Anti-angiogenic Isoform Research Articles

Synthetic Antiangiogenic Vascular Endothelial Growth Factor-A Splice Variant Revascularizes Ischemic Muscle in Peripheral Artery Disease.

Alternative splicing in the eighth exon C-terminus of VEGF-A (vascular endothelial growth factor-A) results in the formation of proangiogenic VEGF165a and antiangiogenic VEGF165b isoforms. The only known difference between these 2 isoform families is a 6-amino acid switch from CDKPRR (in VEGF165a) to SLTRKD (in VEGF165b). We have recently shown that VEGF165b can induce VEGFR2-activation but fails to induce VEGFR1 (VEGF receptor 1)-activation. The molecular mechanisms that regulate VEGF165b's ability toward differential VEGFR2 versus VEGFR1 activation/inhibition are not yet clear. Hypoxia serum starvation was used as an invitro peripheral artery disease model. Unilateral single ligation of the femoral artery was used as a preclinical peripheral artery disease model. VEGFR1 activating ligands have 2 arginine (RR) residues in their eighth exon C-terminus, that were replaced by lysine-aspartic acid (KD) in VEGF165b. A synthetic anti-angiogenic VEGF165b splice variant in which the KD residues were switched to RR (VEGF165bKD→RR) activated both VEGFR1- and VEGFR2-signaling pathways to induce ischemic-endothelial cell angiogenic capacity invitro and enhance perfusion recovery in a severe experimental-peripheral artery disease model significantly higher than VEGF165a. Phosphoproteome arrays showed that the therapeutic efficacy of VEGF165bKD→RR over VEGF165a is due to its ability to induce P38-activation in ischemic endothelial cells. Our data shows that the KD residues regulate VEGF165b's VEGFR1 inhibitory property but not VEGFR2. Switching these KD residues to RR resulted in the formation of a synthetic/recombinant VEGF165bKD→RR isoform that has the ability to activate both VEGFR1- and VEGFR2-signaling and induce ischemic-endothelial cell angiogenic and proliferative capacity that matched the angiogenic requirement necessary to achieve perfusion recovery in a severe experimental-peripheral artery disease model.

Inhibiting anti-angiogenic VEGF165b activates a miR-17-20a-Calcipressin-3 pathway that revascularizes ischemic muscle in peripheral artery disease

BackgroundVEGF165a increases the expression of the microRNA-17-92 cluster, promoting developmental, retinal, and tumor angiogenesis. We have previously shown that VEGF165b, an alternatively spliced anti-angiogenic VEGF-A isoform, inhibits the VEGFR-STAT3 pathway in ischemic endothelial cells (ECs) to decrease their angiogenic capacity. In ischemic macrophages (Møs), VEGF165b inhibits VEGFR1 to induce S100A8/A9 expression, which drives M1-like polarization. Our current study aims to determine whether VEGF165b inhibition promotes perfusion recovery by regulating the microRNA(miR)-17-92 cluster in preclinical PAD.MethodsFemoral artery ligation and resection was used as a preclinical PAD model. Hypoxia serum starvation (HSS) was used as an in vitro PAD model. VEGF165b was inhibited/neutralized by an isoform-specific VEGF165b antibody.ResultsHere, we show that VEGF165b-inhibition induces the expression of miR-17-20a (within miR-17-92 (miR-17-18a-19a-19b-20a-92) cluster) in HSS-ECs and HSS-Møs vs. respective normal and/or isotype-matched IgG controls to enhance perfusion recovery. Consistent with the bioinformatics analysis that revealed RCAN3 as a common target of miR-17 and miR-20a, Argonaute-2 pull-down assays showed decreased miR-17-20a expression and higher RCAN3 expression in the RNA-induced silencing complex of HSS-ECs and HSS-Møs vs. respective controls. Inhibiting miR-17-20a induced RCAN3 levels to decrease ischemic angiogenesis and promoted M1-like polarization to impair perfusion recovery. Finally, using STAT3 inhibitors, S100A8/A9 silencers, and VEGFR1-deficient ECs and Møs, we show that VEGF165b-inhibition activates the miR-17-20a-RCAN3 pathway independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 in ischemic-ECs and ischemic-Møs respectively.ConclusionsOur data revealed a hereunto unrecognized therapeutic ‘miR-17-20a-RCAN3’ pathway in the ischemic vasculature that is VEGFR1-STAT3/S100A8/A9 independent and is activated only upon VEGF165b-inhibition in PAD.

