Evaluation of Variances in VEGF-A-D and VEGFR-1-3 Expression in the Ishikawa Endometrial Cancer Cell Line Treated with Salinomycin and Anti-Angiogenic/Lymphangiogenic Effect.

BACKGROUND: In cancer, an excessive and uncontrolled process of creating new blood and lymphatic vessels that play a key role in the metastasis process can be observed. The Vascular Endothelial Growth Factor (VEGF-A,-B,-C,-D) family together with their specific receptors (VEGFR-1,-2,- 3) plays a key role in these processes, therefore, it would be reasonable to determine the correct pattern of their expression. OBJECTIVES: The study aimed to assess the use of salinomycin as an anti-angiogenic and anti-lymphangiogenic drug during endometrial cancer by examining changes in the expression pattern of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2 and VEGFR-3 depending on the treatment period of the Ishikawa endometrial cancer cells with salinomycin in comparison to the control culture. MATERIALS AND METHODS: To determine how influential salinomycin was on the expression of both mRNAs, 1 µM of the drug was added to the cell culture and then it was cultured all together for 12, 24 and 48 hour periods. The cells that made up the control culture were not treated with salinomycin. To determine the changes in the expression profile of the selected genes, we used the microarray, techniques: RTqPCR and ELISA (p<0.05). RESULTS: For all isoforms of VEGF-A-D as well as receptors of VEGFR-1-3, a decrease in expression under the influence of salinomycin was noted. For VEGF-A and VEGFR-1, the difference in the expression between the culture treated with salinomycin in comparison to the control was statistically significant (p=0.0004). In turn, for VEGF-B, the difference between the culture exposed for 24 hours in comparison to the control (p=0.00000) as well as the comparison between H48 vs. C (p=0.00000) was statistically significant. In reference to VEGF-C, VEGFR-2 and VEGFR-3, the statistical analysis showed the significant difference in expression between the culture incubated with the drug for 12, 24 and 48 hours in comparison to the control as well as between the selected times. For all of these comparisons, p=0.00000 was utilized. CONCLUSION: Salinomycin changes the expression pattern of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2, and VEGFR-3 in endometrial cancer cells. The obtained results suggest that salinomycin might exert the effect via VEGF signaling pathways.

Effect of 532nm photodynamic therapy with hemoporfin on the expression of vascular endothelial growth factor in cultured human vascular endothelial cells.

BACKGROUND: Photodynamic therapy (PDT) with hemoporfin (hematoporphyrinmonomethyl ether, HMME) and 532 nm continuous-wave lasers is effective for port-wine stains (PWS) and is considered as a promising treatment modality. The vascular endothelial growth factor (VEGF) is involved in the development of PWS. This study aimed to investigate the effect of 532 nm-HMME-PDT on the in vitro expression of VEGF in human umbilical vein endothelial cells (HUVECs) exposed to different power levels of 532 nm laser and different concentrations of HMME. METHOD: The CCK-8 assay was performed to assess cell viability. Enzyme-linked immunosorbent assay (ELISA) was performed to measure VEGF secretion. VEGF and VEGF receptor (VEGFR) mRNA expression levels were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Within 72 h after HMME-PDT, cell viability was inhibited, secretion and expression of VEGF was decreased, and expression of VEGFR mRNA was significantly decreased with the increase of HMME concentration. CONCLUSION: 532 nm-HMME-PDT can downregulate the expression of VEGF and VEGFR mRNA. Future studies are necessary to examine the HMME doses to achieve better efficacy with fewer adverse effects.

Increased protein expression of VEGF-A, VEGF-B, VEGF-C and their receptors in the temporal neocortex of pharmacoresistant temporal lobe epilepsy patients.

The vascular endothelial growth factor (VEGF) system has been shown to play a crucial role in several neuropathological processes. Temporal lobe epilepsy (TLE) is the most common focal epilepsy type in adult humans. We assessed the protein expression levels of VEGF-A, VEGF-B, and VEGF-C, their specific receptors VEGFR-2 and -3, their accessory receptors neuropilins 1 and 2, and PI3 and Akt kinases, in temporal neocortex from pharmacoresistant TLE (PR-TLE) patients and control subjects by western blotting. All proteins were found to be significantly overexpressed in samples of PR-TLE patients, indicating that the VEGF system contributes to PR-TLE pathogenesis and should be further studied.

