Expression of Lymphangiogenic Vascular Endothelial Growth Factor Family Members in Bovine Corpus Luteum

The aim of this study was to evaluate mRNA expression, protein concentration and localization of the assumedly important lymphangiogenic factors VEGFC and VEGFD and the receptor FLT4 in bovine corpora lutea (CL) during different physiological stages. In experiment 1, CL were collected in a slaughterhouse and stages (days 1–2, 3–4, 5–7, 8–12, 13–16, >18) of oestrous cycle and month <3, 3–5, 6–7 and >8 of pregnancy. In experiment 2, prostaglandin F2α (PGF)‐induced luteolysis was performed in 30 cows, which were injected with PGF analogue on day 8–12 (mid‐luteal phase), and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF injection. The mRNA expression was characterized by RT‐qPCR. All three factors were clearly expressed and showed significant changes during different groups and periods examined in both experiments. Protein concentrations of VEGFD and FLT4 measured by ELISA were not detectable in early cyclic CL but increased to higher plateau levels during pregnancy. After PGF‐induced luteolysis FLT4 protein showed an increase within 2–24 h after the injection. FLT4 localization by immunohistochemistry in the cytoplasm of luteal cells was relatively weak in early CL. It increased in late CL and especially in CL during pregnancy. During pregnancy, a positive FLT4 staining in both the nucleus and cytoplasm of lymphatic endothelial cells in peripheral tissue was observed. In conclusion, our results lead to the assumption that lymphangiogenic factors are produced and regulated in CL and may be involved in mechanisms regulating CL function, especially during pregnancy.     

血管内皮生长因子D基因的原核表达及纯化

利用GST融合基因表达系统原核表达血管内皮生长因子D,并进行纯化。诱导重组质粒pGEX-VEGF-D在大肠杆菌BL21(DE3)中表达,经超声破菌后,采用GST蛋白纯化系统进行纯化,并用3C-Protease酶切去标签GST,所得产物进行15%SDS-PAGE电泳。结果表明:大肠杆菌经诱导,高效表达出分子质量约56 ku的GST-VEGF-D融合蛋白;纯化后的蛋白经酶切后电泳显示只有一条带,纯度相对较高,可基本满足生物学活性测定,为研究VEGF-D蛋白的结构和功能提供了有利的条件。     

血管内皮生长因子在绵羊肺脏中的表达分布研究

目的研究血管内皮生长因子（vascular endothelial growth factor,VEGF）在绵羊肺脏中的表达分布特征。方法取成年绵羊肺脏组织,制备石蜡切片,利用HE染色法观察绵羊肺脏组织的形态结构,采用免疫组织化学方法检测VEGF在绵羊肺脏组织中的分布。结果肺脏的各类型细胞均可见VEGF表达,在绵羊肺脏导气部的细支气管和终末细支气管的上皮细胞,呼吸部的肺泡管和呼吸性细支气管的上皮细胞,以及肺的血管内皮细胞均可检测到VEGF的强阳性表达信号。结论VEGF广泛分布于绵羊肺脏组织中,对其形态结构和功能的维持具有重要作用。     

Expression Pattern of Vascular Endothelial Growth Factor in Canine Folliculogenesis and its Effect on the Growth and Development of Follicles after Ovarian Organ Culture

In this study, the expressions of VEGF in dog follicles were detected by immunohistochemistry and the effects of VEGF treatment on the primordial to primary follicle transition and on subsequent follicle progression were examined using a dog ovary organ culture system. The frozen‐thawed canine ovarian follicles within slices of ovarian cortical tissue were cultured for 7 and 14 days in presence or absence of VEGF. After culture, the ovaries were fixed, sectioned, stained and counted for morphologic analysis. The results showed that VEGF was expressed in the theca cells of antral follicles and in the granulosa cells nearest the oocyte in preantral follicle but not in granulosa cells of primordial and primary follicles; however, the VEGF protein was expressed in CL. After in vitro culture, VEGF caused a decrease in the number of primordial follicles and concomitant increase in the number of primary follicles that showed growth initiation and reached the secondary and preantral stages of development after 7 and 14 days. Follicular viability was also improved in the presence of VEGF after 7 and 14 days in culture. In conclusion, treatment with VEGF was found to promote the activation of primordial follicle development that could provide an alternative approach to stimulate early follicle development in dogs.     

