Angiogenic Growth Research Articles

PI3K-C2β limits mTORC1 signaling and angiogenic growth.

Phosphoinositide 3-kinases (PI3Ks) phosphorylate intracellular inositol lipids to regulate signaling and intracellular vesicular trafficking. Mammals have eight PI3K isoforms, of which class I PI3Kα and class II PI3K-C2α are essential for vascular development. The class II PI3K-C2β is also abundant in endothelial cells. Using in vivo and in vitro approaches, we found that PI3K-C2β was a critical regulator of blood vessel growth by restricting endothelial mTORC1 signaling. Mice expressing a kinase-inactive form of PI3K-C2β displayed enlarged blood vessels without corresponding changes in endothelial cell proliferation or migration. Instead, inactivation of PI3K-C2β resulted in an increase in the size of endothelial cells, particularly in the sprouting zone of angiogenesis. Mechanistically, we showed that the aberrantly large size of PI3K-C2β mutant endothelial cells was caused by mTORC1 activation, which sustained growth in these cells. Consistently, pharmacological inhibition of mTORC1 with rapamycin normalized vascular morphogenesis in PI3K-C2β mutant mice. Together, these results identify PI3K-C2β as a crucial determinant of endothelial signaling and illustrate the importance of mTORC1 regulation during angiogenic growth.

Safety and efficacy review of aflibercept for the treatment of metastatic colorectal cancer

ABSTRACT Introduction Anti-angiogenic drugs are an efficacious class of therapy in the treatment of patients with metastatic colorectal cancer (mCRC). Aflibercept, a vascular endothelial growth factor (VEGF) trap which binds the angiogenic factors VEGF-A, VEGF-B, and placental growth factor (PIGF) is approved in combination with FOLFIRI chemotherapy following progression after an oxaliplatin-containing regimen. Areas covered This report provides a review of the practice-changing clinical studies which have established the use of anti-angiogenic therapy as second-line therapy in mCRC including aflibercept with FOLFIRI (5FU, leucovorin, irinotecan). This review also evaluates aflibercept with other chemotherapy regimens as well as efficacy and safety data from real-world studies. Expert opinion Aflibercept in combination with FOLFIRI chemotherapy is an established safe and efficacious regimen for the treatment of mCRC as second-line chemotherapy. Although several toxicities have been described, the majority are either low grade or manageable by drug cessation and supportive therapies. For optimal outcomes, patient selection and close observation of toxicities is essential.

NR4A2 expression is not altered in placentas from cases of growth restriction or preeclampsia, but is reduced in hypoxic cytotrophoblast

Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) transcripts are elevated in the circulation of individuals whose pregnancies are complicated by preterm fetal growth restriction (FGR). In this paper, we show that the cases with preeclampsia (PE) have increased circulating NR4A2 transcripts compared to those with normotensive FGR. We aimed to establish whether the dysfunctional placenta mirrors the increase in NR4A2 transcripts and further, to uncover the function of placental NR4A2. NR4A2 expression was detected in preterm and term placental tissue; expressed higher at term. NR4A2 mRNA expression and protein were not altered in placentas from preterm FGR or PE pregnancies. Hypoxia (1% O2 compared to 8% O2) significantly reduced cytotrophoblast NR4A2 mRNA expression, but not placental explant NR4A2 expression. Silencing cytotrophoblast NR4A2 expression under hypoxia (via short interfering (si)RNAs) did not alter angiogenic Placental Growth Factor, nor anti-angiogenic sFlt-1 mRNA expression or protein secretion, but increased expression of cellular antioxidant, oxidative stress, inflammatory, and growth genes. NR4A2 expression was also not altered in a model of tumour necrosis factor-α-induced endothelial dysfunction, or with pravastatin treatment. Further studies are required to identify the origin of the circulating transcripts in pathological pregnancies, and investigate the function of placental NR4A2.