Effects of hypoxia and hyperoxia on the differential expression of VEGF-A isoforms and receptors in Idiopathic Pulmonary Fibrosis (IPF).

Dysregulation of VEGF-A bioavailability has been implicated in the development of lung injury/fibrosis, exemplified by Idiopathic Pulmonary Fibrosis (IPF). VEGF-A is a target of the hypoxic response via its translational regulation by HIF-1α. The role of hypoxia and hyperoxia in the development and progression of IPF has not been explored. In normal lung (NF) and IPF-derived fibroblasts (FF) VEGF-Axxxa protein expression was upregulated by hypoxia, mediated through activation of VEGF-Axxxa gene transcription. VEGF-A receptors and co-receptors were differentially expressed by hypoxia and hyperoxia. Our data supports a potential role for hypoxia, hyperoxia and VEGF-Axxxa isoforms as drivers of fibrogenesis.

All-trans retinoic acid suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and metastasis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the second common cancer in Henan province and is well-known for aggressiveness and dismal prognosis. Adjuvant therapies, chemotherapy, radiotherapy and endoscopic treatment have not improved survival rates in patients with late stage esophageal carcinoma. All-trans retinoic acid (ATRA) is the active ingredient of Vitamin A and affects a wide spectrum of biological processes including development, growth, neural function, immune function, reproduction, and vision. It is one of the most potent therapeutic agents used for treating cancers, especially lung adenocarcinomas. ATRA inhibits metastatic potential and angiogenesis in several tumor models. We investigated the effects of ATRA on the expression of angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2) and receptor Tie-2 in EC1 cells in vitro. We also assessed the growth and migration of EC1 cells in vitro. ATRA treatment caused 29.5% and 40.3% reduction of the growth of EC1 cells after 24 hours and 48 hours, relative to the control. ATRA plus fluorouracil treatment reduced the viability more strongly than either drug alone, indicating an additive effect. Moreover, ATRA decreased EC1 migration by 87%. Furthermore, ATRA treatment led to a marked decrease of the transcript levels of Ang-1, Ang-2, Tie-2, VEGF, and VEGF receptors, as assessed by real-time RT-PCR. Importantly, the protein levels of Ang-1, Ang-2 and Tie-2 were reduced by ATRA treatment. In vivo, we found ATRA treatment suppressed the tumor growth and improved the cachexia of mice. Importantly, ATRA treatment decreased the expression of CD31, Ang-1, Ang-2 and Tie-2 in subcutaneous tumors of EC1 cells. Collectively, our findings demonstrate that ATRA exhibits a dose- and temporal-dependent effect on the metastatic behavior, suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and the progression of xenograft tumors of EC1 cells.

Spatholobi Caulis extracts promote angiogenesis in HUVECs in vitro and in zebrafish embryos in vivo via up-regulation of VEGFRs.

