Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with its functional ability to inhibit angiogenesis and tumor growth in vivo. We have shown that the soluble anti-angiogenic domain of BAI1 (termed Vstat120) requires CD36, a cell surface glycoprotein expressed on microvascular endothelial cells (MVECs), for it to elicit an anti-angiogenic response. We now report that Vstat120 binding to CD36 on MVECs activates a caspase-mediated pro-apoptotic pathway, and this effect is abrogated by histidine-rich glycoprotein (HRGP). HRGP is a circulating glycoprotein previously shown to function as a CD36 decoy to promote angiogenesis in the presence of thrombospondin-1 or −2. Data here show that Vstat120 specifically binds HRGP. Under favorable MVEC growth conditions this interaction allows chemotactic-directed migration as well as endothelial tube formation to persist in in vitro cellular systems, and increased tumor growth in vivo as demonstrated in both subcutaneous and orthotopic brain tumor models, concomitant with an increase in tumor vascularity. Finally, we show that HRGP expression is increased in human brain cancers, with the protein heavily localized to the basement membrane of the tumors. These data help define a novel angiogenic axis that could be exploited for the treatment of human cancers and other diseases where excess angiogenesis occurs.Angiogenesis is the process through which new blood vessels are formed from pre-existing capillaries. In adulthood the vasculature is normally quiescent, a homeostatic state that is maintained by a precise balance of pro-angiogenic inducers such as vascular endothelial growth factor and anti-angiogenic inhibitors such as thrombospondin (TSP)−1 and −2. This angiostatic balance can be physiologically regulated in favor of new blood vessel formation during processes such as menstruation and wound healing allowing normal tissue regeneration; while pathological disruption of this balance is associated with many disease states, including diabetic retinopathy, atherosclerosis-induced tissue ischemia, chronic inflammation, tumor growth and metastasis, obesity, asthma, and several autoimmune diseases.¹ Therefore, it is extremely important to identify and dissect the pathways involved in vessel growth in both normal and aberrant conditions.Neovascularization is a major component of malignant tumor growth and many therapeutic strategies have been developed to inhibit tumor angiogenesis, including antibodies or decoys that bind and neutralize vascular endothelial growth factor,^2,3 small molecule inhibitors of growth factor signaling pathways,⁴ and peptides based on the anti-angiogenic type I repeat domains (TSR) of TSP-1.^5,6,7 Studies on the mechanisms of TSP-mediated anti-angiogenesis revealed that the TSR domains play an essential role⁸ and that the type B scavenger receptor CD36 functions as the critical endothelial cell surface receptor.^9,10 Using mouse corneal pocket angiogenesis assays we recently demonstrated that CD36 also functions as the receptor for a 120 kDa anti-angiogenic fragment derived from an unrelated TSR-containing protein, Brain Angiogenesis Inhibitor 1 (BAI1). This fragment, known as vasculostatin or Vstat120, suppressed neovessel formation in corneas from wild-type mice yet no effect was observed in CD36 null animals, showing for the first time that a TSR-containing protein distinct from TSP-1 and −2 mediates its anti-angiogenic functions through interactions with CD36.¹¹BAI1 is a 1584-aa brain-specific protein predicted to have seven transmembrane segments and a large extracellular domain. The extracellular domain contains an RGD integrin recognition motif, a putative hormone receptor (HomR) domain, and five TSR domains. Its expression is down-regulated in glioblastomas¹² and inversely correlated with vascularity and metastasis in colorectal cancer,¹³ consistent with an anti-angiogenic role. Kaur et al¹⁴ have shown that the TSR-containing fragment, Vstat120, was released from the cell membrane via proteolytic cleavage at a G protein–coupled receptor cleavage site and that this fragment inhibited microvascular endothelial cell (MVEC) proliferation, migration, and tube formation equivalent to that seen with full-length BAI1. Strikingly, restoration of Vstat120 expression in human glioma cells suppressed tumorigenicity and vascularity and enhanced animal survival in subcutaneous and orthotopic tumor implantation models in nude mice.