Levels Of Angiogenic Cytokines Research Articles

MiR-143-5p regulates the proangiogenic potential of human dental pulp stem cells by targeting HIF-1α/RORA under hypoxia: A laboratory investigation in pulp regeneration.

Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR-143-5p in the process. Human dental pulp stem cells were isolated, cultured and characterized invitro. Cobalt chloride (CoCl2) was used to induce hypoxia in hDPSCs. CCK-8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF-1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured invitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR-143-5p in hDPSCs, and the proangiogenic effects were assessed using qRT-PCR, WB, and tube formation assays. miR-143-5p target genes were identified and verified using bioinformatics prediction tools, dual-luciferase reporter assays and RNA pull-down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR-143-5p overexpression/inhibition in hDPSCs in dental pulp regeneration. Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The invivo experiments showed that hypoxia treatment (50 and 100 μM CoCl2) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR-143-5p levels decreased with increasing CoCl2 concentration. miR-143-5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR-143-5p overexpression inhibited angiogenesis invitro. Dual-luciferase reporter assay identified retinoic acid receptor-related orphan receptor alpha (RORA) as an miR-143-5p target gene in hDPSCs. RNA pull-down experiments demonstrated that HIF-1α and RORA were pulled down by biotin-labelled miR-143-5p, and the levels of HIF-1α and RORA bound to miR-143-5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR-143-5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration. CoCl2-induced hypoxia promotes hDPSCs-driven paracrine angiogenesis and pulp regeneration. The inhibition of miR-143-5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF-1α and RORA.

Contemporaneous Perioperative Inflammatory and Angiogenic Cytokine Profiles of Surgical Breast, Colorectal, and Prostate Cancer Patients: Clinical Implications.

Surgery-induced tumor growth acceleration and synchronous metastatic growth promotion have been observed for decades. Surgery-induced wound healing, orchestrated through growth factors, chemokines, and cytokines, can negatively impact patients harboring residual or metastatic disease. We provide detailed clinical evidence of this process in surgical breast, prostate, and colorectal cancer patients. Plasma samples were analyzed from 68 cancer patients who had not received treatment before surgery or adjuvant therapy until at least four weeks post-surgery. The levels of plasma cytokines, chemokines, and growth factors were simultaneously quantified and profiled using multiplexed immunoassays for eight time points sampled per patient. The immunologic processes are induced immediately after surgery in patients, characterized by a drastic short-term shift in the expression levels of pro-inflammatory and angiogenic molecules and cytokines. A rapid and significant spike in circulating plasma levels of hepatocyte growth factor (HGF), interleukin-6 (IL-6), placental growth factor (PLGF), and matrix metalloproteinase-9 (MMP-9) after surgery was noted. The rise in these molecules was concomitant with a significant drop in transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF-AB/BB), insulin-like growth factor-1 (IGF-1), and monocyte chemoattractant protein-2 (MCP-2). If not earlier, each plasma analyte was normalized to baseline levels within 1-2 weeks after surgery, suggesting that surgical intervention alone was responsible for these effects. The effects of surgical tumor removal on disrupting the pro-inflammatory and angiogenic plasma profiles of cancer patients provide evidence for potentiating malignant progression. Our findings indicate a narrow therapeutic window of opportunity after surgery to prevent disease recurrence.

Effects of 1-year tofacitinib therapy on angiogenic biomarkers in rheumatoid arthritis.

