Vascular Endothelial Growth Factor Splice Research Articles

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.

TARDBP promotes ovarian cancer progression by altering vascular endothelial growth factor splicing.

Disruptions in alternative splicing regulation play an essential role in ovarian cancer progression. However, the underlying mechanism remains unclear. TAR DNA-binding protein (TARDBP) plays a crucial role in alternative splicing regulation. Herein we found that TARDBP expression was significantly upregulated in OC tissue samples, particularly in cases of metastasis; further, TARDBP expression was markedly upregulated in OC patients with poor prognosis. These findings were validated by extensive tissue microarray data. TARDBP was also found to promote tumorigenesis and metastasis of OC cells in vitro and in vivo. Mechanistically, TARDBP increased the binding of the splicing factor serine/arginine-rich splicing factor 1 (SRSF1) to intron 7 of vascular endothelial growth factor (VEGF), increasing the formation of the proangiogenic VEGF165 isoform and decreasing that of the antiangiogenic VEGF165b isoform. The abnormal alternative splicing event was responsible for the activation of angiogenesis and contributed to the progression of OC. To conclude, TARDBP was found to regulate the alternative splicing of VEGF via SRSF1, induce the formation of VEGF165 but inhibit that of VEGF165b, and promote OC angiogenesis. Hence, TARDBP can serve as an independent prognostic factor and new target for OC cancer therapy.

Protein-protein interaction inhibitor of SRPKs alters the splicing isoforms of VEGF and inhibits angiogenesis

SummarySerine-arginine (SR) protein kinases (SRPKs) regulate the functions of the SR-rich splicing factors by phosphorylating multiple serines within their C-terminal arginine-serine-rich domains. Dysregulation of these phosphorylation events has been implicated in many diseases, suggesting SRPKs are potential therapeutic targets. In particular, aberrant SRPK1 expression alters the balances of proangiogenic (VEGF165) and antiangiogenic (VEGF165b) splicing isoforms of the key angiogenesis factor, vascular endothelial growth factor (VEGF), through the phosphorylation of prototypic SR protein SRSF1. Here, we report a protein-protein interaction (PPI) inhibitor of SRPKs, docking blocker of SRPK1 (DBS1), that specifically blocks a conserved substrate docking groove unique to SRPKs. DBS1 is a cell-permeable inhibitor that effectively inhibits the binding and phosphorylation of SRSF1 and subsequently switches VEGF splicing from the proangiogenic to the antiangiogenic isoform. Our findings thus provide a new direction for the development of SRPK inhibitors through targeting a unique PPI site to combat angiogenic diseases.

Altered VEGF Splicing Isoform Balance in Tumor Endothelium Involves Activation of Splicing Factors Srpk1 and Srsf1 by the Wilms' Tumor Suppressor Wt1.

Angiogenesis is one hallmark of cancer. Vascular endothelial growth factor (VEGF) is a known inducer of angiogenesis. Many patients benefit from antiangiogenic therapies, which however have limitations. Although VEGF is overexpressed in most tumors, different VEGF isoforms with distinct angiogenic properties are produced through alternative splicing. In podocytes, the Wilms’ tumor suppressor 1 (WT1) suppresses the Serine/arginine-rich protein-specific splicing factor kinase (SRPK1), and indirectly Serine/arginine-rich splicing factor 1 (Srsf1) activity, and alters VEGF splicing. We analyzed VEGF isoforms, Wt1, Srpk1, and Srsf1 in normal and tumor endothelium. Wt1, Srpk1, Srsf1, and the angiogenic VEGF164a isoform were highly expressed in tumor endothelium compared to normal lung endothelium. Nuclear expression of Srsf1 was detectable in the endothelium of various tumor types, but not in healthy tissues. Inducible conditional vessel-specific knockout of Wt1 reduced Wt1, Srpk1, and Srsf1 expression in endothelial cells and induced a shift towards the antiangiogenic VEGF120 isoform. Wt1(−KTS) directly binds and activates both the promoters of Srpk1 and Srsf1 in endothelial cells. In conclusion, Wt1 activates Srpk1 and Srsf1 and induces expression of angiogenic VEGF isoforms in tumor endothelium.

