Trisomy 21 causes changes in the circulating proteome indicative of chronic autoinflammation.

Trisomy 21 (T21) causes Down syndrome (DS), but the mechanisms by which T21 produces the different disease spectrum observed in people with DS are unknown. We recently identified an activated interferon response associated with T21 in human cells of different origins, consistent with overexpression of the four interferon receptors encoded on chromosome 21, and proposed that DS could be understood partially as an interferonopathy. However, the impact of T21 on systemic signaling cascades in living individuals with DS is undefined. To address this knowledge gap, we employed proteomics approaches to analyze blood samples from 263 individuals, 165 of them with DS, leading to the identification of dozens of proteins that are consistently deregulated by T21. Most prominent among these proteins are numerous factors involved in immune control, the complement cascade, and growth factor signaling. Importantly, people with DS display higher levels of many pro-inflammatory cytokines (e.g. IL-6, MCP-1, IL-22, TNF-α) and pronounced complement consumption, resembling changes seen in type I interferonopathies and other autoinflammatory conditions. Therefore, these results are consistent with the hypothesis that increased interferon signaling caused by T21 leads to chronic immune dysregulation, and justify investigations to define the therapeutic value of immune-modulatory strategies in DS.

Selected neuroendocrine tumour markers, growth factors and their receptors in typical and atypical bronchopulmonary carcinoids.

INTRODUCTION: Bronchopulmonary neuroendocrine tumours (BP NET) cause many diagnostic and therapeutic problems. There is an ongoing search for biochemical markers of activity of these tumours. The use of polypeptide growth factors seems potentially feasible in establishing the diagnosis, prognosis and treatment of these tumours. MATERIAL AND METHODS: We included 41 patients aged 25 to 78 years with histopathologically confirmed typical and atypical bronchopulmonary carcinoid tumours and 20 healthy volunteers. We assessed the levels of specific and non-specific markers of these tumours and of selected growth factors relative to TNM classification. RESULTS: The levels of specific markers (serotonin and its metabolite, 5-hydroxyindoleacetic acid [5HIAA]) and non-specific markers (chromogranin A [CgA]) were significantly higher in patients with atypical carcinoid tumours. The serum levels of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and VEGF receptor-1 (VEGFR-1) were significantly higher in patients with carcinoid tumours versus the control group. The levels of VEGFR-1 closely correlated with TNM classification. No such correlation could, however, be confirmed for the levels of HGF, VEGF or VEGFR-2. CONCLUSIONS: Determination of CgA, serotonin and 5HIAA may be useful in the diagnosis of BP NET, particularly in atypical carcinoid tumours, and their levels depend on the presence of distant metastases. Determination of growth factors (VEGF and its receptor, VEGFR­1, and HGF) may prove useful in the clinical diagnosis of these tumours, while the assessment of VEGFR­1 expression may be helpful in tumour staging.

Biomarkers to predict response to sunitinib therapy and prognosis in metastatic renal cell cancer.

Sunitinib is an orally-administered, multitargeted tyrosine kinase inhibitor. The main targets are vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3, platelet-derived growth factor receptor (PDGFR)-α, and PDGFR-ß. Among therapeutic targeting agents, it is the best available in the USA for patients with metastatic renal cell cancer (RCC). Well-constructed clinical trials have led to the worldwide approval of various agents for RCC. However, in clinical practice, it remains difficult to determine the best treatment strategy with these agents. Therefore, the identification of biomarkers to predict response and side-effects and to select optimal dosages is urgently needed. Potential mechanisms of action and resistance need to be understood in order to make accurate predictions. This article briefly reviews candidate biomarkers of sunitinib therapy in terms of clinical variables, genetic factors, and circulating proteins and endothelial cells. Although further validation and implementation is necessary, if validated, biomarkers will help measure the therapeutic response in individual patients and establish treatment strategies for metastatic RCC.

Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid.

Dietary conjugated linoleic acid (CLA) has been shown previously to inhibit rat mammary carcinogenesis. In addition to direct effects on mammary epithelial cells,including decreased proliferation and induction of apoptosis, CLA may exert its effects indirectly by inhibiting the differentiation of mammary stromal cells to an endothelial cell type. Specifically, CLA was found to decrease the ability of mammary stromal cells to form complex anastomosing microcapillary networks in vitro on Engelbreth-Holm-Swarm-derived reconstituted basement membrane. This suggested that CLA might inhibit angiogenesis in vivo. To test this possibility, CD2/F(1) mice were placed on synthetic diets containing 0, 1, or 2% CLA for 6 weeks, before angiogenic challenge by s.c. injection with an angiogenic gel substrate (Matrigel pellet assay). After 7 days, the pellets from animals fed the control diet were infiltrated by abundant branching networks of blood vessels with patent lumen-containing RBCs. In contrast, pellets from the CLA-fed animals contained fewer infiltrating cells, which formed limited branching cellular networks, the majority of which had collapsed lumen and no RBCs. Both levels of dietary CLA showed similar effects, with the number of RBC-containing vessels per 20x field decreased to a third of that seen in control. Dietary CLA decreased serum levels of vascular endothelial growth factor (VEGF) and whole mammary gland levels of VEGF and its receptor Flk-1. Both cis-9, trans-11 and trans-10, cis-12 CLA isomers were effective in inhibiting angiogenesis in vitro in a dose-dependent fashion. The ability of CLA to inhibit angiogenesis may contribute to its efficacy as a chemopreventive agent.

