Stathmin 在微血管内皮细胞的表达与胶质瘤恶性程度关系

目的:检测Stathmin在正常脑组织及不同级别胶质瘤微血管内皮细胞中的表达情况。方法:利用结合CD105单克隆抗体的免疫磁珠内皮细胞分选系统特异性分选出68例胶质瘤微血管内皮细胞(其中低级别胶质瘤(WHO分级Ⅰ-Ⅱ)24例,高级别胶质瘤(WHO分级Ⅲ-Ⅳ)44例)和20例正常脑组织微血管内皮细胞。应用免疫组化、RT-PCR和Western blot检测Stathmin在胶质瘤微血管内皮细胞和正常脑组织微血管内皮细胞中的表达。结果:免疫组化证实Stathmin在正常脑组织微血管内皮细胞、低级别胶质瘤微血管内皮细胞和高级别胶质瘤微血管内皮细胞的表达百分率分别是20%,66%和95%(P<0.05)。RT-PCR和Western blot法检测显示,Stathmin在胶质瘤微血管内皮细胞中的表达明显增高。低级别胶质瘤组、高级别胶质瘤组分别与正常组比较,均有显著性差异(P<0.01);且低级别胶质瘤组与高级别胶质瘤组比较,有显著性差异(P<0.01),随着胶质瘤恶性程度的增加,Stathmin表达上调,具有统计学意义。结论:Stathmin在脑胶质瘤微血管内皮细胞中表达随肿瘤恶性程度增高而增加,可能为脑胶质瘤的生物治疗提供一个新靶点。     

A Role for the Endothelial Glycocalyx in Regulating Microvascular Permeability in Diabetes Mellitus

Diabetic angiopathy is a major cause of morbidity and mortality in diabetes mellitus. Endothelial dysfunction and associated alterations in blood flow, pressure and permeability are widely accepted phenomena in the diabetic milieu and are understood to lead to microangiopathy. Despite the clinical importance of diabetic microangiopathy, the mechanisms of pathogenesis remain elusive. In particular, much is yet to be understood about the nature of the putative increased permeability with respect to diabetes. Microvessel permeability is intrinsically difficult to measure and a surrogate (solute or solvent flux) is usually reported, the measurement of which is hampered by haemodynamic factors, such as flow rate, hydrostatic pressure gradient, solute concentration and surface area available for exchange. Very few studies describing the measurement of permeability with respect to diabetes have controlled for all these factors. As a result, the nature of the increased microvessel permeability in diabetes mellitus and indeed its causes are poorly understood. Recent studies have shown that hyperglycaemia can alter the glycocalyx structure, and parallel findings have shown that the apparent increase in permeability demonstrated in hyperglycaemia may be due to an increase in the permeability of the vessels to water, and not an increase in protein permeability, an effect attributable to altered glycocalyx. This review focuses on the current understanding of microvascular permeability in terms of the endothelial glycocalyx- fibre-matrix theory, those methods used to determine permeability in the context of diabetes, and the more recent developments in our understanding of elevated microvascular permeability in the diabetic circulation.     

Obesity Is Associated with Lower Coronary Microvascular Density

Background

Obesity is associated with diastolic dysfunction, lower maximal myocardial blood flow, impaired myocardial metabolism and increased risk of heart failure. We examined the association between obesity, left ventricular filling pressure and myocardial structure.

Methods

We performed histological analysis of non-ischemic myocardium from 57 patients (46 men and 11 women) undergoing coronary artery bypass graft surgery who did not have previous cardiac surgery, myocardial infarction, heart failure, atrial fibrillation or loop diuretic therapy.

