mPGES-1-expressing bone marrow-derived cells enhance tumor growth and angiogenesis in mice

Microsomal prostaglandin (PG) E synthase 1 (mPGES-1) is a major PGE synthase and has recently been reported to be expressed at high levels in several cancer types. We previously reported that the PGE receptor EP3 is expressed in bone marrow (BM) derived cells, enriched in stromal tissue, and enhances the potential for tumor angiogenesis. In the present study, we examined the role of mPGES-1-expressing BM cells on tumor angiogenesis using BM chimeric mice. After lethal radiation, wild-type (WT) BMs were excised and replaced with BM cells isolated from mPGES-1 knockout mice (mPGES-1^−/−). Lewis lung carcinoma cell were implanted subcutaneously and the levels of neoangiogenesis were measured tumor growth in mPGES-1^−/− BM chimeric mice was significantly reduced compared to that observed in WT BM chimeric mice. Tumor-associated angiogenesis as measured by histological analysis was localized to tumor stroma, and was significantly lower in mPGES-1^−/− BM chimeric mice compared to that in WT BM chimeric mice. Tumor sections probed by immunohistochemistry revealed that vascular endothelial growth factor (VEGF) that was present in the stromal tissue was markedly reduced in mPGES-1^−/− BM chimeric mice compared to WT BM chimeras. These results suggest that the recruitment of mPGES-1-expressing BM cells to tumor-associated stromal tissue is crucial for tumor growth and angiogenesis, and correlates with gene expression of host VEGF in stroma. Taken together, these data suggest that regulation of mPGES-1-expressing BM cell recruitment to the site of primary tumors may be a novel strategy for the treatment of solid tumors.     

Expression of COX-1 and COX-2 in a clinical model of acute inflammation.

Cyclooxygenase (COX) plays an important role in the induction of pain and inflammation as well as the analgesic actions of NSAIDs and coxibs. This study evaluates the expression of the two isoforms COX-1 and COX-2 in a clinical model in which the surgical removal of impacted third molars is used to evaluate the analgesic activity of anti-inflammatory drugs. A 3-mm punch biopsy was performed on the oral mucosa overlying 1 impacted third molar immediately before extraction of 2 impacted lower third molars. After the second tooth was extracted, a second biopsy was performed adjacent to the surgical site either immediately after surgery or 30, 60, or 120 minutes after surgery. RNA was extracted from the biopsy specimens, and RT-PCR was performed to assess mRNA levels of COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The RT-PCR products in the biopsy specimens were normalized to G3PDH and compared with baseline. COX-2 mRNA was progressively increased at 30, 60, and 120 minutes after surgery (P<.05); COX-1 mRNA was transiently decreased at 60 minutes during the postsurgical period (P<.05). The results demonstrate peripheral elevation of COX-2 after tissue injury, which may contribute to increased prostaglandin E(2) at the site of injury, pain onset, and the analgesic activity of both nonselective NSAIDs and selective COX-2 inhibitors. PERSPECTIVE: This clinical study uses a physiologically relevant model to determine the time course of expression of COX-1 and COX-2 in acute inflammation of the human oral mucosa. This study furthers our understanding of the contribution of the COX isoforms to acute pain.     

LYVE1+ macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth

Two resident macrophage subsets reside in peritoneal fluid. Macrophages also reside within mesothelial membranes lining the peritoneal cavity, but they remain poorly characterized. Here, we identified two macrophage populations (LYVE1^hi MHC II^lo-hi CX₃CR1gfp^lo/− and LYVE1^lo/− MHC II^hi CX₃CR1gfp^hi subsets) in the mesenteric and parietal mesothelial linings of the peritoneum. These macrophages resembled LYVE1⁺ macrophages within surface membranes of numerous organs. Fate-mapping approaches and analysis of newborn mice showed that LYVE1^hi macrophages predominantly originated from embryonic-derived progenitors and were controlled by CSF1 made by Wt1⁺ stromal cells. Their gene expression profile closely overlapped with ovarian tumor-associated macrophages previously described in the omentum. Indeed, syngeneic epithelial ovarian tumor growth was strongly reduced following in vivo ablation of LYVE1^hi macrophages, including in mice that received omentectomy to dissociate the role from omental macrophages. These data reveal that the peritoneal compartment contains at least four resident macrophage populations and that LYVE1^hi mesothelial macrophages drive tumor growth independently of the omentum.     

