VEGF Production Research Articles

Production of factors involved into fibrosis regulation by various types of human macrophages

Macrophages (Mφ) play a key role in regulation of fibrogenesis, including proliferation of fibroblasts and myofibroblasts, differentiation of progenitor cells into myofibroblasts, as well as synthesis and secretion of the extracellular matrix, mainly collagen. The direction of the Mφ effects (stimulation or suppression) is determined by a number of factors, including the stage of the fibrotic process and the Mφ functional phenotype dependent on the signals of microenvironment. One of the feasible ways of the fibrogenesis regulating is the secretion of pro- or antifibrotic factors such as matrix metalloproteinases, inhibitors of metalloproteinases and some cytokines. However, existing data on ability to secrete these factors by various subpopulations of human Mφ are rare and controversial. The aim of this study was to characterize the ability of human M1, M2a, and M2c Mφ differentiating in the presence of GM-CSF to produce matrix metalloproteinases (MMP-9) and their tissue inhibitors (TIMP-1), as well as some cytokines and growth factors. As compared to M2 macrophages, the M1 macrophages polarized by lipopolysaccharide produced significantly more TNFα, IL-6 and IL-2 that have pro-inflammatory activity and are able to initiate a fibrotic process. In turn, M2a Mφ stimulated by IL-4 were characterized by a high level of VEGF production and, at the same time, low levels of TNFα and IL-6, which may determine the important role of these cells at the proliferative stage of fibrosis and stimulation of extracellular matrix deposition. Finally, M2c Mφ polarized by dexamethasone, exhibited the М2а-like cytokine profile, i.e., VEGF was actively produced against the background of low TNFα and IL-6 synthesis. Moreover, all three Mφ subpopulations did actively secrete MMP-9 and TIMP-1, without significant difference in production of these factors. However, M2c Mφ differed by a significantly higher MMP-9/TIMP-1 ratio index compared to M1 and M2a Mφ, and it is crucial at the rearrangement stage of the fibrotic process. Thus, the production of MMP-9 and TIMP-1, together with other pleiotropic cytokines and growth factors by various Mφ subtypes may reflect their role in regulation of fibrotic process at various stages.

Rac Signaling Drives Clear Cell Renal Carcinoma Tumor Growth by Priming the Tumor Microenvironment for an Angiogenic Switch.

Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of antiangiogenic therapies that block the blood vessels that supply tumors with nutrients for growth and metastasis. Unfortunately, most ccRCC patients eventually become resistant to initial treatments, creating a need for alternative treatment options. We investigated the role of the small GTPase Rac1 in ccRCC. Analysis of ccRCC clinical samples indicates that Rac signaling drives disease progression and predicts patients with poorer outcomes. Investigation of Rac1 identifies multiple roles for Rac1 in the pathogenesis of ccRCC. Rac1 is overexpressed in RCC cell lines and drives proliferation and migratory/metastatic potential. Rac1 is also critical for endothelial cells to grow and form endothelial tubular networks potentiated by angiogenic factors. Importantly, Rac1 controls paracrine signaling of angiogenic factors including VEGF from renal carcinoma cells to surrounding blood vessels. A novel Rac1 inhibitor impaired the growth and migratory potential of both renal carcinoma cells and endothelial cells and reduced VEGF production by RCC cells, thereby limiting paracrine signaling both in vitro and in vivo Lastly, Rac1 was shown to be downstream of VEGF receptor (VEGFR) signaling and required for activation of MAPK signaling. In combination with VEGFR2 inhibitors, Rac inhibition provides enhanced suppression of angiogenesis. Therefore, targeting Rac in ccRCC has the potential to block the growth of tumor cells, endothelial cell recruitment, and paracrine signaling from tumor cells to other cells in the tumor microenvironment.

Long Non-Coding RNA (lncRNA) NEAT1 Aggravates Cerebral Ischemia-Reperfusion Injury by Suppressing the Inhibitory Effect of miR-214 on PTEN.

BackgroundCerebral ischemia-reperfusion injury is a form of serious nervous system injury. Activation of the PI3K/Akt pathway can effectively relieve cerebral ischemia-reperfusion injury. miR-214 can target and inhibit the expression of PTEN, thereby alleviating its inhibitory effect on the PI3K/Akt pathway. Moreover, lncRNA NEAT1 was reported to affect proliferation and metastasis of tumor cells by targeting and suppressing the expression of miR-214. However, whether lncRNA NEAT1 affects the cerebral ischemia-reperfusion-induced damage by regulating the miR-214/PTEN/PI3K/Akt pathway is unclear.Material/MethodsThe miR-214 agomir and miR-214 antagomir were designed and injected into the encephalocele of MCAO rats. Next, the production of oxidative stress kinase and apoptosis of brain cells were detected using commercial kits. The levels of PTEN, PI3K, Akt, p-Akt, and VEGF in brain tissues were determined. Next, the targeting effect of lncRNA NEAT1 and miR-214 was determined with luciferase reporter assay.ResultsOverexpression of miR-214 relieved the apoptosis and oxidative stress of brain tissues. Overexpression of miR-214 promoted the expression of PI3K, Akt, p-Akt, and VEGF by inhibiting the production of PTEN. However, overexpression of lncRNA NEAT1 repressed the remission effect of miR-214 on cerebral ischemia-reperfusion-induced damage and inhibited the production of PI3K, Akt, p-Akt, and VEGF by rescuing the levels of PTEN.ConclusionslncRNA NEAT1 aggravates cerebral ischemia-reperfusion injury by abolishing the activation effect of miR-214 on the PI3K/Akt pathway.

