Vascular Endothelial Growth Factor Splice Variants Research Articles

A Splice Form of VEGF, a Potential Anti-Angiogenetic Form of Head and Neck Squamous Cell Cancer Inhibition.

Angiogenesis, primarily mediated by vascular endothelial growth factor (VEGF), is a fundamental step in the progression and metastasis of head and neck squamous cell carcinoma (HNSCC). Traditional anti-angiogenic therapies that target the VEGF pathway have shown promise but are often associated with significant side effects and variable efficacy due to the complexity of the angiogenic signaling pathway. This review highlights the potential of a specific VEGF splice form, VEGF165b, as an innovative therapeutic target for HNSCC. VEGF165b, unlike standard VEGF, is a natural inhibitor that binds to VEGF receptors without triggering pro-angiogenic signaling. Its distinct molecular structure and behavior suggest ways to modulate angiogenesis. This concept is particularly relevant when studying HNSCC, as introducing VEGF165b's anti-angiogenic properties offers a novel approach to understanding and potentially influencing the disease's dynamics. The review synthesizes experimental evidence suggesting the efficacy of VEGF165b in inhibiting tumor-induced angiogenesis and provides insight into a novel therapeutic strategy that could better manage HNSCC by selectively targeting aberrant vascular growth. This approach not only provides a potential pathway for more targeted and effective treatment options but also opens the door to a new paradigm in anti-angiogenic therapy with the possibility of reduced systemic toxicity. Our investigation is reshaping the future of HNSCC treatment by setting the stage for future research on VEGF splice variants as a tool for personalized medicine.

The VEGF/VEGFR Axis Revisited: Implications for Cancer Therapy

The vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) axis is indispensable in the process of angiogenesis and has been implicated as a key driver of tumor vascularization. Consequently, several strategies that target VEGF and its cognate receptors, VEGFR-1 and VEGFR-2, have been designed to treat cancer. While therapies targeting full-length VEGF have resulted in an improvement in both overall survival and progression-free survival in various cancers, these benefits have been modest. In addition, the inhibition of VEGFRs is associated with undesirable off-target effects. Moreover, VEGF splice variants that modulate sprouting and non-sprouting angiogenesis have been identified in recent years. Cues within the tumor microenvironment determine the expression patterns of these variants. Noteworthy is that the mechanisms of action of these variants challenge the established norm of VEGF signaling. Furthermore, the aberrant expression of some of these variants has been observed in several cancers. Herein, developments in the understanding of the VEGF/VEGFR axis and the splice products of these molecules, as well as the environmental cues that regulate these variants are reviewed. Furthermore, strategies that incorporate the targeting of VEGF variants to enhance the effectiveness of antiangiogenic therapies in the clinical setting are discussed.

In Vitro Evaluation of Vegf-Pseudomonas Exotoxin: A Conjugated on Tumor Cells.

Background:Angiogenesis which occurs mandatory in solid tumors, is a critical step in malignancy progression. Vascular endothelial growth factor (VEGF) is mainly responsible for angiogenesis process and facilitates the formation of new vessels. Distribution of monoclonal antibodies against VEGF or VEGF receptor (VEGFR) into the solid tumors is limited because of their huge dimensions. Moreover, many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors.Materials and Methods:We designed, expressed and evaluated the cytotoxicity of a novel nano-immunotoxin composed of VEGF splice variant containing 121 amino acids (VEGF121) and truncated the exotoxin A of Pseudomonas aeruginosa (PE38-KDEL). The fusion protein VEGF121-PE38 was successfully cloned and expressed in Escherichia coli, purified by Ni+ 2 affinity chromatography. The fusion protein was subsequently subjected to refolding using the reduced and oxidized glutathione.Results:The expression level of the fusion protein reached to 1 mg/ml. The VEGF121-PE38 immunotoxin showed a 59 KDa MW which had cytotoxic effect on HUVEC and 293/KDR cells as low and high expressing VEGFR2 cells, respectively. But the cytotoxicity on 293/KDR was 100 folds more than that of VEGFR2 low expressing cell HUVEC.Conclusion:The designed immunotoxin showed more selectivity for higher VEGFR2 expressing cells in vitro.

Clinicopathological implications of vascular endothelial growth factor 165b expression in oral squamous cell carcinoma stroma.

Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. VEGF165b was recently isolated as the anti-angiogenic VEGF splice variant. In the present study, we examined the association between VEGF165b expression and clinicopathological characteristics in order to determine how VEGF165b produced from oral squamous cell carcinoma (OSCC) affects the stromal cell biological activity. We examined the relationships between the expressions of both VEGF isoforms in normal human dermal fibroblasts (NHDFs) and OSCC cell lines (HSC2, 3, 4 and SAS). Our analyses indicated that both the mRNA and protein expression levels of VEGF165b in the HSC2 and SAS cells were higher than those in the NHDFs. VEGF165b did not promote cell growth or invasive capabilities, but it induced the cell adhesive capabilities to ECM. Although strong expression of the VEGF165 isoforms in tumor cells of OSCC tissues was observed, there was no significant difference in the VEGF165b expression level among the various degrees of malignancy. OSCC cells secrete VEGF165b into the stroma, and this factor may contribute to the process of anti-angiogenesis by inhibiting gelatinase-expressing cells and activating cell adhesive capabilities to ECM, such as that of fibroblasts surrounding tumor cells.

VEGF Splicing and the Role of VEGF Splice Variants: From Physiological-Pathological Conditions to Specific Pre-mRNA Splicing.

During this past decade, the vascular endothelial growth factor (VEGF) pathway has been extensively studied. VEGF is a paradigm of molecular regulation since its expression is controlled at all possible steps including transcription, mRNA stability, translation, and pre-mRNA splicing. The latter form of molecular regulation is probably the least studied. This field has been neglected; yet different forms of VEGF with different sizes and different physiological properties issued from alternative splicing have been described a long time ago. Recently a new level of complexity was added to the field of splicing of VEGF pre-mRNA. Whereas thousands of publications have described VEGF as a pro-angiogenic factor, an alternative splicing event generates specific anti-angiogenic forms of VEGF that only differ from the others by a modification in the last six amino acids of the protein. According to the scientists who discovered these isoforms, which are indistinguishable from the pro-angiogenic ones with pan VEGF antibodies, some of the literature on VEGF is at least inexact if not completely false. Moreover, the presence of anti-angiogenic forms of VEGF may explain the disappointing efficacy of anti-VEGF therapies on the overall survival of patients with different forms of cancers and with wet age-related macular degeneration. This review focuses on the existence of the different alternative splice variants of VEGF and the molecular mechanisms associated with their expression and function.

Reversal of vasohibin-driven negative feedback loop of vascular endothelial growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver diseases

Reversal of vasohibin-driven negative feedback loop of vascular endothelial growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver diseases

Soluble fms-like tyrosine kinase-1 and atherosclerosis in chronic kidney disease

Vascular endothelial growth factor (VEGF) and its cousins, such as placental growth factor, have moved into the daily clinical arena. One-step-removed regulators, such as the VEGF splice variant soluble fms-like tyrosine kinase-1 (sFlt-1), now clinically emerge. sFlt-1 keeps our corneas 'clean.' However, sFlt-1 could also cause disease, such as preeclampsia. The report by Matsui et al. on ApoE and sFlt-1 gene-deleted mice could foster important subsequent research. We are limited only by our imaginations.

Enhanced secretion of biologically active, non-glycosylated VEGF from Saccharomyces cerevisiae

Vascular endothelial growth factor (VEGF) mediates angiogenesis, which plays a critical role in the development and differentiation of the vascular system. VEGF is a homodimeric glycoprotein that contains one N-glycosylation site. In this study, we evaluated Saccharomyces cerevisiae expression systems producing glycosylated and non-glycosylated splice variants of human VEGF, VEGF121, and VEGF165. The pre region of the mating factor α1 (MFα1) signal sequence was found to perform better than the entire MFα1 prepro signal sequence in secreting glycosylated VEGF. Secretion of non-glycosylated VEGF165 was completely blocked, indicating the importance of glycosylation in VEGF165 secretion. Interestingly, non-glycosylated VEGF165 was secreted when guided by the MFα1 prepro signal sequence, albeit to a lesser degree, compared to glycosylated VEGF165. N-glycosylation in the pro region was required for the prepro sequence to promote VEGF secretion. Furthermore, substitution of asparagine at the VEGF glycosylation site with lysine or glutamic acid increased secretion of non-glycosylated VEGF, a finding not previously reported. Our findings suggest that S. cerevisiae could be a suitable host for secreting biologically active, non-glycosylated VEGF for clinical use.

