Molecular mechanisms leading to myocardial injury during warm or cold ischemia are insufficiently understood. Although proteasomes are thought to contribute to myocardial ischemia-reperfusion injury, their roles during the ischemic period remain elusive. Because donor hearts are commonly exposed to prolonged global cold ischemia prior to cardiac transplantation, we evaluated the role and regulation of the proteasome during cold ischemic storage of rat hearts in context of the myocardial ATP content. When measured at the actual tissue ATP concentration, cardiac proteasome peptidase activity increased by 225% as ATP declined during cold ischemic storage of hearts in University of Wisconsin (UW) solution for up to 48h. Addition of the specific proteasome inhibitor epoxomicin to the UW solution inhibited proteasome activity in the cardiac extracts, significantly reduced edema formation and preserved the ultrastructural integrity of the cardiomyocyte. Utilizing purified 20S/26S proteasome enzyme preparations, we demonstrate that this activation can be attributed to a subset of 26S proteasomes which are stable at ATP concentrations far below physiological levels, that ATP negatively regulates its activity and that maximal activation occurs at ATP concentrations in the low mol/L range. These data suggest that proteasome activation is a pathophysiologically relevant mechanism of cold ischemic myocardial injury. A subset of 26S proteasomes appears to be a cell destructive protease that is activated as ATP levels decline. Proteasome inhibition during cold ischemia preserves the ultrastructural integrity of the cardiomyocyte.

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Objective To determine whether treatment with the CXC chemokine receptor (CXCR) 4 agonist ubiquitin results in beneficial effects in a polytrauma model consisting of bilateral femur fractures plus blunt chest trauma (Injury Severity Score 18-25). Design Treatment study. Setting Research Laboratory. Subjects Seventeen Yorkshire pigs. Interventions Intravenous (i.v.) injection of 1.5 mg/kg ubiquitin or albumin (=control) at 60 min after polytrauma. Measurements and Main Results Anesthetized, mechanically ventilated pigs underwent polytrauma, followed by a simulated 60 min shock phase. At the end of the shock phase ubiquitin or albumin were administered and animals were resuscitated to a mean arterial blood pressure of 70 mmHg until t = 420 min. After i.v. ubiquitin, ubiquitin plasma concentrations increased sixteen-fold to 2870 ± 1015 ng/mL at t = 90 min and decreased with t1/2 = 60 min. Endogenous plasma ubiquitin increased two-fold in the albumin group with peak levels of 359 ± 210 ng/mL. Plasma levels of the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α were unchanged in both groups. Ubiquitin treatment reduced arterial lactate levels and prevented a continuous decrease in arterial oxygenation, which occurred in the albumin group during resuscitation. Wet weight to dry weight ratios of the lung contralateral from the injury, heart, spleen and jejunum were lower with ubiquitin. With ubiquitin treatment, tissue levels of IL-8, IL-10, TNFα and SDF-1α were reduced in the injured lung and of IL-8 in the contralateral lung, respectively. Conclusions Administration of exogenous ubiquitin modulates the local inflammatory response, improves resuscitation, reduces fluid shifts into tissues and preserves arterial oxygenation after blunt polytrauma with lung injury. This study further supports the notion that ubiquitin is a promising protein therapeutic and implies CXCR4 as a drug target after polytrauma.

Objective To define systemic release kinetics of a panel of cytokines and heat shock proteins (HSP) in porcine polytrauma/hemorrhage models and to evaluate whether they could be useful as early trauma biomarkers. Design and Setting Prospective study in a research laboratory. Subjects Twenty-one Yorkshire pigs. Measurements and Main Results Pigs underwent polytrauma (femur fractures/lung contusion, P), hemorrhage (mean arterial pressure 25-30mmHg, H), polytrauma plus hemorrhage (P/H) or sham procedure (S). Plasma was obtained at baseline, in 5-15min intervals during a 60min shock period without intervention and in 60-120min intervals during fluid resuscitation for up to 300min. Plasma was assayed for IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/IL-23p40, IL-13, IL-17, IL-18, IFNγ, TGFβ, TNFα, HSP40, HSP70 and HSP90 by ELISA. All animals after S, P and H survived (n=5/group). Three of six animals after P/H died. IL-10 increased during shock after P and this increase was attenuated after H. TNFα increased during the shock period after P, H and also after S. P/H abolished the systemic IL-10 and TNFα release and resulted in 20-30% increased levels of IL-6 during shock. As fluid resuscitation was initiated TNFα and IL-10 levels decreased after P, H and P/H, HSP 70 increased after P, IL-6 levels remained elevated after P/H and also increased after P and S. Conclusions Differential regulation of the systemic cytokine release after polytrauma and/or hemorrhage, in combination with the effects of resuscitation, can explain the variability and inconsistent association of systemic cytokine/HSP levels with clinical variables in trauma patients. Insults of major severity (P/H) partially suppress the systemic inflammatory response. The plasma concentrations of the measured cytokines/HSPs do not reflect injury severity or physiological changes in porcine trauma models and are unlikely to be able to serve as useful trauma biomarkers in patients.

