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Although dual X-ray absorptiometry (DXA) has been used to determine total body volume, using DXA to determine regional (i.e., arm and leg) volumes needs further assessment. Thus, the aim of the present study is to evaluate the validity of total and regional DXA-derived body volume compared to a traditional method for measuring body volume.A total of 30 males and females (Age: 25.9 ± 4.0 yrs; Height: 1.75 ± 0.10 m; Weight: 70.98 ± 14.02 kg) underwent one whole body DXA scan, underwater weighing, and regional measures of volume via water displacement. Manually created DXA region of interest boxes were used to determine regional DXA body composition. Total body volume was calculated by taking the participant's dry weight and dividing it by the average density from underwater weighing. Linear regression models with body volume from underwater weighing for total body volume and water displacement for regional volume as the dependent variable and DXA lean mass, fat mass, and bone mass as independent variables created total and regional DXA-derived body volume. T-tests assessed DXA-derived body volume to the traditional method of body volume assessment. Regression models were cross-validated using the Repeated k-fold Cross Validation method.DXA-derived total body volume was not significantly (p = 0.999) different from total body volume measured via total body water displacement. In addition, both arm and leg regional DXA-derived volume was not significantly different (p = 0.999) compared to regional volume measured by regional water displacement. Cross-validation of each model produced RThe DXA may be used as valid method for estimating total and regional body volume. Thus, these results expand the DXA's capabilities and potentially allow for a convenient regional four-compartment model with DXA-derived regional volume.

The gold standard cell therapy for repair of articular cartilage defects is autologous chondrocyte implantation, with good outcomes long-term. Mesenchymal stromal/stem cells (MSCs) from bone marrow or connective tissues such as fat are being pursued as alternatives for cartilage repair, and are trialled via intra-articular administration in patients with knee osteoarthritis. Early-phase clinical studies concur on safety and provide some promising insight into efficacy, but the mechanism of action remains unclear. Recent studies implicate extracellular vesicles as important mediators of MSC action, offering exciting therapeutic prospects. Our increasing understanding of the mechanisms underlying intrinsic articular cartilage maintenance and repair fosters hope that novel/repurposed therapeutics could elicit repair through activation of endogenous stem/progenitor cells to maintain healthy joints and prevent osteoarthritis.

The purpose of this study was to evaluate the reliability of ultrasound measures of muscle cross-sectional area (mCSA) and echo intensity (EI) in overweight subjects. A secondary purpose was to evaluate racial differences in EI, mCSA and body composition. In 33 overweight subjects, mCSA and EI were determined from a panoramic ultrasound scan of the vastus lateralis. Body composition was determined using dual-energy X-ray absorptiometry (DXA). Reliability of mCSA and EI was calculated from the intra-class correlation coefficient (ICC), standard error of the measurement (SEM) and minimal difference (MD). The ICC, SEM and MD for mCSA and EI were 0.87, 2.12, 5.89 and 0.74, 4.58, 12.69, respectively. There were no significant racial differences in body composition (p > 0.05); however, EI was significantly lower for black subjects (p = 0.018). These results suggest ultrasound measures of mCSA and EI are reliable in overweight subjects, and EI may provide additional muscle composition evaluations, beyond DXA measures.

Background Estrogen receptor alpha (ERα) has been identified in the vessel wall, offering vasoprotective effects when upregulated. Estrogens are known to mediate the inflammatory milieu, and inflammation has long been associated with abdominal aortic aneurysm (AAA) formation. Therefore, it is theorized that increased estrogen receptor in females contributes to their relative resistance to AAAs. The objective of this study was to determine gender differences in ERα levels during experimental AAA formation. Methods Infrarenal aortas of male and female C57 mice (n = 18 and n = 16, respectively) were infused with 0.4% elastase. Diameters were measured at days 0 and 14. Aortic messenger RNA expression of ERα was determined on day 3 by reverse transcription–polymerase chain reaction, whereas ERα protein levels were measured via Western blot. Immunohistochemistry using rabbit antibody for ERα was performed on day 14 samples and quantified. Zymography was done for matrix metalloproteinases (MMP)2 and 9 activity levels. Samples of human AAAs were collected and Western blot performed. Data were compared for significance using a student t-test. Results Infrarenal aortic diameter increased in elastase-perfused males (ME) by 80% at 14 days after perfusion, whereas females (FE) increased by only 35% (P = 0.0012). FE had ×10 greater ERα messenger RNA expression compared with ME at day 3 (P = 0.003). Similarly, ERα protein levels were 100% higher in FE compared with those in ME on day 14 (P = 0.035). ERα protein levels were 80% higher in female human patients with AAA than those in their male counterparts (P = 0.029). ERα visualized via immunohistochemistry was 1.5 fold higher in FE than ME (P = 0.029). MMP2 and 9 activity levels were decreased in female compared with male aortas. Conclusions This study demonstrates an increase in aortic wall ERα in females compared with males that correlates inversely with MMP activity and AAA formation. These findings, coupled with observations that exogenous estrogen inhibits AAA formation in males, further suggest that estrogen supplementation may be important to prevent AAA formation and growth.