早期生长反应基因1对巨噬细胞分泌肿瘤坏死因子α的影响

背景：体内外研究已证实肺巨噬细胞合成和分泌肿瘤坏死因子α 等参与肺组织局部损伤和炎症反应，但具体发生机制仍不明确。 目的：观察核转录因子早期生长反应基因1对巨噬细胞分泌肿瘤坏死因子α的影响。设计、时间及地点：随机分组设计，于2004-06／2006—12在湘雅医学院病理学系完成。材料：标准石英粉尘（SiO2）为Sigma公司产品：早期生长反应基因1抗体为Santa Cruz公司产品：小鼠巨噬细胞系RAW264．7购自中科院上海生物细胞研究所细胞库。方法：实验将巨噬细胞分为正常对照组、二氧化硅刺激组、Egr-1＋二氧化硅组和IgG＋二氧化硅组，前两组加无血清培养基，后两组分别加入5，10，20mg／L不同浓度的Egr-1和IgG抗体干预。 主要观察指标：酶联免疫吸附实验检测各组细胞上清中肿瘤坏死因子α蛋白的水平；反转录-聚合酶链反应检测细胞内肿瘤坏死因子amRNA的表达。结果：①与二氧化硅刺激组相比，不同浓度Egr-1＋二氧化硅组细胞上清中肿瘤坏死因子α蛋白水平均下降（P〈0．01），且10mg／L组与5，20mg／L组相比，差异有显著性意义（P〈0．05）。不同浓度IgG＋二氧化硅组相比，上清液中肿瘤坏死因子α蛋白水平差异无显著性意义（F=1．008，P=0．438）。②二氧化硅刺激组肿瘤坏死因子ⅡmRNA表达高于正常对照组（P〈0．01），而Egr-1＋二氧化硅组mRNA表达低于二氧化硅刺激组和IgG＋二氧化硅组（P〈0．01）。结论：二氧化硅致巨噬细胞分泌肿瘤坏死因子α增加可能通过激活核转录因子早期生长反应基因1介导的信号通路而实现。     

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.     

Evidence that peptidoleukotriene is a prerequisite for antigen-dependent thromboxane synthesis in IgG1-passively sensitized guinea pig lungs

Lungs from guinea pigs passively sensitized with an affinity-purified IgG1 antibody produce both leukotriene (LT)D4 and thromboxane (Tx)B2 upon ex vivo antigen challenge. This study was undertaken to determine the possibility of endogenously generated peptido-LTs being a prerequisite for Tx synthesis. In immunoglobulin G1-sensitized lungs, exogenous LTD4 induced TxB2 production with a median effective dose of 4.1 nM, whereas the response to LTE4, LTB4 or platelet-activating factor was relatively weak. Although LTC4 was as potent as LTD4 in stimulating TxB2 generation, LTC4's dose-response curve was shifted significantly to the right by AT-125, an irreversible gamma-glutamyl transpeptidase inhibitor, suggesting that at least a part of LTC4 sensitized lungs with antigen (0.01-30 micrograms/ml ovalbumin) for 20 min precipitated a significant amount of LTD4 production. The levels of LTD4 range from 8 to 26 nM (without taking LTD4 recovery into consideration). This level is 2- to 7-fold greater than the median effective dose value observed with exogenous LTD4. Moreover, pretreatment of sensitized lungs with ICI-198,615 a specific LTD4 antagonist, blocked equally both antigen (IC50 = 0.01 microM)- and LTD4 (IC50 = 0.017 microM)-induced TxB2 production. When sensitized lung fragments were treated with 5 mM AT-125, ICI-198,615 was effective in preventing not only antigen-but also LTC4-dependent production of TxB2 (IC50 = 0.018 and 0.021 microM, respectively). In contrast, neither WEB-2086, a platelet-activating factor antagonist, nor pyrilamine, a histamine antagonist, inhibited antigen and LTD4 responses (IC50 greater than 30 microM). Unlike its effect on antigen response, ICI-198,615 was unable to block Ca2+ ionophore-induced TxB2 production.2     

Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin

BACKGROUND: The mechanisms for the variability in antiplatelet effects of aspirin are unclear. Immature (reticulated) platelets may modulate the antiplatelet effects of aspirin through uninhibited cyclooxygenase (COX)-1 and COX-2. Objectives: To evaluate the role of reticulated platelets in the antiplatelet effects of aspirin. METHODS: Sixty healthy volunteers had platelet studies performed before and 24 h after a single 325-mg dose of aspirin. Platelet studies included light transmission aggregometry; P-selectin and integrin alpha(IIb)beta(3) expression, and serum thromboxane B(2) (TxB(2)) levels. Reticulated platelets and platelet COX-2 expression were measured using flow cytometry. RESULTS: Subjects were divided into tertiles based on the percentage of reticulated platelets in whole blood. Baseline platelet aggregation to 1 microg mL(-1) collagen, and postaspirin aggregations to 5 microm and 20 microm ADP and collagen, were greater in the upper than in the lower tertile of reticulated platelets. Stimulated P-selectin and integrin alpha(IIb)beta(3) expression were also higher in the upper tertile both before and after aspirin. Platelet COX-2 expression was detected in 12 +/- 7% (n = 10) of platelets in the upper tertile, and in 7 +/- 3% (n = 12) of platelets in the lower two tertiles (P = 0.03). Postaspirin serum TxB(2) levels were higher in the upper (5.5 +/- 4 ng mL(-1)) than in the lower tertile (3.2 +/- 2.5 ng mL(-1), P = 0.03), and decreased even further with ex vivo additional COX-1 and COX-2 inhibition. The incidence of aspirin resistance (>or= 70% platelet aggregation to 5 microm ADP) was significantly higher in the upper tertile (45%) than in the lower tertile (5%, P < 0.0001). CONCLUSIONS: Reticulated platelets are associated with diminished antiplatelet effects of aspirin and increased aspirin resistance, possibly because of increased reactivity, and uninhibited COX-1 and COX-2 activity.     

Serum levels of tumor necrosis factor and IL-1 alpha and IL-1 beta in diabetic patients

OBJECTIVE: To determine whether chronic hyperglycemia causes increased levels of serum tumor necrosis factor (TNF) and interleukin 1 alpha (IL-1 alpha) and IL-1 beta. RESEARCH DESIGN AND METHODS: Sera were obtained from 59 diabetic patients, 44 chronically ill nondiabetic patients, and 34 age-matched healthy control subjects. Mononuclear cells were isolated from a subgroup of diabetic patients and healthy control subjects. RESULTS: Except for a modest increase in the prevalence of detectable serum TNF levels in diabetic patients, the serum cytokines measured in this study did not appear to be altered in diabetes. In vitro TNF production by mononuclear cells was not altered in diabetic patients. However, in vitro IL-1 beta secretion, in response to lipopolysaccharides, was reduced. CONCLUSIONS: Diabetes mellitus is not associated with significant changes in serum levels of TNF, IL-1 alpha, or IL-1 beta. In vitro secretion of IL-1 beta in response to lipopolysaccharides may be reduced in diabetes.     

Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme

Proliferating embryonic and cancer cells preferentially use aerobic glycolysis to support growth, a metabolic alteration commonly referred to as the "Warburg effect." Here, we show that the glycolytic enzyme hexokinase 2 (HK2) is crucial for the Warburg effect in human glioblastoma multiforme (GBM), the most common malignant brain tumor. In contrast to normal brain and low-grade gliomas, which express predominantly HK1, GBMs show increased HK2 expression. HK2 expression correlates with worse overall survival of GBM patients. Depletion of HK2, but neither HK1 nor pyruvate kinase M2, in GBM cells restored oxidative glucose metabolism and increased sensitivity to cell death inducers such as radiation and temozolomide. Intracranial xenografts of HK2-depleted GBM cells showed decreased proliferation and angiogenesis, but increased invasion, as well as diminished expression of hypoxia inducible factor 1α and vascular endothelial growth factor. In contrast, exogenous HK2 expression in GBM cells led to increased proliferation, therapeutic resistance, and intracranial growth. Growth was dependent on both glucose phosphorylation and mitochondrial translocation mediated by AKT signaling, which is often aberrantly activated in GBMs. Collectively, these findings suggest that therapeutic strategies to modulate the Warburg effect, such as targeting of HK2, may interfere with growth and therapeutic sensitivity of some GBMs.     