Background The most important purpose of reconstruction is to increase or restore the patient's quality of life (QOL). The purpose of our study was to evaluate the QOL and aesthetic outcomes of patients after autologous versus implant-based breast reconstruction. Methods Patients who underwent breast reconstruction between 2009 and 2011 were included. The Breast-Q, a validated breast reconstruction QOL questionnaire, was used along with postoperative photographs panel analyses using a multiparameter breast-specific aesthetic outcome scale and retrospective evaluation of demographic and treatment data. Results Of 820 patients, 261 complete questionnaires were evaluated. On the multivariable linear regression, the “satisfaction with breasts” was positively influenced by autologous and bilateral reconstructions, whereas radiation therapy (RTx), the time between the reconstruction and the questionnaire, and the number of surgeries due to complications were negative factors (adjusted R 2 = 0.183; P < 0.001). The same factors influenced the “satisfaction with the outcomes.” The mean “overall breast appearance” was also positively influenced by autologous and bilateral reconstructions, and RTx and the total number of surgeries were negative predictive factors (adjusted R 2 = 0.311, P < 0.001). Conclusions The aesthetic result and QOL after breast reconstruction for breast cancer treatment are positively influenced by the use of autologous tissue and bilaterality. Factors that negatively influenced the aesthetic result and the QOL include use of RTx, a higher number of surgeries needed for the reconstruction, reoperations due to complications, higher body mass index, and a longer time elapsed between reconstruction and the questionnaire.

CXC chemokine receptor (CXCR)-4 agonists have been shown to attenuate inflammation and organ injury in various disease models, including trauma/hemorrhage. The pathophysiological role of CXCR4 during the early response to tissue injury, however, remains unknown. Therefore, we investigated the effects of AMD3100, a drug that antagonizes binding of stromal cell-derived factor (SDF)-1α and ubiquitin to CXCR4 during the initial response to polytrauma in pigs. Fifteen minutes before polytrauma (femur fractures/lung contusion; control: sham), 350 nmol/kg AMD3100, equimolar AMD3100 and ubiquitin (350 nmol/kg each) or vehicle were administered intravenously. After a 60-min shock period, fluid resuscitation was performed for 360 min. Ubiquitin binding to peripheral blood mononuclear cells was significantly reduced after intravenous AMD3100. SDF-1α plasma levels increased transiently >10-fold with AMD3100 in all animals. In injured animals, AMD3100 increased fluid requirements to maintain hemodynamics and enhanced increases in peripheral blood granulocytes, lymphocytes and monocytes, compared with its effects in uninjured animals. Cytokine release from leukocytes in response to Toll-like receptor (TLR)-2 and TLR-4 activation was increased after in vitro AMD3100 treatment of normal whole blood and after in vivo AMD3100 administration in animals subjected to polytrauma. Coadministration of AMD3100/ubiquitin reduced lactate levels, prevented AMD3100-induced increases in fluid requirements and sensitization of the tumor necrosis factor (TNF)-α and interleukin (IL)-6 release upon TLR-2/4 activation, but did not attenuate increases in leukocyte counts and SDF-1α plasma levels. Our findings suggest that CXCR4 controls leukocyte mobilization after trauma, regulates leukocyte reactivity toward inflammatory stimuli and mediates protective effects during the early phase of trauma-induced inflammation.

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Recently, we provided evidence for a possible role of the cardiac proteasome during ischemia, suggesting that a subset of 26S proteasomes is a cell-destructive protease, which is activated as the cellular energy supply declines. Although proteasome inhibition during cold ischemia (CI) reduced injury of ischemic hearts, it remains unknown whether these beneficial effects are maintained throughout reperfusion, and thus, may have pathophysiological relevance. Therefore, we evaluated the effects of epoxomicin (specific proteasome inhibitor) in a rat heterotopic heart transplantation model. Donor hearts were arrested with University of Wisconsin solution (UW) and stored for 12h/24h in 4°C UW ± epoxomicin, followed by transplantation. Efficacy of epoxomicin was confirmed by proteasome peptidase activity measurements and analyses of myocardial ubiquitin pools. After 12hCI, troponin I content of UW was lower with epoxomicin. Although all hearts after 12hCI started beating spontaneously, addition of epoxomicin to UW during CI reduced cardiac edema and preserved the ultrastructural integrity of the post-ischemic cardiomyocyte. After 24hCI in UW ± epoxomicin, hearts did not regain contractility. When hearts were perfused with epoxomicin during cardioplegia, the cardiac proteasome was inhibited immediately, all of these hearts started beating after 24hCI in UW plus epoxomicin and cardiac edema and myocardial ultrastructure were comparable to hearts after 12hCI. Epoxomicin did not affect markers of lipidperoxidation or neutrophil infiltration in post-ischemic hearts. These data further support the concept that proteasome activation during ischemia is of pathophysiological relevance and suggest proteasome inhibition as a promising approach to improve organ preservation strategies.