¹¹The binding site on CD36 for the TSR domains of TSP-1 and −2 and Vstat120 has been localized to amino acids 93 to 120^11,15,16 within a highly conserved region termed the CLESH domain (CD36, LIMP2, EMP structural homology – see Figure 1A). CLESH domains are also found in proteins genetically distinct from the CD36 family,¹⁷ including some, such as HIV gp120 and histidine-rich glycoprotein (HRGP) which are known to bind TSP-1. HRGP, a 75-kDa glycoprotein synthesized in the liver, circulates in the blood at moderately high concentrations (approximately 100 to 150 μg/ml), and is secreted from activated platelets. In addition to TSP-1 and −2, it binds a wide range of ligands including divalent metal cations and several components of the provisional matrix laid down by healing wounds and tumors where angiogenesis is prevalent (for review see¹⁸). It has been implicated in the coagulation and fibrinolytic systems, and high levels have been associated with thrombotic disorders.^19,20 Work in our lab has shown that HRGP acts as a CD36 decoy to promote angiogenesis by binding TSP-1 and −2 and thereby preventing them from binding to CD36 on the endothelial cell surface.^21,22 Because both HRGP and CD36 compete for the identical binding region in TSP-1 and −2, we hypothesized that the relative concentrations of ligands, receptor, and decoy within a given microenvironment would ultimately determine the degree of angiogenesis. Within tumors and other areas of chronic inflammation, HRGP can be released from activated platelets as well as deposited from plasma that gained access via poorly organized hyperpermeable vessels. The angiogenic balance would then be tipped to favor angiogenesis by HRGP binding to TSPs as well as potentially other TSR containing anti-angiogenic proteins.Open in a separate windowFigure 1HRGP CLESH domain fusion protein specifically precipitates Vstat120 from tumor cell postculture media. A: HRGP/GST fusion proteins. Three recombinant GST fusion proteins were designed using fragments of the HRGP protein spanning amino acids 155 to 213 (within the second cystatin domain), amino acids 330 to 389 (the HRR), and amino acids 428 to 502 (CLESH domain). The linear structure of CD36 with the CLESH domain highlighted is shown below for reference. B: Coomassie stained gel (top panel) showing the purified GST-HRGP protein fragments after glutathione sepharose chromatography. The bottom panel shows Western blot analysis of each fusion protein probed with anti-GST antibody. C: Western blot analysis of a GST pull-down assay. The three GST-HRGP fusion proteins and GST alone were bound to glutathione sepharose beads, and CM from LN229V120 glioma cells were added to each sample. Each precipitate was then probed using the Vstat120/BAI1-specific antibody. Vstat120 was only detected when the CLESH domain (HRGP 428 to 502) was used in the pull-down and not the cystatin (HRGP 155 to 213) or the HRR (HRGP 330 to 389) domains. HRGP 428 to 502 only precipitated Vstat120 when CM from Vstat120-transfected LN229 cells (+ lanes) was used. No protein was detected when CM from the nontransfected parent cells (− lane) was used. This is a representative image from n = 3 experiments.In this article we show that the CLESH domain of HRGP binds Vstat120 and suppresses its anti-angiogenic activity by reversing inhibition of endothelial cell migration and tube formation. Furthermore, we show in both subcutaneous and orthotopic brain tumor models that HRGP exacerbates glioblastoma tumor growth and enhances tumor vascularity. We also show that the amount of HRGP present in human brain is increased in patients with primary tumors with the protein localized predominantly within the basement membrane. Finally, we offer some insight into the mechanism of action of Vstat120 by showing caspase-3 activation and endothelial cell apoptosis on Vstat120 addition. Together these results suggest that deposition of HRGP into angiogenic microenvironments, perhaps as the result of the inherent “leakiness” of the neo-vasculature and/or of platelet granule release, can modulate the anti-angiogenic processes mediated by the general family of TSR-containing proteins, and may shed light on the mechanism of angiogenesis regulation in the brain.     