转化生长因子β1在白细胞介素1β诱导大鼠肾小管上皮细胞转分化及大黄素干预中的作用

目的:大黄素对白细胞介素1β诱导NRK52E细胞转分化有显著抑制作用。实验拟进一步观察转化生长因子β1在白细胞介素1β诱导大鼠肾小管上皮细胞-肌成纤维细胞转分化及大黄素抑制作用中的意义。方法:实验于2006-10/2007-05在泸州医学院附属医院免疫实验室完成。⑴实验材料及分组:以培养的大鼠肾小管上皮细胞株(NRK52E)为观察对象,按如下分组分别添加不同处理因素:①对照组:仅加入体积分数为0.05小牛血清的高糖DMEM培养基。②白细胞介素1β诱导组:加含白细胞介素1β终浓度为10μg/L的高糖DMEM培养基。③SB431542阻断组:加含白细胞介素1β终浓度为10μg/L及SB431542终浓度为10μmol/L的高糖DMEM培养基。④白细胞介素1β 大黄素组:同时加分别含白细胞介素1β终浓度为10μg/L及大黄素终浓度为25mg/L的高糖DMEM培养基。⑵实验评估:培养48h后用倒置相差显微镜观察细胞形态,细胞免疫化学染色法检测肌酸激酶、α-平滑肌肌动蛋白及转化生长因子β1的表达。结果:①白细胞介素1β可诱导部分细胞由卵圆形转变为梭形,且肌酸激酶表达减弱(P<0.01),α-平滑肌肌动蛋白及转化生长因子β1表达显著增强(P<0.01)。②SB431542特异性抑制转化生长因子β1作用后,白细胞介素1β诱导的细胞形态改变受抑,同时肌酸激酶表达增强(P<0.01),α-平滑肌肌动蛋白表达减弱(P<0.01),但转化生长因子β1的表达却无明显变化。③大黄素对白细胞介素1β诱导的细胞形态改变及肌酸激酶、α-平滑肌肌动蛋白的表达有明显抑制作用,其抑制作用与SB431542的作用相比无显著差异;同时,大黄素对白细胞介素1β诱导的转化生长因子β1的表达也有明显抑制作用(P<0.01)。结论:转化生长因子β1可能介导了白细胞介素1β诱导大鼠肾小管上皮细胞-肌成纤维细胞转分化,并参与了大黄素抑制白细胞介素1β诱导大鼠肾小管上皮细胞转分化的作用。     

肥大细胞、巨噬细胞及转化生长因子在IgA肾病患者肾组织中的浸润及意义

目的:观察肥大细胞、巨噬细胞、转化生长因子β1在IgA肾病患者肾组织中的浸润表达,并分析其意义。方法:①收集2004-06/2006-02青岛大学医学院附属医院肾内科收治的26例IgA肾病住院患者的肾活检标本,均经肾活检确诊肾小球系膜区存在以IgA为主的免疫复合物,并经临床及实验室检查排除紫癜性肾炎、狼疮性肾炎等继发性IgA肾病,临床资料及病理资料完整,患者全部签署肾穿刺知情同意书。另取5例配型不合的移植肾或肾穿刺后病理结果正常的肾组织(捐献者均签署捐献协议)作为正常对照组。②两组标本均行常规组织化学染色及特染,采用免疫组织化学技术检测肥大细胞、巨噬细胞在肾组织中的浸润及转化生长因子β1的表达,肥大细胞与巨噬细胞阳性呈黄褐色,转化生长因子β1阳性呈深黄色。③IgA肾病活动性指标包括每系膜区系膜细胞增生数、间质炎性细胞浸润细胞性新月体占肾小球面积的百分比3项。慢性化指标包括纤维性新月体占肾小球面积、局灶性节段性硬化占肾小球面积、肾小管萎缩占肾小管间质面积、间质纤维化程度占肾小管间质面积4项。④单盲法分析肥大细胞、巨噬细胞及转化生长因子β1的相关性及其与IgA肾病活动性指标、慢性化指标、血肌酐、尿蛋白之间的关系。结果:①肥大细胞、巨噬细胞的浸润及转化生长因子β1的表达:正常肾组织中偶见或无肥大细胞、巨噬细胞和转化生长因子β1的表达,而患肾中肥大细胞、巨噬细胞的浸润数量和转化生长因子β1的表达均明显增强(t=3.82～7.27,P<0.01)。②肥大细胞、巨噬细胞、转化生长因子β1与临床指标的相关性:相应部位肥大细胞、巨噬细胞的浸润数量及转化生长因子β1的表达呈显著正相关(r=0.51～0.91,P均<0.01)。肥大细胞数量与慢性化指标、血肌酐呈显著正相关(r=0.87,0.69,P均<0.01),与活动性指标和尿蛋白定量无明显相关性(r=0.30,0.21,P均>0.05);巨噬细胞数量和转化生长因子β1与活动性指标、慢性化指标、血肌酐、尿蛋白定量均呈显著正相关(r=0.39～0.90,P均<0.01;r=0.34～0.68,P<0.01或0.05)结论:①肥大细胞可能是IgA肾病肾间质纤维化的重要参与者,其数量增加可能预示着IgA肾病的慢性化和预后不良。②巨噬细胞可能是IgA肾病发生发展过程的全程参与者,它的浸润可能是肥大细胞在肾间质中浸润的前提条件之一。③转化生长因子β1不仅是贯穿始终的重要的致纤维化因子,而且可能是巨噬细胞和肥大细胞的重要趋化因子,并能促进它们的增殖、活化、发挥生物学作用。     

