Biology subjects

Sudhahar, V.

Publications and source records attributed to Sudhahar, V..

5 recordsLinked to original sources

bioRxiv2026

Myeloid DRP1 Sulfenylation Drives Reparative Macrophage Polarization and Neovascularization in Ischemic Muscle

Reparative macrophage polarization and macrophage-derived reactive oxygen species (ROS) are required for ischemia-induced revascularization in peripheral artery disease (PAD). Our previous study showed that mitochondrial fission protein DRP1 promotes reparative polarization and metabolic reprogramming in macrophages and post-ischemic neovascularization. However, the redox-dependent mechanism governing DRP1 activation in this context remains elusive. Here, using a mouse hindlimb ischemia (HLI) model of PAD, we identify cysteine sulfenylation (CysOH) of DRP1 as a critical redox modification induced in ischemic bone marrow (BM)-derived cells. BM chimeric mice reconstituted with CRISPR/Cas9-generated "redox-dead" DRP1-C631A knock-in mutant (Drp1C/A) BM exhibited markedly reduced limb perfusion recovery and CD31 capillary density in ischemic muscles following HLI. These defects were associated with enhanced Ly6G neutrophil accumulation, pro-inflammatory F4/80CD80 M1 macrophages and reduced anti-inflammatory F4/80CD206 M2 macrophages in ischemic muscle. Mechanistically, using an in vitro PAD model, hypoxia-serum starvation (HSS) rapidly induced cytosolic ROS production and DRP1-CysOH formation in wild type macrophages. In contrast, Drp1C/A macrophages failed to undergo DRP1-CysOH-dependent mitochondrial fission under HSS, resulting in aberrant metabolic reprogramming characterized by enhanced glycolysis and mitochondrial ROS, pro-inflammatory p-NF-{kappa}B and M1-genes, and suppressed anti-inflammatory p-AMPK and M2-genes. Thus, our findings establish DRP1 sulfenylation as a previously unrecognized redox-sensing mechanism that links ischemia-induced ROS to reparative macrophage reprogramming and revascularization, identifying a novel therapeutic target for PAD.

cell biology↗

bioRxiv2025

Novel Role of Copper Transporter CTR1 and Therapeutic Potential of Copper Chelators in Retinal Ischemia-Reperfusion Injury

BackgroundRetinal ischemia contributes to vision loss in ischemic and diabetic retinopathies through oxidative stress, neurovascular injury, and inflammation. Copper (Cu), while essential, can be toxic in excess and is regulated by Cu transporters such as CTR1. However, the role of CTR1 in ischemic retinopathy remains unclear. Methods and ResultsRetinal ischemia-reperfusion (IR) injury was induced by elevating intraocular pressure to 110 mmHg for 40 minutes in the right eye of Ctr1 heterozygous (Ctr1/-) and wild-type (WT) mice. In WT mice, IR triggered rapid CTR1 upregulation and increased retinal Cu levels (measured by ICP-MS). IR injury caused retinal ganglion cell loss, inner retinal thinning, vascular degeneration, and apoptosis, all of which were significantly attenuated in Ctr1/- mice. Ctr1/- mice also exhibited reduced microglial (Iba1) and glial cells (GFAP) activation and preserved visual function, as assessed by electroretinography. Mechanistically, IR-induced reactive oxygen species (O2-) production (DHE staining), upregulation of NADPH oxidase components (NOX2, p47phox), and NF-{kappa}B activation were markedly suppressed in Ctr1/- mice. Treatment with the Cu chelator tetrathiomolybdate (TTM) similarly reduced retinal thinning, neurovascular damage, apoptosis, gliosis, and oxidative stress after IR injury. ConclusionsCTR1 plays a central role in mediating Cu-dependent oxidative stress, neurovascular degeneration, and inflammation following retinal IR injury. Targeting the CTR1- Cu axis may represent a novel therapeutic strategy for ischemic retinopathy.

cell biology↗

bioRxiv2025

Endothelial Cu Uptake Transporter CTR1 Senses Disturbed Flow to Promote Atherosclerosis through Cuproptosis