Zinc Finger Protein-activating Transcription Factor Up-regulates Vascular Endothelial Growth Factor-A Expression in Vitro

To construct the zinc finger protein-activating transcription factor (ZFP-ATF) plasmid and evaluate its efficacy in inducing vascular endothelial growth factor (VEGF) expression in EY.HY926 endothelial cells. Firstly, we constructed the ZFP-ATF plasmid, then testified the quantity of VEGF protein in EY.HY926 endothelial cells after transfected with ZFP-ATP plasmid by Western blot, finally, we used the RT-PCR to testify whether the ZFP-ATF can stimulate expression of VEGF splice variants. The ZFP-ATF DNA sequences were located the multiclone sites of PVAX1 vector between the site of BamH1and Xhol.Western blot result showed VEGF expression in EY.HY926 endothelial cells transfected with ZFP-ATF plasmid was significantly higher than that in cells transfected with VEGF165 (19.95±3.95 vs.12.15±1.55 μg÷μL, P<0.01).RT-PCR result showed VEGF-A mRNA expression level induced by ZFP-ATF was high than that induced by VEGF165. ZFP-ATF can up-regulate the VEGF-A expression in comparison with VEGF165, which might have beneficial effects in angiogenesis process.

Activated Pericytes and the Inhibition of Renal Vascular Stability

The hallmark feature of CKD and its progression to end stage is the presence and severity of interstitial fibrosis.1,2 This long-recognized association is the prime driving force of research to understand the nature of renal scarification. Much of the research in this area can be grouped into one of two broad categories: efforts geared toward understanding the biochemical and pathophysiological factors that govern the degree of progression and efforts aimed at defining the cellular and molecular elements that comprise renal scars. With regard to the former, it is clear that a profibrotic environment is promoted by activity from a variety of factors with activity driving the production of extracellular matrix (ECM). Of these, TGFβ and other profibrotic cytokines have been widely studied, and these are enhanced by local tissue injury, epithelial cell cycle arrest, cell differentiation status, or hypoxia.1,3

VEGF Spliced Variants: Possible Role of Anti-Angiogenesis Therapy

Angiogenesis has been targeted in retinopathies, psoriasis, and a variety of cancers (colon, breast, lung, and kidney). Among these tumour types, clear cell renal cell carcinomas (RCCs) are the most vascularized tumours due to mutations of the von Hippel Lindau gene resulting in HIF-1 alpha stabilisation and overexpression of Vascular Endothelial Growth Factor (VEGF). Surgical nephrectomy remains the most efficient curative treatment for patients with noninvasive disease, while VEGF targeting has resulted in varying degrees of success for treating metastatic disease. VEGF pre-mRNA undergoes alternative splicing generating pro-angiogenic isoforms. However, the recent identification of novel splice variants of VEGF with anti-angiogenic properties has provided some insight for the lack of current treatment efficacy. Here we discuss an explanation for the relapse to anti-angiogenesis treatment as being due to either an initial or acquired resistance to the therapy. We also discuss targeting angiogenesis via SR (serine/arginine-rich) proteins implicated in VEGF splicing.

Breaking barriers: insight into the pathogenesis of neovascular age-related macular degeneration.

Neovascular age-related macular degeneration (AMD) is a leading cause of central visual acuity loss in a growing segment of the population, those over the age of 60 years. Treatment has improved over the last decade, with the availability of agents that inhibit the bioactivity of vascular endothelial growth factor (VEGF), but it is still limited, because of tachyphylaxis and potential risk and toxicity of anti-VEGF agents. The authors have sought to understand the mechanisms of choroidal endothelial cell (CEC) activation and transmigration of the retinal pigment epithelium (RPE) and of RPE barrier dysfunction, events preceding vision-threatening neovascular AMD. The authors developed physiologically relevant human RPE and CEC coculture and transmigration models that have been important in helping to understand causes of events in human neovascular AMD. The authors can control for interactions between these cells and can separately assess activation of signaling pathways in each cell type relevant during CEC transmigration. Using these models, it was found that VEGF, particularly the cell-associated VEGF splice variant VEGF189, accounts for about 40% of CEC transmigration across the RPE. This percentage is in the range of similar reports following clinical inhibition of VEGF in neovascular AMD. RPE VEGF189 working through CEC VEGF receptor 2 activates the small guanosine triphosphatase (GTPase) of the Rho family, Rac1, in CECs, which in turn facilitates CEC transmigration. Conversely, inhibition of Rac1 activity prevents CEC transmigration. Once activated, Rac1 aggregates with subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in the generation of reactive oxygen species. Activated NADPH oxidase increases choroidal neovascularization in animal models of laser-induced injury. Rac1 is also downstream of the eotaxin-CCR3 pathway, another pathway important in human neovascular AMD. Studies also suggest that active Ras-related protein 1 (Rap1), another small GTPase, in RPE can strengthen the RPE barrier integrity and can resist CEC transmigration of the RPE, suggesting Rap1 activation may be another potential target for preventing neovascular AMD.

