Matrix metalloproteinases as target genes for gene regulatory networks driving molecular and cellular pathways related to a multistep pathogenesis of cerebrovascular disease

The present study investigated a joint contribution of matrix metalloproteinases (MMPs) genes to ischemic stroke (IS) development and analyzed interactions between MMP genes and genome-wide associated loci for IS. A total of 1288 unrelated Russians (600 IS patients and 688 healthy individuals) from Central Russia were recruited for the study. Genotyping of seven single nucleotide polymorphisms (SNPs) of MMP genes (rs1799750, rs243865, rs3025058, rs11225395, rs17576, rs486055, and rs2276109) and eight genome-wide associated loci for IS were done using Taq-Man–based assays and MALDI-TOF mass spectrometry iPLEX platform, respectively. Allele − 799T at rs11225395 of the MMP8 gene was significantly associated with a decreased risk of IS after adjustment for sex and age (OR = 0.82; 95%CI, 0.70-0.96; P = 0.016). The model-based multifactor dimensionality reduction method has revealed 21 two-order, 124 three-order, and 474 four-order gene-gene (G×G) interactions models meaningfully (P_perm < 0.05) associated with the IS risk. The bioinformatic analysis enabled establishing the studied MMP gene polymorphisms possess a clear regulatory potential and may be targeted by gene regulatory networks driving molecular and cellular pathways related to the pathogenesis of IS. In conclusion, the present study was the first to identify an association between polymorphism rs11225395 of the MMP8 gene and IS risk. The study findings also indicate that MMPs deserve special attention as a potential class of genes influencing the multistep mechanisms of cerebrovascular disease including atherosclerosis in cerebral arteries, acute cerebral artery occlusion as well as the ischemic injury of the brain and its recovery.     

Platycodin D protects cortical neurons against oxygen-glucose deprivation/reperfusion in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is one of the leading causes of death in infants. Increasing evidence indicates that oxidative stress and apoptosis are major contributors to hypoxic-ischemic injury and can be used as particularly promising therapeutic targets. Platycodin D (PLD) is a triterpenoid saponin that exhibits antioxidant properties. The aim of this study was to evaluate the effects of PLD on hypoxic-ischemic injury in primary cortical neurons. We found that oxygen-glucose deprivation/reperfusion (OGD/R) induced inhibition of cell viability and cytotoxicity, which were attenuated by PLD treatment. PLD treatment inhibited oxidative stress induced by OGD/R, which was evidenced by the reduced level of reactive oxygen species and increased activities of catalase, superoxide dismutase, and glutathione peroxidase. Histone-DNA enzyme-linked immunosorbent assay revealed that apoptosis was significantly decreased after PLD treatment in OGD/R-treated cortical neurons. The increased bax expression and decreased bcl-2 expression induced by OGD/R were reversed by PLD treatment. Furthermore, PLD treatment caused the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in OGD/R-stimulated cortical neurons. Suppression of this pathway blocked the protective effects of PLD on OGD/R-induced cell injury. These findings suggested that PLD executes its protective effects on OGD/R-induced cell injury via regulating the PI3K/Akt/mTOR pathway in cortical neurons.     

Lentiviral-mediated silencing of mast cell-expressed membrane protein 1 promotes angiogenesis of rats with cerebral ischemic stroke

Cerebral ischemic stroke is a devastating neurological disease with high rates of morbidity, disability, and mortality. Lentiviral-mediated mast cell-expressed membrane protein 1 (MCEMP1) has been shown to function in ischemic stroke. Hence, this study aims to explore the function of MCEMP1 specifically in angiogenesis, neuronal proliferation, and apoptosis in rats with cerebral ischemic stroke. Initially, stroke-related genes were obtained through microarray-based gene expression analysis, followed by the construction of a lentiviral vector for MCEMP1 shRNA and establishment of the middle cerebral artery occlusion model. After rats were transfected with MCEMP1 shRNA lentivirus, microvessel density (MVD), expression of MCEMP1, caspase-3, and vascular endothelial growth factor (VEGF), and neuronal proliferation and apoptosis were measured to explore the role of MCEMP1 in cerebral ischemic stroke. MCEMP1 was found to be highly expressed in rats with cerebral ischemic stroke. Silencing of MCEMP1 led to upregulation of VEGF, while downregulation of caspase-3, and resulted in the promotion of MVD in rats with ischemic stroke. Moreover, MCEMP1 silencing could increase Ki67 positive cells and reduce terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells in the marginal zone of cortical infarction in rats. Our study provides evidence that silenced MCEMP1 could enhance angiogenesis and suppress neuronal apoptosis in rats with cerebral ischemic stroke, highlighting that MCEMP1 silencing could serve as a therapeutic target for cerebral ischemic stroke treatment.     