Abstract 18844: LncRNA MIR17HG and Peripheral Arterial Disease

Introduction: VEGF-A, a potent angiogenic growth factor has been extensively investigated as a therapeutic for Peripheral artery Disease (PAD). Recently, we have shown that increased levels of the anti-angiogenic VEGF-A isoform (VEGF165b) decreases perfusion recovery by inhibiting a truncated miR-17-20a cluster in ischemic endothelial cells and macrophages. Since miR-17-20a cluster is produced from the lncRNA-MIR17HG, we wanted to determine the role of VEGF165b-MIR17HG axis in regulating ischemic angiogenesis and perfusion recovery in PAD Methods: Hypoxia serum starvation in Human Umbilical vein endothelial cells (HUVECs) and Human microvascular endothelial cells (HMEC-1) was used as an in vitro PAD model. Results: Since LncRNAs have multiple transcript variants, we first determined if a specific MIR17HG transcript is present in human chronic limb threatening ischemia (CLTI) vs. normal. Bioinformatics analysis of a recently published bulk RNA-Seq data showed that among 17 annotated transcripts, transcript-1 (MIR17HG-Tr1) is specifically expressed in normal and CLTI muscle biopsies. Analysis of FPKMs showed a numerical increase in MIR17HG-Tr1 in CLTI vs. normal biopsies. qPCR analysis using primers specific for MIR17HG-Tr1 showed significantly higher expression of MIR17HG-Tr1 in HSS-HUVECs and HSS-HMEC-1 vs. their respective controls. VEGF165b inhibition in HSS-HUVECs and HSS-HMECs significantly decreased MIR17HG-Tr1 expression compared to IgG controls. To determine whether MIR17HG functions independent of its miRNA cluster, we silenced MIR17HG-Tr1 and examined miR17-92 cluster expression. Silencing MIR17HG-Tr1 didn’t significantly affect the expression of miR-17-92 cluster members either under normoxic or HSS conditions. In vitro tube formation assay showed that silencing MIR17HG-Tr1 significantly increased the angiogenic capacity of HSS-HUVECs vs. control. Conclusion: This data indicates VEGF165b inhibits MIR17HG-Tr1 to induce ischemic angiogenesis independently of its miR17-92 cluster. Ongoing studies are aimed at understanding the mechanism by which 1) VEGF165b regulates MIR17HG-Tr1 and 2) MIR17HG-Tr1 regulates revascularization in PAD.

Hypoxia-induced loss of SRSF2-dependent DNA methylation promotes CTCF-mediated alternative splicing of VEGFA in breast cancer

Alternative splicing of vascular endothelial growth factor A (VEGFA) generates numerous isoforms with unique roles in tumor angiogenesis, and investigating the underlying mechanism during hypoxia necessitates diligent pursuance. Our research systematically demonstrated that the splicing factor SRSF2 causes the inclusion of exon-8b, leading to the formation of the anti-angiogenic VEGFA-165b isoform under normoxic conditions. Additionally, SRSF2 interacts with DNMT3A and maintains methylation on exon-8a, inhibiting CCCTC-binding factor (CTCF) recruitment and RNA polymerase II (pol II) occupancy, causing exon-8a exclusion and decreased expression of pro-angiogenic VEGFA-165a. Conversely, SRSF2 is downregulated by HIF1α-induced miR-222-3p under hypoxic conditions, which prevents exon-8b inclusion and reduces VEGFA-165b expression. Furthermore, reduced SRSF2 under hypoxia promotes hydroxymethylation on exon-8a, increasing CTCF recruitment, pol II occupancy, exon-8a inclusion, and VEGFA-165a expression. Overall, our findings unveil a specialized dual mechanism of VEGFA-165 alternative splicing, instrumented by the cross-talk between SRSF2 and CTCF, which promotes angiogenesis under hypoxic conditions.

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.

Hypophysial angiogenesis decodes annual time and underlies physiological adaptation to seasonal changes in the environment.