ETHNOPHARMACOLOGICAL RELEVANCE: Spatholobi Caulis is a traditional blood-activating and stasis-dispelling herb medicine, which has been used to treat diseases related to blood stasis syndrome (BSS) by inhibiting platelet aggregation, stimulate hematopoiesis, etc. It has been demonstrated that pro-angiogenesis could improve BSS. However, the pro-angiogenic activity of Spatholobi Caulis was not well elucidated AIM OF STUDY: To determine the potential pro-angiogenic activity of Spatholobi Caulis and elucidate its underlying mechanism. The active fractions of Spatholobi Caulis were further screened. MATERIAL AND METHODS: Gelatin precipitation and reversed-phase liquid chromatography (RPLC) were used to purify the methanol extracts of Spatholobi Caulis, respectively. The RPLC was also used to prepare fractions. Total flavonoids of purified methanol extracts of Spatholobi Caulis (PSC) were determined using ultraviolet spectrophotometry. The morphological observation of subintestinal vessel plexus (SIVs) and tyrosine kinase inhibitor II (VRI)-induced intersegmental blood vessels (ISVs) loss in transgenic zebrafish Tg(fli-1a: EGFP)y1 were selected to evaluate the pro-angiogenic activity of PSC in vivo. Cell proliferation by MTT assay and cell migration assay were used to evaluate the pro-angiogenesis effect of PSC in human umbilical vein endothelial cells (HUVECs) in vitro. Both zebrafish and HUVECs were used in screening active fractions of PSC. The mechanism of PSC promoting angiogenesis were studied by real-time PCR in zebrafish and western blotting in HUVECs. RESULTS: Co-treatment PSC dramatically rescued VRI-induced ISVs loss in zebrafish embryos in a dose-dependent manner and 80% of the defective vascular recovered at the concentration of 30µg/ml compared with VRI-only group. PSC also concentration-dependently increased average sprouting number and diameter of SIVs in zebrafish embryo. Real-time PCR assay proved that PSC significantly restored the down regulation of VEGFRs including Flt-1, Kdr and Kdrl induced by VRI in zebrafish (P<0.001). Furthermore, PSC not only promoted proliferation and migration of normal HUVECs but also ameliorated VRI-induced HUVECs cytotoxicity. Western blotting assay showed that co-treatment of PSC increased the expression of VEGFRs and phosphorylation of MAPKs which decreased by VRI treatment. In addition, quality evaluation experiments showed that the content of total flavonoids of PSC reached 56.36% and the main pro-angiogenic fractions of PSC were F3, F4 and F5 both in zebrafish and HUVECs. CONCLUSIONS: Our data demonstrated that PSC presented pro-angiogenic activity both in zebrafish and HUVECs, and principal pro-angiogenic active components were likely flavonoids. Thus, the current study provided evidence for the clinical usage of Spatholobi Caulis in promoting blood circulation and removing stasis in traditional Chinese medicine (TCM).

Preoperative Treatment With Pazopanib in a Case of Chemotherapy-Resistant Infantile Fibrosarcoma.

Clinical and radiological diagnosis of infantile fibrosarcoma (IFS) is challenging because of its similarity to vascular origin tumors. Treatment involves complete resection. Although chemotherapy may allow more conservative resection, treatment guidelines are not strictly defined. One IFS patient with an unresectable tumor had disease progression during chemotherapy. A primary tumor sample showed high VEGFR-1/2/3 and PDGFR-α/ß expression. After pazopanib therapy, most tumor showed necrosis within 29 days and could be removed completely, with no relapse in 8 months post-resection. When IFS features hypervascularity, VEGFR and PDGFR expression may be high, thus allowing consideration of VEGFR inhibitors such as pazopanib.

Expression of Vascular Endothelial Growth Factor Receptors in Benign Vascular Lesions of the Orbit: A Case Series.

PURPOSE: Vascular lesions of the orbit, although not malignant, can cause morbidity because of their location near critical structures in the orbit. For the same reason, they can be challenging to remove surgically. Anti-vascular endothelial growth factor (VEGF) drugs are increasingly being used to treat diseases with prominent angiogenesis. Our study aimed to determine to what extent VEGF receptors and their subtypes are expressed on selected vascular lesions of the orbit. DESIGN: Retrospective case series of all orbital vascular lesions removed by one of the authors (JAG) at the Mayo Clinic. PARTICIPANTS: A total of 52 patients who underwent removal of vascular orbital lesions. METHODS: The pathology specimens from the patients were retrieved, their pathologic diagnosis was confirmed, demographic and clinical information were gathered, and sections from vascular tumors were stained with vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor type 1 (VEGFR1), vascular endothelial growth factor receptor type 2 (VEGFR2), and vascular endothelial growth factor receptor type 3 (VEGFR3). MAIN OUTCOME MEASURES: The existence and pattern of staining with VEGF and its subtypes on these lesions. RESULTS: There were 28 specimens of venous malformations, 4 capillary hemangiomas, 7 lymphatic malformations, and 6 lymphaticovenous malformations. All samples stained with VEGF, 55% stained with VEGFR1, 98% stained with VEGFR2, and 96% stained with VEGFR3. Most (94%) of the VEGFR2 staining was diffuse. CONCLUSIONS: Most orbital vascular lesions express VEGF receptors, which may suggest a future target for nonsurgical treatment.