Upregulation of vascular endothelial growth factor receptors is associated with advanced neuroblastoma.

BACKGROUND: Angiogenesis is essential for tumor growth and relies on the production of angiogenic factors. Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis that binds to tyrosine kinase receptors Flt-1 and KDR. The interaction of VEGF and its receptors at gene and protein levels in neuroblastoma remains widely unknown. METHODS: Tumor biopsy specimens and serum were obtained from 37 neuroblastoma patients; adrenal biopsy sections and sera of 7 normal children served as controls. Biopsy specimens were examined by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting; serum was analyzed by enzyme-linked immunosorbent assay (ELISA). VEGF-A(165), B, C, Flt-1, and KDR were analyzed. RESULTS: VEGF isoforms and its receptors' mRNA were expressed in neuroblastoma and control tissues. Whereas the ligands were increased in stages III and IV, the receptors were upregulated in stage III only. At protein level, VEGF-B and C, Flt-1, and KDR were not detectable in tissue lysates, whereas VEGF-A was increased in stages III and IV. Serum VEGF protein levels were upregulated in stage III. CONCLUSIONS: VEGF-A(165) is one of the major angiogenesis regulators among the ligands' family of VEGF, whereas its receptors KDR, and most probably Flt-1, may contribute to a poor prognosis (angiogenic) phenotype, as indicated by their upregulated MRNA levels in stage III neuroblastoma. VEGF-A(165) mainly contributes to increased serum VEGF levels and may serve as a diagnostic tool in advanced-stage neuroblastoma.

Vascular endothelial growth factor and its receptor, Flt-1, in the plasma of patients with coronary or peripheral atherosclerosis, or Type II diabetes.

Since atherosclerosis is characterized by endothelial damage, re-growth seems likely to be occurring in order to repair or replace injured cells. Angiogenic vascular endothelial growth factor (VEGF), a likely mediator of these events, acts on the endothelium via a specific receptor, Flt-1. We hypothesized that patients with different manifestations of atherosclerosis, and others with diabetes, would have altered plasma levels of VEGF and Flt-1 compared with healthy individuals. Accordingly, 70 patients with peripheral artery disease (PAD), 70 patients with coronary artery disease (CAD), and 70 age- and sex-matched healthy controls were recruited. We also recruited 14 patients with diabetes asymptomatic for atherosclerosis, 14 patients with diabetes and atherosclerosis, and 14 age- and sex-matched controls. VEGF and soluble Flt-1 (sFlt-1) were measured by ELISA. In the main study of PAD and CAD, VEGF was raised in both patient groups (P<0.05) compared with the controls, but was not different between the patient groups. sFlt-1 was lower in patients with PAD (P<0.05), but not in those with CAD, compared with the controls. VEGF was raised in the patients with diabetes plus atherosclerosis (P<0.05), but not in the group with diabetes alone; levels of sFlt-1 were unaltered in both diabetes groups. Our data point to changes in plasma levels of VEGF and its receptor sFlt-1 in diabetes and atherosclerosis that may have relevance for therapy and angiogenesis in these conditions.

Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth.

Many solid tumors produce vascular endothelial growth factor C (VEGF-C), and its receptor, VEGFR-3, is expressed in tumor blood vessels. To study the role of VEGF-C in tumorigenesis, we implanted MCF-7 human breast carcinoma cells overexpressing recombinant VEGF-C orthotopically into severe combined immunodeficient mice. VEGF-C increased tumor growth, but unlike VEGF, it had little effect on tumor angiogenesis. Instead, VEGF-C strongly promoted the growth of tumor-associated lymphatic vessels, which in the tumor periphery were commonly infiltrated with the tumor cells. These effects of VEGF-C were inhibited by a soluble VEGFR-3 fusion protein. Our data suggest that VEGF-C facilitates tumor metastasis via the lymphatic vessels and that tumor spread can be inhibited by blocking the interaction between VEGF-C and its receptor.

