E‐selectin gene in essential hypertension: a case‐control study

Background

Hypertension is associated with endothelial cell dysfunction. E‐selectin, an endothelial cell adhesion molecule, is specific for endothelial cell activation. Polymorphism in E‐selectin gene has recently been identified among which Leu554Phe E‐selectin gene polymorphism is least investigated in essential hypertension. This study reports the association of E‐selectin gene Leu554Phe polymorphism and the expression of E‐selectin gene in patients with essential hypertension.

Materials and methods

We analysed the Leu554Phe polymorphism and expression of E‐selectin gene in 250 patients with essential hypertension and 250 normal healthy controls. Genotyping of Leu554Phe polymorphism was performed by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), and the expression of E‐selectin gene at mRNA and protein levels were carried out by real‐time PCR and Western blot, respectively.

Results

A significant association of E‐selectin genotypes (CT + TT) with essential hypertension (P < .0001, Odds ratio = 2.2 [1.58‐3.24] at 95% CI) was observed. The expression of mRNA for E‐selectin gene in patients with essential hypertension was ~12‐fold higher as compared to control. We observed an elevated level of E‐selectin protein expression (up to 1.9 times) in patients as compared to controls.

Conclusions

A significant association of E‐selectin (Leu554Phe) gene and increased expression of E‐selectin gene at mRNA and protein levels in patients might be related to the genetic predisposition to develop essential hypertension.     

Elevated plasma levels of P‐selectin glycoprotein ligand‐1‐positive microvesicles in patients with unprovoked venous thromboembolism

Essentials

• PSGL‐1⁺ microvesicles (MVs) may be important in venous thromboembolism (VTE).
• We measured plasma levels and parental origin of PSGL‐1⁺ MVs in patients with unprovoked VTE.
• VTE patients had higher plasma levels of PSGL‐1⁺ MVs than healthy controls.
• The PSGL‐1⁺ MVs originated mainly from monocytes and endothelial cells.

Summary

Background

Microvesicles (MVs) express antigens from their parental cells and have a highly procoagulant surface. Animal studies suggest that P‐selectin glycoprotein ligand‐1‐positive (PSGL‐1⁺) MVs play a role in the pathogenesis of venous thromboembolism (VTE).

Objective

The aim of this study was to determine plasma levels, the cellular origin and the morphological characteristics of PSGL‐1⁺ MVs in patients with unprovoked VTE.

Methods

We conducted a population‐based case–control study in 20 patients with a history of unprovoked VTE and 20 age‐ and sex‐matched healthy controls recruited from the general population. Plasma levels, the cellular origin and the morphological characteristics of PSGL‐1⁺ MVs were evaluated using flow cytometry, electron microscopy and confocal microscopy.

Results

Plasma levels of PSGL‐1⁺ MVs were associated with increased risk of VTE. The odds ratio per one standard deviation increase in PSGL‐1⁺ MVs was 3.11 (95% confidence interval [CI], 1.41–6.88) after adjustment for age and sex, and 2.88 (95% CI, 1.29–6.41) after further adjustment for body mass index. The PSGL‐1⁺ MVs originated mainly from monocytes and endothelial cells determined by double staining with markers of parental cells using flow cytometry and transmission electron microscopy. Scanning electron microscopy of PSGL‐1‐labeled plasma‐derived MVs displayed dominantly spherical vesicles that varied between 50 and 300 nm in diameter.

Conclusions

Increased plasma levels of PSGL‐1⁺ MVs are associated with the risk of unprovoked VTE. Large population‐based prospective studies are required to validate our findings.

