Acute resistance exercise increases skeletal muscle angiogenic growth factor expression

AIMS: Both aerobic and resistance exercise training promote skeletal muscle angiogenesis. Acute aerobic exercise increases several pro-angiogenic pathways, the best characterized being increases in vascular endothelial growth factor (VEGF). We hypothesized that acute resistance exercise also increases skeletal muscle angiogenic growth factor [VEGF and angiopoietin (Ang)] expression. METHODS: Seven young, sedentary individuals had vastus lateralis muscle biopsies and blood drawn prior to and at 0, 2 and 4 h post-resistance exercise for the measurement of VEGF; VEGF receptor [KDR, Flt-1 and neuropilin 1 (Nrp1)]; Ang1 and Ang2; and the angiopoietin receptor--Tie2 expression. Resistance exercise consisted of progressive knee extensor (KE) exercise to determine one repetition maximum (1-RM) followed by three sets of 10 repetitions (3 x 10) of KE exercise at 60-80% of 1-RM. RESULTS: Resistance exercise significantly increased skeletal muscle VEGF mRNA and protein and plasma VEGF protein at 2 and 4 h. Resistance exercise increased KDR mRNA and Tie2 mRNA at 4 h and Nrp1 mRNA at 2 and 4 h. Skeletal muscle Flt-1, Ang1, Ang2 and Ang2/Ang1 ratio mRNA were not altered by resistance exercise. CONCLUSIONS: These findings suggest that acute resistance exercise increases skeletal muscle VEGF, VEGF receptor and angiopoietin receptor expression. The increases in muscle angiogenic growth factor expression in response to acute resistance exercise are similar in timing and magnitude with responses to acute aerobic exercise and are consistent with resistance exercise promoting muscle angiogenesis.     

去铁胺对新生大鼠缺氧缺血性脑的保护作用

目的： 研究去铁胺对新生大鼠缺氧缺血性脑病(HIE)模型新生血管形成的影响。方法：新生7 d SD大鼠建立HIE模型，模型组和治疗组建模前24 h分别用去铁胺或生理盐水注射。第1 d、3 d、7 d和14 d处死大鼠，检测缺氧缺血（左）侧脑组织毛细血管密度(BCDI)、增生毛细血管数、脑含水量、脑萎缩程度及其血管内皮生长因子(VEGF)和低氧诱导因子-1α (HIF-1α) mRNA表达。结果：治疗组左脑含水量和左脑萎缩比显著低于模型组(P<0.01)；左侧脑组织增生毛细血管数目显著高于模型组［(2.01±0.31)条/HPF vs (0.90±0.25)条/HPF ,P<0.01］。去铁胺显著上调左侧脑组织VEGF 和HIF-1α mRNA表达［12 h时VEGF：(1.41±0.07) vs (1.10±0.15)，P<0.05；HIF-1α：(1.49±0.12) vs (1.11±0.16)，P<0.05］。结论:去铁胺可能通过上调脑组织HIF-1α和VEGF表达，促进缺氧缺血脑组织新生血管形成，减轻缺氧缺血性脑损伤。     

Effects of cold‐water immersion on VEGF mRNA and protein expression in heart and skeletal muscles of rats

Aim: The effects of cold exposure on gene and protein expression of vascular endothelial growth factor (VEGF), in heart and skeletal muscles, were studied in male adult Wistar rats. Methods: Cold immersion was accomplished by submerging the rats in shoulder‐deep water maintained at ～18 °C, either acutely (1 h) or chronically (1 h day^?1, 5 days week^?1 for 20 weeks). The expressions of VEGF mRNA and protein in heart, gastrocnemius, and soleus muscles were examined by Northern and Western blotting and competitive‐polymerase chain reaction techniques. Results: The expressions of VEGF mRNA and protein were markedly increased in cardiac muscle of the cold‐immersed group, particularly in the 1‐hour exposure group, whereas VEGF mRNA and protein in gastrocnemius were decreased significantly after an acute exposure. Although the protein level in gastrocnemius remained low in the chronically exposed group, the expression of mRNA of VEGF₁₆₅ with chronic exposure in this group returned to the control level and that of VEGF₂₀₆ was 15% greater than that in controls. The expression of mRNA for VEGF₁₆₅ in soleus was also lowered by acute cold exposure, although that for VEGF₂₀₆ was stable. However, VEGF protein was increased by 50%. After 20 weeks, all of these parameters were increased over the levels found in the controls. Conclusion: These results suggest that the VEGF gene may be a major regulatory factor in cardiac and skeletal muscle adaptation to the cold environment stimulating angiogenesis and thermogenesis.     

