Histomorphological and immunohistochemical study of angiogenesis and angiogenic factors in the ovary of the mare

Cyclical ovaries of 18 mares were examined histologically and immunohistochemically for vascular endothelial growth factor A and B (VEGF A; VEGF B), angiopoietin1 and 2 (Ang1; Ang2), vascular endothelial growth factor receptor 1 and 2 (VEGF-R1; VEGF-R2), angiopoietin receptor (Tie2) and von Willebrand factor. The most intensive coexpression of the examined factors and receptors was detected in the periovulatory period, when a distinctive ovarian angiogenesis takes place, being essential for tertiary follicle maturation and for the endocrine function of the Corpus luteum. Based on the immunohistochemical results, VEGF A, Ang2, VEGF-R2 and Tie2 in particular seem to play a significant role on angiogenesis during follicular and luteal development in the mare, while Ang1 supports vessel stabilisation. The findings of luteal regression and follicular atresia showed that, in the absence of VEGF A, Ang2 and its receptor Tie2 contribute substantially to vessel regression and therefore to luteolysis and follicular atresia.     

Angiogenesis in Developing Follicle and Corpus Luteum

Angiogenesis is a process of vascular growth that is mainly limited to the reproductive system in healthy adult animals. The development of new blood vessels in the ovary is essential to guarantee the necessary supply of nutrients and hormones to promote follicular growth and corpus luteum formation. In developing follicles, the pre-existing endothelial cells that form the vascular network in the theca layer markedly develop in response to the stimulus of several growth factors, mainly produced by granulosa cells, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The angiogenic factors also promote vessel permeability, thus favouring the antrum formation and the events inducing follicle rupture. After ovulation, newly formed blood vessels cross the basement membrane between theca and granulosa layers and continue a rapid growth to sustain corpus luteum development and function. The length of luteal vascular growth varies in cycling and pregnant animals and among species; both angiogenesis and subsequent angioregression are finely regulated by systemic and local factors. The control of angiogenic development in the ovary could be a useful tool to improve animal reproductive performances.     

Messenger ribonucleic acid expressions of hepatocyte growth factor, angiopoietins and their receptors during follicular development in gilts

Angiogenic factors are associated with angiogenesis during follicular development in the mammalian ovary. The aim of the present study was to determine the relationships between the vascular network and mRNA expressions of angiopoietins (Ang)-1, Ang-2 and hepatocyte growth factor (HGF), and their receptors in follicles at different developmental stages during follicular development. Ovaries in gilts were collected 72 h after equine chorionic gonadotropin (eCG, 1250 IU) treatment for histological observation of the capillary network. Granulosa cells and thecal tissues in small (<4 mm), medium (4-5 mm) or large (>5 mm) individual follicles were collected for detection of mRNA expression of HGF, Ang-1 and Ang-2 in granulosa cells, and HGF receptor (HGF-R) and Tie-2 in the theca cells by semi-quantitative RT-PCR. The number of capillaries in the thecal cell layer increased significantly in healthy follicles at all developmental stages in the eCG group compared with those in controls. The expression of Ang-1 mRNA declined in granulosa cells of medium and large follicles and the level of Ang-2 mRNA increased in granulosa cells of small follicles after eCG treatment. The ratio of Ang-2/Ang-1 increased in small, medium and large follicles from ovaries after eCG treatment, but Tie-2 mRNA expression in the theca cells did not change. The level of HGF mRNA increased in granulosa cells of small follicles after eCG treatment but HGF-R in theca cells was not increased by eCG. These data suggested that the angiopoietins might be associated with thecal angiogenesis during follicular development in eCG-treated gilts.     

Vascular Mimicry of Granulosa Cells: a New Concept of Corpeus Luteum Development?