The objective of this study was to test a novel model of inducing abdominal aortic aneurysms (AAAs) in different mouse strains and genders.Male and female C57BL/6 and B6129 mice (n = 5 per group) underwent periaortic dissection and porcine pancreatic elastase (30 μL) or inactivated elastase application (5 min) to the aorta. Aortic measurements were taken on days 0 and 14. Aortic samples were analyzed for histology and zymography for matrix metalloproteinase (MMP) activity. Comparison statistics were performed using unpaired t-test.AAA phenotype (50% aortic increase) occurred in external elastase-treated males (100%) and females (90%). No control animals developed AAAs. The aortic diameter was larger in C57BL/6 and B6129 elastase-treated versus control males (P = 0.0028 and P0.0001, respectively) and females (P0.0001 and P = 0.0458, respectively). Histology verified phenotype via disrupted internal elastic laminae. Macrophage counts in elastase-treated animals were6-fold higher than in controls (all groups significant). MMP9 activity was greater in elastase-treated males and females in C57BL/6 (P = 0.0031, P = 0.0004) and B6129 (P = 0.025, P = 0.2) mice; MMP2 activity was greater in C57BL/6 versus B6129 male elastase-treated mice.This rodent model produced AAAs in both genders and strains of mice. This model is simple, has little variability, and occurs in the infrarenal aorta, substantiating the external elastase model for future studies.

In humans, there is a 4:1 male:female ratio in the incidence of abdominal aortic aneurysms (AAAs). c-Jun-N-terminal kinase (JNK) is an important upstream regulator of several enzymes involved in AAA formation, including the matrix metalloproteinases (MMPs). The purpose of this study was to determine if there is a gender difference between males and females in JNK during AAA formation.Male and female C57/B6 mice underwent aortic perfusion with elastase or heat inactivated elastase with aortas harvested at d 3 and 14 for phenotype determination, RT-PCR, Western blot, and zymography. Additionally, in vitro experiments using siRNA were conducted to define JNK regulation of matrix metalloproteinases (MMPs). A t-test was used to compare between groups.Males formed larger AAAs at d 14 compared with females (P0.001), with significantly higher levels of JNK1 protein, proMMP9, proMMP2, and active MMP2. At d 3, males had more JNK1 mRNA, protein, and MMP activity. Knockdown of JNK 1 or 2 in vitro decreased MMP activity, while knockdown of JNK 1 and 2 together blocked all MMP activity.Alterations in JNK between genders is partially responsible for the differential rates of experimental AAA formation, likely through differential regulation of MMPs.

The number of residue measurements in an individual field trial, carried out to provide data for a pesticide registration for a particular crop, is generally too small to estimate upper tails of the residue distribution for that crop with any certainty. We present a new method, using extreme value theory, which pools information from various field trials, with different crop and pesticide combinations, to provide a common model for the upper tails of residue distributions generally. The method can be used to improve the estimation of high quantiles of a particular residue distribution. It provides a flexible alternative to the direct fitting of a distribution to each individual dataset, and does not require strong distributional assumptions. By using a hierarchical Bayesian model, our method also accounts for parameter uncertainty. The method is applied to a range of supervised trials containing residues on individual items (e.g. on individual apples), and the results illustrate the variation in tail properties amongst all commodities and pesticides. The outputs could be used to select conservative high percentile residue levels as part of a deterministic risk assessment, taking account of the variability between crops and pesticides and also the uncertainty due to relatively small datasets.

Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumor-bearing Tax+ mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src(-/-) mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclast-independent role for N-BP therapy in bone metastasis.