Results

Non-obese (body mass index, BMI, ≤30 kg/m², n=33) and obese patients (BMI >30 kg/m², n=24) did not differ with respect to myocardial total, interstitial or perivascular fibrosis, arteriolar dimensions, or cardiomyocyte width. Obese patients had lower capillary length density (1145±239, mean±SD, vs. 1371±333 mm/mm³, P=0.007) and higher diffusion radius (16.9±1.5 vs. 15.6±2.0 μm, P=0.012), in comparison with non-obese patients. However, the diffusion radius/cardiomyocyte width ratio of obese patients (0.73±0.11 μm/μm) was not significantly different from that of non-obese patients (0.71±0.11 μm/μm), suggesting that differences in cardiomyocyte width explained in part the differences in capillary length density and diffusion radius between non-obese and obese patients. Increased BMI was associated with increased pulmonary capillary wedge pressure (PCWP, P<0.0001), and lower capillary length density was associated with both increased BMI (P=0.043) and increased PCWP (P=0.016).

Conclusions

Obesity and its accompanying increase in left ventricular filling pressure were associated with lower coronary microvascular density, which may contribute to the lower maximal myocardial blood flow, impaired myocardial metabolism, diastolic dysfunction and higher risk of heart failure in obese individuals.     

Nanomechanics of the Endothelial Glycocalyx in Experimental Sepsis

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.     

Neutrophil-to-Lymphocyte Ratio Is Associated with Impaired Interferon-Gamma Release to Phytohemagglutinin

ObjectiveThe neutrophil-to-lymphocyte ratio (NLR) has been shown to predict adverse outcomes in several pathologic conditions. The majority of indeterminate interferon (IFN)-γ release assays were due to inadequate IFN-γ response to the phytohemagglutinin. We sought to study the value of NLR to predict an indeterminate result of QuantiFERON-TB Gold In-Tube (QFT-GIT) performed in routine laboratory practice.MethodsResults from 2,773 QFT-GIT assays were analyzed. Data collection included demographic data, the level of IFN-γ to nil, mitogen, and TB antigen of QFT-GIT, total WBC, and a differential count. We calculated the absolute neutrophil count, lymphocyte count, and NLR.ResultsOf the total, 224 (8.1%) indeterminate results were observed. Twelve (1.8%) showed indeterminate results in the NLR range from 1.71 to 2.84, but 132 (19.2%) had indeterminate results in NLR≥5.18 (p<0.0001). The likelihood ratio for indeterminate results were 2.70 (95% CI, 2.36-3.08) in NLR ≥5.18 and 1.93 (95% CI, 1.64-2.27) in lymphocyte count ≤1050/μL. NLR and neutrophil count were independent predictors for indeterminate QFT-GIT result in multiple regression analysis. The IFN-γ response to PHA was negatively associated with NLR (r=-0.33, p<0.001).ConclusionWe showed that the NLR is an independent predictor of indeterminate QFT-GIT result. Low frequency of indeterminate results in group with normal NLR may imply the importance of a balance between two cellular compartments in physiological and pathological conditions.     

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography ³, that topography limits adhesive bond formation with other surfaces ⁹, and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface ^{3, 7}. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface ⁶. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface ⁴. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo ². The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa ⁵. The thickness and structure of the glycocalyx have been studied using electron microscopy ⁸, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.     

Slower Uncoating Is Associated with Impaired Replicative Capability of Simian-Tropic HIV-1

Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116^th position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus.     

Skin Autofluorescence Is Associated with Endothelial Dysfunction in Uremic Subjects on Hemodialysis

Background

Elevated levels of advanced glycation end products (AGEs) within tissues may contribute to endothelial dysfunction, an early indicator of atherosclerosis. We aimed to investigate whether levels of skin AGEs could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.

Methods and Results

One hundred and nineteen uremic patients on hemodialysis and 57 control subjects with moderate-to-high cardiovascular risk factors and without chronic kidney disease (CKD) were enrolled. We used ultrasound to measure flow-mediated vasodilation (FMD). An AGE reader measured skin autoflurorescence (AF). We then compared differences in FMD and skin AF values between the two groups. The uremic subjects had significantly higher levels of skin AF (3.47±0.76 AU vs. 2.21±0.45 arbitrary units; P<0.01) and significantly lower levels of FMD (4.79%±1.88% vs. 7.19%±2.17%; P<0.01) than the non-CKD subjects. After adjusting for all potential covariates, we found that skin AF level independently predicted FMD in both the hemodialysis and the non-CKD groups. In the hemodialysis group, skin AF ≥ 3.05 arbitrary units predicted abnormal FMD at a sensitivity of 87.9% and a specificity of 78.6% (P<0.01).