Adaptation to annual changes in the environment is controlled by hypophysial hormones. In temperate zones, photoperiod is the primary external cue that regulates annual biological cycles and is translated by the pattern of melatonin secretion acting primarily in the hypophysial pars tuberalis. Angiogenic mechanisms within this tissue contribute to decode the melatonin signal through alternative splicing of the vascular endothelial growth factor A (VEGF-A) gene in both the pars tuberalis and the capillary loops of the infundibulum. The resulting melatonin-evoked differential productions of VEGF-A isoforms will induce seasonal remodeling of the vascular connection between the hypothalamus and hypophysis, and act as paracrine messengers in the pars distalis to generate the required seasonal endocrine response. Specifically, the long melatonin signal in winter upregulates antiangiogenic VEGF-A isoforms, which will reduce the number of vascular loops and the density of VEGF receptors in endocrine and folliculo-stellate (FS) cells, inhibit prolactin secretion, and stimulate FSH. In contrast, the short melatonin signal in summer upregulates proangiogenic VEGF-A isoforms that will increase the number of vascular loops and the density of VEGF receptors in endocrine and FS cells, stimulate prolactin secretion, and suppress FSH. A similar system has been identified in long day seasonal breeders, revealing that this is a conserved mechanism of adaptation across species. Thus, an angiogenesis-based, intrahypophysial system for annual time measurement controls local microvascular plasticity and conveys the photoperiodic signal readout from the melatonin sensitive pars tuberalis to the endocrine cells of the pars distalis to regulate seasonal adaptation to the environment.

Macrophage IL-1β promotes arteriogenesis by autocrine STAT3- and NF-κB-mediated transcription of pro-angiogenic VEGF-A.

SUMMARYPeripheral artery disease (PAD) leads to considerable morbidity, yet strategies for therapeutic angiogenesis fall short of being impactful. Inflammatory macrophage subsets play an important role in orchestrating post-developmental angiogenesis, but the underlying mechanisms are unclear. Here, we find that macrophage VEGF-A expression is dependent upon the potent inflammatory cytokine, IL-1β. IL-1β promotes pro-angiogenic VEGF-A165a isoform transcription via activation and promoter binding of STAT3 and NF-κB, as demonstrated by gene-deletion, gain-of-function, inhibition, and chromatin immunoprecipitation assays. Conversely, IL-1β-deletion or inhibition of STAT3 or NF-κB increases anti-angiogenic VEGF-A165b isoform expression, indicating IL-1β signaling may also direct splice variant selection. In an experimental PAD model of acute limb ischemia, macrophage IL-1β expression is required for pro-angiogenic VEGF-A expression and for VEGF-A-induced blood flow recovery via angio- or arteriogenesis. Though further study is needed, macrophage IL-1β-dependent transcription of VEGF-A via STAT3 and NF-κB may have potential to therapeutically promote angiogenesis in the setting of PAD.

Abstract 10451: A Potential Therapeutic Antibody to Induce Collateral Formation in Cardiovascular Diseases

Introduction: Lack of efficient collateralisation during vascular occlusion leads to tissue ischemia in coronary, peripheral and cerebrovascular disease. Patients with peripheral, coronary, and cerebrovascular disease have elevated levels of the anti-angiogenic isoform of VEGF (VEGF-A 165 b), which correlate with poorer outcomes. In experimental animals, VEGF-A 165 b neutralisation improves blood flow and collateralisation after femoral or coronary artery ligation. Hypothesis: We tested the hypothesis that a potent, high affinity, humanised neutralising antibody to VEGF-A 165 b could have therapeutic potential in models of cardiovascular disease. Methods: A mouse monoclonal antibody to VEGF-A 165 b was humanised by cloning and site directed mutagenesis. Multiple clones were screened, and one with high affinity (measured by biolayer interferometry) was amplified. The efficacy in vitro was tested using endothelial cell migration assays and in vivo using the hindlimb ischemia (HLI) model in C57Bl6 mice fed a high fat high sucrose (HFHS) diet, and in Zucker Diabetic Fatty rats. Results: One clone (HC4LC2) had a VEGF-A 165 b affinity of 600pM. 0.3μg/ml of this clone reversed the inhibitory effect of recombinant human VEGF-A 165 b (31±4%, N=6 of VEGF-A 165 a alone) on migration of endothelial cells to 40ng/ml rhVEGF-A 165 a (89±9% N=3). Human monocyte dependent inhibition of VEGF-A 165 a mediated migration was dose dependently reversed by HC4LC2 (EC50, 55ng/ml, N=9 per concentration). HLI in HFHS mice impaired blood flow after 28 days in IgG treated mice (70±7.7% of pre-ischemic blood flow, N=6), which was restored to normal (93.3±5.5, p<0.05, N=8) by intraperitoneal injection of 1mg/kg HC4LC2. Blood flow recovery after HLI was also seen in ZDF rats treated with HC4LC2 (106±16% of pre-ischemic flow, N=5) compared with IgG treated animals (76±.9%, N=5, p<0.05, two-way ANOVA, all values mean ±SEM). Conclusions: These results indicate that HC4LC2 is a humanised, high affinity neutralising antibody to VEGF-A 165 b that could have therapeutic potential for patients with cardiovascular disease, by enhancing collateral formation.

