Vascular Endothelial Growth Factor Transcript Levels Research Articles

Inhibitory Effects of Pectic Polysaccharide Isolated from Diospyros kaki Leaves on Tumor Cell Angiogenesis via VEGF and MMP-9 Regulation.

Persimmon leaves are an attractive source of phytochemicals with potential health benefits. However, there are only a few reports on the chemical properties and biological activity of the polysaccharide fractions (PLE-I–III) of persimmon leaves. We evaluated the angiogenesis-inhibiting ability of pectic-polysaccharides. The molecular weight of PLEs was determined using a high-performance size-exclusion chromatography system. Tube formation assay of human umbilical vein endothelial cells (HUVECs) was performed using Matrigel-coated 96-well plates. Matrix metalloproteinase (MMP-9), vascular endothelial growth factor (VEGF), PI3K, Akt, and p38 phosphorylation levels were determined using Western blotting; VEGF and MMP-9 transcript levels were measured using SYBR Green qRT-PCR. PLE-I–III significantly inhibited HUVEC tube formation at 12.5 and 25 μg/mL. Among them, PLE-II showed the strongest anti-tube formation activity, and the mRNA/protein expression of angiogenesis-related factors (VEGF/MMP-9) was significantly reduced by PLE-II. PLE-II also suppressed the phosphorylation of PI3K/AKT and p38, JNK, and NF-κB p65 in HUVECs. These results suggest that the polysaccharide PLE-II isolated from persimmon leaves inhibited VEGF and MMP-9 expression in HUVECs via regulation of PI3K/AKT, p38, JNK, and NF-κB p65 signaling pathways.

Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion.

Vascular endothelial growth factor (VEGF) has been proposed as an important regulator of amniotic fluid absorption across the amnion into the fetal vasculature on the surface of the placenta. However, the activators of VEGF expression and action in the amnion have not been identified. Using the pregnant sheep model, we aimed to investigate the presence of the retinoic acid (RA) pathway in ovine amnion and to determine its effect on VEGF expression. Further, we explored relationships between RA receptors and VEGF and tested the hypothesis that RA modulates intramembranous absorption (IMA) through induction of amnion VEGF in sheep fetuses subjected to altered IMA rates. Our study showed that RA receptor isoforms were expressed in sheep amnion, and RA response elements (RAREs) were identified in ovine RARβ and VEGF gene promoters. In ovine amnion cells, RA treatment upregulated RARβ messenger RNA (mRNA) and increased VEGF transcript levels. In sheep fetuses, increases in IMA rate was associated with elevated VEGF mRNA levels in the amnion but not in the chorion. Further, RARβ mRNA was positively correlated with VEGF mRNA levels in the amnion and not chorion. We conclude that an RA pathway is present in ovine fetal membranes and that RA is capable of inducing VEGF. The finding of a positive relationship between amnion VEGF and RARβ during altered IMA rate suggests that the retinoid pathway may play a role through VEGF in regulating intramembranous transport across the amnion.

Abstract B33: The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells.

