Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy

PURPOSE: To determine levels of the chemokines CCL1/I-309, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL8/MCP-2, CXCL5/ENA-78, CXCL6/GCP-2, CXCL10/IP-10, and CXCL11/I-TAC in the vitreous humor and serum, from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD), and to investigate the expression of MCP-1, CXCL12/SDF-1, and the chemokine receptor CXCR3 in epiretinal membranes. METHODS: Paired vitreous humor and serum samples were obtained from patients undergoing vitrectomy for the treatment of RD (57 specimens), PVR (32 specimens), and PDR (88 specimens). The levels of chemokines were measured by enzyme-linked immunosorbent assays. Eighteen PDR and 5 PVR membranes were studied by immunohistochemical techniques. RESULTS: Of all the chemokines studied, only MCP-1 and IP-10 were detected in vitreous humor samples. MCP-1 levels in vitreous humor samples were significantly higher than in serum samples (p < 0.001). MCP-1 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0002). MCP-1 levels in vitreous humor samples from patients with active PDR were significantly higher than in inactive PDR cases (p = 0.0224). IP-10 levels in vitreous humor samples were significantly higher than in serum samples (p = 0.0035). IP-10 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0083). The incidence of IP-10 detection in vitreous humor samples was significantly higher in active PDR cases compared with inactive cases (p = 0.0214). There was a significant association between the incidence of IP-10 detection and increased levels of MCP-1 in vitreous humor samples from all patients, and patients with RD and PDR (p < 0.001 for all comparisons). MCP-1, and SDF-1 were localized in myofibroblasts in PVR and PDR membranes and in vascular endothelial cells in PDR membranes. CXCR3 was expressed by vascular endothelial cells in PDR membranes. CONCLUSION: MCP-1, IP-10 and SDF-1 may participate in pathogenesis of PVR and PDR. Myofibroblasts and vascular endothelial cells are the major cell types expressing MCP-1, SDF-1, and CXCR3 in epiretinal membranes.     

Comprehensive Analysis of Inflammatory Immune Mediators in Vitreoretinal Diseases

Inflammation affects the formation and the progression of various vitreoretinal diseases. We performed a comprehensive analysis of inflammatory immune mediators in the vitreous fluids from total of 345 patients with diabetic macular edema (DME, n = 92), proliferative diabetic retinopathy (PDR, n = 147), branch retinal vein occlusion (BRVO, n = 30), central retinal vein occlusion (CRVO, n = 13) and rhegmatogenous retinal detachment (RRD, n = 63). As a control, we selected a total of 83 patients with either idiopathic macular hole (MH) or idiopathic epiretinal membrane (ERM) that were free of major pathogenic intraocular changes, such as ischemic retina and proliferative membranes. The concentrations of 20 soluble factors (nine cytokines, six chemokines, and five growth factors) were measured simultaneously by multiplex bead analysis system. Out of 20 soluble factors, three factors: interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were significantly elevated in all groups of vitreoretinal diseases (DME, PDR, BRVO, CRVO, and RRD) compared with control group. According to the correlation analysis in the individual patient''s level, these three factors that were simultaneously increased, did not show any independent upregulation in all the examined diseases. Vascular endothelial growth factor (VEGF) was significantly elevated in patients with PDR and CRVO. In PDR patients, the elevation of VEGF was significantly correlated with the three factors: IL-6, IL-8, and MCP-1, while no significant correlation was observed in CRVO patients. In conclusion, multiplex bead system enabled a comprehensive soluble factor analysis in vitreous fluid derived from variety of patients. Major three factors: IL-6, IL-8, and MCP-1 were strongly correlated with each other indicating a common pathway involved in inflammation process in vitreoretinal diseases.     