CircSMARCA5-SRSF1 interaction is functionally involved in GBM pathogenesis

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor with a median survival of 15 months. The standard treatments of GBM and the total medical resection are unable to contrast this mortal cancer. For these reason new diagnostic approaches and therapies are needed. The identification of molecular features of this cancer may allow to create a personalized therapy. Circular RNAs (circRNAs) are a new class of non-coding RNAs (ncRNAs) highly enriched in brain, stable within the cells, detectable in body fluids and having a potential role and biological importance still object of debate. This thesis investigated the putative involvement of circRNAs in GBM pathogenesis. It has been demonstrated that circSMARCA5 is downregulated in GBM biopsies, its expression is associated to the glioma grade malignancy and it negatively regulates migration of U87MG cells. Moreover, it has been proved the physical interaction between circSMARCA5 and one of its predicted interactor Serine/arginine-rich splicing factor 1 (SRSF1). Interesting splicing targets of SRSF1 are the serine and arginine rich splicing factor 3 (SRSF3) and the vascular endothelial growth factor A (VEGFA). It has been proposed that circSMARCA5 may regulate the alternative splicing of SRSF3 in favor of the formation of a stable oncoprotein in GBM. It also regulates the alternative splicing of VEGFA mRNA through the binding to SRSF1. In addition, blood vascular microvessel density evaluated in GBM negatively correlates with the expression of circSMARCA5, while positively correlates with that of SRSF1 and pro-to-anti-angiogenic VEGFA isoform ratio. GBM patients with low circSMARCA5 expression have lower overall and progression free survival rates. Based on these findings, CircSMARCA5 could be considered a promising druggable tumor suppressor in GBM. Moreover, its interaction with SRSF1 makes circSMARCA5 an upstream regulator of pro- to anti-angiogenic VEGFA isoforms ratio within GBM cells and a highly promising GBM prospective anti-angiogenic molecule.

The CXCL12/CXCR4 and VEGF165/Nrp1 Pathways Have Differential Effects on Pathological Neo-Angiogenesis and Vascular Remodeling in Hemophilic Arthropathy