Placental growth factor signaling regulates isoform splicing of vascular endothelial growth factor A in the control of lung cancer cell metastasis.

Vascular endothelial growth factor (VEGF) family members play critical and complex roles in the regulation of cancer vascularization and metastasis. The exact molecular control of lung cancer metastasis by VEGF family members is not completely understood. Here, we showed that specimens from non-small cell lung cancer (NSCLC) contained significantly higher levels of placental growth factor (PlGF) than paired non-cancer tissue (p<0.05, N=25). Moreover, higher levels of PlGF were detected in NSCLC specimens from the patients who had distal metastases than those who had not. High-PlGF levels appeared to be associated with poor patient survival. In vitro, PlGF dose-dependently increased the ratio of pro-angiogenic VEGF isoform (VEGF165) versus anti-angiogenic VEGF isoform (VEGF165b), seemingly through induction of expression of splicing regulatory factor SRp40, resulting in the enhancement of the cancer cell metastatic potential. Higher levels of SRp40 were detected in NSCLC specimens, compared to paired non-cancer tissue (p<0.05, N=25). Finally, a strong correlation was detected between the levels of PlGF and SRp40 in NSCLC specimens (r=0.83, p<0.0001, N=25). Together, these data suggest that PlGF may increase NSCLC metastasis through SRp40-mediated mRNA splicing of VEGF.

RNA-Sequencing data supports the existence of novel VEGFA splicing events but not of VEGFAxxxb isoforms

Vascular endothelial growth factor (VEGFA), a pivotal regulator of angiogenesis and valuable therapeutic target, is characterised by alternative splicing which generates three principal isoforms, VEGFA121, VEGFA165 and VEGFA189. A second set of anti-angiogenic isoforms termed VEGFAxxxb that utilise an alternative splice site in the final exon have been widely reported, with mRNA detection based principally upon RT-PCR assays. We sought confirmation of the existence of the VEGFAxxxb isoforms within the abundant RNA sequencing data available publicly. Whilst sequences derived specifically from each of the canonical VEGFA isoforms were present in many tissues, there were no sequences derived from VEGFAxxxb isoforms. Sequencing of approximately 50,000 RT-PCR products spanning the exon 7–8 junction in 10 tissues did not identify any VEGFAxxxb transcripts. The absence or extremely low expression of these transcripts in vivo indicates that VEGFAxxxb isoforms are unlikely to play a role in normal physiology. Our analyses also revealed multiple novel splicing events supported by more reads than previously reported for VEGFA145 and VEGFA148 isoforms, including three from novel first exons consistent with existing transcription start site data. These novel VEGFA isoforms may play significant roles in specific cell types.

RhoGTPase stimulation is associated with strontium chloride treatment to counter simulated microgravity-induced changes in multipotent cell commitment

Microgravity-related cytoskeletal disorganization is associated with an altered balance between osteoblastogenesis and adipogenesis of multipotent cells. Strontium chloride is known to increase osteoblastogenesis and repress adipogenesis, but its effects in microgravity-related conditions have not been established. Our goal was to investigate early events in this process, focusing on RhoGTPases as controllers of cytoskeletal organization leading to stem cell commitment. We cultivated C3H10T1/2 on microspheres using a rotating wall vessel bioreactor (NASA) in order to simulate microgravity-related conditions in adipogenesis and osteoblastogenesis conditions independently. We observed that rotating wall vessel cultures presented increased adipogenesis, while osteoblastogenesis was reduced. Strontium-treated multipotent cells presented a significant repression in adipogenesis (−90 %, p < 0.001 PPARyD8) and an activation of osteoblastogenesis (+95 %, p < 0.001 bone sialoprotein and osteopontin D8), even in gravity altered conditions. We established that concomitant RhoA/Rac1 activations were associated with osteoblastogenesis enhancement and adipogenesis limitation in uncommitted cells. As vascular endothelial growth factor splicing is mechanosensitive and its signaling is central to stem cell commitment, we investigated vascular endothelial growth factor production, isoforms and receptors expressions in our conditions. We observed that vascular endothelial growth factor and receptors expressions were not significantly affected, but we found that presence of soluble vascular endothelial growth factor was associated with RhoA/Rac1 activations, whereas sequestration of vascular endothelial growth factor by cells was associated with RhoA/Rac1 inhibitions. We propose that strontium triggers secretion of vascular endothelial growth factor and the subsequent Rac1 and RhoA activations leading to repression of adipogenesis and osteogenesis stimulation validating strontium as a counter measure for microgravity-induced alteration of cell commitment.