The endothelium–bone axis in development, homeostasis and bone and joint disease

Blood vessels form a versatile transport network that is best known for its critical roles in processes such as tissue oxygenation, metabolism and immune surveillance. The vasculature also provides local, often organ-specific, molecular signals that control the behaviour of other cell types in their vicinity during development, homeostasis and regeneration, and also in disease processes. In the skeletal system, the local vasculature is actively involved in both bone formation and resorption. In addition, blood vessels participate in inflammatory processes and contribute to the pathogenesis of diseases that affect the joints, such as rheumatoid arthritis and osteoarthritis. This Review summarizes the current understanding of the architecture, angiogenic growth and functional properties of the bone vasculature. The effects of ageing and pathological conditions, including arthritis and osteoporosis, are also discussed.

Capturing membrane trafficking events during 3D angiogenic development in vitro.

Vesicular trafficking dictates protein localization, functional activity, and half-life, providing a critically important regulatory step in tissue development; however, there is little information detailing endothelial-specific trafficking signatures. This is due, in part, to limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3-dimensional (3D) in vitro sprouting model to image endothelial membrane trafficking events. Endothelial cells were challenged to grow sprouts in a fibrin bead assay. Thereafter, spouts were transfected with fluorescent proteins and stained for various cell markers. Sprouts were then imaged for trafficking events using live and fixed-cell microscopy. Our results demonstrate that fibrin bead sprouts have a strong apicobasal polarity marked by apical localization of proteins moesin and podocalyxin. Comparison of trafficking mediators Rab27a and Rab35 between 3D sprouts and 2D culture showed that vesicular carriers can be imaged at high resolution, exhibiting proper membrane polarity solely in 3D sprouts. Lastly, we imaged exocytic events of von Willebrand Factor and demonstrated a distinct imaging advantage for monitoring secretion events in 3D sprouts as compared with 2D culture. Our results establish that the fibrin bead sprouting assay is well-suited for imaging of trafficking events during angiogenic growth.

Low-dose metronomic topotecan and pazopanib in children with recurrent or refractory solid tumors: A C17 Canadian phase I trial (TOPAZ).

10020 Background: Low-dose metronomic topotecan (mTP) represents a novel approach to chemotherapy delivery which, in preclinical models, may work synergistically with pazopanib (PZ) in targeting angiogenesis. This study was designed to determine the recommended phase 2 dose (RP2D) of mTP/PZ in pediatric patients with solid tumors, while describing the safety and toxicity of this regimen. Methods: A phase I dose-escalation, pharmacokinetic (PK) and pharmacodynamic (PD) study of mTP/PZ was conducted at ten sites across Canada, enrolling pediatric patients aged 2-21 years with relapsed/refractory solid tumors. Patients were treated with oral mTP and PZ suspension daily without interruption in 28-day cycles, with dose escalation in accordance with the rolling-six design. Five dose levels (0.12/125, 0.16/125, 0.22/125, 0.22/160, and 0.3/160 mg/m2/day of mTP/PZ) were evaluated. PK studies were performed on day 1 and at steady state, and PD studies included circulating angiogenic factors VEGFR1, VEGFR2, VEGF, endoglin and placental growth factor. Results: Thirty patients (pts) were enrolled, of whom 26 were evaluable for dose-limiting toxicity (DLT), with median age 12 years (3-20). The most common diagnoses included osteosarcoma (8), neuroblastoma (NB, 7), Ewing sarcoma/PNET (4), and rhabdomyosarcoma (4). The most common grade 3/4 adverse events (AEs) related to protocol therapy were neutropenia (18%), thrombocytopenia (11%), lymphocytopenia (11%), AST elevation (11%), and lipase elevation (11%). Only 2 cycle-1 DLTs were observed on study, both at the 0.3/160 mg/m2 mTP/PZ dose level (2/5 pts) comprising persistent grade 3 thrombocytopenia and grade 3 ALT elevation. No AEs experienced beyond cycle-1 required treatment discontinuation. Best response was stable disease in 10/25 pts (40%) for a median duration of 6.4 months (1.7-45.1). One patient with refractory NB achieved stable disease for 45 months and continued on mTP/PZ via compassionate access after study closure. PK and PD results are pending at this time. Conclusions: The combination of oral mTP and PZ is safe and tolerable in pediatric patients with solid tumors, with a RP2D of mTP 0.22 mg/m2/day and PZ suspension 160 mg/m2/day. Ten patients achieved stable disease for a median of 6 months. The lack of objective responses suggests that this combination is likely of limited benefit for relapsed disease, but may play a role as maintenance therapy. Clinical trial information: NCT02303028.