The renin-angiotensin system (RAS) has been implicated in vessel wall remodeling. This investigation tested the hypothesis that the RAS is altered during experimental rodent aneurysm formation.Rat aortas were perfused with saline (controls, N = 45) or elastase (6 U/ml, N = 45). At 4, 7, and 14 days after aortic perfusion, aortic diameters were measured (n = 15/time point/group) and aortic wall mRNA and protein were extracted. Real time polymerase chain reation (PCR) measured RNA levels of angiotensin, angiotensin converting enzyme (ACE), angiotensin II receptor 1 (AT(1)), and angiotensin II receptor 2 (AT(2)). Western blot analysis measured ACE protein levels. Immunohistochemical studies localized ACE within the aortic wall. Statistical analyses were performed with the unpaired t-test and ANOVA.Elastase perfusion significantly increased aortic diameter (P0.01), with no significant changes in saline control aortic diameters. ACE mRNA did not become elevated in elastase-perfused aortas, yet ACE protein levels were elevated on days 4 and 7 of perfusion (P0.01) compared to controls, and ACE staining was noted in these aortas. This difference resolved by 14 days. In neither group were there significant alterations in AT(1), AT(2), or An mRNA levels, although ACE mRNA was elevated in controls after 7 days of perfusion compared to elastase perfused aortas (P0.005).Experimental aortic aneurysm formation may be associated with increased aortic wall ACE protein levels. The mechanisms by which these proteins contribute to, or serve as markers of, aneurysm formation in vivo requires further intervention.

BackgroundSelective estrogen receptor modulators (SERMs), similar to estrogens, possess vasoprotective effects by reducing release of reactive oxygen species. Little is known about the potential effects of SERMs on the pathogenesis of abdominal aortic aneurysms (AAAs). This study's objective was to investigate the growth of experimental AAAs in the setting of the SERM tamoxifen.MethodsIn the first set of experiments, adult male rats underwent subcutaneous tamoxifen pellet (delivering 10 mg/kg/day) implantation (n = 14) or sham operation (n = 16). Seven days later, all animals underwent pancreatic elastase perfusion of the abdominal aorta. Aortic diameters were determined at that time, and aortas were harvested 7 and 14 days after elastase perfusion for immunohistochemistry, real-time polymerase chain reaction, Western blot analysis, and zymography. In the second set of experiments, a direct irreversible catalase inhibitor, 3-amino-1,2,4-triazole (AT), was administered intraperitoneally (1 mg/kg) daily to tamoxifen-treated (n = 6) and control rats (n = 6), starting on day 7 after elastase perfusion. Aortic diameters were measured on day 14. In a third set of experiments, rats were perfused with catalase (150 mg/kg) after the elastase (n = 5), followed by daily intravenous injections of catalase (150 mg/kg/day) administered for 10 days. A control group of rats (n = 7) received 0.9% NaCl instead of catalase.ResultsMean AAA diameters were approximately 50% smaller in tamoxifen-treated rats compared with sham rats 14 days after elastase perfusion (P = .002). The tamoxifen-treated group's aortas had a five-fold increase in catalase mRNA expression (P = .02) on day 7 and an eight-fold increase in catalase protein on day 14 (P = .04). Matrix metalloprotroteinase-9 activity was 2.4-fold higher (P = .01) on day 7 in the aortas of the controls compared to the tamoxifen-treated group's aortas. Tamoxifen-treated rats had approximately 40% fewer aortic polymorphonuclear neutrophils compared to controls on day 7 (P = .05). Administration of the direct catalase inhibitor AT to tamoxifen-treated rats partially reversed the aneurysm inhibitory effect of tamoxifen by nearly 30% (P = .02). In contrast, catalase administration inhibited AAA formation by 44% (P = .002).ConclusionsThe selective estrogen receptor modulator tamoxifen inhibits the development of AAAs in male rats in association with an up-regulation of catalase and inhibition of aortic wall neutrophil infiltration.Clinical relevanceA group of medications with estrogen-like properties, the SERMs, act as either an estrogen receptor agonist or antagonist in a tissue-selective manner. SERM use has increased rapidly because they have been found to be beneficial in treatment of breast cancer, prevention of osteoporosis, and treatment of desmoid tumors and retroperitoneal fibrosis. The effects of SERMs on the formation of experimental AAAs have not been studied to date. This study was designed to determine the effect of tamoxifen on the experimental development of AAAs in a rodent model.