So far, it was generally accepted that newly formed blood vessels are exclusively comprised of endothelial cells, and complemented by pericyte and myocyte recruitment during vessel maturation. Accordingly, participation of non-endothelial cells in the formation of blood vessels has rarely been suggested. Recently, evidence supporting the existence of tumour vessels lined by non-endothelial cells has emerged. Consequently, the concept of the inherent capacity of non-endothelial cells to behave like endothelial cells has been discussed for tumours, and this pathomechanism has been termed vascular mimicry. The corpus luteum is one of the most intensely vascularized tissues, and angiogenesis in the corpus luteum is more effective than in highly malignant tumours. Our results indicate active involvement of granulosa cells in luteal angiogenesis, and the aim of this study was to shed more light on this exciting prospect. The study was based on cultured granulosa cells isolated from the bovine ovary in different stages of follicle maturation. Morphology of angiogenic granulosa cells was studied by phase contrast, transmission electron and scanning electron microscopy. Expression of angiogenesis-regulating factors and their receptors was demonstrated by polymerase chain reaction (RT-PCR). Cultured granulosa cells underwent changes reminiscent of endothelial angiogenesis, i.e., migration, proliferation, differentiation and three-dimensional organization, and expressed angiogenesis-regulating factors and their receptors. Our results suggest a tight regulatory and structural association of endothelial and granulosa cells in luteal angiogenesis, suggesting physiological vascular mimicry in the ovary.     

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.     

Promotion of ovarian follicular development by injecting vascular endothelial growth factor (VEGF) and growth differentiation factor 9 (GDF-9) genes

Ovarian follicular development in mammals is the complex process including endocrine, paracrine and autocrine. There is the development of four basic stages of ovarian follicles, i.e. the primordial, primary, secondary and tertiary or Graafian follicles. There are few blood vessels in the cortical area where primordial and primary follicles are assembled. The development of these follicles is stimulated by oocytes derived factor including growth differentiation factor 9 (GDF-9) or bone morphogenetic protein 15 (BMP-15). Porcine GDF-9 complementary DNA (cDNA) cloned, and then injected its gene into the ovary in gilts. The injection of porcine GDF-9 gene resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles, indicating that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary. On the other hand, the development of antral follicles is associated with increased density of blood vessels within the theca cell layers surrounding the follicles. A recent study reported that vascular endothelial growth factor (VEGF) play an important role in the process of thecal angiogenesis during follicular development. To investigate whether additional induction of thecal angiogenesis would support subsequent follicular development, miniature gilts were directly injected VEGF gene into the ovary. Injection of VEGF gene increased the levels of mRNA expression of VEGF 120 and VEGF 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene compared with those treated with eCG alone, indicating that the regulation of thecal angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. This technique may be an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.     

Endothelial cells and angiogenesis in the horse in health and disease—A review

The cardiovascular system is the first functional organ in the embryo, and its blood vessels form a widespread conductive network within the organism. Blood vessels develop de novo, by the differentiation of endothelial progenitor cells (vasculogenesis) or by angiogenesis, which is the formation of new blood vessels from existing ones. This review presents an overview of the current knowledge on physiological and pathological angiogenesis in the horse including studies on equine endothelial cells. Principal study fields in equine angiogenesis research were identified: equine endothelial progenitor cells; equine endothelial cells and angiogenesis (heterogeneity, markers and assessment); endothelial regulatory molecules in equine angiogenesis; angiogenesis research in equine reproduction (ovary, uterus, placenta and conceptus, testis); angiogenesis research in pathological conditions (tumours, ocular pathologies, equine wound healing, musculoskeletal system and laminitis). The review also includes a table that summarizes in vitro studies on equine endothelial cells, either describing the isolation procedure or using previously isolated endothelial cells. A particular challenge of the review was that results published are fragmentary and sometimes even contradictory, raising more questions than they answer. In conclusion, angiogenesis is a major factor in several diseases frequently occurring in horses, but relatively few studies focus on angiogenesis in the horse. The challenge for the future is therefore to continue exploring new therapeutic angiogenesis strategies for horses to fill in the missing pieces of the puzzle.     

The plant alkaloid sanguinarine is a potential inhibitor of follicular angiogenesis

Sanguinarine (SA), a phytobiotic from Sanguinaria Canadensis, has been demonstrated to inhibit vessel growth. Current restrictions on the use of antibiotic growth promoters have motivated addition of this alkaloid as a naturally appetizing feed additive for farm animals. However, concern may araise since angiogenesis is a fundamental event in ovarian follicle growth. Therefore, the aim of this study was to evaluate the potential negative role of SA in follicular angiogenesis. For this purpose, we studied the effect of 300 nM SA on the production of vascular endothelial growth factor (VEGF) by swine granulosa cells from follicles >5 mm and on the activation of Akt, the main effector of the VEGF signalling pathway. In addition, the potential interference of SA in vessel development was tested in an in vitro angiogenesis bioassay. SA inhibited both VEGF production and VEGF-induced Akt activation in swine granulosa cells. Moreover, it was able to block vessel growth induced by VEGF. Taken together, our results suggest that SA could be detrimental to follicular angiogenesis, and therefore supplementation of feed with this alkaloid should be carefully considered.     