四种中成药对气血双虚模型小鼠血象及免疫水平的影响

目的:为艾滋病抗病毒疗法所致的骨髓不良反应筛选疗效确切的中成药,观察分析参芪颗粒、复方阿胶浆、贞芪扶正颗粒、复方皂矾丸四种中成药对放血和注射环磷酰胺联合复制的气血双虚模型小鼠血象及免疫水平的影响。方法:实验于2005-08/09在河南中医学院药理实验室完成。①参芪颗粒(江西山高制药有限公司生产,批号040702);复方阿胶浆(山东东阿阿胶股份有限公司生产,批号050446);贞芪扶正颗粒(甘肃扶正药业科技股份有限公司生产,批号040803);复方皂矾丸(陕西郝其军制药有限责任公司生产,批号041014);当归补血口服液(郑州市协和制药厂生产,批号041122);环磷酰胺(上海华联制药有限公司生产,批号050101)。②选取清洁级昆明种小鼠150只,随机数字表法分为15组,10只/组:1～3组分别灌服参芪颗粒混悬液3,2,1g/kg;4～6组分别灌服复方阿胶浆30,20,10mL/kg;7～9组分别灌服贞芪扶正颗粒混悬液15,10,5g/kg;10～12组分别灌服复方皂矾丸混悬液2.4,1.6,0.8g/kg;第13组灌服当归补血口服液10g/kg;剩余2组为空白对照组和模型对照组,分别给于同体积生理盐水10g/kg。各组给药1次/d,连续给药10d。③除空白对照组外,其他各组从给药第1天开始建立气血双虚模型。每只鼠尾部放血0.25mL/10g,然后分别于第2,4,6,8天腹腔注射环磷酰胺80,40,40,40mg/kg。空白对照组同时间点仅腹腔注射等体积生理盐水。末次注射环磷酰胺后2h,眼眶取血,一部分用于血象测定,另一部分离心取血清,测定血细胞比容及血清中巨噬细胞集落刺激因子水平;解剖取胸腺和脾脏,检测胸腺皮质厚度、胸腺淋巴细胞数、脾小结大小、脾脏淋巴细胞数病理学指标的变化。结果:150只小鼠全部进入结果分析,放血和注射环磷酰胺并用可成功建立小鼠气血双虚模型。①与模型对照组比较,参芪颗粒3g/kg组、贞芪扶正颗粒10,5g/kg组、复方皂矾丸1.6g/kg组均可升高气血双虚模型小鼠白细胞水平(t=2.18～2.74,P<0.05),贞芪扶正颗粒15g/kg组作用更为显著(t=2.98,P<0.01);参芪颗粒1g/kg组、复方阿胶浆20mL/kg组、贞芪扶正颗粒15,10g/kg组均可升高红细胞水平(t=2.44～2.69,P<0.05),复方阿胶浆30mL/kg组、贞芪扶正颗粒5g/kg组、复方皂矾丸2.4,1.6g/kg组作用更为显著(t=2.91～3.66,P<0.01);当归补血口服液组、复方阿胶浆20mL/kg组、参芪颗粒3,1g/kg组、贞芪扶正颗粒15,10,5g/kg组均可升高血红蛋白水平(t=2.27～2.85,P<0.05),复方阿胶浆30mL/kg组、复方皂矾丸2.4,1.6g/kg组作用更为显著(t=3.07～4.04,P<0.01);当归补血口服液组、参芪颗粒3,2g/kg组均可升高血小板水平(t=2.20～2.41,P<0.05)。②与模型对照组比较,参芪颗粒2g/kg组、贞芪扶正颗粒5g/kg组均可升高气血双虚模型小鼠血细胞比容(t=2.01～2.62,P<0.05),参芪颗粒1g/kg组、复方阿胶浆30,20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4,1.6,0.8g/kg组作用更为显著(t=3.18～4.36,P<0.01);参芪颗粒2g/kg组、复方阿胶浆30,20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4,1.6g/kg组均可显著升高巨噬细胞集落刺激因子水平(t=3.60～6.80,P<0.01)。③与模型对照组比较,当归补血口服液组、参芪颗粒3,2,1g/kg组、复方阿胶浆30,20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4,1.6,0.8g/kg组均可显著增加气血双虚模型小鼠胸腺皮质厚度(t=3.71～9.34,P<0.01),增大脾小结(t=3.36～11.97,P<0.01),增加脾脏淋巴细胞数(t=4.29～10.44,P<0.01);复方阿胶浆30mL/kg组可明显增加小鼠胸腺淋巴细胞数(t=2.45,P<0.05),当归补血口服液组、参芪颗粒3,2,1g/kg组、复方阿胶浆20,10mL/kg组、贞芪扶正颗粒15,10,5g/kg组、复方皂矾丸2.4g/kg组作用更为显著(t=3.22～8.20,P<0.01)。结论:①四种中成药对气血双虚模型小鼠血红蛋白升高作用相近,以复方阿胶浆和贞芪扶正颗粒对白细胞和红细胞水平升高作用为强,以复方阿胶浆和贞芪扶正颗粒对血小板水平升高作用为优。②四种中成药对气血双虚模型小鼠血细胞比容的影响无差异,以贞芪扶正颗粒和复方皂矾丸对巨噬细胞集落刺激因子水平的升高作用为优。③以参芪颗粒、复方阿胶浆、贞芪扶正颗粒对胸腺皮质厚度和淋巴细胞数的促进作用为优,以参芪颗粒、贞芪扶正颗粒、复方皂矾丸对脾小结和脾脏淋巴细胞数的促进作用为优。     