Abstract The Wilms tumor gene, WT1, encodes multiple splice isoforms with varying functions including both transcriptional and post-transcriptional regulation mediated by DNA, RNA and protein binding domains. Involvement of WT1 in regulation of various growth control genes has been widely studied in many solid tumor types. However, evidence of its role in leukemia has been contradictory; although WT1 is both overexpressed and/or mutated in different subsets of leukemia patients, and these aberrations are linked to poor or intermediate prognosis. Our working hypothesis is that in leukemia WT1 regulates expression of genes that play mitogenic roles, such as Cyclin A1 (CCNA1) and Vascular Endothelial Growth Factor (VEGF). To demonstrate that WT1 regulates these genes we first identified potential WT1 binding sites in both CCNA1 and VEGF promoters, using the MatInspector Software. Three potential WT1 binding sites were located within both the CCNA1 and VEGF gene promoters and their functional significance was validated by chromatin immunoprecipitation (ChIP) assays. ChIP analysis of chromatin from K562 cells revealed WT1 binding at 2 of 3 sites within the CCNA1 promoter; and in chromatin of 293 Kidney cells and LNCaP prostate cancer (PC) cells, WT1 binding was observed in VEGF promoter. To demonstrate that these functional sites are involved in modulating transcription from these promoters, we performed luciferase assays using reporter constructs containing CCNA1 (-1180/+145) and VEGF (-411/+50) promoter regions. Co-transfection of WT1 with these reporter constructs demonstrated that both gene promoters were activated by WT1 over-expression in K562 cells. Transcriptional regulation of the endogenous genes was confirmed by Quantitative PCR in K562 cells transfected with WT1. Although CCNA1 mRNA levels increased as expected, no significant changes in the VEGF mRNA levels were observed. This absence of VEGF mRNA up-regulation in K562 cells differs from that observed in kidney and PC cells, and could be attributed to many factors, including a lack of necessary co-activators. To determine the biological relevance of WT1 mediated regulation, we measured the levels of WT1, CCNA1 and VEGF mRNA levels in pediatric leukemia bone marrow (BM) samples using Quantitative PCR. Overall, WT1 levels were higher in Acute Myelogeneous Leukemia- M3 than in Acute Lymphoblastic Leukemia BM samples and WT1 levels were low or undetected in non-neoplastic BM. The AML-M3s samples with high WT1 levels also had higher expression of CCNA1. Conversely, in ALL samples variation was seen in the expression of WT1 and CCNA1. VEGF transcript levels in leukemia BM were generally near or below those in normal BM. Taken together, these results suggest that WT1 transcriptionally up-regulates CCNA1, but the regulation of VEGF may be cell specific. Since CCNA1 is primarily expressed in leukemias and normal testes, this suggests that WT1 could be a significant factor controlling expression of this proliferative gene. Conversely,VEGF is expressed in many different tissues and under many different conditions, including hypoxia, suggesting that WT1 may be part of the many factors contributing to the complex orchestration of VEGF expression. Nonetheless when these proliferative factors are up-regulated in leukemia, WT1 may contribute to their altered expression levels, and therefore be a leukemogenic factor. Citation Format: Sony Pandey, Shorog Al omair, Mustafa Moazam, Kurtis Eisermann, Steven J. Kuerbitz, Gail C. Fraizer. The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B33.

Dynamic biological changes in fibroblasts during hypertrophic scar formation and regression.

The human hypertrophic scar undergoes hyperplasia and regression during progression. This study aimed to investigate whether fibroblasts in scar tissue undergo biological changes during the formation and regression of human hypertrophic scar. Using 32 scar samples, we measured collagen production by Masson's staining and the expression levels of transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) by immunohistochemistry. In addition, fibroblasts from scar tissue were isolated and cultured, and total RNA was extracted for measurement of TGF-β1, VEGF and collagen transcript levels by reverse transcription-polymerase chain reaction (RT-PCR). Masson's staining showed that the number of fibroblasts and microvessels increased gradually in early and proliferative scars but decreased in regressive scars. Immunohistochemistry revealed that the expression of TGF-β1 and VEGF increased in early scars, peaked in proliferative scars and decreased in regressive scars. Moreover, the expression of TGF-β1, VEGF, collagen I and collagen III mRNAs also increased in early and proliferative scars and decreased significantly in regressive scars. Dynamic changes in fibroblast biology correlated with the formation and progression of hypertrophic scar.

Differential regulation of HIF‐1 alpha and VEGF in an anoxia tolerant brain

We examined vascular endothelial growth factor (VEGF) expression during anoxia and related it to the binding activity and expression of HIF‐1alpha in the anoxia tolerant turtle Trachemys scripta. Whole animals and neuronal cultures were exposed to anoxia and reoxygenation to determine changes in mRNA and protein levels; immunohistochemistry showed VEGF in brain slices while EMSA was performed for HIF binding. High constitutive VEGF transcript and protein levels increased 2‐fold during anoxia, returning to basal upon reoxygenation. Brain slice VEGF‐positive cells also increased following 24h anoxia; VEGF colabelled with NeuN in the cortex while ependymal cells were positive for VEGF only. By contrast, high basal HIF protein levels did not increase in anoxia or reoxygenation, and decreased in whole brain nuclear extracts, as did DNA binding activity. As the results suggested a suppression of HIF in anoxia, we exposed cells to a HIF stimulator, which significantly increased cell death over 4h anoxia. Thus while VEGF is upregulated in this anoxia tolerant model, it is unlikely to be regulated by HIF. As HIF binding in hypoxic conditions promotes many functions known to be actively downregulated in the anoxic turtle, it is not surprising that nuclear levels and HIF binding decrease in this model, to the point where increased HIF activity decreases cell survival. This work was funded by NIH grant R15 S048909–01 to SLM.