Prognostic impact of elevation of vascular endothelial growth factor family expression in patients with non-small cell lung cancer: an updated meta-analysis.

BACKGROUND: The vascular endothelial growth factor family has been implicated in tumorigenesis and metastasis. The prognostic value of each vascular endothelial growth factor family member, particular VEGF/ VEGFR co-expression, in patients with non-small lung cancer remains controversial. MATERIALS AND METHODS: Relevant literature was identified by searching PubMed, EMBASE and Web of Science. Studies evaluating expression of VEGFs and/or VEGFRs by immunohistochemistry or ELISA in lung cancer tissue were eligible for inclusion. Hazard ratios (HRs) and 95% confidence intervals (CIs) from individual study were pooled by using a fixed- or random-effect model, heterogeneity and publication bias analyses were also performed. RESULTS: 74 studies covering 7,631 patients were included in the meta-analysis. Regarding pro-angiogenesis factors, the expression of VEGFA (HR=1.633, 95%CI: 1.490-1.791) and VEGFR1 (HR=1.924, 95%CI: 1.220-3.034) was associated separately with poor survival. Especially, VEGFA over-expression was an independent prognostic factor in adenocarcinoma (ADC) (HR=1.775, 95%CI: 1.384-2.275) and SCC (HR=2.919, 95%CI: 2.060-4.137). Co-expression of VEGFA/VEGFR2 (HR=2.011, 95%CI: 1.405-2.876) was also significantly associated with worse survival. For lymphangiogenesis factors, the expression of VEGFC (HR=1.611, 95%CI: 1.407-1.844) predicted a poor prognosis. Co-expression of VEGFC/VEGFR3 (HR=2.436, 95%CI: 1.468-4.043) emerged as a preferable prognostic marker. CONCLUSIONS: The expression of VEGFA (particularly in SCC and early stage NSCLC), VEGFC, VEGFR1 indicates separately an unfavorable prognosis in patients with NSCLC. Co-expression VEGFA/ VEGFR2 is comparable with VEGFC/VEGFR3, both featuring sufficient discrimination value as preferable as prognostic biologic markers.

Recombinant hirudin suppresses the viability, adhesion, migration and invasion of Hep-2 human laryngeal cancer cells.

Recombinant hirudin (rH) is a highly potent and specific inhibitor of thrombin, and has been shown to inhibit the growth and metastasis of several types of cancers in experimental tumor models. The objective of this study was to evaluate the antitumor effects and explore the underlying mechanisms of rH in Hep-2 human laryngeal carcinoma (LC) cells. Hep-2 cells were treated with various concentrations of rH for 24 h. The cell viability was evaluated by a water-soluble tetrazolium salt (WST) assay. The adhesion ability of the cells was evaluated by cell adhesion to fibronectin. Cell migration and invasion were measured with the Boyden chamber assay. Cell apoptosis was detected by Hoechst 33324 fluorescence staining. A chicken chorioallantoic membrane (CAM) assay was used to assess the effects of rH on angiogenesis in vivo. Western blotting was used to detect the expression levels of vascular endothelial growth factor receptor (VEGF-R), focal adhesion kinase (FAK), Bcl-2-associated agonist of cell death (Bad) and B-cell CLL/lymphoma 2 (Bcl-2) proteins. rH significantly inhibited the cell viability and induced apoptosis in LC Hep-2 cells in a dose-dependent manner, as compared with phosphate-buffered saline (PBS) as control. These results were accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bad. Moreover, rH dose-dependently inhibited the adhesion, migration and invasion of the Hep-2 cells, compared to the vehicle PBS. In addition, rH robustly suppressed angiogenesis in the CAM assay. Importantly, the expression of adhesion and angiogenesis-associated proteins FAK and VEGF-R was significantly downregulated by rH in a dose-dependent manner. The present findings demonstrate that rH exerts antitumor effects in Hep-2 human laryngeal cancer cells via multiple mechanisms and suggests that targeting thrombin by rH is a potential strategy for the treatment of LC.

Ephrin B2 and EphB4 selectively mark arterial and venous vessels in cerebral arteriovenous malformation.