Cardiovascular (CV) morbidity and mortality, and perpetuated synovial angiogenesis have been associated with RA. In our study we evaluated angiogenic factors in relation to vascular inflammation and function, and clinical markers in RA patients undergoing 1-year tofacitinib therapy. Thirty RA patients treated with either 5 mg or 10 mg twice daily tofacitinib were included in a 12-month follow-up study. Eventually, 26 patients completed the study and were included in data analysis. Levels of various angiogenic cytokines (TNF-α, IL-6), growth factors [VEGF, basic fibroblast (bFGF), epidermal (EGF), placental (PlGF)], cathepsin K (CathK), CXC chemokine ligand 8 (CXCL8), galectin-3 (Gal-3) and N-terminal prohormone brain natriuretic peptide (NT-proBNP) were determined at baseline, and at 6 and 12 months after initiating tofacitinib treatment. In order to assess flow-mediated vasodilation, common carotid intima-media thickness (ccIMT) and carotid-femoral pulse-wave velocity, ultrasonography was performed. Synovial and aortic inflammation was also assessed by 18F-fluorodeoxyglucose-PET/CT. One-year tofacitinib therapy significantly decreased IL-6, VEGF, bFGF, EGF, PlGF and CathK, while it increased Gal-3 production (P < 0.05). bFGF, PlGF and NT-proBNP levels were higher, while platelet-endothelial cell adhesion molecule 1 (PECAM-1) levels were lower in RF-seropositive patients (P < 0.05). TNF-α, bFGF and PlGF correlated with post-treatment synovial inflammation, while aortic inflammation was rather dependent on IL-6 and PECAM-1 as determined by PET/CT (P < 0.05). In the correlation analyses, NT-proBNP, CXCL8 and Cath variables correlated with ccIMT (P < 0.05). Decreasing production of bFGF, PlGF or IL-6 by 1-year tofacitinib therapy potentially inhibits synovial and aortic inflammation. Although NT-proBNP, CXCL8 and CathK were associated with ccIMT, their role in RA-associated atherosclerosis needs to be further evaluated.

Synergistic Therapeutic Potential of Dual 3D Mesenchymal Stem Cell Therapy in an Ischemic Hind Limb Mouse Model.

Three-dimensional (3D) culture systems have been widely used to promote the viability and metabolic activity of mesenchymal stem cells (MSCs). The aim of this study was to explore the synergistic benefits of using dual 3D MSC culture systems to promote vascular regeneration and enhance therapeutic potential. We used various experimental assays, including dual 3D cultures of human adipose MSCs (hASCs), quantitative reverse transcription polymerase chain reaction (qRT-PCR), in vitro cell migration, Matrigel tube network formation, Matrigel plug assay, therapeutic assays using an ischemic hind limb mouse model, and immunohistochemical analysis. Our qRT-PCR results revealed that fibroblast growth factor 2 (FGF-2), granulocyte chemotactic protein-2 (GCP-2), and vascular endothelial growth factor-A (VEGF-A) were highly upregulated in conventional 3D-cultured hASCs (ASC-3D) than in two-dimensional (2D)-cultured hASCs. Hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and stromal-cell-derived factor-1 (SDF-1) showed higher expression levels in cytokine-cocktail-based, 3D-cultured hASCs (ASC-3Dc). A conditioned medium (CM) mixture of dual 3D ASCs (D-3D; ASC-3D + ASC-3Dc) resulted in higher migration and Matrigel tube formation than the CM of single 3D ASCs (S-3D; ASC-3D). Matrigel plugs containing D-3D contained more red blood cells than those containing S-3D. D-3D transplantation into ischemic mouse hind limbs prevented limb loss and augmented blood perfusion when compared to S-3D transplantation. Transplanted D-3D also revealed a high capillary density and angiogenic cytokine levels and transdifferentiated into endothelial-like cells in the hind limb muscle. These findings highlight the benefits of using the dual 3D culture system to optimize stem-cell-based therapeutic strategies, thereby advancing the therapeutic strategy for ischemic vascular disease and tissue regeneration.

Altered angiogenic and neuroinflammatory markers in peripartum individuals with and without severe preeclampsia compared to non-pregnant adults (P3-5.008)

