Changes of mRNA expression of vascular endothelial growth factor, angiopoietins and their receptors during the periovulatory period in eCG/hCG-treated immature female rats

Angiogenic factors can induce the perifollicular capillary network in the theca interna that shows marked changes in and around the preovulatory luteinizing hormone (LH) surge. To get more information on their functional crosstalk, the aim of the present study was to investigate the manner of mRNA expression of vascular endothelial growth factors (VEGFs) 120, 164, angiopoietin (Ang)-1, Ang-2 and their specific receptors during the periovulatory phase. We used an established equine and human chorionic gonadotropins (eCG/hCG)-derived experimental model capable of stimulating naturally occurring follicular maturation, ovulation and corpus luteum (CL) formation. On day 28 postpartum, immature female rats were administrated s.c. with 10 IU of eCG to promote follicular development, followed 48 hr later by i.p. administration of 20 IU of hCG. Ovaries were dissected at 0, 6, 12, 18 and 24 hr after hCG treatment, and were obtained on day 30 in the untreated control. After induction of follicular growth by the eCG treatment, each mRNA expression of VEGF 120, VEGF 164, Neuropilin-1 and Flt-1 significantly increased. The peaks in mRNA expressions of VEGF120 and VEGF164 were both found at 18 hr after hCG treatment. Flk-1 mRNA expression maintained up to 6 hr after hCG treatment, and then decreased at 12, 18 and 24 hr after hCG treatment. Ang-2 mRNA expression increased in the ovaries at 6 and 12 hr after hCG treatment. Tie-2 mRNA expression decreased at 24 hr after the treatment of gonadotropins. Our findings suggest that ovarian vascular formation during the periovulatory period including preovulatory follicles, ovulation and CL formation may develop via crosstalk of the VEGF-Flt-1 and Ang-Tie2 systems.     

Placental growth throughout the last two thirds of pregnancy in sheep: vascular development and angiogenic factor expression

Morphometric methodologies were developed and applied to investigate the patterns of vascular development in maternal (caruncular; CAR) and fetal (cotyledonary; COT) sheep placentas throughout the last two thirds of gestation. We also examined the expression levels of the major angiogenic factors and their receptors in CAR and COT sheep placentas. Although the vascularity of the CAR tissues increased continuously from Day 50 through Day 140 of pregnancy, those of the COT tissues increased at about twice the instantaneous rate (i.e., the proportionate increase/day) of the CAR. For CAR, vascularity increased 2-fold from Day 50 through Day 140 via relatively small increases in capillary number and 2- to 3-fold increases in capillary diameter. For COT, the increased vascularity resulted from a 12-fold increase in capillary number associated with a concomitant 2-fold decrease in capillary diameter. This large increase in fetal placental capillary number, which was due to increased branching, resulted in 6-fold increases in total capillary cross-sectional area and total capillary surface, per unit of COT tissue. Different patterns of expression of the mRNAs for angiogenic factors and their receptors were observed for CAR and COT. The dilation-like angiogenesis of CAR was correlated with the expression of vascular endothelial growth factor receptor-1 (FLT1), angiopoietin-2 (ANGPT2), and soluble guanylate cyclase (GUCY1B3) mRNAs. The branching-like angiogenesis of COT was correlated with the expression of vascular endothelial growth factor (VEGF), FLT1, angiopoietin-1 (ANGPT1), ANGPT2, and FGF2 mRNAs. Monitoring the expression of angiogenic factors and correlating the levels with quantitative measures of vascularity enable one to model angiogenesis in a spatiotemporal fashion.     

Signaling via vascular endothelial growth factor receptors

Angiogenesis, or development of blood vessels from preexisting vasculature, has important functions under both normal and pathophysiological conditions. Vascular endothelial growth factor receptors 1-3, also known as flt-1, KDR, and flt-4, are endothelial cell-specific receptor tyrosine kinases which serve as key mediators of the angiogenic responses. The review focuses on the signaling pathways that are initiated from these receptors and the recently identified VEGF coreceptor neuroplilin-1.     