OBJECTIVES: Ephrin type B receptor 4 (EphB4, Eph receptor) selectively binds ephrin B2 (Eph ligand). EphB4/ephrin B2 is involved in embryonic vessel development, vascular remodelling and pathological vessel formation in adults (including tumour angiogenesis). Binding of vascular endothelial growth factor (VEGF)-A to the endothelial-specific receptor VEGF receptor-2 is the main extracellular signal triggering angiogenic response. Little is known about the role of EphB4/ephrin B2 during angiogenesis and arteriovenous plasticity in cerebral arteriovenous malformation (cAVM). This study investigated EphB4 and ephrin B2 expression in cAVM. METHODS: Haemorrhagic (H-AVM) and nonhaemorrhagic (NH-AVM) specimens of AVM nidus, obtained after microsurgical cAVM resection, and normal superficial temporal artery (STA) specimens, were analysed retrospectively. VEGF-A, EphB4 and ephrin B2 expression were studied by immunohistochemistry and immunoblotting. RESULTS: In cAVM (10 H-AVM; 10 NH-AVM), VEGF-A was immunocytochemically localized to endothelial cells; strong endothelial cell staining was found for EphB4 in veins and ephrin B2 in arteries. Normal STA (n = 10) did not express EphB4 or ephrin B2. EphB4 and ephrin B2 expression was greater in H-AVM than in NH-AVM. CONCLUSIONS: Endothelial cells are more active in H-AVM than NH-AVM. EphB4 and ephrin B2 play important roles in neovascularization and arteriovenous differentiation/plasticity. These data provide new insights into the aetiology of cAVM and lay a foundation for further study. The notch pathway induced by VEGF-A may be a key signalling pathway in this process.

Valproic acid inhibits tumor angiogenesis in mice transplanted with Kasumi­1 leukemia cells.

Histone deacetylase (HDAC) inhibitors have been reported to inhibit tumor angiogenesis via the downregulation of angiogenic factors. Our previous in vitro studies demonstrated that valproic acid (VPA) exerted antitumor effects on Kasumi­1 cells, which are human acute myeloid leukemia cells with an 8;21 chromosome translocation. In the present study, the effects of VPA on tumor angiogenesis were investigated in mice transplanted with Kasumi­1 cells. Semi­quantitative reverse transcription­polymerase chain reaction, western blotting and immunohistochemistry were used to detect the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR2) and basic fibroblast growth factor (bFGF). The tumor microvessel density was measured following staining with an anti­CD34 antibody. Chromatin immunoprecipitation was used to study the effect of VPA­induced histone hyperacetylation on VEGF transcription. An intraperitoneal injection of VPA inhibited tumor growth and angiogenesis in mice transplanted with Kasumi­1 cells. The mRNA and protein expression of VEGF, VEGFR2 and bFGF were inhibited by VPA treatment. In addition, VPA downregulated HDAC, increased histone H3 acetylation and enhanced the accumulation of hyperacetylated histone H3 on the VEGF promoters. The findings of the present study indicate that VPA, an HDAC inhibitor, exerts an antileukemic effect through an anti­angiogenesis mechanism. In conclusion, the mechanism underlying VPA­induced anti­angiogenesis is associated with the suppression of angiogenic factors and their receptors. VPA may increase the accumulation of acetylated histones on the VEGF promoters, which possibly contributes to the regulation of angiogenic factors.

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.

Assessment of angiogenic markers and female sex hormone receptors in pregnancy tumor of the gingiva.

PURPOSE: Oral pregnancy tumors (OPTs) arise on the inflamed gingiva of women after the first trimester of pregnancy. The expression of angiogenic markers and female hormone receptors was assessed. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the expression of estrogen and progesterone receptors and the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) and its receptor, fibroblast growth factor (FGF), and hypoxia inducible factors 1α and 3α (HIF1α and HIF3α). Experimental groups included 9 OPTs, 10 oral pyogenic granulomas from nonpregnant women of the same age, and 9 oral pyogenic granulomas from postmenopausal women. RESULTS: VEGF expression in stromal histiocytes and endothelial cells of small vessels was positively correlated in the OPT group (P < .05 by χ(2) test). VEGF receptor also was overexpressed in stromal histiocytes and endothelial cells of OPTs compared with oral pyogenic granulomas from nonpregnant and postmenopausal women (P < .005 by χ(2) test). No correlation was detected among estrogen and progesterone receptors, FGF and HIF1α and HIF3α (ER and PgR respectively) in the 3 experimental groups. CONCLUSIONS: VEGF-associated angiogenesis is most likely involved in the pathogenesis of the lesion. These results imply that local inhibition of VEGF activity could be an adjuvant therapeutic approach for OPTs to control hemorrhage, which can be massive at the surgical excision of such lesions during pregnancy.

