Increased VEGF Secretion Research Articles

Development of a novel high-throughput culture system for hypoxic 3D hydrogel cell culture.

Animal models lack physiologic relevance to the human system which results in low clinical translation of results derived from animal testing. Besides spheroids or organoids, hydrogel-based 3D in vitro models are used to mimic the in vivo situation increasing the relevance while reducing animal testing. However, to establish hydrogel-based 3D models in applications such as drug development or personalized medicine, high-throughput culture systems are required. Furthermore, the integration of oxygen-reduced (hypoxic) conditions has become increasingly important to establish more physiologic culture models. Therefore, we developed a platform technology for the high-throughput generation of miniaturized hydrogels for 3D cell culture. The Oli-Up system is based on the shape of a well-plate and allows for the parallel culture of 48 hydrogel samples, each with a volume of 15µl. As a proof-of-concept, we established a 3D culture of gelatin-methacryloyl (GelMA)-encapsulated mesenchymal stem/stromal cells (MSCs). We used a hypoxia reporter cell line to establish a defined oxygen-reduced environment to precisely trigger cellular responses characteristicof hypoxia in MSCs. In detail, the expression of hypoxia response element (HRE) increased dependent on the oxygen concentration and cell density. Furthermore, MSCs displayed an altered glucose metabolism and increased VEGF secretion upon oxygen-reduction. In conclusion, the Oli-Up system is a platform technology for the high-throughput culture of hydrogel-based 3D models in a defined oxygen environment. As it is amenable for automation, it holds the potential for high-throughput screening applications such as drug development and testing in more physiologic 3D in vitro tissue models.

VEGF Secretion Drives Bone Formation in Classical MAP2K1+ Melorheostosis.

Patients with classical melorheostosis exhibit exuberant bone overgrowth in the appendicular skeleton, resulting in pain and deformity with no known treatment. Most patients have somatic, mosaic mutations in MAP2K1 (encoding the MEK1 protein) in osteoblasts and overlying skin. As with most rare bone diseases, lack of affected tissue has limited the opportunity to understand how the mutation results in excess bone formation. The aim of this study was to create a cellular model to study melorheostosis. We obtained patient skin cells bearing the MAP2K1 mutation (affected cells), and along with isogenic control normal fibroblasts reprogrammed them using the Sendai virus method into induced pluripotent stem cells (iPSCs). Pluripotency was validated by marker staining and embryoid body formation. iPSCs were then differentiated to mesenchymal stem cells (iMSCs) and validated by flow cytometry. We confirmed retention of the MAP2K1 mutation in iMSCs with polymerase chain reaction (PCR) and confirmed elevated MEK1 activity by immunofluorescence staining. Mutation-bearing iMSCs showed significantly elevated vascular endothelial growth factor (VEGF) secretion, proliferation and collagen I and IV secretion. iMSCs were then differentiated into osteoblasts, which showed increased mineralization at 21 days and increased VEGF secretion at 14 and 21 days of differentiation. Administration of VEGF to unaffected iMSCs during osteogenic differentiation was sufficient to increase mineralization. Blockade of VEGF by bevacizumab reduced mineralization in iMSC-derived affected osteoblasts and in affected primary patient-derived osteoblasts. These data indicate that patient-derived induced pluripotent stem cells recreate the elevated MEK1 activity, increased mineralization, and increased proliferation seen in melorheostosis patients. The increased bone formation is driven, in part, by abundant VEGF secretion. Modifying the activity of VEGF (a known stimulator of osteoblastogenesis) represents a promising treatment pathway to explore. iPSCs may have wide applications to other rare bone diseases. © 2023 American Society for Bone and Mineral Research (ASBMR).

Hyperglycemia-induced miR182-5p drives glycolytic and angiogenic response in Proliferative Diabetic Retinopathy and RPE cells via depleting FoxO1