Characterization of the receptors for vascular endothelial growth factor

Vascular endothelial growth factor (vEGF) is a recently discovered mitogen for endothelial cells. It is also a potent angiogenic factor. We have characterized the vEGF receptors of endothelial cells using both binding and cross-linking techniques. Scatchard analysis of equilibrium binding experiments revealed two types of high-affinity binding sites on the cell surfaces of bovine endothelial cells. One of the sites has a dissociation constant of 10(-12) M and is present at a density of 3 x 10(3) receptors/cell. The other has a dissociation constant of 10(-11) M, with 4 x 10(4) receptors/cell. A high molecular weight complex containing 125I-vEGF is formed when 125I-vEGF is cross-linked to bovine endothelial cells. This complex has an apparent molecular mass of 225 kDa. Two other faintly labeled complexes with apparent molecular masses of 170 and 195 kDa also are detected. Reduction in the presence of dithiothreitol causes a substantial increase in the labeling intensity of the 170- and 195-kDa complexes, suggesting that these complexes are derived from the 225-kDa complex by reduction of disulfide bonds. The labeling of the vEGF receptors was inhibited by an excess of unlabeled vEGF but not by high concentrations of several other growth factors. Suramin and protamine, as well as several species of lectins, inhibited the binding. The expression of functional vEGF receptors was inhibited when the cells were preincubated with tunicamycin, indicating that glycosylation of the receptor is important for the expression of functional vEGF receptors. Pretreatment with swainsonine on the other hand, did not prevent formation of functional receptors. However, the mass of the 225-kDa complex is decreased by 20 kDa when 125I-vEGF is cross-linked to swainsonine-treated endothelial cells.     

Double-antiangiogenic protein DAAP targeting vascular endothelial growth factor A and angiopoietins attenuates collagen-induced arthritis

Introduction

Angiogenesis plays a critical role in synovial inflammation and joint destruction in rheumatoid arthritis (RA). Vascular endothelial growth factor A (VEGF-A) and angiopoietins are two important mediators of synovial angiogenesis. We have previously developed a novel chimeric decoy receptor, namely, double-antiangiogenic protein (DAAP), which can both bind VEGF-A and angiopoietins and block their actions. This study was performed to evaluate the antiarthritic effect of DAAP and the combination effect with the tumor necrosis factor α (TNF-α) inhibitor in collagen-induced arthritis (CIA).

Methods

Recombinant DAAP, VEGF-Trap, Tie2-Fc and dimeric Fc proteins were produced and purified from CHO cells in large-scale bioreactors. CIA was induced in DBA/1 mice with type II collagen. The preventive effect of DAAP was determined and compared with other decoy receptors such as VEGF-Trap or Tie2-Fc, which block VEGF-A or angiopoietins, respectively. The clinical, radiographic, pathologic and immunohistochemical analyses were performed in CIA mice. The levels of matrix metalloprotease 3 (MMP-3) and interleukin 1β (IL-1β) were quantified by enzyme-linked immunosorbent assay, and receptor activator of nuclear factor κB ligand (RANKL) mRNA levels were measured by polymerase chain reaction. Finally, we investigated the combination effects of DAAP with a low dose of TNF-α decoy receptor (etanercept 10 mg/kg).

Results

On the basis of clinical and radiographic evaluation, DAAP had a much greater inhibitory effect than VEGF-Trap or Tie2-Fc on arthritis severity and bone destruction. These inhibitory effects were accompanied by significantly diminishing pathologic abnormalities, CD31-positive vasculature and synovial infiltration by F4/80-positive macrophages. The levels of MMP-3, IL-1β and RANKL were much lower in the DAAP-injected group than those of the control. Furthermore, DAAP showed a therapeutic effect and a combination effect with etanercept when injected after arthritis onset in established CIA.

Conclusions

DAAP has not only potent prophylactic effects on both inflammation and bone destruction but also therapeutic effects, alone and in combination with a TNF-α inhibitor in CIA mice. These results suggest that DAAP could be used as an effective new therapeutic agent for RA.     

Vascular endothelial growth factor, vascular endothelial growth factor receptor-3 and cyclooxygenase-2 expression in psoriasis