AB0153 ADIPOSE-DERIVED STROMAL/STEM CELLS FROM SYSTEMIC SCLEROSIS PATIENTS SUCCESSFULLY EXERT A PARACRINE ANTI-FIBROTIC ACTIVITY AND INDUCE A PRO-ANGIOGENIC PHENOTYPE OF SCLERODERMA FIBROBLASTS IN VITRO

Background:Adipose-derived stromal/stem cells (ADSCs) are multipotential non-hematopoietic progenitor cells with anti-inflammatory, immunomodulatory and regenerative effects. They have the advantage of accessibility and potent pro-angiogenic effects when compared with other stem cells, such as bone-marrow derived stem cells. Recent studies have shown that autologous fat grafting may be effective in the treatment of fibrotic and vascular complications in systemic sclerosis (SSc), despite a pro-fibrotic signature.Objectives:Aim of the study was to better characterize the proliferative and secretive profile of ADSCs in normoxic and hypoxic conditions, and to evaluate the mechanisms by which ADSCs exert these observed clinical effects.Methods:Adipose tissue samples were obtained by liposuction from 12 SSc patients and 10 healthy donors (HD). ADSCs were purified according to their adherence to the plastic and characterized to express specific MSC surface antigens by flow cytometry analysis. Proliferation of ADSCs from SSc patients and normal controls was evaluated in normoxic and hypoxic conditions. Fibroblasts and ADSCs derived from SSc patients were co-cultured in direct and indirect culture systems and compared to HD. Fibroblasts proliferation, mRNA expression and protein secretion of VEGF and known fibrotic mediators including TGF β-1, TGFR, CTGF, Collagen type I (Col I) were analyzed in the same conditions.Results:Normoxic and hypoxic culture conditions did not modify the proliferation rate of both normal and SSc ADSCs. Hypoxia significantly increased mRNA expression of VEGF by HD and SSc ADSCs but had no effect on the mRNA expression of pro-fibrotic mediators, ie TGFβ and TGFR. Normal and SSc fibroblast proliferation was significantly reduced in both co-culture systems (p < 0.001) and by treatment with normoxic and hypoxic conditioned medium (CM) (p=0.001 and p=0.002). In the same conditions, mRNA expression and protein secretion of TGF-β1, CTGF and Col I were significantly reduced (p = 0.003, p =0.02, p=0.04). These results were confirmed when normal and SSc fibroblasts were cultured in the presence of ADSCs normoxic and hypoxic CM (p=0.02 and p=0.01). Furthermore, a significant increase in mRNA expression and production of VEGF was observed in SSc fibroblasts cultured in the presence of normoxic and hypoxic CM (p = 0.002 and p< 0.0001, respectively).Conclusion:We found that treatment with the medium from normoxic and hypoxic preconditioned ADSCs has an anti-fibrotic effect through both the inhibition of fibroblast proliferation and key mediators of fibrosis. The increased expression of VEGF by SSc fibroblasts in the presence of normoxic and, even more, hypoxic ADSCs CM, suggests that ADSCs can induce a paracrine pro-angiogenic phenotype, even in fibroblasts with a pro-fibrotic signature. Altogether these data show that ADSCs may exert their anti-fibrotic and pro-angiogenetic effects on SSc fibroblasts by the secretion of paracrine factors, partly explaining the mechanisms underlining beneficial clinical results of fat graft in SSc patients.Disclosure of Interests:Nicoletta Del Papa: None declared, Maurizio Lorini: None declared, Vincenzo Carbonelli: None declared, Antonina Minniti: None declared, Francesca Pignataro: None declared, Wanda Maglione: None declared, Gabriele Di Luca: None declared, Nicola Montano: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB

Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization

SummaryEndothelial cell (EC)-derived signals contribute to organ regeneration, but angiocrine metabolic communication is not described. We found that EC-specific loss of the glycolytic regulator pfkfb3 reduced ischemic hindlimb revascularization and impaired muscle regeneration. This was caused by the reduced ability of macrophages to adopt a proangiogenic and proregenerative M2-like phenotype. Mechanistically, loss of pfkfb3 reduced lactate secretion by ECs and lowered lactate levels in the ischemic muscle. Addition of lactate to pfkfb3-deficient ECs restored M2-like polarization in an MCT1-dependent fashion. Lactate shuttling by ECs enabled macrophages to promote proliferation and fusion of muscle progenitors. Moreover, VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis. Finally, increasing lactate levels during ischemia rescued macrophage polarization and improved muscle reperfusion and regeneration, whereas macrophage-specific mct1 deletion prevented M2-like polarization. In summary, ECs exploit glycolysis for angiocrine lactate shuttling to steer muscle regeneration from ischemia.