     

Serum brain‐derived neurotrophic factor and platelet activation evaluated by soluble P‐selectin and soluble CD‐40‐ligand in patients with acute myocardial infarction

Little is known about the role of neurotrophins (NT) under adult vascular homeostasis in normal and pathological conditions. The NT family, including nerve growth factor and brain‐derived neurotrophic factor (BDNF) are expressed in atherosclerotic vessels. Previous studies demonstrated that plasma BDNF levels were increased in the coronary circulation in patients with unstable angina. However, the role of BDNF during the onset and evolution of unstable angina remains to be elucidated. The objective of this study was to evaluate the relationship between BDNF, functional parameters and biological markers associated with inflammatory processes and platelet activation. BDNF serum levels were assessed in patients with acute myocardial infarction (MI) (n = 20) or stable angina pectoris (SAP) (n = 20) who underwent coronary angiography. Serum levels of IL‐6, MCP1, sVCAM, soluble CD‐40‐ligand (sCD40L) and soluble P‐selectin (sP‐selectin) were measured simultaneously by flux cytometry. Median BDNF levels were higher in the MI than in the SAP group (1730 vs. 877 pg/mL, respectively; P = 0.025). In MI patients, we observed a significant correlation between BDNF and sP‐selectin (r = 0.58, P = 0.023), although we found a non‐significant trend between BDNF and sCD40L (r = +0.35, P = 0.144). By contrast, no such correlation was observed in SAP patients (r = ?0.22, P = 0.425). No difference was observed between the two groups regarding baseline demographics, risk factors, biological data and angiographic findings. The study suggests that BDNF serum levels in MI patients could be related to platelet activation and the inflammatory response. Further studies are needed to investigate the role of NT in the setting of acute MI.     

P—选择素凝集样区和绿色荧光蛋白融合基因的构建

目的 构建P-选择素凝集样区与绿色荧光蛋白融合基因（pEGFP-N1/L）,为进一步明确P-选择素不同表位与功能间的关系,为阐明P-选择素生理病理意义奠定基础。方法 应用PCR技术扩增质粒pCDM1/cDNA凝集素校区,用T4连接酶将其插入载体pEGFP-N1;经酶切和测序对插入片段进行分析、鉴定。结果 通过酶切和序列分析证实插入片段序列正确。结论 应用基因工程技术构建pEGFP-N1/L融合基因成功,为绿色荧光蛋白作为主生物标记分子来研究P-选择素分子的构效关系打下良好的基础。     

Specific E‐selectin targeting with a superparamagnetic MRI contrast agent

Targeting of the endothelial inflammatory adhesion molecule E‐selectin by magnetic resonance imaging (MRI) was performed with a superparamagnetic contrast agent in the context of in vitro and in vivo models of inflammation. The specific contrast agent was obtained by grafting a synthetic mimetic of sialyl Lewis^x (sLe^x), a natural ligand of E‐selectin expressed on leukocytes, on the dextran coating of ultrasmall particles of iron oxide (USPIO). This new contrast agent, called USPIO‐g‐sLe^x, was tested, in vitro, on cultured human umbilical vein endothelial cells (HUVECs) stimulated to express inflammatory adhesion molecules, and in vivo, on a mouse model of hepatitis. In vitro, HUVECs were stimulated with the pro‐inflammatory cytokine tumor necrosis factor alpha (TNF‐α) and were then incubated with USPIO‐g‐sLe^x or ungrafted USPIO. In vivo, hepatitis was induced on NMRI mice by injection of concanavalin A (Con A). USPIO‐g‐sLe^x and ungrafted USPIO were injected intravenously. In vitro results showed an extensive retention of USPIO‐g‐sLe^x on TNF‐α stimulated HUVECs. Image intensity and R₂ measurements performed on T₂‐weighted MR images demonstrated a significantly higher binding of USPIO‐g‐sLe^x on stimulated HUVECs. In vivo, USPIO are known to pass through the fenestrae of the liver and to be captured by Kupffer cells, inducing a loss of signal intensity on T₂‐weighted MR images. Unexpectedly, when injected to Con A‐treated mice, USPIO‐g‐sLe^x induced a significantly lower attenuation of liver signal intensity than USPIO or USPIO‐g‐sLe^x injected to healthy mice, or USPIO injected to Con A‐treated mice, suggesting that the specific contrast media is retained extracellularly by an interaction with E‐selectin overexpressed on the vascular endothelium. Both in vitro and in vivo results therefore indicate that USPIO‐g‐sLe^x is recognizing endothelial E‐selectin. USPIO‐g‐sLe^x is thus well suited for the MRI diagnosis of inflammation and for the in vitro evaluation of endothelial cells activation. Copyright © 2006 John Wiley & Sons, Ltd.     