Abstract 5140: Development and evaluation of a liquid bead array assay for the expression of VEGF121, VEGF165 and VEGF189 splice variants in non-small cell lung cancer (NSCLC)

Abstract Introduction. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key regulator of angiogenesis. The aim of our study was to develop and validate a novel liquid bead array assay for the expression of VEGF121, VEGF165 and VEGF189 splice variants, based on the Luminex technology and evaluate it in NSCLC. Materials and methods. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of VEGF splice variants VEGF121, VEGF165 and VEGF189. We included PBGD as a reference gene, in order to evaluate sample RNA quality. The developed assay is based on liquid bead array technology (Luminex® xMAP® and xTAG®). The assay is based on the following procedure: total RNA isolation from samples, cDNA synthesis, multiplex PCR, treatment with ExoSAP-IT® reagent, target specific primer extension (TSPE), biotinylation of PCR products, hybridization of the biotinylated products to xMAP microspheres, addition of detection reagent (Streptavidin- phycoerythrin) and measurement in the Luminex analyzer. Before processing to patients samples an extensive optimization of every step of the method was performed in terms of analytical sensitivity, specificity, repeatability and reproducibility. The clinical performance of the assay was evaluated in 20 pairs of fresh frozen cancerous and corresponding noncancerous adjacent tissue samples originating from patients with NSCLC. The results were compared with RT-qPCR using our previously developed methodology for VEGF splice variants, (Zygalaki et al, Clin Chem, 2007). Results. When using the tumor cell line MCF-7 the developed method can detect down to 10 cells, is highly specific and is characterized by satisfactory repeatability and reproducibility for each VEGF splice variant. Comparison between the developed liquid bead array for VEGF splice variants and RT-qPCR has shown an accordance of 16/20 pairs (80%) for PBGD, 16/20 for VEGF121 (80%), 18/20 for VEGF165 (90%) and 17/20 for VEGF189 (85%). Conclusions. The developed liquid bead array can be used to detect VEGF splice variants simultaneously in clinical samples with high specificity and sensitivity. The method can be extended to analyze additional gene targets, while the use of an internal control could further enable quantification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5140. doi:10.1158/1538-7445.AM2011-5140

Association of Retinal Vascular Endothelial Growth Factor With Avascular Retina in a Rat Model of Retinopathy of Prematurity

To study the effects of oxygen fluctuations on rat vascular endothelial growth factor (VEGF), VEGF receptor 1(VEGFR1), and VEGFR2 in a model of retinopathy of prematurity (ROP). Retinas at several postnatal days (p) were analyzed for VEGF splice variants, VEGFR1 and VEGFR2 messenger RNAs (mRNAs) using real-time polymerase chain reaction or for VEGF protein using enzyme-linked immunosorbent assay. Older developmental age was associated with VEGFR1 (P < .001), VEGF(120) (P < .001), and VEGF(188) (P = .03) mRNA overexpression. Expression of VEGFR2 and VEGF(164) mRNAs were associated with older age (P < .001) or exposure to the ROP model (P = .02 and P < .001, respectively). Expression of VEGF protein was greater at p14, when 30% avascular retina existed in the ROP model, compared with room air, when no avascular retina existed, and at p18, when intravitreous neovascularization existed in the model but not in room air (P < .001 for both). Unlike models of oxygen-induced retinopathy that describe ROP before implementation of oxygen regulation, the ROP model re-creates oxygen stresses relevant to preterm infants with severe ROP today. Expression of VEGF(164) and VEGFR2 mRNAs and VEGF protein were increased in association with the ROP model and older developmental age and at time points when not only intravitreous neovascularization but also avascular retina were present in the ROP model and not in room air. Clinical Relevance Regulation of VEGF may have a role in the development of avascular retina and intravitreous neovascularization in some forms of severe ROP.

