Attenuation of the exercise-induced increase in skeletal muscle Flt-1 mRNA by nitric oxide synthase inhibition

We investigated the vascular endothelial growth factor (VEGF) receptor [fms-like-tyrosine kinase (Flt-1 and fetal liver kinase-1 (Flk-1)] response to acute exercise. In female Wistar rats, the VEGF receptor messenger RNA (mRNA) response to a single acute exercise bout was examined using semi-quantitative Northern blot from the left gastrocnemius muscles at rest and post-exercise at 0, 1, 2, 4, 8, 16, 24 and 48 h. Exercise altered both Flt-1 and Flk-1 mRNA, with significant increases in Flt-1 mRNA at 1 and 24 h. However, post-hoc analysis was unable to discern the time point where a significant increase in Flk-1 mRNA occurred. To investigate the regulation of Flt-1 mRNA by exercise we examined if nitric oxide synthase (NOS) inhibition alters the Flt-1 mRNA response. Eight groups [Condition: Rest or Exercise; Drug: Saline, 30 mg kg(-1)N(omega)-nitro-L-arginine methyl ester (L-NAME), 300 mg kg(-1) L-NAME or 300 mg kg(-1) D-NAME] were used to determine the effect of NOS inhibition on the Flt-1 mRNA response to exercise. L-NAME, a known NOS inhibitor, attenuated the exercise-induced increase in Flt-1 mRNA by approximately 50%. These findings suggest that: (1) exercise alters Flt-1 and Flk-1 gene expression; and (2) NO is important in the regulation of the Flt-1 gene response to exercise.     

Adenosine and nitric oxide in exercise‐induced human skeletal muscle vasodilatation

The vasoactive substances adenosine and nitric oxide (NO) are credible candidates in the local regulation of skeletal muscle blood flow. Adenosine and NO have both been shown to increase in skeletal muscle cells and interstitial fluid during exercise and the enzymes responsible for their formation, AMP 5′‐nucleotidase and NO synthase (NOS), have been shown to be activated upon muscle contraction. In vitro as well as in vivo evidence suggest that the contraction‐induced increase in interstitial adenosine concentration largely stems from extracellular formation via the membrane‐bound ecto‐form of AMP 5′‐nucleotidase. It remains unclear whether the exercise‐induced NO formation in muscle originates from endothelial NOS in the microvascular endothelium, or from neuronal NOS (nNOS) in nerve cells and muscle fibres. Functional evidence for the role of adenosine in muscle blood flow control stems from studies using adenosine receptor agonists and antagonsits, adenosine deaminase or adenosine uptake inhibitors. The majority of these studies have been performed on laboratory animals and, although the results show some discrepancy, the majority of studies indicate that adenosine does participate in the regulation of muscle blood flow. In humans, evidence is lacking. The role of NO in the regulation of skeletal muscle blood flow has mainly been studied using NOS inhibitors. Despite a large number of studies in this area, the role of NO for the contraction‐induced increase in skeletal muscle blood flow is uncertain. The majority, but not all, human and animal studies show that, whereas blockade of NOS reduces muscle blood flow at rest and in recovery from exercise, there is no effect on the exercise‐induced increase in muscle perfusion. Conclusive evidence for the mechanisms underlying the precise regulation of the multiphased increase in skeletal muscle blood flow during exercise and the role and potency of various vasoactive substances, remain missing.     

Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors,Flk‐1 and Flt‐1

The biological effects of vascular endothelial growth factor A (VEGF‐A) are mediated by fetal liver kinase‐1 (Flk‐1) and fms‐like tyrosine kinase‐1 (Flt‐1). In lung tissue, VEGF‐A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk‐1 and Flt‐1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk‐1 and Flt‐1 of embryonic mouse lung tissue. When Flk‐1 was predominantly expressed (embryonic day [E] 9.5–E13.5), then vascular endothelial cells actively proliferated. When Flt‐1 was enhanced (E14.5–E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk‐1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt‐1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt‐1 promoted Flk‐1 expression. These results suggest that the two VEGF‐A receptors regulate pulmonary vascular development by modulating the VEGF‐A signaling. Anat Rec, 290:958–973, 2007. © 2007 Wiley‐Liss, Inc.     