Magnetic resonance molecular imaging of post‐stroke neuroinflammation with a P‐selectin targeted iron oxide nanoparticle

We have developed a magnetic resonance molecular imaging method using a novel iron‐oxide contrast agent targeted towards P‐selectin – MNP‐PBP (magnetic nanoparticle‐P‐selectin binding peptide) – to image endothelial activation following cerebral ischemia/reperfusion. MNP‐PBP consists of ～1000 PBP ligands (primary sequence: GSIQPRPQIHNDGDFEEIPEEYLQ GGSSLVSVLDLEPLDAAWL) conjugated to a 50 nm diameter aminated dextran iron oxide particle. In vitro P‐ and E‐selectin binding was assessed by competition ELISA. Transient focal cerebral ischemia was induced in male C57/BL 6 mice followed by contrast injection (MNP‐PBP; MNP‐NH2; Feridex; MNP‐PBP‐FITC) at 24 h after reperfusion and T₂ magnetic resonance imaging at 9.4 T was performed. Infarction and microvasculature accumulation of contrast agent was assessed in coronal brain sections. MNP‐PBP attenuated antibody binding to P‐selectin by 34.8 ± 1.7%. P‐selectin was preferentially increased in the infarct hemisphere and MNP‐PBP‐FITC accumulation in the infarct hemisphere microvasculature was observed. Compared with the nontargeted iron oxide agents MNP‐NH2 and Feridex, MNP‐PBP showed a significantly greater T₂ effect within the infarction. MR imaging of P‐selectin expression with a targeted iron oxide nanoparticle contrast agent may reveal early endothelial activation in stroke and other neuroinflammatory states. Copyright © 2009 John Wiley & Sons, Ltd.     

从结核病和肺癌患者外周血中检测结核分枝杆菌及其L型

目的 评价从外周血中检测结核分枝杆菌(MTB)及其L型(MTB-L)的临床应用价值.方法 用溶血离心滴片法IK抗酸染色(IK染色)及溶血离心培养法(培养法)检测结核病组(156例)、肺癌组(147例)和非结核病/非肺癌对照组(42例)外周血中的MTB及其L型,用TaqMan-PCR技术分别检测上述各组标本的单个核细胞、全血及血浆中的MTB DNA.结果 IK染色结核病组、肺癌组和非结核病/非肺癌对照组的MTB阳性率分别为1.3%(2/156)、0.7%(1/147)和0(0/42),培养法分别为0.6%(1/156)、0(0/147)和0(0/42).IK染色MTB-L阳性率分别为41.0%(64/156)、32.7%(48/147)和7.2%(3/42),培养法分别为25.6%(40/156)、39.5%(58/147)和0(0/42).结核病组、肺癌组的MTB与MTB-L检测结果 差异均有统计学意义(X2值分别为73.87、44.35、57.41、76.68,均P＜0.01).培养出98株L型菌中,人型81株,牛型17株,返祖为原菌型者44株.结核病组单个核细胞、全血TaqMan-PCR阳性率分别为77.6%(121/156)和68.6%(107/156),均高于血浆的阳性率的20.5%(32/156,X2值分别为112.96、82.18,均P＜0.05),肺癌组单个核细胞、全血阳性率分别为59.2%(87/147)和48.3%(52/147),均高于血浆阳性率的12.2%(18/147,X2值分别为70.53、21.68,P均＜0.05).两组各血液成分(除外血浆)的检测结果 与对照组比较差异均有统计学意义(X2值分别为37.50、48.30、44.37、11.31,P均＜0.01).结论 结核病、肺癌患者外周血中存在MTB-L播散;溶血离心培养法检测外周血中MTB-L简便易行;TaqMan-PCR技术检测单个核细胞和全血标本中MTB DNA的阳性率高于血浆的阳性率.     