Xenoestrogens modulate vascular endothelial growth factor secretion in breast cancer cells through an estrogen receptor-dependent mechanism

Environmental chemicals may affect human health by disrupting endocrine function. Their possible role in the mammary gland and breast tumors is still unknown. Previous studies have demonstrated that vascular endothelial growth factor (VEGF), a key factor in angiogenesis and tumor progression, is an estrogen-regulated gene. We analyzed whether VEGF expression is regulated by different xenoestrogens in several breast cancer cells, MELN (derived from MCF-7) and MELP (derived from MDA-MB-231) and stably expressing estrogen receptor alpha (ERalpha); these cell lines stably express estrogen response element (beta-globin)-luciferase. Genistein, bisphenol A (BPA), 4-(tert-octyl)phenol (OP), dieldrin, and several phthalates, including benzyl butyl phthalate (BBP) and di-ethyl-2-hexyle phthalate (DEHP), were first shown to be estrogenic. These compounds induced a dose-dependent increase of VEGF secretion in MELN and MCF-7 cells; maximal effect was observed at 1-10 microM non-cytotoxic concentrations and was inhibited by the antiestrogen ICI 182 780. VEGF increase was not observed in ERalpha-negative MDA-MB-231 cells. Most substances increased VEGF transcript levels in MELN cells. In contrast, gamma-hexachlorocyclohexane, vinclozolin, and the phthalates (mono-n-butyl ester phthalic acid, di-isononyle phthalate, and di-isodecyle phthalate) were ineffective on both VEGF secretion and estrogenic luciferase induction in these cell lines. Specific kinase inhibitors PD98059, SB203580, or LY294002 suppressed the xenoestrogen-induced VEGF response, suggesting activation of MEK, p38 kinase, and phosphatidylinositol-3-kinase pathways. Our in vitro results show for the first time that genistein and xenoestrogens (BPA, OP, dieldrin, BBP, and DEHP at high concentrations) up-regulate VEGF expression in MELN cells by an ER-dependent mechanism. Since VEGF increases capillary permeability and breast tumor angiogenesis in vivo, the physiological relevance of these findings is discussed.

Vascular endothelial growth factor mRNA and protein do not change in parallel during non‐inflammatory skeletal muscle ischaemia in rat

Impaired blood flow is thought to induce a pro-angiogenic environment due to local hypoxia, yet prolonged mild ischaemia induces only modest capillary growth. We compared the expression of vascular endothelial growth factor (VEGF) mRNA and protein with capillary to fibre ratio (C: F) and muscle blood flow in extensor digitorum longus of rats that had undergone unilateral ligation of the common iliac artery. Resting blood flow during the first two weeks after ligation (3, 7 and 14 days) was decreased by approximately 60% but recovered partially after 5 weeks (36% reduction). Functional hyperaemia (9-fold increase in blood flow during contractions) was eliminated in the first week after ligation, with a moderate recovery seen after 14 and 35 days. Muscle histology confirmed the absence of tissue necrosis or inflammation. Both VEGF mRNA (60%, P<0.05) and protein levels (700%, P<0.01) increased during the initial phase of ischaemia (at 1 and 3 days), well before any overt angiogenesis, and both declined towards control levels by 7 days. A secondary increase in VEGF protein (by 60% at 14 days, P<0.05) preceded the 20% increase in C: F seen after 5 weeks, but occurred while VEGF transcript levels continued to decline (to 50% of control at 35 days, P<0.05). Thus, evaluation of neither VEGF mRNA nor protein is an adequate index of angiogenic potential in response to ischaemia. We conclude that VEGF alone is insufficient to induce angiogenesis in ischaemic conditions, and that effective angiotherapy requires intervention aimed at other cytokines.