VEGFR‐2 expression in malignant tumours of the canine mammary gland: a prospective survival study

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is the main receptor activated by vascular endothelial growth factor ‐A (VEGF‐A) to promote tumour angiogenesis. Its clinical prognostic value has not been studied in canine mammary tumours (CMTs). Dogs with mammary cancer were enrolled in a survival study and the immunohistochemical expressions of VEGFR‐2 and VEGF‐A were analysed and associated with clinicopathological features. VEGFR‐2 expression was associated with VEGF immunoreactivity in cancer cells, supporting the presence of an autocrine loop that may be involved in CMTs growth and survival. VEGFR‐2 was also expressed by endothelial cells from tumour vasculature and positively associated with stromal matrix metalloproteinase‐9 (MMP‐9), suggesting the existence of a link between endothelial cells activation and up‐regulation of matrix degrading proteins. Carcinosarcomas exhibited high VEGFR‐2 expression suggesting that it may be one of the activated molecular pathways in this aggressive histological type and that VEGFR‐2 inhibitors may constitute a potential treatment to improve the prognosis of these patients. Both VEGF and VEGFR‐2 immunoreactivities were independent of patients' overall survival (OS) and disease‐free survival (DFS).     

Angiogenic activity of swine granulosa cells: effects of hypoxia and vascular endothelial growth factor Trap R1R2, a VEGF blocker

The possible role played by hypoxia and vascular endothelial growth factor (VEGF) in the regulation of follicular angiogenesis was studied in a three-dimensional fibrin gel model. Granulosa cells from follicles >5mm were subjected to normoxia (19% O2), partial (5% O2) or total (1% O2) hypoxia and their culture media were collected and used to stimulate porcine Aortic Endothelial Cells (AOC) included in the fibrin matrix. A suspension of AOC on microcarrier beads was pipetted in a fibrinogen solution (1 mg/ml PBS) before the addition of 1250 IU thrombine (250 microl) to catalize the gel formation. Granulosa cell conditioned media were tested in the presence or absence of VEGF Trap R1R2 (150 ng/ml), a potent VEGF inhibitor, that had its efficacy tested by adding VEGF (100 ng/ml) to AOC culture. Endothelial cell proliferation was measured at 48, 96, 144, 192 h by means of Scion Image Beta. A significant (p < 0.01) increase of AOC proliferation at each time of measurement was induced by culture media from granulosa cells subjected to partial (except at the end of the first 48 h) and total hypoxia compared to control and normoxia conditions, and by VEGF. VEGF Trap significantly (p < 0.01) inhibited the stimulatory effect of media conditioned by granulosa cells cultured in hypoxic conditions. These data suggest that hypoxia stimulates angiogenic activity of granulosa cells possibly by means of VEGF which could represent the main effector in promoting endothelial cell proliferation.     

Isolation, characterization and establishment of an equine retinal glial cell line: a prerequisite to investigate the physiological function of Müller cells in the retina

Retinal Müller glial cells are of vital importance for maintaining a physiological environment within the retina. To this end, they provide highly specialized physiological properties to support neurons in structure, nutrition and metabolism. The purpose of this study was to isolate Müller cells from the equine retina, determine their characteristics and subsequently establish a stable equine Müller cell line (eqMC) that will provide a prerequisite for investigations on their physiological properties. Dissociated retinal cells were obtained from equine retinas by a papain digestion technique followed by trituration and a cell attachment method by which pure Müller cell cultures were achieved. Morphological examination was performed using phase-contrast microscopy, and further characterization of different subcultures was accomplished by immunocytochemistry. Cells of passage 1 showed distinct signals for glutamine synthetase and vimentin, whereas glial fibrillary acidic protein expression was almost absent. Characteristic expression patterns remained unaltered in all subcultures. Furthermore, cultured Müller cells stably expressed the microfilament alpha-smooth muscle actin, the proliferation marker Ki67 and the membrane channels Kir4.1 and aquaporin 4. The present study introduces the eqMC-7 that will facilitate studies investigating the physiological role of Müller cells within the equine retina.     