A New ELISA for Use in a 3-ELISA System to Assess Concentrations of VEGF Splice Variants and VEGF110 in Ovarian Cancer Tumors

AbstractBackground: Vascular endothelial growth factor (VEGF), which affects tumor angiogenesis, is expressed as different splice variants, including the major isoforms VEGF165 and VEGF121, and can be cleaved by plasmin to generate VEGF110. The amount of VEGF121 and VEGF110 in biological samples has not been well studied.Methods: We developed an ELISA that detects VEGF165 and VEGF121 equally, but does not detect VEGF110. We used this ELISA together with 2 other ELISAs, one detecting VEGF165 and the other detecting VEGF165, VEGF121, and VEGF110 equally, to assess the concentrations of VEGF121 and VEGF110 in ovarian cancer tumors.Results: The median concentrations in ovarian cancer tumor lysates were 0.61 (range &amp;lt;0.055–74) fmol/mg protein for VEGF165, 1.4 (range &amp;lt;0.20–500) fmol/mg protein for VEGF165 plus VEGF121, and 2.3 (range &amp;lt;0.079–520) fmol/mg protein for total VEGF including VEGF110 (n = 248). VEGF concentrations measured by the 3 ELISAs were highly correlated (r = 0.91–0.94). Median estimated VEGF121 and VEGF110 concentrations were 0.77 and 0.58 fmol/mg protein, respectively. In lysates with measurable VEGF165 and total VEGF concentrations, mean VEGF165 was approximately 31% (SD 23%) of the total VEGF (n = 217). In contrast, VEGF165 constituted approximately half of the total circulating VEGF.Conclusion: VEGF165, VEGF121, and VEGF110 may be present at significant amounts in ovarian cancer tumors.

Adenoviral expression of vascular endothelial growth factor splice variants differentially regulate bone marrow‐derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) are being explored for clinical applications, and genetic engineering represents a useful strategy for boosting the therapeutic potency of MSCs. Vascular endothelial growth factor (VEGF)-based gene therapy protocols have been used to treat tissue ischemia, and a combined VEGF/MSC therapeutics is appealing due to their synergistic paracrine actions. However, multiple VEGF splice variants exhibit differences in their mitogenicity, chemotactic efficacy, receptor interaction, and tissue distribution, and the differential regulatory effects of multiple VEGF isoforms on the function of MSCs have not been characterized. We expressed three rat VEGF-A splice variants VEGF120, 164, and 188 in MSCs using adenoviral vectors, and analyzed their effects on MSC proliferation, differentiation, survival, and trophic factor production. The three VEGF splice variants exert common and differential effects on MSCs. All three expressed VEGFs are potent in promoting MSC proliferation. VEGF120 and 188 are more effective in amplifying expression of multiple growth factor and cytokine genes. VEGF164 on the other hand is more potent in promoting expression of genes associated with MSC remodeling and endothelial differentiation. The longer isoform VEGF188, which is preferentially retained by proteoglycans, facilitates bone morphogenetic protein-7 (BMP7)-mediated MSC osteogenesis. Under serum starvation condition, virally expressed VEGF188 preferentially enhances serum withdrawal-mediated cell death involving nitric oxide production. This work indicates that seeking the best possible match of an optimal VEGF isoform to a given disease setting can generate maximum therapeutic benefits and minimize unwanted side effects in combined stem cell and gene therapy.

Expression profile of total VEGF, VEGF splice variants and VEGF receptors in the myocardium and arterial vasculature of diabetic and non-diabetic patients with coronary artery disease

Background: Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and is implicated in the development of diabetic microvascular and macrovascular disease. Methods: The expression of total VEGF, VEGF splice variants (VEGF 121, VEGF 145, VEGF 148, VEGF 165, VEGF 183 and VEGF 189), VEGFR-1 and VEGFR-2, was investigated in biopsies from the right atrium and left internal mammary artery (LIMA) of 32 non-diabetic and 20 diabetic patients undergoing coronary artery bypass grafting. Results: Diabetes was independently negatively correlated to total VEGF mRNA expression in atrium. Total VEGF, VEGF 121 and VEGF 165 mRNA levels were upregulated in the LIMA of diabetics vs. non-diabetics. The expression of VEGF receptors in atrium and LIMA was similar between these groups. VEGF 121 and VEGF 165 were the major variants expressed, followed by VEGF 189 and VEGF 183, while VEGF 148 and VEGF 145 were detected in small amounts. The expression profile of VEGF splice variants displayed significant heterogeneity between the examined tissues. Conclusions: This is the first study to quantify VEGF splice variants expression in cardiac and vascular tissue. Our results could help elucidate the role of VEGF splice variants in diabetic complications.