免疫磁珠分选骨髓衍生肝干细胞亚群c-Kit+lin-

目的:利用免疫磁性细胞分选系统分离纯化骨髓衍生肝干细胞亚群c-Kit lin-。方法:实验于2006-07/08在南方医科大学实验动物中心完成。6～8周龄的SPF级纯系BALB/C雄性小鼠10只,体质量18～20g。收集小鼠股骨骨髓细胞,利用免疫磁性细胞分选系统,通过两步法分选纯化c-Kit lin-:将获取的lin-细胞悬液8℃条件下1500r/min离心10min,弃上清,按80μL/107加入Buffer重悬细胞。按20μL/107加生物素抗体磁珠,混匀,4℃冰箱孵育15min,按1mL/107加入Buffer洗细胞1次,8℃条件下1500r/min离心10min,弃上清,按500μL/108加入Buffer重悬细胞。Buffer500μL润MS柱,悬液过柱后,Buffer500μL/次洗柱3次,柱子脱离磁场,加1mLBuffer,用配套柱塞推出柱中的c-Kit lin-细胞,收集到c-kit lin-细胞,细胞计数。取2.0×106个细胞分成10等份,流式细胞仪分析c-Kit lin-细胞纯度,计算回收率,评估纯化效率,苔盼兰染色检测纯化前后的细胞活力。计算活细胞的百分率。细胞纯度和细胞回收率的计算:细胞纯度=分离产物中的阳性细胞数/分离细胞的总细胞数×100%,细胞回收率=分离产物中的阳性细胞数/起始标本阳性细胞总数×100%。结果:10只小鼠均进入结果分析。利用免疫磁性细胞分选系统分选出的骨髓衍生肝干细胞亚群c-Kit lin-细胞纯度和回收率分别为(77.98±2.34)%,75.40%,纯化前后细胞活力不受影响。结论:免疫磁性细胞分选系统能有效分选骨髓衍生肝干细胞亚群c-Kit lin-,纯度和回收率高,且不影响细胞活力。     