Introduction: Joint hypervascularity is characteristic to hemophilic arthropathy (HA), which is a hallmark complication in patients with hemophilia. Angiogenesis in the hemophilic joint is triggered by bleeding and results in excessive vascular remodeling with enlarged irregularly shaped vessels that appear fragile, potentially causing re-bleeding. Therefore, targeting angiogenesis may offer novel treatment avenues in HA. Vascular endothelial growth factor A (VEGF-A) is an important angiogenic mediator implicated in the pathogenesis of HA, but a complete blockade may cause vascular instability with more bleeding, or inhibition of anti-angiogenic isoforms, thereby decreasing therapeutic efficacy. An alternative approach may be to target the co-receptor Neuropilin 1 (Nrp1) that facilitates binding of the pro-angiogenic isoform VEGF165 to its signaling receptor, VEGFR2. Here, we determine whether blocking the binding of VEGF165 to Nrp1 can curb the pathological vascular remodeling associated with hemophilic joint bleeding.Methods and results: Joint bleeding was induced by subpatellar needle puncture in FVIII-/- mice, resulting in pronounced intra- and peri-articular bleeding, evidenced by a drop in hematocrit from ~45% to 28 ± 10% 2 days after injury. The following antibody treatments were initiated 2 days after injury and were administered at 5 mg/kg, twice weekly for 4 weeks: B20-4 targeting murine VEGF-A, and YW107.4.87 blocking the VEGF165 binding site on Nrp1. The CXCR4 antagonist, AMD3100 (300 µg/day by subcutaneous osmotic pump) was included as positive control as it corrects hypervascularity induced by hemophilic joint bleeding (Wyseure et al . ISTH 2017).Four weeks after injury blood flow in the joint, measured by ultrasound-Power Doppler (PD), was increased 2.8-fold in injured vs uninjured FVIII-/- mice (p<0.001; n=13). Anti-VEGF treatment resulted in a similar 3.2-fold increase in PD signal (n=8), indicating that total inhibition of VEGF had no effect on the aberrant blood flow. Inhibition of Nrp1 had a partial effect (1.9-fold increase over baseline; p=0.16 vs no treatment; n=7), whereas inhibition of CXCL12-mediated vascular cell recruitment by the CXCR4 antagonist significantly blunted aberrant PD signals (p<0.05 vs no treatment; n=10). On histology, the total vessel count after anti-Nrp1 treatment was similar to no treatment and significantly greater than baseline (86 anti-Nrp1 vs 80 no treatment vs 42 baseline; p<0.01). Similarly, anti-Nrp1 treatment had no effect on the number of abnormally enlarged vessels (with a diameter ≥ 20 µm) compared to no treatment (15 anti-Nrp1 vs 13 no treatment vs 3 baseline; p<0.01 vs baseline). In contrast, with CXCR4 antagonist treatment both the total vessel count (64; p=0.3 vs baseline) and the number of abnormally enlarged vessels (7; p=0.1 vs baseline) remained low. However, anti-Nrp1 treatment did prevent the 2.5-fold increased perivascular expression of smooth muscle cell actin (αSMA; p<0.01) that typically accompanies the abnormally enlarged vessels in injured FVIII-/- mice. Thus, inhibition of Nrp1 lowered αSMA expression after hemophilic bleeding, similar to baseline and CXCR4 antagonist treatment, although it had no effects on vessel number and enlargement. This suggests that anti-Nrp1 treatment prevents the maturation during vascular remodeling in HA, potentially leaving the vessels destabilized and prone to vascular permeability. Towards that end, staining with Prussian Blue demonstrated increased hemosiderin deposits in anti-Nrp1 treated mice, similar to untreated injured FVIII-/- mice, whereas no signs of macroscopic joint bleeding or hemosiderin deposits were observed after treatment with the CXCR4 antagonist.Conclusions: Abrogating the VEGF165-Nrp1 interaction was not sufficient to prevent neo-angiogenesis and vessel lumen expansion in a murine hemophilic joint injury model. However, inhibition of Nrp1 did reduce perivascular remodeling that may allow regression of the aberrant vasculature over time, but also may promote further vessel instability and additional hemorrhaging. Thus, inhibition of the CXCL12/CXCR4 pathway affects neo-angiogenesis, whereas inhibition of the VEGF165/Nrp1 pathway predominantly affects perivascular maturation, but not neo-angiogenesis. These findings provide novel insights on the potential benefits and pitfalls of anti-angiogenic treatments in HA. Disclosuresvon Drygalski:Hematherix LLC: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novo Nordisk: Honoraria; CSL Behring: Honoraria; Bioverativ: Honoraria; Bayer: Honoraria; Shire: Honoraria. Mosnier:Hematherix LLC: Membership on an entity's Board of Directors or advisory committees.

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.

Vascular endothelial growth factor (VEGF)-A and VEGF-A165b are associated with time to remission of granulomatosis with polyangiitis in a nationwide Japanese prospective cohort study.