Micro-RNA miR-542-3p suppresses decidualization by targeting ILK pathways in human endometrial stromal cells

Decidualization of human endometrial stromal cells (HESCs) is a vital step for successful pregnancy. However, the process by which micro-RNAs (miRNAs) regulate decidualization remains elusive. Our current study was designed to identify the mechanism of miRNA miR-542-3p and its potential targets in regulating decidualization. The results showed that miR-542-3p was down-regulated in HESCs. Luciferase assay confirmed that integrin-linked kinase (ILK) is a direct target of miR-542-3p. Overexpression of miR-542-3p resulted in decreased ILK and downstream transforming growth factor β1 (TGF-β1) and SMAD family member 2 (SMAD2) expression. Additional expression of ILK attenuates the miR542-3p-induced down-regulation of TGF-β1 and SMAD2, changes properties such as suppression of proliferation and invasion, and induction of apoptosis, thereby affecting the differentiation of HESCs. Moreover, miR-542-3p overexpression caused down-regulation of the angiogenic factors vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9), and the supernatant of HESCs overexpressing miR-542-3p inhibited the formation of tubular structures in human umbilical vein endothelial cells (HUVECs), suggesting that miR-542-3p inhibits angiogenesis of HUVECs. Furthermore, in our mouse model, following injection of miR-542-3p mimic into the endometrium of mice at pregnancy day 8 (D8), we found decreased miR-542-3p expression and loss of embryo implantation sites. In conclusion, miR-542-3p can affect the process of endometrial decidualization by down-regulating ILK. The present study adds further understanding of the process and regulation of decidualization.

Pantoprazole impairs fracture healing in aged mice

Proton pump inhibitors (PPIs) belong to the most common medication in geriatric medicine. They are known to reduce osteoclast activity and to delay fracture healing in young adult mice. Because differentiation and proliferation in fracture healing as well as pharmacologic actions of drugs markedly differ in the elderly compared to the young, we herein studied the effect of the PPI pantoprazole on bone healing in aged mice using a murine fracture model. Bone healing was analyzed by biomechanical, histomorphometric, radiological and protein biochemical analyses. The biomechanical analysis revealed a significantly reduced bending stiffness in pantoprazole-treated animals when compared to controls. This was associated with a decreased amount of bone tissue within the callus, a reduced trabecular thickness and a higher amount of fibrous tissue. Furthermore, the number of osteoclasts in pantoprazole-treated animals was significantly increased at 2 weeks and decreased at 5 weeks after fracture, indicating an acceleration of bone turnover. Western blot analysis showed a lower expression of the bone morphogenetic protein-4 (BMP-4), whereas the expression of the pro-angiogenic parameters was higher when compared to controls. Thus, pantoprazole impairs fracture healing in aged mice by affecting angiogenic and osteogenic growth factor expression, osteoclast activity and bone formation.