P1133XYLITOL-BASED PERITONEAL DIALYSIS SOLUTIONS MITIGATED IN VITRO MESOTHELIAL AND ENDOTHELIAL TO MESENCHYMAL TRANSITION

Abstract Background and Aims Fibrosis, angiogenesis and microvascular alteration are main pathogenetic mechanisms involved in the progressive loss of the peritoneal ultrafiltration capacity in patients undergoing peritoneal dialysis (PD). Main cause of this condition is the continuous peritoneal exposure to hyperosmotic and hyperglycaemic agents. High glucose level activates the Mesothelial to Mesenchymal Transition (MMT) and the Endothelial-to-Mesenchymal (EndMT) program, which are responsible for the development of fibrosis/chronic peritoneal damage. Moreover, the high glucose content of PD solution may induce the VEGF production with consequent neo-angiogenesis. Therefore, the introduction of more biocompatible solutions in clinical practice is necessary for preserving the long-term peritoneal membrane. To this purpose we tested the in vitro effects of a new commercially available PD solutions containing xylitol, carnitine and reduced glucose, at comparable osmotic strength (XyloCore). Method Human vein microvascular endothelial cells (HMVEC) were cultured in EGM™-2MV medium (Lonza) and Human peritoneal mesothelial cell line (HMrSV5) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) containing 10% fetal bovine serum. Cells were cultured to confluence and then treated for 3 hours with serum free medium, XyloCore 0.7 (0.5% Glucose, 0.7% Xylitol and 0.02% L-carnitine), XyloCore 1.5 (0.5% Glucose, 1.5% Xylitol and 0.02% L-carnitine) and commercially available glucose-based solutions (Fixioneal) 1.36% and 2.27% Glucose. Gene expression of MMT/EndMT, apoptosis, inflammation, extracellular remodeling and angiogenesis markers was evaluated by real-time PCR. Cell viability was assayed by MTS assay. Results Our in vitro results demonstrated that XyloCore solutions, by influencing only partially the mesothelial and endothelial cells viability, demonstrated a good biocompatible profile. Then, gene expression analysis of HMVEC and HMrSV5 treated with XyloCore solutions revealed a significant down-regulation of transcripts encoding for MMT and EndMT biomarkers (Zinc finger protein SNAI1, TGF-beta, alpha-SMA and vimentin), and pivotal biological elements involved in apoptosis (Bcl-2), extracellular matrix remodeling (matrix metallopeptidases), inflammation (IL-1beta, IL-6) and angiogenesis (Vascular endothelial growth factor) compared to glucose-based solutions with comparable osmotic strength. Conclusion These in vitro results demonstrated, for the first time, that XyloCore solutions have a better biocompatible impact and less pro-fibrotic potentials compared to conventional glucose-based solutions. These effects, if confirmed in in vivo studies, could have interesting clinical potentials.

591-P: Macrophage IL-1beta–Dependent VEGF-A Expression Is Required for Inflammatory Arteriogenesis and Wound Healing in Type 2 Diabetes

Impaired wound healing in patients with diabetes mellitus (DM) is a major source of morbidity and mortality. Current treatments for diabetic wounds are modestly effective at promoting healing. Recently, we identified an important role for inflammatory macrophage VEGF-A production in consequent angiogenesis/arteriogenesis required for adequate wound healing. We sought to understand the impact of diabetes on inflammatory angiogenesis and consequent healing. We hypothesized that diabetes mellitus (DM) results in reduced inflammatory macrophage VEGF-A expression with consequent impairments in angiogenesis-dependent wound healing. Mice with experimental diabetes demonstrated profound delays both in wound healing using a dermal punch biopsy model and in perfusion recovery using the femoral artery ligation model of hind limb ischemia. VEGF-A expression is highly upregulated in “classic inflammatory” macrophages relative to “alternatively activated” or wound healing macrophages. This is supported by a strong association between IL-1β and VEGF-A expression. In order to understand whether this relationship between IL-1β and VEGF-A was causal, we generated a novel mouse with a LoxP flanked IL-1β allele to create an inducible macrophage deletion of IL-1β. Myeloid IL-β deletion resulted in severely impaired macrophage VEGF-A expression and consequent decreases in angiogenesis and wound healing, consistent with the experimental model of DM. Moreover, macrophages from diabetic mice demonstrated reduced expression of IL-1R signaling complex components along with consequent reductions in VEGF-A expression. Defining inflammatory macrophages as key, early drivers of angiogenesis supports a paradigm shift away from inflammation-suppression for adequate wound healing, allowing for macrophage reprograming strategies that promote appropriate inflammation-dependent healing responses. Disclosure C.S. Mantsounga: None. Funding National Institutes Health (1R01HL139795)

ESM-1 Overexpression is Involved in Increased Tumorigenesis of Radiotherapy-Resistant Breast Cancer Cells.