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.

VEGF121 and VEGF165 differentially promote vessel maturation and tumor growth in mice and humans.

Tumor angiogenesis depends on the vascular endothelial growth factor (VEGF), which exists in multiple splicing isoforms, including the most abundant VEGF165 and VEGF121. We have previously shown that the differential capacity of these two VEGF isoforms to bind Neuropilin-1 accounts for their diverse ability to recruit Nrp1-expressing monocytes (NEMs), resulting in a different arteriogenic potential. Here we measure the expression of VEGF165 and VEGF121 in human cancer and their influence on tumor growth and vascularization. We measured the expression levels of VEGF165 and VEGF121 in human colorectal cancer and found that VEGF121 was more expressed than VEGF165, particularly in patients with extensive lymph node infiltration. Overexpressing either VEGF165 or VEGF121 in a cancer mouse model, we observed that the former decreased, whereas the latter increased tumor growth. In both clinical and experimental tumors, VEGF165 expression resulted in the recruitment of NEMs, paralleled by maturation of the tumor vascular network. Finally, hypoxia induced a shift toward the VEGF165 isoform in the central core of human cancers, as well as in various types of cultured cells. These results demonstrate that the two VEGF splicing isoforms are differentially expressed in colorectal cancers, exerting opposite effects on tumor growth and vessel maturation.

Abstract A14: Use of VEGF-based splicing-sensitive fluorescent reporters to screen for anti-angiogenic molecules

Abstract Two vascular endothelial growth factor (VEGF) protein families are generated via alternative splicing of the VEGF gene terminal exon. Use of the canonical proximal 3' splice site results in pro-angiogenic VEGF isoforms (VEGFxxx). Anti-angiogenic (VEGFxxxb) isoforms are produced through usage of a distal 3' splice site. In a quest to discover novel anti-angiogenic compounds we performed a screen using molecular tools designed to mimic VEGF splicing. Splicing-sensitive fluorescent reporters (SSFRs) can be used to study specific splicing events. A bichromatic SSFR has been engineered to mimic VEGF terminal exon splicing. Endogenous VEGF intron 7 and parts of exon 8 have been inserted into the reporter. These sequences are followed by the coding sequences for two fluorescent proteins. Depending on which alternative 3' splice site is used, a different fluorescent protein is expressed. This splicing reporter allows VEGF alternative splicing to be followed in vitro and in vivo. Reporter validation was achieved by transfection of the reporter in several cell lines with differential endogenous VEGF splicing as well as by treating stable cell lines expressing the reporter with growth factors or small molecule inhibitors known to alter VEGF alternative splicing. Flow cytometric analysis was used to evaluate the alternative splicing pattern. Following successful validation, several small pilot screens looking for novel regulators of the pro/anti-angiogenic splicing switch were undertaken with growth factors and various chemicals. Following validation and pilot experiments, a large screen of 1280 pharmacologically active compounds (LOPAC library) was performed. Changes to reporter splicing were measured using a fluorescent plate reader. Hits from the screen were investigated further using flow cytometry to confirm alteration in splicing pattern. Several compounds of interest were identified and their effect on endogenous pro/anti-angiogenic VEGF being confirmed by RTPCR. Anti-angiogenic activity is being assessed by several in vitro and in vivo angiogenic assays. Citation Format: Eleanor Star, Steven Harper, David Bates, Sebastian Oltean. Use of VEGF-based splicing-sensitive fluorescent reporters to screen for anti-angiogenic molecules. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A14.

