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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...

We recently identified hepatoma-derived growth factor (HDGF) as a nuclear targeted vascular smooth muscle cell (VSM) mitogen that is expressed in developing vascular lesions. In the present study, VSM in culture express endogenous HDGF only in the nucleus and target a green fluorescent protein (GFP)-HDGF fusion to the nucleus. To define the features of the HDGF molecule that are essential for nuclear localization and mitogenic function, deletion and site-directed mutagenesis were performed. Deletion analysis identified the carboxyl-terminal half of HDGF to be responsible for nuclear targeting in VSM. Overexpression of tagged HDGF proteins with point mutations in the putative bipartite nuclear localization sequence in the carboxyl terminus demonstrated that single Lys 3 Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, the Lys 3 Asn mutation of all three lysines blocked nuclear entry. Point mutation of a p34 cdc2 kinase consensus motif within the nuclear localization sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple Lys 3 Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that HDGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is required for HDGF stimulation of DNA replication in VSM.Growth factors play a key role in vascular injury-induced smooth muscle cell (VSM) 1 proliferation leading to neointimal formation and narrowing of the vascular lumen (1-4). However, the mechanisms of growth factor-stimulated VSM proliferation are still not fully understood. After binding and receptor activation, most receptor-growth factor complexes are thought to be internalized, transported to lysosomes, and degraded (5). However examples have emerged where growth factors exert their effect directly on the nucleus. For example, exogenously applied PDGF and bFGF accumulate in the nucleus of endothelial and mesenchymal cells (6, 7), and bFGF can regulate gene transcription in a cell-free system (8). These findings suggest that growth factors may function within the nucleus (9).Nuclear targeting has a structural basis as exemplified by the well characterized basic cluster of seven amino acids that comprise the nuclear localization sequence (NLS) of the SV40 large T antigen. The SV40 sequence has served as a NLS consensus and has allowed the identification of a more complex NLS in vertebrate nuclear targeted proteins (reviewed in Ref. 10). Studies of nucleoplasmin, a major Xenopus nuclear protein, revealed that the NLS was bipartite, containing two required basic amino acid clusters of lysines separated by a variable spacer of 8 -10 amino acids (11). Mutation and deletion studies of nucleoplasmin have demonstrated that the basic amino acid cluster is necessary for nuclear targeting whereas the compo...

To define whether angiotensin I-converting enzyme (ACE) inhibition affects the distribution of renin gene-expressing cells within the kidney, a control group of adult male Wistar-Kyoto rats (C, n = 7) was compared with a group of rats treated with enalapril (E, n = 6) for 5 days. Renin mRNA distribution was assessed using in situ hybridization to a 35S-labeled 28 mer oligonucleotide complementary to rat renin mRNA. Whereas in control rats renin mRNA was confined to a juxtaglomerular location, in enalapril-treated rats, renin mRNA extended proximally along the length of the afferent arteriole. The percent of visible afferent arteriolar length containing renin mRNA was higher in enalapril-treated (71.7 +/- 2.8%) than in control (49.6 +/- 2.1%) rats (P less than 0.0001). These findings were accompanied by an increase in the percent of juxtaglomerular apparatuses (JGAs) containing renin mRNA (71 +/- 2.2 vs. 49 +/- 2.9%; E vs. C, P less than 0.0001). Also, the intensity of the JGA hybridization signals was higher in enalapril-treated (757 +/- 59 grains/JGA) than in control (167 +/- 11 grains/JGA) rats (P less than 0.00001). We conclude that the increased kidney renin gene expression elicited by ACE inhibition is the result of an increase in renin mRNA content per JGA, an increase in the number of JGAs expressing the renin gene, and a recruitment of renin gene-expressing cells along the afferent arteriole.

The role of angiotensin II via the angiotensin type 1 or type 2 receptor in the development of cardiac hypertrophy was determined in adult male Sprague-Dawley rats subjected to coarctation of the abdominal aorta. Five groups of animals were studied: coarctation, coarctation plus DuP 753, coarctation plus PD 123319, sham plus DuP 753, or sham operation. Type 1 receptor blockade was accomplished with DuP 753 given in the drinking water and type 2 blockade with PD 123319 delivered by osmotic minipumps beginning with the day of surgery until 72 hours after aortic coarctation. Mean carotid blood pressures and the carotid-femoral artery blood pressure gradients were not different among coarctation, coarctation plus DuP 753, and coarctation plus PD 123319 animals. However, ratios of heart weight to body weight were higher in coarctation (4.95 ±0.8) or coarctation plus PD 123319 (4.52±0.5) than in sham animals (3.6±0.4; P<.005 and .05, respectively). In coarctation plus DuP 753-treated animals

