Cédric Broussard

Identification of Target Antigens of Antifibroblast Antibodies in Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension (PAH) may be classified as idiopathic (IPAH) or familial (FPAH) or associated with various conditions and exposures such as dexfenfluramine intake (Dex-PAH) or systemic sclerosis (SSc-PAH). Because fibroblast dysfunction has been identified in SSc and IPAH and antifibroblast antibodies (AFAs) with a pathogenic role have been detected in the serum of SSc patients, we used a proteomic approach combining two-dimensional electrophoresis and immunoblotting to identify the target antigens of AFAs in such patients. OBJECTIVES To identify target antigens of antifibroblast antibodies in pulmonary arterial hypertension. METHODS Sera from 24 patients with IPAH, 6 with FPAH, 6 with Dex-PAH, and 12 with SSc-PAH were collected. We pooled sera from sets of three patients with PAH classification and SSc-PAH based on autoantibody profile. Sera from 14 healthy blood donors were also pooled and used as a control. MEASUREMENTS AND MAIN RESULTS Serum IgG antibodies in the pools of patients with IPAH (n = 8), FPAH (n = 2), Dex-PAH (n = 2), and SSc-PAH (n = 4) recognized 103 +/- 31, 63 +/- 20, 78 +/- 11, and 81 +/- 12 protein spots, respectively, whereas serum IgG antibodies from healthy control subjects recognized 43 +/- 22 protein spots. Twenty-one protein spots were specifically recognized by the serum IgG antibodies from patients with PAH. We identified 16 of the protein spots as vimentin, calumenin, tropomyosin 1, heat shock proteins 27 and 70, glucose-6-phosphate-dehydrogenase, phosphatidylinositol 3-kinase, DAP kinase, and others. These proteins are involved in regulation of cytoskeletal function, cell contraction, oxidative stress, cell energy metabolism, and other key cellular pathways. CONCLUSIONS AFAs detected in patients with PAH recognize cellular targets playing key roles in cell biology and maintenance of homeostasis.

Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma

Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected. Target antigens were identified by two-dimensional electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry. Serum immunoglobulin G from patients with SSc with or without PAH and patients with iPAH specifically recognised 110, 82 and 37 protein spots, respectively. Among others, target antigens of AECAs included lamin A/C, tubulin &bgr;-chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin &bgr;-chain. In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension, and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin &bgr;-chain.

Differential Proteomic Analysis of Human Glioblastoma and Neural Stem Cells Reveals HDGF as a Novel Angiogenic Secreted Factor

Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem‐like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio‐chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two‐dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma‐derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC‐conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC‐conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti‐HDGF antibodies. In vivo, GSC‐conditioned medium induced neoangiogenesis, whereas HDGF‐targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high‐grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties. STEM CELLS 2012;30:845–853

Proteomic analysis of β-catenin activation in mouse liver by DIGE analysis identifies glucose metabolism as a new target of the Wnt pathway

The Wnt/β‐catenin signaling pathway has been increasingly implicated in liver development and physiology. Aberrant activation of this pathway is one of the major genetic events observed during the process of human HCC development. To gain insight into the mechanism underlying β‐catenin action in the liver, we conducted a quantitative differential proteomic analysis using 2‐D DIGE combined with MS, in mice with liver‐specific deletion of Apc resulting in acute activation of β‐catenin signaling (ApcKOliv mice). We identified 94 protein spots showing differential expression between mutant ApcKOliv and control mice, corresponding to 56 individual proteins. Most of the proteins identified were associated with metabolic pathways, such as ammonia and glucose metabolism. Our analysis showed an increase in lactate dehydrogenase activity together with a downregulation of two mitochondrial ATPase subunits (ATP5a1 and ATP5b). These observations indicate that β‐catenin signaling may induce a shift in the glucose metabolism from oxidative phosphorylation to glycolysis, known as the “Warburg effect”. Imaging with 18F‐fluoro‐2‐deoxy‐D‐glucose‐positron emission tomography suggests that the specific metabolic reprogramming induced by β‐catenin in the liver does not imply the first step of glycolysis. This observation may explain why some HCCs are difficult to assess by fluoro‐2‐deoxy‐D‐glucose‐positron emission tomography imaging.

Antifibroblast antibodies from systemic sclerosis patients bind to α-enolase and are associated with interstitial lung disease

Objective: To identify target antigens of antifibroblast antibodies (AFA) in systemic sclerosis (SSc) patients. Patients and Methods: In the first part, sera from 24 SSc patients (12 with pulmonary arterial hypertension (PAH) and 12 without) and 36 idiopathic PAH patients, tested in pooled sera for groups of three, were compared with a sera pool from 14 healthy controls (HC). Serum IgG reactivity was analysed by the use of a two-dimensional electrophoresis and immunoblotting technique with normal human fibroblasts antigens. In the second part, serum IgG reactivity for two groups: 158 SSc, 67 idiopathic PAH and 100 HC; and 35 SSc and 50 HC was tested against α-enolase from Saccharomyces cerevisiae and recombinant human (rHu) α-enolase, respectively, on ELISA. Results: In the first part, α-enolase was identified as a main target antigen of AFA from SSc patients. In the second part, 37/158 (23%) SSc patients, 6/67 (9%) idiopathic PAH patients and 4/100 (4%) HC (p<0.001) had anti-S cerevisiae α-enolase antibodies; 12/35 (34%) SSc patients and 3/50 (6%) HC had anti-rHu α-enolase antibodies (p = 0.001). In SSc, the presence of anti-S cerevisiae α-enolase antibodies was associated with interstitial lung disease (ILD), decreased total lung capacity (73.2% vs 89.7%; p<0.001) and diffusion capacity for carbon monoxide (47.4% vs 62.3%; p<0.001), and antitopoisomerase 1 antibodies (46% vs 21%; p = 0.005) but not anticentromere antibodies (11% vs 34%; p = 0.006). Results were similar with rHu α-enolase testing. Conclusion: In SSc, AFA recognise α-enolase and are associated with ILD and antitopoisomerase antibodies.

