Abstract-Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 months) and old (30 months) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II and monocyte chemoattractant protein (MCP)-1 within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VSMCs from young aorta to angiotensin II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity.The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the angiotensin II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis. Key Words: MFG-E8 Ⅲ angiotensin II Ⅲ monocyte chemoattractant protein-1 Ⅲ vascular smooth muscle cells Ⅲ aging P roinflammatory processes and associated elevated invasion capacity of vascular smooth muscle cells (VSMCs) are increased within the diffuse thickening of the arterial wall that evolves with advancing age. [1][2][3][4] In humans, this ageassociated arterial remodeling is an independent risk factor for the epidemic of quintessential human cardiovascular diseases, ie, atherosclerosis, hypertension, and stroke. 1,3,5,6 The age-associated arterial wall thickening and other aspects of arterial remodeling are evolutionarily conserved in various mammalian species, including rodents, nonhuman primates, and humans. 1,3,[7][8][9][10][11][12][13][14] The thickened arterial intima is formed because of VSMCs invasion and proliferation and is not limited to secretion within the subendothelial space. 2,3,7,14 A growing body of evidence indicates that VSMCs within the arterial media begin to express and activate proteases such as matrix metalloprotease (MMP)2 and calpain-1, which enable cytoskeletal remodeling and degradation of (1) basement membranes that surround VSMCs, (2) adjacent matrix, and (3) elastic lamina. Thus VSMCs invasion into the subendothelial space is driven, at least in part, by angiotensin (Ang) Original received September 8, 2008; revision re...
In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation (undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic "cellular" proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present. There has been a surge of interest in the proteomic analysis of plasma and serum in the search for clinically relevant biomarkers of disease. In biomarker discovery, it is necessary to maximize the observation of the plasma or serum proteome to detect proteins with low abundance. This can be achieved by optimization of protein separation methods as well as selective depletion of the proteins at high abundance such as immunoglobins (e.g. IgG) and albumin. There are a large number of proteomic technologies that separate either proteins or peptides prior to MS (1). A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatographic focusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. To date, assessment and optimization of either plasma or serum separation by the PF2D has not been undertaken. Generally LC proteomic methods have focused on separating complex mixtures of peptides obtained following digestion of the serum proteome (peptide LC, shotgun), whereas separation of proteins has been relegate...
Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (twodimensional liquid chromatography using the ProteomeLab TM PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one-and two-dimensional LC allowed for the separation of the ␣-subunit of F 1 F 0 ATP synthase that differed due to a change in pI or hydrophobicity. The eukaryotic proteome is a compilation of proteins that represents the integration of numerous cellular processes that begin with the variable transcription of genes to mRNA. These products are then translated to proteins, which may in turn be potentially co-and/or post-translationally modified to produce an array of proteins (1, 2). Due to the large number of unique protein species produced coupled with differences in their relative abundance, there is as of yet no single proteomics technology that has the analytical capacity or sensitivity to realize the goal of complete proteome coverage. One strategy to maximize proteome coverage is to combine synergistic proteomics technologies, particularly if each technology reveals a unique subset of proteins. Using the inner mitochondrial membrane as a model subproteome, we compared the ability of three protein separation methods (two-dimensional LC (2-DLC 1 with the PF2D), one-dimensional reversed phase HPLC (1-DLC; reversed phase high performance liquid chromatography (RP-HPLC)), and two-dimensional gel electrophoresis (2-DE) to determine the relative overlap in protein separation for these technologies.2-DE, a classical proteomics technology that separates proteins based on their pI and molecular weight, has a practical dynamic range of 10 4 orders of magnitude (for r...
The ability to decipher the dynamic protein component of any system is determined by the inherent limitations of the technologies used, the complexity of the sample, and the existence of an annotated genome. In the absence of an annotated genome, large-scale proteomic investigations can be technically difficult. Yet the functional and biological species differences across animal models can lead to selection of partially or nonannotated organisms over those with an annotated genome. The outweighing of biology over technology leads us to investigate the degree to which a parallel approach can facilitate proteome coverage in the absence of complete genome annotation. When studying species without complete genome annotation, a particular challenge is how to ensure high proteome coverage while meeting the bioinformatic stringencies of high-throughput proteomics. A protein inventory of Oryctolagus cuniculus mitochondria was created by overlapping "protein-centric" and "peptide-centric" one-dimensional and two-dimensional liquid chromatography strategies; with additional partitioning into membraneenriched and soluble fractions. With the use of these five parallel approaches, 2934 unique peptides were identified, corresponding to 558 nonredundant protein groups. 230 of these proteins (41%) were identified by only a single technical approach, confirming the need for parallel techniques to improve annotation. To determine the extent of coverage, a side-by-side comparison with human and mouse cardiomyocyte mitochondrial studies was performed. A nonredundant list of 995 discrete proteins was compiled, of which 244 (25%) were common across species. The current investigation identified 142 unique protein groups, the majority of which were detected here by only one technical approach, in particular peptide-and protein-centric two-dimensional liquid chromatography. Although no single approach achieved more than 40% coverage, the combination of three approaches (protein-and peptide-centric two-dimensional liquid chromatography and subfractionation) contributed 96% of all identifications. Parallel techniques ensured minimal false discovery, and reduced single peptide-based identifications while maximizing sequence coverage in
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.