Unfractionated heparin displaces sFlt-1 from the placental extracellular matrix

Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is an anti-angiogenic protein which is secreted by numerous cell types and acts as a decoy receptor for the angiogenic protein vascular endothelial growth factor (VEGF). Despite its physiologic importance in maintaining angiogenic balance, excess sFlt-1 levels are associated with the pathogenesis of many diseases, especially those with angiogenic imbalance, endothelial dysfunction, and hypertension. Although sFlt-1 is a soluble protein, it contains a binding site for the extracellular matrix component heparan sulfate. This allows cells to retain and localize sFlt-1 in order to prevent excessive VEGF signaling. During pregnancy, placental syncytiotrophoblasts develop a large extracellular matrix which contains significant amounts of heparan sulfate. Consequently, the placenta becomes a potential storage site for large amounts of sFlt-1 bound to extracellular heparan sulfate. Additionally, it should be noted that sFlt-1 can bind to the anticoagulant unfractionated heparin due to its molecular mimicry to heparan sulfate. However, it remains unknown whether unfractionated heparin can compete with heparan sulfate for binding of localized sFlt-1. In this study, we hypothesized that administration of unfractionated heparin would displace and solubilize placental extracellular matrix(ECM)-bound sFlt-1. If unfractionated heparin can displace this large reservoir of sFlt-1 in the placenta and mobilized it into the maternal circulation, we should be able to observe its effects on maternal angiogenic balance and blood pressure. To test this hypothesis, we utilized in vitro, ex vivo, and in vivo methods. Using the BeWo placental trophoblast cell line, we observed increased sFlt-1 in the media of cells treated with unfractionated heparin compared to controls. The increase in media sFlt-1 was found in conjunction with decreased localized cellular Flt (sFlt-1 and Flt-1) as measured by total cell fluorescence. Similar results were observed using ex vivo placental villous explants treated with unfractionated heparin. Real-time quantitative PCR of the explants showed no change in sFlt-1 or heparanase-1 mRNA expression, eliminating increased production and enzymatic cleavage of heparan sulfate as causes for sFlt-1 media increase. Timed-pregnant rats given a continuous infusion of unfractionated heparin exhibited an increased mean arterial pressure as well as decreased bioavailable VEGF compared to vehicle-treated animals. These data demonstrate that chronic unfractionated heparin treatment is able to displace matrix-bound sFlt-1 into the maternal circulation to such a degree that mean arterial pressure is significantly affected. Here we have shown that the placental ECM is a storage site for large quantities of sFlt-1, and that it should be carefully considered in future studies concerning angiogenic balance in pregnancy.

Immunohistochemical and gene expression analysis of autologous platelet rich fibrin for distal limb wound defects healing in donkeys (Equus asinus).

Objective: To evaluate the effect of platelet-rich fibrin (PRF) in the promotion of distal limb wound defects healing in donkeys. Design: A randomized experimental design Animals: Twelve clinically healthy male donkeys, weighing, 130–230 kg and aged 4 –5 years were allocated into three groups(4 animals/each) and undergo a 6cm2 (2cm X 3cm) 2 wound defects on the dorsolateral surface of right metacarpal and metatarsal regions for each donkey. Control (group A): the wound defects were left for spontaneous healing. In groups B and C, the wound defects were treated with either one application of PRF (B) or with three consecutive applications of PRF (a week interval) (C). Wound defects healing were evaluated clinically, histologically and immunohistochemically, in addition to gene expression patterns of angiogenic and myofibroblastic genes vascular endothelial growth factor (VEGF-A), collagen type 3 α1 (COL3α1), and fibroblast growth factor 7 (FGF-7) and tissue growth factor β1 (TGFβ1) were performed. Results: The healing percentage of single and three PRF applications was significantly higher (P <0.05) (84.6%, and 93.7% respectively) than in control one (66.7%). The number of days needed for complete wound healing was considerably shorter in repeated PRF treated wound defects (63.2±2.8) compared with single PRF and untreated wound defects (71.6±3 and 86.3±3, respectively). Semi-quantitative evaluation of histological sections at 15 and 45 days post-operative showed a significant difference (P<0.05) in epithelization, PMNL, fibroblasts, tissue macrophages, neo-angiogenesis and new collagen scores in both PRF groups compared to control one. Qualitative analysis of immunohistochemical views of the wound defects showed a significant immunostaining difference against EGFR, VEGF, and TGFβ stain between both PRF treated groups and control one. Immunohistochemical analysis of cells stained for epidermal growth factor receptor (EGFR), VEGF, and TGFβ at 15 and 45 days after interference was higher in both PRF treated groups compared to control one, but three PRF application showed the highest rates. The relative expression of FGF-7, TGFβ1, VEGF-A, and COL3α1 genes was higher in both PRF groups compared to control one, but the triple PRF group revealed the highest expression. Conclusion and clinical relevance: Application of PRF could improve the healing of distal limb wound defects in donkeys.