<h3>Objective:</h3> To characterize angiogenic and neuroinflammatory cytokine levels in serum and cerebrospinal fluid (CSF) of peripartum individuals with and without preeclampsia. <h3>Background:</h3> Preeclampsia, a hypertensive disorder of pregnancy associated with systemic endothelial dysfunction, carries high risk of neurovascular complications. Preliminary studies suggest maternal blood-brain barrier (BBB) dysfunction in preeclampsia. <h3>Design/Methods:</h3> We analyzed serum from 26 peripartum participants (13 severe preeclampsia, 13 normotensive controls), of whom 13 participants (7 cases, 6 controls) also had CSF available for analysis. ProcartaPlex and ELISA immunoassays were used to quantify angiogenic and neuroinflammatory proteins implicated in BBB dysfunction in serum and separately CSF. We used t-test or Wilcoxon tests to compare levels between participants with and without preeclampsia. We next compared our values to published normative values for healthy non-pregnant adults. We did not correct for multiple comparisons for this exploratory analysis. <h3>Results:</h3> We found no statistically significant difference in serum or CSF levels of angiogenic (VEGF, PlGF, and sFLT1) or neuroinflammatory (CCL5/RANTES, GM-CSF, CXCL10) markers between normotensive peripartum controls and those with severe preeclampsia. Compared to historical non-pregnant controls, serum levels in both cases and controls were lower of VEGF (p&lt;0.005 for both comparisons), CCL5 (p&lt;0.002 for both comparisons), and CXCL10 (p&lt;0.0.002 for both comparisons), and sFLT1 and GM-CSF were higher (p&lt;0.02 for all comparisons). In CSF analyses, the concentration of CXCL10 was significantly lower (p&lt;0.002 for both comparisons) and sFLT1 was significantly higher (p&lt;0.03 for both comparisons), compared with non-pregnant adult reference values. <h3>Conclusions:</h3> In this pilot study, angiogenic and neuroinflammatory profiles in the serum and CSF of peripartum individuals with and without severe preeclampsia were not significantly different. However, when compared with normative values for healthy non-pregnant adults, peripartum individuals demonstrate alterations in factors regulating BBB permeability. Our results suggest that the peripartum state may be associated with changes in BBB regulation, regardless of preeclampsia diagnosis. <b>Disclosure:</b> Miss Haghighi has nothing to disclose. Ms. Tuohy has nothing to disclose. Natalie Bello has nothing to disclose. Dr. Booker has nothing to disclose. Dr. Miltiades has nothing to disclose. Ronald Wapner has nothing to disclose. The institution of Dr. Marshall has received research support from NIH. Dr. Marshall has received publishing royalties from a publication relating to health care. Dr. Miller has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Elsevier. Dr. Miller has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Argionis and Associates LLC. The institution of Dr. Miller has received research support from National Institutes of Health.

VEGF and IL-6 As Markers of Severity and Response to Treatment in Waldenstrom Macroglobulinemia and IgM-Monoclonal Gammopathy of Undetermined Significance: A Monocentric Experience

Background: Angiogenesis plays an important role in the development and the maintenance of hematolymphoid malignancies. Serum levels of angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6), are known to be increased in patients with Waldenstrom Macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS). Even if the role of IL-6 and VEGF as markers of disease status and severity has been recognized, only a limited number of studies have addressed the role of cytokines in WM and there is no proof about any correlation between their serum levels in WM and IgM-MGUS patients. Nevertheless, their dosage at various stages of the disease aims to understand if they have a comparable pattern, and therefore they could be used in clinical practice for disease monitoring. Aims: This study aims to find a correlation of serum levels of VEGF and IL-6 with the burden of disease in our patients with IgM-MGUS and WM and to evaluate a possible relationship between the two angiogenic cytokines, in an attempt to identify potential markers of severity. Methods: We performed a retrospective monocentric study of 18 patients (ten with WM and eight with IgM-MGUS) with a median age of 73 years (range: 52-90), whose 66% were male. Patients with WM were at different stages of disease (four were naive, four in remission, two relapsed/refractory). A total of 18 controls with a similar age and gender distribution were also tested. Controls were selected among patients with neuropathies in the absence of any hematological condition. We used the 1:1 ratio for the selection of controls: for every male or female WM patient, one control of the same gender and of similar age was included. In order to find a relationship of VEGF and IL-6 with a laboratory parameter, IgM levels dosage for each patient have been measured at the same time-point of the measurement of the cytokines. In all patients and controls, we evaluated serum levels of VEGF and IL-6 using Immunoassay panels (i.e., anti-IL-6 and anti-VEGF ELISA kit). Results: All patients showed elevated values of angiogenic cytokines when compared with controls (P value < 0.05). Both VEGF and IL-6 serum levels (median values: 623,5 pg/mL and 4,5 pg/mL, respectively; ranges: 113,4-1981 pg/mL and 0,3-21,9 pg/mL, respectively) showed a positive correlation with IgM serum levels (median value: 2055 mg/dL, range: 300-6123 pg/mL) (Fig. 1a and 1b). Furthermore, the relationship between IL-6 and VEGF was statistically significant and could be explained by a linear model (P value = 0,007) (Fig. 1c). No statistically significant differences were observed between the different phases of WM. Conclusions: Evidence from our sample shows that serum levels of angiogenic cytokines as VEGF and IL-6 have a comparable pattern with IgM levels. Therefore, it seems reasonable to suggest that they could be used in clinical practice as markers of severity of disease and response to treatment. Moreover, since the cytokines profiles are comparable, IL-6 can be used interchangeably with VEGF, allowing the monitoring of the disease with easily availability and unexpensive techniques. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A nonrandomized phase I and biomarker trial of regorafenib in advanced myeloid malignancies.