Endothelial cells (ECs) lining blood vessels sense disturbed blood flow (D-flow), which drives mitochondrial dysfunction and atherosclerosis. Copper (Cu) is an essential micronutrient, and its disruption of homeostasis has been implicated in atherosclerosis. Cellular Cu levels are tightly controlled by Cu transport proteins including the Cu importer CTR1. Cuproptosis is a recently discovered form of regulated cell death triggered by mitochondrial Cu accumulation, but its endogenous stimulants and role in atherosclerosis remain unknown. Using EC-specific CTR1-deficient mice and cultured ECs, we show that endothelial CTR1 responds to D-flow by increasing mitochondrial Cu levels through its interaction with the mitochondrial Cu transporter SLC25A3 at caveolae/lipid rafts. This leads to the aggregation of lipoylated mitochondrial proteins, mitochondrial dysfunction, and cuproptosis, thereby exacerbating atherosclerosis. Importantly, mitochondria-targeted Cu-chelating nanoparticles effectively mitigate D-flow-induced cuproptosis and atherosclerosis, highlighting the endothelial CTR1-SLC25A3-mitochondrial Cu axis as a potential therapeutic target.

cell biology↗

bioRxiv2024

Endothelial Drp1 Couples VEGF-induced Redox Signaling with Glycolysis Through Cysteine Oxidation to Drive Angiogenesis

Angiogenesis plays a vital role for postnatal development and tissue repair following ischemia. Reactive oxygen species (ROS) generated by NADPH oxidases (NOXes) and mitochondria act as signaling molecules that promote angiogenesis in endothelial cells (ECs) which mainly relies on aerobic glycolysis for ATP production. However, the connections linking redox signaling with glycolysis are not well understood. The GTPase Drp1 is a member of the dynamin superfamily that moves from cytosol to mitochondria through posttranslational modifications to induce mitochondrial fission. The role of Drp1 in ROS-dependent VEGF signaling and angiogenesis in ECs has not been previously described. Here, we identify an unexpected function of endothelial Drp1 as a redox sensor, transmitting VEGF-induced H2O2 signals to enhance glycolysis and angiogenesis. Loss of Drp1 expression in ECs inhibited VEGF-induced angiogenic responses. Mechanistically, VEGF rapidly induced the NOX4-dependent sulfenylation (CysOH) of Drp1 on Cys644, promoting disulfide bond formation with the metabolic kinase AMPK and subsequent sulfenylation of AMPK at Cys299/304 via the mitochondrial fission-mitoROS axis. This cysteine oxidation of AMPK, in turn, enhanced glycolysis and angiogenesis. In vivo, mice with EC-specific Drp1 deficiency or CRISPR/Cas9-engineered "redox-dead" (Cys to Ala) Drp1 knock-in mutations exhibited impaired retinal angiogenesis and post-ischemic neovascularization. Our findings uncover a novel role for endothelial Drp1 in linking VEGF-induced mitochondrial redox signaling to glycolysis through a cysteine oxidation-mediated Drp1-AMPK redox relay, driving both developmental and reparative angiogenesis.

cell biology↗

bioRxiv2023

Myeloid Drp1 Deficiency Limits Revascularization in Ischemic Muscles via Inflammatory Macrophage Polarization and Metabolic Reprograming

In the preclinical model of peripheral arterial disease (PAD), M2-like anti-inflammatory macrophage polarization and angiogenesis are required for revascularization. The regulation of cell metabolism and inflammation in macrophages is tightly linked to mitochondrial dynamics. Drp1, a mitochondrial fission protein, has shown context-dependent macrophage phenotypes with both pro- and anti-inflammatory characteristics. However, the role of macrophage Drp1 in reparative neovascularization remains unexplored. Here we show that Drp1 expression was significantly increased in F4/80+ macrophages within ischemic muscle at day 3 after hindlimb ischemia (HLI), an animal model of PAD. Myeloid-specific Drp1-/- mice exhibited reduced limb perfusion recovery, angiogenesis and muscle regeneration after HLI. These effects were associated with an increase in pro-inflammatory M1-like macrophages, p-NFkB and TNF, and reduced anti-inflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1-/- mice. In vitro, Drp1-/- macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction and excessive mitochondrial ROS, resulting in increased M1-gene and reduced M2-gene expression. Conditioned media from HSS-treated Drp1-/- macrophages exhibited increased secretion of pro-inflammatory cytokines and suppressed angiogenic responses in cultured endothelial cells. Thus, Drp1 deficiency in macrophages under ischemia drives inflammatory metabolic reprogramming and macrophage polarization, thereby limiting revascularization in experimental PAD.

cell biology↗