The key barrier to the effectiveness of radiotherapy remains the radioresistance of breast cancer cells, resulting in increased tumor recurrence and metastasis. Thus, in this study, we aimed to clarify the difference between radiotherapy-resistant (RT-R) breast cancer (BC) and BC, and accordingly, analyzed gene expression levels between radiotherapy-resistant (RT-R) MDA-MB-231 cells and MDA-MB-231 cells. Gene expression array showed that ESM-1 was the most upregulated in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells. Then, we aimed to investigate the role of ESM-1 in the increased tumorigenesis of RT-R-BC cells. RT-R-MDA-MB-231, which showed an increased expression level of ESM1, exhibited significantly enhanced proliferation, colony forming ability, migration, and invasion compared to MDA-MB-231 cells, and ESM-1 knockdown effectively reversed these effects. In addition, compared to MDA-MB-231 cells, RT-R-MDA-MB-231 cells displayed improved adhesion to endothelial cells (ECs) due to the induction of adhesion molecules and increased MMP-9 activity and VEGF-A production, which were decreased by ESM-1 knockdown. Moreover, the expression of HIF-1α and activation of NF-κB and STAT-3 were increased in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells, and these effects were abolished by the knockdown of ESM-1. Finally, we confirmed the role of ESM-1 in tumorigenesis in an in vivo mouse model. Tumor volume, lung metastasis, and tumorigenic molecules (VEGF-A, HIF-1α, MMP-9, ICAM-1, VCAM-1, and phospho-NF-κB and phospho-STAT-3) were significantly induced in mice injected with ESM-1-overexpressing 4T1 cells and greatly enhanced in those injected with ESM-1-overexpressing RT-R-4T1 cells. Taken together, these results suggest for the first time that ESM-1 plays a critical role in tumorigenesis of breast cancer cells, especially RT-R-breast cancer cells, through the induction of cell proliferation and invasion.

Low-dose oral etoposide is an active option for patients with heavily pre-treated thymic epithelial tumors.

9074 Background: Platinum based regimens are used in the first line setting for advanced Thymic Epithelial Tumors (TETs). Angiogenesis plays an important role in TETs: VEGF is overexpressed in TETs, and associated with aggressiveness and advanced stage. Etoposide inhibits angiogenesis in vitro and in vivo by decreasing VEGF production and microvessel density. The aim of this study is to assess the activity of metronomic oral etoposide, with identification of circulating predictive and pharmacodynamics biomarkers. Methods: Patients with advanced platinum pretreated TET referred from 2014 to 2019 at Rare Tumors Reference Center of Naples, were prospectively enrolled in this study. Oral etoposide 50 mg daily for 3 weeks on and 1 week off every 28 days, has been delivered until progression of disease, complete response or unacceptable toxicity. Response rate (RR), progression free survival (PFS), toxicity and ratio between time to etoposide progression (TTPe) and time to previous best treatment progression (TTPp) were evaluated. Serum samples were prospectively obtained from ten patients with simultaneously radiological assessment. cfDNA quantification was assessed using Qubit Fluorometric Quantitation. Results: 21 patients were enrolled: median age 59 years range (41 - 88); 70% male, 60% T (4 B1, 3 B2, 4 B3, 1 B1-B2); 40% had TC. A median of 5 (range 1-9) prior therapy regimens had been administered. Median follow-up since etoposide was 5 years (range 0.5-5). Obtaining an overall response rate of 85%, 3 patients achieved complete response and 15 partial response. Median PFS was 16 months [95%CI 3-60] with respectively a median PFS of 12 for T (95%CI 3-38) and 19 for TC (95%CI 6-60). No grade 3-4 related events occurred, G1-2 myelotoxicity has been registered in 20% of patients. Therapy is still ongoing for 15 patients and all are still alive. Median TTPe was 16 months, TTPp was 9 months and TTPe / TTPp ratio equal to 1.7. The median cfDNA of 8 responder patients, before starting therapy, was 2.2 ng/μl (0.178-5.24), dropping dramatically at radiological response to 0.5 ng/μl (0.323-2.56). 2 out of 3 non-responder patients had a median baseline value of 2.49 ng/μl, increasing to 4.6 ng/μl at progression. Variation of circulating VEGF correlates with radiological response. Conclusions: Taking into account that other antiangiogenic drugs, showing some activity in second and further lines treatment, are very expensive and associated with several side effects, we suggest that low dose oral etoposide might become the preferred treatment option in heavily pretreated TETs.

