Tumour Cells Expressing Single VEGF Isoforms Display Distinct Growth,Survival and Migration Characteristics

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions.     

Vascular endothelial growth factor and angiopoietin in liver regeneration

Liver architecture remodeling following partial hepatectomy (PHx) involves the formation of a complex network of liver sinusoids through which the blood flows. The present study examines the involvement of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ang-1) during liver regeneration. Following PHx, VEGF and ang-1 mRNA levels increase, followed by gradual return to baseline levels. RT-PCR analysis of VEGF mRNA reveals three isoforms, VEGF120, VEGF164 and VEGF188. Of the three, VEGF188 is the predominant isoform, VEGF120 being the less abundant. Although VEGF mRNA fluctuates following PHx, the relative expression of each isoform remains the same throughout the recovery process. The level of neuropilin-1, an accessory receptor of VEGF to main receptor corresponds with that of VEGF and ang-1. We have previously demonstrated the capacity of exogenous VEGF165 to stimulate liver cell proliferation following PHx. We now report similar effect using VEGF121, further demonstrating the benefit of manipulating growth factors where such an intervention is required.     

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.     

Isoforms of vascular endothelial growth factor act in a coordinate fashion To recruit and expand tumor vasculature

Vascular endothelial growth factor (VEGF) is an essential regulator of vascularization. It is expressed as several splice variants; the major forms contain 120 amino acids, 164 amino acids, and 188 amino acids. We utilized transformed cells nullizygous for VEGF to specifically express each of these isoforms in isolation, in order to determine the role of each in tumorigenic neo-vascularization. We found that only the intermediate isoform, VEGF164, could fully rescue tumor growth; VEGF120 partially rescued tumor growth, and VEGF188 failed completely to rescue tumor expansion. Surprisingly, the vascular density of VEGF188 isoform-expressing tumors is significantly greater than that of wild-type VEGF cells and the other isoform-specific tumors. The failure of the hypervascular VEGF188-expressing tumors to grow may be due to inadequate perfusion of the massive number of microvessels in these tumors; three-dimensional imaging of the tumorigenic vasculature indicated little or no recruitment of the peripheral vasculature. This demonstrates that the VEGF isoforms perform unique functions which together enable tumorigenic vascularization.     

Vascular endothelial growth factor (VEGF) isoform regulation of early forebrain development

This work was designed to determine the role of the vascular endothelial growth factor A (VEGF) isoforms during early neuroepithelial development in the mammalian central nervous system (CNS), specifically in the forebrain. An emerging model of interdependence between neural and vascular systems includes VEGF, with its dual roles as a potent angiogenesis factor and neural regulator. Although a number of studies have implicated VEGF in CNS development, little is known about the role that the different VEGF isoforms play in early neurogenesis. We used a mouse model of disrupted VEGF isoform expression that eliminates the predominant brain isoform, VEGF164, and expresses only the diffusible form, VEGF120. We tested the hypothesis that VEGF164 plays a key role in controlling neural precursor populations in developing cortex. We used microarray analysis to compare gene expression differences between wild type and VEGF120 mice at E9.5, the primitive stem cell stage of the neuroepithelium. We quantified changes in PHH3-positive nuclei, neural stem cell markers (Pax6 and nestin) and the Tbr2-positive intermediate progenitors at E11.5 when the neural precursor population is expanding rapidly. Absence of VEGF164 (and VEGF188) leads to reduced proliferation without an apparent effect on the number of Tbr2-positive cells. There is a corresponding reduction in the number of mitotic spindles that are oriented parallel to the ventricular surface relative to those with a vertical or oblique angle. These results support a role for the VEGF isoforms in supporting the neural precursor population of the early neuroepithelium.     

L-Maf,a downstream target of Pax6, is essential for chick lens development

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform (VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate. Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Co‐ordinated expression of the VEGF system components in granulosa cells to develop a proangiogenic autocrine milieu during ovarian follicle development

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine–arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine–arginine‐rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC‐like kinases‐1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.     

Relationship between vascular endothelial growth factor and angiogenesis in spontaneous and indomethacin-delayed healing of acetic acid-induced gastric ulcers in rats.

Angiogenesis is an important event for gastric ulcer healing. Vascular endothelial growth factor (VEGF) is known to be a potent stimulator of angiogenesis. This study consequently examined VEGF production, VEGF mRNA expression and angiogenesis during the spontaneous and indomethacin-delayed healing of acetic acid-induced ulcers in rats. The production of VEGF, taking place in the normal mucosa, was significantly elevated by ulceration. The mRNA expression of three isoforms of VEGF (VEGF188, VEGF164 and VEGF120) was also detected. Following the increase in VEGF production, angiogenesis was significantly promoted in the ulcer base. VEGF-immunoreactivity was observed in granulocytes, fibroblasts and regenerated epithelial cells. Indomethacin markedly inhibited prostaglandin E2 synthesis in the ulcer base, resulting in the prevention of ulcer healing. Angiogenesis was also significantly inhibited by indomethacin, but neither VEGF production nor VEGF mRNA expression was reduced. Such results suggest that VEGF might play a role in angiogenesis in the spontaneous healing of gastric ulcers in rats. However, the inhibition of angiogenesis in indomethacin-delayed ulcer healing is not explainable on VEGF expression.     

磷胁迫诱导大豆叶片酸性磷酸酶同工酶的表达

通过田间试验对两种磷处理的274个大豆基因型叶片酸性磷酸酶活性进行筛选,并将其中8个进行营养液栽培试验以研究磷胁迫对其叶片酸性磷酸酶同工酶表达的影响.结果表明,大豆叶片酸性磷酸酶活性存在着明显的基因型差异,不施磷处理提高了大部分(约60%)供试基因型叶片酸性磷酸酶的活性.营养液栽培试验表明,低磷处理普遍提高了所有8个供试大豆基因型叶片酸性磷酸酶的活性.等电聚焦电泳结果表明,供试大豆基因型的老叶和新叶中均有6条酸性磷酸酶的同工酶带.低磷处理显著增加了叶片酸性磷酸酶酶带的活性,但是没有诱导新的酸性磷酸酶酶带产生.研究发现叶片酸性磷酸酶活性可作为反映大豆磷胁迫的酶学指标;磷胁迫诱导大豆叶片酸性磷酸酶活性的增加是由于已有同工酶活性的提高而不是由于特异性酶带的产生.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.