Adipose tissue angiogenesis

A review of adipose tissue angiogenesis includes the morphological and cytochemical development of adipose tissue vasculature and the concept of primitive fat organs. Spatial and temporal relationships between fetal vascular and fat cell development are discussed, including depot- and genetic-dependent arteriolar differentiation. The relationship between connective tissue deposition and elaboration of adipose tissue vasculature is discussed with respect to regulating adipocyte development in a depot-dependent manner. In vitro studies indicated that depot-dependent vascular traits may be attributable to intrinsic growth characteristics of adipose tissue endothelial cells. These studies indicate that adipogenesis may be regulated by factors that drive angiogenesis. Fundamental aspects of angiogenesis, including basement membrane breakdown, vasculogenesis, angiogenic remodeling, vessel stabilization, and vascular permeability were reviewed. Critical angiogenic factors include vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins (Ang), ephrins, matrix metalloproteinases, and the plasminogen enzymatic system. Vascular endothelial growth factor is the most critical factor because it initiates the formation of immature vessels and disruption of a single VEGF allele leads to embryonic lethality in mice. Expression of VEGF is influenced by hypoxia, insulin, growth factors, and several cytokines. Angiogenic factors secreted and/or produced by adipocytes or preadipocytes are discussed. Vascular endothelial growth factor expression and secretion by adipocytes is regulated by insulin and hypoxia, and is associated with adipose tissue accretion. Vascular endothelial growth factor accounts for most of the angiogenic activity of adipose tissue. The proposed role of leptin as an adipogenic factor is reviewed with respect to efficacy on various aspects of angiogenesis relative to other angiogenic factors. The VEGF and leptin genes are both hypoxia inducible, but potential links between VEGF and leptin gene expression have not been examined. Finally, several studies including a study of mice treated with antiangiogenic factors indicate that adipose tissue accretion can be controlled through the vasculature per se.     

Angiogenesis and vascular regression in the ovary

Angiogenesis is prominent during development and downregulated in the adult. Strictly controlled angiogenesis in the healthy adult occurs cyclically in the ovary and corpus luteum, which therefore make an excellent model with which to study vascular growth. Dysfunctional or uncontrolled angiogenesis is involved in a number of diseases and is responsible for growth and dissemination of tumours. This review focuses on the following aspects of the ovary: the gross and microscopical anatomy of the blood vessels, described mainly--but not exclusively--in the bovine; vascularization of the follicle before and after ovulation; angiogenesis in the developing and the mature corpus luteum as well as in the corpus luteum of pregnancy. The potential mechanisms of vascular regression during luteolysis and the potential role of vascular growth in dominance and atresia of follicles will be described. Furthermore, recent research on ovarian angiogenic and potential anti-angiogenic factors including fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), angiopoietin and metalloproteinase inhibitor will be presented. Finally, the role of hormones including FSH, LH, sexual steroids, prostaglandins, prolactin, oxytocin and activin/inhibin in ovarian angiogenesis will be summarized. Future research is likely to yield valuable information that can contribute to the development of novel therapeutic strategies for the treatment of diseases characterized by disregulated angiogenesis and vascular regression.     

Intraovarian localization of growth factors in induced cystic ovaries in rats

We hypothesized that the special hormonal environment present in animals with cystic ovarian disease (COD) interferes with cellular production of growth factors (GFs). The objective of the present study was to characterize the expression of insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in induced COD using immunohistochemistry. We used an experimental model based on the exposure to constant light of adult rats during 15 weeks. We quantified the expression of GFs in cystic and normal ovaries by the Immunohistochemical Stained Area (IHCSA). In animals with COD, a significant reduction in the IHCSA of IGF-I in the follicular fluid, theca and granulosa layers of cysts occurred; and an increase in the interstitial tissue with regard to the control group. We found moderate immunoreactivity of FGF-2 in granulosa and theca layers of secondary and tertiary follicles and lower expression in the granulosa and theca interna layers of cystic follicles. Immunoexpression of VEGF was found in granulosa and theca cells of secondary and tertiary follicles. This study shows changes in the ovarian expression of IGF-I, FGF-2 and VEGF in induced COD. We can propose that an alteration in the control of the follicular dynamic, through the GFs, added to other features, could be involved in the ovarian cyst pathogenesis.     

