Angiogenic Cytokines Research Articles

MicroRNA-646 inhibits angiogenesis of endothelial progenitor cells in pre-eclamptic pregnancy by targeting the VEGF-A/HIF-1α axis.

Pre-eclampsia is a complication that occurs during pregnancy, the pathological feature of which is a change in vascular endothelial homeostasis. microRNA (miR)-646 is an anti-angiogenic miRNA that has been indicated to exhibit potential anti-angiogenic effects in endothelial cells cultured in vitro and in ischemia-induced angiogenesis. However, whether miR-646 has therapeutic potential in placental angiogenesis in pre-eclampsia remains to be determined. In the current study, human peripheral blood-derived endothelial progenitor cells (EPCs) were isolated to study the coordination between miR-646, vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor (HIF)-1α expression in preeclampsia EPCs. EPCs were isolated from human peripheral blood to demonstrate a potential interaction between miR-646 and targets (VEGF-A) in vitro. The number of EPCs and the expression of miR-646 in patients with preeclampsia was detected, and the effects of miR-646 on EPC function and preeclampsia angiogenesis was assessed. Clinical specimens demonstrated that miR-646 expression was enhanced in pregnancy with preeclampsia. The results indicated that miR-646 suppressed EPCs multiplication, differentiation and migration. miR-646 was observed to exert an anti-angiogenic function by suppressing the expression of angiogenic cytokines VEGF-A and HIF-1α. Additionally, luciferase results displayed that miR-646 downregulated VEGF-A expression by directly binding to a specific sequence in its 3'-untranslated region. The results of the current study demonstrated that the miR-646/VEGF-A/HIF-1α axis is significant for angiogenic properties of EPCs in vitro and in vivo placental vasculogenesis. The results of the present study provide a new insight into microRNA regulation of vessel homeostasis and angiogenesis, and a basis for alternative treatments for patients with pre-eclampsia.

Protein-Bound Polysaccharides from Coriolus Versicolor Fungus Disrupt the Crosstalk Between Breast Cancer Cells and Macrophages through Inhibition of Angiogenic Cytokines Production and Shifting Tumour-Associated Macrophages from the M2 to M1 Subtype.

The tumour microenvironment is rich in multiple cells that influence cancer development. Among them, macrophages are the most abundant immune cells, which secrete factors involved in carcinogenesis. Since protein-bound polysaccharides (PBP) from the Coriolus versicolor fungus are believed to inhibit the growth of cancers, in the present study, we investigated whether these PBP influence crosstalk between triple-negative 4T1 breast cancer cells and RAW 264.7 macrophages. 4T1 cells were cultured in conditioned media (CM) collected after: stimulation of the macrophages with PBP (CM-PBP) or incubation of non-treated macrophages (CM-NT). A co-cultured model of both cell lines was also employed to investigate the crosstalk between the cells. Cell viability was measured using the MTT assay. The levels of cytokines and chemokines were determined by ELISA methods. Commercial assay kits were used to assess the activity of both arginase 1 and inducible nitric oxide synthase (iNOS) and the level of cell migration. The results revealed that CM-NT promotes proliferation and migration of 4T1 cells, and increases the secretion of pro-angiogenic factors (VEGF, MCP-1) by cancer cells. In contrast, CM-PBP inhibits 4T1 cell growth and migration, decreases the secretion of pro-angiogenic factors (VEGF, MCP-1) and upregulates the production of pro-inflammatory mediators (IL-6, TNF-α) with certain anti-tumoral properties Moreover, PBP-treated CM significantly decreases the level of M2 macrophage markers (arginase 1 activity, IL-10 and TGF-β concentrations), but upregulates iNOS activity and IL-6 and TNF-α production, which are M1 cell markers. The results suggest that PBP suppress the favourable tumour microenvironment by inhibiting the crosstalk between 4T1 cells and macrophages through the regulation of production of angiogenic and inflammatory mediators, and modulating the M1/M2 macrophage subtype.

Level of VEGF-A and interleukin 6 in lacrimal fluid of patients with diabetic retinopathy

Diabetic retinopathy (DR) is an ocular manifestation characterized by microvascular complications. The aim of this study was to investigate the relationship between diabetic retinopathy (DR) and two lacrimal angiogenic cytokines, interleukin-6 (Il-6) and vascular endothelial growth factor-A (VEGF-A). A cross-sectional study was conducted at Yaoundé Central Hospital from February,1 to June, 15, 2019. Twenty-six diabetic patients with retinopathy and 55 diabetics without retinopathy were enrolled. Tears were collected with Schirmer strips. The concentrations of VEGF-A and Il-6 were determined by the ELISA test. Statistical analysis was performed using Epi Data version 7.1.5.2. and GraphPad 5.0. The mean VEGF-A concentration was 163.4 ± 45.1 and 149± 81.8 pg/ml in patients with and without DR respectively (p= 0.292) while tear Il-6 level was 118.5±103 and 119±79.43 pg/ml in patients with and without DR respectively (p= 0.497). The mean tear VEGF level was 165.2 ± 76.10 and 153.50 ± 57.94 pg/mL in patients with and without diabetic maculopathy (DM) respectively (p=0.343) while tear Il-6 level was significantly lower in DM (54.92±32.68) compared to patients without DM (159.10±89.78) (p=0.047). Tear Il-6 level was significantly low in patients with DM. There was no association between the 2 lacrimal biomarkers and diabetic retinopathy. Further analysis is needed for this type of screening in diabetic patients.Keywords: eye, retina, tear, diabetes, biomarker; cytokine.