We conducted a single‐center, open‐label, dose escalation, and expansion phase I trial of the antiangiogenic multikinase inhibitor regorafenib in patients with advanced myeloid neoplasms. We enrolled 16 patients with relapsed/refractory acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), or myelodysplastic syndrome (MDS). A 3 + 3 dose escalation design was used with two planned dose levels (120 or 160 mg daily) and one de‐escalation level (80 mg daily). An additional 10 patients were treated on an expansion cohort. The recommended phase two dose of regorafenib was 160 mg daily, with no dose‐limiting toxicities. The best overall disease response by International Working Group criteria included one partial and stable disease in 11 patients. Tissue studies indicated no change in Ras/mitogen‐activated protein kinase (MAPK) pathway activation in responders. Pharmacodynamic changes in plasma VEGF, PlGF, and sVEGFR2 were detected during treatment. Baseline proinflammatory and angiogenic cytokine levels were not associated with clinical response. Single‐agent regorafenib demonstrated an acceptable safety profile in relapsed/refractory myeloid malignancy patients. Most patients achieved stable disease, with modest improvements in cell counts in some MDS patients. Biomarker studies were consistent with on‐target effects of regorafenib on angiogenesis. Future studies should investigate the role of regorafenib in combination therapy approaches.

Vitreous cytokine levels following the administration of a single 0.19 mg fluocinolone acetonide (ILUVIEN®) implant in patients with refractory diabetic macular edema (DME)—results from the ILUVIT study

PurposeTo investigate the changes in vitreous inflammatory and angiogenic cytokine levels, primarily interleukin-(IL)-6, following intravitreal injection of the 0.19 mg fluocinolone acetonide (FAc, ILUVIEN®) implant in patients with diabetic macular edema.MethodsA single-center phase IV study involving 12 patients’ eyes with diabetic macular edema. Vitreous fluid samples were obtained prior to intravitreal injection of the fluocinolone acetonide implant and then again over a 6-month period. Vitreous samples were examined using a cytometric bead array to measure IL-6, IL-8, IP-10, MCP-1, VEGF, and CD54. PIGF and PEDF were measured using an enzyme-linked immunosorbent assay. Changes in the cytokine and chemokine expression patterns were analyzed. Clinical parameters such as BCVA and center point thickness (CPT) were also examined.ResultsThere were mean reductions in all parameters between baseline and month 6. Significant changes (p < 0.05 versus baseline) were observed in the expression of IL-6, IP-10, MCP-1, and CD54 following the administration of fluocinolone acetonide implant. VEGF and PIGF increased at month 1 before declining at month 6, though this trend was not significant. CPT decreased rapidly between screening and the first follow-up visit, and this decrease was sustained. BCVA remained relatively stable throughout.ConclusionThis study demonstrated changes in vitreous inflammatory and angiogenic cytokine levels following intravitreal injection of the FAc implant in patients with diabetic macular edema. Data show that the fluocinolone acetonide implant led to rapid and sustained reductions of some inflammatory cytokines with improvement of the overall clinical picture.

Phenotypic, trophic, and regenerative properties of mesenchymal stem cells from different osseous tissues.