PurposeDiabetic Retinopathy (DR) is associated with metabolic dysfunction in cells such as retinal pigmented epithelium (RPE). Small molecular weight microRNAs can simultaneously regulate multiple gene products thus having pivotal roles in disease pathogenesis. Since miR182-5p is involved in regulating glycolysis and angiogenesis, two pathologic processes of DR, we investigated its status in DR eyes and in high glucose model in vitro. Methodology: Total RNA was extracted from vitreous humor of PDR (n = 48) and macular hole (n = 22) subjects followed by quantification of miR182-5p and its target genes. ARPE-19 cells, cultured in DMEM under differential glucose conditions (5 mM and 25 mM) were used for metabolic and biochemical assays. Cells were transfected with miRNA182 mimic or antagomir to evaluate the gain and loss of function effects. ResultsPDR patient eyes had high levels of miR182-5p levels (p < 0.05). RPE cells under high glucose stress elevated miR182-5p expression with altered glycolytic pathway drivers such as HK2, PFKP and PKM2 over extended durations. Additionally, RPE cells under high glucose conditions exhibited reduced FoxO1 and enhanced Akt activation. RPE cells transfected with miR182-5p mimic phenocopied the enhanced basal and compensatory glycolytic rates observed under high glucose conditions with increased VEGF secretion. Conversely, inhibiting miR182-5p reduced Akt activation, glycolytic pathway proteins, and VEGF while stabilizing FoxO1. ConclusionGlycolysis-associated proteins downstream of the FoxO1-Akt axis were regulated by miR182-5p. Further, miR182-5p increased expression of VEGFR2 and VEGF levels, likely via inhibition of ZNF24. Thus, the FoxO1-Akt-glycolysis/VEGF pathway driving metabolic dysfunction with concurrent angiogenic signaling in PDR may be potentially targeted for treatment via miR182-5p modulation.

The Involvement of Unfolded Protein Response in the Mechanism of Nitrogen Mustard-Induced Ocular Toxicity.

Nitrogen mustard (NM) is a known surrogate of sulfur mustard, a chemical-warfare agent that causes a wide range of ocular symptoms, from a permanent reduction in visual acuity to blindness upon exposure. Although it has been proposed that the two blistering agents have a similar mechanism of toxicity, the mode of NM-induced cell death in ocular tissue has not been fully explored. Therefore, we hypothesized that direct ocular exposure to NM in mice leads to retinal tissue injury through chronic activation of the unfolded protein response (UPR) PERK arm in corneal cells and VEGF secretion, eventually causing cell death. We topically applied NM directly to mice to analyze ocular and retinal tissues at 2 weeks postexposure. A dramatic decline in retinal function, measured by scotopic and photopic electroretinogram responses, was detected in the mice. This decline was associated with enhanced TUNEL staining in both corneal and retinal tissues. In addition, exposure of corneal cells to NM revealed 228 differentially and exclusively expressed proteins primarily associated with the UPR, ferroptosis, and necroptosis. Moreover, these cells exhibited activation of the UPR PERK arm and an increase in VEGF secretion. Enhancement of VEGF staining was later observed in the corneas of the exposed mice. Therefore, our data indicated that the mechanism of NM-induced ocular toxicity should be carefully examined and that future research should identify a signaling molecule transmitted via a prodeath pathway from the cornea to the retina. SIGNIFICANCE STATEMENT: This study demonstrated that NM topical exposure in mice results in dramatic decline in retinal function associated with enhanced TUNEL staining in both corneal and retinal tissues. We also found that the NM treatment of corneal cells resulted in 228 differentially and exclusively expressed proteins primarily associated with ferroptosis. Moreover, these cells manifest the UPR PERK activation and an increase in VEGF secretion. The latter was also found in the corneas of the cexposed mice.

P-445 Recovery of metabolic activity and VEGF secretion after thawing of cryopreserved, slow frozen ovarian tissue

Abstract Study question Is slow-frozen ovarian cortical tissue limited in its metabolical activity after thawing, especially with respect to VEGF secretion? Summary answer Slow-frozen ovarian cortical tissue showed decreased metabolic activity and VEGF directly after thawing, but recovered in the course of 48h in in vitro culture. What is known already Cryopreservation of ovarian cortical tissue has become an important method in fertility preservation. Freezing and thawing processes had been optimized to preserve tissue viability. However, the major obstacle of retransplantation is the rapid angiogenesis to accomplish sufficient blood supply. As freezing and thawing entails constraints in cellular metabolic activity, we aimed to assess the metabolic activity and the secretion of the angiogenic factor VEGF of frozen-thawed ovarian cortical tissue over time. Study design, size, duration Ex vivo gained ovarian tissue was cultured over 48h. Thereby, non-frozen fresh tissue was compared to slow-frozen-thawed ovarian tissue. Participants/materials, setting, methods Provided a signed consent, biopsies of ovarian tissue from patients undergoing fertility preservation were cultured without freezing (n = 13) or after slow-freezing and thawing (n = 12). VEGF secretion during 48h was measured by ELISA. Applying CellTiterBlue assay, metabolic activity was assessed in biopsies without freezing or after thawing from the same patients (n = 4-7) after 0, 24 and 48h. Similarly, VEGF secretion was measured after 0, 24 and 48h as well (n = 7) to capture temporarily changes. Main results and the role of chance Although VEGF secretion varies among patients and biopsies, there was a significant lower VEGF content in supernatant from frozen-thawed tissue compared to fresh ovarian cortical tissue after 48h of culture (p &amp;lt; 0.01). Directly after thawing and 24h thereafter, metabolic activity was significantly reduced (p &amp;lt; 0.05) compared to fresh ovarian cortex. In the course of 48h, the metabolic activity recovered (p &amp;lt; 0.01). Similarly, VEGF secretion increased dramatically during 24h after thawing (p &amp;lt; 0.05). Limitations, reasons for caution This in vitro study might not resemble the metabolic and secretory recovery after retransplantation. Wider implications of the findings We showed that ovarian tissue recovers metabolically over 48h. This recovery included an increase in VEGF secretion. As latter induces angiogenesis a culture step after thawing might support VEGF secretion and angiogenesis. However, the recovery period could also be useful as a temporal bridge until sufficient supplies are available. Trial registration number not applicable

