LCZ696 Possesses a Protective Effect Against Homocysteine (Hcy)-Induced Impairment of Blood-Brain Barrier (BBB) Integrity by Increasing Occludin, Mediated by the Inhibition of Egr-1.

Homocysteine (Hcy) is a non-essential amino acid produced from methionine. It has been reported that high concentrations of Hcy are related to the pathogenesis of neurodegenerative diseases and induce the disruption of the blood-brain barrier (BBB) by triggering oxidative stress and inflammation. LCZ696 is a novel antihypertensive agent that has been recently reported to possess promising anti-inflammatory properties. However, whether it has a protective effect on the BBB disruption is still unknown. For the first time, in this study, we aim to investigate whether LCZ696 exerts anti-inflammatory effects on Hcy-induced injury in brain endothelial cells and explore its neuroprotective properties. In in vivo experiments, we found that treatment with LCZ696 ameliorated oxidative stress by reducing malondialdehyde (MDA) and increasing glutathione (GSH). Furthermore, LCZ696 downregulated the excessive release of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) at mRNA and protein levels. Importantly, it reversed the disruption of the BBB induced by Hcy stimulation. In the in vitro human brain microvascular endothelial cell (HBMVEC) experiments, compared to the control, the permeability of the endothelial monolayer was significantly enlarged, the expression level of occludin declined, and Egr-1 upregulated by the introduction of Hcy, and these were all reversed by the treatment with LCZ696. Lastly, we found that the protective effects of LCZ696 against Hcy-induced reduction of occludin and hyper-permeability of the endothelial monolayer were greatly abolished by the overexpression of Egr-1. Taken together, we found that LCZ696 protected against Hcy-induced impairment of BBB integrity by increasing the expression of occludin, all mediated by the inhibition of Egr-1.

Azilsartan ameliorates ox-LDL-induced endothelial dysfunction via promoting the expression of KLF2.

BACKGROUND: Oxidized LDL(Ox-LDL) mediated endothelial dysfunction is involved in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Azilsartan is a potent agent for the treatment of hypertension as the antagonist of the angiotensin II receptor. This study will investigate whether Azilsartan possesses a beneficial effect against endothelial cell dysfunction induced by ox-LDL and explore the underlying preliminary mechanism. METHODS: Ox-LDL was applied to construct an in vitro endothelial dysfunction model in human umbilical vascular endothelial cells (HUVECs). The expression of lectin-type oxidized LDL receptor 1 (LOX-1), endothelial nitric oxide synthase (eNOS), tight junction protein occludin, and transcriptional factor Krüppel-like factor 2 (KLF2) was detected using qRT-PCR and Western blot. ELISA and qRT-PCR were utilized to evaluate the production of chemokine monocyte chemotactic protein 1 (MCP-1) and chemokine (C-X-C motif) Ligand 1 Protein (CXCL1) in treated HUVECs. The generation of nitro oxide (NO) was determined using DAF-FM DA staining assay. KLF2 was silenced by transfecting the cells with specific Small interfering RNA (siRNA). FITC-dextran permeation assay was used to check the endothelial monolayer permeability of treated HUVECs. RESULTS: Firstly, the elevated expressions of LOX-1, MCP-1, and CXCL-1 induced by stimulation with ox-LDL were significantly suppressed by Azilsartan. The downregulated eNOS and reduced production of NO induced by ox-LDL were reversed by the introduction of Azilsartan. Secondly, enlarged endothelial monolayer permeability and decreased expression of occludin stimulated with ox-LDL were greatly reversed by treatment with Azilsartan but were abolished by silencing the expression of KLF2. Lastly, the inhibited expression of KLF2 induced by ox-LDL was significantly elevated by the introduction of Azilsartan. CONCLUSION: Azilsartan might ameliorate ox-LDL-induced endothelial damage via elevating the expression of KLF2.

GPR120 Agonist GW9508 Ameliorated Cellular Senescence Induced by ox-LDL.

