Vascular smooth muscle-specific LRRC8A knockout ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting the WNK1/FOXO3a/MMP signaling pathway.

Hypertensive cerebrovascular remodeling involves the enlargement of vascular smooth muscle cells (VSMCs), which activates volume-regulated Cl- channels (VRCCs). The leucine-rich repeat-containing family 8 A (LRRC8A) has been shown to be the molecular identity of VRCCs. However, its role in vascular remodeling during hypertension is unclear. In this study, we used vascular smooth muscle-specific LRRC8A knockout (CKO) mice and an angiotensin II (Ang II)-induced hypertension model. The results showed that cerebrovascular remodeling during hypertension was ameliorated in CKO mice, and extracellular matrix (ECM) deposition was reduced. Based on the RNA-sequencing analysis of aortic tissues, the level of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-14, were reduced in CKO mice with hypertension, which was further verified in vivo by qPCR and immunofluorescence analysis. Knockdown of LRRC8A in VSMCs inhibited the Ang II-induced upregulation of collagen I, fibronectin, and matrix metalloproteinases (MMPs), and overexpression of LRRC8A had the opposite effect. Further experiments revealed an interaction between with-no-lysine (K)-1 (WNK1), which is a "Cl--sensitive kinase", and Forkhead transcription factor O3a (FOXO3a), which is a transcription factor that regulates MMP expression. Ang II induced the phosphorylation of WNK1 and downstream FOXO3a, which then increased the expression of MMP-2 and MMP-9. This process was inhibited or potentiated when LRRC8A was knocked down or overexpressed, respectively. Overall, these results demonstrate that LRRC8A knockout in vascular smooth muscle protects against cerebrovascular remodeling during hypertension by reducing ECM deposition and inhibiting the WNK1/FOXO3a/MMP signaling pathway, demonstrating that LRRC8A is a potential therapeutic target for vascular remodeling-associated diseases such as stroke.

Two- and three-dimensional QSAR studies on hURAT1 inhibitors with flexible linkers: topomer CoMFA and HQSAR.

hURAT1 (human urate transporter 1) is a successful target for hyperuricemia. Recently, the modification work on hURAT1 inhibitors showed that the flexible linkers would benefit biological activity. The study aimed to investigate the contribution of the linkers and give modification strategies on this kind of structures based on QSAR models (HQSAR and topomer CoMFA). The most effective HQSAR and topomer CoMFA models were generated by applying the training set containing 63 compounds, with the cross-validated q2 values of 0.869/0.818 and the non-cross-validated correlation coefficients r2 of 0.951/0.978, respectively. The Y-randomization test was applied to ensure the robustness of the models. The external predictive correlation coefficient (rpred2) grounded on the external test set (21 compounds) of two models was 0.910 and 0.907, respectively. In addition, the models were validated by Golbraikh-Tropsha and Roy methods, as well as other statistical metrics. The results showed that both models were reliable. Topomer CoMFA steric/electrostatic contours and HQSAR atomic contribution maps illustrated the structural features which governed their inhibitory potency. The dependable results could provide important insights to guide the designing of more potential hURAT1 inhibitors.

Low Chloride-Regulated ClC-5 Contributes to Arterial Smooth Muscle Cell Proliferation and Cerebrovascular Remodeling.

BACKGROUND: Low serum chloride (Cl-) level is considered an independent predictor of cardiovascular mortality associated with chronic hypertension. However, the underlying mechanisms are unknown. ClC-5, a member of the Cl- channel family, is sensitive to changes in intracellular and extracellular Cl- concentration and conducts outwardly rectifying Cl- currents. The aims of this study were to determine if ClC-5 is regulated by low extracellular Cl-, clarify its putative roles in hypertension-induced cerebrovascular remodeling, and elucidate the associated underlying mechanisms. METHODS: Whole-cell patch technique, intracellular Cl- concentration measurements, flow cytometry, Western blot, Clcn5 knockdown (Clcn5-/y), and adenovirus-mediated ClC-5 overexpression mice, 2-kidney, 2-clip, and angiotensin II infusion-induced hypertensive models were used. RESULTS: We found that low extracellular Cl- evoked a ClC-5-dependent Cl- current that was abolished by ClC-5 depletion in basilar artery smooth muscle cells (BASMCs). ClC-5 was upregulated in the arterial tissues of rats and patients with hypertension. Low Cl--induced current and ClC-5 protein expression positively correlated with basilar artery remodeling during hypertension. ClC-5 knockdown ameliorated hypertension-induced cerebrovascular remodeling and smooth muscle cell proliferation, whereas ClC-5 overexpression mice exhibited the opposite phenotype. ClC-5-dependent Cl- efflux induced by low extracellular Cl- activated WNK1 (lysine-deficient protein kinase 1) which, in turn, activated AKT (protein kinase B), and culminated in BASMC proliferation and vascular remodeling. CONCLUSIONS: ClC-5 mediates low extracellular Cl-induced Cl- currents in BASMCs and regulates hypertension-induced cerebrovascular remodeling by promoting BASMC proliferation via the WNK1/AKT signaling pathway.

SGK1 mediates hypotonic challenge-induced proliferation in basilar artery smooth muscle cells via promoting CREB signaling pathway.

Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl- concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G0/G1 phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl- channel to further verify the role of SGK1 in low Cl- environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl--sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.

Fluorescent film sensors based on SAMs of pyrene derivatives for detecting nitroaromatics in aqueous solutions.