TKI-Resistant Renal Cancer Secretes Low-Level Exosomal miR-549a to Induce Vascular Permeability and Angiogenesis to Promote Tumor Metastasis.

Tyrosine kinase inhibitors (TKI)-resistant renal cancer is highly susceptible to metastasis, and enhanced vascular permeability promotes the process of metastasis. To evaluate the effect of cancer-derived exosomes on vascular endothelial cells and clarify the mechanism of metastasis in TKI-resistant renal cancer, we studied the crosstalk between clear cell renal cell carcinoma (ccRCC) cells and human umbilical vein endothelial cells (HUVECs). Exosomes from ccRCC cells enhanced the expression of vascular permeability-related proteins. Compared with sensitive strains, exosomes from resistant strains significantly enhanced vascular endothelial permeability, induced tumor angiogenesis and enhanced tumor lung metastasis in nude mice. The expression of miR-549a is lower in TKI-resistant cells and exosomes, which enhanced the expression of HIF1α in endothelial cells. In addition, TKI-resistant RCC cells reduced nuclear output of pre-miR-549a via the VEGFR2-ERK-XPO5 pathway, and reduced enrichment of mature miR-549a in cytoplasm, which in turn promoted HIF1α expression in RCC, leading to increased VEGF secretion and further activated VEGFR2 to form a feedback effect. miR-549a played an important role in the metastasis of renal cancer and might serve as a blood biomarker for ccRCC metastasis and even had the potential of becoming a new drug to inhibit TKI-resistance.

Polycaprolactone Electrospun Fiber Mats Prepared Using Benign Solvents: Blending with Copper(II)-Chitosan Increases the Secretion of Vascular Endothelial Growth Factor in a Bone Marrow Stromal Cell Line.

Inducing the formation of new blood vessels (angiogenesis) is an essential requirement for successful tissue engineering. Approaches have been proposed to enhance angiogenesis using growth factors and other biomolecules; however, this approaches present drawbacks in terms of high cost and patient safety. Copper is known to effectively regulate angiogenesis and can offer a more cost-effective alternative than the direct use of growth factors. With this study, a strategy to incorporate copper in electrospun fibrous scaffolds with pro-angiogenic properties is presented. Polycaprolactone (PCL) and copper(II)-chitosan are electrospun using benign solvents. The morphological and physicochemical properties of the fiber mats are investigated through scanning electron microscopy (SEM), static contact angle measurements, energy dispersive X-ray, and Fourier-transform infrared spectroscopies. Scaffold stability in phosphate buffered saline at 37 °C is monitored over 1 week. A bone marrow stromal cell line (ST-2) is cultured for 7 days and its behavior is evaluated using SEM, fluorescence microscopy and a tetrazolium salt-based colorimetric assay. Results confirm that PCL/copper(II)-chitosan is suitable for electrospinning. The fiber mats are biocompatible and favor cell colonization and infiltration. Most notably, the angiogenic potential of PCL/copper(II)-chitosan blends is confirmed by a three-fold increase in VEGF secretion by ST-2 cells in the presence of copper(II)-chitosan.

Comparison of adipose particle size on autologous fat graft retention in a rodent model