Deficiency of the mitochondrial sulfide regulator ETHE1 disturbs cell growth,glutathione level and causes proteome alterations outside mitochondria

The mitochondrial enzyme ETHE1 is a persulfide dioxygenase essential for cellular sulfide detoxification, and its deficiency causes the severe and complex inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of well-described clinical symptoms of the disease, detailed cellular and molecular characterization is still ambiguous. Cellular redox regulation has been described to be influenced in ETHE1 deficient cells, and to clarify this further we applied image cytometry and detected decreased levels of reduced glutathione (GSH) in cultivated EE patient fibroblast cells. Cell growth initiation of the EE patient cells was impaired, whereas cell cycle regulation was not. Furthermore, Seahorse metabolic analyzes revealed decreased extracellular acidification, i. e. decreased lactate formation from glycolysis, in the EE patient cells. TMT-based large-scale proteomics was subsequently performed to broadly elucidate cellular consequences of the ETHE1 deficiency. More than 130 proteins were differentially regulated, of which the majority were non-mitochondrial. The proteomics data revealed a link between ETHE1-deficiency and down-regulation of several ribosomal proteins and LIM domain proteins important for cellular maintenance, and up-regulation of cell surface glycoproteins. Furthermore, several proteins of endoplasmic reticulum (ER) were perturbed including proteins influencing disulfide bond formation (e.g. protein disulfide isomerases and peroxiredoxin 4) and calcium-regulated proteins. The results indicate that decreased level of reduced GSH and alterations in proteins of ribosomes, ER and of cell adhesion lie behind the disrupted cell growth of the EE patient cells.     

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.     

大鼠肢体预缺血减小心肌缺血-再灌注后的梗塞范围

在氨基甲酸乙酯麻醉大鼠上观察肢体预缺血(limb ischemic preconditioning,LIP)对缺血-再灌注(ischemia—reperfusion,IR)心肌的影响,旨在探讨LIP对IR心肌有无保护效应,并明确腺苷和神经通路是否参与此效应。所得结果如下:(1)在心脏缺血30 min和再灌注120 min过程中,梗塞心肌占缺血心肌的51.48±0.82％。(2)LIP时心肌梗塞范围为35.14±0.88％,较单纯心肌缺血-再灌注时显著减小(P<0.01),表明LIP对心肌有保护作用。(3)事先切断股神经可取消LIP的保护效应。(4)股动脉内局部给予腺苷(10nmol/kg),可模拟LIP对心肌的保护作用;心肌梗塞范围是37.28±1.68％,而股静脉内注射同等剂量腺苷则无保护作用。(5)股动脉内预先应用腺苷A.受体拈抗剂8-环戊-1,3-二丙基嘌呤(DPCPX)(32 nmol/kg)可部分阻断LIP诱发的心肌保护效应。以上结果表明,肢体短暂预缺血可减小心肌缺血-再灌注后的梗塞范围,而局部释放的腺苷和由此所激活的相关的神经通路在LIP的心肌保护中起重要作用。     

Comparison of pulmonary vascular response to endogenous nitric oxide inhibition in sheep and pigs living at 2,300 m

To compare the role of nitric oxide in an adaptive process to chronic hypoxia, we examined the effects of endogenous nitric oxide synthase inhibition on pulmonary vascular tone in conscious sheep and pigs living at high altitude. Unanesthetized male sheep (n=6) and pigs (n=5), born and residing in the highlands of Qinghai Province, China (2,300–3,000 m a.s.l.) were studied at that altitude. Pulmonary artery pressure (P_pa), pulmonary artery wedge pressure (P_cwp), and cardiac output (CO) were measured. Pulmonary vascular resistance (PVR) was calculated as (P_pa—P_cwp)/CO. Using a climatic chamber, hemodynamic measurements during exposures to atmospheric pressures corresponding to altitudes of 0, 2,300, and 4,500 m a.s.l. were performed with and without NO inhibition, using N^w-nitro-l-argine (NLA; 20 mg kg^–1), a potent stereospecific competitive inhibitor of nitric oxide synthase. P_pa and PVR at baseline (2,300 m) and during hypoxic exposure (4,500 m) were significantly higher in pigs than in sheep. After NLA administration, P_pa increased and CO decreased in both animals, resulting in significantly increased PVR at baseline and during hypoxic exposure. However, there were no significant differences in the percent increase in basal or hypoxic PVR after NLA administration between sheep and pigs. We conclude that augmented endogenous NO production could contribute to the regulation of pulmonary vascular tone at high altitude in sheep and pigs. However, it is unlikely that NO is responsible for the different pulmonary vascular tones between sheep and pigs at basal condition at moderately high altitude.Communicated by G. Heldmaier     