Effective prognostic markers are needed for antineutrophil cytoplasmic antibody-associated vasculitis (AAV). This study evaluated the clinical associations of serum vascular endothelial growth factor-A (sVEGF-A) and sVEGF-A165b (an antiangiogenic isoform of VEGF-A) concentrations with time to remission of AAV in a nationwide Japanese prospective follow-up cohort. We collected samples from patients with AAV who were enrolled in the nationwide Japanese cohort study (RemIT-JAV-RPGN). We measured sVEGF-A and sVEGF-A165b concentrations using enzyme-linked immunosorbent assays in 57 serum samples collected 6 months before and after initiation of AAV treatment. Patients were classified based on AAV disease subtypes: microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis (EGPA). Results revealed significant reductions in sVEGF-A and sVEGF-A165b concentrations in patients with microscopic polyangiitis and EGPA, respectively. However, despite the comparable concentrations of sVEGF-A and sVEGF-A165b during the 6 months of treatment in granulomatosis with polyangiitis patients, correlation analysis revealed that the differences in log2-transformed concentrations of sVEGF-A and sVEGF-A165b were inversely correlated with time to remission in granulomatosis with polyangiitis patients. These results suggest that sVEGF-A and -A165b can serve as potential markers of time to remission in patients with granulomatosis with polyangiitis.

Implication of anti-angiogenic VEGF-A165b in angiogenesis and systolic function after reperfused myocardial infarction

Abstract Background Angiogenesis participates in re-establishing microcirculation after myocardial infarction (MI). Purpose In this study, we aim to further understand the role of the anti-angiogenic isoform vascular endothelial growth factor (VEGF)-A165b after MI and explore its potential as a co-adjuvant therapy to coronary reperfusion. Methods Two mice MI models were formed: 1) permanent coronary ligation (non-reperfused MI), 2) transient 45-min coronary occlusion followed by reperfusion (reperfused MI); in both models, animals underwent echocardiography before euthanasia at day 21 after MI induction. Serum and myocardial VEGF-A165b levels were determined. In both experimental MI models, functional and structural implication of VEGF-A165b blockade was assessed. In a cohort of 104 ST-segment elevation MI patients, circulating VEGF-A165b levels were correlated with cardiovascular magnetic resonance-derived left ventricular ejection fraction at 6-months and with the occurrence of adverse events (death, heart failure and/or re-infarction). Results In both models, circulating and myocardial VEGF-A165b presence was increased 21 days after MI induction. Serum VEGF-A165b levels inversely correlated with systolic function evaluated by echocardiography. VEGF-A165b blockage increased capillary density, reduced infarct size, and enhanced left ventricular function in reperfused, but not in non-reperfused MI experiments. In patients, higher VEGF-A165b levels correlated with depressed ejection fraction and worse outcomes. Conclusions In experimental and clinical studies, higher serum VEGF-A165b levels associates with a worse systolic function. Its blockage enhances neoangiogenesis, reduces infarct size, and increases ejection fraction in reperfused, but not in non-reperfused MI experiments. Therefore, VEGF-A165b neutralization represents a potential co-adjuvant therapy to coronary reperfusion. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This study was funded by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (Exp. PIE15/00013, PI17/01836, PI18/00209 and CIBERCV16/11/00486).

VEGF165b augments NK92 cytolytic activity against human K562 leukemia cells by upregulating the levels of perforin and granzyme B via the VEGR1-PLC pathway

Pro-angiogenic Vascular endothelial growth factors (VEGFs) exert immunosuppressive functions on some immune cells by interacting with VEGF receptors. Blocking the VEGF/VEGFR pathway could reverse the tumor immunosuppressive microenvironment to some degree. We recently demonstrated that the anti-angiogenic VEGF isoform VEGF165b, similar to other anti-angiogenic agents, inhibit the accumulation immunosuppressive cells such as Tregs and MDSCs. However, whether VEGF165b affects the functions of immune effector cells remain unclear. Here, NK92 cell line was utilized as an immune effector cell model. Our results verified that NK92 cells endogenously express VEGF165 and VEGFR1. Further investigation showed that NK92 treatment with VEGF165b augments its killing ability against human K562 leukemia cells by upregulating perforin and granzyme B through the VEGFR1-PLC pathway, whereas VEGF165b had no impact on the proliferation of NK92 cells in vitro. The results of this study improve our understanding of the immunomodulatory function of VEGF165b, which may help in enhancing the efficacy of NK92-based cancer immunotherapy.