The definition and classification of ventricular septal defects have been fraught with controversy. The International Society for Nomenclature of Paediatric and Congenital Heart Disease is a group of international specialists in pediatric cardiology, cardiac surgery, cardiac morphology, and cardiac pathology that has met annually for the past 9 years in an effort to unify by consensus the divergent approaches to describe ventricular septal defects. These efforts have culminated in acceptance of the classification system by the World Health Organization into the 11th Iteration of the International Classification of Diseases. The scheme to categorize a ventricular septal defect uses both its location and the structures along its borders, thereby bridging the two most popular and disparate classification approaches and providing a common language for describing each phenotype. Although the first-order terms are based on the geographic categories of central perimembranous, inlet, trabecular muscular, and outlet defects, inlet and outlet defects are further characterized by descriptors that incorporate the borders of the defect, namely the perimembranous, muscular, and juxta-arterial types. The Society recognizes that it is equally valid to classify these defects by geography or borders, so the emphasis in this system is on the second-order terms that incorporate both geography and borders to describe each phenotype. The unified terminology should help the medical community describe with better precision all types of ventricular septal defects.

Objective To determine if, in children, plasma glial fibrillary acidic protein (GFAP) is associated with brain injury during extracorporeal membrane oxygenation (ECMO) and with mortality. Design Prospective, observational study. Setting Pediatric intensive care unit in an urban tertiary care academic center. Patients Neonatal and pediatric ECMO patients (n=22). Interventions Serial blood sampling for GFAP measurements. Measurements and Main Results Prospective patients age 1 day-18 years who required ECMO from April 2008 to August 2009 were studied. GFAP was measured using an electrochemiluminescent immunoassay developed at Johns Hopkins. Control samples were collected from 99 healthy children (0.5-16 years) and 59 NICU infants without neurologic injury. In controls, the median GFAP concentration was 0.055 ng/mL (IQR: 0-0.092 ng/mL) and the 95th percentile of GFAP was 0.436ng/mL. In ECMO patients, plasma GFAP was measured at 6, 12 and every 24 hours after cannulation. We enrolled 22 children who underwent ECMO. Median age was 7 days (IQR, 2 days-9 years), and primary ECMO indication was: cardiac failure, 6/22 (27.3%), respiratory failure, 12/22 (54.5%), ECPR, 3/22 (13.6%), and sepsis, 1/22 (4.6%). Seven of 22 (32%) patients developed acute neurologic injury (intracranial hemorrhage, brain death or cerebral edema diagnosed by imaging). Fifteen of 22 (68%) survived to hospital discharge. In the ECMO group, peak GFAP levels were higher in children with brain injury than those without (median, 5.9 vs 0.09ng/mL, p=0.04) and in non-survivors compared to survivors to discharge (median, 5.9 vs 0.09ng/mL, p=0.04). The odds ratio (OR) for brain injury for GFAP >0.436ng/mL vs normal was 11.5 (95%CI: 1.3-98.3) and the OR for mortality was 13.6 (95%CI: 1.7-108.5). Conclusions High GFAP during ECMO is significantly associated with acute brain injury and death. Brain injury biomarkers may aid in outcome prediction and neurologic monitoring of ECMO patients to improve outcomes and benchmark new therapies.

To define whether intrarenal renin and angiotensinogen synthesis and distribution are affected by angiotensin-converting enzyme (ACE) inhibition, a control group of adult, male Wistar-Kyoto rats (n = 7) was compared with a group of rats treated with enalapril (n = 8) for 5 days. Kidney renin and angiotensinogen mRNA levels were detected by Northern and dot blot analysis, using full-length rat renin and angiotensinogen cDNAs. Renin mRNA levels in the enalapril-treated group were 4.6-fold higher than in the control group (P less than 0.05). Angiotensinogen mRNA levels were not significantly different. The intrarenal distribution of renin assessed by immunocytochemistry was markedly different between the two groups of rats. Whereas in the control kidney renin was localized in a juxtaglomerular position, in the kidneys from enalapril-treated rats, renin immunoreactivity of the afferent arteriole extended well beyond the juxtaglomerular loci in the direction of the interlobular artery. The percent of afferent arteriolar length immunostained for renin was higher in the enalapril-treated (53 +/- 17%) than in the control (33 +/- 15) group. Similarly, the ratio of immunostained juxtaglomerular apparatuses (JGA) over total number of JGA and the ratio of immunostained arteries over total number of arteries were higher in the enalapril-treated (0.84 +/- 0.017; 0.68 +/- 0.03) than in the control (0.67 +/- 0.034; 0.43 +/- 0.045) group (P less than 0.05). We conclude that chronic ACE inhibition enhances intrarenal renin synthesis and increases renin expression upstream from the glomerulus and in new sites in blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)