DETERMINING THE EFFECTS OF PRO‐ANGIOGENIC ELP‐VEGF THERAPY ON TUMOR GROWTH AND PROGRESSION

The members of the Vascular Endothelial Growth Factor (VEGF) family are endogenous growth factors with a central role in angiogenesis and endothelial cell health. We recently showed the therapeutic potential of renal VEGF delivery using a fusion protein of VEGF bound to the carrier protein Elastin‐like Polypeptide (ELP‐VEGF) designed in our laboratory, to promote angiogenesis in chronic kidney diseases. Indeed, we showed that ELP‐VEGF therapy improves renal vascular density, decreased renal fibrosis, and improved glomerular filtration rate (GFR) in swine models of chronic renovascular disease and chronic kidney disease at therapeutic doses of 0.1 – 1.0 mg/kg.A potential limitation of the use of ELP‐VEGF as a therapeutic is the possibility of promoting uninhibited off‐target angiogenic growth which may be undesirable in certain disease processes such as cancer. In order to determine whether ELP‐VEGF therapy may be harmful for a patient with an undiagnosed tumor, the effects of a single administration of escalating doses (0.1 m/kg, 1 mg/kg, 10 mg/kg, 10 times the highest therapeutic dose) of ELP‐VEGF on tumor size and vascular density were determined in a mouse xenograft model of breast cancer.Human triple‐negative breast cancer cells (MDA‐MB‐231) that stably express a luciferase reporter were implanted into the mammary fat pad of athymic nude mice. Tumors were monitored thrice weekly for 7 weeks with external caliper measurements (blind) and bioluminescence imaging to assess tumor growth. Body weights of the mice were also recorded 3 times per week. At the end of the 7‐week period, mice were fully perfused with heparinized saline and microfil. Tumors were removed and scanned using micro‐computed tomography (u‐CT), and 3D reconstructions of the tumor vascular network were used to blindly quantify tumor vascular density.Caliper measurements and bioluminescence imaging showed no significant difference in tumor size or growth rate amongst the groups. There were also no significance differences in mouse body weights throughout the study or final weights of the tumors (0.1–10mg/kg 1.12±0.39 g, 1.233±0.46 g, 1.247±0.52 g, respectively; p=0.8695). However, analysis of the renal vasculature by u‐CT revealed a significant increase in total microvascular density after a single injection of ELP‐VEGF at 1mg/kg (78.88 ± 34.34 vessels/mm2; p = 0.0489 vs. controls) and 10mg/kg (97.17 ± 48.65 vessels/mm2; p = 0.0012 vs. controls). In conclusion, we determined that one‐time injection of ELP‐VEGF had no toxic effects on body weights and did not influence tumor growth rate, but it did significantly and dose‐dependently increase tumor vasculature density in a breast cancer xenograft model.Support or Funding InformationR01HL095638 and IPA34170267Figure 2. Analysis and results from ELP‐VEGF‐A administration to a rodent model of breast cancer. Results include body weight (a), tumor caliper measurements (b) tumor bioluminescence (c), final tumor mass(d).Figure 1Figure 3. Vasculature of each tumor was visualized using uCT scanner after microfi fixation. Blood vessels were divided and quantified by diameter between different ranges (a). Kidneys from these mice were also perfused with microfil, scanned through uCT scanner, and quantified by vessel diameter range (b).Figure 2

No association between early antiretroviral therapy during pregnancy and plasma levels of angiogenic factors: a cohort study

BackgroundEarly antiretroviral therapy (ART) during pregnancy has dramatically reduced the risk of perinatal HIV transmission. However, studies have shown an association between premature delivery and the use of ART during pregnancy (particularly protease inhibitor (PI)-based therapies), which could be explained by placental dysfunction. The objective of this study was to evaluate the association of ART (class, duration of exposure and time of initiation) with placental function by using angiogenic factors placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) as biomarkers.MethodsClinical and biological data from 159 pregnant women living with HIV were analyzed. Levels of each biomarker were measured in the first and second trimester of pregnancy. After logarithmic transformation, we compared these using generalized estimating equations according to (a) the type of ART; (b) the duration of exposure to ART; and (c) the time of initiation of ART.ResultsAfter adjusting for variables such as ethnicity, maternal age, gestational age, body mass index, parity, smoking status, and sex of the fetus, we found no significant association between the class of ART (PI-based or not) and serum concentrations of PlGF or sFlt-1. Furthermore, no significant association was found between biomarker levels and the duration of ART exposure or the timing of ART initiation (pre- or post-conception).ConclusionsThis study suggests that first and second trimester angiogenic factor levels are not significantly associated with ART, regardless of the duration or type (with or without PI). These observations seem reassuring when considering the use of ART during early pregnancy.