OBJECTIVE: To investigate expression patterns and relationship of vascular endothelial growth factor (VEGF), vascular endothelial receptor-3 (VEGF-R3) (FLT-4) and cyclooxygenase-2 (COX-2) in psoriasis. STUDY DESIGN: Forty-three patients were included in this study. The clinical severity of psoriasis was assessed using the psoriasis area and severity index (PASI). Punch biopsy samples both from psoriatic and nonlesional skin were taken and VEGF, VEGF-R3 and COX-2 expressions determined. RESULTS: VEGF, VEGF-R3 and COX-2 expressions were detected in 90.9%, 78.0% and 86.4% of psoriatic and 84.1%, 71.8%, and 84.1% of nonlesional skin, respectively. Epidermal VEGF, VEGF-R3 and COX-2 expressions were detected in 56.8%, 77.8% and 34.1 of psoriatic and 75%, 78.1% and 65.9% of nonlesional skin, respectively. In dermis, VEGF, VEGF-R3 and COX-2 expression was observed in 88.6%, 77.5% and 84.1% of psoriatic and 81.8%, 64.1% and 77.3% of nonlesional skin, respectively. Among the PASI subgroups no statistically significant differences were detected for VEGF, VEGF-R3 and COX-2 expression. CONCLUSION: Our study demonstrated that VEGF, VEGF-R3 and COX-2 expression in psoriatic and nonlesional skin is significantly high in epidermis and dermis. Although there was significant concordance between VEGF and VEGF-R3 expressions in psoriatic lesions, there seems to be no concordance between the others.     

Signal transduction by vascular endothelial growth factor receptors

VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.     

Circulating and exhaled vascular endothelial growth factor in asthmatic pregnancy

Context: Vascular endothelial growth factor (VEGF) plays a role in asthma and pathological pregnancies.

Objective: This is the first study assessing plasma and exhaled breath condensate VEGF levels in asthmatic pregnancy.

Material and methods: Thirty-one asthmatic pregnant, 29 asthmatic nonpregnant, 28 healthy pregnant and 22 healthy nonpregnant women were enrolled. Plasma was collected in all subjects, EBC in 57 volunteers for VEGF measurements.

Results: Plasma VEGF decreased in both pregnant groups (p < 0.01), without any differences between the asthmatic and the respective nonasthmatic groups (p > 0.05). VEGF was undetectable in EBC.

Conclusion: Concomitant asthma does not affect plasma VEGF during pregnancy.     

Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung

Background

Chronic alveolar hypoxia, due to residence at high altitude or chronic obstructive lung diseases, leads to pulmonary hypertension, which may be further complicated by right heart failure, increasing morbidity and mortality. In the non-diseased lung, angiogenesis occurs in chronic hypoxia and may act in a protective, adaptive manner. To date, little is known about the behaviour of individual vascular endothelial growth factor (VEGF) family ligands in hypoxia-induced pulmonary angiogenesis. The aim of this study was to examine the expression of placenta growth factor (PlGF) and VEGFB during the development of hypoxic pulmonary angiogenesis and their functional effects on the pulmonary endothelium.

Methods

Male Sprague Dawley rats were exposed to conditions of normoxia (21% O₂) or hypoxia (10% O₂) for 1-21 days. Stereological analysis of vascular structure, real-time PCR analysis of vascular endothelial growth factor A (VEGFA), VEGFB, placenta growth factor (PlGF), VEGF receptor 1 (VEGFR1) and VEGFR2, immunohistochemistry and western blots were completed. The effects of VEGF ligands on human pulmonary microvascular endothelial cells were determined using a wound-healing assay.

Results

Typical vascular remodelling and angiogenesis were observed in the hypoxic lung. PlGF and VEGFB mRNA expression were significantly increased in the hypoxic lung. Immunohistochemical analysis showed reduced expression of VEGFB protein in hypoxia although PlGF protein was unchanged. The expression of VEGFA mRNA and protein was unchanged. In vitro PlGF at high concentration mimicked the wound-healing actions of VEGFA on pulmonary microvascular endothelial monolayers. Low concentrations of PlGF potentiated the wound-healing actions of VEGFA while higher concentrations of PlGF were without this effect. VEGFB inhibited the wound-healing actions of VEGFA while VEGFB and PlGF together were mutually antagonistic.

Conclusions

VEGFB and PlGF can either inhibit or potentiate the actions of VEGFA, depending on their relative concentrations, which change in the hypoxic lung. Thus their actions in vivo depend on their specific concentrations within the microenvironment of the alveolar wall during the course of adaptation to pulmonary hypoxia.     