康柏西普联合视网膜激光光凝对缺血型视网膜中央静脉阻塞患者球结膜微循环指标和血管内皮功能的影响

摘要 目的：探讨康柏西普联合视网膜激光光凝对缺血型视网膜中央静脉阻塞患者球结膜微循环指标和血管内皮功能的影响。方法：选取2016年8月到2019年9月我院收治的缺血型CRVO患者90例,按信封抽签法分为对照组（n=45,视网膜激光光凝治疗）和研究组（n=45,视网膜激光光凝联合康柏西普治疗）,比较两组患者黄斑中心视网膜厚度(CMT)、球结膜微循环指标、最佳矫正视力(BCVA)、疗效、血清血管内皮生长因子（VEGF）、内皮素-1（ET-1）、一氧化氮（NO）水平以及不良反应。结果：治疗后6个月研究组临床总有效率高于对照组（P<0.05）。治疗后6个月,研究组的BCVA升高程度以及细动脉管径、细静脉管径扩大程度均大于对照组（P<0.05）,CMT以及红细胞聚集积分降低程度大于对照组（P<0.05）。治疗后3 d,研究组的VEGF、ET-1降低程度大于对照组（P<0.05）,NO升高程度大于对照组（P<0.05）。两组不良反应发生率对比未见差异（P>0.05）。结论：康柏西普联合视网膜激光光凝治疗缺血型CRVO疗效较好,可有效改善患者视力及球结膜微循环,同时还可改善血管内皮功能,安全可靠。     

MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.

Methods and Findings

We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DL_CO%).

Conclusions

Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.     

Interleukin-1β Level Is Increased in Vitreous of Patients with Neovascular Age-Related Macular Degeneration (nAMD) and Polypoidal Choroidal Vasculopathy (PCV)

PurposeTo examine the expression of pro-interleukin-1β (pro-IL-1β) and interleukin-1β (IL-1β) in the vitreous body of patients with neovascular age-related macular degeneration(nAMD), polypoidal choroidal vasculopathy (PCV), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO) or Eales’ disease to further elucidate the role of IL-1β and inflammation in the pathogenesis of neovascular retinal disease.DesignProspective clinical laboratory investigation study.MethodsAll patients enrolled had vitreous hemorrhage due to nAMD, PCV, PDR, RVO or Eales’ disease that required vitrectomy. Patients were excluded for any history of active intraocular inflammation, or other ophthalmic surgery besides vitrectomy. Control samples were obtained from patients with idiopathic macular epiretinal membrane. A total of fifty vitreous samples were collected from patient during vitrectomy. Pro-IL-1β and IL-1β expression were measured by enzyme-linked immunosorbent assay (ELISA). Results were analyzed statistically using nonparametric tests.ResultsExpression of pro-IL-1β protein was increased by 2.83-fold and 9.19-fold in PCV and nAMD vitreous samples relative to control, respectively. Expression of IL-β protein was increased by 10-fold and 4.83-fold in PCV and nAMD vitreous samples relative to control, respectively.ConclusionsOur results demonstrate that expression of pro-IL-1β and IL-1β proteins is higher in PCV and nAMD. The roles of pro-IL-1β and IL-1β as inflammatory mediators in the development of PCV and nAMD may be associated with photoreceptor degeneration and neovascularization which necessitates further study.     

Iatrogenic chorioretinal venous anastomosis in the treatment of central retinal vein occlusion

This paper discusses the clinical application of an iatrogenic chorioretinal venous anastomosis for experimental treatment of non-ischemic central retinal vein occlusions (CRVO). The creation of an iatrogenic chorioretinal venous anastomosis offers a potential means of by-passing the obstructed central retinal vein and allowing venous blood to exit the retinal circulation through the choroid. A successful anastomosis may allow improved venous drainage and faster resolution of macular edema. This procedure may also prevent the conversion of a non-ischemic CRVO to the ischemic type. The natural history of a CRVO, the procedure for anastomosis creation, the patients most likely to benefit, and complications of chorioretinal anastomosis are discussed.     

