in vivo. Targeting this pathway may offer therapeutic potential for preventing endothelial dysfunction and atherosclerosis.

Background: Platelet factor 4 (PF4/CXCL4) is a chemokine with reported anti-angiogenic and immunomodulatory properties; however, the role of PF4 in neovascular age-related macular degeneration (nAMD) remains unclear. Thus, this study aimed to evaluate the therapeutic potential of PF4 in experimental models of ocular pathological neovascularization and explored the underlying mechanisms. Methods: PF4 expression was assessed in a laser-induced choroidal neovascularization (CNV) mouse model using quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. Recombinant PF4 was administered intravitreally in laser-induced CNV mice and very low-density lipoprotein receptor knockout (Vldlr⁻/⁻) mice, a model of spontaneous retinal neovascularization with retinal angiomatous proliferation (RAP)-like lesions. Pathological neovascularization and vascular leakage were quantified by fundus fluorescein angiography and choroidal/retinal flat-mount analyses. Immunofluorescence, qRT-PCR, and RNA sequencing were employed to evaluate inflammatory responses. Moreover, the effects of PF4 on vascular endothelial growth factor (VEGF)-induced proliferation, migration, and tube formation of human retinal microvascular endothelial cells were examined in vitro, and VEGF-mediated signaling was analyzed by Western blotting. Ocular safety was assessed by optical coherence tomography (OCT), electroretinography (ERG), hematoxylin and eosin (H&E) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: Intravitreal PF4 significantly reduced pathological neovascularization and vascular leakage in both models and attenuated intraocular inflammation, as indicated by decreased expression of proinflammatory cytokines and reduced microglial/macrophage recruitment. PF4 inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation in vitro. Mechanistically, PF4 downregulated VEGF expression in CNV lesions in vivo and suppressed VEGF-induced activation of vascular endothelial growth factor receptor 2 (VEGFR2) and downstream extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and signal transducer and activator of transcription 3 (STAT3) signaling in vivo and in vitro. PF4 administration was well tolerated, with no detectable adverse effects on retinal structure or function. Conclusions: PF4 effectively inhibits ocular pathological neovascularization and inflammation by modulating the VEGF/VEGFR2 signaling pathway. These findings support PF4 as a promising therapeutic candidate for nAMD and warrant further investigation.

Membrane mechanics play a crucial role in cellular signaling and fate determination, yet their impact on angiogenesis remains poorly understood. Here, we identify Kindlin-2 as a key regulator of sprouting angiogenesis via regulating endothelial membrane tension through its interaction with Moesin, a crucial linker protein between the cell membrane and the actin cortex. Mechanistically, Kindlin-2 binds to the N62 residue of Moesin, limiting its overactivation and maintaining proper membrane tension to facilitate VEGFR2 endocytosis and downstream signaling. Using both developmental and pathological models, we demonstrate that the interaction of Kindlin-2 and Moesin is enhanced in high angiogenic conditions, and endothelial Kindlin-2 deletion reduces angiogenesis. Furthermore, mutation of Moesin at N62 phenocopies the effects of Kindlin-2 loss. Together, these findings uncover a previously unrecognized mechanism linking membrane tension regulation to angiogenesis and provide new insights into targeting the Kindlin-2–Moesin axis for therapeutic intervention in neovascular diseases.

Background: Angiogenesis plays a crucial role in organ development. However, aberrant blood vessel growth is involved in various diseases, including tumors and neovascular eye diseases. Angiotensin-converting enzyme 2 (ACE2) is a critical enzyme regulating the health of cardiovascular system, and its post-translational modifications (PTMs) are crucial to determine ACE2 expression level and activity. Here, we studied how the PTM of ACE2 in vascular endothelial cells (ECs) affect pathological retinal neovascularization and tumor angiogenesis. Methods: The angiogenic capabilities of ECs were assessed by tube formation, sprouting assays, and 5-ethynyl-2-deoxyuridine (EdU) and filopodia staining. EC angiogenesis was examined by poteome profiler array and aortic ring assays in vitro and by the oxygen-induced retinopathy (OIR) and tumor angiogenesis models in vivo. High-throughput screening involving data from RNA-seq, ATAC-seq, and ChIP-seq were used to explore the epigenetic and transcriptional regulations of pro-angiogenic genes regualtged by ACE2 PTMs. Results: ACE2 Ser-680 dephosphorylation in connection with Lys-788 ubiquitination increased EC angiogenic phenotype, which were manifested by aberrant vascularization in mouse OIR and tumor models. ACE2 Ser-680 dephosphorylation led to the activation of activator protein-1 (AP-1), which transactivated multiple genes involved in angiogenesis. AP- 1 inhibition mitigated such angiogenesis in vivo. Conclusion: Our findings show a novel PTM mechanism of ACE2 involved in pathological angiogeneis. Specifically, ACE2 Ser-680 dephosphorylation facilitated AP-1 transactivation of the downstream pro-angiogenic genes in ECs.