Quantification and cell-to-cell variation of vascular endothelial growth factor receptors

The vascular endothelial growth factor receptors (VEGFR) play a significant role in angiogenesis, the formation of new blood vessels from existing vasculature. Systems biology offers promising approaches to better understand angiogenesis by computational modeling the key molecular interactions in this process. Such modeling requires quantitative knowledge of cell surface density of pro-angiogenic receptors versus anti-angiogenic receptors, their regulation, and their cell-to-cell variability. Using quantitative fluorescence, we systematically characterized the endothelial surface density of VEGFRs and neuropilin-1 (NRP1). We also determined the role of VEGF in regulating the surface density of these receptors. Applying cell-by-cell analysis revealed heterogeneity in receptor surface density and VEGF tuning of this heterogeneity. Altogether, we determine inherent differences in the surface expression levels of these receptors and the role of VEGF in regulating the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) receptors.     

Expression of vascular endothelial growth factor and basic fibroblast growth factor receptors in lung cancer

OBJECTIVE: To determine the expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor receptors (FGFR), in non-small cell lung carcinoma (NSCLC) in relation to tumor stage (TN0, TN1, TN2) and in association with the expression of p53 protein, a potential suppressor of tumor angiogenesis. STUDY DESIGN: The immunohistochemical (IHC) expression of VEGF and FGFR was examined in paraffin sections of 56 NSCLC in relation to the presence of lymph node metastases and p53 expression. Nodal status of NSCLC determined: 27 tumors, N0; 16, N1; and 13, N2 stage. Semiquantitative analysis with a score corresponding to IHC staining intensity and percentage of positive cells was used. Statistical analysis was performed with the chi 2 test. RESULTS: A significant association was noted between VEGF and FGFR expression in NSCLC. No relation was found between VEGF, FGFR expression and lymph node metastasis or p53 expression. CONCLUSION: We assume that VEGF and FGFR act in a synergistic manner in NSCLC and that their expression is not related to lymph node metastases. Angiogenesis is a very complex phenomenon and heterogeneous within tumors. Also, it is affected by microenviromental factors.     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in the chick embryo area vasculosa

Vascular endothelial growth factor is an angiogenic factor in vivo and in vitro that plays a crucial role in the control of blood vessel development and in pathological angiogenesis. The vascularized extraembryonic membranes of the chick embryo include the area vasculosa and the chorioallantoic membrane. In this study, we investigated the expression of vascular endothelial growth factor and of its receptor-2, specifically expressed by the endothelial cells, in the chick area vasculosa at days 6, 10 and 14 of incubation. Our results indicate that, in all the three developmental stages examined, vascular endothelial growth factor is clearly expressed in the endodermal cells immediately adjacent to the mesodermal endothelial cells which, in turn, expressed vascular endothelial growth factor receptor-2. These observations suggest that during the development of the vascular system, endodermal cells, expressing vascular endothelial growth factor, initiate angiogenesis by stimulating directly mesodermal cells, which express vascular endothelial growth factor receptor-2. Moreover, our data demonstrate that vascular endothelial growth factor receptor-2 expression is also maintained by endothelial cells in the later stages of development, until day 14 of incubation. In accord with other literature data, this suggests that vascular endothelial growth factor is required not only for proliferation, but also for the survival of endothelial cells.     

Expression of vascular endothelial growth factor receptors in smooth muscle cells

Vascular Endothelial Growth Factor (VEGF) has been typically considered to be an endothelial-specific growth factor. However, it was recently demonstrated that VEGF can interact with non endothelial cells. In this study, we tested whether vascular smooth muscles cells (VSMCs) can express VEGF receptors, such as flk-1, flt-1, and neuropilin (NP)-1, and respond to VEGF in vitro. In cultured VSMCs, flk-1 and flt-1 expression was inversely related to cell density. The expression of flk-1 was down-regulated with increasing passage numbers. However, NP-1 levels were not affected by cell density or passage numbers. Flk-1, Flt-1, and NP-1 protein levels were confirmed by Western Blotting. Although the functional mature form of Flk-1 protein is expressed at low levels in VSMCs, phosphorylation of Flk-1 following VEGF(165) stimulation was still observed. SMCs migrated significantly in response to VEGF(165) and VEGF-E, whereas Placenta Growth Factor (PlGF) induced migration only at higher concentrations. Since VEGF-E is a specific activator of flk-1 while PlGF specifically activates only flt-1, SMC migration induced by VEGF(165) is likely to be mediated primarily through the flk-1 receptor. VSMCs did not significantly proliferate in response to VEGF(165), PlGF, and VEGF-E. In conclusion, our studies demonstrate the presence of VEGF receptors on VSMCs that are functional. These studies also indicate that in vivo, VEGF may play a role in modulating the response of VSMCs.     