Conclusions

Skin AF could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.     

Attenuated XPC Expression Is Not Associated with Impaired DNA Repair in Bladder Cancer

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors.     

Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Dihydrobiopterin in Adult Falciparum Malaria: Association with Disease Severity,Impaired Microvascular Function and Increased Endothelial Activation

Tetrahydrobiopterin (BH₄) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH₄ is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH₂), which inhibits NOS. Depending on BH₄ availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH₄/BH₂ ratio decreases, NO production falls and is replaced by superoxide. In African children and Asian adults with severe malaria, NO bioavailability decreases and plasma phenylalanine increases, together suggesting possible BH₄ deficiency. The primary three biopterin metabolites (BH₄, BH₂ and B₀ [biopterin]) and their association with disease severity have not been assessed in falciparum malaria. We measured pterin metabolites in urine of adults with severe falciparum malaria (SM; n=12), moderately-severe malaria (MSM, n=17), severe sepsis (SS; n=5) and healthy subjects (HC; n=20) as controls. In SM, urinary BH₄ was decreased (median 0.16 ¼mol/mmol creatinine) compared to MSM (median 0.27), SS (median 0.54), and HC (median 0.34)]; p<0.001. Conversely, BH₂ was increased in SM (median 0.91 ¼mol/mmol creatinine), compared to MSM (median 0.67), SS (median 0.39), and HC (median 0.52); p<0.001, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4 in severe malaria. Overall, the median BH₄/BH₂ ratio was lowest in SM [0.18 (IQR: 0.04-0.32)] compared to MSM (0.45, IQR 0.27-61), SS (1.03; IQR 0.54-2.38) and controls (0.66; IQR 0.43-1.07); p<0.001. In malaria, a lower BH₄/BH₂ ratio correlated with decreased microvascular reactivity (r=0.41; p=0.03) and increased ICAM-1 (r=-0.52; p=0.005). Decreased BH4 and increased BH₂ in severe malaria (but not in severe sepsis) uncouples NOS, leading to impaired NO bioavailability and potentially increased oxidative stress. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.     

Adiposity Is Associated with Gender-Specific Reductions in Left Ventricular Myocardial Perfusion during Dobutamine Stress

Background

Obesity and visceral adiposity are increasingly recognized risk factors for cardiovascular disease. Visceral fat may reduce myocardial perfusion by impairing vascular endothelial function. Women experience more anginal symptoms compared to men despite less severe coronary artery stenosis, as assessed by angiography. Women and men have different fat storage patterns which may account for the observed differences in cardiovascular disease. Therefore, our objective was to evaluate the relationship between visceral adipose tissue distributions and myocardial perfusion in men and women.

Methods

Visceral and subcutaneous fat distributions and myocardial perfusion were measured in 69 men and women without coronary artery disease using magnetic resonance imaging techniques. Myocardial perfusion index was quantified after first-pass perfusion with gadolinium contrast at peak dose dobutamine stress.

Results

We observed inverse relationships between female gender (r = -0.35, p = 0.003), pericardial fat (r = -0.36, p = 0.03), intraperitoneal fat (r = -0.37, p = 0.001), and retroperitoneal fat (r = -0.36, p = 0.002) and myocardial perfusion index. Visceral fat depots were not associated with reduced myocardial perfusion at peak dose dobutamine in men. However, in women, BMI (r = -0.33, p = 0.04), pericardial fat (r = -0.53, p = 0.02), subcutaneous fat (r = -0.39, p = 0.01) and intraperitoneal fat (r = -0.30, p = 0.05) were associated with reduced myocardial perfusion during dobutamine stress.

Conclusions

Higher visceral fat volumes are associated with reduced left ventricular myocardial perfusion at peak dose dobutamine stress in women but not in men. These findings suggest that visceral fat may contribute to abnormal microcirculatory coronary artery perfusion syndromes, explaining why some women exhibit more anginal symptoms despite typically lower grade epicardial coronary artery stenoses than men.     