Objective. The objective of this study was to determine the significance of membrane type 1 matrix metalloproteinase (MT1-MMP) activation of MMP-2 in experimental abdominal aortic aneurysms. Methods. Rat aortas were perfused with either saline as a control or elastase, and harvested on 2, 4, or 7 days after perfusion (n = 5 per treatment group/day). Aortic MT1-MMP and MMP-2 expression and protein were determined by real time polymerase chain reaction and Western blotting, respectively. Aortic explants were used to measure MMP-2 activity by zymography. Rat aortic smooth muscle cells in vitro were exposed to increasing doses of elastase and analyzed for MT-1 MMP expression. Results. Aneurysms formed in 80% of the elastase-perfused aortas at 7 days, whereas none formed in the saline-perfused aortas. Significantly increased MT1-MMP expression was observed only on day 4, when levels were 6.5-fold higher in elastase-perfused aortas compared with saline-perfused aortas (P

Objective. Nitric oxide (NO) may mediate vessel wall remodeling by regulating expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). This study tested the hypothesis that nitric oxide synthase (NOS) inhibition in whole aortic wall causes increases in cytokine-stimulated MMP and TIMP expression. Methods. Cultured infrarenal aortic segments from Sprague-Dawley rats were exposed to increasing concentrations (0, 0.1, 0.5, 1, and 5 mM; n = 6 per concentration) of NG-monomethyl-l-arginine (L-NMMA), a known inhibitor of NOS. This was in the presence of 2 ng/ml of interleukin-1β, a known inducer of NOS, MMP, and TIMP expression. Media nitrate and nitrite (NOx) were measured at 72 h using the Saville method. Media MMP activity was measured using gelatin zymography. MMP-2 and -9 protein and mRNA levels were determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). TIMP activity and mRNA levels were evaluated by reverse zymography and RT-PCR. Data were analyzed using ANOVA. Results. Increasing concentrations of L-NMMA produced a dose-dependent decrease in NOx (2214 ± 405 to 347 ± 37 ng/mg, P < 0.001). Zymography demonstrated a dose-dependent increase in 92-kDa MMP (pro-MMP-9) activity (P < 0.001) with corresponding increases in pro-MMP-9 protein (P = 0.03) and mRNA levels (P = 0.004). While there was a dose-dependent increase in 72-kDa MMP (pro-MMP-2) activity (P = 0.001), pro-MMP-2 protein and mRNA levels were unchanged. Reverse zymography demonstrated a dose-dependent increase in 29-kDa TIMP-1 activity (P = 0.01), but there was no change in TIMP-1 mRNA levels. Conclusions. NOS inhibition in ex vivo aortic tissue causes a dose-dependent increase in MMP-9 expression and activity. It is speculated that deficiencies of NO in vivo alter MMP and TIMP homeostasis, favoring matrix degradation.

The importance of pancreatic versus intestinal-derived GLP-1 for glucose homeostasis is controversial. We detected active GLP-1 in the mouse and human pancreas, albeit at extremely low levels relative to glucagon. Accordingly, to elucidate the metabolic importance of intestinal proglucagon-derived peptides (PGDPs), we generated mice with reduction of Gcg expression within the distal (Gcg(DistalGut−/−)) or entire (Gcg(Gut−/−)) gut. Substantial reduction of gut Gcg expression markedly reduced circulating levels of GLP-1, and impaired glucose homeostasis, associated with increased levels of GIP, and accelerated gastric emptying. Gcg(DistalGut−/−) mice similarly exhibited lower circulating GLP-1 and impaired oral glucose tolerance. Nevertheless, plasma levels of insulin remained normal following glucose administration in the absence of gut-derived GLP-1. Collectively, our findings identify the essential importance of gut-derived PGDPs for maintaining levels of circulating GLP-1, control of gastric emptying, and glucose homeostasis.

Background Treatment with low-molecular-weight heparin (LMWH) favorably alters the vein wall response to deep venous thrombosis (DVT), although the mechanisms remain unclear. Previous studies have suggested that LMWH alters the levels of circulating plasminogen activator inhibitor 1 (PAI-1), a known mediator of fibrosis, and may improve endogenous fibrinolysis. We hypothesized that LMWH favorably alters the vein wall response by binding of PAI-1 and acceleration of fibrinolysis. Methods Wild-type and PAI-1 −/− mice underwent treatment with LMWH after induction of occlusive DVT. Vein wall and plasma were harvested and analyzed by enzyme-linked immunosorbent assay, zymography, real-time polymerase chain reaction, and immunohistochemistry. Results Wild-type mice treated with LMWH exhibited diminished vein wall fibrosis (0.6 ± 0.6 vs 1.4 ± 0.2; P P −/− mice treated with LMWH were not similarly protected from fibrosis, despite improved thrombus resolution. Treatment with LMWH was associated with decreased intrathrombus interleukin-1β (68.6 ± 31.0 vs 223.4 ± 28.9 ρg/mg total protein; P −/− mice exhibited significantly elevated intrathrombus (257.2 ± 51.5 vs 14.3 ± 3.8 ρg/mg total protein; n = 5) and vein wall interleukin-13 (187.2 ± 57.6 vs 9.9 ± 1.1 ρg/mg total protein; P P Conclusions LMWH did not accelerate venous thrombosis resolution but did protect against vein wall fibrosis in a PAI-1-dependent manner in an occlusive DVT model. Lack of PAI-1 correlated with accelerated venous thrombosis resolution but no protection from fibrosis. PAI-1 inhibition as a treatment strategy for DVT is likely to accelerate clearance of the thrombus but may come at the expense of increased vein wall fibrosis.