Aim: Unpredictable retention outcomes remain a significant issue in autologous fat grafting procedures. Liposuction cannula variation leads to variability in fat particle size. Recent data suggest that the size of fat particles is closely related to graft healing outcomes; however, this remains a point of contention due to potential confounding variables such as tissue trauma with harvest. The aim of this study was to compare autologous fat grafting outcomes with variable fat particle sizes in an animal model which isolated fat particle size as the primary experimental variable. The overall goal of this work was to determine if reducing fat particle size is an effective method for enhancing graft retention in autologous fat grafting. Methods: The range of fat particle diameter harvested by four common liposuction cannulas was quantified to define relevant small and large particle target diameters. To determine if particle size impacted nutrient and oxygen permeability, small and large particles were incubated in vitro in a spinner flask with an abundance of culture media and vascular endothelial growth factor secretion was measured with enzyme-linked immunosorbent assay. Finally, small and large fat grafts were prepared from subcutaneous mouse fat pads and grafted in syngeneic Balb/CJ mice. Weight and volume retention were evaluated at 1, 4, 8, and 12 weeks. Histological analysis with Masson’s trichrome and perilipin immunofluorescent staining was performed. Real-time quantitative polymerase chain reaction was performed for adipogenic, inflammatory and apoptotic genes. Results: The range of fat particle diameters harvested with four commonly used cannulas was 2-7 mm. In vitro studies showed that 5-7-mm particles had significantly increased VEGF secretion normalized to weight, indicating increased tissue hypoxia in these particles compared to 2-4-mm particles. Surprisingly, in vivo comparison in two unique studies showed 2-4-mm and 5-7-mm fat particles had comparable graft retention (P = 0.5329). Masson’s trichrome staining revealed increased extracellular matrix and fibrosis in the 5-7-mm particle group (P = 0.0115). Adipocyte survival with perilipin demonstrated comparable viability. Gene expression showed large particles experienced increased inflammation and apoptosis at one week after grafting, but overall there were no significant differences between groups. Conclusion: The ideal fat particle size should be large enough to contain adequate mesenchyme while not so thick as to preclude imbibition. This study suggests that, despite changes in hypoxia and VEGF levels, differing fat particles (2-4-mm and 5-7-mm) can achieve similar graft retention.

Abstract 3596: The combinatory effect of bevacizumab and metabolic inhibitors on angiogenesis and autophagy in gastrointestinal cancer cells

Abstract Background: Bevacizumab is anti-VEGF drug that has shown therapeutic. However, treatment with bevacizumab has been associated to the development of resistance in several types of cancer. Glycolysis has been considered as a potential target for anti-cancer therapies and several molecules that target this pathway have been developed, including 2-Deoxy-D-glucose (2-DG) and 3-Bromopyruvate (3-BP). Autophagy is a process that degrades cellular organelles and proteins and maintains cellular biosynthesis during nutrient deprivation or metabolic stress. Since anti-angiogenic therapy and metabolic inhibitors both lead to nutrient deprivation in cancers, we investigated how a combination therapy would affect angiogenesis and autophagy in gastrointestinal cancer cells. Methods: AGS and Caco-2 cells were treated with glycolysis inhibitors 2-DG (4 mM) and 3-BP (20 µM), in the presence or in the absence of bevacizumab at 100 µg/ml. Cell proliferation was assessed using tetrazolium salt. VEGF protein levels were quantified by ELISA. Autophagy and apoptosis markers’ expression was assessed by quantitative real time PCR. Tube formation by human umbilical vein endothelial cells (HUVECs) was assessed using ECMatrix™. Results: Our results showed that combining bevacizumab to 2-DG decreased AGS and Caco-2 cells’ proliferation rates compared to each of these compounds alone. Interestingly, VEGF quantification showed that 2-DG increased VEGF secretion, in contrary to 3-BP. Moreover, our results showed that 3-BP reduced tube formation by HUVECs and amplified bevacizumab’s anti-angiogenic effect when combined together. In autophagy markers’ assessment, our results showed that 2-DG, 3-BP, and bevacizumab treatment increased FOXO1, FOXO3, LC3II, and HIF-1α expression. However, Beclin-1 expression, a protein implicated in the autophagic programmed cell death, decreased following bevacizumab treatment while it was upregulated by 2-DG and 3-BP. Hence, it was important to evaluate apoptotic markers’ expression, caspase-3 and BAX. Our results showed that bevacizumab decreased caspase-3 and BAX expression while their expression was upregulated by 2-DG and that its combination to bevacizumab reestablished their levels. Conclusions: Taken together, our results suggest that 2-DG and 3-BP, both improve cancer cells response to bevacizumab treatment by increasing its antiangiogenic outcome and reversing its anti-apoptotic effect. Moreover, it was interesting to find that bevacizumab treatment decreased Beclin-1, caspase-3, and BAX expression, suggesting a possible implication of autophagy in the development of resistance to bevacizumab. Hence, further studies are required in order to uncover the means by which bevacizumab modulates cancer cells autophagy and metabolism, and the effect of these changes on cancer cells survival. Citation Format: Nadine Mahfouz, Rita Ammoury, Mayssam Moussa, Charbel Khalil, Joe Aoun, George Hilal. The combinatory effect of bevacizumab and metabolic inhibitors on angiogenesis and autophagy in gastrointestinal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3596.