Low-Density Lipoprotein Receptor-Related Protein 6 Is Essential for Trophoblast Survival and Invasion.

Remodeling of maternal spiral artery after the implantation of embryo relies on tightly regulated trophoblast functions and is pivotal to ensure the success of pregnancy. Low-density lipoprotein receptor-related protein 6 (LRP6) is implicated in angiogenesis and several vascular diseases, but its function in trophoblast regulation is still not fully understood. We aimed to investigate the involvement of LRP6 in trophoblast functions during maternal spiral artery remodeling. HTR-8/SVneo cells, a trophoblast cell line, were employed to examine the impact of LRP6 knockdown on proliferation, apoptosis, and migration, as well as invasion and tube formation. Expression of matrix metalloproteinases (MMPs), angiogenic factors placental growth factor (PlGF), and vascular endothelial growth factor (VEGF), as well as activation of canonical Wnt/β-catenin signaling pathway, which was involved in artery remodeling process, was also analyzed. Lipoprotein receptor-related protein 6 knockdown suppressed proliferation, migration, invasion, and tube formation, as well as induced apoptosis in HTR-8/SVneo cells. In addition, LRP6 knockdown also significantly decreased expression of MMPs, PlGF, and VEGF and activation of Wnt/β-catenin signaling pathway. Lipoprotein receptor-related protein 6 might positively regulate the remodeling of the maternal artery through both endovascular formation and trophoblast invasion.

190. Combination sulfasalazine and metformin are additive in reducing placental secretion of antiangiogenic factor sFlt

Introduction Preeclampsia is associated with increased placental secretion of antiangiogenic factors sFlt-1 and sENG and a reduction in angiogenic proteins placental growth factor secretion (PlGF) and vascular endothelial growth factor (VEGF). We have identified two medications, safe in pregnancy, that mitigate key aspects of preeclampsia in vitro. We have demonstrated metformin and sulfasalazine independently reduce placental sFlt-1 secretion and vessel dysfunction. In this study we investigate the possibility of combining these medications to reduce the dose required to mitigate key aspects of preeclampsia; sFlt-1 and sENG secretion and PlGF and VEGF expression, using primary human tissues. Methods Metformin and sulfasalazine, at low doses individually and in combination, were administered to primary trophoblasts and placental explants and sFlt-1 secretion assessed. At the mRNA level there are two sFlt-1 splice variants; the primate and placental specific isoform sFlt-1 e15a and the predominately vascular isoform sFlt-1 i13. Both were measured following combination treatment. In addition the expression of angiogenic factors PlGF and VEGFα were assessed. Results Combining low-dose metformin and sulfasalazine additively decreased sFlt-1 secretion from primary trophoblast compared to either drug alone. Similarly, an additive decrease in sFlt-1 e15a and i13 was identified following combination treatment. In placental explants, combination treatment also additively decreased sFlt-1 secretion. Low dose sulfasalazine potently upregulated PlGF expression in primary trophoblasts and this was maintained with the addition of metformin. Metformin and sulfasalazine individually increased VEGFα, and this effect was additive when the two drugs were combined. Conclusion Combination metformin and sulfasalazine additively reduced sFlt-1 secretion and the expression of its splice variants, sFlt-1 e15a and i13. Furthermore, in combination metformin and sulfasalazine upregulate angiogenic factor VEGFα. Therefore we conclude that they might be promising combination therapeutics that could reduce the burden of this devastating disease.

Upregulation of angiostatic chemokines IP-10/CXCL10 and I-TAC/CXCL11 in human obesity and their implication for adipose tissue angiogenesis.