Aqueous humor nitric oxide in patients with central retinal vein occlusion

PurposeIt has been suggested that nitric oxide (NO) has a role in ischemic retinopathies. Since retinal ischemia may develop in retinal vein occlusion, we investigated the presence of nitric oxide in the pathogenesis of central retinal vein occlusion (CRVO).MethodsEighteen consecutive patients with CRVO were included in this study. Aqueous humor specimens were obtained within 21 days of diagnosis. Samples of aqueous humor were also collected from 20 control patients undergoing cataract surgery. For each sample after reduction of nitrate to nitrite with vanadium chloride (VCl₃), we used spectrophotometric method for simultaneous detection of nitrate and nitrite (NO_x).ResultsMean level of aqueous humor NO_x in CRVO and control group was 94.1 ± 23.2 μmol/l and 55.6 ± 11.0 μmol/l, respectively. The difference between two groups was statistically significant (p < 0.0001).ConclusionsOur results may support involvement of nitric oxide in the pathogenesis of CRVO.     

Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer''s disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer''s Disease Research Center (ADRC) and Alzheimer''s Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10⁻¹⁰) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.     

Clinical Relevance of Increased Endothelial and Mesothelial Expression of Proangiogenic Proteases and VEGFA in the Omentum of Patients with Metastatic Ovarian High-Grade Serous Carcinoma

Epithelial ovarian cancer (EOC) metastasis to the omentum requires implantation and angiogenesis. We propose that prometastatic changes in the omental endothelium (for angiogenesis) and mesothelium (for implantation) are critical. We investigated the expression of angiogenic proteases [cathepsin D (CD), cathepsin L (CL), and matrix metalloproteinase 2 (MMP2) and MMP9] and vascular endothelial growth factor A (VEGFA) in the mesothelium and endothelium of omentum from patients with EOC with omental metastases and control patients with benign ovarian tumors. Endothelial expression of CL, VEGFA, and MMP9 and mesothelial expression of VEGFA, MMP9, and CD were significantly increased in patients with metastasized EOC. High expression of MMP9 and VEGFA in endothelium and mesothelium and CD in mesothelium was positively associated with poor disease-specific survival (DSS). High MMP9 expression in either endothelium or mesothelium and presence of ascites prospectively showed the greatest risk of shorter DSS [hazard ratio (HR)= 6.16, 95% confidence interval (CI) = 1.76-21.6, P = .0045; HR = 11.42, 95% CI = 2.59-50.35, P = .0013; and HR = 6.35, 95% CI = 2.01-20.1, P = .002, respectively]. High endothelial MMP9 expression and ascites were independent predictors of reduced DSS and overall survival, together resulting in worst patient prognosis. Our data show that omental metastasis of EOC is associated with increased proangiogenic protein expression in the omental endothelium and mesothelium.     

Methamphetamine induces thoracic aortic aneurysm/dissection through C/EBPβ

AimsThoracic aortic aneurysm/dissection (TAAD) is a life-threatening disease with diverse clinical manifestations. Although the association between methamphetamine (METH) and TAAD is frequently observed, the causal relationship between METH abuse and aortic aneurysm/dissection has not been established. This study was designed to determine if METH causes aortic aneurysm/dissection and delineate the underlying mechanism.Methods and resultsA new TAAD model was developed by exposing METH to SD rats pre-treated with lysyl oxidase inhibitor β-aminopropionitrile (BAPN). Combination of METH and BAPN caused thoracic aortic aneurysm/dissection in 60% of rats. BAPN+METH significantly increased the expression and activities of both matrix metalloproteinase MMP2 and MMP9, consistent with the severe elastin breakage and dissection. Mechanistically, METH increased CCAAT-enhancer binding protein β (C/EBPβ) expression by enhancing mothers against decapentaplegic homolog 3 (Smad3) and extracellular regulated protein kinase (ERK1/2) signaling. METH also promoted C/EBPβ binding to MMP2 and MMP9 promoters. Blocking C/EBPβ significantly attenuated METH+BAPN-induced TAAD and MMP2/MMP9 expression. Moreover, BAPN+METH promoted aortic medial smooth muscle cell (SMC) apoptosis through C/EBPβ-mediated IGFBP5/p53/PUMA signaling pathways. More importantly, the expression of C/EBPβ, MMP2/MMP9, and apoptosis-promoting proteins was increased in the aorta of human patients with thoracic aortic dissection, suggesting that the mechanisms identified in animal study could be relevant to human disease.ConclusionsOur study demonstrated that METH exposure has a casual effect on TAAD. C/EBPβ mediates METH-introduced TAAD formation by causing elastin breakage, medial cell loss and degeneration. Therefore, C/EBPβ may be a potential factor for TAAD clinical diagnosis or treatment.     