Interleukin-4 gene, but not the interleukin-1 beta gene polymorphism, is associated with oral cancer

We aimed to evaluate whether polymorphisms of the interleukin-4 (IL-4) gene promoter and intron 3 regions, and polymorphisms of the IL-1 beta gene promoter and exon 5 regions are associated with oral cancer. This study included 130 patients with oral cancer and 105 age-matched healthy controls who lived in the same area as the patients. Each genetic polymorphism was typed by polymerase chain reaction (PCR)-based restriction analysis. We then compared the genotype distribution and allelic frequencies of each polymorphism between the oral cancer patients and the controls. The CC homozygote genotype of the IL-4 gene promoter -590 region differed significantly between the patients with oral cancer and the controls (odds ratio (OR)=6.0, 95% confidence interval (CI)=1.2-30.7, chi-square test, P=0.044). No significant difference in either the genotype distribution or the allelic frequencies of the IL-1 beta gene polymorphisms was observed between patients with oral cancer and controls. The IL-4 gene -590 C/T polymorphism is associated with oral cancer and is a suitable genetic marker for screening for oral cancer. However, whether the -590 C/T polymorphism of the IL4 gene plays a role in oral cancer remains unclear. Further substantiation based on larger patient samples is needed.     

Angiostatin K1‐3 induces E‐selectin via AP1 and Ets1: a mediator for anti‐angiogenic action of K1‐3

Summary. Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1‐3 included. We previously showed that K1‐3 was the most potent angiostatin to induce E‐selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1‐3‐induced E‐selectin expression and investigate the role of E‐selectin in the anti‐angiogenic action of K1‐3. Methods and results: Quantitative real time RT‐PCR and Western blotting analyses confirmed a time‐dependent increase of E‐selectin mRNA and protein induced by K1‐3. Subcellular fractionation and immunofluorescence microscopy showed the co‐localization of K1‐3‐induced E‐selectin with caveolin 1 (Cav1) in lipid rafts in which E‐selectin may behave as a signaling receptor. Promoter‐driven reporter assays and site‐directed mutagenesis showed that K1‐3 induced E‐selectin expression via promoter activation and AP1 and Ets‐1 binding sites in the proximal E‐selectin promoter were required for E‐selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1‐3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E‐selectin by K1‐3. A modulatory role of E‐selectin in the anti‐angiogenic action of K1‐3 was manifested by both overexpression and knockdown of E‐selectin followed by cell proliferation assay. Conclusions: We show that K1‐3 induced E‐selectin expression via AP1 and Ets‐1 binding to the proximal E‐selectin promoter (?356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E‐selectin as a novel target for the anti‐angiogenic therapy.     

复发性口腔溃疡5-羟色胺转运体启动子基因多态性研究

目的：探讨5-羟色胺转运体启动子区域（5-HTTLPR）基因多态性和复发性口腔溃疡之间是否存在关联性。方法：运用聚合酶链反应技术（PCR）检测分析T87例复发性口腔溃疡患者和96例健康人群的5-HTTLPR基因多态性。结果：两组5-HTTLPR基因型Short／Short（s／s）差异有显著性（x^2-6．423,P=0．044）,提示s／s基因型携带者比L／L和S／L基因型的携带者患RAV的相对风险显著增高。结论：5-羟色胺转运蛋白启动子区的s／s纯合子基因型可能是RALL的易感基因之一。     

P-选择素和L-选择素在大鼠脑缺血/再灌注损伤中表达的实验研究

目的研究P-选择素和L-选择素在大鼠局灶性脑缺血／再灌注后的表达规律，探讨它们在脑缺血／再灌注损伤中的作用。方法72只雄性SD大鼠随机分为假手术组、P-选择素组、L-选择素组和持续缺血组，采用尼龙线栓法建立大鼠局灶性脑缺血／再灌注模型，用免疫组化方法检测脑组织缺血／再灌注不同时间点P-选择素和L-选择素的表达。结果假手术组未见P-选择素和L-选择素的表达，P-选择素在脑缺血／再灌注后3h出现少量表达，12h达高峰，48h仍有表达；L-选择素在脑缺血／再灌注后2h出现少量表达，6h达高峰，24h仍有表达。结论大鼠局灶性脑缺血／再灌注损伤中，P-选择素和L-选择素的表达明显上调，两者介导了白细胞、脑微血管内皮细胞的黏附，参与了脑缺血／再灌注损伤的病理过程。