WT1 activates transcription of the splice factor kinase SRPK1 gene in PC3 and K562 cancer cells in the absence of corepressor BASP1

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF165b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth.

Therapeutic angiogenesis using autologous adipose-derived regenerative cells in patients with critical limb ischaemia in Japan: a clinical pilot study

Adipose-derived regenerative cell (ADRC) is a promising alternative source of autologous somatic stem cells for the repair of damaged tissue. This study aimed to assess the safety and feasibility of autologous ADRC implantation for therapeutic angiogenesis in patients with critical limb ischaemia (CLI). A clinical pilot study—Therapeutic Angiogenesis by Cell Transplantation using ADRCs (TACT-ADRC) study—was initiated in Japan. Adipose tissue was obtained by ordinary liposuction method. Isolated ADRCs were injected into the ischaemic limb. We performed TACT-ADRC procedure in five patients with CLI. At 6 months, no adverse events related to the TACT-ADRC were observed. No patients required major limb amputation, and ischaemic ulcers were partly or completely healed during the 6-month follow-up. In all cases, significant clinical improvements were seen in terms of rest pain and 6-min walking distance. Numbers of circulating CD34+ and CD133+ cells markers of progenitor cell persistently increased after ADRC implantation. The ratio of VEGF-A165b (an anti-angiogenic isoform of VEGF) to total VEGF-A in plasma significantly decreased after ADRC implantation. In vitro experiments, cultured with ADRC-conditioned media (CM) resulted in increased total VEGF-A and decreased VEGF-A165b in C2C12 cells, but not in macrophages. ADRC-CM also increased CD206+ cells expression and decreased TNF-α in macrophages. Autologous ADRC implantation was safe and effective in patients with CLI and could repair damaged tissue via its ability to promote angiogenesis and suppress tissue inflammation.

The Production of Pro-angiogenic VEGF-A Isoforms by Hypoxic Human NK Cells Is Independent of Their TGF-β-Mediated Conversion to an ILC1-Like Phenotype.

Circulating natural killer (NK) cells have been shown to adopt a type 1 innate lymphoid cell (ILC1)-like phenotype in response to TGF-β and secrete VEGF-A when exposed to hypoxia. Although these changes are often considered to be linked attributes of tissue residency, it has yet to be determined if TGF-β and hypoxia work in concert to coordinate NK cellular phenotype and angiogenic potential. Examination of human circulating NK cells treated with TGF-β demonstrated heterogeneity in their potential to adopt an ILC1-like phenotype, as indicated by the upregulation of CD9 and CD103 on only a subset of cells in culture. Culturing NK cells in chronic hypoxia did not induce a similar ILC1-like conversion and did not enhance the degree of conversion for cells exposed to TGF-β. Similarly, hypoxic culture of circulating NK cells induced VEGF-A secretion, but this production was not enhanced by TGF-β. Fluorescent in-situ hybridization flow cytometry demonstrated that hypoxia-induced VEGF-A production was uniform across all NK cells in culture and was not a selective feature of the cellular subset that adopted an ILC1-like phenotype in response to TGF-β. Examination of VEGF-A isoforms demonstrated that hypoxia induces the production of pro-angiogenic VEGF-A isoforms, including VEGF-A165 and VEGF-A121, and does not stimulate any meaningful production of anti-angiogenic isoforms, such as VEGF-Ab transcriptional variants or VEGF-Ax. In summary, TGF-β-mediated ILC1-like conversion and hypoxia-induced VEGF-A production are discrete processes in NK cells and are not part of a linked cellular program associated with tissue residency.

Implicación de la isoforma antiangiogénica VEGF-A165b en la angiogénesis y la función sistólica tras un infarto de miocardio reperfundido