Quantitative Real-Time Reverse Transcription–PCR Study of the Expression of Vascular Endothelial Growth Factor (VEGF) Splice Variants and VEGF Receptors (VEGFR-1 and VEGFR-2) in Non–Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and its expression is increased in non-small cell lung cancer (NSCLC). We aimed to determine the expression pattern of VEGF splice variants in NSCLC and its correlation with the clinicopathological characteristics of tumors. We used real-time reverse transcription PCR to quantify the mRNA expression of total VEGF, 4 VEGF splice variants (VEGF(121), VEGF(165), VEGF(183), and VEGF(189)), and 2 VEGF receptors (VEGFR-1 and VEGFR-2) in 27 pairs of cancerous and adjacent noncancerous tissues originating from patients with NSCLC. Total VEGF, VEGF(121), and VEGF(165) were expressed in all specimens, whereas VEGF(183) and VEGF(189) were present in small amounts in certain samples. Total VEGF, VEGF(121), and VEGF(165) mRNA was upregulated in cancerous compared with healthy tissues, whereas VEGF(183) and VEGF(189) expression tended to be higher in healthy tissues. The expression of VEGFRs was similar between matched specimens. No correlation was found between the expression of total VEGF or VEGF splice variants and the clinicopathological characteristics of tumors. The expression patterns of VEGF splice variants differed between tissue pairs. VEGF(121) was the major variant expressed in all samples; however, its relative expression was higher in cancerous tissues. The relative expression of VEGF(183) and VEGF(189) was upregulated in healthy lung tissues, whereas the ratio of VEGF(165) to total VEGF was similar between matched specimens. The expression pattern of certain VEGF splice variants is altered during tumorigenesis. Our data support the hypothesis that during malignant progression an angiogenic switch favoring the shorter diffusible isoforms occurs.

Vascular Endothelial Cell Growth Factor-A: Not Just for Endothelial Cells Anymore

Vascular Endothelial Cell Growth Factor-A: Not Just for Endothelial Cells Anymore

Mast cell degranulation in rat uterine cervix during pregnancy correlates with expression of vascular endothelial growth factor mRNA and angiogenesis

Vascular growth of the uterine cervix during pregnancy is associated with mast cell (MC) degranulation. To better understand the mechanism underlying this process, uterine cervices of intact pregnant rats were dissected and endothelial cell proliferation was measured by a bromodeoxyuridine incorporation technique. Total vascular endothelial growth factor (VEGF) mRNA expression and the relative abundance of VEGF splice variants (120, 164, and 188) were determined by RT-PCR. VEGF protein expression was evaluated by immunohistochemistry. To investigate the role of MCs on cervical angiogenesis, a second set of pregnant animals were treated with an MC stabilizer (disodium cromoglycate) to inhibit MC degranulation. Furthermore, 17beta-estradiol (E(2)) serum levels were established by RIA. In intact pregnant rats, VEGF mRNA expression was positively correlated with endothelial cell proliferation and circulating E(2) levels. All selected splice variants of VEGF gene were detected and their relative abundance did not show any change throughout pregnancy. Animals treated with disodium cromoglycate showed a decrease in endothelial cell proliferation and in VEGF mRNA expression compared with controls. Relative abundance of VEGF mRNA splice variants and E(2) serum levels showed no differences between these experimental groups. These results show a time-dependent correlation between VEGF mRNA expression and E(2) serum levels in the uterine cervix of intact pregnant rats, while MC stabilizer-treated animals reduced the VEGF expression without modifying E(2) serum levels. We suggest that cervical angiogenesis during pregnancy could be regulated by a mechanism which involves endogenous E(2) and chemical mediators stored in MC granules via a VEGF-dependent pathway.