HO-1 and VGEF gene expression in human arteries with advanced atherosclerosis

OBJECTIVES: Both heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) have been shown to be involved in the progression of atherosclerosis. The relationship between HO-1 and VEGF gene expression and their proteins in endothelial cells from human atherosclerotic arterial specimens was investigated. DESIGN AND METHODS: The study included seventeen human arterial specimens with early and six specimens with advanced atherosclerotic lesions. Ten specimens were obtained from healthy young adults undergoing arterial reconstruction for trauma and were considered as non-atherosclerotic control. HO-1 and VEGF expressions as well as HO activity and VEGF protein content were measured in isolated endothelial cells (ECs). RESULTS: HO-1 expression and activity (5.3+/-2.1 nmol bilirubin/mg protein/h) were only present in ECs from advanced atherosclerotic lesions. VEGF expression was more strongly expressed in ECs from advanced lesion compared with early lesions and was absent in healthy arteries. VEGF protein (1.35+/-0.69 ng/mg) was only detected in advanced lesions. A significant positive correlation (r=0.9, p<0.01) exists between HO activity and VEGF protein content in ECs of advanced lesions. CONCLUSIONS: This study demonstrated that HO-1 expression and activity in ECs are present only in advanced atherosclerosis whereas, VEGF expression is present in early as well as in advanced atherosclerosis and the degree of its expression increases with severity of atherosclerosis. This study suggests an association between HO activity and VEGF protein in human ECs from advanced atherosclerotic lesions.     

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1alpha and CA-IX in the menstrual and early proliferative phases. HIF1alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase.     

Effects of peritoneal fluid from endometriosis patients on the release of vascular endothelial growth factor by neutrophils and monocytes

BACKGROUND: An increase in the level of the vascular endothelial growth factor (VEGF) production has been reported in the peritoneal fluid (PF) of endometriosis patients. This suggests that changes in the vascular permeability and angiogenesis play an important role in the pathophysiology of this disease. This study examined the effects of the PF obtained from endometriosis patients on the release of VEGF by neutrophils and monocytes. METHODS: Neutrophils and monocytes were obtained from young healthy volunteers and cultured with the PF obtained from either endometriosis patients (EPF) (n=18) or a control group (CPF) (n=4). A human monocyte/macrophage cell line, THP-1, was cultured with either 10% EPF or 10% CPF. The PF and culture supernatants were assayed for VEGF using ELISA. Real-time PCR and Western blotting were used to measure the VEGF mRNA and protein expression level, respectively. RESULTS: The VEGF levels were higher in the EPF than in the CPF (591+/-75 versus 185+/-31 pg/ml, P<0.05). However, the level of VEGF released by THP-1 cells in CPF and EPF was similar. The EPF induced the release of VEGF by neutrophils, but no VEGF was released by monocytes. The VEGF mRNA expression levels in the neutrophils were higher in the EPF, which was abrogated by cycloheximide, suggesting that the EPF induces the production of VEGF in neutrophils. Neutralizing antibodies against IL-8 and TNF-alpha did not completely prevent the EPF-induced release of VEGF by the neutrophils, even though these growth factors stimulated the release of VEGF by neutrophils. There was a positive correlation between the VEGF and IL-10 concentrations in the EPF (correlation coefficient=0.549, P=0.012, n=18), but the neutralizing antibody of IL-10 did not affect the release of VEGF by the EPF-treated neutrophils. CONCLUSION: The EPF induced the production and release of VEGF by neutrophils, suggesting that neutrophils may be a source of peritoneal VEGF. In addition, neutrophil-derived VEGF might be a marker for diagnosing endometriosis.     

沙利度胺对A549细胞体外增殖的影响及其作用机制

目的探讨沙利度胺对人非小细胞肺癌细胞株A549体外增殖和其对A549细胞中血管内皮生长因子(VEGF)和血管内皮生长因子C(VEGF-C)表达的影响。方法体外培养A549细胞株,分为对照组和沙利度胺组,后者分别加入不同质量浓度(5,10,20,40,60 mg/L)的沙利度胺体外培养24 h。采用MTT法检测对A549细胞增殖的抑制率,采用RT-PCR和Western-blot方法分别检测各组VEGF和VEGF-C mRNA和蛋白水平的表达。结果沙利度胺体外能抑制A549细胞增殖,呈浓度依赖性(P<0.05)。不同浓度沙利度胺作用于A549细胞后,VEGF和VEGF-C mRNA和蛋白水平的表达均降低(P<0.05)。结论沙利度胺体外能抑制A549细胞的增殖,呈浓度依赖性,其作用机制可能与沙利度胺下调VEGF及VEGF-C的表达有关。     

VEGF183(132-158)肽段作为蛋白缓释药物载体的细胞外基质结合研究

人血管内皮生长因子VEGF183(132-158)肽段含有细胞外基质结合序列与纤溶酶、基质金属蛋白酶以及uPA酶裂解位点,研究该肽段作为一种蛋白药物的缓释载体,在体内既可和细胞外基质结合,又可在体内的相应酶作用下,缓慢释放。该研究以绿色荧光蛋白GFP为指示分子,进一步采用加端PCR方法将VEGF183(132-158)肽段连到GFP的C端,原核表达并纯化该融合蛋白,通过与琼脂糖肝素柱结合实验与冷冻切片的荧光显微镜观察,发现该肽段赋予了GFP蛋白结合琼脂糖肝素柱与细胞外基质的功能,GFP融合蛋白的获得为下一步的体内释放研究打下了基础。     