Retinal angiogenesis is mediated by an interaction between the angiotensin type 2 receptor,VEGF, and angiopoietin

There is evidence that angiotensin II, vascular endothelial growth factor (VEGF), angiopoietins, and their cognate receptors participate in retinal angiogenesis. We investigated whether angiotensin type 2-receptor blockade (AT2-RB) reduces retinal angiogenesis and alters the expression of VEGF/VEGF-R2 and angiopoietin-Tie2. Retinopathy of prematurity (ROP) was induced in Sprague Dawley (SD) rats by exposure to 80% oxygen from postnatal (P) days 0 to 11, followed by 7 days in room air. ROP shams were in room air from P0-18. A group of ROP rats received the AT2-RB, PD123319, by mini-osmotic pump (5 mg/kg/day) from P11-18 (angiogenesis period). Evaluation of the retinal status of the AT2 receptor indicated that this receptor, as assessed by real-time PCR, immunohistochemistry, and in vitro autoradiography, was present in the retina, was more abundant than the AT1 receptor in the neonatal retina, and was increased in the ROP model. AT2-RB reduced retinal angiogenesis. VEGF and VEGF-R2 mRNA were increased in ROP and localized to blood vessels, ganglion cells, and the inner nuclear layer, and were decreased by PD123319. Angiopoietin2 and Tie2, but not angiopoietin1 mRNA were increased with ROP, and angiopoietin2 was reduced with PD123319. This study has identified a potential retinoprotective role for AT2-RB possibly mediated via interactions with VEGF- and angiopoietin-dependent pathways.     

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.     

Norrin在氧诱导的视网膜病变中的时空表达及其意义

目的：探讨视网膜新生血管疾病动物模型-氧诱导的视网膜病变（OIR）小鼠视网膜中Norrin的时空表达变化及意义。方法：荧光素灌注铺片观察OIR小鼠视网膜血管分布和形态。RT－PCR检测对照组P7、P10、P12、P17小鼠和OIR P7、P8、P12、P12.5、P13、P14、P15、P17、P19小鼠视网膜中Norrin、VEGF mRNA的表达。免疫组化法检测Norrin蛋白在视网膜中的表达和分布位置。结果：OIR小鼠P12时在视网膜后极部形成无灌注区，P17时有大量新生血管形成。Norrin mRNA在正常小鼠视网膜中表达水平低且稳定。缺氧12 h后，Norrin mRNA有显著升高，缺氧1 d到达高峰，然后缓慢下降，其变化趋势与VEGF mRNA一致。Norrin蛋白在正常小鼠各时点免疫组化均未能检出，在OIR模型中P15见弱表达，P17-P19表达明显增强。主要分布在视网膜外层，以内外节层、外丛状层为主。结论：Norrin在mRNA和蛋白水平都在缺氧视网膜中明显升高，主要分布在视网膜外层。Norrin很可能参与新生血管的形成，并起着重要的调控作用。     

Peritumoral brain edema in angiomatous supratentorial meningiomas: an investigation of the vascular endothelial growth factor A pathway