Amount of mRNA and localization of vascular endothelial growth factor and its receptors in the ovarian follicle during estrous cycle of water buffalo (Bubalus bubalis).

The objective of the present study was to characterize the temporal patterns of gene expression for vascular endothelial growth factors (VEGF) and VEGF receptors during ovarian follicular growth, development and maturation in buffalo (Bubalus bubalis). Follicles were classified into four groups according to size and the concentration of estradiol-17ß (E2) in follicular fluid (FF): Group I (small), 4-6mm diameter, E2>0.5ng/ml of FF; Group II (medium), 7-9mm, E2=0.5-5ng/ml; Group III (large), 10-13mm, E2=5-40ng/ml; Group IV(pre-ovulatory), >13mm, E2>180ng/ml). The mRNAs for FSH receptor (FSHR), LH receptor (LHR) and aromatase (CYP19A1) in theca interna and granulosa layers were also determined, further defining the maturational state of each group. The relative expression of VEGF isoforms (120, 164, and 188 amino acid forms), as determined by quantitative real-time PCR (qRT-PCR), increased during follicular development in both the granulosa (P<0.05) and theca layers. Relative amounts of VEGF receptors (VEGFR-1 and VEGFR-2) were least in granulosa cell (GC) and theca interna cell (TI) layers of Gp-I follicles. The amount of VEGFR-2 transcripts increased in the granulosa layer throughout development, reaching a maximum in Gp-IV follicles (P<0.05). The relative amount of VEGF isoforms and receptors in follicle lysates, as determined by western blotting, increased throughout follicular maturation to maximum amounts in pre-ovulatory follicles. Immunohistochemistry revealed a clear localization of VEGF isoforms and receptors in both steroidogenic cell types (GC and TI) and of VEGF receptors in the vascular endothelial cells of the thecal blood vessels. The most intense immunofluorescence was evident in pre-ovulatory follicles compared to other smaller follicles. These data provide evidence that the VEGF may contribute to the extensive capillary proliferation associated with the increase in size, selection, and maturation of the pre-ovulatory follicle. This may facilitate follicle maturation by enhancing the supply of nutrients, hormones, and other essential blood-borne signals to the follicle. VEGF may also promote maturation of follicles through recently recognized, non-angiogenic mechanisms.

Role of vascular endothelial growth factor in maintenance of pregnancy in mice.

It is well known that withdrawal of progesterone from the maternal circulation is a critical stimulus to parturition in rodents, such as rats and mice. However, mechanisms that determine the timing of progesterone withdrawal are not completely understood. In the present study, we examined whether the vascular endothelial growth factor (VEGF) system in the corpus luteum (CL) contributes to the regulation of circulating progesterone levels and acts as a determinant of the timing of parturition in mice. We found that reduction in the expression levels of VEGF and VEGF receptor-2 in the CL precedes the impairment of luteal circulation and a series of events leading to parturition (i.e., reduction of plasma progesterone, enhancement of myometrium contractility, and onset of parturition). Blocking of VEGF signaling by using the inhibitor of VEGFR tyrosine kinase KRN633 at mid-pregnancy caused a similar sequence of events and induced preterm birth. These results suggest that the VEGF system in the CL plays a critical role in maintaining a high level of circulating progesterone, and determining the timing of parturition in mice.

Vascular endothelial growth factor--marker for proliferation in thyroid diseases?

