Vascular Endothelial Growth/permeability Factor Research Articles

Regulation of Placental Vascular Endothelial Growth/Permeability Factor Expression and Angiogenesis by Estrogen during Early Baboon Pregnancy

We have recently shown that there was a developmental increase in placental trophoblast vascular endothelial growth/permeability factor (VEG/PF) expression and vascularization that closely paralleled maternal serum estrogen levels during advancing baboon gestation. The present study determined whether estrogen regulates these important aspects of primate development. VEG/PF mRNA levels were determined by competitive RT-PCR in isolated villous placental cells, and placental vascularization was assessed by image analysis. Placentas were obtained on d 60 of gestation (length of gestation is 184 d) from baboons in which estrogen levels on d 25-59 were increased by daily administration of aromatizable androstenedione or decreased by aromatase inhibitor CGS 20267. Androstenedione treatment increased maternal serum estradiol levels 3-fold (P < 0.01) and placental villous cytotrophoblast VEG/PF mRNA level to a value (mean +/- se, 26,836 +/- 5,625 attomoles/microg total RNA) 2.5-fold greater (P < 0.05) than that in untreated animals (11,645 +/- 1,746 attomoles/microg RNA). In contrast, administration of CGS 20267 decreased serum estradiol (P < 0.01) and placental cytotrophoblast mRNA (2,912 +/- 693 attomoles/microg RNA; P < 0.05) levels by 75%, effects prevented by concomitant administration of CGS 20267 and estradiol. VEG/PF mRNA levels in inner villous cells were unaltered. Coinciding with the increase in placental VEG/PF expression, the percent vascularized area (3.46 +/- 0.23) and vessel density (493 +/- 34 vessels/mm(2)) of the villous placenta in untreated baboons on d 60 were increased (P < 0.01) in baboons in which estrogen levels were elevated by androstenedione treatment (6.54 +/- 0.56 and 743 +/- 27 vessels/mm(2), respectively). It is concluded that estrogen has an important role in stimulating trophoblast VEG/PF expression and consequently villous placental angiogenesis to promote fetal growth and development in early primate pregnancy.

Acute Temporal Regulation of Placental Vascular Endothelial Growth/Permeability Factor Expression in Baboons by Estrogen1

Vascular endothelial growth/permeability factor (VEG/PF) has an established role in angiogenesis, however, the regulation of placental VEG/PF expression during primate pregnancy is incompletely understood. A temporal study was conducted in baboons to determine the effect of acute administration of estradiol on the expression of VEG/PF by cells of the villous placenta. VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in isolated placental cell fractions of baboons after acute i.v. and i.m. administration of estradiol. Within 2 h of estradiol treatment, VEG/PF mRNA (attomoles/ micrograms total RNA) increased within villous cytotrophoblasts to a level (mean +/- SEM, 12,612 +/- 2419) that was almost 2-fold greater (P < 0.05) than in untreated controls (6810 +/- 1368). Cytotrophoblast VEG/PF mRNA levels remained elevated (P < 0.01) 6 h after estradiol treatment (15,006 +/- 506), but were not different from controls 18 h after estradiol administration. VEG/ PF mRNA levels in whole villous tissue also were greater 6 h (12,667 +/- 2284, P < 0.05) and 18 h (16,080 +/- 3816, P < 0.01) after estradiol treatment than in untreated animals (3380 +/- 594). In contrast, VEG/PF mRNA levels in cells of the inner villous core were not altered by estradiol treatment. Expression of both the VEG/PF(121) and VEG/PF(165) mRNA species appeared to increase in the placenta 6 h after estradiol treatment of baboons. We propose that estrogen regulates VEG/PF expression within the placenta in a cell-specific manner, providing a paracrine system to promote vascularization of the villous placenta during the first half of primate pregnancy.

Acute temporal regulation of vascular endothelial growth/permeability factor expression and endothelial morphology in the baboon endometrium by ovarian steroids.