The aim of this work was to study the vascular endothelial growth factor A (VEGF‐A) pathway and peritumoral brain edema (PTBE) through comparison of non‐angiomatous and angiomatous meningiomas. Meningiomas are common intracranial tumors, which often have PTBE. VEGF‐A is an integral part of PTBE formation and angiogenesis, and the capillary‐rich angiomatous meningiomas are known for their PTBE. The VEGF‐A receptor VEGFR‐2 is responsible for the angiogenic effect of VEGF‐A on endothelial cells, which is enhanced by the co‐receptor neuropilin‐1. Forty non‐angiomatous, 22 angiomatous meningiomas, and 10 control tissue samples were collected for the study. Magnetic resonance images were available for 40 non‐angiomatous and 10 angiomatous meningiomas. Tissue sections were immunostained for CD34, MIB‐1, estrogen‐ and progesterone receptors. ELISA, chemiluminescence, and RT‐qPCR were used for VEGF‐A, VEGFR‐2, and neuropilin‐1 protein and mRNA quantification. Angiomatous meningiomas had larger PTBE (695 vs 218 cm³, p = 0.0045) and longer capillary length (3614 vs 605 mm/mm³, p < 0.0001). VEGF‐A mRNA, neuropilin‐1 mRNA, and VEGFR‐2 protein levels were higher in angiomatous meningiomas independently of the capillary length (p < 0.05). Neuropilin‐1 protein levels were lower in angiomatous meningiomas (p < 0.0001). The VEGF‐A pathway and tumor capillary length may be essential for PTBE‐formation in meningiomas. Further investigations of this pathway could lead to earlier therapy and targeted pharmacological treatment options.     

Inhibition of platelet-derived growth factor promotes pericyte loss and angiogenesis in ischemic retinopathy

We investigated whether inhibition of platelet-derived growth factor (PDGF) receptor tyrosine kinase activity would affect pericyte viability, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR-2) expression and angiogenesis in a model of retinopathy of prematurity (ROP). ROP was induced in Sprague Dawley rats by exposure to 80% oxygen from postnatal (P) days 0 to 11 (with 3 hours/day in room air), and then room air from P12-18 (angiogenesis period). Shams were neonatal rats in room air from P0-18. STI571, a potent inhibitor of PDGF receptor tyrosine kinase, was administered from P12-18 at 50 or 100 mg/kg/day intraperitoneal (i.p.). Electron microscopy revealed that pericytes in the inner retina of both sham and ROP rats appeared normal; however STI571 induced a selective pericyte and vascular smooth muscle degeneration. Immunolabeling for caspase-3 and alpha-smooth muscle cell actin in consecutive paraffin sections of retinas confirmed that these degenerating cells were apoptotic pericytes. In all groups, VEGF and VEGFR-2 gene expression was located in ganglion cells, the inner nuclear layer, and retinal pigment epithelium. ROP was associated with an increase in both VEGF and VEGFR-2 gene expression and blood vessel profiles in the inner retina compared to sham rats. STI571 at both doses increased VEGF and VEGFR-2 mRNA and exacerbated angiogenesis in ROP rats, and in sham rats at 100 mg/kg/day. In conclusion, PDGF is required for pericyte viability and the subsequent prevention of VEGF/VEGFR-2 overexpression and angiogenesis in ROP.     

Hypoxia and expression of VEGF-A protein in relation to capillary growth in electrically stimulated rat and rabbit skeletal muscles

To investigate the role of hypoxia as a stimulus to the early upregulation of vascular endothelial growth factor (VEGF) in fast skeletal muscles during chronic low frequency stimulation, blood flow, oxygen consumption, VEGF expression and capillary : fibre ratio were measured in chronically stimulated tibialis anterior and extensor digitorum longus (EDL) muscles in rabbits and rats. No differences were found in blood flow, oxygen consumption and extraction between rabbit muscles stimulated for 2 or 4 days (8 h on-16 h off) and controls. Muscle P(O(2)) polarographically measured immediately at the end of stimulation on day 2 was also no different from control under resting conditions (10.7 +/- 1.6 vs. 9.5 +/- 1.2 Torr, n.s.). Unlike control muscles, however, P(O(2)) in 2 day stimulated muscles did not increase immediately after a further acute bout of contractions. This difference was not apparent after similar acute contractions in 4 day stimulated muscles. The involvement of VEGF in early angiogenesis in stimulated muscles was studied in serial cryosections of rat EDL. The proportion of capillaries positively immunostained for VEGF increased from 25 +/- 1 % to 40 +/- 1 % (P < 0.05) in muscles removed on day 2 immediately at the end of chronic stimulation; it decreased slightly after 16 h rest, and increased again after 4 days of stimulation. Capillary : fibre ratio was unchanged throughout the experiment. Capillary cell proliferation increased only after the rest period on day 2 (20-fold increase) and day 4 (12-fold increase), indicating angiogenesis in progress. Thus the timing of transient hypoxia and increase in capillary-linked VEGF in stimulated muscles, albeit in different species, was similar, and increased VEGF staining and capillary cell proliferation occurred even after the hypoxia had resolved. This suggests (1) a connection between hypoxia and VEGF during the early stages of stimulation, although ensuing capillary proliferation may thereafter rapidly correct for local hypoxia, and (2) that the subsequent angiogenesis and VEGF expression are dependent on factors other than hypoxia.     