p38 MAPKα/β和ERK1/2在心肌缺氧预处理信号传递中的不同作用

建立培养乳鼠心肌细胞的缺氧／复氧(A／R)损伤模型和缺氧预处理(APC)模型,以细胞存活率、细胞内超氧化物趋化酶(SOD)活性、丙二醛(MDA)含量、培养上清液乳酸脱氢酶(LDH)活性作为反映心肌细胞损伤的指标。采用细胞外信号调节蛋白激酶(ERK1／2)抑制剂PD98059及丝裂素活化蛋白激酶p38α/β(p38α/β)阻滞剂SB203580干预模型,并以胶内原位磷酸化法测定ERK1／2和p38活性,借以探讨ERK1／2和p38α/β在缺氧预处理保护机制中的作用。结果表明：(1)在APC组,于预处理的缺氧时相给予PD98059,可以完全消除APC的延迟保护作用;在A／R组的缺氧时相加入PD98059对细胞损伤无影响;(2)在APC组的预处理缺氧时相给予p38α／β抑制剂SB203580并不能消除APC的保护作用,而在A／R组的持续缺氧时相给予SB203580则可显著减轻缺氧对细胞的损伤;(3)ERK1／2和p38总活性测定表明,缺氧可激活ERK1／2和p38,它们的活性在缺氧后4h时达到高峰,而经过APC处理后,两者活性高峰提前于缺氧后3h时出现,且峰值显著降低。上述结果提示,预处理过程中ERK1／2的激活可能是缺氧预处理延迟保护机制中细胞信号传递的重要环节,预处理阶段p38α/β的活化不参与APC诱导的延迟保护信号传递过程,p38的过度激活可能是缺氧／复氧损伤过程中的一个致损伤参与因素,而预处理抑制随后持续缺氧阶段p38的过度激活可能是其保护机制的一个环节。     

Kappa 阿片受体的抗缺血性心脏保护作用--信息机制

有证据表明,心脏细胞产生强腓肽和强腓肽类多肽,它们是kappa阿片受体(κ-0R)的激动剂。κ-0R是心脏一种优势的阿片受体,其激活可改变在体和离体心脏的功能。在正常和病理情况下,内源性κ-阿片肽可能通过自分泌或旁分泌的方式调节心脏功能。心肌缺血是导致心脏功能紊乱的一个常见原因,主要表现为心肌功能减弱,心律失常及心肌梗塞等。心肌缺血时,交感神经发放增强,从而增加作功负荷及氧消耗量;而这又使缺血引发的状况更为恶化。机体抵抗缺血引发心肌损害／心律失常的保护机制之一是抑制β-肾上腺素受体(β—AR)的兴奋。κ-0R确实能抑制β-AR的激动。这种抑制主要是由于GS蛋白受到抑制,也在较小程度上由于信息通路的腺苷酸环化酶的抑制。因为该种酶能通过对百日咳毒素敏感的G蛋白转导β—AR的激动。另一保护心肌对抗缺血性损害的机制是预处理。预处理是指预先受到缺血等损伤使心脏对随后更严重的损伤产生较强的耐受能力。这种保护作用可以在预处理后即时产生,也可延至预处理后1—3天。在采用缺血或其产生的后果之一——代谢抑制作为预处理而致的心脏保护中,κ-OR参与媒介预处理的作用。用κ—OR的特异性激动剂U50488H激活κ—OR(U50488H药理性预处理,UP)可激活蛋白激酶C(PKC),开放ATY敏感的钾通道(KATP channels)及增加热休克蛋白(HSP)的产生。阻断PKC的作用,关闭KATP通道或抑制HSP的合成,均可消除UP的心脏保护作用。这些发现表明,PKC、KATP通道和HSP在UP的心脏保护中均具重要作用。此外,UP也能减低缺血造成心肌损害的因素之一,即Ca^2 的超负荷。这个事实表明UP发挥心脏保护作用至少部分地是通过减低Ca^2 的超负荷。最有趣的是,以阻断剂阻塞KATP通道,在消除UP的延迟性心脏保护作用的同时也降低了UP对Ca^2 超负荷的抑制作用。这个事实揭示了KATP通道开放所致的心脏保护作用至少部分地可能是由于防止或减低了Ca^2 的超负荷。