Genes that regulate metabolism and energy partitioning have the potential to influence economically important traits in farm animals, as do polymorphisms within these genes. In the current study, SNP in the bovine neuropeptide Y (NPY), growth hormone receptor (GHR), ghrelin (GHRL), uncoupling proteins 2 and 3 (UCP2 and UCP3), IGF2, corticotrophin-releasing hormone (CRH), cocaine and amphetamine regulated transcript (CART), melanocortin-4 receptor (MC4R), proopiomelanocortin (POMC), and GH genes were evaluated for associations with growth, feed efficiency, and carcass merit in beef steers. In total, 24 SNP were evaluated for associations with these traits and haplotypes were constructed within each gene when 2 or more SNP showed significant associations. An A/G SNP located in intron 4 of the GHR gene had the largest effects on BW of the animals (dominance effect P < 0.01) and feed efficiency (allele substitution effect P < 0.05). Another A/G SNP located in the promoter region of GHR had similar effects but the haplotypes of these 2 SNP reduced the effects of the SNP located in intron 4. Three SNP in the NPY gene showed associations to marbling (P < 0.001) as well as with ADG, BW, and feed conversion ratio (FCR; P < 0.05). The combination of these 3 SNP into haplotypes generally improved the association or had a similar scale of association as each single SNP. Only 1 SNP in UCP3, an A/G SNP in intron 3, was associated with ADG (P = 0.025), partial efficiency of growth, and FCR (P < 0.01). Three SNP in UCP2 gene were in almost complete linkage disequilibrium and showed associations with lean meat yield, yield grade, DMI, and BW (P < 0.05). Haplo-types between the SNP in UCP3 and UCP2 generally reduced the associations seen individually in each SNP. An A/G SNP in the GHRL gene tended to show effects on residual feed intake, FCR, and partial efficiency of growth (P < 0.10). The IGF2 SNP most strongly affected LM area (P < 0.01), back fat, ADG, and FCR (P < 0.05). The SNP in the CART, MC4R, POMC, GH, and CRH genes did not show associations at P < 0.05 with any of the traits. Although most of the SNP that showed associations do not cause amino acid changes, these SNP could be linked to other yet to be detected causative mutations or nearby QTL. It will be very important to verify these results in other cattle populations.

These results confirm our hypothesis that the source of cells resulting in venous stenosis formation is derived from the adventitia and media, with cell migration being greatest within the first two weeks after graft placement with translocation of these cells into the intima at four weeks. MMP-2 activity peaks at day seven in the adventitia and again at days 19 to 26 in the intima. A key to limiting venous stenosis formation may lie in inhibiting MMP-2 by adventitial and medial targeting.

Venous injury and subsequent venous stenosis formation are responsible for hemodialysis graft failure. Our hypothesis is that these pathological changes are in part related to changes in wall shear stress (WSS) that results in the activation of matrix regulatory proteins causing subsequent venous stenosis formation. In the present study, we examined the serial changes in WSS, blood flow, and luminal vessel area that occur subsequent to the placement of a hemodialysis graft in a porcine model of chronic renal insufficiency. We then determined the corresponding histological, morphometric, and kinetic changes of several matrix regulatory proteins including VEGF-A, its receptors, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and TIMP-2. WSS was estimated by obtaining blood flow and luminal vessel area by performing phase-contrast MRI with magnetic resonance angiography in 21 animals at 1 day after graft placement and prior to death on day 3 ( n = 7), day 7 ( n = 7), and day 14 ( n = 7). At all time points, the mean WSS at the vein-to-graft anastomosis was significantly higher than that at the control vein ( P < 0.05). WSS had a bimodal distribution with peaks on days 1 and 7 followed by a significant reduction in WSS by day 14 ( P < 0.05 compared with day 7) and a decrease in luminal vessel area compared with control vessels. By day 3, there was a significant increase in VEGF-A and pro-MMP-9 followed by, on day 7, increased pro-MMP-2, active MMP-2, and VEGF receptor (VEGFR)-2 ( P < 0.05) and, by day 14, increased VEGFR-1 and TIMP-1 ( P < 0.05) at the vein-to-graft anastomosis compared with control vessels. Over time, the neointima thickened and was composed primarily of α-smooth muscle actin-positive cells with increased cellular proliferation. Our data suggest that hemodialysis graft placement leads to early increases in WSS, VEGF-A, and pro-MMP-9 followed by subsequent increases in pro-MMP-2, active MMP-2, VEGFR-1, VEGFR-2, and TIMP-1, which may contribute to the development of venous stenosis.

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Purpose Hemodialysis grafts fail because of venous neointimal hyperplasia formation caused by adventitial fibroblasts which have become myofibroblasts (α-smooth muscle actin positive cells) and migrate to the neointima. There is increased expression of hypoxia inducible factor-1 alpha (HIF-1α in venous neointimal hyperplasia formation in experimental animal model and clinical samples. We hypothesized that under hypoxic stimulus (HIF-1α fibroblasts will convert to myofibroblasts through a matrix metalloproteinase-2 (MMP-2) mediated pathway. Materials and methods Murine AKR-2B fibroblasts were made hypoxic or normoxic for 24, 48, and 72 hours. Protein expression for HIF-1α, α-smooth muscle actin, MMP-2, MMP-9, TIMP-1, and TIMP-2 was performed to determine the kinetic changes of these proteins. Immunostaining for α-smooth muscle actin, collagen, and fibronectin was performed. Results At all time points, there was significantly increased expression of HIF-1α in the hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was significantly increased expression α-smooth muscle actin at all time points which peaked by 48 hours in hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was a significant increase in the expression of active MMP-2 by 48-72 hours and a significant increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) by 48-72 hours by hypoxic fibroblasts (P<0.05). By 72 hours, there was significant increase in TIMP-2 expression (P<0.05). Immunohistochemical analysis demonstrated increased expression for α-smooth muscle actin, collagen, and fibronectin as the length of hypoxia increased. Conclusions Under hypoxia, fibroblasts will convert to myofibroblasts through a MMP-2 mediated pathway which may provide insight into the mechanism of venous neointimal hyperplasia.

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Isotope-coded affinity tags (cICAT) coupled with mass spectrometric analysis is one of the leading technologies for quantitative proteomic profiling and protein quantification. We performed proteomic analysis of bovine aortic endothelial cells (BAEC) in response to laminar shear stress using cICAT labeling coupled with LC-MS/MS. Protein expressions in BAEC under 15 dynes/cm2 of shear stress for 10 min, 3 h, and 6 h were compared with matched stationary controls. Analysis of each sample produced 1800-2400 proteins at >or=75% confidence level. We found 142, 213, and 186 candidate proteins that were up- or down-regulated by at least two-fold after 10 min, 3 h, and 6 h of shear stress, respectively. Some of these proteins have known cellular functions and they encompass many signaling pathways. The signaling pathways that respond to shear stress include those of integrins, G-protein-coupled receptors, glutamate receptors, PI3K/AKT, apoptosis, Notch and cAMP-mediated signaling pathways. The validity of the mass spectrometric analysis was also confirmed by Western blot and confocal immunofluorescence microscopy. The present quantitative proteomic analysis suggests novel potential regulatory mechanisms in vascular endothelial cells in response to shear stress. These results provide preliminary footprints for further studies on the signaling mechanisms induced by shear stress.