No difference in the skeletal muscle angiogenic response to aerobic exercise training between young and aged men

Ischaemia-induced skeletal muscle angiogenesis is impaired in aged compared with young mice. In humans, vascular endothelial growth factor (VEGF) mRNA and protein following an acute exercise bout are lower in aged compared with young untrained men. We hypothesized that exercise-induced skeletal muscle angiogenesis would be attenuated in aged compared with young men. In eight aged (mean age: 64 years) and six young (mean age: 25 years) sedentary men, muscle biopsies were obtained from the vastus lateralis prior to (Pre), after 1 week and after 8 weeks of an aerobic exercise training program for the measurement of capillarization and VEGF mRNA. Dialysate VEGF protein collected from the muscle interstitial space was measured at rest and during submaximal exercise at Pre, 1 week and 8 weeks. Exercise training increased capillary contacts (CC) and capillary-to-fibre perimeter exchange index (CFPE) of type I and IIA fibres similarly in young and aged. The CC of type IIA and IIB fibres was lower in aged compared with young independent of training status. Exercise-induced interstitial VEGF protein was lower in aged compared with young independent of training status. In untrained, greater exercise-induced interstitial VEGF protein during exercise was associated with greater type I, IIA and IIB CC. Exercise training increased VEGF mRNA similarly in young and aged. These results demonstrate that the angiogenic response to aerobic exercise training is not altered during the ageing process in humans. In addition, muscular activity-associated increases in interstitial VEGF protein may play an important role in the maintenance of skeletal muscle capillarization across the life span.     

Rat sponge implant model: a new system for evaluating angiogenic gene transfer

Therapeutic angiogenesis, either by protein injection or gene therapy, holds considerable promise for the treatment of coronary and peripheral artery diseases. Given the large number of angiogenic genes available, a simple, well defined, standard system to compare the relative angiogenic efficacy of such genes would be valuable. We have employed a replication-deficient adenovirus vector (complete E1a-, partial E1b- and partial E3-) to deliver the beta-galactosidase (beta-gal, AdLacZ) reporter gene or the human VEGF121 gene (AdGV VEGF121.10) to a rat sponge implant model of angiogenesis. beta-gal staining results reveal a transfection efficiency as high as 60% 24 h after 2x1010 particle units AdLacZ injection. Our results also indicate that a single injection of 2x1010 particle units of AdGVVEGF121.10 in the sponge results in >10, 000 pg VEGF protein expression per milligram of sponge tissue 24 h later. VEGF121 protein concentrations decreased 10-fold within 3 days and 100-fold within 7 days after injection. Significant VEGF121 protein levels were still detectable 14 days after initial virus injection. The high level of gene transfection efficiency was accompanied by enhanced angiogenesis in the sponge, a tissue devoid of any vessels before implantation. Compared to control (AdNull: adenovirus vector without the VEGF gene), AdGVVEGF121.10 induced a 2- to 3-fold up-regulation of angiogenesis at 7 and 14 days post vector injection as determined by both increased capillary number and increased tissue ingrowth. The angiogenic effects of AdGVVEGF121. 10 were dose-related in this model system. These findings demonstrate a dose-related angiogenic response to adenovirus-mediated gene therapy in this model.     

