SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

Astrocyte Signaling in the Neurovascular Unit After Central Nervous System Injury.

Astrocytes comprise the major non-neuronal cell population in the mammalian neurovascular unit. Traditionally, astrocytes are known to play broad roles in central nervous system (CNS) homeostasis, including the management of extracellular ion balance and pH, regulation of neurotransmission, and control of cerebral blood flow and metabolism. After CNS injury, cell⁻cell signaling between neuronal, glial, and vascular cells contribute to repair and recovery in the neurovascular unit. In this mini-review, we propose the idea that astrocytes play a central role in organizing these signals. During CNS recovery, reactive astrocytes communicate with almost all CNS cells and peripheral progenitors, resulting in the promotion of neurogenesis and angiogenesis, regulation of inflammatory response, and modulation of stem/progenitor response. Reciprocally, changes in neurons and vascular components of the remodeling brain should also influence astrocyte signaling. Therefore, understanding the complex and interdependent signaling pathways of reactive astrocytes after CNS injury may reveal fundamental mechanisms and targets for re-integrating the neurovascular unit and augmenting brain recovery.

HDAC3 inhibition prevents oxygen glucose deprivation/reoxygenation-induced transendothelial permeability by elevating PPARγ activity in vitro.

Histone deacetylase 3 (HDAC3), a member of class I HDAC, regulates a wide variety of normal and abnormal physiological functions. Recent experimental studies suggested that inhibition of HDAC3 may increase acetylation of certain key signaling regulating proteins such as peroxisome proliferator-activated receptor γ (PPARγ), which plays a crucial role in modulating cerebrovascular function and integrity. However, the role of HDAC3 inhibition in cerebrovascular endothelium function under pathological condition has not been fully investigated. In this study, we tested the hypothesis that inhibition of HDAC3 by RGFP966, a highly selective HDAC3 inhibitor, promotes PPARγ activation by enhancing its protein acetylation, resulting in protection of oxygen glucose deprivation and reoxygenation (OGD/R)-induced increase of transendothelial cell permeability. In cultured primary human brain microvascular endothelial cells, our experimental results show that OGD/R increases transendothelial cell permeability and down-regulates junction protein expression. While we also detected HDAC3 activity increase and PPARγ activity decline after OGD/R. However, treatment with RGFP966 significantly attenuated the OGD/R-induced increase of transendothelial cell permeability and down-regulation of tight junction protein Claudin-5. These effects were observed to be dependent on HDAC3 activity inhibition-mediated PPARγ protein acetylation/activation. Lastly, HDAC3 small interfering RNA mimics the protective effects of RGFP966 on human brain microvascular endothelial cells. Taken together, our data indicate that HDAC3 inhibition might comprise a new therapeutic target for reducing blood-brain barrier integrity disruption and vascular dysfunctions in neurological disorders.

Annexin A2 is a Robo4 ligand that modulates ARF6 activation-associated cerebral trans-endothelial permeability.

Blood-brain barrier (BBB) disruption in neurological disorders remains an intractable problem with limited therapeutic options. Here, we investigate whether the endothelial cell membrane protein annexin A2 (ANXA2) may play a role in reducing trans-endothelial permeability and maintaining cerebrovascular integrity after injury. Compared with wild-type mice, the expression of cerebral endothelial junctional proteins was reduced in E15.5 and adult ANXA2 knockout mice, along with increased leakage of small molecule tracers. In human brain endothelial cells that were damaged by hypoxia plus IL-1ß, treatment with recombinant ANXA2 (rA2) rescued the expression of junctional proteins and decreased trans-endothelial permeability. These protective effects were mediated in part by interactions with F-actin and VE-cadherin, and the ability of rA2 to modulate signaling via the roundabout guidance receptor 4 (Robo4)-paxillin-ADP-ribosylation factor 6 (ARF6) pathway. Taken together, these observations suggest that ANXA2 may be associated with the maintenance of endothelial tightness after cerebrovascular injury. ANXA2-mediated pathways should be further explored as potential therapeutic targets for protecting the BBB in neurological disorders.