Expression of Angiogenic Proteins in Tumor and Stroma Affects Survival in Patients With Gastric Cancer

BackgroundGastric cancer is one of the most frequent malignancies worldwide. Angiogenic growth factors play a crucial role in mediating the crosstalk between cancer cells and the surrounding microenvironment. In this exploratory study, we investigate the impact of angiogenic proteins within the tumor cell or stroma compartment on survival of patients with gastric cancer. Materials and methodsIn 29 patients, tumor and stromal compartments were separated using laser capture microdissection. Angiogenic protein expression was measured using a bead-based immunoassay and correlated with tumor stage and overall survival. ResultsOverall survival was significantly shorter in patients with a high stroma concentration of vascular endothelial growth factor (VEGF)-A (23.5 (±17.6) versus 33.6 (±21.0) mo; P = 0.009) and stem cell factor (22.2 (±18.5) versus 33.6 (±21.8) mo; P = 0.01) compared with patients with a low stroma concentration. High stromal VEGF-D showed a trend toward worse survival (26.8 (±22.0) versus 37.2 (±19.0) mo; P = 0.09). We did not observe any significant correlation between tumor-specific expression of angiogenic cytokines and survival. ConclusionsThis translational study highlights the difference in clinical impact between tumor and stromal expression of angiogenic proteins. Compartment-specific concentrations of VEGF-A and stem cell factor affect the clinical prognosis and help to identify the best therapy for patients with gastric cancer.

FRI0053 PROLIFERATIVE SYNOVITIS, AN ULTRASOUND PATTERN ASSOCIATED WITH ACPA POSITIVE RHEUMATOID ARTHRITIS

Background:Seronegative (sero-) and seropositive (sero+) Rheumatoid Arthritis (RA) have different genetic, immunopathological and vascular morphology features, but no previous studies have analyzed if US characteristics differ between sero+ or sero- RA. Our preliminary studies suggest that sero+ RA is associated with an expansive synovitis pattern that we have called “proliferative synovitis” (PS)Objectives:To analyze potential differences between patients with RA according to their autoantibody status by using ultrasonography (US). We aimed to assess whether PS is associated with ACPA+ ptsMethods:We collected clinical, epidemiological data and bilateral carpal and hand US images of pts with RA. Synovial hypertrophy (SH), Power Doppler signal (PD) and total score (sum of scores of SH and PD) in wrist and hand (1-5 metacarpophalangeal) were assessed. We evaluated the presence of PS, defined as expansive synovial growth encompassing the concepts of synovial SH grade II and III. We performed synovial biopsies of a subgroup of pts using arthroscopy or US guided in order to see immunohistochemistry differences between “proliferative” and “flat” (non-proliferative) synovitis. Serum levels of angiogenic and inflammatory biomarkers were performedResults:Two hundred and five RA patients were collected. Overall, 173 (84.8%) pts were sero+ for RF (68.7%) or ACPA (74.6%), general characteristics are summarized in Table. No significant differences between sero+ and sero- pts in terms of disease activity or therapy were found. PS was present in 55.5% of sero+ pts (55.3% in RF+ and 58.2% in ACPA+ pts) and 16.1% of sero- pts (p=0.0001). Globally, 101 pts (49.2%) had PS. Ninety-six (95.0%) were RF or ACPA positive. Only 5 pts with sero- RA had PS (p=0.001). In the univariate analysis, significantly more pts with PS had erosive disease (72.3% vs 35.0% p=0.0001), higher US scores (p=0.0001) and more of them were taking conventional synthetic Disease-modifying anti-rheumatic drugs (csDMARD) (81.8% vs 69.6% p=0.05). No differences regarding disease activity were found.In the multivariate analysis erosions [OR 4.90 CI 95% (2.17-11.07) p=0.0001] and ACPA [OR 3.5 CI 95% (1.39-10.7) p=0.09] but not RF status [OR 0.74 CI 95% (0.31-1.71) p=0.483] were independently associated with the presence of PS.We immunostained synovial biopsies from 23 pts with PS (13 pts) or non-PS (10 pts). PS was significantly associated with higher density of vessels (p=0.042) and a strong trend to a higher density of B, T, Mast cells and macrophages (figure 1). Significantly higher serum levels of angiogenic (Activin A, bFGF, IL18, IL20, PIGF, SDF-1 and VEGF-D) and pro-inflammatory (IL23) cytokines were found in patients with PS (figure 2).Conclusion:The presence of “proliferative Synovitis” was significantly associated with ACPA and erosive disease in patients with RA. PS pattern also was associated with higher density of synovial vessels and higher serum levels of angiogenic and inflammatory mediatorsTable .Total US pattern p valueN=205Proliferative (N=101)Non proliferative (N=104)Female, n (%)162 (79.4)79 (78.2)83 (80.6)0.57Age, mean (SD) years57.1 (± 14,1)56.3 (± 12.0)58.0 (± 15.9)0.40Current Smoker, n (%)47 (26.9)22 (25.6)25 (28.1)0.73Disease duration, mean (SD) months113.3 (± 105.7)127.7 (± 111.1)99.3 (± 99.3)0.05Erosion, n (%)108 (53.7)73 (72.3)35 (35.0)0.00ACPA, n (%)153 (75.4)89 (89)64 (62.1)0.00RF, n (%)99 (68.3)78 (78)63 (61.2)0.01DAS 28–CRP, mean (SD)2.55 (±1.03)2.66 (±1.04)2.44 (±1.02)0.17GC, n (%)99 (49.3)45 (45.5)54 (52.9)0.32cDMARDs, n (%)152 (75.6)81 (81.8)71 (69.6)0.05bDMARD, n (%)69 (34.3)35 (35.4)34 (33.3)0.76Total US score14.9 (± 11.5)18.8 (± 11.8)11.1 (± 9.9)0.00*ACPA anti-citrullinated protein antibodies, RF rheumatoid factor, DAS28-CRP Disease Activity Score 28-joint count, CRP C-reactive protein, GC glucocorticoids, bDMARD biological disease-modifying antirheumatic drugsDisclosure of Interests:Ana Belén Azuaga-Piñango: None declared, Beatriz Frade-Sosa: None declared, Roberto Gumucio: None declared, Katherine Cajiao: None declared, Andrea Cuervo: None declared, Raquel Celis: None declared, Jose A. Gómez-Puerta Speakers bureau: Abbvie, BMS, GSK, Lilly, Pfizer, Roche, Raimón Sanmartí Speakers bureau: Abbvie, Eli Lilly, BMS, Roche and Pfizer, Juan de Dios Cañete: None declared, Julio Ramirez: None declared