Minocycline modulates NF\u03baB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells

BackgroundMesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds.MethodsModulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague–Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student’s t test was used to determine p values for all assays with multiple or two conditions, respectively.ResultsMinocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial activity against SA via an increased production of IL-6 and SA internalization. MSC and antibiotic-loaded hydrogels reduced SA bioburden in inoculated wounds over 3 days and accelerated reepithelialization.ConclusionsMinocycline modulates the NFκB pathway in MSCs that leads to an enhanced production of IL-6 and internalization of SA. This mechanism may have contributed to the in-vivo antibacterial efficacy of MSC and antibiotic-loaded hydrogels.

Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses.

Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is considered potentially effective for cartilage repair and in-vitro chondrogenesis. We thus aimed to harness chondro-promotive effects of PRP and hypertrophic-suppressive effects of AC:MSC co-culture to achieve a more functional cartilage neo-tissue. We used PRP or conventional-differentiation chondrogenic media (ConvDiff) in MSC mono-cultures and AC:MSC co-cultures. We assessed gene expression of chondrogenic and hypertrophic markers using real-time RT-PCR and immunostaining. Alkaline-phosphatase activity (ALP) and calcium content of the pellets were quantified. We also measured VEGF and TNF-α secretion via ELISA. We showed PRP had higher chondrogenic potential (in mRNA and protein level) and hypertrophic-suppressive effects than Conv-Diff (mRNA level). Co-culturing reduced ALP while PRP increased calcium deposition. In all four groups, TNF-α was down-regulated compared to MSC controls, with co-cultures receiving ConvDiff media secreting the least. Meanwhile, the only group with increased VEGF secretion was PRP-mono-cultures. We observed synergistic effects for PRP and AC:MSC co-culture in enhancing chondrogenesis. Inclusion of AC reduced hypertrophic markers and angiogenic potential in PRP groups. We thus propose that combination of PRP and co-culture would favor chondrogenesis while alleviate but not totally eradicate undesired hypertrophic and pathologic responses.

Effect of Glucose on Retinal Endothelial Cell Viability and VEGF Secretion

Previous studies have shown that in diabetic patients, there is an increase of retinal capillaries associated with the development of diabetic retinopathy in the eye. The objective of current study is to investigate the effect of glucose on retinal endothelial cell viability and VEGF secretion. 20,000 cells per well were treated without glucose or with 5.5mM (euglycemic), 18.5mM and 30mM (hyperglycemic) glucose for 24 hours. Viable cells were counted using Trypan blue dye exclusion method. ELISA was used to measure VEGF secretion from cells into the cell medium. The number of viable cells incubated with 5.5mM glucose (physiological control) increased by 53.7% after 24 hours. In comparison, cells treated with 18.5mM glucose decreased by 2.8% while cells treated with 30mM glucose decreased by 20% after 24 hours of incubation. Cells without glucose treatment (0mM control) decreased by 33.3%. In contrast to the decrease of viable cell numbers after treatment with high glucose, there is an increase in VEGF secretion (pg/mL) to the cell medium with increase in glucose concentration from 5.5mM to 0, 18.5, and 30mM. The amount of VEGF secreted per cell also increased with increasing glucose concentrations. Our results show that viability of retinal endothelial cells and VEGF release are highly responsive to changes in glucose concentration. Such glucose-induced changes in retinal endothelial cells may negatively impact the integrity of the microvasculature in the diabetic retina leading to angiogenesis and microaneursyms.

Triiodothyronine Attenuates Prostate Cancer Progression Mediated by β-Adrenergic Stimulation.

Prostate cancer cells are responsive to adrenergic and thyroid stimuli. It is well established that β-adrenergic activation (protein kinase A [PKA]/cAMP response element binding protein [CREB]) promotes cancer progression, but the role of thyroid hormones is poorly understood. We analyzed the effects of β-adrenergic stimulation (isoproterenol [ISO]) and/or thyroid hormone on neuroendocrine (NE) differentiation and cell invasion, using in vivo (LNCaP tumor) and in vitro models (LNCaP and DU145 human cells). Nude mice were inoculated with LNCaP cells and were treated for 6 wks with ISO (200 μg/d), triiodothyronine (T3, 2.5 μg/d) or both. ISO alone reduced tumor growth but increased tumor expression of cAMP response element (CRE)-dependent genes (real-time polymerase chain reaction, chromogranin A, neuron-specific enolase, survivin, vascular endothelial growth factor [VEGF], urokinase plasmin activator [uPA] and metalloproteinase-9 [MMP-9]) and some proteins related to NE differentiation and/or invasiveness (synaptophysin, VEGF, pCREB). T3 reduced tumor growth and prevented the overexpression of ISO-stimulated factors through a pCREB-independent mechanism. In low invasive LNCaP cells, 50 μmol/L ISO or 100 nmol/L thyroxine (T4) induced the acquisition of NE-like morphology (phase-contrast microscopy), increased VEGF secretion (ELISA) and invasive capacity (Transwell assay), but no synergistic effects were observed after the coadministration of ISO + T4. In contrast, 10 nmol/L T3 alone had no effect, but it prevented the NE-like morphology and invasiveness stimulated by ISO. None of these treatments had any effect on highly invasive DU145 cells. In summary, this study showed that ISO and T4 increase cancer progression, and T3 attenuates ISO-stimulated progression. Further studies are required to determine if changes in the ratio of T4/T3 could be relevant for prostate cancer progression.

