Muscone and (+)-Borneol Cooperatively Strengthen CREB Induction of Claudin 5 in IL-1ß-Induced Endothelium Injury.

Claudin 5 is one of the major proteins of tight junctions and is responsible for cerebrovascular integrity and BBB function. Muscone and (+)-borneol is the major ingredient of moschus and borneolum, respectively, with antioxidative and anti-inflammatory activities. This study investigated whether muscone and (+)-borneol combination protected claudin 5 by targeting ROS-mediated IL-1ß accumulation. Muscone and (+)-borneol reduced cerebral infarct volume and cerebrovascular leakage with claudin 5 protection in mice after stroke, largely due to inhibiting ROS accumulation and inflammatory infiltrate of microglia. Muscone reduced ROS and then blocked the CaN/Erk1/2 pathway to decrease IL-1ß release, while (+)-borneol removed mitochondrial ROS and attenuated the SDH/Hif-1α pathway to inhibit IL-1ß transcription, thereby jointly reducing IL-1ß production. Accumulated IL-1ß disrupted cAMP/CREB activation and attenuated transcriptional regulation of claudin 5. Muscone and (+)-borneol combination cooperatively protected BBB function by blocking IL-1ß-mediated cAMP/CREB/claudin 5 cascades. Mutation of Ser133 site of CREB or knockdown of claudin 5 weakened the effects of muscone and (+)-borneol on upregulation of TEER value and downregulation of FITC-dextran permeability, suggesting that targeting CREB/claudin 5 was an important strategy to protect vascular integrity. This study provided ideas for the studies of synergistic protection against ischemic brain injury about the active ingredients of traditional Chinese medicines (TCMs).

Hydroxysafflor Yellow A Blocks HIF-1α Induction of NOX2 and Protects ZO-1 Protein in Cerebral Microvascular Endothelium.

Zonula occludens-1 (ZO-1) is a tight junction protein in the cerebrovascular endothelium, responsible for blood-brain barrier function. Hydroxysafflor yellow A (HSYA) is a major ingredient of safflower (Carthamus tinctorius L.) with antioxidative activity. This study investigated whether HSYA protected ZO-1 by targeting ROS-generating NADPH oxidases (NOXs). HSYA administration reduced cerebral vascular leakage with ZO-1 protection in mice after photothrombotic stroke, largely due to suppression of ROS-associated inflammation. In LPS-stimulated brain microvascular endothelial cells, HSYA increased the ratio of NAD+/NADH to restore Sirt1 induction, which bound to Von Hippel-Lindau to promote HIF-1αdegradation. NOX2 was the predominant isoform of NOXs in endothelial cells and HIF-1α transcriptionally upregulated p47phox and Nox2 subunits for the assembly of the NOX2 complex, but the signaling cascades were blocked by HSYA via HIF-1α inactivation. When oxidate stress impaired ZO-1 protein, HSYA attenuated carbonyl modification and prevented ZO-1 protein from 20S proteasomal degradation, eventually protecting endothelial integrity. In microvascular ZO-1 deficient mice, we further confirmed that HSYA protected cerebrovascular integrity and attenuated ischemic injury in a manner that was dependent on ZO-1 protection. HSYA blocked HIF-1α/NOX2 signaling cascades to protect ZO-1 stability, suggestive of a potential therapeutic strategy against ischemic brain injury.

Latent HIV-Exosomes Induce Mitochondrial Hyperfusion Due to Loss of Phosphorylated Dynamin-Related Protein 1 in Brain Endothelium.

Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders. To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of primary human brain microvascular endothelial cells (HBMVECs) by inducing mitochondrial hyperfusion, and reducing the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS). The quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. It was further supported by the detection of exosomal markers, and the absence of large EV-related protein in the isolated EVs. The exosomes were readily taken up by primary HBMVECs. HIV-exosomes induce cellular and mitochondrial superoxide production but reduce mitochondrial membrane potential in HBMVECs. HIV-exosomes increase mitochondrial hyperfusion, possibly due to loss of phosphorylated dynamin-related protein 1 (p-DRP1). HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein reduce the expression of p-DRP1 and p-eNOS, and accelerate brain endothelial dysfunction. Finally, exosomes isolated from HIV-1 infected primary human peripheral blood mononuclear cells (hPBMCs) produce more exosomes than uninfected controls and reduce both p-DRP1 and p-eNOS expressions in primary HBMVECs. Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium.

Clostridium perfringens type D epsilon toxin produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro.

Clostridium perfringens type D epsilon toxin (ETX) is responsible for a severe and frequently fatal neurologic disorder in ruminant livestock. Light microscopic, immunohistochemical, and ultrastructural studies have suggested that ETX injury to the cerebral microvasculature, with subsequent severe, generalized vasogenic edema and increased intracranial pressure, is critically important in producing neurologic dysfunction. However, the effect of ETX on brain capillary endothelial cells in vitro has not been examined previously, to our knowledge. We exposed a well-characterized human blood-brain barrier cell line to increasing concentrations of ETX, and demonstrated a direct and dose-dependent endotheliotoxic effect. Our findings are concordant with the primacy of vasculocentric brain lesions in the diagnosis of acute epsilon toxin enterotoxemia in ruminant livestock.