CLEC-2-dependent platelet subendothelial accumulation by flow disturbance contributes to atherogenesis in mice.

Rationale: Platelets play an essential role in atherosclerosis, but the underlying mechanisms remain to be addressed. This study is to investigate the role of platelets in d-flow induced vascular inflammation and the underlying mechanism. Methods: We established a disturbed blood flow (d-flow) model by partial carotid ligation (PCL) surgery using atherosclerosis-susceptible mice and wild-type mice to observe the d-flow induced platelet accumulation in the subendothelium or in the plaque by immunostaining or transmission electron microscopy. The mechanism of platelet subendothelial accumulation was further explored by specific gene knockout mice. Results: We observed presence of platelets in atherosclerotic plaques either in the atheroprone area of aortic arch or in carotid artery with d-flow using Ldlr-/- or ApoE-/- mice on high fat diet. Immunostaining showed the subendothelial accumulation of circulating platelets by d-flow in vivo. Transmission electron microscopy demonstrated the accumulation of platelets associated with monocytes in the subendothelial spaces. The subendothelial accumulation of platelet-monocyte/macrophage aggregates reached peak values at 2 days after PCL. In examining the molecules that may mediate the platelet entry, we found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) reduced the subendothelial accumulation of platelets and monocytes/macrophages by d-flow, and ameliorated plaque formation in Ldlr-/- mice on high fat diet. Supportively, CLEC-2 deficient platelets diminished their promoting effect on the migration of mouse monocyte/macrophage cell line RAW264.7. Moreover, monocyte podoplanin (PDPN), the only ligand of CLEC-2, was upregulated by d-flow, and the myeloid-specific PDPN deletion mitigated the subendothelial accumulation of platelets and monocytes/macrophages. Conclusions: Our results reveal a new CLEC-2-dependent platelet subendothelial accumulation in response to d-flow to regulate vascular inflammation.

Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow.

Disturbed blood flow (d-flow) has been known to induce changes of the cells in the arterial wall, increasing the risk of atherosclerosis. However, the heterogeneity of the vascular cell populations under d-flow remains less understood. To generate d-flow in vivo, partial carotid artery ligation (PCL) was performed. Seven days after ligation, single-cell RNA sequencing of nine left carotid arteries (LCA) from the PCL group (10,262 cells) or control group (14,580 cells) was applied and a single-cell atlas of gene expression was constructed. The integrated analysis identified 15 distinct carotid cell clusters, including 10 d-flow-relevant subpopulations. Among endothelial cells, at least four subpopulations were identified, including Klk8hi ECs, Lrp1hi ECs, Dkk2hi ECs, and Cd36hi ECs. Analysis of GSVA and single-cell trajectories indicated that the previously undescribed Dkk2hi ECs subpopulation was mechanosensitive and potentially transformed from Klk8hi ECs under d-flow. D-flow-induced Spp1hi VSMCs subpopulation that appeared to be endowed with osteoblast differentiation, suggesting a role in arterial stiffness. Among the infiltrating cell subpopulations, Trem2hi Mφ, Birc5hi Mφ, DCs, CD4+ T cells, CXCR6+ T cells, NK cells, and granulocytes were identified under d-flow. Of note, the novel Birc5hi Mφ was identified as a potential contributor to the accumulation of macrophages in atherosclerosis. Finally, Dkk2hi ECs, and Cd36hi ECs were also found in the proatherosclerotic area of the aorta where the d-flow occurs. In conclusion, we presented a comprehensive single-cell atlas of all cells in the carotid artery under d-flow, identified previously unrecognized cell subpopulations and their gene expression signatures, and suggested their specialized functions.

Acid-sensing ion channel 1 (ASIC1) mediates weak acid-induced migration of human malignant glioma cells.

Glioblastoma is the most aggressive and lethal tumor in the central nervous system in adult and has poor prognosis due to strong proliferation and aggressive invasion capacity. Acidic microenvironment is commonly observed in tumor tissues but the exact role of acidosis in the pathophysiology of glioblastoma and underlying mechanisms remain unclear. Acid-sensing ion channels (ASICs) are proton-gated cation channels activated by low extracellular pH. Recent studies have suggested that ASICs are involved in the pathogenesis of some tumors, such as lung cancer and breast cancer. But the effect of acidosis and activation of ASICs on malignant glioma of the central nervous system has not been reported. In this study, we investigated the expression of ASIC1 in human glioma cell lines (U87MG and A172) and its possible effect on the proliferation and migration of these cells. The results demonstrated that ASIC1 is functionally expressed in U87MG and A172 cells. Treatment with extracellular weak acid (pH 7.0) has no effect on the proliferation but increases the migration of the two cell lines. Application of PcTX1, a specific inhibitor of ASIC1a and ASIC1a/2b channels, or knocking down ASIC1 by siRNA, can abolish the effect of weak acid-induced cell migration. Together, our results indicate that ASIC1 mediates extracellular weak acid induced migration of human malignant glioma cells and may therefore serve as a therapeutic target for malignant glioma in human.

The crosstalk between autonomic nervous system and blood vessels.

