Biology subjects

ZHENG, Y.

Publications and source records attributed to ZHENG, Y..

5 recordsLinked to original sources

bioRxiv2025

Evaluating Microglial Contributions to the Neurovascular Unit in Health and Neurodegeneration Using Human In Vitro Models

BackgroundMicroglia are emerging as critical regulators of neurovascular function in health and Alzheimers disease (AD), yet their interactions with the human neurovascular unit (NVU), particularly brain endothelial cells, remain incompletely understood. Current in vitro NVU platforms typically exclude microglia and lack perfusable vascular networks with physiologically relevant architecture. Here, we established complementary two-dimensional (2D) and three-dimensional (3D) NVU models to investigate microglia-endothelial and microglia-neurovascular interactions. MethodsHuman induced pluripotent stem cell derived-neurons (iNs), astrocytes (iAs), and microglia-like cells (iMGLs) were incorporated into a soft-lithography based engineered microvessel system to establish a multicellular neuroimmune-vascular model. To specifically evaluate iMGL-endothelial cell (EC) interactions, iMGL were co-cultured with primary human brain microvascular endothelial cells (HBMECs) and junctional protein localization was evaluated using immunofluorescence. The barrier integrity of engineered microvessels containing iMGL was evaluated using dextran permeability. Our 2D and 3D systems were stimulated with tumor necrosis factor- (TNF) to evaluate whether iMGL would promote or attenuate EC inflammation and barrier breakdown. ResultsIncorporation of iNs, iAs, and iMGLs into a perfusable vascular model enabled a more complete representation of NVU cellular diversity and promoted neuronal health. In monolayer co-culture with iMGL, HBMECs enhanced the junctional localization of tight and adherens junction proteins through both contact-dependent and paracrine mechanisms. Following an inflammatory challenge, iMGLs reduced endothelial inflammatory activation, suggesting a protective role in response to AD-relevant inflammatory conditions. Finally, when embedded in 3D collagen matrices surrounding perfusable endothelialized lumen networks, iMGLs reduced dextran permeability and preserved endothelial barrier integrity following TNF challenge. ConclusionsTogether, these findings establish a 3D perfusable neuroimmune-vascular model that enables the dissection of microglial contributions to neurovascular function in health and disease.

cell biology↗

bioRxiv2025

A Machine Learning- 3D Microvessel Platform Identifies Kinase Targets Restoring Blood-Brain-Barrier Endothelial Integrity.

Disruption of the brain endothelial barrier is a hallmark of traumatic brain injury (TBI), and contributes to cerebral edema, coagulopathy, and delayed neurological deficits, yet there are no mechanism-guided therapies that directly stabilize human brain vessels. Here we used a three-dimensional (3D) human brain endothelial vessel platform with quantifiable macromolecular barrier function to assess responses to thrombin and plasma from patients with TBI. This endothelial-focused 3D system isolates the central cellular regulator of blood-brain barrier function and enables mechanistic analysis of endothelial signaling and drug responses as a foundation for personalized vascular therapeutics. By integrating this platform with machine learning-guided kinase analysis, we identified kinase pathways associated with barrier stabilization versus disruption and nominated inhibitors that reversed thrombin- and TBI plasma-induced leakage as well as compounds that exacerbated endothelial loss. This work establishes an important first step toward precision vascular therapeutics in TBI, by linking patient-derived plasma phenotypes to specific endothelial barrier responses and pharmacologically tractable kinase signaling, and it provides a generalizable framework for mechanistic drug discovery across neurovascular disorders. SIGNIFICANCE STATEMENTBlood-brain barrier (BBB) disruption drives cerebral edema, coagulopathy, ischemia, and neurological deficits after TBI. Targeting kinases as therapeutics is promising but challenged by complex inhibitor profiles and patient variability. We present a machine learning-guided platform built on 3D perfusable human brain endothelial-centric microvessels to systematically screen and select kinase inhibitors that reverse TBI-induced endothelial barrier breakdown. By pinpointing key kinase and gene regulators and demonstrating restoration of endothelial barrier integrity in patient-derived plasma models, our approach provides a transformative strategy for stabilizing the endothelial barrier after TBI and offers a versatile framework for therapeutic discovery across neurovascular disorders.