Targeting podocyte-associated diseases

Injury to the podocytes is the initiating cause of many renal diseases, leading to proteinuria with possible progression to end-stage renal disease. Podocytes are highly specialized cells, with an important role in maintaining the glomerular filtration barrier and producing growth factors for both mesangial cells and endothelial cells. With their foot processes they cover the glomerular basement membrane, and form slit diaphragms with neighboring podocytes.Human podocytopathies include focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy, collapsing glomerulopathy and diabetic nephropathy. Research in the last two decades has demonstrated great progress in understanding the molecular mechanisms leading to podocytopathies. These include single gene defects in slit diaphragm proteins, but also discovery of apoptotic, enzymatic and other pathways involved in podocyte injury. With this progress, a great number of animal models is now available to study either specific podocytopathies, e.g. in mouse models with single gene mutations, or more general podocyte injury patterns, such as the lipopolysaccharide or protamine sulfate model of foot process effacement.In this review, the morphology of the glomerulus will be discussed, with a focus on the podocyte, its interactions with surrounding cells, and the highly differentiated slit diaphragm separating the apical from the basal membrane. We also provide an overview of human podocytopathies and animal models to study these diseases. In the last part we discuss targeted therapies addressing pathways and proteins affected in podocyte injury.     

Vascular endothelial growth factor in amyotrophic lateral sclerosis and other neurodegenerative diseases

The angiogenic activity of vascular endothelial growth factor (VEGF) is well known. Recently, it has become evident that VEGF is involved in central nervous system physiology and may play a role in the pathogenesis of neurological diseases. In particular, it may be involved in the mechanism of motor neuron degeneration in amyotrophic lateral sclerosis (ALS), and has been hypothesized to be implicated in the pathogenesis of peripheral neuropathies such as occur in the so-called POEMS syndrome and diabetes. VEGF is also being studied as a possible treatment option in some of these disorders. In this review we critically analyze the data supporting the notion that VEGF is a factor involved in motor neuron degeneration and review the studies linking VEGF to other diseases of the peripheral and central nervous systems.     

Cigarette Smoke Differently Alters Normal and Ovalbumin-Sensitized Bronchial Epithelial Cells from Rat

Background. Smoking is a common habit in the general population, even in asthmatic patients. Bronchial epithelial cells are the first cellular elements exposed to environmental stimuli such as cigarette smoke. These cells produce a wide range of mediators involved in inflammation and remodeling processes. However, the effects of chronic smoke exposure on the release and production of these mediators remain unclear. Objectives. To investigate the effects of repeated exposure to cigarette smoke extract on mediator released by bronchial epithelial cells isolated from control and asthmatic rats. Methods. Bronchial epithelial cells were isolated from normal (NRBE) and asthmatic rats (ARBE) (ovalbumin (OVA)-sensitized rat). Cells were exposed to cigarette smoke extract (CSE) obtained by impacting cigarette smoke with an AGI-30. A concentration of 3% CSE was added in the medium daily, for 5 consecutive days. Supernatant was recovered at baseline and after the 5 days. Levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-10, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF), IL-1α, and interferon (IFN)-γ were measured using enzyme-linked immunosorbent assay (ELISA). Results. TNF, IL-1α, and IFN-γ were lower than the detection limit of our methods. At the baseline, NRBE produced less MCP-1 but more IL-10 and VEGF when compared with ARBE. CSE exposure reduced NRBE IL-10 production but did not significantly alter MCP-1 and VEGF production. Interestingly, bronchial epithelial cells of asthmatic rats responded differently to CSE. MCP-1 level was decreased and VEGF increased after CSE exposure, whereas IL-10 level did not change in ARBE. Conclusion. Cells isolated from asthmatic rats produced distinct levels of mediators compared with cells isolated from control rats. Furthermore, these cells react differently to CSE exposure.     

VEGF overexpression improves mesenchymal stem cell sheet transplantation therapy for acute myocardial infarction

Cell sheet‐based tissue engineering shows great potential in the treatment of ischaemic heart disease. However, treatment efficacy is compromised by low blood and nutrient supply. The aim of this study was to investigate the effect of pro‐angiogenic vascular endothelial growth factor (VEGF)‐modified mesenchymal stem cell (MSC) sheet transplantation therapy in ischaemic heart failure. Rat MSCs were manipulated to overexpress the VEGF gene. In vitro, the antiapoptotic and paracrine effects were assessed under hypoxic conditions. In vivo, we evaluated the therapeutic effect of VEGF‐modified MSC sheet therapy in a rat model of acute myocardial infarction (AMI). Forty‐five Wistar rats were divided into three groups; one group underwent AMI (control), another underwent AMI and WT sheet transplantation (WT‐MSC) and a third group underwent AMI and VEGF sheet transplantation (VEGF‐MSC). Echocardiography was performed after 3, 10 and 28 days. Samples for histological analysis were collected at the end of the study. The VEGF gene protected MSCs against apoptosis. In vitro, VEGF overexpression significantly reduced MSC apoptosis compared with wild‐type and enhanced VEGF secretion under hypoxic conditions. Capillary density in the infarct border zone was higher in VEGF‐MSC‐transplanted animals than in WT‐MSC‐treated animals. Furthermore, VEGF‐MSC‐transplanted animals had a smaller infarct size than WT‐MSC‐treated animals and exhibited remarkable functional recovery. These findings support the premise that transplantation of proangiogenic gene‐modified MSCs may be valuable for mediating substantial functional recovery after AMI. Copyright © 2012 John Wiley & Sons, Ltd.