Nucleoside Reverse Transcriptase Inhibitors Suppress Laser-Induced Choroidal Neovascularization in Mice.

To evaluate the efficacy of nucleoside reverse transcriptase inhibitors (NRTIs) in the laser-induced mouse model of choroidal neovascularization (CNV). We evaluated the NRTIs lamivudine (3TC), zidovudine (AZT), and abacavir (ABC) and the P2X7 antagonist A438079. Choroidal neovascularization was induced by laser injury in C57BL/6J wild-type, Nlrp3-/-, and P2rx7-/- mice, and CNV volume was measured after 7 days by confocal microscopy. Drugs were administered by intravitreous injection immediately after the laser injury. Vascular endothelial growth factor-A in RPE-choroid lysates was measured 3 days after laser injury by ELISA. HEK293 cells expressing human and mouse P2X7 were exposed to the selective P2X7 receptor agonist 2', 3'-(benzoyl-4-benzoyl)-ATP (Bz-ATP) with or without 3TC, and VEGF-A levels in media were measured by ELISA. Intravitreous injection of 3TC, AZT, and ABC significantly suppressed laser-induced CNV in C57BL/6J wild-type and Nlrp3-/- mice (P < 0.05) but not in P2rx7-/- mice. Intravitreous injection of A438079 also suppressed the laser-induced CNV (P < 0.05). The NRTIs 3TC, AZT, and ABC blocked VEGF-A levels in the RPE/choroid after laser injury in wild-type (P < 0.05) but not P2rx7-/- mice. Moreover, there was no additive effect of 3TC on CNV inhibition when coadministered with a neutralizing VEGF-A antibody. Stimulation of human and mouse P2X7-expressing HEK293 cells with Bz-ATP increased VEGF secretion (P < 0.001), which was abrogated by 3TC (P < 0.001). Stimulation of primary human RPE cells with Bz-ATP increased VEGFA and IL6 mRNA levels, which were abrogated by 3TC. Multiple clinically relevant NRTIs suppressed laser-induced CNV and downregulated VEGF-A, via P2X7.

Abstract 4183: Differential expression of VEGF in breast cancer cells induced by gp130 cytokines

Abstract Glycoprotein 130 (gp130) is a glycosylated type 1 membrane protein and functions as a cytokine receptor modulating activity of the interleukin-6 (IL-6) family cytokines, which include IL-6, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine (CLC), and neuropoietin (NP). This study was designed to determine differential expression of VEGF in breast cancer cells by IL-6 family cytokines, and to elucidate the molecular mechanisms and signaling pathways involved in cytokine-mediated VEGF induction. Triple negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor positive/progesterone receptor positive/Her2 negative (ER+/PR+/Her2-) T47D cells were treated with three cytokines (25 ng/mL), OSM, IL-6, and LIF, and analyzed. Our results showed that all three cytokines increased levels of HIF1α by Western blot analysis. OSM lead to a greater production of VEGF compared to IL-6 and LIF, as measured by enzyme-linked immunosorbent assay (ELISA) and as seen by luciferase reporter assay. Of the three cytokines, OSM alone induced VEGF in TNBC MDA-MB-231 cells, and this effect was mediated by signal transducer activator of transcription 3 (STAT3) signaling. However, both OSM and IL-6 increased VEGF secretion levels in ER+/PR+/Her2- T47D breast cancer cells, and this signaling was mediated by both HIF1α and pSTAT3, utilizing ELISA and Western blot analysis. These results implicate OSM as the key gp130 cytokine inducing VEGF secretion, and also suggest that this induction is not always HIF1α dependent and is cell-line specific. Citation Format: Danielle S. Hedeen, Ken Tawara, Madhuri Nandakumar, Ryan Fox, David Chang, Alex Ide, Andrew Oler, Dollie LaJoie, Cheryl L. Jorcyk. Differential expression of VEGF in breast cancer cells induced by gp130 cytokines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4183. doi:10.1158/1538-7445.AM2015-4183