Ki-67 and Vascular Endothelial Growth Factor Expression in Intracranial Meningiomas in Dogs

Background: Tumor proliferation in human intracranial meningiomas can be defined by the reactivity of the monoclonal antibody MIB-1 to the Ki-67 antigen. Vascular endothelial growth factor (VEGF), a pro-angiogenic factor, is a predictive marker for survival of dogs with intracranial meningiomas.
Hypothesis: Ki-67 is expressed in canine intracranial meningiomas and is associated with VEGF expression. Ki-67 expression is a prognostic marker for patient outcome.
Animals: Seventy client-owned dogs with WHO grade I intracranial meningiomas.
Methods: Retrospective study assessing the degree of immunostaining for Ki-67 by MIB-1 and VEGF expression in intracranial meningioma tissue from dogs. MIB-1 Labeling Index (LI) was calculated with Image J NIH-software. Extent, intensity, and distribution of VEGF-expression was assessed semiquantitatively. Cross tabulations with Fisher's exact tests and nonparametric Spearman's rank correlations were performed to identify associations between VEGF expression and MIB-1 LI. Fifteen dogs underwent postsurgical radiotherapy and were included in survival analysis. The effect of MIB-1 LI on survival was examined by Kaplan-Meier and Cox proportional hazards regression procedures.
Results: Ki-67 staining was positive in 91% (64/70) and VEGF expression was detected in 96% (67/70). There was no significant association between VEGF expression and MIB-1 LI. MIB-1 LI was not associated with survival.
Conclusions and Clinical Importance: MIB-1 antibody can be used to document cell proliferation in intracranial meningiomas in dogs, but does not predict outcome. No association between VEGF as a marker of angiogenesis and tumor proliferation was found. Angiogenesis might be a more important predictor of meningioma activity in dogs than is Ki-67.     

藏鸡血管内皮生长因子基因表达与低氧适应

比较藏鸡在常氧(O2,21%)和低氧(O2,13%)浓度孵化环境中,胚胎尿囊绒毛膜(CAM)组织VEGF基因mRNA表达变化及其与低地矮小隐性白鸡的差别,分析VEGF表达与低氧适应的关系.结果发现低氧刺激使藏鸡和矮小隐性白鸡CAM组织VEGF表达均上调,矮小隐性白鸡上调程度明显大于藏鸡.结果说明在低氧环境中藏鸡CAM组织VEGF表达表现一定程度的增加,有利于血管形成,表现对低氧环境的适应;而低地鸡胚胎VEGF可能表现异常表达,不利于胚胎发育和存活.     

Expression and Localization of Vascular Endothelial Growth Factor and its Receptors in the Corpus Luteum During Oestrous Cycle in Water Buffaloes (Bubalus bubalis)

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid‐luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non‐angiogenic function.     

血管内皮细胞生长因子在鸡马立克病肝脏肿瘤中的表达

为研究血管内皮细胞生长因子(VEGF)在鸡马立克病肝脏肿瘤中的表达,探索其在马立克病(MD)肿瘤形成中的作用与机制。采用实时荧光定量PCR技术、Western blot技术,检测MD病毒组鸡肝脏肿瘤及正常组鸡肝脏中VEGF mRNA和蛋白质的表达。结果表明,MD病毒组鸡肝脏VEGF的mRNA和蛋白表达水平极显著高于正常组(P<0.01)。表明VEGF参与了MD肿瘤的形成。     

EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子基因表达的影响

[目的]观察EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子(vascular endothelial growth factor,VEGF)基因表达的影响,探讨前列腺素类化合物对奶牛子宫内膜组织修复的机制。[方法]分离培养奶牛子宫内膜上皮细胞,采用荧光定量PCR技术检测EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中VEGFmRNA表达的影响。[结果]10-6mol/L的Butaprost作用于奶牛子宫内膜上皮细胞4、8、16、24h可显著(P<0.05)或极显著(P<0.01)地促进VEGFmRNA的表达。[结论]EP2受体激动剂Butaprost能够促进奶牛子宫内膜上皮细胞中VEGF基因的表达。     

Expression of the Vascular Endothelial Growth Factor (VEGF‐A) and its Receptors in the Umbilical Cord in the Course of Pregnancy in the Pig

The umbilical cord (UC) and the placenta are important organs through which respiratory gases, nutrients, wastes and biologically active substances are exchanged between the maternal and the foetal system. A rapid placental vascularization observed in the second half of pig pregnancy is positively correlated with the mRNA expression of the vascular endothelial growth factor (VEGF). Based on these findings, we hypothesized that VEGF may have a stimulatory effect in the dynamically growing UC. To further understand the role of the VEGF–VEGFR system during UC development, mRNA and protein expression as well as the cellular localization of VEGF‐A, VEGFR‐1 and VEGFR‐2 in UC were examined on days 40, 60, 75 and 90 of pregnancy and after physiological delivery in the pig (day 114 of pregnancy). Real Time RT‐PCR analysis showed an increase in the mRNA levels of VEGF120 and VEGF164 from day 90 of pregnancy. VEGFR‐1 mRNA expression was significantly increased on day 75 of pregnancy. No significant changes in VEGFR‐2 mRNA expression were detected. In turn, western blot analysis revealed an increase in VEGF‐A protein expression on day 40, compared to the later days of pregnancy. A rapid increase in the VEGFR‐1 protein level was noted on day 75 and 90 of gestation. No significant changes in VEGFR‐2 protein expression were detected on any of the analysed days of pregnancy. Immunohistochemical staining enabled detection of VEGF–VEGFR system, in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium on all analysed days of pregnancy. Positive reactions for VEGF‐A and VEGFR‐1, but not VEGFR‐2, were also observed in myofibroblasts. In conclusion, this data shows that members of the VEGF–VEGFR system are temporally and spatially well localized for playing key roles during umbilical cord formation and its intensive growth observed after day 75 of pregnancy.     