绵羊发情周期中VEGF在卵巢中的表达

应用免疫组化方法结合计算机图像分析技术,分析同期发情后0、5、9、12、15d的绵羊卵巢中血管内皮生长因子（Vascular endothelial growth factor,VEGF）表达强度变化,以期了解VEGF在绵羊卵巢发情周期不同时期的表达规律。结果显示：VEGF阳性目标主要出现于卵泡膜与颗粒细胞。原始卵泡、初级卵泡、次级卵泡VEGF表达依次增强（P〈0．05）。发情周期0～5d,大窦腔卵泡（颗粒细胞4～8层）VEGF表达量骤然上升（P〈0．05）,而9d开始显著下降,与5d比较差异显著（P〈0．05）。12d继续下降（P〈0．05）且为最低值,15d又明显上升（P〈0．05）。VEGF在卵巢间质呈弱表达,各个时期之间差异不显著（P〉0．05）。结果表明,绵羊卵巢存在着血管周期性新生的变化特点,而VEGF在这种周期性血管新生过程中起着重要的调控作用。     

Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1-3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P<0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.     

Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRa and c-Met in canine primary brain tumours

Inhibition of tumour growth and angiogenesis by targeting key growth factor receptors is a promising therapeutic strategy for central nervous system tumours. Characterization of these growth factor receptors in canine primary brain tumours has not been done. Using quantitative real‐time TaqMan polymerase chain reaction (PCR), we evaluated the expression of messenger RNA (mRNA) for five tyrosine kinase growth factor receptors (vascular endothelial growth factor receptor [VEGFR]‐1, VEGFR‐2, endothelial growth factor receptor [EGFR]‐1, platelet‐derived growth factor receptor a [PDGFRa], and c‐Met) relative to normal cerebral cortex in 66 spontaneous canine primary brain tumours. Increased expression of VEGFR‐1 and VEGFR‐2 mRNA was greatest in grade IV astrocytomas (glioblastoma multiforme) and grade III (anaplastic) oligodendrogliomas. EGFR‐1 mRNA expression was more consistently increased than the other receptors in all tumour types, while increased PDGFRa mRNA expression was mostly restricted to oligodendrogliomas. The similarities in increased expression of these tyrosine kinase growth factor receptors in these canine tumours, as compared to data from their human counterparts, suggest that common molecular mechanisms may be present.     

Immunolocalization of angiogenic growth factors in the ovine uterus during the oestrus cycle and in response to Steroids

The vascular changes associated with endometrial maturation in preparation for embryo implantation depend on numerous growth factors, known to regulate key angiogenic events. Primarily, the vascular endothelial growth factor (VEGF) family promotes vascular growth, whilst the angiopoietins maintain blood vessel integrity. The aim was to analyse protein levels of VEGFA ligand and receptors, Angiopoietin‐1 and 2 (ANG1/2) and endothelial cell receptor tyrosine kinase (TIE‐2) in the ovine endometrium in the follicular and luteal phases of the oestrus cycle and in response to ovarian steroids. VEGFA and its receptors were localized in both vascular cells and non‐vascular epithelium (glandular and luminal epithelium) and stroma cells. VEGFA and VEGFR2 proteins were elevated in vascular cells in follicular phase endometrium, compared to luteal phase, most significantly in response to oestradiol. VEGFR1 was expressed by epithelial cells and endothelial cells and was stimulated in response to oestradiol. In contrast, Ang‐1 and Ang‐2 proteins were elevated in luteal phase endometrium compared to follicular phase, and in response to progesterone, evident in vascular smooth muscle cells and glands which surround TIE‐2‐expressing blood vessels. Our findings indicate that VEGFA is stimulated by oestradiol, most predominantly in follicular phase endometrium, and Ang‐1 and 2 are stimulated by progesterone and were increased during the luteal phase of the oestrus cycle, during the time of vascular maturation.