We recently showed that endometrial vascular endothelial growth/permeability factor (VEG/PF) mRNA expression was decreased by ovariectomy of baboons and restored by chronic administration of estrogen. However, it remains to be determined whether this effect of estrogen reflects genomic up-regulation of VEG/PF and leads to an increase in microvascular permeability, an early physiological event in angiogenesis. Therefore, we determined the temporal expression of VEG/PF mRNA in glandular epithelial and stromal cells isolated by laser capture microdissection from and width of microvascular paracellular clefts that regulate vessel permeability in the endometrium of ovariectomized baboons after acute estradiol and/or progesterone administration. Endometrial VEG/PF mRNA levels were increased in five of five animals within 2 h of estradiol administration and remained elevated at 4 and 6 h. The net increase in glandular epithelial (7.31 +/- 2.72 attomol/fmol 18S ribosomal rRNA) and stromal (3.13 +/- 0.36) cell VEG/PF mRNA levels after estradiol administration was over 8-fold (P < 0.05) and 2.6-fold (P < 0.01) greater, respectively, than after vehicle (0.90 +/- 0.30, glands and 1.20 +/- 0.33, stroma). In contrast, endometrial VEG/PF mRNA expression was unaltered by progesterone. After estradiol treatment, endometrial paracellular cleft width was increased (P < 0.01) from a mean (+/-SE) of 71.6 +/- 4.6 nm at 0 h to 101.1 +/- 6.4 nm at 6 h, whereas vehicle or progesterone had no effect. We suggest that estrogen has a major role in regulating VEG/PF synthesis and early events in angiogenesis in the primate endometrium.

Effect of estrogen on vascular endothelial growth/permeability factor expression by glandular epithelial and stromal cells in the baboon endometrium.

The ovarian steroid hormones, estrogen and progesterone, have important roles in establishing the new vascular bed within the endometrium during each menstrual cycle; however, little is known about the mechanisms underlying this process. We recently showed that mRNA and protein levels for the angiogenic factor vascular endothelial growth/permeability factor (VEG/PF) in endometrial glandular epithelial and stromal cells of baboons were decreased to very low levels by ovariectomy, and we proposed that the levels of estrogen and progesterone exhibited during the menstrual cycle regulate endometrial VEG/PF expression in the primate. To test this hypothesis, VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in glandular epithelial and stromal cells isolated by laser-capture microdissection from, and VEG/PF protein was determined by immunocytochemistry in the endometrium of baboons after ovariectomy and chronic administration of estradiol and progesterone in levels designed to replicate the hormonal profiles that are characteristic of the proliferative and secretory phases of the menstrual cycle. Administration of estradiol to ovariectomized baboons in levels that replicated the late-proliferative phase of the menstrual cycle (209 +/- 40 pg/ml serum) increased/restored VEG/PF mRNA to levels in the glands (5.57 +/- 1.53 amol/fmol 18S rRNA, P < 0.01) and stroma (2.61 +/- 1.57 amol/fmol 18S rRNA, P < 0.02) that were approximately 10-fold greater than those observed after ovariectomy alone (0.52 +/- 0.21 and 0.22 +/- 0.11 amol/fmol 18S rRNA, respectively) and were similar to those previously shown in intact baboons. Concomitant administration of estradiol and progesterone to ovariectomized baboons in levels that replicated the midsecretory phase of the menstrual cycle (44 +/- 15 pg/ml serum and 9.8 +/- 2.2 ng/ml serum, respectively) resulted in glandular epithelial (3.65 +/- 1.42 amol/fmol 18S rRNA) and stromal (1.25 +/- 0.77 amol/fmol 18S rRNA) VEG/PF mRNA levels that were not significantly different from those exhibited after ovariectomy or ovariectomy and estradiol treatment. Comparable results were obtained for VEG/PF mRNA expression in whole-endometrial tissue, although the relative 2-fold increase (P < 0.03) in VEG/PF mRNA levels induced by estrogen in mixed endometrial cells of ovariectomized baboons appeared to be less marked than that in isolated glandular epithelial and stromal cells. After ovariectomy, endometrial width (0.98 +/- 0.09 mm) was approximately one-third of that in intact baboons (3.58 +/- 0.32 mm), and endometrial VEG/PF protein expression was low. Estradiol restored endometrial width (3.00 +/- 0.12 mm, P < 0.01) and VEG/PF protein expression to normal. In summary, estrogen has a significant role in regulating and maintaining VEG/PF expression by glandular epithelial and stromal cells of the endometrium during the menstrual cycle.