Expression of Total Vascular Endothelial Growth Factor and the Anti-angiogenic VEGF165b Isoform in the Vitreous of Patients with Retinopathy of Prematurity

Background:This study was to examine the expression of total vascular endothelial growth factor (VEGF) and the anti-angiogenic VEGF165b isoform in the vitreous body of retinopathy of prematurity (ROP) patients, and to further study the role of the VEGF splicing in the development of ROP.Methods:This was a prospective clinical laboratory investigation study. All patients enrolled received standard ophthalmic examination with stage 4 ROP that required vitrectomy to collect the vitreous samples. The control samples were from congenital cataract patients. The expression of total VEGF and the anti-angiogenic VEGF165b were measured by enzyme-linked immunosorbent assay. Results were analyzed statistically using nonparametric tests.Results:The total VEGF level was markedly elevated in ROP samples while VEGF165b was markedly decreased compared to control group. The relative protein expression level of VEGF165b isoform was significantly decreased in ROP patients which were correlated with the ischemia-induced neovascularization.Conclusions:There was a switch of VEGF splicing from anti-angiogenic to pro-angiogenic family in ROP patients. A specific inhibitor that more selectively targets VEGF165and controls the VEGF splicing between pro- and anti-angiogenic families might be a more effective therapy for ROP.

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.

Abstract P5-07-05: Oncogene PELP1 regulates alternative splicing in breast cancer through PRMT6

Abstract Background: Alternative splicing (AS) is essential in regulating gene expression and protein diversity; however, dysregulation leads to cancer development with tumor-specific splice variants that act by disrupting a tumor suppressor function. Protein arginine methylation is a modification involved in signal transduction, pre-RNA metabolism and transcriptional activation and deregulation of arginine methylase expression or functions can contribute to cancer progression via alternate splicing. PELP1 is a proto-oncogene, whose expression is associated with higher histological grade and shorter breast cancer specific survival, and PELP1 localization is a determinant of hormone sensitivity. This study focuses on examining the role of PELP1 in regulating gene expression and its possible role in alternative splicing. Methods: ZR75 and MCF7 cells stably expressing PELP1-shRNA or overexpressing PELP1 were used in RNASeq and ChipSeq analysis. Gene differential expression lists were generated using DEseq and PELP1 regulated pathways were analyzed using Ingenuity Pathway Analysis (IPA). Cell proliferation, migration and ERE reporter gene assays were used to test the role of PRMT6 in PELP1's oncogenic functions. Immunoprecipitation, confocal microscopy, ChIP, and GST pull down assays were used to demonstrate PELP1-PRMT6 interactions. Exon specific RTqPCR assays and CD44 minigene reporter assays were used to demonstrate PELP1's role in splicing. Results: RNA- and ChIP-sequencing results showed that the ERa coregulator PELP1 is a novel regulator of alternative splicing in breast cancer through the modulation of several genes involved in splicing including protein arginine methyltransferase 6 (PRMT6). PRMT6 plays a key role in coupling of transcription and alternative splicing by functioning as a transcriptional coactivator that can also regulate alternative splicing in a hormone independent manner. We discovered the novel complex of PELP1 and PRMT6 by co-immunoprecipitation. Biochemical assays revealed that PELP1 binds RNA, colocalizes with the splicing factor SC35 and promotes alternative splicing in reporter gene assays. PELP1 regulates the exon inclusion:skipping ratio of alternatively spliced exons in endogenous vascular endothelial growth factor (VEGF). PELP1's regulation of VEGF splicing is affected by the status of PRMT6. Interestingly, PRMT6 is needed for optimal oncogenic functions of PELP1. Further PELP1 has the potential to regulate the histone methyltransferase activity of PRMT6 and chromatin immunoprecipitation showed that PELP1 affects the enzyme recruitment and enrichment of the histone modification H3R2me2 at estrogen responsive genes. Conclusions: Our findings show a role of PELP1-PRMT6 axis in ERα-mediated RNA splicing and their deregulation has implications for targeted therapeutics in ERα-driven breast cancer. This work was supported by the NIH grant CA095681 and NIH F31 Fellowship 1F31CA173909-01A1. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-05.

