Junhao Hu

Glucagon-like peptide-1 (GLP-1) is a key incretin hormone rapidly degraded by circulating proteases such as DPP-4. The metabolism of GLP-1 by other proteases, particularly tissue-resident proteases, remains largely unexplored. Here, we identify insulin-degrading enzyme (IDE) as a previously unknown GLP-1-degrading protease with two cleavage sites. We show that IDE-mediated degradation of GLP-1, but not insulin, represents a major mechanism regulating glucose control. To resist IDE, we engineered GLP-1 and Semaglutide with D-amino acid substitutions at these sites. These peptides exhibit enhanced stability in plasma, liver and intestinal secretomes, peritoneal fluid, and central nervous system (CNS). D-Ser ¹⁸ -Semaglutide shows prolonged plasma retention and sustained glucose-lowering effects in mice. Moreover, IDE knockdown and intracerebral injection of D-Ser ¹⁸ -Semaglutide confirm IDE’s physiological role in GLP-1 degradation, particularly in the CNS. These findings reveal a previously unidentified regulatory mechanism of GLP-1 metabolism and provide a strategy for designing long-acting agonists with improved metabolic and CNS therapeutic potential.

Current single-cell metabolomics approaches are limited by insufficient sensitivity, robustness and metabolite coverage. We present an ion mobility-resolved mass cytometry technology that integrates high-throughput single-cell injection with ion mobility–mass spectrometry for multidimensional metabolomic profiling. Ion mobility-enabled selective ion accumulation and cell superposition-based amplification strategies substantially enhance sensitivity, robustness and overall analytical performance. Combined with our computational tool, MetCell, this technology allows high-throughput analysis while achieving exceptional profiling depth, detecting over 5,000 metabolic peaks and annotating approximately 800 metabolites per cell—representing a 3-fold to 10-fold improvement over existing methods. It offers attomole-level sensitivity and captures a broad dynamic range of metabolites within individual cells. Applied to 45,603 primary liver cells from aging mice, it enabled accurate cell-type and cell-subtype annotation and revealed distinct metabolic states and heterogeneity in hepatocytes during aging. This platform sets a new benchmark for high-throughput single-cell metabolomics, advancing our understanding of metabolic heterogeneity at single-cell resolution.

Malignant serous effusion (MSE), including malignant pleural effusion (MPE) and malignant ascites (MA), is a common and severe complication in advanced malignancies, associated with poor prognosis and high recurrence rates. Currently, no standardized treatments are available for MSE management, posing significant clinical challenges. Here, we identify elevated LIN28B expression and dysregulation of DNA repair pathways as two major features associated with MSE from patient and preclinical samples. We develop a targeted siRNA nanoparticle delivery system (siLin28B/DSSP@lip‐PEG‐FA) in combination with the PARP inhibitor BMN673, providing a synergistic therapeutic strategy against MSE. This combination significantly alleviated MA accumulation and prolonged survival in a preclinical ovarian cancer (OC) model without causing systemic cytotoxicity. Mechanistically, single‐cell RNA sequencing (scRNA‐seq) revealed that this combination therapy markedly remodeled the immune microenvironment by decreasing M2 macrophages and neutrophil populations with altered subtypes. Notably, Arg1‐positive neutrophils, producing pro‐inflammatory cytokines to increase vascular permeability, were diminished after the combination treatment. Furthermore, in vitro and in vivo experiments demonstrated that suppression of PARP and LIN28B inhibited vascular leakage and reinforced tight junction integrity. Collectively, our findings highlight dual targeting of PARP and LIN28B as a promising MA management approach in patients with advanced cancers, with the potential to improve patient quality of life.

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.

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.