Abstract 1867: Enrichment of CD44high stem-cell-like cells as a possible mechanism of progestin-dependent progression of human breast cancer

Abstract Combined hormone replacement therapy (HRT) containing progestins (P) and estrogens (E) has been shown to increase the risk of breast cancer in post-menopausal women (JNCI, 2000, 92:328). We showed that the commonly used P, medroxyprogesterone acetate (MPA), increases production of the potent angiogenic growth factor VEGF in breast cancer cells (Int J Cancer, 2001, 92:469). Furthermore, it has been established that P drives tumor progression in an in-vivo xenograft model (Can Res 2007; 67:9929). CD44 has recently been shown to be expressed by cancer stem cells (CSC) (Nature Reviews Cancer 2011; 11:254), and has therefore been used as a marker for detecting and enriching of tumor initiating cells. CD44 binds potent growth factors such as VEGF and acts as a co-receptor that helps mediate proliferative and migratory signaling for a variety of receptor tyrosine kinases. Thus, we hypothesized that MPA increases CD44 levels in breast cancer cells and promotes the emergence of self-renewing stem-cell-like-cells, which support MPA-accelerated tumors in vivo. Human T47-D human breast cancer cells were treated with MPA for 24h. Subsequent FACS analysis showed that MPA caused a significant increase in CD44 protein; CD44 was induced 10-fold (Control 5.9%, MPA 60.7%). A less robust, but significant increase also occurred in BT474 cells (Control 4.0%, MPA 11.2%). Further studies showed that MPA-induced CD44high cells also had high levels of ALDH enzyme activity, a characteristic of stem cells. CD44high cell induction was completely repressed by the anti-progestin RU-486, indicating involvement of progesterone receptors. Induction of CD44 protein occurred in a time-dependent manner, with a significant increase 6h after MPA treatment and reaching a peak after 24h. Interestingly, CD44high expression remained elevated for at least another 24 hours, even though MPA was removed from the treatment media, indicating that once induced, CD44 may be a stable protein. Importantly, various natural and synthetic P that are used widely in HRT prescriptions (Progesterone, Norethindrone, Norgestrel), brought about a similar increase in CD44high cells. Considering P has previously been linked to increased VEGF secretion by hormone-responsive cancer cells, we tested whether VEGF signaling plays a role in the elevation of CD44high cells. However, this process was found to be VEGF-independent. Our results show that natural and synthetic progestins induce a CD44high cell population which is likely associated with development of MPA-induced tumors. Thus targeted therapies against CD44 may be useful in controlling progression of P-driven tumors. Supported in part by COR award from the College of Veterinary Medicine, University of Missouri. Citation Format: Sandy Goyette, Benford Mafuvadze, Matthew T. Cook, Yayun Liang, Salman M. Hyder. Enrichment of CD44high stem-cell-like cells as a possible mechanism of progestin-dependent progression of human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1867. doi:10.1158/1538-7445.AM2015-1867

Cyclic phosphatidic acid inhibits the secretion of vascular endothelial growth factor from diabetic human coronary artery endothelial cells through peroxisome proliferator-activated receptor gamma

Atherosclerosis is a disease characterized by building up plaques formation and leads to a potentially serious condition in which arteries are clogged by fatty substances such as cholesterol. Increasing evidence suggests that atherosclerosis is accelerated in type 2 diabetes. Recent study reported that high level of alkyl glycerophosphate (AGP) was accumulated in atherosclerotic lesions. The presence of this phospholipid in mildly oxidized low-density lipoprotein (LDL) is likely to be involved in atherogenesis. It has been reported that the activation of peroxisome proliferator-activated receptor gamma plays a key role in developing atherosclerosis. Our previous result indicates that cyclic phosphatidic acid (cPA), one of bioactive lipids, potently suppresses neointima formation by inhibiting the activation of peroxisome proliferator-activated receptor gamma (PPARγ). However, the detailed mechanism is still unclear. In this study, to elucidate the mechanism of the cPA-PPARγ axis in the coronary artery endothelium, especially in patients with type 2 diabetes, we investigated the proliferation, migration, and secretion of VEGF in human coronary artery endothelial cells from diabetes patients (D-HCAECs). AGP induced cell growth and migration; however, cPA suppressed the AGP-elicited growth and migration in D-HCAECs. Moreover, AGP increased VEGF secretion from D-HCAECs, and this event was attenuated by cPA. Taken together, these results suggest that cPA suppresses VEGF-stimulated growth and migration in D-HCAECs. These findings could be important for regulatory roles of PPARγ and VEGF in the vascular processes associated with diabetes and atherosclerosis.