Histomorphological, VEGF and TGF-β immunoexpression changes in the diabetic rats' ovary and the potential amelioration following treatment with metformin and insulin.

Diabetes mellitus (DM) affects the ovary by reducing the number and diameters of ovarian follicles and increasing atretic follicles. Follicular growth and diameters depend on VEGF production. Hyperglycemia causes ovarian stromal and follicular degeneration then fibrosis by activating TGF-β. Insulin and metformin promote development of ovarian follicles and reduce atretic follicles. Therefore, the present study investigates the ovarian VEGF and TGF-β immune-expression and its variations in diabetic, insulin and metformin-treated rats. Forty adult female albino rats were divided equally into four groups: control, diabetic (STZ-induced diabetes), diabetic metformin-treated group (100 mg/kg/day orally/eight weeks) and diabetic insulin-treated group (5 U insulin /day). Ovarian sections were stained with hematoxylin and eosin, Masson's trichrome, immunohistochemistry for VEGF and TGF-β. The diabetic group showed noticeable atrophic and degenerative changes in cortex and medulla as well as increased density and distribution of the collagenous fibers. The number and diameter of primary, secondary and tertiary follicles were decreased. However, the number of atretic follicles and corpus luteum was increased. Significant decrease in the surface area percentageof VEGF immuno-expression and significant increase in TGF-β immuno-expression surface area percentage were detected. By treating animals with metformin and insulin, there was restoration of the ovarian histological structure more or less as in control. DM negatively affects the histological and morphometric parameters of ovaries. Furthermore, insulin showed more beneficial effects than metformin in hindering these complications by modifying the expression of VEGF and TGF-β.

Imperatorin alleviates ROS-mediated airway remodeling by targeting the Nrf2/HO-1 signaling pathway.

In this study, we investigated the role and mechanism of imperatorin (IMP) in chronic inflammation and airway remodeling. The levels of TNF-α, IL-1β, IL-6, IL-8, VEGF, α-SMA, and ROS were detected by ELISA, immunohistochemistry (IHC), immunofluorescence, and Western blot. In addition, we evaluated the effect of IMP on MAPK, PI3K/Akt, NF-κB, and Nrf2/HO-1 signaling pathways. IMP treatment obviously attenuated the production of inflammatory cytokines and inflammatory cells in bronchoalveolar lavage fluid of OVA-induced airway remodeling model. Meanwhile, it significantly inhibited inflammatory cell infiltration, goblet cell hyperplasia, collagen deposition, VEGF production, α-SMA, and ROS expression. Our study has shown that IMP could regulate the signaling pathways including MAPK, PI3K/Akt, NF-κB, and Nrf2/HO-1 to release the inflammatory responses. IMP might attenuate airway remodeling by the down-regulation of Nrf2/HO-1/ROS/PI3K/Akt, Nrf2/HO-1/ROS/MAPK, and Nrf2/HO-1/ROS/NF-κB signaling pathways.

Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF-1α-independent VEGF expression and angiogenesis in hepatocellular carcinoma.

Background and PurposeAs a typical hypervascular tumour, hepatocellular carcinoma (HCC) is predominantly grown through angiogenesis. Geniposide is a promising anti‐inflammatory compound found in Gardenia jasminoides, but its effects on the progression of HCC remain untested.Experimental ApproachThe anti‐HCC effects of geniposide was investigated in cellular models and orthotopic HCC mice. Transcriptional regulation of the VEGF promoter was measured by dual‐luciferase reporter assay. The anti‐angiogenic action of geniposide was measured by tube formation assay. Both surface plasmon resonance techniques and human phospho‐kinase array analysis were utilized to validate the relationship between targets of geniposide and hepatocarcinogenesis.Key ResultsGeniposide exhibited significant disruption of HCC proliferation, invasion, angiogenesis and lung metastasis in orthotopic HCC mice. Geniposide inhibited secretion of VEGF by HCC and suppressed the migration of endothelial cells and the formation of intra‐tumour blood vessels, without cytotoxicity and independently of the transcription factor HIF‐1α. Direct inhibition of TLR4 by geniposide led to the shutdown of the TLR4/MyD88 pathway and STAT3/Sp1‐dependent VEGF production. However, LPS, an agonist of TLR4, restored STAT3/Sp1‐related VEGF production in geniposide‐inhibited HCC angiogenesis.Conclusion and ImplicationsThe direct inhibitory effect of geniposide on TLR4/MyD88 activation contributes to the suppression of STAT3/Sp1‐dependent VEGF overexpression in HCC angiogenesis and pulmonary metastasis. This action of geniposide was not affected by stabilization of HIF‐1α. Our study offers a novel anti‐VEGF mechanism for the inhibition of HCC.

Expression of pro-angiogenic factors as potential biomarkers in experimental models of colon cancer.