Pericytes and endothelial cells (ECs) constitute the fundamental components of blood vessels. While the role of ECs in tumor angiogenesis and the tumor microenvironment is well appreciated, pericyte function in tumors remains underexplored. In this study, we used pericyte-specific deletion of the nitric oxide (NO) receptor, soluble guanylate cyclase (sGC), to investigate via single-cell RNA sequencing how pericytes influence the vascular niche and the tumor microenvironment. Our findings demonstrate that pericyte sGC deletion disrupts EC–pericyte interactions, impairing Notch-mediated intercellular communication and triggering extensive transcriptomic reprogramming in both pericytes and ECs. These changes further extended their influence to neighboring cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) through paracrine signaling, collectively suppressing tumor growth. Inhibition of pericyte sGC has minimal impact on quiescent vessels but significantly increases the vulnerability of angiogenic tumor vessels to conventional anti-angiogenic therapy. In conclusion, our findings elucidate the role of pericytes in shaping the tumor vascular niche and tumor microenvironment and support pericyte sGC targeting as a promising strategy for improving anti-angiogenic therapy for cancer treatment.

Disruption of endothelial cell (ECs) and pericytes interactions results in vascular leakage in acute lung injury (ALI). However, molecular signals mediating EC–pericyte crosstalk have not been systemically investigated, and whether targeting such crosstalk could be adopted to combat ALI remains elusive. Using comparative genome-wide EC–pericyte crosstalk analysis of healthy and LPS-challenged lungs, we discovered that crosstalk between endothelial nitric oxide and pericyte soluble guanylate cyclase (NO–sGC) is impaired in ALI. Indeed, stimulating the NO–sGC pathway promotes vascular integrity and reduces lung edema and inflammation-induced lung injury, while pericyte-specific sGC knockout abolishes this protective effect. Mechanistically, sGC activation suppresses cytoskeleton rearrangement in pericytes through inhibiting VASP-dependent F-actin formation and MRTFA/SRF-dependent de novo synthesis of genes associated with cytoskeleton rearrangement, thereby leading to the stabilization of EC–pericyte interactions. Collectively, our data demonstrate that impaired NO–sGC crosstalk in the vascular niche results in elevated vascular permeability, and pharmacological activation of this crosstalk represents a promising translational therapy for ALI.

Background: Arteriogenesis plays a critical role in maintaining adequate tissue blood supply and is related to a favorable prognosis in arterial occlusive diseases. Strategies aimed at promoting arteriogenesis have thus far not been successful because the factors involved in arteriogenesis remain incompletely understood. Previous studies suggest that evolutionarily conserved KANK4 (KN motif and ankyrin repeat domain-containing proteins 4) might involve in vertebrate vessel development. However, how the KANK4 regulates vessel function remains unknown. We aim to determine the role of endothelial cell-specifically expressed KANK4 in arteriogenesis. Methods: The role of KANK4 in regulating arteriogenesis was evaluated using Kank4 ^−/− and KANK4 ^iECOE mice. Molecular mechanisms underlying KANK4-potentiated arteriogenesis were investigated by employing RNA transcriptomic profiling and mass spectrometry analysis. Results: By analyzing Kank4-EGFP reporter mice, we showed that KANK4 was specifically expressed in endothelial cells. In particular, KANK4 displayed a dynamic expression pattern from being ubiquitously expressed in all endothelial cells of the developing vasculature to being explicitly expressed in the endothelial cells of arterioles and arteries in matured vessels. In vitro microfluidic chip-based vascular morphology analysis and in vivo hindlimb ischemia assays using Kank4 ^−/− and KANK4 ^iECOE mice demonstrated that deletion of KANK4 impaired collateral artery growth and the recovery of blood perfusion, whereas KANK4 overexpression leads to increased vessel caliber and blood perfusion. Bulk RNA sequencing and Co-immunoprecipitation/mass spectrometry (Co-IP/MS) analysis identified that KANK4 promoted EC proliferation and collateral artery remodeling through coupling VEGFR2 (vascular endothelial growth factor receptor 2) to TALIN-1, which augmented the activation of the VEGFR2 signaling cascade. Conclusions: This study reveals a novel role for KANK4 in arteriogenesis in response to ischemia. KANK4 links VEGFR2 to TALIN-1, resulting in enhanced VEGFR2 activation and increased EC proliferation, highlighting that KANK4 is a potential therapeutic target for promoting arteriogenesis for arterial occlusive diseases.