Plasma levels of vascular endothelial growth factor and its soluble receptor (SFlt-1) in essential hypertension.

In this study of 27 untreated patients with uncomplicated essential hypertension, we report on elevated plasma levels of vascular endothelial growth factor and its soluble receptor Flt-1 compared with healthy controls; these increased levels are reduced by the treatment of hypertension. This raises the possibility that abnormal angiogenesis may contribute to the pathogenesis of complications related to hypertension and merits exploration in larger studies.

Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery.

In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell factor (SCF), Flt3 ligand (Flt3) and fetal antigen 1 (FA1)) and three lineage-specific growth factors (EPO, G-CSF and thrombopoietin (Tpo)) were analyzed by ELISA in 16 patients with multiple myeloma (MM) and 16 patients with non-Hodgkin's lymphoma (NHL). The relative number of SCF, Flt3, Tpo and G-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need to be confirmed in prospectively designed studies.

Vascular endothelial growth factor and its receptor, Flt-1, in smokers and non-smokers.

Raised levels of plasma vascular endothelial growth factor (VEGF) are found in some cancers, diabetes, and certain other conditions, but levels of its receptor, soluble Flt-1 (sFlt-1), in these diseases have yet to be reported. We hypothesised that smoking would influence levels of these molecules. Consequently, we measured VEGF and sFlt-1 by enzyme-linked immunosorbent assay (ELISA) in plasma from 92 non-smokers and 35 smokers. No difference in VEGF was seen between the groups but, despite considerable overlap, sFlt-1 was significantly lower in smokers (P = 0.027). VEGF and sFlt-1 correlated strongly with each other (P < 0.001). Although VEGF may arise from a number of cell types, including endothelial cells, the primary source of sFlt-1 is thought to be the endothelium; however, neither VEGF nor sFlt-1 correlated with levels of the endothelial cell activation/damage marker soluble thrombomodulin. Our data point to changes in levels of the VEGF receptor, sFlt-1--but not VEGF itself--in smokers, which appears to be unrelated to endothelial cell function.

Plasma VEGF and soluble VEGF receptor FLT-1 in proliferative retinopathy: relationship to endothelial dysfunction and laser treatment.

PURPOSE: To study plasma levels of vascular endothelial growth factor (VEGF, an index of angiogenesis), its soluble receptor (sFlt-1) and von Willebrand factor (vWf, an index of endothelial damage or dysfunction) in patients with proliferative retinopathy and corresponding changes in plasma levels after pan-retinal photocoagulation (PRP). METHODS: Eighteen patients (10 men; age, 57+/-16 years, mean +/- SD) with proliferative retinopathy secondary to diabetes (n = 13) and ischemic retinal vein occlusion (n = 5) with no previous PRP therapy were studied. Blood samples were obtained before and at 4 months after the last PRP session. Baseline (prelaser) plasma levels of VEGF, sFlt-1, and vWf (all by ELISA) were compared with levels in 16 diabetic patients with background retinopathy ("hospital controls"), and 18 healthy, age- and sex-matched "healthy controls." RESULTS: Patients with proliferative retinopathy had significantly raised plasma VEGF when compared with both control groups (P = 0.001). Patients with proliferative retinopathy and hospital controls had significantly raised plasma vWf levels when compared with healthy controls (P = 0.012). There was no difference in sFlt-1 levels between patients and controls (P = 0.162). After PRP, there was a significant reduction in plasma VEGF levels at 4 months' follow-up (P < 0.001), but no significant changes in plasma sFlt-1 or vWf levels. Patients with complete resolution of neovascularization had a trend toward lower median VEGF levels (80 versus 150 pg/ml, P = 0.062), but vWf levels (P = 0.50) and sFlt-1 (P = 0.479) were not statistically different. Baseline VEGF and sFlt-1 levels were significantly correlated (Spearman r = 0.505, P = 0.032) but after PRP at 4 months' follow-up, this was no longer significant (r = -0.269, P = 0.28). CONCLUSIONS: In this pilot study, patients with proliferative retinopathy demonstrate elevated peripheral markers of angiogenesis and endothelial dysfunction, suggesting a role for these processes in the pathogenesis of this condition. A fall in levels of VEGF after successful laser treatment may provide an opportunity for monitoring disease progression or relapse via a blood sample.

Expression of hematopoietic growth factor receptors on early hematopoietic precursors: detection and regulation.