血管生成因子在糖尿病大鼠心肌组织的表达

 目的：检测糖尿病大鼠心肌组织中血管生成因子的表达。方法：选择雄性SD大鼠一次性腹腔注射链脲佐菌素制造1型糖尿病模型,糖尿病成功诱导后12周,运用MPA心功能分析系统评估心功能改变,Masson染色计算心肌胶原容积分数（collagen volume fraction,CVF）,CD31免疫组织化学法测定心肌毛细血管并计算心肌毛细血管/心肌细胞比值（capillary/myocyte,C/M）,Western blotting检测血管内皮生成因子（VEGF）、血管生成素1（angiopoietin-1,Ang-1）、血管生成素2（angiopoietin-2,Ang-2）和内皮抑素(endostatin) 在心肌组织中的表达。结果：与正常对照组比较,糖尿病大鼠左心室舒张末期压力(LVEDP)明显增高(P<001),而左心室压力上升最大速率(+dp／dtmax)和左心室压力下降最大速率(-dp／dtmax)均明显下降（P<0.05）;心肌C/M比值、 VEGF和Ang-1表达明显减少（P<0.05）;而心肌CVF含量和endostatin表达显著增高（P<0.05）,但Ang-2表达无明显差异。结论：VEGF和Ang-1在糖尿病大鼠心肌组织表达下调,endostatin表达显著上调,可能是糖尿病心肌病发生的重要机制。     

Activity-independent modulation of acetylcholine receptor levels in rat skeletal muscle following neonatal denervation

Following denervation of adult muscle, levels of acetylcholine receptor (AChR) increase; normal, low levels are restored only after muscle reinnervation. After neonatal denervation, we found a large initial increase in AChR levels during the first days postsurgery, as in adult denervated muscle. However, 1 week after denervation, total AChR levels decreased in the absence of any sign of reinnervation. By 3 weeks after surgery, near-normal levels of AChR were restored and extrajunctional AChR had disappeared. Thus, in sharp contrast to adult muscle, in young denervated muscle a down-regulation of AChR occurs without recovery of innervation and normal muscle contractile activity. These results suggest that different mechanisms regulate the levels of AChR in developing and adult skeletal muscle.     

Myostatin在小鼠腓肠肌失神经支配萎缩过程中的表达

目的:分析myostatin在腓肠肌失神经支配萎缩过程中的表达变化及其在肌萎缩过程中的作用。方法:采用坐骨神经横断术制备小鼠腓肠肌失神经支配模型,实时荧光定量PCR和Western印迹法分别检测失神经支配不同时段腓肠肌myostatin mRNA和蛋白表达水平,并对失神经前后肌肉湿重比、肌纤维横截面积进行比较。结果:失神经支配1 d时,腓肠肌myostatin mRNA迅速上升,3 d达到高峰,随后逐渐下降,而相应蛋白水平逐渐增高,7 d达到高峰继而逐渐下降,至56 d时mRNA和蛋白水平仍略高于正常水平。结论:腓肠肌失神经支配萎缩过程中myostatin的表达变化是一重要的分子事件。     