Mesenchymal stem cells (MSCs) have broad-based therapeutic potential in regenerative medicine. However, a major barrier to their clinical utility is that MSCs from different tissues are highly variable in their regenerative properties. In this study, we defined the molecular and phenotypic identities of different MSC populations from different osseous tissue sites of different patients and, additionally, determined their respective regenerative properties. MSCs from 6 patients were isolated from either bone marrow of the iliac crest (BMSCs) or alveolar bone tissue (aBMSCs), and flow cytometry revealed that regardless of the tissue source, MSC immunotypes had the same expression of MSC markers CD73, CD90, and CD105. However, transcriptomic analyses revealed 589 genes differentially expressed (DE) between BMSCs and aBMSCs, including eightfold higher levels of bone morphogenetic protein 4 (BMP-4) in aBMSCs. In striking contrast, gene expression of MSCs derived from the same tissue, but between different patients (i.e., BMSCs to BMSCs, aBMSCs to aBMSCs), showed only 38 DE BMSC genes and 51 DE aBMSC genes. A protein array showed that aBMSC and BMSC produced equivalent levels of angiogenic cytokines; however, when placed in angiogenesis model systems, aBMSCs induced significantly more capillaries in vitro and in vivo. Finally, cell transplantation of MSCS into osseous defects showed that the bone regenerative capacity of aBMSCs was significantly greater than that of BMSCs. This study is the first to link the molecular, phenotypic, and regenerative properties of different MSCs from different patients and provides novel insights toward MSC differences based on the osseous tissue origin.

Human adipose tissue-derived stem cell paracrine networks vary according metabolic risk and after TNFα-induced death: An analysis at the single-cell level

ObjectiveAdipose tissue-derived stem cells (ASCs) might play an important role in adipose microenvironment remodelling during tissue expansion through their response to hypoxia. We examined the cytokine profiles of hypoxic visceral ASCs (hypox-visASCs) from subjects with different metabolic risk, the interactions between cytokines as well as the impact of TNFα-induced death in the behavior of surviving hypoxic subcutaneous ASCs (hypox-subASCs) both at bulk population and single-cell level. Materials/methodsVisceral adipose tissue was processed to isolate the ASCs from 33 subjects grouped into normal weight, obese with and without metabolic syndrome. Multiplex assay was used to simultaneously measure multiple inflammatory, anti-inflammatory and angiogenic cytokines in hypox-visASCs from these patients and to elucidate cytokine profiles of hypox-subASCs upon stimulation with IL1β or TNFα and after TNFα-induced death. qPCR and single-cell RNA-sequencing were also performed to elucidate transcriptional impact in surviving hypox-subASCs after TNFα-induced apoptosis. ResultsHypox-visASCs from subjects without metabolic syndrome showed greater secretion levels of inflammatory, anti-inflammatory and angiogenic cytokines compared with those from patients with metabolic syndrome. While IL-1β stimulation was sufficient to increase the secretion levels of these cytokines in hypox-subASCs, TNFα-induced apoptosis also increased their levels and impacted on the expression levels of extracellular matrix proteins, acetyl-CoA producing enzymes and redox-balance proteins in surviving hypox-subASCs. TNFα-induced apoptosis under different glucose concentrations caused selective impoverishment of cell clusters and differentially influenced gene expression profiles of surviving hypox-subASCs. ConclusionsImmunoregulatory and angiogenic functions of hypox-visASCs from patients with metabolic syndrome could be insufficient to promote healthy adipose tissue expansion. TNFα-induced apoptosis may impact on functionality of hypox-subASC populations, whose differential metabolic sensitivity to death could serve to manipulate individual populations selectively in order to elucidate their role in shaping adipose heterogeneity and treating metabolic disorders.

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.

Efficacy and Tolerability of Daratumumab with Ixazomib and Dexamethasone in Patients with One Prior Lenalidomide-Based Regimen: Preliminary Results of the Phase 2 Daria Study

Efficacy and Tolerability of Daratumumab with Ixazomib and Dexamethasone in Patients with One Prior Lenalidomide-Based Regimen: Preliminary Results of the Phase 2 Daria Study

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.

Comparative Analysis of Multiplex Platforms for Detecting Vitreous Biomarkers in Diabetic Retinopathy.