Periatrial Epicardial Fat Is Associated with Markers of Endothelial Dysfunction in Patients with Atrial Fibrillation

Background

Epicardial adipose tissue (EAT) is associated to atrial fibrillation (AF) burden and outcome after AF ablation. We intended to determine whether global or local EAT is associated with systemic and/or left atrial (LA) inflammation and markers of endothelial dysfunction in AF patients.

Methods and Results

Total, atrial, and ventricular EAT volume (EATtotal, EATatrial, EATventricular) were measured by multislice cardiac CT in 49 patients with paroxysmal (PAF, n=25) or persistent AF (PeF, n=24). Periatrial epicardial fat thickness at the esophagus (LA-ESO) and thoracic aorta (LA-ThA) were also measured. Vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), soluble intercellular adhesion molecule 1 (sICAM-1), transforming growth factor-β1 (TGF-β1), and von Willebrand Factor (vWF) levels were measured in peripheral and LA blood samples obtained during catheterization during AF ablation. Patients with PeF had higher EATatrial (P<0.05) and LA-ESO (P=0.04) than patients with PAF. VEGF, IL-8, and TGF-β1 were not associated with EAT. In contrast, after adjusting for LA volume and body mass index, higher LA-ThA was significantly associated with higher sICAM-1 and vWF levels, both in peripheral blood (P<0.05) and in LA (P<0.05). Similar results were found with LA-ESO. Body mass index, EATtotal and EATventricular were not associated with sICAM-1 and vWF.

Conclusions

Periatrial epicardial fat showed a significant positive association with increased levels of sICAM-1 and vWF, which are biomarkers of endothelial dysfunction. No such associations were found when considering body mass index or EATtotal. These results suggest that local EAT rather than regional or total adiposity may modulate endothelial dysfunction in patients with AF.     

Reactive Oxygen Species Modulate the Barrier Function of the Human Glomerular Endothelial Glycocalyx

Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S³⁵ and ³H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.     

Periodontitis Is Associated with Endothelial Dysfunction in a General Population: A Cross-Sectional Study

A large body of evidence underlines an association between periodontal disease and cardiovascular disease. In contrast, data on its relation with endothelial dysfunction as a marker of early subclinical atherosclerosis is inconclusive and limited to patient-cohort studies. We therefore investigated the association between periodontal disease and flow-mediated dilation of the brachial artery (FMD) as a measure of endothelial dysfunction in a general population, and also addressed a possible mediation via inflammation. The study population comprised 1,234 subjects (50.5% men) aged 25–85 years from the 5-year follow-up of the Study of Health in Pomerania, a population-based cohort study. Clinical attachment loss (CAL) and pocket probing depth (PPD) as measures of periodontal disease were assessed half-mouth at four sites per tooth. Subjects were classified according to the periodontitis case definition proposed by Tonetti and Claffey (2005). Measurements of FMD and nitroglycerin-mediated dilation (NMD) were performed using standardized ultrasound techniques. High-sensitive C-reactive protein, fibrinogen and leukocyte count were measured. Fully adjusted multivariate linear regression analyses revealed significant associations of the percentage of sites with PPD ≥6 mm with FMD (p_trend=0.048), with subjects within the highest category having a 0.74% higher FMD compared to subjects within the lowest category (p<0.05). Consistently, FMD values increased significantly across categories of the percentage of sites with CAL ≥6 mm (p_trend=0.01) and the periodontitis case definition (p_trend=0.006). Restrictions to subjects without antihypertensive or statin medication or current non-smokers confirmed previous results. Systemic inflammation did not seem to mediate the relation. Both PPD and CAL were not consistently associated with NMD. In contrast to previous studies, high levels of periodontal disease were significantly associated with high FMD values. This association was not mediated via systemic inflammation. This study revives the discussion on whether and how periodontitis contributes to endothelial dysfunction.     