Vascular Endothelial Growth Factor (VEGF) Is Suppressed in WT1-Transfected LNCaP Cells

The Wilms' tumor suppressor gene product (WT1) regulates expression of growth control genes. Microarray analysis of gene expression profiles of hormone-treated LNCaP prostate cancer cell lines transfected with either wild-type WT1 or a zinc finger mutant form, DDS (R394W), revealed significantly altered patterns of expression. Validation studies using quantitative real-time PCR confirmed the differential expression of the tumor progression gene, vascular endothelial growth factor (VEGF). WT1-LNCaP cells had significantly reduced levels of VEGF mRNA when compared to vector control cells; in contrast, DDS-LNCaP cells showed elevated levels of VEGF transcripts. To address a functional role for WT1 overexpression, we investigated whether induction of VEGF expression, by the synthetic androgen R1881, would be disrupted in wild-type or mutant WT1-transfected LNCaP cells. Hormone treatment failed to elevate VEGF transcript levels above uninduced baseline levels in WT1-LNCaP cells, despite 48 h of treatment with 5 nM R1881. Consistent with our quantitative real-time PCR analysis, immunofluorescent staining of VEGF protein was reduced in WT1-LNCaP cells in both the presence and absence of R1881 treatment. Conversely, VEGF levels increased in vector control and DDS-LNCaP cells treated with 5 nM R1881. Not only do these studies point out the regulatory potential of WT1 for VEGF, but they also indicate an altered function for the mutant DDS isoform. Because VEGF is associated with neovascularization and promotion of metastasis in a variety of solid tumors including prostate cancer, a better understanding of the regulation of VEGF expression by transcription factors, such as WT1, is important for halting disease progression.

Relaxin Stimulates Multiple Signaling Pathways: Activation of cAMP, PI3K, and PKCζ in THP‐1 Cells

Relaxin has been shown previously to stimulate cyclic AMP production and the activation of MAPK. We reported that phosphoinositide-3 kinase (PI3K) activity is required for biphasic stimulation of cAMP by relaxin and that relaxin treatment increased PI3K activity in THP-1 cells. A downstream target of PI3K is protein kinase C zeta (PKCzeta). Relaxin stimulated translocation of PKCzeta to the plasma membrane in THP-1, MCF-7, pregnant human myometrial (PHM1-31), and mouse mesangial (MMC) cells. PKCzeta translocation is PI3K dependent and independent of cAMP production. Pharmacological and antisense approaches, utilized to inhibit or knock down PKCzeta, resulted in a 40% inhibition of relaxin-stimulated cAMP production. The stimulation of PKCzeta by relaxin therefore is downstream of PI3K leading to increased cAMP production. To determine the role of PI3K/PKCzeta stimulation by relaxin on downstream-mediated events, we examined the increase in vascular endothelial growth factor (VEGF) gene expression by relaxin. Treatment of THP-1 or MMC cells with the PI3K inhibitor, LY294002, abolished the relaxin-mediated stimulation of VEGF transcript levels. In summary, relaxin has pleiotropic signaling effects in THP-1 cells activating ERK1/2, cAMP, PI3K, and PKCzeta. We have described a novel bifurcated pathway by which relaxin stimulates Gs alpha and PI3K/PKCzeta leading to increased cAMP production and increased VEGF gene expression. Some, but not all, of these pathways are detected in other cell lines which may cause the unique diversity of downstream responses from this interesting hormone.

VEGF, FGF2, TGFB1 and TGFBR1 mRNA expression levels correlate with the malignant transformation of the uterine cervix