PurposeTo evaluate the feasibility of using the Proximity Extension Assay (PEA) platform to detect biomarkers in vitreous and to compare the findings with results obtained with an electrochemiluminescent (ECL) sandwich immunoassay.MethodsVitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic controls were tested using two different proteomics platforms. Forty-one assays were completed with the ECL platform and 459 with the PEA platform. Spearman's rank correlation coefficient (rs) was used to determine the direction and strength of the relationship between protein levels detected by both platforms.ResultsThree hundred sixty-six PEA assays detected the tested protein in at least 25% of samples, and the difference in protein abundance between PDR and controls was statistically significant for 262 assays. Seventeen ECL assays yielded a detection rate ≥ 25%, and the difference in protein concentration between PDR and controls was statistically significant for 13 proteins. There was a subset of proteins that were detected by both platforms, and for those the Spearman's correlation coefficient was higher than 0.8.ConclusionsPEA is suitable for the analysis of vitreous samples, showing a strong correlation with the ECL platform. The detection rate of PEA panels was higher than the panels tested with ECL. The levels of several proinflammatory and angiogenic cytokines were significantly higher in PDR vitreous compared to controls.Translational RelevanceThis study provides new information on the yields of small-volume assays that can detect proteins of interest in ocular specimens, and it identifies patterns of cytokine dysregulation in PDR.

A modified laser-induced choroidal neovascularization animal model with intravitreal oxidized low-density lipoprotein.

To investigate whether intravitreal injection of oxidized low-density lipoprotein (OxLDL) can promote laser-induced choroidal neovascularization (CNV) formation in mice and the mechanism involved, thereby to develop a better animal model. C57BL6/J mice were randomized into three groups. Immediately after CNV induction with 532 nm laser photocoagulation, 1.0 µL of OxLDL [100 µg/mL in phosphate-buffered saline (PBS)] was intravitreally injected, whereas PBS and the same volume low-density lipoprotein (LDL; 100 µg/mL in PBS) were injected into the vitreous as controls. Angiogenic and inflammatory cytokines were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) after 5d, and CNV severity was analyzed by choroid flat mount and immunofluorescence staining after 1wk. In vitro, retinal pigment epithelial (RPE) cell line (ARPE19) were treated with OxLDL (LDL as control) for 8h. Angiogenic and inflammatory cytokine levels were measured. A specific inhibitor of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) was used to evaluate the role of LOX1 in this process. At 7d after intravitreal injection of 1 µL (100 µg/mL) OxLDL, T15-labeled OxLDL was mainly deposited around the CNV area, and the F4/80-labeled macrophages, the CD31-labeled vascular endothelial cells number and CNV area were increased. Meanwhile, WB and qRT-PCR results showed that vascular endothelial growth factor (VEGF), CC chemokine receptor 2 (CCR2), interleukin-6 (IL-6), IL-1β, and matrix metalloproteinase 9 (MMP9) expressions were increased, which was supported by in vitro experiments in RPE cells. LOX1 inhibitors significantly reduced expressions of inflammatory factors IL-1β and VEGF. A modified laser-induced CNV animal model is established with intravitreal injection of 1 µL (100 µg/mL) of OxLDL at 7d, which at least partially through LOX1. This animal model can be used as a simple model for studying the role of OxLDL in age-related macular degeneration.

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

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.

Association between the fetal cerebroplacental ratio and biomarkers of hypoxia and angiogenesis in the maternal circulation at term

ObjectivesA low fetal cerebroplacental ratio (CPR) in late pregnancy is a marker of a fetus that has failed to reach its growth potential and is associated with a variety of perinatal and pregnancy complications. It is not known if it is also correlated with aberrations in angiogenic, hypoxia-responsive or inflammatory cytokine levels in the maternal circulation. We investigated if there were any differences in levels of biomarkers of angiogenesis, endothelial cell dysfunction, hypoxia and/or inflammation in term pregnancies with a low fetal CPR compared to controls. We hypothesized that as the CPR is a marker of suboptimal growth, this would be reflected in a shift towards upregulation of hypoxia-responsive factors even in non-small for gestational age fetuses. Study designWe used Multiplex ELISA to measure a panel of 28 candidate biomarkers of angiogenesis and/or hypoxia in pre-labour maternal plasma from 113 women at term, stratified for CPR <10th centile vs. CPR >10th centile. Plasma levels of the biomarkers were measured using 2 multiplex Luminex assays - a commercially available human angiogenesis/growth factor panel (R&D Systems®), comprising 15 analytes and an in-house custom panel of a further 13 candidate biomarkers. ResultsOf the 28 candidate biomarkers investigated, we found significantly elevated levels of Carbonic Anhydrase 9 and soluble Fms-like tyrosine kinase (Vascular Endothelial Growth Factor Receptor 1), and lower levels of Placental Growth Factor in plasma from women with a low fetal CPR. The soluble Fms-like tyrosine kinase-1/Placental Growth Factor ratio was also markedly elevated in this cohort. We also demonstrated significant inverse correlations between the fetal CPR and Carbonic Anydrase 9, soluble Fms-like tyrosine kinase and Hepatocyte Growth Factor. ConclusionsA low fetal CPR is associated with changes in some hypoxia-responsive and angiogenesis factors in the maternal circulation in pregnancies with normally grown fetuses.