The autonomic nervous system (ANS), comprised of two primary branches, sympathetic and parasympathetic nervous system, plays an essential role in the regulation of vascular wall contractility and tension. The sympathetic and parasympathetic nerves work together to balance the functions of autonomic effector organs. The neurotransmitters released from the varicosities in the ANS can regulate the vascular tone. Norepinephrine (NE), adenosine triphosphate (ATP) and Neuropeptide Y (NPY) function as vasoconstrictors, whereas acetylcholine (Ach) and calcitonin gene-related peptide (CGRP) can mediate vasodilation. On the other hand, vascular factors, such as endothelium-derived relaxing factor nitric oxide (NO), and constriction factor endothelin, play an important role in the autonomic nervous system in physiologic conditions. Endothelial dysfunction and inflammation are associated with the sympathetic nerve activity in the pathological conditions, such as hypertension, heart failure, and diabetes mellitus. The dysfunction of the autonomic nervous system could be a risk factor for vascular diseases and the overactive sympathetic nerve is detrimental to the blood vessel. In this review, we summarize findings concerning the crosstalk between ANS and blood vessels in both physiological and pathological conditions and hope to provide insight into the development of therapeutic interventions of vascular diseases.

GYY4137, an H2S Slow-Releasing Donor, Prevents Nitrative Stress and α-Synuclein Nitration in an MPTP Mouse Model of Parkinson's Disease.

The neuromodulator hydrogen sulfide (H2S) was shown to exert neuroprotection in different models of Parkinson's disease (PD) via its anti-inflammatory and anti-apoptotic properties. In this study, we evaluated the effect of an H2S slow-releasing compound GYY4137 (GYY) on a mouse PD model induced by acute injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). GYY was intraperitoneally (i.p.) injected once daily into male C57BL/6J mice 3 days before and 2 weeks after MPTP (14 mg/kg, four times at 2-h intervals, i.p.) administration. Saline was given as a control. Behavioral tests (rotarod, balance beam, and grid walking) showed that 50 mg/kg GYY significantly ameliorated MPTP-caused motor impairments. At lower doses (12.5 and 25 mg/kg) GYY exhibited a less obvious effect. Consistent with this, immunohistochemistry and western blot analysis demonstrated that 50 mg/kg GYY attenuated the loss of tyrosine hydroxylase (TH) positive neurons in the substantia nigra and the decrease of TH expression in the striatum of MPTP-treated mice. Moreover, at this regimen GYY relieved the nitrative stress, as indicated by the decreases in nitric oxide (NO) generation and neuronal NO synthase (nNOS) upregulation elicited by MPTP in the striatum. The suppression of GYY on nNOS expression was verified in vitro, and the results further revealed that Akt activation may participate in the inhibition by GYY on nNOS upregulation. More important, GYY reduced the nitrated modification of α-synuclein, a PD-related protein, in MPTP-induced mice. Overall, our findings suggest that GYY attenuated dopaminergic neuron degeneration and reduced α-synuclein nitration in the midbrain, thus exerting neuroprotection in MPTP-induced mouse model of PD.

Urate promotes SNCA/α-synuclein clearance via regulating mTOR-dependent macroautophagy.

Serum urate levels are reported to be significantly lowered in patients with Parkinson's disease (PD) and inversely correlated to the risk and progression of PD. However, the mechanism by which urate affects PD is poorly understood. Here we showed that treatment with uric acid (UA) resulted in an autophagy activity enhancement in PC12 cells in dose- and time-dependent manners, as indicated by LC3-II increase and P62 decrease. Moreover, UA was still able to increase the LC3-II level and the number of LC3 puncta in the presence of Bafilomycin A1, a lysosomal inhibitor. These changes of autophagic markers were preceded by mTOR inhibition and ULK1 activation. Co-treatment with 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO), an mTOR activator, abolished the UA-induced LC3-II increase. More importantly, UA reduced SNCA/α-synuclein accumulation in PC12 cells that overexpress wildtype or A53T mutant SNCA, and this was blocked by Bafilomycin A1 co-treatment. The in vivo study showed that UA administration was able to modulate the levels of autophagy markers, increase the autophagosome/autolysosome formation, and reduce SNCA accumulation in the midbrain of SNCAA53T transgenic mice. Taken together, our findings suggest that UA could induce autophagy activation via an mTOR-dependent signaling and ameliorate SNCA accumulation. This implicates that urate-elevating agent may become a potential strategy for PD therapy.

Development and evaluation of an efficient HPLC/MS/MS method for the simultaneous determination of pseudoephedrine and cetirizine in human plasma: application to phase-I pharmacokinetic study.

A sensitive, simple and highly selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and evaluated to determine simultaneously the concentrations of pseudoephedrine and cetirizine in human plasma. The chief benefit of the present method is the minimal sample preparation, as the procedure is only one-step protein precipitation. Two drugs were separated on a C(8) column and analyzed by LC/MS/MS using positive electrospray ionisation (ESI). The method had a chromatographic run time of 12.0 min and a linear calibration curve over the concentration range of 1.0-800 ng/ml for pseudoephedrine and 1.0-400 ng/ml for cetirizine, respectively. The lower limit of quantification of the two drugs was 1.0 ng/ml, respectively. The intra- and inter-batch precisions were less than 9.7%. The method described herein has been first used to reveal the pharmacokinetic characters in healthy Chinese volunteers treated with oral administration of different dosages of cetirizine dihydrochloride and controlled-released pseudoephedrine hydrochloride compound tablet, and approached the influence of a standard meal on the extent and rate of absorption of the combination tablet.