UNLABELLED: Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis and is involved in tumor development. To date, the role of VEGF in benign diseases of the thyroid is not well known. The purpose of the present study is to determine the expression of VEGF and its receptors in primary cultures of human thyrocytes. METHODS: 50 patients with uninodular (n=11), multinodular (n=15), recurrent goiter (n=14) and Graves' disease (n=10) were enrolled. Nodular and corresponding paranodular tissue was obtained after surgery and investigated. RNA and protein were extracted from primary thyrocyte cultures. PCR, western blot and ELISA were performed to evaluate VEGF isoforms and VEGF receptor 1 and 2. RESULTS: Significantly increased transcription and protein expression of VEGF and its receptors were detected in nodular tissue of uninodular and recurrent goiter compared to the corresponding normal tissue. Active secretion of VEGF by thyrocytes was confirmed by ELISA. In multinodu-lar goiter, no difference could be found between nodular and corresponding paranodular tissue in terms of expression of VEGF or its receptors. Furthermore, we found the highest levels of VEGF and its receptors in tissue obtained from patients with Graves' disease. CONCLUSION: Increased expression of VEGF and its receptors might be crucial in the proliferation of thyrocytes and therefore may contribute to the development of goiter and goiter recurrence.

Increased expression of pro-angiogenic factors and vascularization in thyroid hyperfunctioning adenomas with and without TSH receptor activating mutations.

Autonomously functioning thyroid nodules (AFTN) are known to receive an increased blood influx necessary to sustain their high rate of growth and hormone production. Here, we investigated the expression of hematic and lymphatic vases in a series of 20 AFTN compared with the contralateral non-tumor tissues of the same patients, and the transcript levels of proteins involved in the control of vascular proliferation, including the vascular endothelial growth factor (VEGF) and platelet-derived growth factors (PDGF) and their receptors and the endothelial nitric oxide synthase (eNOS). In parallel, the expression of the differentiation markers sodium/iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (Tg), and TSH receptor (TSHR) was also investigated. The data were further analyzed comparing subgroups of tumors with or without mutations in the TSHR gene. Analysis by means of CD31 and D2-40 immunostaining showed in AFTN an increased number of hematic, but not lymphatic, vessels in parallel with an enhanced proliferation rate shown by increased Ki67 staining. Quantitative RT-PCR analysis revealed an increase of VEGF, VEGFR1 and 2, PDGF-A, PDGF-B, and eNOS expression in tumor versus normal tissues. Also, higher transcript levels of NIS, TPO, and Tg were detected. Comparison of the two subgroups of samples revealed only few differences in the expression of the genes examined. In conclusion, these data demonstrate an increased expression of angiogenesis-related factors associated with an enhanced proliferation of hematic, but not lymphatic, vessels in AFTNs. In this context, the presence of TSHR mutations may only slightly influence the expression of pro-angiogenic growth factors.

Differential expression of vascular endothelial growth factor isoforms and receptor subtypes in the infarcted heart.

AIMS: The vascular endothelial growth factor (VEGF) family contains four major isoforms and three receptor subtypes. The expressions of each VEGF isoform and receptor subtype in cardiac repair/remodeling after myocardial infarction (MI) remain uncertain and are investigated in the current study. METHODS AND RESULTS: Temporal and spatial expressions of VEGF isoforms and VEGFR subtypes were examined in the infarcted rat heart. Sham-operated rats served as controls. We found that the normal myocardium expressed all VEGF isoforms. Following MI, VEGF-A was only increased in the border zone at day 1 and was significantly decreased in the infarcted heart during the 42 day observation period afterwards. VEGF-B was significantly suppressed in the infarcted heart. VEGF-C and VEGF-D were markedly increased in the infarcted heart in both early and late stages of MI. VEGFR-1 and 2 were significantly decreased in the infarcted heart, while VEGFR-3 was significantly increased, which was primarily expressed in blood vessels and myofibroblasts (myoFb). CONCLUSIONS: VEGF isoforms and VEGFR subtypes are differentially expressed in the infarcted heart. Increased VEGF-A in the very early stage of MI suggests the potential role in initiating the cardiac angiogenic response. Suppressed cardiac VEGF-B postMI suggests that it may not be critical to cardiac repair. The presence of enhanced VEGF-C and VEGF-D along with its receptor, VEGFR-3, in various cell types of the infarcted heart suggest that these isoforms may regulate multiple responses during cardiac repair/remodeling.