Co-expression of endothelin-1 and vascular endothelial growth factor mediates increased vascular permeability in lung grafts before reperfusion

Endothelin-1 (ET-1) protein levels increase in lung transplant recipients, although the source of ET-1 remains uncertain. Evidence is accumulating that ET-1 coregulates the expression of vascular endothelial growth/permeability factor (VEGF-A). By using real-time polymerase chain reaction and Western blotting, we describe upregulated ET-1 mRNA and VEGF-A protein levels as well as increased fluid content in donor lung-tissue biopsies before reperfusion. Hypoxia and ET-1 co-upregulate VEGF-A overexpression, therefore temporary VEGF-A antagonism during graft preservation might be of benefit in lung transplantation.

Vascular endothelial growth-permeability factor in granulation tissue of chronic subdural haematomas.

Vascular Endothelial Growth/Permeability Factor (VEG/PF) has been correlated with angiogenesis and vascular leakage in diverse pathological conditions of the brain. On the other hand, neoformation of capillaries and vascular hyperpermeability have been related to progressive enlargement of chronic subdural haematomas (CSDH). The purpose of the present study is to verify the presence of VEG/PF in tissue of CSDH, with the hypothesis that neovascularization and vascular hyperpermeabitity in CSDH can be related to the presence of this cytokine. Immunohistochemical studies and Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis were performed in the neomembranes of 12 CSDH to verify presence of VEG/PF in this tissue. Strong expression of VEG/PF was immunohistochemically disclosed in neomembranes of CSDH, as well as in inflammatory cells currently infiltrating this tissue. Furthermore, RT-PCR studies showed the presence of VEG/PF in all the studied samples. These findings suggest a role for inflammatory cells, currently present in the granulation tissue of CSDH, in the release of VEG/PF and in the enlargement of these lesions, stating the possibility of a new therapeutic approach to CSDH, based on blocking the physiological effects of VEG/PF.

Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy.

Vascular endothelial growth/permeability factor (VEG/PF) has an important role in angiogenesis; however, very little is known about the developmental regulation of VEG/PF and the vascular system within the placenta during human pregnancy. In the present study, therefore, a developmental approach was used in the baboon to determine the placental source of VEG/PF and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors, and whether the rise in estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization. VEG/PF messenger RNA (mRNA) levels were determined by competitive RT-PCR in villous cell fractions isolated by Percoll gradient centrifugation from placentas obtained on days 45 and 54 (very early), 60 (early), 100 (mid), and 165-170 (late) of baboon pregnancy (term = 184 days). Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15-0.20 ng/ml) to an early surge of over 2.5 ng/ml on days 60-85, and peak levels of 4-6 ng/ml late in baboon pregnancy. VEG/PF mRNA was expressed in low level in the syncytiotrophoblast (<2,000 attomol/microgram total RNA), and values in this fraction did not change significantly with advancing gestation. VEG/PF mRNA expression was slightly greater in the inner villous core cell fraction; however, levels decreased (P < 0.05) between early and late gestation. Cytotrophoblasts were a major source of VEG/PF mRNA and levels increased (P < 0.01) from 3,631 +/- 844 attomol/microgram total RNA on day 45 to 25,807 +/- 5,873 attomol/microgram total RNA on day 170. VEG/PF protein expression determined by immunocytochemistry was abundant in cytotrophoblasts and lower in the syncytiotrophoblast and inner villous core cells. The flt-1 and KDR/flk-1 receptors were expressed in the vascular endothelial cells of the baboon villous placenta. The percentage of villous placenta occupied by blood vessels and the number of vessels/mm(2) villous tissue, determined by image analysis, progressively increased (P < 0.001; r = 0.97) from 3.4 +/- 0.2% and 447 +/- 29, respectively, on day 54 to 15.9 +/- 0.9% and 1,375 +/- 71, respectively, on day 170. In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta, and that cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta closely paralleled the increase in estradiol concentrations of advancing pregnancy. These results are consistent with the concept that estrogen has an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.

Expression of vascular permeability factor in craniopharyngioma.