Serum and Plasma Vascular Endothelial Growth Factor Levels in Various Cat Populations

Introduction:  Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Elevated levels within tumors, and in serum, plasma, and tumor effusion have been correlated with the development of metastatic disease, recurrence, and poor prognosis in many tumors in humans. Canine VEGF has been sequenced as homologous with the human form, and elevated serum and plasma VEGF have been found in dogs with hemangiosarcoma. Feline VEGF has also been sequenced, and shares homology with the human and canine forms.
Materials and Methods:  Stored serum and plasma samples from normal cats, cats with various neoplasms, and cats with non‐neoplastic disease were evaluated with a commercial ELISA kit (R&D Systems, Minneapolis MN). Samples were run in duplicate, and a standard curve was performed for each plate. The data were analyzed for differences between populations, and between serum and plasma levels in the same patient to determine the optimal sample for evaluating VEGF in cats.
Results:  In seven apparently healthy cats mean plasma VEGF was 95.6 pg/mL. In non‐neoplastic disease (7 cases), mean plasma VEGF was 117.3 pg/mL and mean serum VEGF level was 219.7 pg/mL. In ten tumor‐bearing patients mean plasma VEGF was 247.1 pg/mL, and mean serum VEGF was 322.3 pg/mL. Statistical analysis showed no significant difference between mean serum and plasma VEGF concentrations within each group or between groups (p > 0.05).
Discussion:  Serum and plasma VEGF levels could not be used to distinguish between healthy cats and cats with neoplastic or non‐neoplastic disease.     

Localization of Vascular Endothelial Growth Factor and Fibroblast Growth Factor 2 in the Ovary of the Ostrich (Struthio camelus)

Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF‐2) play a paramount role in the regulation of normal and pathologic angiogenesis in the ovary of mammals. Very little is known on the expression of these two growth factors in the avian ovary. The aim of this study was to determine for the first time the localization of VEGF and FGF‐2 in the ovary of the ostrich using immunohistochemical techniques to investigate the vascularization of the rapidly growing huge ostrich oocyte. At the oocyte periphery, distinct VEGF‐positive granules are visible. In our opinion, the expression of VEGF in the growing oocytes, which does not occur in mammals such as bovines, does not significantly contribute to angiogenesis in the theca interna and externa, where all the original and developing vessels are located, but may contribute to the mitoses and survival of granulosa cells during folliculogenesis. A different immunostaining can be demonstrated for FGF‐2: from late pre‐vitellogenic follicles, FGF‐2 immunopositivity can be observed at the inner perivitelline layer area. In the stroma, the smooth muscle cells of small arteries and the endothelial cells of venules and veins are positively stained for FGF‐2. Another interesting finding of this study is the occurrence of a significant number of VEGF‐ and FGF‐2 positive heterophilic granulocytes within the ovarian stroma, which migrate from the periphery of the ovary towards the growing follicles. We assume that the growth factors of the heterophilic granulocytes contribute significantly to the angiogenesis seen in both theca layers.     

Altered Expression of Transforming Growth Factor‐Beta Isoforms in Bovine Cystic Ovarian Disease

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors may contribute to follicular persistence. Transforming growth factor‐beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)‐induced COD. In the oestrous‐synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous‐synchronized control group with that in ACTH‐induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH‐induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.     