Angiogenesis is a complex procedure induced by the secretion of numerous growth factors from endothelial cells. Vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (FGF2), transforming growth factor-β1, 2, 3 (TGFB1, 2, 3), and transforming growth factor-β receptors (TGFBR1, 2, 3) mRNA expression pattern was evaluated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer, compared to that of normal cervical tissues, and correlated to the clinical stage of the disease. Transcript levels of the above genes were assessed by RT-PCR analysis in a total of 44 cervical specimens. VEGF, TGFB1, TGFBR1, and FGF2 transcript levels were significantly different in the normal, CIN and cancer specimen groups ( P=0.015, 0.001, 0.008, and 0.029, respectively). Higher TGFBR1 mRNA levels were observed in parallel with increased severity of the lesion, whereas FGF2 exhibited lower transcript levels. A highly significant increase of VEGF mRNA expression was found upon cervical neoplastic transformation ( P<0.0001). High-grade squamous intraepithelial lesions exhibited higher VEGF mRNA levels than low-grade lesions ( P=0.039). TGFBR1 and TGFBR3 receptors demonstrated significant co-expressions with TGFB2 ( P<0.0001), and TGFB1 ( P=0.005 and 0.002, respectively) in normal cervical specimens. However, a disruption of co-expression patterns was observed in the groups of CIN and cancer cases, compared to normal tissues. Our findings show that VEGF, FGF2, TGFB1 and TGFBR1 mRNA expression levels correlate with the malignant transformation of the uterine cervix. The involvement of the examined markers in cervical carcinogenesis is furthermore supported by the observed disruption of their mRNA co-expression patterns.

Exercise-induced expression of vascular endothelial growth factor mRNA in rat skeletal muscle is dependent on fibre type.

In this study, we quantified the expression of the vascular endothelial growth factor (VEGF) gene in individual muscle fibres at the end of a single 90 min run of 20-25 m min-1, at 10 % incline. In addition, we evaluated the co-ordinated expression of several hypoxia-sensitive genes, including the ORP-150 gene. Individual fibres were taken from rat plantaris muscle, either at the end of a single bout of exercise or at rest, and classified as Type I, IIa, IIx or IIb, according to the expression of myosin heavy chain (MHC) isoforms. VEGF mRNA levels increased by 90 % in exercising whole plantaris in comparison with those in control muscle (P < 0.001), while the VEGF protein content increased by 72 % (P < 0.05). Using real-time PCR analysis, an accurate and reproducible method for quantification of mRNA levels, a marked rise in VEGF transcript levels was observed at the end of exercise in individual myofibres (P < 0.05), providing the first direct evidence that VEGF transcripts increase in muscle cells after a single bout of exercise. This exercise-induced increase in VEGF transcript levels was specifically observed in type IIb myofibres, which are predominantly glycolytic and more susceptible to local hypoxia than oxidative myofibres such as type I or IIa fibres (110 %, P < 0.05). Moreover, treadmill exercise increased the expression of two hypoxia-sensitive genes. The levels of mRNA for Flt-1, a VEGF-specific receptor, and those for ORP-150, a chaperone essential for the secretion of mature VEGF, increased in whole plantaris muscles (108 and 92 %, respectively, P < 0.05). Taken together, these findings are consistent with the suggestion that hypoxia could be one of the mechanisms involved in exercise-induced capillary growth.

Pharmacological and endogenous progestins induce vascular endothelial growth factor expression in human breast cancer cells.

Tumor expansion is dependent on angiogenesis, which is regulated by peptide growth factors of which vascular endothelial growth factor (VEGF) is one of the most selective and potent. VEGF expression is regulated by steroid hormones in a number of systems, including T47-D human breast cancer cells in which VEGF protein levels are elevated by progestins. In the present study, we investigated the effect of progestins on VEGF mRNA levels in human breast cancer cells. For these experiments, T47-D cells were exposed to progestins, RNA was prepared for measurement of VEGF transcript levels by Northern blot analysis and VEGF protein in the cell culture media was measured by enzyme-linked immunosorbent assay. Basal expression of VEGF mRNA is low in these cells, and is rapidly induced following exposure to progestins, reaching a maximum induction of 2- to 5-fold between 3 and 6 hr after hormone addition. This induction was inhibited by the antiprogestin RU-486 indicating that it is progesterone receptor (PR) dependent. Transcripts for VEGF165 and VEGF121 were the two major spliced forms of VEGF mRNA that were detected by reverse transcription-polymerase chain reaction in basal and progestin-stimulated T47-D cells. Maximum induction of VEGF mRNA was achieved with 10(-8) M progesterone, and induction was hormone specific, as estrogens, glucocorticoids, and androgens were without effect. Actinomycin D completely abolished the induction of VEGF transcript levels by progestins, suggesting that this response involves de novo mRNA synthesis, but puromycin did not inhibit induction, suggesting that this effect does not require protein synthesis. This report demonstrates that progestins stimulate VEGF mRNA levels and raises the possibility that anti-progestins may be useful to inhibit proliferation and metastasis in some human breast cancers by blocking VEGF production.