Ocular fibrosis, a severe consequence of excessive retinal wound healing, can lead to vision loss following retinal injury. Proliferative vitreoretinopathy (PVR), a common form of ocular fibrosis, is a major cause of blindness, characterized by the formation of extensive fibrous proliferative membranes. Understanding the cellular origins of PVR-associated fibroblasts (PAFs) is essential to decipher the mechanisms of ocular wound healing. In this study, we combined single-cell transcriptomics with genetic lineage tracing to map the contributions of retinal pigment epithelial (RPE) cells, immune cells, and Müller cells to disease progression. RPE cells were found to constitute the largest fraction of cells within PVR lesions, transitioning through metabolic, proliferative, and epithelial-to-mesenchymal transition stages during their conversion to PAFs. These cells exhibited remarkable plasticity and heterogeneity. Notably, Pdgfrb ⁺ RPE cells demonstrated significant morphological plasticity, transitioning toward a fibroblast-like phenotype, while macrophage-like RPE cells acquired inflammation-related functions post-PVR. Cell communication network analysis identified Thbs1 (encoding TSP-1) as a key hub gene driving RPE cell fate transitions during PVR. Importantly, therapeutic antibodies targeting TSP-1 significantly mitigated PVR progression. This study provides a detailed roadmap of fibrosis formation during ocular wound healing and highlights the therapeutic potential of targeting TSP-1 in the management of PVR.

Age-related macular degeneration (AMD) is a prevalent neuroinflammation condition and the leading cause of irreversible blindness among the elderly population. Smoking significantly increases AMD risk, yet the mechanisms remain unclear. Here, we investigate the role of Sema4D-PlexinB1 axis in the progression of AMD, in which Sema4D-PlexinB1 is highly activated by smoking. Using patient-derived samples and mouse models, we discover that smoking increases the presence of Sema4D on the surface of CD8⁺ T cells that migrate into the choroidal neovascularization (CNV) lesion via CXCL12-CXCR4 axis and interact with its receptor PlexinB1 on choroidal pericytes. This leads to ROR2-mediated PlexinB1 phosphorylation and pericyte activation, thereby disrupting vascular homeostasis and promoting neovascularization. Inhibition of Sema4D reduces CNV and improves the benefit of anti-VEGF treatment. In conclusion, this study unveils the molecular mechanisms through which smoking exacerbates AMD pathology, and presents a potential therapeutic strategy by targeting Sema4D to augment current AMD treatments.

BACKGROUND: Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability. METHODS: Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice ( Kindlin-2 ^iΔEC ) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (12±4 dynes/cm ² ) or oscillatory shear (0.5±4 dynes/cm ² ) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and OptoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function. RESULTS: We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2 ^iΔEC mice showed heightened vascular permeability. Kindlin-2 ^iΔEC were bred onto ApoE ^−/− mice to generate Kindlin-2 ^iΔEC ; ApoE ^{− /−} mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation. CONCLUSIONS: Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis.

Purpose: Retinal neovascularization poses heightened risks of vision loss and blindness. Despite its clinical significance, the molecular mechanisms underlying the pathogenesis of retinal neovascularization remain elusive. This study utilized single-cell multiomics profiling in an oxygen-induced retinopathy (OIR) model to comprehensively investigate the intricate molecular landscape of retinal neovascularization. Methods: Mice were exposed to hyperoxia to induce the OIR model, and retinas were isolated for nucleus isolation. The cellular landscape of the single-nucleus suspensions was extensively characterized through single-cell multiomics sequencing. Single-cell data were integrated with genome-wide association study (GWAS) data to identify correlations between ocular cell types and diabetic retinopathy. Cell communication analysis among cells was conducted to unravel crucial ligand–receptor signals. Trajectory analysis and dynamic characterization of Müller cells were performed, followed by integration with human retinal data for pathway analysis. Results: The multiomics dataset revealed six major ocular cell classes, with Müller cells/astrocytes showing significant associations with proliferative diabetic retinopathy (PDR). Cell communication analysis highlighted pathways that are associated with vascular proliferation and neurodevelopment, such as Vegfa–Vegfr2, Igf1–Igf1r, Nrxn3–Nlgn1, and Efna5–Epha4. Trajectory analysis identified a subset of Müller cells expressing genes linked to photoreceptor degeneration. Multiomics data integration further unveiled positively regulated genes in OIR Müller cells/astrocytes associated with axon development and neurotransmitter transmission. Conclusions: This study significantly advances our understanding of the intricate cellular and molecular mechanisms underlying retinal neovascularization, emphasizing the pivotal role of Müller cells. The identified pathways provide valuable insights into potential therapeutic targets for PDR, offering promising directions for further research and clinical interventions.

Metabolic disorder significantly contributes to diabetic vascular complications, including diabetic retinopathy, the leading cause of blindness in the working-age population. However, the molecular mechanisms by which disturbed metabolic homeostasis causes vascular dysfunction in diabetic retinopathy remain unclear. O-GlcNAcylation modification acts as a nutrient sensor particularly sensitive to ambient glucose. Here, we observe pronounced O-GlcNAc elevation in retina endothelial cells of diabetic retinopathy patients and mouse models. Endothelial-specific depletion or pharmacological inhibition of O-GlcNAc transferase effectively mitigates vascular dysfunction. Mechanistically, we find that Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, are O-GlcNAcylated in diabetic retinopathy. We identify threonine 383 as an O-GlcNAc site on YAP, which inhibits its phosphorylation at serine 397, leading to its stabilization and activation, thereby promoting vascular dysfunction by inducing a pro-angiogenic and glucose metabolic transcriptional program. This work emphasizes the critical role of the O-GlcNAc-Hippo axis in the pathogenesis of diabetic retinopathy and suggests its potential as a therapeutic target.