Activated macrophages are crucial during acute PM2.5 exposure-induced angiogenesis in lung cancer.

The importance of ambient particulate matter (PM2.5) in lung cancer progression is well established; however, the precise mechanisms by which PM2.5 modulates lung cancer development have not yet been determined. The present study demonstrated increased mRNA and protein expression levels of vascular endothelial growth factor in PM2.5-induced macrophages. However, no significant changes to the expression levels of angiogenic cytokines (vascular endothelial growth factor A, matric metallopeptidase 9, basic fibroblast growth factor and platelet-derived growth factor) were observed in the Lewis lung carcinoma (LLC) cell line in response to acute PM2.5 exposure. PM2.5-induced activated macrophages were revealed to upregulate angiogenic cytokine expression following the acute exposure of LLC cells to PM2.5-induced macrophage supernatant. In vivo, the pro-angiogenic and macrophage accumulation functions of PM2.5 were supported by the establishment of a polyvinyl alcohol sponge implantation mouse model. Furthermore, PM2.5 was demonstrated to increase angiogenesis and macrophage recruitment in mice that were subcutaneously injected with LLCs. These results indicated that PM2.5 increases angiogenesis, and macrophages are crucial mediators of PM2.5-induced angiogenesis in lung cancer. These findings may provide novel insights for the development of lung cancer treatment strategies.

Therapeutic effects of human umbilical cord mesenchymal stem cell-derived microvesicles on premature ovarian insufficiency in mice

BackgroundPremature ovarian insufficiency (POI) is one of the leading causes of female infertility, which is caused by an abnormal ovarian reserve. Currently, there is no effective treatment to restore the fertility of POI patients. Recent studies suggested that microvesicles (MVs) released from mesenchymal stem cells (MSCs) exert therapeutic effects in various degenerative diseases. In this study, the effect of human umbilical cord MSC-derived MVs (HUCMSC-MVs) on the restoration of ovarian function in a chemotherapy-induced POI mouse model is investigated.MethodsMVs were obtained from the supernatant of cultured HUCMSCs. The localization of PKH26-labeled HUCMSC-MVs in ovarian tissues was observed by confocal laser scanning microscopy. Histomorphometric analysis was performed to count the number of ovarian follicles and vessels. The ovarian sections were stained with anti-CD34 to evaluate angiogenesis. The levels of estradiol (E2) and follicle-stimulating hormone (FSH) were measured by enzyme-linked immunosorbent serologic assay. The mRNA expression of angiogenesis-related cytokines and the protein expression of AKT in mouse ovaries were measured by quantitative RT-PCR and western blot analysis. The parametric variables were compared by Student’s t test and analysis of variance. The non-parametric variables were compared by the Mann-Whitney U test. Categorical variables were compared by χ2 test. P < 0.05 was considered statistically significant.ResultsPKH26-labeled HUCMSC-MVs were detectable within the ovaries and migrated to the ovarian follicles 24 h after transplantation. The transplantation of HUCMSC-MVs could increase the body weight and number of ovarian follicles (primordial, developing, and preovulatory follicles), induce ovarian angiogenesis, and recover the disturbed estrous cycle of POI mice. The expression levels of total AKT, p-AKT, and angiogenic cytokines (including VEGF, IGF, and angiogenin) in the ovaries of POI mice were markedly upregulated after HUCMSC-MVs transplantation, suggesting that HUCMSC-MVs transplantation might recover ovarian function by inducing angiogenesis via the PI3K/AKT signaling pathway.ConclusionsThis study provides valuable insight into the effects of HUCMSC-MVs on ovarian tissue angiogenesis and on the restoration of ovarian function in POI mice, which may be helpful to develop a treatment strategy for POI patients.