Biology subjects

Shyy, J. Y.-J.

Publications and source records attributed to Shyy, J. Y.-J..

7 recordsLinked to original sources

bioRxiv2026

Label free multimodal optical imaging of metabolic heterogeneity in aging by integrating SRS, MPF, FLIM, and SHG

Cellular metabolism is governed by the coordinated organization of macromolecules, including lipids and proteins, together with redox-active cofactors such as NADH and FAD. However, resolving these biochemical features quantitatively and spatially at subcellular resolution remains challenging because no single imaging modality can capture molecular composition, redox state, and tissue architecture simultaneously without labeling. Here, we present MANIFEST (Multimodal Analysis of Nonlinear Imaging for Functional Endogenous Spatial correlaTion), a label-free imaging platform that integrates stimulated Raman scattering (SRS), second harmonic generation (SHG), multiphoton fluorescence (MPF), and fluorescence lifetime imaging microscopy (FLIM) with their spatial correlation. MANIFEST combines chemical imaging of lipids with autofluorescence- and lifetime-based quantification of NADH and FAD metabolism, enabling spatially resolved analysis of metabolic heterogeneity at organelle and tissue-compartment levels. We apply this framework to four distinct aging or disease models: amyloid-beta-treated tri-cultured brain cells, high-fat diet mouse liver, human non-ischemic cardiomyopathy tissue, and aging mouse retina. Across these systems, MANIFEST reveals disease-associated lipid remodeling, redox imbalance, disrupted metabolic zonation, collagen reorganization, and layer-specific metabolic changes. By integrating complementary nonlinear optical modalities into a single label-free platform, MANIFEST provides an extensible, proof-of-concept platform for high-resolution metabolic phenotyping in complex biological systems and offers new opportunities for studying disease mechanisms, aging biology, and metabolism-driven tissue pathology.

bioengineering↗

bioRxiv2025

ACE2 Phosphorylation Modulates Angiogenesis via the Activator Protein-1

BackgroundAngiogenesis 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. MethodsThe 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. ResultsACE2 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. ConclusionOur 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.

cell biology↗

bioRxiv2025

Chronic kidney disease exacerbates hyperlipidemia and atherosclerosis via miR-148a-3p targeting SIK1/AMPKα1 in liver

BackgroundCardiovascular complications are the leading cause of death in patients with chronic kidney disease (CKD). As a major risk factor for atherosclerotic cardiovascular disease (ASCVD), hyperlipidemia is a common sequela of CKD, which is linked to increased lipid synthesis, impaired lipoprotein clearance, and maladapted reverse cholesterol transport. Because exosomes can transport miRNAs to modulate intercellular and tissue communication, we hypothesized that exosomal miRNAs can act as endocrine-like molecules to modulate the CKD-associated hyperlipidemia and ASCVD. MethodsSmall RNA-seq and qPCR were used to determine the differential plasma level of exosomal miR-148a-3p in ASCVD patients with and without CKD. RNA-seq and in silico analysis were used to establish the regulation of lipid metabolism via miR-148a-3p/salt-inducible kinase 1 (SIK1)/AMP-activated protein kinase alpha 1 subunit (AMPK1) in the liver. Inhibition of miR-148a-3p or overexpression of SIK1/AMPK1 in ApoE-/- mice with 5/6 nephrectomy was used to elucidate the role of the miR-148a-3p/SIK1/AMPK1 axis in CKD-related hyperlipidemia and atherosclerosis. ResultsThe plasma level of exosomal miR-148a-3p was significantly elevated in patients with ASCVD and CKD (ASCVD/CKD) and ApoE-/- mice with 5/6 nephrectomy. Via circulating exosomes, kidney-generated miR-148a-3p was delivered to the liver, where it targeted SIK1/AMPK1 transcripts, thereby upregulating SREBP2/PCSK9 and reciprocally downregulating LDLR. Levels of SIK1 and AMPK1 were decreased in hepatocytes transfected with miR-148a-3p or treated with plasma exosomes from ASCVD/CKD patients, with attendant SREBP2 activation and attenuated LDL-C binding. The decreased LDL-C binding was rectified by SIK1/AMPK1 or LNA-miR-148a-3p overexpression. LNA-miR-148a-3p or AAV8-SIK1/AMPK administration in 5/6 nephrectomy/ApoE-/- mice significantly ameliorated hyperlipidemia and atherosclerosis. ConclusionIn ASCVD/CKD patients and nephrectomy/ApoE-/- mice, kidney-originating miR-148a-3p targeted hepatic SIK1/AMPK1 mRNA. This exosome-mediated endocrine effect upregulated SREBP2/PCSK9 and reciprocally reduced LDLR level, which elevated plasma LDL-C level and exacerbated ASCVD. These findings underscore a kidney-liver-artery axis involved in cardiovascular-kidney-metabolic syndrome.