Chronic Household Air Pollution Exposure Is Associated with Impaired Alveolar Macrophage Function in Malawian Non-Smokers

Background

Household air pollution in low income countries is an important cause of mortality from respiratory infection. We hypothesised that chronic smoke exposure is detrimental to alveolar macrophage function, causing failure of innate immunity. We report the relationship between macrophage function and prior smoke exposure in healthy Malawians.

Methods

Healthy subjects exposed daily to cooking smoke at home volunteered for bronchoalveolar lavage. Alveolar macrophage particulate content was measured as a known correlate of smoke exposure. Phagocytosis and intraphagosomal function (oxidative burst and proteolysis) were measured by a flow cytometric assay. Cytokine responses in macrophages were compared following re-exposure in vitro to wood smoke, before and after glutathione depletion.

Results

Volunteers had a range of alveolar macrophage particulate loading. The macrophage capacity for phagosomal oxidative burst was negatively associated with alveolar macrophage particulate content (n = 29, r² = 0.16, p = 0.033), but phagocytosis per se and proteolytic function were unaffected. High particulate content was associated with lower baseline CXCL8 release (ratio 0.51, CI 0.29–0.89) and lower final concentrations on re-exposure to smoke in vitro (ratio 0.58, CI 0.34–0.97). Glutathione depletion augmented CXCL8 responses by 1.49x (CI 1.02–2.17) compared with wood smoke alone. This response was specific to smoke as macrophages response to LPS were not modulated by glutathione.

Conclusion

Chronic smoke exposure is associated with reduced human macrophage oxidative burst, and dampened inflammatory cytokine responses. These are critical processes in lung defence against infection and likely to underpin the relationship between air pollution and pneumonia.     

大鼠脑微血管内皮细胞培养及其药物转运体Oatp2和P-gp的表达

贴块法培养脑微血管内皮细胞(BMECs),倒置显微镜动态观察细胞生长及形态,Ⅷ因子相关抗原、CD34免疫细胞化学联合鉴定细胞并确定纯度。免疫细胞化学和Western印迹法检测药物转运体有机阴离子转运多肽亚型2(Oatp2)及P-糖蛋白(P-gp)在培养内皮细胞上的表达。结果显示,获得的BMECs呈多角形或铺路石形,单层贴壁生长;培养细胞Ⅷ因子相关抗原免疫细胞化学、CD34免疫荧光染色均为阳性,细胞纯度90%;培养细胞有Oatp2及P-gp表达,且二者均主要表达于BMECs细胞膜。提示贴块法可获得原代培养BMECs,方法简便易行,细胞纯度较高。原代培养的BMECs上有药物转运体Oatp2及P-gp的表达,为血脑屏障上药物转运体的体外研究提供了可能途径。     

Impaired Cerebral Autoregulation Is Associated with Brain Atrophy and Worse Functional Status in Chronic Ischemic Stroke

Dynamic cerebral autoregulation (dCA) is impaired following stroke. However, the relationship between dCA, brain atrophy, and functional outcomes following stroke remains unclear. In this study, we aimed to determine whether impairment of dCA is associated with atrophy in specific regions or globally, thereby affecting daily functions in stroke patients.We performed a retrospective analysis of 33 subjects with chronic infarctions in the middle cerebral artery territory, and 109 age-matched non-stroke subjects. dCA was assessed via the phase relationship between arterial blood pressure and cerebral blood flow velocity. Brain tissue volumes were quantified from MRI. Functional status was assessed by gait speed, instrumental activities of daily living (IADL), modified Rankin Scale, and NIH Stroke Score.Compared to the non-stroke group, stroke subjects showed degraded dCA bilaterally, and showed gray matter atrophy in the frontal, parietal and temporal lobes ipsilateral to infarct. In stroke subjects, better dCA was associated with less temporal lobe gray matter atrophy on the infracted side ( = 0.029), faster gait speed ( = 0.018) and lower IADL score (0.002). Our results indicate that better dynamic cerebral perfusion regulation is associated with less atrophy and better long-term functional status in older adults with chronic ischemic infarctions.     

Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.