Identification of target antigens of antiendothelial cell antibodies in healthy individuals: A proteomic approach

In order to identify target antigens of anti‐endothelial cell (anti‐EC) antibodies (AECA) in healthy individuals, we have used a proteomic approach combining 2‐DE and immunoblotting. Whole cell protein extracts obtained from human umbilical vein EC (HUVEC) cultures were used as a source of antigens. Serum IgG from 12 healthy blood donors were tested at a concentration of 200 μg/mL. Targeted spots were identified by MS. The HUVEC proteome was composed of 884 protein spots. Among these, 61 ± 25.8 (mean ± SD) spots were recognized by serum IgG from healthy individuals, with marked differences from one individual to another. Among these spots, 11 were recognized by serum IgG from all healthy individuals tested. These spots corresponded to six different proteins with several spots corresponding to different isoforms of the same protein. Target antigens were: cytoskeletal proteins (β‐actin, α‐tubulin, and vimentin); glycolytic enzymes (glucose‐3‐phosphate‐deshydrogenase and α‐enolase); and prolyl‐4‐hydroxylase β subunit, a member of the disulfide isomerase family. This study shows that the repertoire of IgG AECA is heterogeneous among healthy individuals. IgG from all of the healthy individuals tested recognized a restricted set of highly conserved ubiquitous proteins playing key roles in cell biology and maintenance of homeostasis.

Analysis of transcriptomic and proteomic profiles demonstrates improved Madin–Darby canine kidney cell function in a renal microfluidic biochip

We have evaluated the influence of the microfluidic environment on renal cell functionality. For that purpose, we performed a time lapse transcriptomic and proteomic analysis in which we compared gene and protein expressions of Madin–Darby canine kidney cells after 24 h and 96 h of culture in both microfluidic biochips and plates. The transcriptomic and proteomic integration revealed that the ion transporters involved in calcium, phosphate, and sodium homoeostasis and several genes involved in H+ transporters and pH regulation were up‐regulated in microfluidic biochips. Concerning drug metabolism, we found Phase I (CYP P450), Phase II enzymes (GST), various multidrug resistance genes (MRP), and Phase III transporters (SLC) were also up‐regulated in the biochips. Furthermore, the study shows that those inductions were correlated with the induction of the Ahr and Nrf‐2 dependent pathways, which results in a global cytoprotective response induced by the microenvironment. However, there was no apoptosis situation or cell death in the biochips. Microfluidic biochips may thus provide an important insight into exploring xenobiotic injury and transport modifications in this type of bioartificial microfluidic kidney. Finally, the investigation demonstrated that combining the transcriptomic and proteomic analyses obtained from a cell “on chip” culture would provide a pertinent new tool in the mechanistic interpretation of cellular mechanisms for predicting kidney cell toxicity and renal clearance in vitro.

IgG from patients with pulmonary arterial hypertension and/or systemic sclerosis binds to vascular smooth muscle cells and induces cell contraction

Objectives Pulmonary arterial hypertension (PAH) is characterised by remodelling of pulmonary arteries with enhanced vascular smooth muscle cell (VSMC) contraction, migration and proliferation. The authors investigated the presence of antibodies to human VSMCs in the serum of patients with systemic sclerosis with or without PAH and idiopathic PAH (iPAH). Methods and results Antibodies to VSMCs were detected by immunofluorescence in sera from healthy controls and patients with scleroderma without PAH, scleroderma-associated PAH and iPAH. Serum IgG from these patients induced contraction of VSMCs in a collagen matrix in contrast with IgG from healthy controls. Several protein spots of interest and target antigens were identified by two-dimensional immunoblotting and MS, including stress-induced phosphoprotein 1 and α-enolase. Finally, antibodies to stress-induced phosphoprotein 1 were detected by ELISA in sera from 84%, 76% and 24% of patients with scleroderma without PAH, scleroderma-associated PAH and iPAH, respectively, compared with only 3% of healthy controls. Conclusion The authors have identified IgG that binds to VSMCs in the serum of patients with scleroderma and iPAH. These antibodies may be pathogenic by modulating vascular contraction. The target antigens of these antibodies are stress-induced phosphoprotein 1 and α-enolase.

A Generic Approach for the Purification of Signaling Complexes That Specifically Interact with the Carboxyl-terminal Domain of G Protein-coupled Receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His6-tagged baits, immobilized metal affinity chromatography, one- and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT1 and MT2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT1, 14 proteins that interacted with the C-tail of MT2, and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques.

Identification of target antigens of self‐reactive IgG in intravenous immunoglobulin preparations

Intravenous immunoglobulin (IVIg) contains a wide range of self‐reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self‐antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2‐DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp‐2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic‐IVIg) and one with β‐propiolactone (propiolactone‐IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone‐IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self‐reactive IgG. Thus, protein spots recognized by IgG in propiolactone‐IVIg represent the core set of self‐antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as α‐enolase, RNA processing and cytoskeletal proteins such as lamin‐A/C.