白藜芦醇对一次性力竭游泳大鼠肝脏组织保护作用的量效关系

目的:观察白藜芦醇对一次性力竭游泳大鼠肝脏组织的作用及发挥作用的最佳口服剂量。方法:实验于2006-05/07在成都体育学院运动医学系动物实验室完成。①实验分组:选取雄性SD大鼠70只,随机分为7组,每组10只,分别为安静对照组,运动对照组,运动 15mg/kg白藜芦醇组,运动 50mg/kg白藜芦醇组,运动 100mg/kg白藜芦醇组,运动 200mg/kg白藜芦醇组,运动 300mg/kg白藜芦醇组。②实验干预:不同剂量白藜芦醇组每天灌胃15,50,100,200,300mg/kg白藜芦醇,安静对照组和运动对照组分别灌胃相同体积的溶媒(二甲亚砜 生理盐水),连续5周。末次给予实验用样品1h后,各运动组每只鼠尾跟部负荷3%体质量铅皮,置于水深50cm、水温(31±1)℃游泳槽中游泳。游泳力竭后即刻,股动脉取血并迅速取出肝组织。③指标检测:赖氏比色法测定血清中谷丙转氨酶活性;邻苯三酚自氧化法测定肝组织超氧化物歧化酶活性;硫代巴比妥酸法测定肝组织丙二醛含量。结果:纳入动物70只,均进入结果分析。①血清谷丙转氨酶活性和肝组织中丙二醛含量:运动对照组显著高于安静对照组,不同剂量白藜芦醇组低于运动对照组(P<0.05或P<0.01)。运动 100,200,300mg/kg白藜芦醇组低于运动 15,50mg/kg白藜芦醇组[谷丙转氨酶活性:(972.36±121.86),(944.36±105.35),(888.34±88.68),(1773.52±89.35),(1377.78±27.01)nkat/L,P<0.01;丙二醛含量:(7.90±2.56),(7.69±3.69),(7.13±2.62),(19.90±2.21),(12.16±1.78)μmol/g,P<0.05]。100,200,300mg/kg白藜芦醇组间差异无显著性。②肝脏组织中超氧化物歧化酶活性:运动对照组显著低于安静对照组,不同剂量白藜芦醇组高于运动对照组(P<0.05或P<0.01)。运动 100,200,300mg/kg白藜芦醇组高于15,50mg/kg白藜芦醇组[(2325.80±163.37),(2379.14±121.86),(2447.16±89.18),(1096.05±120.19),(1514.64±28.17)μkat/g,P<0.01]。结论:①白藜芦醇对力竭性运动大鼠肝脏组织具有保护作用。②100,200,300mg/kg白藜芦醇对肝脏组织发挥保护作用效果优于15,50mg/kg,建议使用100mg/kg白藜芦醇就能达到理想效果。     

Higher Hb level is associated with better early functional recovery after hip fracture repair

BACKGROUND: The benefits and indications for blood transfusion are controversial. One possible reason to transfuse is to improve functional recovery after major surgery. However, the data linking improved function with higher Hb concentration are limited. STUDY DESIGN AND METHODS: A: retrospective cohort study was performed in 5793 patients at least 60 years old undergoing hip fracture repair at 20 academic and community hospitals. The primary outcome was distance walked at the time of discharge from the hospital. The mean postoperative Hb concentration was the main exposure variable and was defined as the average value from Day 1 after surgery to discharge. We used robust regression to assess the association between postoperative Hb level with distance walked, controlling for other preoperative variables that could influence functional recovery. RESULTS: On bivariate analysis, the predicted distance walked at discharge in feet (95% CI) increased with higher Hb levels (7 g/dL, 56 feet [42-70]; 8 g/dL, 61 feet [54-68]; 9 g/dL, 67 feet [64-70]; 10 g/dL, 74 feet [72-77]; 11 g/dL, 83 feet [80-85]; 12 g/dL, 92 feet [87-96]). After adjustment for other factors associated with ability to walk, higher average postoperative Hb level was independently associated with walking greater distance (p < 0.001). CONCLUSIONS: Higher postoperative Hb level may improve functional recovery after hip fracture repair. If confirmed with clinical trials, this finding would provide a rationale to maintain higher Hb concentrations in elderly patients recovering from surgery.     