pathology↗

bioRxiv2025

Silencing KRIT1 Partially Reverses the Effects of Disturbed Flow onThe Endothelial Cell Transcriptome

BackgroundEndothelial cells respond to forces generated by laminar blood flow with changes in vasodilation, anticoagulant, fibrinolytic, or anti-inflammatory functions which preserve vessel patency. These responses to flow sheer stress are primarily mediated by the modulation of transcription factors Kruppel-like factors 2 and 4 (KLF2 and KLF4). Notably, disturbed flow patterns, which are found in vascular areas predisposed to atherosclerosis, significantly reduce the endothelial expression of KLF2 and KLF4, resulting in changes in the transcriptome that exacerbate inflammation and thrombosis. The endothelial CCM complex, comprising KRIT1, CCM2, and CCM3, suppresses the expression of KLF2 and KLF4. Loss of function of the CCM complex has recently been suggested to protect from coronary atherosclerosis in humans. We thus hypothesized that silencing of KRIT1, the central scaffold of the CCM complex, can normalize the atherogenic effects of disturbed flow on the human endothelial transcriptome MethodsBulk RNA sequencing (RNA-seq) was conducted on human umbilical vein endothelial cells (HUVECs) after the expression of KRIT1 was silenced using specific siRNAs. The endothelial cells were exposed to three different conditions for 24 hours: pulsatile shear stress (laminar flow), oscillatory shear stress (disturbed flow), and static conditions (no flow). ResultsWe found that silencing KRIT1 expression in HUVECs restored the expression of the transcription factors KLF2 and KLF4 under oscillatory shear stress. This treatment resulted in a transcriptomic profile similar to that of endothelial cells under pulsatile shear stress. These findings suggest that inhibition of the CCM complex in endothelium plays a vasoprotective role by reactivating a protective gene program to help endothelial cells resist disturbed blood flow. ConclusionsTargeting CCM genes can activate well-known vasoprotective gene programs that enhance endothelial resilience to inflammation, hypoxia, and angiogenesis under disturbed flow conditions, providing a novel pathway for preventing atherosclerosis. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/642862v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@a20119org.highwire.dtl.DTLVardef@1f62f68org.highwire.dtl.DTLVardef@1a26d71org.highwire.dtl.DTLVardef@bf3c82_HPS_FORMAT_FIGEXP M_FIG C_FIG Endothelial cells respond to blood flow by expressing transcription factors such as Kruppel-Like Factors 2 and 4 (KLF2 and KLF4). Under oscillatory shear stress (OS) conditions, characterized by disturbed flow, KLF2 and KLF4 are suppressed in endothelial cells, which is linked to an atheroprone gene program that includes increased inflammation and risk of atherosclerosis (unhealthy endothelium). Our work shows that silencing of endothelial KRIT1 under OS increases expression of KLF2 and KLF4 mRNA levels as well as the expression of a protective gene program associated with resilience to inflammation, hypoxia, and angiogenesis, similar to those observed under pulsatile shear stress conditions (healthy endothelium).

pathology↗

bioRxiv2025

Obesity Increases Atherosclerosis Susceptibility via Inter-tissue miR-30e-SLC7A11 Axis