RAS mutational status in colorectal cancer (CRC) represents a predictive biomarker of response to anti-EGFR therapy, but to date it cannot be considered an appropriate biomarker of response to anti-VEGF therapy. To elucidate the function of K-Ras in promoting angiogenesis, the effect of conditioned media from KRAS mutated and wild type colon cancer cell lines on HUVECs tubule formation ability and the correspondent production of pro-angiogenic factors have been evaluated by a specific ELISA assay. Ras-activated signaling pathways were compared by western blot analysis and RTq-PCR. In addition, VEGF, IL-8, bFGF and HIF-1α expression was determined in K-RAS silenced cells. Furthermore, we conducted an observational study in a cohort of RAS mutated metastatic CRC patients, treated with first-line bevacizumab-based regimens, evaluating VEGF-A and IL-8 plasma levels at baseline, and during treatment. K-RAS promotes VEGF production by cancer cell lines. At the transcriptional level, this is reflected to a K-RAS dependent HIF-1α over-expression. Moreover, the HIF-1α, VEGF and FGF expression inhibition in KRAS knocked cells confirmed these results. Within the clinical part, no statistically significant correlation has been found between progression-free survival (PFS) and VEGF-A/IL-8 levels, but we cannot exclude that these biomarkers could be further investigated as predictive or prognostic biomarkers in this setting. Our study confirmed the direct involvement of K-Ras in promoting angiogenesis into colon cancer cell lines.

An Extract of Olive Mill Wastewater Downregulates Growth, Adhesion and Invasion Pathways in Lung Cancer Cells: Involvement of CXCR4.

Several diet-derived compounds have been reported to exert antioxidant, anti-proliferative and anti-angiogenic effects in numerous cancers and could be beneficial in cancer prevention. Olive oil production involves the generation of an aqueous phase defined as olive mill wastewater (OMWW), a polluting effluent rich in soluble polyphenols. Here, we assessed the cancer preventive properties exerted by a purified extract of OMWW (A009) for its activity on lung cancer cell lines. Hydroxytyrosol, the most abundant polyphenol present in our A009 extracts, was used as reference molecule in the assays performed. Extracts from OMWW from two different olive oil cultivars were used. We found that the A009 extracts limit lung cancer cell proliferation in a dose and time dependent manner. These effects were associated with the induction of apoptosis. A009 extracts were effective in inhibiting adhesion capabilities on a fibronectin layer accompanied with a reduction in their ability to generate invasive sprouts in a Matrigel layer. The production of chemokine CXCL12 and CXCR4 receptor were reduced by treatment with the extracts. Also, A009 interfered with the production of proangiogenic and pro-inflammatory VEGF, CXCL8, and CCL2 (as detected by FACS analysis) in the lung cell lines. A009 extracts were able to decrease STAT3 phosphorylation in lung cancer cells. Our results show that A009 extracts reduced activities related to tumor cell behavior in lung cancer cell lines, suggesting that they could have a potential cancer preventive role.

Regorafenib sensitizes human breast cancer cells to radiation by inhibiting multiple kinases and inducing DNA damage

Purpose Triple-negative breast cancer (TNBC) is the most challenging and aggressive subtype of breast cancer with limited treatment options because of tumor heterogeneity, lack of druggable targets and therapy resistance. TNBCs are characterized by overexpression of growth factor receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) making them promising therapeutic targets. Regorafenib is an FDA approved oral multi-kinase inhibitor that blocks the activity of multiple protein kinases including those involved in the regulation of tumor angiogenesis [VEGFR1-3, TIE2], tumor microenvironment [PDGFR-β, FGFR] and oncogenesis (KIT, RET, RAF-1, BRAF). In the current study, we examined the radiosensitizing effects of Regorafenib on TNBC cell lines and explored the mechanism by which Regorafenib enhances radiosensitivity. Methods MDA-MB-231 and SUM159PT (human TNBC cell lines) and MCF 10a (human mammary epithelial cell line) were treated with Regorafenib, ionizing radiation or a combination of both. Following treatment with Regorafenib and radiation we conducted clonogenic assay to determine radiosensitivity, immunoblot analysis to assess the effect on key signaling targets, tube formation to evaluate effect on angiogenesis and comet assay as well as western blot for γH2AX to assess DNA damage response (DDR). Results Regorafenib reduced cell proliferation and enhanced radiosensitivity of MDA-MB-231 and SUM159PT cell lines but had no effect on the MCF 10a cells. Clonogenic survival assays showed that the surviving fraction at 2 Gy for both MDA-MB-231 and SUM159PT was reduced from 66.4 ± 8.9 and 88.2 ± 1.7 in controls to 38.1 ± 4.9 and 75.1 ± 1.1 following a 24 hr pretreatment with 10 μM and 5 μM Regorafenib, respectively. A marked reduction in the expression of VEGFR, PDGFR, EGFR and the downstream target, ERK, was observed with Regorafenib treatment alone or in combination with radiation. We also observed a significant inhibition of VEGF-A production in the TNBC cell lines following treatment with Regorafenib. Further, the addition of conditioned medium from Regorafenib-treated tumor cells onto human umbilical vein endothelial cells (HUVEC) suppressed tube formation, indicating an inhibition of tumor angiogenesis. Regorafenib also decreased migration of TNBC cells and suppressed radiation-induced DNA damage repair in a time-dependent manner. Conclusions Our findings demonstrate that Regorafenib enhanced radiosensitivity of breast cancer cells by inhibiting the expression of multiple receptor tyrosine kinases, VEGF-mediated angiogenesis and DNA damage response in TNBC. Therefore, combining Regorafenib with radiation and antiangiogenic agents will be beneficial and effective in controlling TNBC.