Effect of thyroidectomy on circulating angiogenic cytokines in papillary thyroid carcinoma and benign goiter: Potential for new biomarkers?

BackgroundCirculating angiogenic factors have been associated with clinical outcomes of papillary thyroid carcinoma, although they may also be released in the context of benign multinodular goiter. We sought to investigate the effect of thyroidectomy on the activity and importance of multiple circulating angiogenic factors in papillary thyroid carcinoma and benign multinodular goiter. MethodsBetween May 2015 and December 2016, patients scheduled for total thyroidectomy for papillary thyroid carcinoma or benign multinodular goiter were offered to enroll in this study. Serum levels of angiopoietin-2, fibroblast growth factor-2, hepatocyte growth factor, platelet-derived growth factor-BB, placenta growth factor, heparin-binding epidermal growth factor, and vascular endothelial growth factor-A and -C were collected preoperatively and 2 weeks postsurgery. These levels were measured by enzyme-linked immunosorbent assay and compared with those of 35 healthy control subjects. ResultsSixty patients with a median age of 52 years, 37 of whom were females, were included: 36 had papillary thyroid carcinoma, and 24 had benign multinodular goiter. In both benign multinodular goiter and papillary thyroid carcinoma, preoperative, circulating angiogenic factors levels were increased with respect to controls (P < .0001), and a decrease after total thyroidectomy was observed in the levels of angiopoietin-2 (P < .0001), fibroblast growth factor-2 (P < .0001), hepatocyte growth factor (P < .001), and heparin-binding epidermal growth factor (P < .01 each). Only patients with papillary thyroid carcinomas, however, showed decrease in the postoperative levels of platelet-derived growth factor-BB and vascular endothelial growth factor-A (P = .001 each). ConclusionResults from this study raise the potential for vascular endothelial growth factor-A and platelet-derived growth factor-BB to be used as biomarkers of the effectiveness of treatment of papillary thyroid carcinoma. These results warrant further investigation and may have potential prognostic implications.

Vascular Regeneration in Peripheral Artery Disease.

Peripheral artery disease is a common disorder and a major cause of morbidity and mortality worldwide. Therapy is directed at reducing the risk of major adverse cardiovascular events and at ameliorating symptoms. Medical therapy is effective at reducing the incidence of myocardial infarction and stroke to which these patients are prone but is inadequate in relieving limb-related symptoms, such as intermittent claudication, rest pain, and ischemic ulceration. Limb-related morbidity is best addressed with surgical and endovascular interventions that restore perfusion. Current medical therapies have only modest effects on limb blood flow. Accordingly, there is an opportunity to develop medical approaches to restore limb perfusion. Vascular regeneration to enhance limb blood flow includes methods to enhance angiogenesis, arteriogenesis, and vasculogenesis using angiogenic cytokines and cell therapies. We review the molecular mechanisms of these processes; briefly discuss what we have learned from the clinical trials of angiogenic and cell therapies; and conclude with an overview of a potential new approach based upon transdifferentiation to enhance vascular regeneration in peripheral artery disease.