VEGF在家畜黄体早期发育过程中对血管生成的调控作用

对黄体时期VEGF依赖性血管生成的分子机制研究,将有助于我们开发新的策略用于治疗黄体相关的不孕症,以及改善动物的繁殖性能。论文对VEGF在家畜黄体血管生成过程中的调控作用进行综述,旨在为临床研究及畜牧生产提供理论依据及参考资料。     

Expression of Osteopontin (OPN) mRNA in Bovine Ovarian Follicles and Corpora Lutea

The matricellular protein osteopontin (OPN) plays a role in various physiological processes, including angiogenesis and tissue remodelling. As these processes are essential for the maintenance of ovarian physiology, the aim of the study was to investigate the expression of OPN (mRNA) in ovarian cells and to evaluate whether it can be regulated by gonadotrophins. Using conventional RT‐PCR and real‐time PCR, we have detected and quantified OPN mRNA as well as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA expression in bovine granulosa, theca and luteal cells. In all cells examined, both genes were found in equal amounts and no striking variations in the expression could be observed between granulosa, theca and luteal cells. Furthermore, no effect on either OPN or GAPDH mRNA expression was evident after culturing ovarian cells in the presence of gonadotrophic hormones, although the cells were still highly responsive in terms of cAMP formation. Although neither variations between different cell types nor a regulation of OPN mRNA expression by gonadotrophic hormones could be detected, the high and unambiguous mRNA expression in steroidogenic cells suggests that OPN should be added to the growing list of intraovarian factors which may be involved in ovarian physiology.     

Vascular Endothelial (VEGF) and Epithelial Growth Factor (EGF) as Well as Platelet‐Activating Factor (PAF) and Receptors are Expressed in the Early Pregnant Canine Uterus

The aim of this study was to investigate the course of expression of platelet‐activating factor (PAF), PAF‐receptor (PAF‐R), epidermal growth factor (EGF), EGF‐R, vascular endothelial growth factor (VEGF), VEGF‐R1 and VEGF‐R2 in uterine tissue during canine pregnancy. For this purpose, 20 bitches were ovariohysterectomized at days 10–12 (n = 10), 18–25 (n = 5) and 28–45 (n = 5) days after mating, respectively. The pre‐implantation group was proven pregnant by embryo flushing of the uterus after the operation, the others by sonography. Five embryo negative, that is, non‐pregnant, bitches in diestrus (day 10–12) served as controls. Tissue samples from the uterus (placentation sites and horn width, respectively) were excised and snap‐frozen in liquid nitrogen after embedding in Tissue Tec^®. Extraction of mRNA for RT‐PCR was performed with Tri‐Reagent. In the embryos, mRNA from all factors except VEGF was detected. In the course of pregnancy, significantly higher expression of PAF and PAFR as well as VEGF and VEGFR2 during the pre‐implantation stage than in all other stages and a strong upregulation of EGF during implantation were characteristic. The course of EGF was in diametrical opposition to the course of the receptor. These results point towards an increased demand for VEGF, EGF and PAF during the earliest stages of canine pregnancy.     

促卵泡素对体外培养牛卵泡类固醇合成相关基因表达的影响

本研究旨在探究促卵泡素（follicle stimulating hormone,FSH）处理对体外培养的牛有腔卵泡颗粒细胞和膜细胞类固醇激素合成相关基因表达的影响。采集牛卵巢表面直径9~11 mm的有腔卵泡,用含不同浓度FSH的DMEM/F12体外培养牛有腔卵泡24 h。提取卵泡颗粒细胞、膜细胞RNA并反转录成cDNA,利用实时荧光定量PCR检测卵泡颗粒细胞、膜细胞中类固醇激素合成酶基因（CYP11A1、3β-HSD、CYP17A1、CYP19A1、17β-HSD）和促性腺激素受体基因（FSHR、LHR）的表达水平。结果显示,FSH处理上调了颗粒细胞中CYP11A1、3β-HSD和CYP19A1基因表达,其中,25 ng/mL FSH处理极显著上调了CYP11A1基因表达（P<0.01）,10 ng/mL FSH处理显著上调了3β-HSD基因表达（P<0.05）,50 ng/mL FSH处理显著上调了CYP19A1基因表达（P<0.05）;50 ng/mL FSH处理显著或极显著上调了膜细胞中CYP11A1、3β-HSD和CYP17A1基因表达（P<0.05;P<0.01）,但在10和25 ng/mL FSH处理组中CYP11A1、3β-HSD和CYP17A1基因表达显著或极显著下调（P<0.05;P<0.01）。对FSHR、LHR基因研究结果显示,不同浓度FSH处理对颗粒细胞中FSHR、LHR基因的表达均无显著影响（P>0.05）,只有25和50 ng/mL FSH处理显著或极显著上调了膜细胞中LHR基因表达水平（P<0.05;P<0.01）,且不同处理组之间膜细胞中CYP11A1、3β-HSD和CYP17A1基因的表达变化与LHR基因表达变化趋势较一致。结果表明,FSH处理可提高牛有腔卵泡颗粒细胞中CYP11A1、3β-HSD和CYP17A1基因的表达,膜细胞中CYP11A1、3β-HSD和CYP17A1基因对LH的刺激更敏感,FSH可能通过影响LHR基因的表达来调节膜细胞中类固醇合成酶基因的表达。