Accuracy of estimates of premorbid intelligence based on demographic variables

Regression equations have been presented for estimating premorbid IQs on Wechsler's intelligence scales, using the demographic variables employed to stratify the various standardization samples as predictors. The accuracy of the resulting estimates for classifying patients into Wechsler's seven IQ categories (from very superior to mentally retarded) was evaluated, and it was found that even the best estimates can be expected to misclassify more than half of all patients. Such estimates still can contribute to valid inferences about a patient's premorbid intelligence, but only when they are used in conjunction with information from other sources.     

Equal weighting vs. differential weighting of subtest scores on short forms of Wechsler's Intelligence Scales

Data from the standardization samples for the WISC-R, the WPPSI, and the WAIS-R were employed to compare the validities and reliabilities of each of several short forms, using (a) the multiple regression weights based on those data, and (b) equal weights. For the short forms of the WISC-R, comparisons were also made using (c) the multiple regression weights reported by the investigators who proposed the short forms, based on their data. The validities and reliabilities varied little from one set of weights to another, so that a strong case can be made for the use of equal weights, which also possess the advantages of simpliciity and robustness.     

The diagnosis of polyarteritis nodosa

We investigated diagnostic testing in polyarteritis nodosa (PAN) by calculating, from published data, the sensitivity and specificity of visceral angiography and muscle, nerve, testicle, kidney, and liver biopsy. Test sequence strategies were constructed by Bayesian inference using a computer program written for this purpose. Test sequences were compared with an aggressive strategy consisting of repeated tests until there was a positive finding or until the available tests were exhausted, and a conservative strategy consisting of 1 biopsy procedure plus angiography. The Bayesian analysis agreed most closely with the conservative approach for most prior probabilities (degree of suspicion) that a patient had PAN. The aggressive strategy had an overall sensitivity of 90% and specificity of 91%, whereas the conservative strategy was 85% sensitive and 96% specific. Furthermore, the aggressive strategy was more costly ($2,986 versus $1,961) and had a higher rate of morbidity (3.8 versus 2.7 days of hospitalization per patient evaluated) than did the conservative strategy. The mortality rates of both strategies were equivalent (∼0.05 deaths per hundred patients evaluated). The per-case cost of diagnosis increased as prevalence decreased, and at 10% prevalence, the aggressive strategy cost more than $17,000 per case diagnosed. Sensitivity analysis revealed that the strategies were moderately affected by the test characteristics, within reasonable assumptions, but that the differences in conservative and aggressive approaches remained. Thus, our analysis based on available data and the assumption of test independence suggests that the preferred diagnostic evaluation of patients with symptoms suggestive of PAN consists, in most cases, of a single biopsy procedure, with angiographic evaluation if necessary.     

Effect of hydrazine sulfate on glucose-regulating enzymes in the normal and cancerous rat

Glucokinase, hexokinase, fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase specific activities were monitored in liver cytosol from rats that had been made cancerous with 1,2-dimethylhydrazine and then treated with hydrazine sulfate. The presence of intestinal cancer, specifically, was confirmed by laparotomy and by histological analysis. Sustained changes in hexokinase and glucokinase specific activities were first evident during the latter weeks that the carcinogen was being administered. Upon subsequent treatment with hydrazine sulfate, glucokinase activity further decreased, and liver cytosolic phosphoenolpyruvate carboxykinase activity increased. Liver cytosolic hexokinase and fructose 1,6-bisphosphatase specific activities were not appreciably affected by the hydrazine sulfate treatment. These results indicate that hydrazine sulfate may influence carbohydrate metabolism at the level of selected liver enzymes not only with respect to gluconeogenesis, but also in terms of glucose uptake.