人血管内皮生长因子受体Flt—l胞外配体结合域的筛选及其结构分析

目的 应用酵母双杂交系统筛选人血管内皮生长因子受体Flt－1胞外最小配合结合域。方法 采用PCR技术,从人胎盘cDNA文库扩增出4个截短的Flt-1cDNA,分别含胞外第2、1、－2、2－3和1－3个loop,构建酵母双杂交系统配合表达质粒,并将pGB59／hVEGF165与pGAD424／Flt-ls两两配对转化酵母菌SFY526。采用滤纸法和液体培养法对阳性克隆进行β－半乳糖苷酶活性分析。结果 Flt－1胞外loop2－3与loop1-3的配体结合能力相关不大,loop1-2的结合力较弱,单独第2个loop无配体结合域－2－3loop,这对研制分子量更小、安全性高的可溶性Flt-1片段,开发其在肿瘤、糖尿病性网膜病变等血管生成相关疾病基因治疗中的应用具有重要的指导意义。     

原发性肝癌患者手术前后血清SICAM-1、VEGF水平检测的临床意义

目的:分析了31例原发性肝癌患者手术前后血清SICAM-1、VEGF水平的变化。方法:采用双抗 体夹心法测定了31例原发性肝癌患者手术前后血清SICAM-1、VEGF含量,并与35名正常人作比较。结果: 手术前血清SICAM-1、VEGF水平非常显著地高于正常人组(P<0.01),手术后6个月,复发者SICAM-1、 VEGF水平持续异常,未复发者SICAM-1、VEGF水平恢复正常。结论:观察原发性肝癌患者血清SICAM-1 和VEGF水平的变化与原发性肝癌患者的病情和预后密切相关。     

Calorie restriction increases insulin‐stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate‐1 in rat skeletal muscle

A moderate reduction in calorie intake (calorie restriction, CR) improves insulin‐stimulated glucose transport in skeletal muscle. Therefore, we studied muscle insulin signalling in ad libitum (AL) and CR (~60% AL intake for 20 days) fed rats, which received a control injection (sterile water) or an insulin injection (30 U kg^–1 body weight). In control (not insulin‐treated) rats, there was no detectable tyrosine phosphorylation of insulin receptor (IR), regardless of diet; no diet effect on tyrosine phosphorylation of insulin receptor substrate‐1 (IRS1) or IRS1‐associated phosphatidylinositol 3‐kinase (PI3K) protein and 21% higher IRS1‐associated PI3K activity in AL vs. CR. In insulin‐treated rats, tyrosine‐phosphorylated IR was 79% higher for CR vs. AL; tyrosine‐phosphorylated IRS1 was 109% higher for CR vs. AL; IRS1‐associated PI3K protein and IRS1‐associated PI3K activity were unaffected by diet. Calorie restriction amplifies early insulin signalling steps without changing IRS1‐associated PI3K, suggesting enhanced glucose transport is mediated by altering: IRS1‐PI3K localization, PI3K associated with proteins other than IRS1 or post‐PI3K events.     

Prognostic role of insulin‐like growth factor receptor‐1 expression in small cell lung cancer