The detection of nitroaromatics in aqueous solutions by a novel pyrene-functionalized film has been investigated in the present study. The pyrene moieties were attached on the glass surface via a long flexible spacer based on self-assembled monolayer technique. Steady-state fluorescence measurements revealed that these surface-attached pyrene moieties exhibited both monomer and excimer emission. Nitroaromatics such as 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,4,6-trinitrophenol (picric acid) were found to efficiently quench the fluorescence emission of this film. The quenching results demonstrated that the excimer emission of these surface-confined pyrene moieties is more sensitive to the presence of nitroaromatics than the monomer emission. The quenching mechanism was examined through fluorescence lifetime measurement and it revealed that the quenching is static in nature and may be caused by electron transfer from the polycyclic aromatics to the nitroaromatics. Furthermore, the response of the film to nitroaromatics is fast and reversible, and the obtained film shows promising potentials in detecting explosives in aqueous environment.

Fluorescent film sensor for copper ion based on an assembled monolayer of pyrene moieties.

We previously reported the construction of a family of fluorescent film sensors for organic copper salts by covalently coupling polycyclic aromatic hydrocarbons on epoxy-terminated self-assembled monolayers on glass plate surfaces. Here we investigate the sensing properties and mechanism of covalently coupling pyrene on a glass plate surface via a long flexible "Y" type spacer. X-ray photoelectron spectroscopy (XPS) and fluorescence spectra measurements demonstrate the covalent attachment of pyrene in our adlayer. Compared with those results obtained in the previous studies, this new film sensor did not show highly selectivity for organic copper salts, which can be attributed to the introduction of sulfonyl groups connecting the pyrene moieties and the spacers. The presence of sulfonyl units made the microenvironments of pyrene relatively hydrophilic and thus showed less screening effect for inorganic ions. The specificity and reversibility of the film sensor toward Cu (II) made it attractive for sensing applications.

Probing the microenvironment of surface-attached pyrene formed by a thermo-responsive oligomer.

We have constructed a novel molecular assembly attached to quartz, oxidized silicon and indium-doped tin oxide coated substrates, where a tethered pyrene derivative is co-immobilized with oligo-N-isopropyl acrylamide (oligo-NIPAM). The addition of tethered oligo-NIPAM to the adlayer creates two different, temperature-dependent microenvironments for the surface-bound pyrene. X-ray photoelectron spectroscopy (XPS) and ellipsometry measurements demonstrate the covalent attachment of both oligo-NIPAM and pyrene in our adlayers. Contact angle results confirm the thermo-responsive nature of the oligo-NIPAM on the substrate surface. Steady-state fluorescence data show that the presence of oligo-NIPAM moieties reduces the extent of pyrene excimer formation and provides different environments for the chromophore at temperatures above and below the phase transition. Fluorescence lifetime decay data on surface-bound pyrene are biexponential, consistent with multiple local environments, regardless of whether tethered oligo-NIPAM is present or not. Quenching studies reveal that we can manipulate the sensing properties of this new film simply by adjusting the conformations of oligo-NIPAM.

Surface-confined energy transfer in mixed self-assembled monolayers.

We have fabricated a set of self-assembled monolayers consisting of naphthalene and dansyl derivatives in a range of surface loading ratios for the purpose of examining excitation transport in mixed self-assembled monolayer systems. Both tethered chromophores were immobilized on an epoxide-terminated adlayer on silica via an identical spacer, where the linking chemistry produced an amide linkage. X-ray photoelectron spectroscopy (XPS), ellipsometry, and contact angle measurements were used to characterize these chromophore-containing layers. The excitation transfer behavior of these monolayers has been examined using steady-state and time-resolved fluorescence spectroscopy. Steady-state fluorescence measurements show that excitation transfer from the naphthalene to dansyl chromophores occurs, with the efficiency of excitation transport scaling with chromophore surface loading densities, as expected. The donor lifetimes decrease with increasing acceptor loading density, and the functional form of the acceptor decay was independent of the donor/acceptor ratio. Our findings are not consistent with a homogeneous adlayer, but do provide information on the structural heterogeneity that is characteristic of these interfaces.

Fluorescent sensors for nitroaromatic compounds based on monolayer assembly of polycyclic aromatics.

A class of fluorescent films in which pyrene was assembled, in a monolayer manner, on glass slide surfaces via various flexible spacers of different lengths and substructures was used for the detection of nitroaromatic compounds (NACs) in vapor phase. This design strategy offers several advantages for thin film fluorescent sensory materials. These advantages have been demonstrated experimentally by the sensitive response of the films to the presence of trace amounts of NACs in vapor phase. The fluorescence quenching of the films upon exposure to NACs vapors depends on several factors, including the evaporate rate of the NAC detected, the length of the spacers connecting the sensing element and the substrate surface, and the density of the sensing element on the substrate surface. Further experimentation showed that the sensing process is reversible and free of commonly encountered interference. The sensitive response, reversibility of the sensing process, and freedom from commonly encountered interference of the specially designed films to NACs qualify these materials as promising NACs fluorescent sensory materials.

Spacer layer screening effect: a novel fluorescent film sensor for organic copper(II) salts.

A fluorescent film sensor was prepared by chemical assembly of pyrene on a glass plate surface via a long flexible spacer. It was found that the film is highly selective for some organic Cu2+ salts, such as copper acetate and copper propionate. The presence of inorganic Cu2+ salts and other metal(II) acetates, including Ni2+, Co2+, Pb2+, Cd2+, Zn2+, etc., had little effect upon the sensing behavior of the film for copper acetate or copper propionate. The observation was explained by employing a proposed "two-dimensional solution" model. The quenching by copper acetate of the emission of the film is static in nature due to complexation of the spacers to the metal ions. Furthermore, the response of the film sensor to copper acetate is fully reversible. To the best of our knowledge, this film sensor may be the first one that can differentiate greasy copper salts from inorganic copper salts.