2808 Proteomics 2012, 12, 2808–2821 DOI 10.1002/pmic.201100586 RESEARCH ARTICLE Proteomic proﬁling of high glucose primed monocytes identiﬁes cyclophilin A as a potential secretory marker of inﬂammation in type 2 diabetes Surya Ramachandran 1 , Anila Venugopal 1 , Sathisha K. 2 , Reshmi G. 3 , Sona Charles 3 , Divya G. 1 , N. S. Pratap Chandran 4 , Ajit Mullassari 5 , M. Radhakrishna Pillai 3 and C. C. Kartha 1 1 Cardiovascular Disease Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India 2 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India 3 Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India 4 Indian Institute of Diabetes, Thiruvananthapuram, India 5 Madras Medical Mission, Chennai, India Hyperglycemia is widely recognized to be a potent stimulator of monocyte activity, which is a crucial event in the pathogenesis of atherosclerosis. We analyzed the monocyte proteome for potential markers that would enhance the ability to screen for early inﬂammatory status in Type 2 diabetes mellitus (T2DM), using proteomic technologies. Monocytic cells (THP-1) were primed with high glucose (HG), their protein proﬁles were analyzed using 2DE and the downregulated differentially expressed spots were identiﬁed using MALDI TOF/MS. We selected ﬁve proteins that were secretory in function with the help of bioinformatic programs. A predominantly downregulated protein identiﬁed as cyclophilin A (sequence coverage 98%) was further validated by immunoblotting experiments. The cellular mRNA levels of cyclophilin A in various HG-primed cells were studied using qRT-PCR assays and it was observed to decrease in a dose-dependent manner. LC-ESI-MS was used to identify this protein in the conditioned media of HG-primed cells and conﬁrmed by Western blotting as well as ELISA. Cyclophilin A was also detected in the plasma of patients with diabetes. We conclude that cyclophilin A is secreted by monocytes in response to HG. Given the paracrine and autocrine actions of cyclophilin A, the secreted immunophilin could be signiﬁcant for progression of atherosclerosis in type 2 diabetes. Our study also provides evidence that analysis of monocyte secretome is a viable strategy for identifying candidate plasma markers in diabetes. Keywords: Cell biology / Hyperglycemia / Monocytes / Type 2 diabetes mellitus Received: November 9, 2011 Revised: June 5, 2012 Accepted: June 14, 2012 1 Introduction Diabetes is a proinﬂammatory state and the proinﬂammatory phenotype in diabetes is characterized by elevated plasma Correspondence: Professor C. C. Kartha, Disease Biology and Molecular Medicine, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, INDIA E-mail: cckartha@rgcb.res.in Fax: +91-471-234-9303 Abbreviations: BiNGO, biological networks gene ontology; HG, high glucose; MRP, myeloid-related protein; PBMCs, peripheral blood mononuclear cells; ROS, reactive oxygen species; T2DM, type 2 diabetes mellitus; VSMCs, vascular smooth muscle cells C-reactive protein, cytokines, chemokines, adhesion molecules, monocytic activity, etc. Monocyte activation and adhesion to the arterial endothelium are key events in the pathogenesis of atherosclerosis [1, 2]. These cellular events are intensiﬁed in type 2 diabetes and lead to accelerated development of atherosclerotic vascular lesions in diabetes. Chronic hyperglycemia leads to an uncontrolled produc- tion of free radicals and increased extracellular as well as intracellular protein glycation. This results in altered function of endothelial and smooth muscle cells in blood vessels. Jointly, these processes cause basement membrane Colour Online: See the article online to view Figs. 2 and 3 in colour. C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com