Chang MH, Lee J, Han J, Park YH, Ahn JS, Park K, Ahn M‐J. Prognostic role of insulin‐like growth factor receptor‐1 expression in small cell lung cancer. APMIS 2009; 117: 861–9. Insulin‐like growth factor receptor‐1 (IGFR‐1) is a cellular membrane receptor which is overexpressed in many tumors and seems to play a critical role in anti‐apoptosis. The insulin‐like growth factor binding protein‐3 (IGFBP‐3) is known as a growth suppressor in multiple signaling pathways. The aim of this study was to determine IGFR‐1 and IGFBP‐3 expression in small‐cell lung cancer (SCLC) and analyze the prognostic value in patients with SCLC. We analyzed IGFR‐1 and IGFBP‐3 expression in 194 SCLC tissues by immunohistochemical staining. Correlative analyses between IGFR‐1 and IGFBP‐3 expression in SCLC and clinicopathologic factors were performed. A total of 117 patients had extensive disease (ED) (60.3%) and 77 had limited disease (39.7%). With the median follow‐up duration of 49.5 months (24–82 months), the median progression‐free survival (PFS) and overall survival (OS) were 7.2 months [95% confidence interval (CI): 6.4–8.0 months] and 14.4 months (95% CI: 12.7–16 months), respectively. IGFR‐1 expression was observed in 154 of the 190 tumor tissues, whereas there was no IGFBP‐3 expression. Multivariate analysis showed that stage (p < 0.001), response rate (p < 0.001), and lactate dehydrogenase (LDH) levels (p < 0.001) were the independent prognostic factors for PFS, and age (p = 0.014), LDH level (p < 0.001), and stage (p < 0.001) for OS. The IGFR‐1 positivity was not associated with PFS or OS in the entire cohort. Subgroup analysis revealed that OS was significantly longer in patients with IGFR‐1‐positive tissue than IGFR‐1‐negative tissue in SCLC‐ED (p = 0.034). These results suggest that IGFR‐1 expression may be useful as a prognostic marker in patients with SCLC‐ED.     

原发性肝癌患者手术前后血清SICAM-1、L-Selection和VEGF检测的临床意义

目的：探讨原发性肝癌患者手术前后血清可溶性细胞间黏附分子-1（SICAM-1）、L-选择素（L—Selection）和血管内皮生长因子（VEGF）水平的变化。方法：采用双抗体夹心法（ELISA）对35例原发性肝癌患者进行手术前后血清SICAM-1、L—Selection和VEGF测定,并与30名正常健康人作比较。结果：手术前血清SICAM-1、L—Selection和VEGF水平非常显著地高于正常人组（P〈0．01）。手术后6个月,复发者血清SICAM-1、L-Selection和VEGF水平持续异常;未复发者SICAM-1、L—Selection和VEGF水平恢复正常。结论：观察原发性肝癌患者血清SICAM-1、L—Selection和VEGF水平的变化与原发性肝癌患者的病情和预后密切相关．     

The role of high‐mobility group box protein 1 in collagen antibody‐induced arthritis is dependent on vascular endothelial growth factor

High‐mobility group box 1 (HMGB1) has been implicated in angiogenesis and rheumatoid arthritis (RA). The aim of this study was to define more clearly the role of HMGB1 in the synovial angiogenesis and pathogenesis of an immune model of arthritis. BALB/c mice were injected with monoclonal anti‐collagen antibody cocktail followed by lipopolysaccharide to induce arthritis. HMGB1 and vascular endothelial growth factor (VEGF) were over‐expressed in the areas of the synovium where more inflammation and neoangiogenesis were present. The selective blockade of HMGB1 or VEGF resulted alternatively in a lower severity of arthritis evaluated by the arthritis index. Furthermore, exogenous HMGB1 administration caused a worsening of arthritis, associated with VEGF up‐regulation and increased synovial angiogenesis. The selective inhibition of VEGF also resulted in no induction of arthritis in mice receiving exogenous HMGB1. Cytokine enzyme‐linked immunosorbent assay (ELISA) analyses performed on peripheral blood and synovial fluid demonstrated a significant reduction of interleukin (IL)?1β, IL‐6 and tumour necrosis factor (TNF)‐α in mice where HMGB1 and VEGF pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL‐17A concentration. The development of arthritis mediated by HMGB1 and the synovial angiogenesis can be blocked by inhibiting the VEGF activity. The proinflammatory and proangiogenic cytokine IL‐17A was increased when HMGB1 is inhibited, but the synovial angiogenesis was nevertheless reduced in this model of arthritis. Taken together, these findings shed new light on the role of this nuclear protein in the pathogenesis of arthritis in an RA‐like model.     