TXNDC5 protects synovial fibroblasts of rheumatoid arthritis from the detrimental effects of endoplasmic reticulum stress.

TXNDC5 is an endoplasmic reticulum (ER)-resident chaperone that protects the endothelium from secondary effects of ER stress. Previous studies by the current authors identified TXNDC5 as a key pathological factor in promoting the inflammatory phenotype of fibroblast-like synoviocytes (FLSs) from rheumatoid arthritis (RA). However, its activity in RA FLSs under ER stress remains unclear. The current study found that TXNDC5 is responsive to ER stress in RA FLSs since its expression was induced by ER stress at both the endogenous and secretory level. A functional study indicated that silencing TXNDC5 reduced the viability of RA FLSs more markedly in the presence of ER stressors. In contrast, rhTXNDC5 attenuated a decrease in cell viability as a result of ER stress. Moreover, silencing TXNDC5 attenuated the induction of IL-6 and IL-8 from RA FLSs in response to ER stress. In addition, rhTXNDC5 induced a greater increase in VEGF production during ER stress. These findings confirm the pro-survival and pro-inflammation roles of TXNDC5 under ER stress in RA FLSs. TXNDC5 appears to act as a mediator linking ER stress and inflammation of RA.

Abstract PD8-10: Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells

Abstract Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a Tie2hi/Vegfhi macrophage and an endothelial cell, creates a micro-anatomic doorway called “tumor microenvironment of metastasis” (TMEM). TMEM are responsible for cancer cell intravasation and dissemination to distant sites. The density of TMEM doorways is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that TMEM doorways create increased localized vascular permeability which cancer cells utilize to intravasate, the precise molecular mechanisms relating TMEM-doorway function and intravasation has not been elucidated. Active TMEM doorways are found in pre-invasive and invasive ductal breast carcinoma as well as in metastatic foci in lymph nodes and lungs, indicating that TMEM-mediated cancer cell dissemination occurs not only at the primary tumor site but also at metastatic sites, which may perpetuate metastatic dissemination even after removal of the primary tumor. Thus it is essential to understand the exact molecular mechanism of TMEM-doorway function so that specific targeted therapies can be developed to intercept systemic cancer cell dissemination. We outline here the exact molecular mechanism of TMEM-doorway functions. TMEM doorway endothelial cell-secreted Ang2 (a Tie2 ligand) stimulates VEGF expression and production by the Tie2hi TMEM macrophage. Subsequently, the TMEM doorway tumor cell- secreted CSF1 stimulates local secretion of VEGF from the Tie2hi TMEM macrophages, leading to dissociation of endothelial adherens and tight junctions near TMEM and cancer cell intravasation. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased tumor cell trans-endothelial migration, and decreased TMEM-dependent vascular permeability, tumor cell dissemination and circulating tumor cells. This is the first description of the molecular mechanisms regulating TMEM doorway function and thus represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors. Citation Format: Chinmay Surve, Allison S. Harney, Mary Chen, Yarong Wang, Xianjun Ye, Yu Lin, Ved Sharma, Richard Stanley, Maja H. Oktay, John S. Condeelis. Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-10.

Gene polymorphisms of CCL3, CCL4, CCL5, and CCR5 network in metastatic colorectal cancer patients treated with regorafenib.