Copper-induced vascular endothelial growth factor expression and wound healing

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. Whereas a direct role of copper to facilitate angiogenesis has been evident two decades ago, the specific targets of copper action remained unclear. This report presents first evidence showing that inducible VEGF expression is sensitive to copper and that the angiogenic potential of copper may be harnessed to accelerate dermal wound contraction and closure. At physiologically relevant concentrations, copper sulfate induced VEGF expression in primary as well as transformed human keratinocytes. Copper shared some of the pathways utilized by hypoxia to regulate VEGF expression. Topical copper sulfate accelerated closure of excisional murine dermal wound allowed to heal by secondary intention. Copper-sensitive pathways regulate key mediators of wound healing such as angiogenesis and extracellular matrix remodeling. Copper-based therapeutics represents a feasible approach to promote dermal wound healing.     

Expression of vascular endothelial growth factor receptors and their ligands in rat uterus during the postpartum involution period

Abstract

Vascular endothelial growth factor (VEGF) and its specific receptors, FLt1/fms, Flk1/KDR and FLt4, play important roles in vasculogenesis, and physiological and pathological angiogenesis. Whether angiogenic growth factors are involved in regulating angiogenic processes during the postpartum involution period (PP) of the rat uterus is unknown. We used immunohistochemistry to analyze the expression levels of VEGF, the fms-like tyrosine kinase 1 (FLt1/fms), the kinase insert domain-containing region 1 (Flk1/KDR), Fms-related tyrosine kinase 4 (FLt4) and vascular endothelial growth inhibitor (VEGI) in the rat uterus during the days 1, 3, 5, 10 and 15 of the PP to determine the temporal and spatial expressions of VEGF and its receptors during the PP. Throughout the PP, cytoplasmic and membrane staining of VEGI, VEGF and their receptors were observed in the lumens, crypts and glandular epithelial cells as well as in connective tissue and vascular endothelial and smooth muscle cells in the endometrium. We found that the intensity of the immunoreactions in the endometrium varied with the morphological changes that occurred during involution. Immunoreactions for VEGI, VEGF and their receptor, Flk1/KDR, in the luminal epithelial cells were stronger than those in the glandular epithelial and stromal cells, particularly during PP 1, 3 and 5, which suggests that these peptides may contribute to re-epithelialization of the endometrium. On the other hand, Flt1/fms immunoreactivity was strong mainly in the stromal cells during the PP. The presence of VEGF and its receptors (FLt1/fms, Flk1/KDR, FLt4) in the stromal cells and blood vessels during the PP suggests that they may contribute to regulating stromal repair and angiogenesis in the involuting uterus of the rat.     

The adaptation of the blood-brain barrier to vascular endothelial growth factor and placental growth factor during pregnancy

Vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) are increased in the maternal circulation during pregnancy. These factors may increase blood-brain barrier (BBB) permeability, yet brain edema does not normally occur during pregnancy. We therefore hypothesized that in pregnancy, the BBB adapts to high levels of these permeability factors. We investigated the influence of pregnancy-related circulating factors on VEGF-induced BBB permeability by perfusing cerebral veins with plasma from nonpregnant (NP) or late-pregnant (LP) rats (n=6/group) and measuring permeability in response to VEGF. The effect of VEGF, PLGF, and VEGF-receptor (VEGFR) activation on BBB permeability was also determined. Results showed that VEGF significantly increased permeability (×10(7) μm(3)/min) from 9.7 ± 3.5 to 21.0 ± 1.5 (P<0.05) in NP veins exposed to NP plasma, that was prevented when LP veins were exposed to LP plasma; (9.7±3.8; P>0.05). Both LP plasma and soluble FMS-like tyrosine-kinase 1 (sFlt1) in NP plasma abolished VEGF-induced BBB permeability in NP veins (9.5±2.9 and 12±2.6; P>0.05). PLGF significantly increased BBB permeability in NP plasma (18±1.4; P<0.05), and required only VEGFR1 activation, whereas VEGF-induced BBB permeability required both VEGFR1 and VEGFR2. Our findings suggest that VEGF and PLGF enhance BBB permeability through different VEGFR pathways and that circulating sFlt1 prevents VEGF- and PLGF-induced BBB permeability during pregnancy.