Impaired angiogenesis is linked to adipose tissue (AT) dysfunction, inflammation, and insulin resistance in human obesity. Chemokine (C-X-C motif) receptor. (CXCR3) ligands are important regulators of angiogenesis in different disease contexts such as cancer; however, their role in human morbid obesity is unknown. We investigated the role of the CXCR3 axis in AT angiogenesis in morbidly obese patients. The study group comprised 50 morbidly obese patients (mean age 44 ± 1 years, body mass index 44 ± 1 kg/m2) who had undergone laparoscopic Roux-Y-gastric bypass surgery, and 25 age-matched non-obese control subjects. We measured the circulating levels of the CXCR3 ligands monokine induced by interferon-γ (MIG/CXCL9), interferon-γ inducible protein 10 (IP-10/CXCL10), and interferon-γ-inducible T-cell alpha chemoattractant (I-TAC/CXCL11) in all studied subjects. Additionally, the expression of CXCR3 ligands was analyzed in paired biopsies of subcutaneous and visceral AT obtained during the laparoscopic procedure in morbidly obese patients. Additionally, we explored the functional role of CXCR3 ligands on angiogenesis in AT from morbidly obese patients using an ex vivo assay. Plasma levels of CXCL10 and CXCL11 were significantly higher in morbidly obese patients than in controls (p < 0.01). In ex vivo assays, angiogenic growth was markedly lower in visceral AT than in subcutaneous AT (p < 0.05), which was related to significant tissue upregulation of CXCL10, CXCL11 and CXCR3 (p < 0.05). CXCL10 or CXCL11 inhibited AT angiogenesis (p < 0.05), and blockade of CXCR3 function significantly increased capillary sprouting in visceral fat deposits (p < 0.05). Western blot analysis showed that the p38 mitogen-activated protein kinase signaling pathway was implicated in the angiostatic effects of CXCR3 in AT. CXCL10 and CXCL11 may play. deleterious role in obesity as potential inhibitors of AT angiogenesis. Accordingly, pharmacological blockade of CXCR3 could represent. therapy to prevent AT dysfunction in obesity.

Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart.

Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

Abstract 5023: Disruption of Monocarboxylate transporter-4 Basigin interaction as an effective strategy to inhibit hypoxic response, tumor growth and vascularization, and stem cell phenotype in human glioblastoma in vitro and in vivo

Abstract Monocarboxylate transporters, constitute a family (SLC16) of proton-linked plasma membrane transporters that carry molecules containing a single carboxylate group across biological membranes. Basigin (CD147), is involved in many physiological functions during early stages of development and in cancer. Basigin has been shown to be required for functional plasma membrane expression of Monocarboxylate transporter-1 and Monocarboxylate transporter-4. Using a cell-based screening assay, we identified acriflavine, a small molecule that inhibits the binding between Basigin and Monocarboxylate transporters in vitro and in vivo. Surface Plasmon Resonance analysis confirmed direct binding of acriflavine to Basigin’s immunoglobulin extracellular domain with a low binding constant (kD) of 0.16µM. Acriflavine inhibits normoxic growth of glioma stem cells in vitro and this activity is augmented by hypoxia or by expression of oxygen-stable mutant forms of HIF-1α or HIF-2α. Treatment of mice bearing established glioma stem cell-derived xenografts resulted in inhibition of tumor growth. Acriflavine treatment inhibited intratumoral expression of the angiogenic cytokine vascular endothelial growth factor and tumor vascularization. Our work shows that disruption of monocarboxylate transporter binding to Basigin is an effective approach to target glioma stem cells. Citation Format: Raffaella Spina, Dillon M. Voss, Kah Suan Lim, Constance J. Jeffery, Eli E. Bar. Disruption of Monocarboxylate transporter-4 Basigin interaction as an effective strategy to inhibit hypoxic response, tumor growth and vascularization, and stem cell phenotype in human glioblastoma in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2017-5023

P 59 The sFlt-1/PlGF-ratio an remaining pregnancy duration in RN patients with suspected preeclampsia &gt;34 weeks of gestation

Abstract Introduction The ratio of the antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and the angiogenic placental growth factor (PlGF) has proven to be a accurate marker for the prediction and diagnosis of pre-eclampsia. The established cut-offs of the ratio provide precise prognoses for the occurrence of especially early-onset pre-eclampsia. However its predictive and diagnostic value is significantly lower in the late-onset disease. Objective To evaluate, whether a high sFlt-1/PlGF-ratio correlates with higher rates of adverse maternal and perinatal outcomes and a shorter remaining time to delivery in patients with late-onset pre-eclampsia. Patients and methods We retrospectively assessed 176 women with suspected pre-eclampsia, bearing a singleton pregnancy, whose first measurement of the sFlt-1/PlGF-ratio took place after 336/7 weeks of gestation. The plasma levels of the ratio were examined for an association with time to delivery as well as adverse maternal and perinatal outcomes until discharge from the hospital. Results We were able to show correlations between the level of sFlt-1/PlGF, adverse maternal and perinatal outcomes and time of delivery. The remaining time to delivery became significantly shorter with a rising sFlt-1/PlGF-ratio ( r = 0,477; p p Conclusion The sFlt-1/PlGF ratio was inversely correlated with the remaining time to delivery and positively correlated with the incidence of maternal and perinatal adverse outcomes. Validating existing cut-offs predicting the remaining time to delivery and finding a specific cut-off on the basis of these data will be the aim of further analyses.