Silencing gastrin-releasing peptide receptor suppresses key regulators of aerobic glycolysis in neuroblastoma cells.

Under normoxic conditions, cancer cells use aerobic glycolysis as opposed to glucose oxidation for energy production; this altered metabolism correlates with poor outcomes in neuroblastoma. Hypoxia-inducible factor-1 alpha (HIF-1α) and pyruvate dehydrogenase kinase 4 (PDK4) regulate aerobic glycolysis, while pyruvate dehydrogenase phosphatase 2 (PDP2) promotes glucose oxidation. Here, we sought to determine whether gastrin-releasing peptide receptor (GRP-R) signaling regulates glucose metabolism. Neuroblastoma cell lines, BE(2)-C and SK-N-AS, were used. PCR microararay for glucose metabolism was performed on GRP-R silenced cells. Target protein expression was validated using Western blotting and VEGF ELISA. Cobalt chloride (CoCl2 ) was used to induce chemical hypoxia. Efficacy of targeting PDK regulation in neuroblastoma was assessed using dichloroacetate (DCA) by conducting cell viability assays and Western blotting for apoptotic markers. Silencing GRP-R decreased HIF-1α expression and blocked VEGF expression and secretion in both normoxic and CoCl2 induced hypoxia. PCR array analysis identified that GRP-R silencing reduced PDK4 and increased PDP2 mRNA expression. These findings were validated by Western blotting. CoCl2 induced hypoxia increased VEGF secretion, HIF-1α, and PDK4 expression. PDK4 silencing decreased HIF-1α expression and VEGF expression and secretion. DCA treatment decreased BE(2)-C and SK-N-AS proliferation while promoting cell death. GRP-R silencing and DCA treatment synergistically halted BE(2)-C proliferation. We report that GRP-R regulates glucose metabolism in neuroblastoma by modulating HIF-1α, PDK4 and PDP2. PDK4 regulates glucose metabolism, in part, via regulation of HIF-1α. Synergistic consequences of GRP-R inhibition and DCA treatment may suggest a novel therapeutic strategy for the treatment of aggressive neuroblastoma.

Abstract 1681: Iron chelation therapy increased the anticancer effect of sorafenib in hepatocarcinoma

Abstract [Purpose] Iron overload is known to associate with the progression of chronic hepatitis and the increase risk of hepatocellular carcinoma (HCC). We have reported that iron depletion inhibited the cancer cell proliferation and reciprocally induced angiogenesis. Anti-angiogenic drug synergistically enhanced the anti-tumor effect in iron depletion condition. Sorafenib is molecular-targeting drug with anti-angiogenic effect in HCC. We hypothesized that iron depletion also enhanced the effect of sorafenib and investigated it. [Experimental Design] HepG2 and HLE (Human HCC cell line) were used in this study. We used sorafenib (NexavarTM) and an oral iron chilator, deferasirox (EXJADETM). The anti-cancer effect and mechanism were determined using cell viability assay, VEGF ELISA, Flow Cytometry, Western blot analysis and in vivo subcutaneous tumor model. In clinical cases, we retrospectively analyzed the relationship between sorafenib response and serum iron-related makers(n=58). [Results] In HepG2 and HLE cells, deferasirox suppressed the cancer cell proliferation by itself and increased the suppression of sorafenib. Deferasirox increased VEGF secretion of supernatant on dose dependent manner. In the analysis of combination therapy, cell cycle arrest and apoptosis was enhanced in the combination therapy by down-regulation of cyclinD1 and cyclinE and up-regulation of cleaved PARP. The suppression of HepG2 subcutaneous tumor was enhanced in the combination therapy. In clinical cases, sorafenib prolonged overall survival in TIBC high and Ferritin low group. [Conclusion] Iron depletion by deferasirox increased the anti-cancer effect of sorafenib in vitro and in vivo experiments. In clinical cases, sorafenib was also effective in iron deficient patients. These results suggested that iron depletion therapy can be a novel combination chemotherapy with sorafenib for hepatocarcinoma patients. Citation Format: Shinichi Urano, Toshiaki Ohara, Ryoichi Katsube, Shinichiro Watanabe, Kazuhiro Noma, Yasuko Tomono, Hiroshi Tazawa, Kazuhiro Nouso, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Iron chelation therapy increased the anticancer effect of sorafenib in hepatocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1681. doi:10.1158/1538-7445.AM2014-1681

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB2 levels were correlated with VEGF (p<0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p<0.0001) and microvessel density (CD-31; p<0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p<0.0001) and female patients (p<0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p<0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion: TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.