Keynote symposium

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.     

Protein expression pattern in experimental pneumococcal meningitis

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.     

Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs.     

Effect of a vascular endothelial growth factor (VEGF) inhibitory treatment on the folliculogenesis and ovarian apoptosis in gonadotropin-treated prepubertal rats

In the present study, we investigated whether vascular endothelial growth factor A (VEGFA) plays a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. The effect of an intrabursal administration of a VEGFA antagonist on follicular development, apoptosis, and levels of pro- and antiapoptotic proteins of BCL2 family members (BAX, BCL2, and BCL2L1), as well as of TNFRSF6 (also known as FAS) and FAS ligand (FASLG), was examined. To inhibit VEGFA, a soluble FLT1/Fc Chimera (Trap) was administered to prepubertal eCG-treated rats. Injection of 0.5 mug of Trap per ovary did not change the number of preantral follicles (PFs) or early antral follicles (EAFs); however, it significantly decreased the number of periovulatory follicles 48 h after surgery and significantly increased the number of atretic follicles. No significant differences were found in any stage of the follicles either 12 or 24 h after injection. Cells undergoing DNA fragmentation were quantified by performing TUNEL on ovarian sections. Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs. DNA isolated from antral follicles incubated for 24 h exhibited the typical apoptotic DNA pattern. Follicles obtained from Trap-treated ovaries showed a significant increase in the spontaneous onset of apoptotic DNA fragmentation. The injection of Trap significantly increased the levels of BAX and decreased the levels of BCL2 protein. The ratio of BCL2L1L:BCL2L1s was significantly diminished in follicles obtained from ovaries treated with Trap. No changes in the levels of TNFRSF6 or FASLG were observed after treatment. We concluded that the local inhibition of VEGFA activity appears to produce an increase in ovarian apoptosis through an imbalance among the BCL2 family members, thus leading a larger number of follicles to atresia.     

Stem cell mobilization and harvesting by leukapheresis alters systemic cytokine levels in patients with multiple myeloma

Background aimsStem cell mobilization and harvesting by peripheral blood leukapheresis in patients with myeloma can alter plasma levels of certain cytokines. In the present study, we investigated the effects of these interventions on a larger group of cytokines.MethodsPlasma cytokine levels were determined in 15 patients with myeloma who were undergoing peripheral blood stem cell harvesting, and we compared the patients with healthy donors who were undergoing platelet apheresis.ResultsSeveral cytokines showed altered levels in patients with myeloma when examined after chemotherapy plus granulocyte colony-stimulating factor–induced stem cell mobilization. The most striking effect was increased levels of several CCL (CCL2/3/4) and CXCL (CXCL5/8/10/11) chemokines as well as increased thrombopoietin, interleukin 1 receptor antagonist, interleukin-4, granulocyte colony-stimulating factor and hepatocyte growth factor. Stem cell harvesting by apheresis altered the plasma levels of several mediators (CD40 ligand, interleukin 1 receptor antagonist, CCL5 and CXCL5/8/10/11). Apheresis in patients with myeloma had divergent effects on these chemokine levels, although they were all still significantly higher than for healthy individuals. Thrombapheresis in healthy individuals had only minor effects on plasma cytokine levels. Stem cell graft supernatants showed high levels of several cytokines, especially CCL and CXCL chemokines. Analyses of chemokine profiles in pre-apheresis plasma and graft supernatants suggested that such profiling can be used to detect prognostically relevant differences between patients.ConclusionsOur results demonstrate that patients with myeloma have an altered cytokine network during stem cell mobilization, and the network is further altered during stem cell harvesting by leukapheresis. These treatment- or procedure-induced alterations involve several mediators known to affect myeloma cell proliferation, migration and survival.     