CKAP4 is a Dickkopf1 receptor and is involved in tumor progression

Dickkopf1 (DKK1) is a secretory protein that antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor–related protein 6 (LRP6). DKK1 may also regulate its own signaling to promote cancer cell proliferation, but the mechanism is not understood. Here, we identified cytoskeleton-associated protein 4 (CKAP4) as a DKK1 receptor and evaluated CKAP4-mediated DKK1 signaling in cancer cell proliferation. We determined that DKK1 binds CKAP4 and LRP6 with similar affinity but interacts with these 2 receptors with different cysteine-rich domains. DKK1 induced internalization of CKAP4 in a clathrin-dependent manner, further supporting CKAP4 as a receptor for DKK1. DKK1/CKAP4 signaling activated AKT by forming a complex between the proline-rich domain of CKAP4 and the Src homology 3 domain of PI3K, resulting in proliferation of normal cells and cancer cells. Expression of DKK1 and CKAP4 was frequent in tumor lesions of human pancreatic and lung cancers, and simultaneous expression of both proteins in patient tumors was negatively correlated with prognosis and relapse-free survival. An anti-CKAP4 antibody blocked the binding of DKK1 to CKAP4, suppressed AKT activity in a human cancer cell line, and attenuated xenograft tumor formation in immunodeficient mice. Together, our results suggest that CKAP4 is a potential therapeutic target for cancers that express both DKK1 and CKAP4.     

Focus on the COX-1 and COX-2 agents: renal events of nonsteroidal and anti-inflammatory drugs-NSAIDs

This article will focus upon some of the cautions used in the process of prescribing NSAIDs with a focus upon renal events, pharmacokinetics of COX-2 agents, and phytopharmaceuticals that present co-prescribing hematologic challenges. Prescribing any pharmacotherapeutic agent presents the clinician with the cognitive challenge between providing a therapeutic balance weighing potential benefits to be achieved through prescribing against the potential liabilities of pharmacokinetic and pharmacodynamic iatrogenic events, and drug interactions. The following information is presented as a brief overview of the familiar arachidonic acid cascade followed by the renal events reported with non-COX-2-specific NSAIDs. The pharmacokinetics of the three currently available COX-2 NSAIDs are presented. Patient-specific risk assessments for renal function/dysfunction should be considered prior to or concurrent with initiation of any NSAID therapy coupled with periodic renal monitoring during treatment of those with patient risk factors. Phytopharmaceuticals, supplements, and over-the-counter agents should be discussed with the patient following patient disclosure of use and not omitted by the patient during presentation of their medication consumption with utilization history to their respective healthcare professionals.     

Distinct isoforms (COX-1 and COX-2) of cyclooxygenase: possible physiological and therapeutic implications

Summary— The discovery of an inducible isoform of cyclooxygenase (COX-2) requires a refinement of the theory that inhibition of cyclooxygenase activity explains both therapeutic and side effects of non-steroidal anti-inflammatory drugs (NSAIDs). Indeed, new pharmacological results suggest that COX-2 inhibition provides the therapeutic (ie, anti-inflammatory) activity of NSAIDs, whereas inhibition of constitutive COX-1 is responsible for their gastric and renal side effects as well as for their antithrombotic activity. However, a role of COX-1 in inflammation cannot be excluded. Furthermore, the functional relevance of COX-2 expression and induction in various tissues warrants further investigation. These studies should help in predicting potential adverse effects as well as new indications for selective COX-2 inhibitors.