Expression of vascular Notch ligands Delta-like 4 and Jagged-1 in glioblastoma

Jubb A M, Browning L, Campo L, Turley H, Steers G, Thurston G, Harris A L & Ansorge O
(2012) Histopathology  60, 740–747
Expression of vascular Notch ligands Delta‐like 4 and Jagged‐1 in glioblastoma Aims: The coordinated expression of the Notch ligands Delta‐like 4 (Dll4) and Jagged (Jag)1 is believed to define appropriate endothelial sensitivity to vascular endothelial growth factor (VEGF). Preclinical data suggest that Dll4‐Notch signalling may confer resistance to anti‐VEGF therapy with bevacizumab, and Jag1 may antagonize Dll4–Notch. The aims of this study were to characterize the expression of Dll4 and Jag1 in primary glioblastomas. Methods and results: Immunohistochemistry was performed on 40 glioblastomas and normal brain using validated antibodies against Dll4 and Jag1. In‐situ hybridization for Dll4 was performed on serial sections and compared with protein expression. Dll4 expression was localized to the cytoplasm and membrane of endothelial cells in all glioblastomas; it was weak or absent in normal brain. Jag1 expression was observed in the cytoplasm and membrane of glomeruloid and non‐glomeruloid endothelial cells from 76% and 67% of glioblastomas, respectively. However, endothelial Jag1 expression was less intense and less prevalent than Dll4. There was no association between Dll4 and Jag1 expression. Conclusions: In summary, Dll4 and Jag1 are expressed in glioblastoma vasculature. These data may define subsets of glioblastoma that might be sensitive (Dll4⁺/Jag1⁺) or resistant (Dll4⁺/Jag1^–) to bevacizumab. Our data also suggest that anti‐Dll4 therapy should be evaluated experimentally in glioblastoma.     

Autologous cytokine‐induced killer (CIK) cell immunotherapy combined with cyclophosphamide in five patients with POEMS syndrome

The primary objective of this study was to evaluate the safety and clinical efficacy of autologous cytokine‐induced killer (CIK) cells combined with cyclophosphamide in the treatment of polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) syndrome patients. We evaluated five POEMS syndrome patients treated with autologous CIK cell immunotherapy combined with cyclophosphamide from 1 May 2012 to 30 November 2014. The Overall Neuropathy Limitation Scale (ONLS), computed tomography of the chest and abdomen, ultrasound of the abdomen, serum vascular endothelial growth factor (VEGF) level and lymphocyte count findings in the five patients were recorded. The median age of the patients was 40 years (range: 25–62), and all the patients were male. CIK cells were generated routinely from peripheral blood mononuclear cells (PBMCs) of all five patients, and the numbers of CIK cells increased by approximately 105‐fold after 14 days of culture. All five patients (100%) responded to their neuropathy treatment, the ONLS scores were reduced by at least 1 and a paired‐sample t‐test revealed a significant difference (t = 5·715, P = 0·003 < 0·01). The extravascular volume overload responses indicated partial remission (PR = 60%) or stable disease (SD = 40%), and no cases of progressive disease (PD) or complete remission (CR) were observed. During clinical treatment, the serum VEGF of patient 5 decreased after one cycle of transfusion within 1 month. The lymphocyte counts of all the patients increased significantly after CIK transfusion, and a paired‐sample t‐test revealed a significant difference (t = 5·101, P = 0·004 < 0·01). Autologous CIK cell infusion combined with cyclophosphamide was found to be highly safe and elicited no adverse reactions. CIK cells can improve both the symptoms and quality of life, decrease serum VEGF levels and increase lymphocyte counts in patients with POEMS syndrome.     