Since the original isolation of colony-stimulating factors from human serum, conditioned medium of murine or human cell lines, or freshly isolated human mononuclear cells, a revolutionary explosion of ideas has occurred in our understanding of molecular controls of the hematopoietic stem cell self-renewal and differentiation. With the availability of techniques of molecular cloning in the early 1 980s, the first hematopoietically activated cytokines led to molecular clones expressed in bacteria, yeast, or mammalian cellular systems. There then followed a development of techniques leading to the molecular cloning and expression of many hematopoietic growth factors and their receptors, as well as the primary, secondary, and tertiary molecules in signal transduction into activation of specific genes for differentiation or self-renewal. The clinical use of these factors in the diagnosis, treatment, and incorporation into new cell therapies for a variety of diseases is a subject of current interest.

Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ.

Antiangiogenic therapy shows promise as a strategy for cancer treatment. We constructed an adenovirus (AdVEGF-ExR) expressing the entire extracellular domain of the human vascular endothelial growth factor (VEGF) receptor (flt-1) fused to the Fc portion of human IgG. The soluble receptor secreted from AdVEGF-ExR-infected cells bound to VEGF and inhibited VEGF-induced DNA synthesis in endothelial cells. When human lung cancer cell line H157, which produces not only VEGF but also fibroblast growth factor 2 and interleukin 8 at substantial levels, was infected with AdVEGF-ExR, cell growth in vitro was not affected. However, when H157 cells infected with AdVEGF-ExR were injected s.c. into nude mice, tumor formation stopped on the 10th day after reaching a certain size (about 100 mm3), and tumor size declined gradually thereafter. When AdVEGF-ExR was injected into skeletal muscle and uninfected H157 cells were injected s.c., the soluble receptor was detectable in the circulating blood for 3 weeks, tumor growth ceased after 10 days, and tumor size declined thereafter. Histological examination revealed that intratumor angiogenesis was markedly suppressed, and apoptosis was enhanced. Using the same experimental protocol, a significant suppression of tumor growth was also seen in four of five other lung cancer cell lines, some of which secreted VEGF at nominal levels, at least under normoxic conditions in vitro. Our results demonstrate that adenovirus-mediated expression of a soluble VEGF receptor in a remote organ could inhibit tumor angiogenesis and enhance apoptosis and thereby suppress tumor growth in vivo. Adenovirus-mediated overexpression of a soluble VEGF receptor in a remote organ may have the potential to be a feasible and effective strategy for cancer treatment.

Reduced expression of hemopoietic cytokine receptors on cord blood progenitor cells in neonates at risk for atopy.

BACKGROUND: Recent findings point to an association between allergic asthma in adults and increased responsiveness of myeloid progenitor cells to certain hemopoietic growth factors. However, it is not clear at what age these changes in progenitor cells first become manifest, although increasing evidence suggests that the allergic phenotype may begin to emerge in very early life. OBJECTIVE: We sought to compare expression of hemopoietic cytokine receptors on CD34(+) progenitor cells in cord blood from normal infants ("low risk" for subsequent atopy) and infants with at least one atopic first degree relative ("at risk" for subsequent atopy). METHODS: Cord blood was obtained from 21 neonates. Nonadherent mononuclear cells were stained with mAbs directed against CD45, CD34, and the alpha-chains of the GM-CSF, IL-3, and IL-5 receptors and analyzed by flow cytometry. RESULTS: No differences in absolute CD34(+) numbers were observed between the 2 groups. However, expression of GM-CSF receptor on CD34(+) cells was reduced in the "at-risk" compared with the "low- risk" group (P =.021), although no significant differences were noted between the 2 groups with respect to IL-3 and IL-5 receptor expression. CONCLUSION: The functional sequelae of reduced GM-CSF receptor expression on CD34(+) cells remain to be determined. Nonetheless, these findings show an association between genetic risk for atopy and changes in the expression of hemopoietic cytokine receptors on cord blood progenitor cells and support the notion that the allergic phenotype may begin to evolve in the perinatal period.

Molecular markers of carcinogenesis.

The protein products of oncogenes and tumor suppressor genes play critical roles in the development of many cancers. The expression of a number of these proteins can be detected in extracellular fluids such as blood. This article reviews the literature on the application of methods for the detection of the proteins of oncogenes and tumor suppressor genes in the blood of humans with cancer or at risk for the development of cancer. The detection of these proteins in blood may be useful molecular markers of carcinogenesis that could play an important part in cancer diagnosis, prognosis, and prevention.

Biomarkers of gene expression: growth factors and oncoproteins.

This article reviews the literature on the application of methods for the detection of growth factors, oncogene proteins, and tumor-suppressor gene proteins in the blood of humans with cancer or who are at risk for the development of cancer. The research summarized here suggests that many of these biomarker assays can be used to distinguish between diseased and nondiseased states and in some instances may be able to predict susceptibility for future disease. Thus, these biomarkers could be valuable tools for monitoring at-risk populations for purposes of disease prevention and control.