Angiogenic cytokines can reflect the synovitis severity and treatment response to biologics in rheumatoid arthritis

Angiogenesis and synoviocyte hyperplasia, called ‘pannus,’ are pathologic hallmarks of rheumatoid arthritis (RA). To determine the clinical significance of angiogenic cytokines in RA, the levels of pro-angiogenic cytokines, including VEGF, placenta growth factor (PlGF), and IL-6, were measured in the synovial fluid (SF, n = 54) and sera of RA patients (n = 157) using ELISA. Patients (n = 103) with disease activity score 28 (DAS28) > 3.2, which indicates moderate to high RA activity, underwent follow-up blood sampling at 6 months after treatment with conventional disease-modifying anti-rheumatic drugs (c-DMARD) or biologic DMARD (b-DMARD) including an anti-TNFα antibody, an anti-IL-6 antibody, and abatacept. Ultrasonography (US) was performed on affected joints to define the synovitis severity at the time of sampling. Consequently, in the SF of RA patients, PlGF and IL-6 levels correlated well with synovitis severity determined by US. In RA sera, VEGF and IL-6 levels were elevated in proportion to synovitis severity, correlating with conventional markers for disease activity, including ESR, CRP, and DAS28. In c-DMARD users (n = 53), serially monitored levels of serum VEGF, IL-6, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) all decreased in good and moderate responders but not in nonresponders. In b-DMARD users (n = 49), only serum VEGF well represented the treatment response, while CRP nonspecifically decreased irrespective of the treatment outcome. By multivariable analysis, serum ΔVEGF, but not ΔESR or ΔCRP, was an independent factor associated with good and moderate responses to DMARD. In summary, the angiogenic cytokines PlGF and VEGF represent the synovitis severity of RA assessed by US. In patients receiving b-DMARD, serum VEGF may be more valuable than CRP in reflecting the treatment response.

A scalable and xeno-free bioreactor system for biomanufacturing of hUC-MSCs

Background & Aim Mesenchymal stem/stromal cells (MSCs) are a prominent cell type in cellular therapies with over 900 registered clinical trials. A critical bottleneck in using MSCs, however, is generating the large number of cells required for both clinical trials and commercial therapies. Many commercial therapies will require 100’s of millions to billions of cells. While 2D cell culture may be able to generate sufficient cells for development and early clinical trials, later clinical trials and commercial therapies will require 3D bioreactors in a scalable manufacturing platform. Thus for consistency in the therapeutic product from the development stage through clinical trials and commercial therapies, it is necessary that the state of the cells be comparable across the transition from 2D to 3D culture and then maintained with scale-up in 3D. Here, we establish a scalable 3D manufacturing process using MSCs isolated from human umbilical cord tissue (hUC-MSCs) to generate cells at a consistent PDL that maintain comparable critical quality attributes (CQAs) to 2D flask culture. Methods, Results & Conclusion hUC-MSCs were isolated from the perivascular region of human umbilical cords. Using a xeno-free culture system (medium, reagents and materials), cells were cryopreserved to create a Working Cell Bank (WCB). WCB vials were expanded either in 2D cell stacks or on microcarriers in a small-scale (0.1L) or larger scale (15L) bioreactor. Cells were then assessed for CQAs: final PDL, typical MSC surface marker expression (positive for CD90 and CD166, negative for CD34 and CD45), trilineage differentiation potential (osteogenesis, adipogenesis, and chondrogenesis), and functional properties including IDO secretion and angiogenic cytokine secretion (VEGF, IL-8, bFGF, HGF, TIMP1 and TIMP2). hUC-MSCs were successfully expanded in 2D flasks, a 0.1L bioreactor, and a 15L bioreactor. Cells maintained all the tested CQAs across both 2D and 3D manufacturing platforms as well as with increases in scale in 3D. Therefore, this study establishes a scalable xeno-free manufacturing paradigm to reproducibly generate populations of hUC-MSCs that have consistent properties. Repeatedly generating cell populations with similar attributes is critical for well-designed studies during the development of cellular therapy approaches and as these approaches move towards clinical trials and commercial therapies.

Characterisation of baseline microbiological and host factors in an inception cohort of people with surgical wounds healing by secondary intention reveals circulating IL-6 levels as a potential predictive biomarker of healing

Background: More than 2 million people per year are treated for surgical wounds in the UK. Over a quarter of these wounds are estimated to heal by secondary intention (from the “bottom up”) resulting in further complications and requiring increased healthcare resources. Identification of microbiological or host biomarkers that can predict healing outcomes may help to optimize the management of surgical wounds healing by secondary intention. However, the microbial and host factor heterogeneity amongst this diverse population is completely unexplored. Methods: We demonstrate feasibility of determining presence and levels of wound microbes and systemic host factors in an inception cohort of 54 people presenting with surgical wounds healing by secondary intention, who were subsequently followed-up for a period of 12-21 months. We present descriptive statistics for plasma levels of inflammatory, angiogenic cytokines and microRNAs, and we identify a range of wound colonizing microbes. We tentatively explore association with healing aiming to generate hypotheses for future research. Results: We report a potential correlation between poor healing outcomes and elevated interleukin (IL)-6 plasma levels at presentation (ρ=0.13) which requires confirmation. Conclusions: This study demonstrates the degree of biological heterogeneity amongst people with surgical wounds healing by secondary intention and proves the feasibility of embedding a biomarker discovery study in a cohort study in surgical wounds. Our results are essential for designing large biomarker discovery studies to further investigate the potential validity of circulating IL-6 or other factors as novel predictive biomarkers of healing for surgical wounds healing by secondary intention.

Exercise and insulin-like growth factor1 supplementation improve angiogenesis and angiogenic cytokines in a rat model of diabetes-induced neuropathy.

What is the central question of this study? Do changes in levels of angiogenesis-related mediators [vascular endothelial growth factor-A (VEGF-A), thrombospondin-1 (TSP-1) and nuclear factor-κB (NF-κB)] in the sciatic nerve mediate diabetic neuropathy in the streptozotocin-induced type1 diabetic male rat? Can exercise and insulin-like growth factor1 (IGF-I) treatment improve the diabetes-related decrease in angiogenesis in sciatic nerve in these animals? What is the main finding and its importance? Levels of VEGF-A, TSP-1 and NF-κB change in the sciatic nerve of diabetic rats and might mediate diabetic neuropathy. Treatment with IGF-I and exercise could increase angiogenesis in the diabetic rats by increasing VEGF-A and decreasing TSP-1 and NF-κB expression in the sciatic nerve. Diabetic neuropathy is a severe complication of diabetes that affects 40-50% of diabetic people in the world. The aim of this study was to characterize alterations in angiogenesis and related molecular mediators in the sciatic nerve in diabetic conditions alone or in diabetes in combination withexercise and/or administration of insulin-like growth factor1 (IGF-I). Forty male Wistar rats were assigned into one of five groups, namely control, diabetes, diabetes+exercise, diabetes+IGF-I and diabetes+exercise+IGF-I. Type1 diabetes was induced by i.p. injection of streptozotocin (60mgkg-1 ). After 30 days of treatment with exercise or IGF-I alone or in combination, diabetic neuropathy was evaluated with a hotplate, glycated haemoglobin was measured, angiogenesis was determined by immunostaining for PECAM-1/CD31, and expressions of vascular endothelial growth factor-A (VEGF-A), thrombospondin-1 (TSP-1) and nuclear factor-κB (NF-κB) were determined by enzyme-linked immunosorbent assay.After 4weeks, the diabetes group showed a significant decrease in capillary density and VEGF-A levels, but a significant increasein glycated haemoglobin in blood, TSP-1 and NF-κB levels in the sciatic nerve compared with the control group, and these effects were ameliorated by exercise and IGF-I. However, simultaneous treatment of diabetic rats with IGF-I and exercise did not have any synergistic effects. These findings indicate that diabetes-induced neuropathy may be associated, in part, with decreasedangiogenesis mediated by overproduction of TSP-1 and NF-κB, in addition to reduced production of VEGF-A. The findings also showed that exercise and IGF-I can reduce neuropathy, followed by increased angiogenesis, by changes in TSP-1, NF-κB and VEGF-A production levels.

The miR-590-3p/VEGFA axis modulates secretion of VEGFA from adipose-derived stem cells, which acts as a paracrine regulator of human dermal microvascular endothelial cell angiogenesis.

The paracrine secretion of angiogenic cytokines from adipose-derived stem cells (ADSCs) might promote endothelial cell angiogenesis, therefore promoting wound healing in injured tissues. Hypoxia is one of the common occurrence in injured tissues, during which angiogenesis is enhanced to improve the oxygen supply. In the present study, miR-590-3p, an anti-angiogenic miRNA, was predicted to target VEGFA, a key factor that can be transcriptionally upregulated by HIF1A during ADSC proliferation and tubule formation in response to hypoxic stimulation. Herein, we found that in response to hypoxic stimuli, HIF1A and VEGFA protein expressions were remarkably induced. In addition, ADSC viability was promoted. Incubation with conditioned medium from ADSCs stimulated by hypoxia significantly enhanced the angiogenic ability of human dermal microvascular endothelial cells (HDMECs), while the conditioned medium from VEGFA-silenced ADSCs significantly reversed the angiogenic ability of HDMECs. Regarding the molecular mechanism, it was verified that miR-590-3p binds to VEGFA; miR-590-3p inhibited VEGFA to affect the paracrine regulation by ADSCs, subsequently hindering the HDMEC angiogenesis. More importantly, the consequences of miR-590-3p-overexpressing conditioned medium on HDMEC angiogenesis were partially reversed by VEGFA-overexpressing conditioned medium. In conclusion, miR-590-3-5p/VEGFA axis modulates the paracrine secretion of VEGFA by ADSCs to affect the angiogenesis of HDMECs.

The Epigenetic Profile of Tumor Endothelial Cells. Effects of Combined Therapy with Antiangiogenic and Epigenetic Drugs on Cancer Progression.

Tumors require a constant supply of nutrients to grow which are provided through tumor blood vessels. To metastasize, tumors need a route to enter circulation, that route is also provided by tumor blood vessels. Thus, angiogenesis is necessary for both tumor progression and metastasis. Angiogenesis is tightly regulated by a balance of angiogenic and antiangiogenic factors. Angiogenic factors of the vascular endothelial growth factor (VEGF) family lead to the activation of endothelial cells, proliferation, and neovascularization. Significant VEGF-A upregulation is commonly observed in cancer cells, also due to hypoxic conditions, and activates endothelial cells (ECs) by paracrine signaling stimulating cell migration and proliferation, resulting in tumor-dependent angiogenesis. Conversely, antiangiogenic factors inhibit angiogenesis by suppressing ECs activation. One of the best-known anti-angiogenic factors is thrombospondin-1 (TSP-1). In pathological angiogenesis, the balance shifts towards the proangiogenic factors and an angiogenic switch that promotes tumor angiogenesis. Here, we review the current literature supporting the notion of the existence of two different endothelial lineages: normal endothelial cells (NECs), representing the physiological form of vascular endothelium, and tumor endothelial cells (TECs), which are strongly promoted by the tumor microenvironment and are biologically different from NECs. The angiogenic switch would be also important for the explanation of the differences between NECs and TECs, as angiogenic factors, cytokines and growth factors secreted into the tumor microenvironment may cause genetic instability. In this review, we focus on the epigenetic differences between the two endothelial lineages, which provide a possible window for pharmacological targeting of TECs.

Association Between EphA1 and Tumor Microenvironment in Gastric Carcinoma and its Clinical Significance.

BackgroundWith the growing global burden of gastric carcinoma (GC) and the urgent need for biomolecular targeted therapies, this study aimed to elucidate the relationship between EphA1 and the tumor microenvironment (focusing primarily on the key inflammatory cytokines IL-6 and tumor angiogenic cytokine VEGF) to identify a new potential therapeutic target.Material/MethodsIHC and qRT-PCR were performed to quantify the protein and gene expression levels of EphA1, IL-6, and VEGF in normal mucosal tissues, carcinoma tissues, and paracarcinomatous tissues from 57 GC patients. Spearman’s rank correlation test was performed to determine the relationship between EphA1, IL-6, and VEGF expression levels. The relationships of EphA1 with clinicopathologic parameter and survival in GC patients were also evaluated.ResultsThe protein and gene expression levels of EphA1 were all attenuated gradually from carcinoma tissues to paracarcinomatous tissues and then to normal mucosal tissues in GC patients. Additionally, significant correlations between the overexpression of EphA1 with aggressive clinicopathological features and shorter survival time of GC patients were verified. In particular, we found a significant positive correlation between the expression of EphA1 and tumor microenvironment hallmark proteins IL-6 and VEGF in carcinoma tissues and paracarcinomatous tissues.ConclusionsEphA1 can promote the occurrence and development of GC by its selective high expression in cancer tissues and its relationship with malignant clinical features and prognosis of GC patients. The underlying potential mechanism appears to involve enhancement of the tumor microenvironment, which via drives the expression of tumor microenvironment hallmark proteins IL-6 and VEGF.

Intraocular tumour necrosis factor ligand related molecule 1 A links disease progression of proliferative diabetic retinopathy after primary vitrectomy.

Tumour necrosis factor ligand related molecule 1 A (TL1A), a member of tumour necrosis factor superfamily, has been identified as a crucial regulator for vascular homeostasis and inflammation. However, the function of TL1A in diabetic retinopathy (DR) is largely unknown. This study aims to examine levels of TL1A in serum and intraocular fluid in patients with proliferative diabetic retinopathy (PDR), and to explore the correlation of intraocular TL1A with the prognosis of PDR progression after primary vitrectomy. Seventy-five patients (75 eyes) with PDR who underwent pars plana vitrectomy (PPV) and 19 patients (19 eyes) who received vitrectomy for idiopathic macular holes (IMH) as non-diabetic control group were enrolled in this prospective study. Serum, aqueous and vitreous fluid samples were collected during cataract and PPV surgery. Protein expressions of TL1A as well as other angiogenic and inflammatory cytokines in serum and intraocular fluid were measured. Correlations of intraocular TL1A concentrations with inflammatory cytokines were analyzed. We found both aqueous and vitreous TL1A levels were significantly higher in the PDR group than in control group (Paqueous =0.026; Pvitreous <0.001). Angiogenic and inflammatory cytokines such as VEGF, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β were significantly higher in intraocular fluid in PDR group than in controls, which MCP-1 and MIP-1α showed positive correlation with intraocular TL1A levels. There is no significant difference in the levels of serum TL1A as well as other inflammatory cytokines between PDR patients and controls. Intraocular levels of TL1A were significantly lower in PDR progression group than in the stable group (Paqueous <0.001; Pvitreous <0.001). Multivariate logistic regression analyses revealed that lower levels of intraocular TL1A was an important risk factor for predicting PDR progression after primary PPV (ORaqueous =0.717, Paqueous =0.001; ORvitreous =0.684; Pvitreous =0.002). In conclusion, TL1A and multiple inflammatory cytokines were highly enriched in the intraocular fluid of PDR patients compared with the controls. Lower levels of intraocular TL1A were associated with development of PDR complications after primary PPV and might be used as prognostic factor in predicting the vitrectomy outcome in PDR patients.

Mesenchymal Stem/Stromal Cells from the Placentae of Growth Restricted Pregnancies Are Poor Stimulators of Angiogenesis.

The extensively branched vascular network within the placenta is vital for materno-fetal exchange, and inadequate development of this network is implicated in the pregnancy disorder fetal growth restriction (FGR), where babies are born pathologically small. Placental mesenchymal stem/stromal cells (pMSCs) and placental macrophages both reside in close proximity to blood vessels within the placenta, where they are thought to promote angiogenesis via paracrine mechanisms. However, the relationship between pMSCs, macrophages and placental vascular development has not yet been examined. We aimed to determine if inadequate paracrine stimulation of placental vascular development by pMSCs and macrophages during pregnancy may contribute to the inadequate vascularisation seen in FGR. Media conditioned by MSCs from FGR placentae significantly inhibited endothelial tube formation, compared to conditioned media derived from normal pMSCs. Similarly, macrophages exposed to media conditioned by FGR pMSCs were less able to stimulate endothelial tube formation in comparison to macrophages exposed to media conditioned by normal pMSCs. While MSCs from normal placentae produce a combination of angiogenic and anti-angiogenic cytokines, there were no significant differences in the secretion of the anti-angiogenic cytokines thrombospondin-1, insulin growth factor binding protein-4, or decorin between normal and FGR pMSCs that could explain how FGR pMSCs inhibited endothelial tube formation. Together, these data suggest a dysregulation in the secretion of paracrine factors by pMSCs in FGR placentae. These findings illustrate how cross talk between pro-angiogenic cell types in the placenta may be crucial for adequate angiogenesis.

Characterization of Naturally Occurring Bioactive Factor Mixtures for Bone Regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.

Circulating IL-8 levels as candidate marker of antiangiogenic therapy for metastatic colorectal cancer.

172 Background: Liver metastasis (LM) confers poor prognosis in metastatic colorectal cancer (mCRC). It has not been well understood whether tumor microenvironment (TM) varies throughout cancer treatment in the presence of LM. We therefore investigated the difference of TM between first-line (FL) and savage-line (SL) anti-angiogenic therapies and tested whether specific angiogenic cytokines could predict clinical outcome in mCRC patients. Methods: A combined cohort consisting of two cohorts (N = 125) was included in this study: 71 patients received first-line treatment with bevacizumab (FL cohort: median age, 60; median follow-up, 28.9 mos) and 54 patients received salvage-line regorafenib (SL cohort: median age, 65; median follow-up, 17.2 mos). Blood samples were obtained from all patients at baseline (BL) and during the treatment, and serum levels of nine angiogenic cytokines were measured using ELISA. Progression-free survival and overall survival (OS) were analyzed using Kaplan-Meier curves, log-rank test, and Cox proportional hazard regression. Results: In univariate analysis for the combined cohort, right-sided CRC, primary unresected, KRAS wild-type, LM, metastatic site ≥2 and SL were associated with shorter OS, and LM and SL remained in multivariable analysis. Ang-2 and IL-8 levels at BL were significantly associated with LM and their cut-off values were 2190.3 pg/ml (COAng2) and 15.1 pg/ml (COIL8), respectively, and no significant interaction was shown between cytokines and cohorts. Patients with Ang-2 levels ≥COAng2 had shorter OS than those with Ang-2 levels &lt; COAng2 (HR2.57, 95%CI: 1.47–4.51, P = 0.001), while IL-8 levels ≥COIL8 was associated with shorter OS than &lt; COIL8 (HR4.31, 95%CI: 2.11–8.79, P &lt; 0.001). IL-8 levels ≥COIL8 remained as significant in multivariable analysis (HR 3.24, 95%CI: 1.47–7.16, P = 0.004). In change analysis, there was no significant correlation of variation of IL-8 and Ang-2 levels with clinical outcome after the first treatment cycle in both cohorts. Conclusions: Circulating IL-8 and Ang-2 levels reflect the TM in the presence of LM, of which IL-8 may serve as solid prognostic marker of anti-angiogenic therapy regardless of the timing of treatment for mCRC.

Abstract TP487: Comprehensive Profiling of Secreted Factors in the Cerebrospinal Fluid of Moyamoya Disease Patients

Background: Moyamoya disease (MMD) is a chronic cerebrovascular disease characterized by progressive occlusion of the internal carotid arteries leading to ischemic and hemorrhagic events. The underlying pathogenesis of MMD remains unclear. In this study we investigated key secreted molecules in the cerebrospinal fluid (CSF) of MMD patients including relation to the extent of neovascularization and eventual functional outcomes. Methods: This study included intraoperative CSF collected from 32 controls (Chiari malformation and cranial nerve hyperactive syndromes) and 73 MMD patients (ischemic: 39; hemorrhagic: 34) that underwent combined direct and indirect superficial temporal artery-middle cerebral artery revascularization procedure. 62 factors were analyzed using multiplex luminex assay, including angiogenic growth factors, cytokines, and chemokines. Cellular retinoic acid binding protein (CRBP1) was also measured using ELISA. Functional outcome was assessed using the modified Rankin scale score (mRS). Matsushima criteria was used for grading the neovascularization. Results: Forty molecules were significantly elevated in both MMD subtypes in comparison with controls. The common top highly secreted CSF proteins included platelet-derived growth factor bb, chemokine ligand 5 and plasminogen activator inhibitor 1 (p&lt;0.001). CRBP1 and other growth factors such as brain-derived nerve growth factor and basic fibroblast growth factor were also elevated in both MMD subtypes (p&lt;0.01). Our results also revealed a number of proteins not previously reported in MMD. Ingenuity pathway analysis indicated that the ischemic and hemorrhagic MMD subtypes exhibited similar cellular/molecular functions and pathways including cellular activation, migration and inflammatory response. Interestingly, while neuro-inflammation and dendritic cell pathways were activated in MMD patients, lipid signaling pathways involving nuclear receptors PPAR and LXR/RXR signaling were inhibited. Conclusions: CSF analysis revealed that a number of cytokines, chemokines and growth factors are elevated in both MMD subtypes. These molecules potentially contribute to the pathogenesis of MMD and could be potential future therapeutic targets.

Modifiers of Endothelial Permeability in the Setting of CAR-t Therapy Related Immune Cell Associated Neurotoxicity Syndrome

Background Chimeric Antigen Receptor T-cell therapy (CAR-t) for patients with relapsed or refractory B cell precursor acute lymphoblastic leukemia (ALL) is associated with high overall remission rates but also with significant complications. Cytokine release syndrome (CRS) and Immune Cell Associated Neurotoxicity Syndrome (ICANS) are life-threatening sequelae that may occur in CAR-t recipients. ICANS presents with varying symptoms ranging from confusion to seizure and cerebral edema. There is no consensus on specific biological events that incite ICANS. Blood brain barrier (BBB) permeability is altered in CD19 CAR-t therapy, but the cause of altered BBB permeability is not clear. IL6 targeted interventions that control symptoms of CRS are ineffective against ICANS, suggesting these entities have separate pathologic mechanisms. Criteria for ICANS severity grading and guidelines for supportive care management are published, but there is no universal predictor of ICANS. Identification of manipulable biologic factors to predict the onset of ICANS would represent significant progress for clinical management of ICANS. Methods We collected plasma samples from 12 adult patients with varying ICANS severity following CAR-t therapy, aged 24-74 years. Diagnoses were diffuse large B cell lymphoma (DLBCL), transformed follicular lymphoma (TFL), and primary mediastinal B cell lymphoma (PMBCL) in varying stages of progression or relapse. Pre-CAR-t, midpoint and 7 or 8 day post CAR-t samples were evaluated with multiplex cytokine arrays to quantify relative levels of 105 angiogenic cytokines. Results were compared between two groups: patients who did not develop ICANS (n=5), and patients who developed any grade of ICANS (n=7; 2 patients grade 0-2, 5 patients grade 3-4). Results There was a trend toward higher baseline relative levels of two angiogenic factors, ApoA1 and Angiogenin, in patients with ICANS relative to those without. Interestingly, ApoA1 increased over time in patients who did not develop ICANS; a 320 +/- 56 % increase in ApoA1 occurred from baseline to day 7/8. In contrast, ApoA1 decreased to 88 +/- 25% of baseline by day 7/8 in patients who developed ICANS (No ICANS vs ICANS; p=0.003). Similarly, Angiogenin increased by 289 +/- 77% over baseline by day 7/8 in patients who did not develop ICANS, while it decreased to 98 +/- 24 % of baseline by day 7/8 in patients who developed ICANS (No ICANS vs ICANS, p = 0.03). Conclusion Our data suggests that ApoA1 and Angiogenin levels at baseline, and changes in ApoA1 or Angiogenin over time, may indicate risk of ICANS. This correlation requires validation in a larger patient population as well as quantification of absolute ApoA1 and Angiogenin concentrations in blood. If ApoA1 and Angiogenin baseline levels are indeed correlated with the risk of ICANS, this may represent major progress in clinical management of ICANS.