The expression of vascular endothelial growth/permeability factor (VEG/PF) has recently been correlated with the presence of tumor-associated cysts in some intracranial tumors. The purpose of this study was to evaluate a possible relationship between the presence of VEG/PF and the formation of cysts in craniopharyngiomas. The expression of VEG/PF was studied in histological specimens from a series of 12 craniopharyngiomas. In this series, the tumors were classified as presenting a mainly solid pattern with small macroscopic cysts (four patients) or a mainly cystic pattern (eight patients). The mainly solid tumors containing small macroscopic cysts showed little or no VEG/PF positivity, which was mainly present in tumor cells surrounding cysts. Nevertheless, mainly cystic craniopharyngiomas showed a moderate or high degree of VEG/PF positivity in tumor cells. These findings indicate that the predominance of a cystic or solid macroscopic appearance of craniopharyngiomas may be influenced by the degree of VEG/PF expression within the tumor cells.

Vascular endothelial growth/permeability factor in spinal cord injury.

Predicated on the hypothesis that this cytokine can contribute to the development of vascular hyperpermeability, leading to tissue edema after trauma, the purpose of this study was to determine the presence in tissue of vascular endothelial growth/permeability factor (VEG/PF) after experimental spinal cord injury. The presence of VEG/PF was studied at 8 hours and 2, 8, and 14 days after a traumatic injury in adult Wistar rats. Studies were conducted in which a monoclonal antibody to the VEG/PF was used. Strong VEG/PF immunoreactivity was detected in the walls of pial and intramedullary vessels and in reactive astrocytes 8 hours posttrauma and was unchanged on Days 2 and 8. By Day 14, immunoreactivity decreased, and most of the arterioles from the pia and gray matter showed no mural VEG/PF. The authors' present findings suggest a role for this cytokine in the development of tissue edema after spinal cord trauma and point to the possible usefulness of a therapeutic approach to spinal cord injury based on blocking the cell expression of VEG/PF or its physiological effects.

Vascular permeability factor expression in cerebellar hemangioblastomas: correlation with tumor-associated cysts.

The presence of vascular endothelial growth/permeability factor (VEG/PF) has been studied in histological specimens from a series of 16 cerebellar hemangioblastomas. In this series, the tumors were classified as presenting a macroscopic solid pattern (4 cases), a mainly solid pattern with macroscopic cysts (4 cases) or a mainly cystic pattern (8 cases). Solid tumors showed a low degree (75% of cases) or moderate degree (25% of cases) of VEG/PF positivity. The mainly solid tumors containing macroscopic cysts showed a moderate degree (50% of cases) or high degree (50% of cases) of VEG/PF positivity. Nevertheless, mainly cystic hemangioblastomas showed a high degree (87.5% of cases) or moderate degree (12.5% of cases) of VEG/PF positivity. These findings suggest that the predominance of a cystic or solid macroscopic appearance of cerebellar hemangioblastomas may be influenced by the degree of VEG/PF expression within the tumor tissue.

Estrogen regulates vascular endothelial growth/permeability factor expression in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

Vascular endothelial growth/permeability factor (VEG/PF) is expressed in some normal tissues and at high levels in a wide range of tumors. This growth factor is believed to be a key mediator of angiogenesis. Recent reports have shown that VEG/PF mRNA in the normal rat uterus is stimulated by estradiol (E2). In this study, we investigated the expression of VEG/PF in the mammary gland and 7,12-dimethylbenz(a)anthracene (DMBA)-induced, hormone-dependent mammary tumor of the rat model, and also whether VEG/PF is regulated by E2. VEG/PF mRNA from tumor extracts was amplified by RT-PCR with VEG/PF primers and generated two main products which corresponded in size to those expected for VEG/PF 164 and 120. In some cases, a third product corresponding in size to that expected for VEG/PF 188 was also generated. No such PCR products were generated from equal amount of RNA from normal mammary tissue, rat brain, or liver. Using immunocytochemistry, VEG/PF expression was detected in the epithelial cells of the tumors. We developed an ELISA assay to measure VEG/PF protein concentrations and found a 4-fold difference between normal mammary glands (1.3 +/- 0.11 ng/mg protein) and tumors (4.44 +/- 0.66) (P < 0.01). E2 treatment (5 microg/rat, s.c.) of rats 24 h after ovariectomy, greatly enhanced the expression of RT-PCR products in tumors within 2 h, which reached a maximum at 6-8 h but declined by 48 h. VEG/PF concentrations were also increased 8-12 h after E2 injection. When rats were given two injections of aromatase inhibitor 4-hydroxyandrostenedione (4-OHA 10 mg/rat s.c.) 24 h apart, to reduce estrogen concentrations, a low level of RT-PCR products was maintained for at least 96 h. After a single injection of 4-OHA, RT-PCR products remained low until 36 h when an increase occurred corresponding with a rise in plasma E2 levels. Injection of E2 2 h after 4-OHA treatment, caused a rise in RT-PCR products in 6-8 h. However, there was no significant change in VEG/PF concentrations. An increase in VEG/PF protein concentrations followed the increase in mRNA levels by 4-6 h. Thus, it appears that E2 causes a rapid induction of VEG/PF expression in mammary tumors that is similar to that observed in the normal uterus. These findings suggest that one mechanism by which estrogen acts as a mammary tumor promotor is by stimulating VEG/PF, leading to increased tumor angiogenesis and/or permeability of the microvessels to allow tumor cell migration.