Objective. Oxidative stress has been implicated in abdominal aortic aneurysm pathogenesis. This study sought to characterize the relevance of specific superoxide dismutases (SOD) within a family of reactive oxygen catalyzing metalloenzymes, in the rodent aneurysm model and rat aortic smooth muscle cells (RA-SMCs), including Manganese SOD (Mn-SOD), Copper-zinc SOD (Cu, Zn-SOD), and Extracellular SOD (ecSOD). Methods. Male rat infrarenal abdominal aortas were measured and perfused with either porcine pancreatic elastase (6 U/mL) or saline (control). Subsequently, aortic diameters were measured and harvested on postperfusion days 1, 2, 4,∗∗ and 7 ( N = 5–6 per treatment group per day). Male RA-SMCs were incubated for 24 and 72 h in the presence of interleukin-1β (IL-1β, 2 ng/mL). MnSOD, Cu,ZnSOD, and ecSOD, and β-actin expression in aortic tissue and in RA-SMCs was determined by quantitative real-time PCR. Data were analyzed by t -test. Results. Elastase perfused aortic diameters were significantly increased compared to control aortas at postperfusion days 2 (76% versus 39%, P = 0.004), 4 (86% versus 50%, P = 0.04), and 7 (263% versus 46%, P = 0.016). MnSOD mRNA levels in elastase perfused aortas were 6.0 ( P = 0.005), 7.4 ( P P = 0.005) greater than control aortas at postperfusion days 1, 2, and 4, respectively. In RA-SMCs, MnSOD mRNA expression in IL-1β stimulated male were 152.2 ( P = 0.029) and 15.3 times ( P = 0.037) greater than control cells at 24 and 72 h, respectively. No significant differences were observed in Cu, Zn-SOD, and ecSOD expression at any time point studied in elastase perfused aortas; however, there was a slight increase in ecSOD and Cu, Zn-SOD expression in RA-SMCs. Conclusion. Elastase perfused aortic aneurysm formation is associated with early increases in MnSOD expression, which primarily occurs in the mitochondria. Strategies aimed at inhibiting oxidative stress during aneurysm formation should focus on this specific SOD isoform.

Objective: The goal of this project is to examine the role of an exogenous NO donor, DETA-NONOate (DETA), on MMP-9, MMP-2, and TIMP-1 expression and activity in interleukin-1β (IL-1β) induced rat aortic explants (RAE). Methods: RAEs were incubated with IL-1β (2 ng/ml) and increasing concentrations of DETA (0, 5.0, 50, 100, and 500 μM) (n = 3 per group). Messenger RNA (mRNA) was extracted from cells after 24 hours and analyzed for MMP-9, MMP-2, and TIMP-1 expression levels by real time RT-PCR. Media at 48 hours was collected and assayed for NO2 and NO3 (NOx) by the Saville Assay, MMP-9 and MMP-2 activity by gelatin zymography, and TIMP-1 activity by reverse zymography. All statistical analyses were performed by ANOVA and Pearson correlation. Results: DETA administration resulted in a dose-dependent increase in media NOx concentration (0.001 +/- 0.0003 ng NOx / mg protein to 0.062 +/- 0.004 ng NOx / mg protein, p < 0.01). In RAE, MMP-9 expression and activity decreased significantly in a dose dependent fashion with increasing DETA concentrations (p < 0.01). At the maximal dose of 500 μM DETA, a 78% decrease in MMP-9 expression (p < 0.05) and a 72% decrease in pro-MMP-9 activity (p < 0.05) was demonstrated compared to RAE treated with IL-1β alone (0 μM DETA). There were no significant differences seen in MMP-2 and TIMP-1 expression or activity in response to DETA exposure. Conclusion: The NO donor DETA-NONOate decreased IL-1β induced MMP-9 expression and activity in RAE in a dose dependent fashion. These data suggest that NO donors may be beneficial in decreasing MMP-9 levels, an enzyme believed to be critical in vessel wall remodeling, and therefore may serve to inhibit MMP-9 dependent vessel wall degradation seen during AAA formation.