Inhibited anabolic effect of insulin‐like growth factor‐I on stromal bone marrow cells in endothelial nitric oxide synthase‐knockout mice

Aims: Insulin‐like growth factor‐I (IGF‐I), parathyroid hormone (PTH) and PTH‐related protein (PTHrP) are hormones that have anabolic effects on bone formation. The aim of this study was to investigate whether production of nitric oxide (NO) is involved in the effect of IGF‐I and PTH/PTHrP on osteoblast‐like cells. Methods: Bone marrow stromal cells from adult endothelial nitric oxide synthase (eNOS)‐knockout (eNOSKO) mice and wild type (WT) counterparts were cultivated with osteogenic substances. The cells showed an osteoblastic phenotype measured as osteocalcin production and alkaline phosphatase activity. DNA synthesis was measured as [³H] thymidine incorporation in the bone marrow cells and in a human osteosarcoma cell‐line (SaOS‐2). Results: The stimulatory effect of IGF‐I on thymidine incorporation seen in WT animals was absent in eNOSKO mice. Addition of a NO donor to eNOSKO cells recovered the effect of IGF‐I on thymidine incorporation. PTH/PTHrP stimulated cell proliferation in both WT and eNOSKO mice. In SaOS‐2 cells, incubation with IGF‐I together with a NOS inhibitor resulted in an inhibition of the anabolic effect of IGF‐I on cell proliferation. Conclusions: The stimulatory effect of IGF‐I on WT cell proliferation was abolished in eNOSKO cells, recovered by an NO donor and inhibited in osteosarcoma cells by a NOS inhibitor. The results indicate that the effect of IGF‐I is dependent on NO production. The impaired IGF‐I response may contribute to the bone defect formation seen in eNOSKO animals.     

sFlt-1mRNA在子痫前期患者胎盘中的表达

目的检测可溶性血管内皮生长因子受体-1（soluble fms-like tyrosine kinase-1,sFlt-1）在子痫前期胎盘组织中的mRNA表达水平,探讨其在子痫前期发生发展中的作用。方法用半定量逆转录聚合酶链反应（RT-PCR）技术检测25例子痫前期患者（轻度（n=8）,重度（n=17））和15例正常妊娠孕妇。结果（1）sFlt-1mRNA在正常孕妇、轻度和重子痫前期患者胎盘组织中均有表达。（2）sFlt-1mRNA的表达水平在轻度和重度子痫前期组均高于正常组,差别有显著性（P〈0.01）。结论胎盘组织中sFlt-1mRNA的过度表达可能与子痫前期的发病有关,并参与了子痫前期的病理生理过程。     

Enhanced beta-adrenergic response in rat papillary muscle by inhibition of inducible nitric oxide synthase after myocardial infarction

Aim: Myocardial infarction (MI) induces a progressive ventricular remodelling leading to a contractility depression. During the acute phase of MI inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production increases in the heart. The aim of this study was to investigate the role of iNOS in the left ventricular contractility at 3 days after MI. Methods: Wistar rats were divided into: sham operated (SHAM, n = 23), infarction (INF, n = 18); sham operated plus the iNOS inhibitor, S‐methylisothiourea (SMT) 5 mg kg^?1 day^?1, i.p. treatment (SHAM‐SMT, n = 26) and infarction plus SMT (INF‐SMT, n = 22). Concentration–response curves for isoprenaline, Ca²⁺ and frequency–force curve were studied in isolated papillary muscle from left ventricle. Results: After 3 days infarct area was similar between groups. SMT treatment reduced the time to peak tension during frequency–force curve in the infarct group (SHAM = 63 ± 3; SHAM‐SMT = 71 ± 3; INF = 90 ± 4; INF‐SMT = 79 ± 4 ms, P < 0.05) and increased the maximal response to isoprenaline (SHAM = 0.93 ± 0.11; SHAM‐SMT = 1.13 ± 0.1; INF =0.84 ± 0.16; INF‐SMT = 1.49 ± 0.15 g mm^?2, P < 0.05). The response to Ca²⁺ was equally reduced in the INF and INF‐SMT groups. SMT treatment did not change the reduced post‐rest potentiation performed by INF group, but attenuated the plasma nitrite and nitrate (NOx) levels in the INF group without any haemodynamic effect. Conclusion: These finding suggest that at 3 days after MI the iNOS modulates the isolated papillary muscle response to isoprenaline and its inhibition improves the β‐adrenergic inotropic responses.