Vascular endothelial growth/permeability factor expression in human glioma specimens: correlation with vasogenic brain edema and tumor-associated cysts

Peritumoral vasogenic brain edema (PVBE) is a common accompaniment of malignant gliomas. It results from microvascular extravasation of plasma fluid and proteins through the interendothelial spaces. Tumor-associated cysts (TACs) are observed more commonly with benign gliomas that are not associated with PVBE. This study investigates the hypothesis that these morphologically distinct epiphenomena of microvascular extravasation are linked by a common pathophysiological mechanism involving vascular endothelial growth/permeability factor (VEG/PF), which has been implicated in vascular leak phenomena including ascites, malignant effusions, and brain edema. Furthermore, VEG/PF has been isolated from cultured glioma cells, and both VEG/PF protein and messenger RNA transcripts are expressed in brain tumor tissue. To further elucidate the relationship of VEG/PF to PVBE and TACs, the authors examined 34 pathological specimens for VEG/PF expression. Nineteen primary low-grade tumors, 11 primary high-grade tumors, and four gliosis controls were immunostained with a polyclonal anti-VEG/PF immunoglobulin G antibody. Magnetic resonance imaging was used to quantitate PVBE and to determine the presence of TACs and tumor enhancement. The study revealed that eight VEG/PF-negative specimens exhibited no significant edema, whereas 26 VEG/PF-positive tumors exhibited either significant PVBE or TACs. Notably, eight of nine benign TACs that were not associated with PVBE immunostained positive for VEG/PF. These data indicate a high degree of correlation between VEG/PF expression by gliomas and the occurrence of PVBE or TACs, irrespective of tumor grade, thus supporting VEG/PF's pivotal role as the common pathophysiological link between these processes.

Increased expression of vascular endothelial growth/permeability factor in the rat ovary following an ovulatory gonadotropin stimulus: potential roles in follicle rupture

Ovulation is accompanied by a large increase in the permeability of the capillaries surrounding the follicle, beginning a few hours after the ovulatory stimulus. The resulting edema may play a role in ovulation as well as in the formation and vascularization of the CL. Vascular endothelial growth/permeability factor (VEG/PF) is both a specific mitogen for endothelial cells and a potent stimulator of vascular permeability. The purpose of this study was to determine whether or not the ovulatory stimulus induces an increase in the expression of VEG/PF in the ovary. Female rats were primed with 8 IU of eCG at 27 days of age. Follicular maturation and ovulation were induced with an injection of hCG (5 IU) in the early afternoon on the second day after priming. Ovaries were removed 0, 1, 2, 4, 8, 10, and 18 h later. VEG/PF mRNA levels were compared through use of quantitative reverse transcription-polymerase chain reaction (RT-PCR). There was a marked increase (approximately 8-fold) in steady state levels of the transcripts for VEG/PF120 and VEG/PF164 between 1 and 4 h after hCG in whole ovaries. Increases were detectable both in granulosa cells and in thecal/stromal tissue. The high level of expression was maintained at 10 and 18 h (Day 1 CL). Thus, the preovulatory increase in follicular vascular permeability is closely associated with a marked, sustained increase in VEG/PF expression. VEG/PF, therefore, may play an important role in that increase and, consequently, in the process of ovulation as well as the subsequent vascularization of the CL.