Epithelial Ovarian Cancer-Induced Angiogenic Phenotype of Human Omental Microvascular Endothelial Cells May Occur Independently of VEGF Signaling

Epithelial ovarian cancer (EOC) metastasizes transcoelomically to the peritoneum and omentum, and despite surgery and chemotherapy, recurrent disease is likely. Metastasis requires the induction of proangiogenic changes in the omental microenvironment and EOC-induced omental angiogenesis is currently a key therapeutic target. In particular, antiangiogenic therapies targeting the vascular endothelial growth factor A (VEGFA) pathway are commonly used, although, with limited effects. Here, using human omental microvascular endothelial cells (HOMECs) and ovarian cancer cell lines as an in vitro model, we show that factors secreted from EOC cells increased proliferation, migration, and tube-like structure formation in HOMECs. However, EOC-induced angiogenic tube-like formation and migration were unaffected by inhibition of tyrosine kinase activity of VEGF receptors 1 and 2 (Semaxanib; SU5416) or neutralization of VEGFA (neutralizing anti-VEGFA antibody), although VEGFA₁₆₅-induced HOMEC migration and tube-like structure formation were abolished. Proteomic investigation of the EOC secretome identified several alternative angiogenesis-related proteins. We screened these for their ability to induce an angiogenic phenotype in HOMECs, i.e., proliferation, migration, and tube-like structure formation. Hepatocyte growth factor (HGF) and insulin-like growth factor binding protein 7 (IGFBP-7) increased all three parameters, and cathepsin L (CL) increased migration and tubule formation. Further investigation confirmed expression of the HGF receptor c-Met in HOMECs. HGF- and EOC-induced proliferation and angiogenic tube structure formation were blocked by the c-Met inhibitor PF04217903. Our results highlight key alternative angiogenic mediators for metastatic EOC, namely, HGF, CL, and IGFBP-7, suggesting that effective antiangiogenic therapeutic strategies for this disease require inhibition of multiple angiogenic pathways.     

Effects of collagen peptides intake on skin ageing and platelet release in chronologically aged mice revealed by cytokine array analysis

Action mechanisms underlying various biological activities of collagen peptides (CPs) remained to be elucidated. Cytokines may play an important role in mediating these health benefits of CPs. This study aimed to systemically examine the cytokines in skin and blood regulated by CPs intake. Thirteen‐month‐old female Kunming mice were administered with CPs for 2 months (0 or 400 mg/kg bodyweight/day). The cytokines in skin and plasma were analysed using a 53‐cytokine array and corresponding ELISA kits. In skin, CPs intake significantly down‐regulated placenta growth factor (PIGF‐2), insulin‐like growth factor (IGF)‐binding protein (IGFBP) ‐2 and IGFBP‐3, and up‐regulated platelet factor 4 (PF4), serpin E1 and transforming growth factor (TGF)‐β₁. CPs treatment also increased the type I collagen mRNA and protein levels and improved the aged skin collagen fibres. In plasma, nine cytokines were significantly down‐regulated by CPs intake compared to the model group: fibroblast growth factor (FGF)‐2, heparin‐binding (HB) epidermal growth factor (EGF)‐like growth factor (HB‐EGF), hepatocyte growth factor (HGF), platelet‐derived growth factor (PDGF)‐AB/BB, vascular endothelial growth factor (VEGF), chemokine (C‐X‐C motif) ligand 1 (KC), matrix metalloproteinase (MMP)‐9, interleukin (IL)‐1α and IL‐10; 2 cytokines were significantly up‐regulated, including TGF‐β₁ and serpin F1. Furthermore, CPs intake significantly decreased the level of platelet release indicators in the plasma and washed platelets, including PF4, granule membrane protein (GMP)‐140, β‐thromboglobulin and serotonin. These results provide a mechanism underlying anti‐skin ageing by CPs intake and highlight potential application of CPs as a healthcare supplement to combat cancer and cardiovascular disease by inhibiting platelet release.     

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.^1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.^1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.⁵ This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.