Introduction Oxidized low-density lipoprotein (ox-LDL)-induced endothelial senescence is involved in the pathogenesis of atherosclerosis and many cardiovascular diseases. G-protein-coupled receptor 120 (GPR120), a type of orphan G-protein-coupled receptors (GPRs), plays a vital role in mediating anti-inflammatory and insulin-sensitizing effects. The biological function of GPR120 in vascular endothelial cells is largely unknown. Methods The human aortic endothelial cells (HAECs) were treated with ox-LDL (100 µg/mL) in the presence or absence of GW9508 (50 µM) or AH9614 (1 µM) for 24 h. The LDH assay was used to determine cell death. The dihydroethidium (DHE) staining assay was used to measure intracellular levels of reactive oxidative species (ROS), and a senescence ß-galactosidase assay kit was used to determine endothelial senescence. Gene and protein expressions were measured using real-time polymerase chain reaction (PCR) and western blot analysis, respectively. Results Ox-LDL treatment decreased the expression of GPR120 by more than half in HAECs. Typically, 100 µg/mL of ox-LDL- induced 35.2% LDH release, which was reduced to 16.9% by 50 µM GW9508, the agonist of GPR120. Importantly, GW9508 relieved cytotoxicity and suppressed the ox-LDL-induced increase in the activity of senescence-associated ß-galactosidase (SA-ß-Gal) (from 3.3-fold to 1.6-fold of the control group) and the generation of cellular reactive oxidative species (ROS) (from 3.8-fold to 1.6-fold of the control group). Furthermore, we found that GW9508 ameliorated ox-LDL-induced endothelial cell cycle arrest at the G0/G1 phase and the expression of key senescence proteins, including p53 and plasminogen activator inhibitor-1(PAI-1). Mechanistically, we showed that GW9508 promoted ox-LDL-induced transcriptional factor NF-E2-related factor 2 (NRF2) (increase by 47.3%) translocation into the nucleus. The effect of GW9508 is dependent on its receptor GPR120, the blockage of which by its specific antagonist, AH7614, abolished the antisenescence effect of GW9508. Conclusion Collectively, this study revealed the protective effect of GPR120 activation in vascular endothelial cells, implying that GPR120 is a promising therapeutic target for the treatment of cardiovascular diseases.

Upconversion Nanoparticles-Based Multiplex Protein Activation to Neuron Ablation for Locomotion Regulation.

The optogenetic neuron ablation approach enables noninvasive remote decoding of specific neuron function within a complex living organism in high spatiotemporal resolution. However, it suffers from shallow tissue penetration of visible light with low ablation efficiency. This study reports a upconversion nanoparticle (UCNP)-based multiplex proteins activation tool to ablate deep-tissue neurons for locomotion modulation. By optimizing the dopant contents and nanoarchitecure, over 300-fold enhancement of blue (450-470 nm) and red (590-610 nm) emissions from UCNPs is achieved upon 808 nm irradiation. Such emissions simultaneously activate mini singlet oxygen generator and Chrimson, leading to boosted near infrared (NIR) light-induced neuronal ablation efficiency due to the synergism between singlet oxygen generation and intracellular Ca2+ elevation. The loss of neurons severely inhibits reverse locomotion, revealing the instructive role of neurons in controlling motor activity. The deep penetrance NIR light makes the current system feasible for in vivo deep-tissue neuron elimination. The results not only provide a rapidly adoptable platform to efficient photoablate single- and multiple-cells, but also define the neural circuits underlying behavior, with potential for development of remote therapy in diseases.

An Upconversion Nanoparticle Enables Near Infrared-Optogenetic Manipulation of the Caenorhabditis elegans Motor Circuit.

Near-infrared (NIR) light penetrates tissue deeply, but its application to motor behavior stimulation has been limited by the lack of known genetic NIR light-responsive sensors. We designed and synthesized a Yb3+/Er3+/Ca2+-based lanthanide-doped upconversion nanoparticle (UCNP) that effectively converts 808 nm NIR light to green light emission. This UCNP is compatible with Chrimson, a cation channel activated by green light; as such, it can be used in the optogenetic manipulation of the motor behaviors of Caenorhabditis elegans. We show that this UCNP effectively activates Chrimson-expressing, inhibitory GABAergic motor neurons, leading to reduced action potential firing in the body wall muscle and resulting in locomotion inhibition. The UCNP also activates the excitatory glutamatergic DVC interneuron, leading to potentiated muscle action potential bursts and active reversal locomotion. Moreover, this UCNP exhibits negligible toxicity in neural development, growth, and reproduction, and the NIR energy required to elicit these behavioral and physiological responses does not activate the animal's temperature response. This study shows that UCNP provides a useful integrated optogenetic toolset, which may have wide applications in other experimental systems.