Regulation of vascular endothelial growth factor expression by estrogens and progestins.

Estrogens increase the expression of vascular endothelial growth factor (VEGF) mRNA in the rodent uterus. This regulatory effect is rapid, beginning within 1 hr after hormone treatment, dose dependent, and blocked by the pure antiestrogen ICI 182,780. The induction of the transcript is blocked by inhibitors of RNA but not of protein synthesis, and we have recently identified estrogen response elements in the VEGF gene. Collectively, these findings indicate that estrogens regulate uterine VEGF expression at the transcriptional level via the classical nuclear estrogen receptor pathway. Estrogen induction of VEGF occurs in the stromal layer of the rodent uterus, and estradiol induces expression of VEGF transcript levels in cultured human uterine stromal cells. Progestins also induce VEGF expression in the rodent uterus, although the effect is less marked and slower in onset than estrogenic effects. The effect of progestins is blocked by the antiprogestin mifepristone (RU-486), suggesting that it is also mediated by a classical nuclear receptor pathway. In addition, progestins regulate expression of VEGF mRNA and protein in cultured human T47-D breast cancer cells. The development of uterine leiomyomas is associated with exposure to ovarian sex steroids, abnormal uterine bleeding is commonly seen in patients with leiomyomas, and fibroids require an increased vascular supply for their growth. These observations suggest that VEGF and other angiogenic factors may represent potential targets for the treatment and prevention of uterine fibroids.

Induction of the angiogenic factor VEGF in the uterus by the antiprogestin onapristone

Onapristone (also referred to as ZK 98,299) is an antiprogestin that shares a number of structural similarities to mifepristone (RU-486) and other drugs in this class. While investigating the actions of antiprogestins on steroid hormone induced gene expression of angiogenic factors such as vascular endothelial growth factor (VEGF), we noted that onapristone alone induces VEGF transcript levels in the immature, ovariectomized rat uterus. In addition, onapristone induces expression of c- fos mRNA, which is induced by estrogens but not progestins in this target tissue. This induction of VEGF and c- fos by onapristone is inhibited by the antiestrogen ICI 182,780, but not by the antiprogestin RU-486. Both transcripts are very rapidly induced by onapristone, with maximal mRNA levels observed 3–6 h after in vivo administration of the drug. This time course is similar to that for induction of these genes by estrogenic hormones. Dose–response studies show that both these genes are maximally induced by a 2.5 mg/kg dose of onapristone following intra peritoneal injection. These results indicate that onapristone rapidly upregulates several genes normally under estrogenic regulation in the immature rat uterus. Importantly, this is the first report of the induction of a major angiogenic factor by an antiprogestin. Since an increase in vascularity increases tumor expansion and metastasis, the induction of angiogenesis and its regulatory factors such VEGF may be an important end-point to consider in the development and use of antiprogestins for the treatment of neoplastic disease.

Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation.

Vascular endothelial growth factor (VEGF) is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells. Such factors are likely candidates for regulatory molecules involved in endothelial growth control. We have characterized the murine VEGF gene and have analysed its expression pattern in embryogenesis, particularly during brain angiogenesis. Analysis of cDNA clones predicted the existence of three molecular forms of VEGF which differ in size due to heterogeneity at the carboxy terminus of the protein. The predicted mature proteins consist of 120, 164 or 188 amino acid residues. Homodimers of the two lower molecular weight forms, but not of the higher molecular weight form, were secreted by COS cells transfected with the corresponding cDNAs and were equally potent in stimulating the growth of endothelial cells. During brain development, VEGF transcript levels were abundant in the ventricular neuroectoderm of embryonic and postnatal brain when endothelial cells proliferate rapidly but were reduced in the adult when endothelial cell proliferation has ceased. The temporal and spatial expression of VEGF is consistent with the hypothesis that VEGF is synthesized and released by the ventricular neuroectoderm and may induce the ingrowth of capillaries from the perineural vascular plexus. In addition to the transient expression during brain development, a persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in choroid plexus and in kidney glomeruli. The data are consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation.