Angiogenic Cytokines Research Articles

VEGF and FGF-2: Promising targets for the treatment of respiratory disorders.

The endothelial cells play a crucial role in the progression of angiogenesis, which causes cell re-modulation, proliferation, adhesion, migration, invasion and survival. Angiogenic factors like cytokines, cell adhesion molecules, growth factors, vasoactive peptides, proteolytic enzymes (metalloproteinases) and plasminogen activators bind to their receptors on endothelial cells and activate the signal transduction pathways like epidermal growth factor receptor (EGFR phosphatidylinositol 3-kinase and (PI3K)/AKT/mammalian target of rapamycin (mTOR) which initiate the process of angiogenesis. Cytokines that stimulate angiogenesis include direct and indirect proangiogenic markers. The direct proangiogenic group of markers consists of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) and hepatocyte growth factor (HGF) whereas the indirect proangiogenic markers include transforming growth factor-beta (TGF-β), interleukin 6 (IL-6), interleukin 8 (IL-8) and platelet-derived growth factor (PDGF). VEGF and FGF-2 are the strongest activators of angiogenesis which stimulate migration and proliferation of endothelial cells in existing vessels to generate and stabilize new blood vessels. VEGF is released in hypoxic conditions as an effect of the hypoxia-inducible factor (HIF-1α) and causes re-modulation and inflammation of bronchi cell. Cell re-modulation and inflammation leads to the development of various lung disorders like pulmonary hypertension, chronic obstructive pulmonary disease, asthma, fibrosis and lung cancer. This indicates that there is a firm link between overexpression of VEGF and FGF-2 with lung disorders. Various natural and synthetic drugs are available for reducing the overexpression of VEGF and FGF-2 which can be helpful in treating lung disorders. Researchers are still searching for new angiogenic inhibitors which can be helpful in the treatment of lung disorders. The present review emphasizes on molecular mechanisms and new drug discovery focused on VEGF and FGF-2 inhibitors and their role as anti-angiogenetic agents in lung disorders.

Abstract 140: Efficacy Evaluation of Transplantation of Three-Dimensional Adipose-derived Stem Cell Sheet With Enhanced Angiogenesis Into Cardiovascular Disease

Integration of adipose-derived stem cells (ASCs) and cell sheet engineering technology has received much interest in the regenerative medicine. First of all, sphere formation of ASCs (sph-ASCs) were successfully produced by poly-2-hydroxyethyl methacrylate (poly-HEMA)-coated plates as we previously reported. Sph-ASCs showed increased expression of angiogenic growth factors and cytokine secretion compared to adherent ASCs. Significantly increased expression of HIF-1α and decreased expression of cleaved caspase-3 were detected in sph-ASCs compared to adherent ASCs. Moreover, ASC sheets were formed using thermo-responsive plates and sph-ASCs were seeded to produce ASC sheets covered with sph-ASCs (sph-ASC sheets). After 48 hrs of incubation, the cross-section of sph-ASC sheets revealed mixed and multi-layered of sph-ASCs and ASC sheets. Sph-ASC sheets showed the positive expression of HIF-1α and angiogenic markers such as FGF-2, CD31, PDGFb, and CD144, especially higher in sph-ASC-covered sites. In addition, cytokine arrays of secretome revealed that sph-ASCs and sph-ASC sheets showed augmented angiogenic potential compared to adherent ASCs. Furthermore, four groups of AMI induction only (sham), intramyocardial injection of sph-ASC, ASC sheet transplantation, and sph-ASC sheet transplantation were compared using acute myocardial infarction (AMI) rat models. After 6 weeks of transplantation, echocardiography revealed that significantly improved ejection fraction and fraction shortening in sph-ASC sheet group compared to sham, sph-ASC, and ASC sheet groups. In conclusion, transplantation of sph-ASC sheets showed enhanced neovascularization in AMI animal models. These results indicated that the use of ASC sheets as a novel tissue engineering approach to improve the cardiac and vascular function.

Abstract 787: Rose - A Novel Vascular Anti-inflammatory Agent

Introduction: Inflammation is common in clinical practice and often results in significant complications. The study was performed in search of a novel anti-inflammatory agent for inflammation modulation which would be useful for cardiovascular disorders and various clinical scenarios. Methods: A crushed red rose extract was prepared from the petals, and it was processed for analysis. The extract was tested on HUVEC cells at various concentrations. By microscopic analysis of cells, a safe concentration was identified, and the levels below the safe limit were tested at 72 hours and seven days for selected cytokines secretion. Results: Majority of the tested Inflammatory cytokine secretion was reduced by the treatment of red rose extract on the cells. VEGF and angiogenic cytokine levels were reduced, but VEGF-R levels were maintained after the cell treatment. Below the safe concentration limit of the extract there were only minimal changes in the cytokines levels tested at various dilutions of the extract.The study results show a significant reduction in the secretion of anti-inflammatory cytokines by red rose extract. The effects of rose extract inhibited major inflammatory cytokines like IL-1, IL-2, RANTES, IGF-1, IL-8 etc. MCP 3 (CCL7) and PDGF BB levels were less than 10% of the baseline values. There was also a tendency in fall at 72 hours, and thereafter a rise at 7 day levels observed in the values of some cytokines. Conclusion: There is potential for a red rose extract treatment in the regulation of inflammatory cytokines secretion. Further studies need to be performed to identify the benefits and side effects profile.

HIF1A/miR-20a-5p/TGFβ1 axis modulates adipose-derived stem cells in a paracrine manner to affect the angiogenesis of human dermal microvascular endothelial cells.

Angiogenic cytokines secreted by the adipose-derived stem cells (ADSCs) might promote the angiogenesis of endothelial cells. In the present study, we hypothesize that miR-20a targets TGFB1 to modulate the transforming growth factor β1 (TGFβ1) secretion by ADSCs, therefore affecting the angiogenesis. We found that hypoxia-inducible factor 1A (HIF1A) and TGFβ1 expressions were increased by hypoxia, accompanied with promoted ADSC cell viability. Incubation with conditioned medium from ADSCs treated with hypoxia significantly enhanced the angiogenesis capacity of human dermal microvascular endothelial cells (HDMECs), while TGFB1-silenced ADSCs medium significantly reverses HDMECs angiogenesis. miR-20a suppresses the expression of TGFB1 and secretion of TGFβ1 by ADSCs via binding to its 3'untranslated region, therefore modulating the HDMEC angiogenesis via affecting the paracrine from ADSCs; the effects of miR-20a-overexpressed conditioned medium on HDMEC angiogenesis were significantly reversed by TGFB1-overexpressed conditioned medium. Finally, HIF1A suppressed the expression of miR-20a via targeting its promoter region, subsequently promoting the paracrine from ADSCs and HDMEC angiogenesis.

Serum PlGF and EGF are independent prognostic markers in non-metastatic colorectal cancer

The aim of this study was to determine the prognostic value of circulating angiogenic cytokines in non-metastatic colorectal cancer (CRC) patients. Preoperative serum samples of a training (TC) (n = 219) and a validation cohort (VC) (n = 168) were analyzed via ELISA to determine PlGF, EGF, VEGF, Ang1, PDGF-A, PDGF-B, IL-8 and bFGF levels. In addition, survival was correlated with PlGF and EGF expression measured by microarray and RNAseq in two publicly available, independent cohorts (n = 550 and n = 463, respectively). Prognostic values for overall (OS) and disease-free survival (DFS) were determined using uni- and multivariate Cox proportional hazard analyses. Elevated PlGF is predictive for impaired OS (TC: HR 1.056; p = 0.046; VC: HR 1.093; p = 0.001) and DFS (TC: HR 1.052; p = 0.029; VC: HR 1.091; p = 0.009). Conversely, elevated EGF is associated with favorable DFS (TC: HR 0.998; p = 0.045; VC: HR 0.998; p = 0.018) but not OS (TC: p = 0.201; VC: p = 0.453). None of the other angiogenic cytokines correlated with prognosis. The prognostic value of PlGF (OS + DFS) and EGF (DFS) was confirmed in both independent retrospective cohorts. Serum PlGF and EGF may serve as prognostic markers in non-metastatic CRC.

Abstract 101: Pentraxin-3 exacerbates glioblastoma invasion and angiogenesis through IL8-VEGF signaling in tumor microenvironment: PTX3 is a potential target for GBM suppression

Abstract Background: Glioblastoma (GBM) is highly invasive and resistant to multimodal treatment due to distorted vasculature and aggravated inflammation in a tumor microenvironment rich in dysregulated cytokines and growth factors. This phenomenon is partly attributed to GBM stem cells (GSC) that release angiogenic and inflammatory cytokines. The glycoprotein pentraxin-3 (PTX3), a modulator of inflammation, is correlated with disease severity in some cancers. However, its role in GBM and the molecular mechanisms of PTX3-mediated oncogenesis remain unclear. In this study, we examined the role of PTX3 in GBM growth and invasion, using in vitro and in vivo models. Methods: Patient derived GSC lines were isolated and validated. Tumor tissue micro arrays (TMA) were used to determine the association of PTX3 expression with patient GBM specimens representing various degree of disease. Intracranial tumor xenograft/orthotopic mouse models, cell culture models, proteomic profiling, immunohistochemistry, molecular and biochemical approaches were used. Results: Proteomic profiling identified increased PTX3 expression in GSCs as compared to GBM lines, and correlated with the degree of GSC invasiveness in an orthotopic tumor xenograft model. Increased PTX3 correlated with cell cycle progression and survival under hypoxic micro-environment. Conditioned media obtained from PTX3-over-expressing U87 cell line enhanced pro-angiogenic tube formation from endothelial cells. TMA screening showed that PTX3 expression is substantially increased in more than 70% of GBM specimans. Furthermore, PTX3 over-expression significantly increased survival of U87 cells under hypoxic conditions and resulted in increased tumor growth and invasion in an intracranial tumor xenograft mouse model. Upregulation of PTX3 in these tumors was associated with increased levels of inflammatory and angiogenic markers including interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), but decreased levels of thrombospondin1, a major anti-angiogenic factor. Downstream activation of PI3K/Akt - NFkB signaling pathways was also observed. Conclusion: PTX3 is dysregulated in GBM, and may augment inflammation and angiogenesis in tumor microenvironment. Thus, PTX3 may serve as an inflammatory marker for GBM microenvironment and also may represent a potential therapeutic target for GBM suppresion. Citation Format: Umadevi V. Wesley, Paul Clark, Ian Sutton, John Kuo, Robert Dempsey. Pentraxin-3 exacerbates glioblastoma invasion and angiogenesis through IL8-VEGF signaling in tumor microenvironment: PTX3 is a potential target for GBM suppression [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 101.

The impact of exercise on growth factors (VEGF and FGF2): results from a 12-month randomized intervention trial

BackgroundVascular endothelial growth factor (VEGF) and Fibroblast growth factor-2 (FGF2) are angiogenic cytokines in normal tissues and tumors. Evidence suggests that increased growth factor expression in adipose tissue leads to improved vascularity and decreased hypoxia, fibrosis, and inflammation, which may, in turn, reduce post-menopausal breast cancer risk.ObjectiveWe investigated whether or not exercise had dose-response effects on levels of plasma VEGF and FGF2 in postmenopausal women.MethodsFour hundred previously inactive but healthy postmenopausal women aged 50–74 years of age were randomized to 150 or 300 min per week of aerobic exercise in a year-long exercise intervention. VEGF and FGF2 were measured from fasting serum samples with a custom-plex multiplex assay.ResultsA high compared to moderate volume of aerobic exercise did not cause chronic changes in plasma VEGF or FGF2 levels in intention-to-treat or per-protocol analyses.ConclusionsWe did not detect differences in growth factor levels related to increasing doses of exercise. It is unlikely that changes in VEGF and FGF2 levels mediate the reduction in risk of post-menopausal breast cancer development in associated with increased levels of exercise.Trial registrationClinicaltrials.gov identifier: NCT01435005.

Gingival fibroblasts and medication-related osteonecrosis of the jaw: Results by real-time and wound healing in vitro assays

ObjectiveThis study investigated the effects of bisphosphonates and denosumab on human gingival fibroblasts (HGFs) that could influence inflammation, wound healing, and angiogenesis in medication-related osteonecrosis of the jaw (MRONJ). MethodsA real-time in vitro assay was performed on HGFs with and without the addition of bacterial lipopolysaccharide and a mononuclear cell co-culture to observe the effects of zoledronate, ibandronate, alendronate, clodronate, denosumab, and combinations of zoledronate and denosumab at varied concentrations. A wound healing assay was performed, and gene and protein expression was analyzed for inflammatory, angiogenic, and osteoclastogenic cytokines and mediators including interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNFα), IL-8, vascular endothelial growth factor (VEGF), RANKL, and osteoprotegerin. ResultsHigher concentrations of antiresorptives resulted in impaired wound healing and HGF death, which also occurred without mechanical damage. These effects were increased with bacterial lipopolysaccharide and mononuclear cells. Increased levels of IL-1β, TNFα, IL-8, VEGF, osteoprotegerin, and decreased levels of IL-6 were observed. ConclusionsAntiresorptive exposure was associated with HGF death and delayed wound healing, which could be attributed to an elevated inflammatory response and immune dysfunction contributing to MRONJ development. There was no evidence of anti-angiogenic effects. Our experiments present the first results of denosumab with HGFs.

Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration

Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

Biodegradable polymerized simvastatin stimulates bone formation

Previous research from our labs demonstrated the synthesis of polymerized simvastatin by ring-opening polymerization and slow degradation with controlled release of simvastatin in vitro. The objective of the present study was to evaluate the degradation and intramembranous bone-forming potential of simvastatin-containing polyprodrugs in vivo using a rat calvarial onlay model. Poly(ethylene glycol)-block-poly(simvastatin) and poly(ethylene glycol)-block-poly(simvastatin)-ran-poly(glycolide) were compared with simvastatin conventionally encapsulated in poly(lactic-co-glycolic acid) (PLGA) and pure PLGA. The rate of degradation was higher for PLGA with and without simvastatin relative to the simvastatin polyprodrugs. Significant new bone growth at the circumference of poly(ethylene glycol)-block-poly(simvastatin) disks was observed beginning at 4 weeks, whereas severe bone resorption (4 weeks) and bone loss (8 weeks) were observed for PLGA loaded with simvastatin. No significant systemic effects were observed for serum total cholesterol and body weight. Increased expression of osteogenic (BMP-2, Runx2, and ALP), angiogenic (VEGF), and inflammatory cytokines (IL-6 and NF-ĸB) genes was seen with all polymers at the end of 8 weeks. Poly(ethylene glycol)-block-poly(simvastatin), with slow degradation and drug release, controlled inflammation, and significant osteogenic effect, is a candidate for use in bone regeneration applications. Statement of SignificanceTraditional drug delivery systems, e.g., drug encapsulated in poly(lactic-co-glycolic acid) (PLGA), are typically passive and have limited drug payload. As an alternative, we polymerized the drug simvastatin, which has multiple physiological effects, into macromolecules (“polysimvastatin”) via ring-opening polymerization. We previously demonstrated that the rate of degradation and drug (simvastatin) release can be adjusted by copolymerizing it with other monomers. The present results demonstrate significant new bone growth around polysimvastatin, whereas severe bone loss occurred for PLGA loaded with simvastatin. This degradable biomaterial with biofunctionality integrated into the polymeric backbone is a useful candidate for bone regeneration applications.

Scaling a xeno-free fed-batch microcarrier suspension bioreactor system from development to production scale for manufacturing XF hMSCs

Background & Aim With over 800 clinical trials investigating the use of mesenchymal stem/stromal cells (hMSCs) for regenerative medicine, there is an urgent need for the development of economical biomanufacturing processes capable of generating billions to trillions of cells per manufacturing lot in order to meet the demand of future commercial applications. Innovative manufacturing technologies, such as suspension bioreactors, show great promise in reaching commercially-viable working volumes; however, scalability of cell production within such systems remain a challenge, hindering widespread adoption of this culture system for hMSCs. The Quality by Design (QbD) approach was used to develop a scalable xeno-free (XF) hMSC bioreactor process that maintains the final cell population doubling level (PDL) within the recommended range (16-20) to ensure product quality, even as the bioreactor culture volume is scaled to hundreds of liters. The strategic XF bioprocess was designed using high volume XF cell banks, an optimized XF fed-batch media system, and XF microcarriers combined with a scalable bioreactor system to meet design criteria and streamlined production at different culture scales. Methods, Results & Conclusion In this study, the ability to scale XF hBM-MSC expansion in low shear single-use, Vertical-Wheel suspension bioreactors (PBS Biotech) was evaluated in systems of different scale including: small scale (0.1L), development scale (3L), pilot scale (15L), and production scale (50L) reactors. Cell densities greater than 500,000 cells/mL (with >95% viability) were achieved within 4 to 5 days of fed-batch culture at each bioreactor scale. Scalability of the bioreactor culture process was supported by comparable nutrient and waste metabolite profiles, cell growth curves, and pH observed in each system. Furthermore, harvested cells from each bioreactor scale demonstrated comparable critical hMSC quality attributes including angiogenic cytokine (FGF, HGF, IL-8, TIMP-1, TIMP-2, and VEGF) secretion, tri-lineage differentiation potential, and inducible immunomodulatory potential (as measured by functional IDO activity) compared to control cells of similar PDL cultured in 2D. The data generated by this study demonstrates the expansion of XF hBM-MSCs in a scalable bioreactor culture system, providing significant time and cost savings as a plug-and-play standardized system for translational researchers and product developers in the regenerative medicine, tissue engineering, and cell therapy fields.

Nailfold capillary changes in adult new-onset dermatomyositis: a prospective cross-sectional study.

It is to prospectively analyze nailfold capillaroscopy (NC) findings in new-onset dermatomyositis (DM) and to correlate NC findings with serum angiogenic cytokines and DM clinical and laboratory features. Twenty-three patients with DM who experienced < 12months of symptoms were included in the study. To assess serum cytokine levels, 23 age-, sex-, and ethnicity-matched healthy volunteers were used. NC characteristics and DM activity parameters were analyzed. Significantly higher serum angiogenin (ANG) and vascular endothelial growth factor-1 (VEGF1) levels were observed in DM patients than in controls. Capillary density and avascular areas correlated positively and negatively, respectively, with serum levels of ANG. Moreover, the capillary density correlated inversely with the number of enlarged and giant capillaries and avascular areas. The number of enlarged capillaries correlated positively with patient and physician visual analogue scales (VAS), the presence of a facial rash, giant capillaries, and microhemorrhages. Giant capillaries had a positive correlation with physician and cutaneous VAS, enlarged capillaries, avascular areas, microhemorrhages and bushy capillaries, and a negative correlation with capillary density. Microhemorrhages correlated positively with the "V-neck" sign and physician VAS. VEGF1 showed no relationship with the NC parameters with DM-related clinical and laboratory features. Additionally, 15 out of 23 patients were assessed prospectively after 3.21years. All patients had a major clinical response with significant improvement in all NC parameters, except for enlarged and bushy capillaries. The NC may be a useful tool to assess disease activity in recent-onset DM, and it can also reinforce the role of ANG in the angiogenesis of this myopathy.

Mussel-Inspired Nanostructures Potentiate the Immunomodulatory Properties and Angiogenesis of Mesenchymal Stem Cells.

The therapeutic effects of mesenchymal stem cells (MSCs)-material constructs mainly come from the secretion of trophic factors from MSCs, especially the immunomodulatory and angiogenic cytokines. Recent findings indicate the significance of topographical cues from these materials in modulating paracrine functions of MSCs. Here, we developed functionalized three-dimensional-printed bioceramic (BC) scaffolds with a mussel-inspired surface coating in order to regulate the paracrine function of adipose-derived MSCs (Ad-MSCs). We found that Ad-MSCs cultured on polydopamine-modified BC scaffolds (DOPA-BC) significantly produced more immunomodulatory and pro-angiogenic factors when compared with those cultured on BC scaffolds or microplates. Functional assays, such as endothelial progenitor cells migration, tube formation, and macrophage polarization, were performed to confirm the enhanced paracrine functions of the secreted trophic factors from Ad-MSCs cultured on DOPA-BC scaffolds. Further investigation identified that both focal adhesion kinase- and extracellular signal-related kinase signaling were the required mechano-transduction pathways through which the mussel-inspired surface stimulated the paracrine effect of Ad-MSCs. In a diabetic skin-defect-healing model in rats, conditioned medium received from the Ad-MSCs cultured on DOPA-BC sped wound closure, enhanced vascularization, and promoted macrophage switching from a proinflammatory M1 to a pro-healing and anti-inflammatory M2 phenotype in the wound bed. These results demonstrate that a bio-inspired coating with polydopamine represents an effective method to enhance the paracrine function of MSCs. Our findings illustrate a novel strategy to accelerate tissue regeneration by guiding the paracrine-signaling network.

Decidual stromal cells promote the differentiation of CD56bright CD16- NK cells by secreting IL-24 in early pregnancy.

Decidual stromal cells (DSCs) are important origins of cytokines to modulate maternal-fetal immunotolerance and provide a feasible environment for embryo implantation and development. Interleukin (IL)-24 is a multifunctional cancer killing cytokine and a pleiotropic immunoregulator with complex potency according to tissue or cell types. Its role in establishment and maintenance of normal pregnancy is largely unknown. The aim of our study was to investigate the function and significance of IL-24 and its receptor in the coordination between DSCs and natural killer cells (NK) in early pregnancy. The levels of IL-24 in DSC, endometrial stromal cell (ESC), peripheral blood NK cells(pNK), or decidual NK cells(dNK) culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA), and the levels of IL-24 receptors were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry assays. The effect of IL-24 on the functions of decidual NK cells was analyzed by flow cytometry assays in vitro. The concentration of IL-24 in culture supernatant of DSCs was significantly higher than that of ESCs. Both eNK (endometrial NK cells) and dNK highly expressed IL-24 receptors (IL-20R1 and IL-22R1), especially on CD56dim eNK. However, there were extremely low levels of IL-20R1 and IL-22R1 on pNK. Recombinant human IL-24 or DSCs-secreted IL-24 downregulated the levels of CD16, Granzyme B, perforin, and interferon (IFN)-γ and upregulated the levels of inhibitory receptors killer-cell immunoglobulin-like receptor (KIR)2DL1 and KIR3DL1, or immunotolerant or angiogenic cytokines (eg, transforming growth factor (TGF)-β, IL-10, and IL-8), and elevated the percentage of CD56bright CD16- dNK in vitro. These data suggest that DSCs promote the differentiation of CD56bright CD16- NK with high levels of inhibitory receptors, immunotolerant, and angiogenic cytokines by secreting IL-24 during decidualization in early pregnancy.

A unique protein kinase C‐dependent pathway for tissue factor downregulation in pericytes

Essentials Many mediators increase tissue factor (TF) expression in a wide variety of cell types. The only known example of TF downregulation is by pericytes during wound healing angiogenesis. Downregulation of TF mRNA and protein in cultured pericytes is Protein Kinase C (PKC) dependent. Pericyte TF regulation is unique, since PKC mediates increased TF in all other cell types tested. SUMMARY: Background Embryonic and tumor-associated angiogenesis are linked to elevated expression of the procoagulant transmembrane receptor tissue factor (TF). In contrast, we have reported that high baseline TF expression by perivascular cells (pericytes) is dramatically reduced during angiogenesis at sites of wound healing. This is the only setting in which active TF downregulation has been reported, thus revealing a novel mechanism of TF regulation. Objectives To define the mechanisms underlying the unique pattern of TF expression in pericytes. Methods TF expression in primary cultures of human pericytes is not altered by angiogenic cytokines or growth factors, but is actively downregulated by phorbol 12-myristate 13-acetate (PMA). We characterized TF transcription, protein stability and trafficking in response to PMA. Results Exposure to PMA reduced TF mRNA synthesis and shortened the half-life of TF protein from 11h to 4.5h. Addition of PMA rapidly triggered endocytosis of cell surface TF, followed by degradation in lysosomes. Cell surface TF coagulant activity was maintained until internal stores were depleted. Reduction of TF transcription, TF endocytosis and enhanced degradation of TF protein were all blocked by broad-spectrum inhibitors of protein kinaseC (PKC). This was a surprising finding, because PKC activation increases TF expression in other cell types that have been tested. Conclusions The unique PKC-dependent pathway of TF downregulation in pericytes suggests that TF downregulation may play a functional role in angiogenesis. Distinct pathways regulating pathological and physiological TF expression could be utilized to modulate TF expression for therapeutic purposes.

Rapamycin Rescues Endoplasmic Reticulum Stress-Induced Dry Eye Syndrome in Mice.

To investigate whether rapamycin protects tear production and the ocular surface during endoplasmic reticulum (ER) stress-induced dry eye syndrome in mice. Tunicamycin was injected intraperitoneally in BALB/c mice without or with rapamycin (TM or RM5 group). Peritoneal injection of PBS performed in vehicle group. Group without injection served as control. Blinking rate, fluorescein staining score (FSS), and phenol red thread tear production test were measured at 4 days, 1 week, and 2 weeks after treatment. Levels of inflammatory and angiogenic cytokines were measured by ELISA. Blinking rate and FSS were elevated, and tear production was decreased in TM group compared with controls (P < 0.05 for all), which was ameliorated by rapamycin at 1 and 2 weeks. Levels of inflammatory and angiogenic cytokines in the cornea and lacrimal glands were higher in the TM group than controls, and lower in the RM5 group than the TM group at 1 and 2 weeks (P < 0.05 for all). Rapamycin protected tear production and the ocular surface against this dry eye syndrome by ameliorating ER stress-induced vascular damage and inflammation of lacrimal glands and the ocular surface.

RRx-001 is a phase III aerospace-derived small molecule that immunonormalizes the tumor microenvironment.

156 Background: Tumor associated macrophages (TAMs) with M2-like phenotypes produce angiogenic factors and cytokines which lead to vascular immaturity. M2 cytokines also impair immune cell activity. CD47 and SIRP⍺ are innate immune checkpoints which inhibit macrophage phagocytosis and enhance M2 polarization. Disruption of the CD47/SIRP⍺ axis polarize TAMs away from an M2-like phenotype to normalize tumor vessels and stimulate antitumor immunity. Normalized vessels facilitate delivery of drugs and immune cells and also sensitizes tumors to therapy and promotes M1-like TAM phenotype. RRx-001 modulates TAM infiltration and phenotype via CD47/SIRP⍺ downregulation, which normalizes both the tumor vasculature, and the immune cell milieu. Methods: The effect of RRx-001 on intratumoral blood flow was evaluated both preclinically and clinically with dynamic contrast MRI. An in vitro phagocytotic assay was used to determine whether RRx-001 promoted engulfment of A549 tumor cells by macrophages. Transcriptional mRNA profiling in murine tumor associated macrophages (TAMs) was performed to analyze the cytokine profile of TAMs in the presence or absence of RRx-001. Athymic mice with intracranial GBM43 were administered RRx-001 (10 mg/kg) followed 4 hours later by 0.4 mg of irinotecan by tail vein on days 12, 18, 24 and 30 after implantation of tumor cells. Mice were euthanized 24 hours after last irinotecan administration, with brains immediately resected and tumor tissue dissected prior to snap-freezing by immersion in liquid nitrogen. Results: RRx-001 was shown to induce: vascular normalization with increased intratumoral perfusion, dual downregulation of CD47 and SIRP⍺, increased phagocytosis, improved delivery of chemotherapy and increased T cell infiltration. Conclusions: RRx-001 leads to vascular normalization and enhanced delivery of chemotherapy and immune cell infiltration. The increase in tumor perfusion diminishes hypoxia and also serves to polarize TAMs away from an M2-like state. These results also suggest and are borne out by clinical biopsies that the effect of RRx-001 depends on the number of TAMs present in the tumor microenvironment with higher numbers leading to better activity.

Angiogenic factor and cytokine analysis among patients with renal cell carcinoma treated with adjuvant VEGFR TKIs.

586 Background: VEGFR TKIs are important therapeutic agents in RCC, but their adjuvant use remains limited. Investigating the effects of these medications on circulating angiogenic factors and cytokines in the adjuvant setting can elucidate whether changes in cytokine levels result from drug-host or drug-tumor interactions, and may reveal biomarkers to guide patient selection for adjuvant treatment. Methods: In the ECOG-ACRIN 2805 (ASSURE) trial, patients with resected RCC were randomized to sunitinib, sorafenib, or placebo. Plasma was collected at start of treatment and 4 or 6 weeks after treatment initiation. All paired samples available by July 2010 were analyzed, corresponding to 413 patients. We analyzed VEGF, PlGF, sFlt1, KDR/sVEGFR-2, Ang2, bFGF, HGF, IFNg, IL8, CXCL9, CXCL10, and CXCL11. Mixed effects models were used to test for changes from baseline. Cox models were used to assess associations between disease-free survival (DFS) and angiogenic factor and cytokine levels. Results: VEGF and PlGF increased after 4 weeks on either treatment (p &lt; 0.0001 for both), and at 6 weeks VEGF and PIGF levels returned to baseline for patients on sunitinib (corresponding to the 2 week break in the sunitinib schedule) but not sorafenib. Levels of sFLT-1 decreased after 4 weeks on suntinib and after 6 weeks on sorafenib (p &lt; 0.0001). sVEGFR2 decreased after both 4 and 6 weeks of treatment on both sunitinib and sorafenib (p &lt; 0.0001). Patients on placebo had no significant changes in circulating angiogenic factor or cytokine levels. CXCL-10 levels increased after 4 weeks on both sunitinib and sorafenib but not on placebo, and remained elevated at 6 weeks on sunitinib. Higher baseline CXCL-10 was associated with worse DFS (HR 1.41 per log increase in CXCL-10, Holm adjusted p-value 0.003, 95% CI 1.18-1.70). This remained significant after adjustment for T-stage, Fuhrman grade, and ECOG PS. Conclusions: Among patients treated with VEGFR TKIs in the adjuvant setting for RCC, drug-host interactions mediate changes in cytokines and angiogenic factors. Elevated CXCL-10 prior to treatment was associated with higher recurrence risk. Studies to understand the functional consequences of these changes are underway.

Associated multiplex biomarker detection for colorectal cancer based on Bio-Plex platform

Background: Associated protein biomarkers are thought to be produced by the tumor or other tissues in response to the existence of cancers or associated conditions. Equally, known examples of cancer protein biomarker are used for the diagnosis of colorectal cancers (CRCs). However, these biomarkers suffer from low diagnostic specificity and sensitivity for CRC patients. Single biomarker detection is rarely useful for clinical applications due to the heterogeneity of cancer. Therefore, new sensitive and specific assays are urgently required for CRC diagnosis at an early stage. Methods: In this study, a total of 58 newly diagnosed primary CRC patients in Dianjiang People’s Hospital of Chongqing were included, while 20 controls were also included. Approximately 2 mL of venous blood samples from 58 CRC patients and 20 controls were collected. Then we detected 16 angiogenic cytokines using the Bio-Plex technology to measure multiple cytokines. Results: The results showed that serum levels of Follistatin, HGF, and Osteopontin were significantly higher in CRC patients, while Leptin and PECAM were significantly decreased. ROC curve analyses indicated that the up-regulated markers, including Follistatin, HGF, and Osteopontin, had diagnostic value for CRC. The down-regulated marker, PECAM, also presented diagnostic value for CRC. To maximize the ability to detect CRC, four biomarkers, as well as CEA, were combined and used for linear discriminant analysis. Using the regression equation, the sensitivity and specificity for CRC were calculated as 93.1% (54/58) and 60.0% (12/20). Conclusions: The study demonstrates that the combined analysis of five cytokines (follistatin, HGF, leptin, PECAM and osteopontin) is more useful for CRC diagnosis than the analysis of any individual marker. The combined detection of these five biomarkers is a valuable method for the diagnosis of CRC.

Microvascular density, macrophages, and mast cells in human clear cell renal carcinoma with and without bevacizumab treatment

BackgroundClear cell renal cell carcinoma (ccRCC) represents a highly vascularized aggressive kidney cancer. Due to ccRCC chemotherapy resistance, antiangiogenesis is one of the most innovative targeted therapies for this tumor. The tumor microenvironment exerts important roles in tumor growth, angiogenesis, and metastatic escape. Materials and methodsIn this study, we investigated the composition of tumor cell microenvironment including mast cells, macrophages, and microvascular density in ccRCC tumor tissues collected from patients who underwent nephrectomy treated or not with bevacizumab as neoadjuvant therapy before surgery. ResultsThe results of this study indicate that bevacizumab-treated ccRCC samples present reduced microvascular density as well as a lower number of CD68-positive macrophages and tryptase-positive mast cells in comparison with the untreated patients. ConclusionsIt follows that the antiangiogenic activity of bevacizumab may be due to a direct effect on angiogenic cytokines released by tumor cells and an indirect effect on the release of pro-angiogenic factors by inflammatory stromal cells.