大鼠脑缺血后血管内皮生长因子和血管生成素的表达变化及其作用

目的 探讨大鼠脑缺血后血管内皮生长因子(VEGF)和血管生成素(angiopoietin,Ang)的表达变化及其在血管生成和血管通透性变化中的作用.方法 选择160只雄性SD大鼠,采用随机区组法分为假手术组,缺血2h、6h、12h、1d、3d、7d和14d组,用线栓法制作大脑中动脉阻塞脑缺血损伤模型,分别于脑缺血后不同时间点处死大鼠.通过逆转录聚合酶链反应(RT-PCR)、Western blot及免疫组织化学方法检测大鼠脑缺血后不同时间点VEGF、Ang-1及Ang-2 mRNA和蛋白的表达变化.CD31标记鼠脑缺血后不同时间点的血管数量.Evans blue检测血脑屏障通透性.结果 大鼠脑缺血后VEGF mRNA表达逐渐增高,12 h达第一个高峰(0.7249 ±0.1933,P＜0.01),7d达第二个高峰(0.5264±0.1519,P＜0.01);VEGF蛋白表达也逐渐增高,于12 h达高峰(1.1017±0.1302,P＜0.01).Ang-2 mRNA和蛋白在脑缺血组织也逐渐增高,均12 h达高峰(0.6747±0.2416,P＜0.01;1.1197±0.1780,P＜0.01).与之相反,Ang-1 mRNA和蛋白表达逐渐降低,分别于3 d(0.3220±0.1427,P＜0.01)和1 d(0.1298±0.0293,P＜0.01)达最低水平,这些因子在脑缺血后的表达促进血管生成.脑缺血后血管通透性逐渐增加,EB含量在缺血1d达高峰[(6.219±0.887) μg/g,P＜0.01].结论 大鼠脑缺血后VEGF、Ang-1和Ang-2共同协调作用促进血管生成,在组织损伤修复过程中发挥着重要的作用.     

参慈胶囊联合顺铂抑制小鼠Lewis肺癌组织血管生成的机制研究

目的:探讨参慈胶囊及顺铂对Lewis肺癌组织生长及血管生成的抑制作用。方法:将40只C57BL/6小鼠自接种Lewis肺癌瘤株细胞建立实体瘤模型,随机分为参慈胶囊组、参慈胶囊+顺铂组、顺铂组、模型组。模型组予等量生理盐水,其余各组荷瘤小鼠均用药21 d,测量肿瘤重量并计算抑瘤率,采用免疫组化技术检测血管内皮生长因子(VEGF)、微血管密度(MVD)表达情况;采用实时荧光定量PCR(FQ-PCR)技术检测Lewis肺癌荷瘤小鼠癌细胞表达血管内皮生长因子受体-2(KDR)mRNA的情况。结果:(1)在抑瘤率方面,参慈胶囊+顺铂组抑瘤率明显高于其它3组,差异显著(P0.01)。(2)参慈胶囊+顺铂组能降低Lewis肺癌组织VEGF、KDR的表达及MVD的形成,差异显著(P0.01)。结论:(1)参慈胶囊联合顺铂能抑制Lewis肺癌组织生长及血管生成,二者联用具有相加或者协同效应;(2)下调VEGF及其受体KDR的表达,可能是二者联合抗肿瘤血管生成的机制之一。     

Effects of cold-water immersion on VEGF mRNA and protein expression in heart and skeletal muscles of rats

AIM: The effects of cold exposure on gene and protein expression of vascular endothelial growth factor (VEGF), in heart and skeletal muscles, were studied in male adult Wistar rats. METHODS: Cold immersion was accomplished by submerging the rats in shoulder-deep water maintained at approximately 18 degrees C, either acutely (1 h) or chronically (1 h day(-1), 5 days week(-1) for 20 weeks). The expressions of VEGF mRNA and protein in heart, gastrocnemius, and soleus muscles were examined by Northern and Western blotting and competitive-polymerase chain reaction techniques. RESULTS: The expressions of VEGF mRNA and protein were markedly increased in cardiac muscle of the cold-immersed group, particularly in the 1-hour exposure group, whereas VEGF mRNA and protein in gastrocnemius were decreased significantly after an acute exposure. Although the protein level in gastrocnemius remained low in the chronically exposed group, the expression of mRNA of VEGF(165) with chronic exposure in this group returned to the control level and that of VEGF(206) was 15% greater than that in controls. The expression of mRNA for VEGF(165) in soleus was also lowered by acute cold exposure, although that for VEGF(206) was stable. However, VEGF protein was increased by 50%. After 20 weeks, all of these parameters were increased over the levels found in the controls. CONCLUSION: These results suggest that the VEGF gene may be a major regulatory factor in cardiac and skeletal muscle adaptation to the cold environment stimulating angiogenesis and thermogenesis.