Introduction and objectivesAngiogenesis helps to reestablish microcirculation after myocardial infarction (MI). In this study, we aimed to further understand the role of the antiangiogenic isoform vascular endothelial growth factor (VEGF)-A165b after MI and to explore its potential as a coadjuvant therapy to coronary reperfusion. MethodsTwo mice MI models were formed: a) permanent coronary ligation (nonreperfused MI); b) transient 45-minute coronary occlusion followed by reperfusion (reperfused MI); in both models, animals underwent echocardiography before euthanasia at day 21 after MI induction. We determined serum and myocardial VEGF-A165b levels. In both experimental MI models, we assessed the functional and structural role of VEGF-A165b blockade. In a cohort of 104 ST-segment elevation MI patients, circulating VEGF-A165b levels were correlated with cardiovascular magnetic resonance-derived left ventricular ejection fraction at 6 months and with the occurrence of adverse events (death, heart failure, and/or reinfarction). ResultsIn both models, circulating and myocardial VEGF-A165b levels were increased 21 days after MI induction. Serum VEGF-A165b levels inversely correlated with systolic function evaluated by echocardiography. VEGF-A165b blockade increased capillary density, reduced infarct size, and enhanced left ventricular function in reperfused, but not in nonreperfused, MI experiments. In patients, higher VEGF-A165b levels correlated with depressed ejection fraction and worse outcomes. ConclusionsIn experimental and clinical studies, higher serum VEGF-A165b levels are associated with worse systolic function. Their blockade enhances neoangiogenesis, reduces infarct size, and increases ejection fraction in reperfused, but not in nonreperfused, MI experiments. Therefore, VEGF-A165b neutralization represents a potential coadjuvant therapy to coronary reperfusion.

Trophoblast lineage specific expression of the alternative splicing factor RBFOX2 suggests a role in placental development

IntroductionRBFOX2, an RNA-binding protein, controls tissue-specific alternative splicing of exons in diverse processes of development. The progenitor cytotrophoblast of the human placenta differentiates into either the syncytiotrophoblast, formed via cell fusion, or the invasive extravillous trophoblast lineage. The placenta affords a singular system where a role for RBFOX2 in both cell invasion and cell fusion may be studied.We investigated a role for RBFOX2 in trophoblast cell differentiation, as a foundation for investigations of RBFOX2 in embryo implantation and placental development. MethodsImmunohistochemistry of RBFOX2 was performed on placental tissue sections from three trimesters of pregnancy and from pathological pregnancies. Primary trophoblast cell culture and immunofluorescence were employed to determine RBFOX2 expression upon cell fusion. Knockdown of RBFOX2 expression was performed with βhCG and syncytin-1 as molecular indicators of fusion. ResultsIn both normal and pathological placentas, RBFOX2 expression was confined to the cytotrophoblast and the extravillous trophoblast, but absent from the syncytiotrophoblast. Additionally, we showed that primary trophoblasts that spontaneously fused in cell culture downregulated RBFOX2 expression. In functional experiments, knockdown expression of RBFOX2 significantly upregulated βhCG, while the upregulation of syncytin-1 did not reach statistical significance. DiscussionRBFOX2, by conferring mRNA diversity, may act as a regulator switch in trophoblast differentiation to either the fusion or invasive pathways. By studying alternative splicing we further our understanding of placental development, yielding possible insights into preeclampsia, where expression of antiangiogenic isoforms produced through alternative splicing play a critical role in disease development and severity.

Modulation of VEGF Expression and Oxidative Stress Response by Iodine Deficiency in Irradiated Cancerous and Non-Cancerous Breast Cells

Breast cancer remains a major concern and its physiopathology is influenced by iodine deficiency (ID) and radiation exposure. Since radiation and ID can separately induce oxidative stress (OS) and microvascular responses in breast, their combination could additively increase these responses. Therefore, ID was induced in MCF7 and MCF12A breast cell lines by medium change. Cells were then X-irradiated with doses of 0.05, 0.1, or 3 Gy. In MCF12A cells, both ID and radiation (0.1 and 3 Gy) increased OS and vascular endothelial growth factor (VEGF) expression, with an additive effect when the highest dose was combined with ID. However, in MCF7 cells no additive effect was observed. VEGF mRNA up-regulation was reactive oxygen species (ROS)-dependent, involving radiation-induced mitochondrial ROS. Results on total VEGF mRNA hold true for the pro-angiogenic isoform VEGF165 mRNA, but the treatments did not modulate the anti-angiogenic isoform VEGF165b. Radiation-induced antioxidant response was differentially regulated upon ID in both cell lines. Thus, radiation response is modulated according to iodine status and cell type and can lead to additive effects on ROS and VEGF. As these are often involved in cancer initiation and progression, we believe that iodine status should be taken into account in radiation prevention policies.