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in Vitro

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.

Abstract 380: Serine-Arginine protein kinase 1 (SRPK1) - A potential target of angiogenesis in prostate cancer.

Abstract Angiogenesis is required for all tumors to grow and metastasize and is mainly induced by the vascular endothelial growth factor (VEGF). Alternative spliced isoforms associated with cancer progression have been described in all areas of hallmarks of cancer, including angiogenesis. VEGF mRNA is alternatively spliced at the terminal exon, to produce two families of isoforms. The pro- angiogenic family, VEGFxxx, was shown to be up-regulated in all cancers studied so far, where as the anti- angiogenic family is preferentially expressed in normal non-angiogenic tissues, but down-regulated in most cancers. One of the factors involved in the regulation of VEGF alternative splicing is SRPK1. This is a protein kinase that phosphorylates the splicing factor SRSF1 and favours the production of the pro-angiogenic VEGF isoform. We therefore tested the hypothesis that down - regulation of SRPK1 in prostate cancer cells would increase the production of the anti-angiogenic isoform, preventing angiogenesis and tumor growth. PC-3, DU145 and LnCap prostate cancer cells showed an increased expression of SRPK1 when compared to Primary Prostate epithelial cells. Stable SRPK1 knock-down (KD) in PC-3 and LnCap prostate cancer cells was obtained using a lentiviral system (Dharmacon). This resulted in a reduction in splice factor expression, mainly SRSF1, and induced a switch in the expression of VEGF towards the anti-angiogenic isoform, VEGF165b, when compared to control cells (transfected with non-targeting shRNA). PC-3 SRPK1 KD and control cell lines were injected subcutaneously in male nude mice. Knock down of SRPK1 resulted in an inhibition of tumour growth, as shown by the significant difference in tumor size between the SRPK1 KD (250.0 ± 82.28 n=9) and control cell line (994.7 ± 243.5 n=9)(Day 29 post-injection). In addition to the reduction in tumor volume, SRPK1 inhibition resulted in decrease in vascular density of the tumors, as shown by the significant reduction in blood vessel number in PC-3 SRPK1 KD (2.714 ± 0.3686 n=7) mice compared with control (4.292 ± 0.5426 n=8). These in vitro and in vivo data suggest that the regulation of VEGF splicing by SRPK1 alters prostate cancer tumour growth and angiogenesis. Citation Format: Athina Mavrou, Sebastian Oltean, Dave Bates. Serine-Arginine protein kinase 1 (SRPK1) - A potential target of angiogenesis in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 380. doi:10.1158/1538-7445.AM2013-380

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.

SRPK1 inhibition in vivo: modulation of VEGF splicing and potential treatment for multiple diseases

SRPK1 (serine-arginine protein kinase 1) is a protein kinase that specifically phosphorylates proteins containing serine-arginine-rich domains. Its substrates include a family of SR proteins that are key regulators of mRNA AS (alternative splicing). VEGF (vascular endothelial growth factor), a principal angiogenesis factor contains an alternative 3' splice site in the terminal exon that defines a family of isoforms with a different amino acid sequence at the C-terminal end, resulting in anti-angiogenic activity in the context of VEGF165-driven neovascularization. It has been shown recently in our laboratories that SRPK1 regulates the choice of this splice site through phosphorylation of the splicing factor SRSF1 (serine/arginine-rich splicing factor 1). The present review summarizes progress that has been made to understand how SRPK1 inhibition may be used to manipulate the balance of pro- and anti-angiogenic VEGF isoforms in animal models in vivo and therefore control abnormal angiogenesis and other pathophysiological processes in multiple disease states.