BackgroundWith obesity as a risk factor for atherosclerotic disease, recent research suggests that adipose tissue in obese animal models and humans can generate endocrine-like molecules that affect arterial health later on. Previous studies showed that microRNA-30e (miR-30e) level is elevated in atherosclerosis and solute carrier family 7 member 11 (SLC7A11), a cystine/glutamate transporter, is involved in atherogenesis. However, whether an endocrine-like link between the adipose-derived miR-30e-5p and SLC7A11 in the vascular endothelium can lead to obesity-caused atherosclerosis is unknown. MethodsMiRNA data mining and RT-PCR validations were used to demonstrate the positive association among serum level of miR-30e-5p, obesity, and coronary arterial disease in human patients. Transcriptomics (RNA-seq and single-nucleus RNA-seq), metabolomics, and in silico analysis were used to establish a miR-30e-5p-SLC7A11 regulation of central carbon metabolism, mitochondrial and endothelial cell (EC) function. Mouse models with EC-specific Slc7a11 knockout (EC-Slc7a11-/-) and gain or loss of function of miR-30e-5p were used to elucidate the detrimental role of this endocrine-like axis in obesity-related atherosclerosis. ResultsThe level of adipocyte-derived miR-30e-5p was significantly upregulated in obese humans with coronary artery disease and obese and atherosclerotic mice. Via serum exosomes, the adipocyte-generated miR-30e-5p targeted SLC7A11 mRNA in vascular ECs. SLC7A11 deficiency due to miR-30e-5p targeting dysregulated glutamate/cystine metabolism increased glycolysis, reduced oxidative phosphorylation, and impaired mitochondrial function. The EC dysfunction could be rectified by SLC7A11 overexpression or miR-30e-5p antagonism. Exogenously delivered miR-30e-5p phenocopied the increased atherosclerosis in EC-Slc7a11-/- mice. In contrast, miR-30e-5p antagomir treatment reduced atherosclerosis in Apoe-/- and ob/ob mice. ConclusionOur multi-omics approaches demonstrates that the adipose-derived miR-30e-5p downregulated SLC7A11 mRNA in ECs via tissue crosstalk. The resulting EC dysfunction led to obesity-related atherosclerosis in mice. These findings underscore a causality between obesity and atherosclerosis in the context of cardiovascular-kidney-metabolic syndrome.

molecular biology↗

bioRxiv2022

Epitranscriptomic Modification of MicroRNA Increases Atherosclerosis Susceptibility

Emerging evidence indicates that oxidative stress causes the hydroxylation of guanine (G) to generate 8-oxo-7,8-dihydro guanosine (8OH-G) in microRNAs (miRs), which induces the guanine-to-uracil (G-to-U) transversion and thus changes the miR targetomes. However, whether and how the 8OH-G-modified miRs are involved in vascular endothelial dysfunction and atherogenesis were unexplored. Using 8OH-G crosslinking immunoprecipitation miR sequencing (8OH-G CLIP-miR-seq), we found that 8OH-G miR-483 were among the most enriched 8OH-G miR species in ECs induced by ox-LDL. Transcriptomic profiling by RNA-seq indicated that the G-to-U transversion of miR-483 altered the original mRNA targeting efficacy and allows 8OH-G miR-483 to recognize new mRNA target sites. A reduced ratio of 8OH-G miR-483 to miR-483 in lung ECs was found in the endothelial-specific miR-483 transgenic (EC-miR-483 Tg) mice. Moreover, reduction of atherosclerosis was significant in EC-miR-483 Tg mice administrated AAV8-PCSK9 and fed an atherogenic diet. In situ miR hybridization revealed an increased 8OH-G miR-483 level in the intima of human atherosclerotic arteries. Collectively, this study demonstrates that the redox burden incurred by cardiovascular risk factors is a culprit of the miR-483 to 8OH-G miR-483 transversion. Such epitranscriptomic modification of miR-483 causes endothelial dysfunction and increases atherosclerosis susceptibility via its targetomes shift.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE2

Coronavirus disease 2019 (COVID-19) includes the cardiovascular complications in addition to respiratory disease. SARS-CoV-2 infection impairs endothelial function and induces vascular inflammation, leading to endotheliitis. SARS-CoV-2 infection relies on the binding of Spike glycoprotein (S protein) to angiotensin converting enzyme 2 (ACE2) in the host cells. We show here that S protein alone can damage vascular endothelial cells (ECs) in vitro and in vivo, manifested by impaired mitochondrial function, decreased ACE2 expression and eNOS activity, and increased glycolysis. The underlying mechanism involves S protein downregulation of AMPK and upregulation of MDM2, causing ACE2 destabilization. Thus, the S protein-exerted vascular endothelial damage via ACE2 downregulation overrides the decreased virus infectivity.

pathology↗