199 Background: We previously reported that genetic variant in the CCL5/CCR5 pathway predict efficacy of regorafenib in metastatic colorectal cancer patients (mCRC). CCL5 rs2280789 G allele and CCL5 rs3817655 T allele were associated with longer overall survival (OS) and severe skin toxicity due to low VEGF-A production via endothelial progenitor cell (EPC). CCL4 rs1634517 G allele and CCL3 rs1130371 C allele correlated with longer Progression-free survival (PFS) and OS. We investigated the biological role of CCL4 and CCL3 gene polymorphisms. Methods: We analyzed genomic DNA extracted from 79 samples of a Japanese cohort receiving regorafenib. Single nucleotide polymorphisms (SNPs) of genes in CCL5/CCR5 pathway were analyzed by PCR-based direct sequencing. Blood samples were obtained from 57 patients at baseline (BL), day 21 and progressive disease (PD), and serum CCR5, CCR5 ligands (CCL3, CCL4, CCL5) and VEGF-A levels were measured using ELISA. PFS and OS were analyzed using Kaplan-Meier curves and log-rank test. Results: Increased CCL3 levels at PD were associated with longer OS than decreased (12.6 vs 4.8 mos, P = 0.003). Patients with decreased CCL4 levels at day 21 had a trend toward longer PFS and tumor shrinkage. Positive correlation was observed between CCL3 and CCR5 throughout the treatment independent of other ligands (change at day 21: r = 0.426, P = 0.009). There was no significant correlation of CCL3 and CCL4 levels with VEGF-A levels. Patients with the G/G variant in CCL3 rs1130371 had increased CCL3, CCR5 and CCL5 levels at day 21 than any A allele. Similarly, patients with the C/C variant had increased CCL3, CCR5 and CCL5 levels at day 21 compared with those with any A allele. In contrast, both CCL5 rs2280789 G allele and CCL5 rs3817655 T allele were associated with increased CCL3 levels and decreased CCL4 levels at day 21 (P = 0.006, P = 0.043; P = 0.006, P = 0.043). Conclusions: Positive correlation of CCL3, CCR5 and CCL5 impact similarly on CCL3 and CCL4 SNPs, while different manner between CCL3 and CCL4 was found in CCL5 SNPs. This suggests an alternative mechanism of action in the network of CCR5 and the ligands except CCL5-VEGF-A signaling via EPC in mCRC patients receiving regorafenib.

Pharmacological Inhibition of Caspase-8 Suppresses Inflammation-Induced Angiogenesis in the Cornea.

Inflammation-induced angiogenesis is closely related to many diseases and has been regarded as a therapeutic target. Caspase-8 has attracted increasing attention for its immune properties and therapeutic potential in inflammatory disorders. The aim of our study is to investigate the clinical application of pharmacological inhibition of caspase-8 and the underlying molecular mechanisms in inflammation-induced angiogenesis in the cornea. A model of alkali burn (AB)-induced corneal neovascularization (CNV) in C57BL/6 wild-type (WT) mice and toll-like receptor 4 knockout (Tlr4-/-) mice was used. We found that AB increased caspase-8 activity and the pharmacological inhibition of caspase-8 exerted substantial inhibitory effects on CNV, with consistent decreases in caspase-8 activity, inflammatory cell infiltration, macrophage recruitment and activation, VEGF-A, TNF-α, IL-1β, MIP-1, and MCP-1 expression in the cornea. In vitro, caspase-8 mediated TLR4–dependent chemokines and VEGF-A production by macrophages. The TLR4 knockout significantly alleviated CNV, suppressed caspase-8 activity and downregulated expression of inflammatory cytokines and chemokines after AB. Taken together, these findings provide the first demonstration that the pharmacological inhibition of caspase-8 suppresses inflammation-induced angiogenesis and support the use of a pharmacological caspase-8 inhibitor as a novel clinical treatment for CNV and other angiogenic disorders.

Titanium implant surface properties enhance osseointegration in ovariectomy induced osteoporotic rats without pharmacologic intervention.

This study determined whether implant surfaces that promote osseointegration in normal rats can promote osseointegration in osteoporotic rats without pharmacologic intervention. Virgin female 8-month-old CD Sprague Dawley rats (N=25) were ovariectomized. At 6weeks, microstructured/non-nanostructured/hydrophobic, microstructured/nanostructured/hydrophobic, or microstructured/nanostructured/hydrophilic Ti implants (Ø2.5×3.5mm; Institut Straumann AG, Basel, Switzerland) were placed in the distal metaphysis of each femur. At 28days, bone quality and implant osseointegration were assessed using microCT, histomorphometrics, and removal torque values (RTVs). Calvarial osteoblasts were isolated and cultured for 7days on Ø15mm Ti disks processed to exhibit similar surface characteristics as the implants used for the in vivo studies. The phenotype was assessed by measuring the production of osteocalcin, osteoprotegerin, osteopontin, BMP2, VEGF, and RANKL. Microstructured/nanostructured/hydrophilic implants promoted increased bone-to-implant contact and RTVs in vivo and increased osteoblastic marker production in vitro compared to microstructured/non-nanostructured/hydrophobic and microstructured/nanostructured/hydrophobic implants, suggesting that osseointegration occurs in osteoporotic animals, and implant surface properties improve its rate. Although all modified implants were able to osseointegrate in rats with OVX-induced osteoporosis without pharmacologic intervention, the degree of osseointegration was greater around microstructured/nanostructured/hydrophilic implant surfaces. These results suggest that when appropriate microstructure is present, hydrophilicity has a greater influence on Ti implant osseointegration compared to nanostructures. Moreover, modified implant surfaces can exert their control over the altered bone turnover observed in osteoporotic patients to stimulate functional osseointegration. These results provide critical insight for developing implants with improved osseointegration in patients with metabolic disorders of bone remodeling.