The prediction, diagnosis and management of complications in monochorionic twin pregnancies: the OMMIT (Optimal Management of Monochorionic Twins) study

BackgroundMonochorionic twin pregnancies are at increased risk of complications due to sharing a single placenta and potentially developing unbalanced vascular anastomoses. Complications include twin-twin transfusion syndrome (TTTS) which affects 10–15% monochorionic twins, and if untreated has a 70–90% perinatal loss rate. We are currently unable to predict which twins will develop complications or to what severity. We have previously shown differences in angiogenic and placental growth factors in maternal blood in pregnancies complicated by TTTS compared to twin pregnancies not complicated by TTTS but matched for gestation. There is also evidence to suggest that abnormal ultrasound measurements recorded in the first trimester (nuchal translucency and crown-rump length) may be associated with severe TTTS later in pregnancy, however the detection rate is only reported as 52%. We hypothesize that if these changes precede the development of the clinical syndrome, this may increase the sensitivity and specificity of detecting adverse pregnancy outcomes.MethodsThis cohort study has retrospective and prospective elements. In the retrospective cohort we will measure factors (decided based on preliminary work and a systematic review and meta-analysis) in stored maternal blood samples taken in the first-trimester, extract first-trimester ultrasound measurements and match these to pregnancy outcome. The prospective cohort will be divided into a “screening” cohort and “complicated” cohort. The screening cohort will undergo serial maternal blood sampling at 12, 16 and 20 weeks; we will extract ultrasound measurements and match to pregnancy outcome. The complicated cohort will comprise of women referred to the Fetal Medicine Centre with complications of monochorionicity. If the decision is taken to undergo fetoscopic laser ablation we will take maternal blood samples and amniotic fluid samples pre- and post-laser treatment. The same factors will be measured in the prospective cohort as informed by the retrospective study.DiscussionThis study aims to increase knowledge surrounding the pathology of complications in monochorionic twins, to aid future diagnosis and management.Trial registrationISRCTN 13114861 (retrospectively registered)

Bevacizumab for Patients with Recurrent Gliomas Presenting with a Gliomatosis Cerebri Growth Pattern.

Bevacizumab has been shown to improve progression-free survival and neurologic function, but failed to improve overall survival in newly diagnosed glioblastoma and at first recurrence. Nonetheless, bevacizumab is widely used in patients with recurrent glioma. However, its use in patients with gliomas showing a gliomatosis cerebri growth pattern is contentious. Due to the marked diffuse and infiltrative growth with less angiogenic tumor growth, it may appear questionable whether bevacizumab can have a therapeutic effect in those patients. However, the development of nodular, necrotic, and/or contrast-enhancing lesions in patients with a gliomatosis cerebri growth pattern is not uncommon and may indicate focal neo-angiogenesis. Therefore, control of growth of these lesions as well as control of edema and reduction of steroid use may be regarded as rationales for the use of bevacizumab in these patients. In this retrospective patient series, we report on 17 patients with primary brain tumors displaying a gliomatosis cerebri growth pattern (including seven glioblastomas, two anaplastic astrocytomas, one anaplastic oligodendroglioma, and seven diffuse astrocytomas). Patients have been treated with bevacizumab alone or in combination with lomustine or irinotecan. Seventeen matched patients treated with bevacizumab for gliomas with a classical growth pattern served as a control cohort. Response rate, progression-free survival, and overall survival were similar in both groups. Based on these results, anti-angiogenic therapy with bevacizumab should also be considered in patients suffering from gliomas with a mainly infiltrative phenotype.