bioengineering↗

bioRxiv2024

Discovery and Engineering of a New BvCas12a Nuclease for Mammalian Genome Editing and Nucleic Acid Detection

Cas12a is an RNA-guided endonuclease that has emerged as a powerful gene-editing tool. We have identified a novel Cas protein, BvCas12a, from Butyricimonas virosa with a 5-TYTN protospacer adjacent motif (PAM). BvCas12a exhibits double-stranded DNA cleavage activity in vitro and genome editing activity in eukaryotic cells. Though the editing efficiency of BvCas12a is marginally lower than that of AsCas12a, the editing specificity of BvCas12a in eukaryotic cells is comparable or superior to that of AsCas12a. Moreover, BvCas12a exhibits substantial collateral activity and can detect HPV DNA effectively and accurately in conjunction with isothermal amplification, highlighting its potential in nucleic acid diagnostics. Furthermore, we have engineered BvCas12a to create two variants, BvCas12a-R (N549R, T606P) and BvCas12a-RVR (N549R, K555V, C559R, T606P). These variants recognize expanded 5-YYN and 5-YN PAM in vitro, respectively. Additionally, they exhibit higher editing activity than wild-type BvCas12a and recognize 5-YYN PAM in vivo at all sites detected. In conclusion, we have identified a novel BvCas12a protein with high specificity and engineered two variants with broader PAM compatibility and improved genome editing efficiency. These findings offer a potent gene editing tool for application in scientific research, gene therapy, and nucleic acid diagnostics.

genomics↗

bioRxiv2024

Under pressure: altered endothelial flow response

Blood flow within the vasculature is a critical determinant of endothelial cell (EC) identity and functionality, yet the intricate interplay of various hemodynamic forces and their collective impact on endothelial and vascular responses are not fully understood. Specifically, the role of hydrostatic pressure in the EC flow response is understudied, despite its known significance in vascular development and disease. To address this gap, we developed in vitro models to investigate how pressure influences EC responses to flow. Our study demonstrates that elevated pressure conditions significantly modify shear-induced flow alignment and increase endothelial cell density. Bulk and single-cell RNA sequencing analyses revealed that, while shear stress remains the primary driver of flow-induced transcriptional changes, pressure modulates shear- induced signaling in a dose-dependent manner. These pressure-responsive transcriptional signatures identified in human ECs were conserved during the onset of circulation in early mouse embryonic vascular development, where pressure was notably associated with transcriptional programs essential to arterial and hemogenic EC fates. Our findings suggest that pressure plays a synergistic role with shear stress on ECs and emphasizes the need for an integrative approach to endothelial cell mechanotransduction, one that encompasses the effects induced by pressure alongside other hemodynamic forces.

bioengineering↗

bioRxiv2022

DNA packaging via hierarchical chromatin structures revealed by live-cell 3D imaging

Single-molecule localization microscopy is a powerful superresolution imaging technique to study biological questions by visualizing subcellular fine structures with nanometer-scale precision. However, its application in live-cell imaging studies has been impeded by the paucity of self-blinking organic fluorophores that enable high spatiotemporal resolution and labeling/localization density at a moderate laser intensity. Herein, we report a self-blinking Si-rhodamine dye 6-HESiR with a suitably increased "ON" fraction and a fluorogenic self-blinking dsDNA probe 6-HoeHESiR as a powerful tool for 3D superresolution imaging of native chromatin in eukaryotes without the use of photoswitching buffer and high laser intensity. With the probe 6-HoeHESiR, 3D superresolution imaging of in vitro reconstituted nucleosomal arrays and chromatin fibers yielded results consistent with EM analysis. Similar euchromatin and heterochromatin structures were visualized in fixed and live cells with high spatiotemporal resolution and labeling density, providing the first live-cell evidence for a hierarchical model of chromatin organization. 3D imaging results obtained in the presence of selective inhibitors of histone deacetylases also corroborate chromatin fiber decompaction upon hyperacetylation of histones.

cell biology↗