The influence of physical and mental health symptoms on Veterans' functional health status.

Veterans who have been deployed to combat often have complex medical histories including some combination of traumatic brain injury (TBI); mental health problems; and other chronic, medically unexplained symptoms (i.e., chronic multisymptom illness [CMI] clusters). How these multiple pathologies relate to functional health is unclear. In the current study, 120 Veterans (across multiple combat cohorts) underwent comprehensive clinical evaluations and completed self-report assessments of mental health symptoms (Patient Health Questionnaire-2 [PHQ-2], PTSD Checklist-Civilian Version [PCL-C]) and functional health (Veterans Rand 36-Item Health Survey). Canonical correlation and regression modeling using split-sample permutation tests revealed that the PHQ-2/PCL-C composite variable (among TBI severity and number of problematic CMI clusters) was the primary predictor of multiple functional health domains. Two subscales, Bodily Pain and General Health, were associated with multiple predictors (TBI, PHQ-2/PCL-C, and CMI; and PHQ-2/PCL-C and CMI, respectively), demonstrating the multifaceted nature of how distinct medical problems might uniquely and collectively impair aspects of functional health. Apart from these findings, however, TBI and CMI were not predictors of any other aspects of functional health. Taken together, our findings suggest that mental health problems might exert ubiquitous influence over multiple domains of functional health. Thus, screening of mental health problems and education and promotion of mental health resources can be important to the treatment and care of Veterans.

Findings from the National Memory Screening Day program.

OBJECTIVES: To report experience with a large, nation-wide public memory screening program. DESIGN: Descriptive study of community-dwelling elderly adults. SETTING: Local community sites (48 sites agreed to provide data) throughout the United States participating in National Memory Screening Day in November 2010. PARTICIPANTS: Of 4,369 reported participants, 3,064 had complete data records and are included in this report. MEASUREMENTS: Participants completed a questionnaire that included basic demographic information and a question about subjective memory concerns. Each site selected one of seven validated cognitive screening tests: Mini-Cog, General Practitioner assessment of Cognition, Memory Impairment Screen, Kokmen Short Test of Mental Status, Mini-Mental State Examination, Montreal Cognitive Assessment, Saint Louis University Mental Status Examination. RESULTS: Overall, 11.7% failed one of the seven screening tests. As expected, failure rates were higher in older and less-educated participants (P's < .05). Subjective memory concerns were associated with a 40% greater failure rate for persons of similar age and education but no memory concerns (odds ratio = 1.4, 95% confidence interval = 1.07-1.78), although only 11.9% of those who reported memory concerns (75% of all participants) had detectible memory problems. CONCLUSION: Screening for cognitive impairment in community settings yielded results consistent with expected effects of age and education. The event attracted a large proportion of individuals with memory concerns; 88.1% were told that they did not have memory problems detectible with the tests used. Further studies are needed to assess how participants respond to and use screening information, whether this information ultimately influences decision-making or outcomes, and whether memory screening programs outside healthcare settings have public health value.

Challenges to be overcome using population-based sampling methods to recruit veterans for a study of post-traumatic stress disorder and traumatic brain injury.

BACKGROUND: Many investigators are interested in recruiting veterans from recent conflicts in Afghanistan and Iraq with Traumatic Brain Injury (TBI) and/or Post Traumatic Stress Disorder (PTSD). Researchers pursuing such studies may experience problems in recruiting sufficient numbers unless effective strategies are used. Currently, there is very little information on recruitment strategies for individuals with TBI and/or PTSD. It is known that groups of patients with medical conditions may be less likely to volunteer for clinical research. This study investigated the feasibility of recruiting veterans returning from recent military conflicts--Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF)--using a population-based sampling method. METHODS: Individuals were sampled from a previous epidemiological study. Three study sites focused on recruiting survey respondents (n = 445) who lived within a 60 mile radius of one of the sites. RESULTS: Overall, the successful recruitment of veterans using a population-based sampling method was dependent on the ability to contact potential participants following mass mailing. Study enrollment of participants with probable TBI and/or PTSD had a recruitment yield (enrolled/total identified) of 5.4%. We were able to contact 146 individuals, representing a contact rate of 33%. Sixty-six of the individuals contacted were screened. The major reasons for not screening included a stated lack of interest in the study (n = 37), a failure to answer screening calls after initial contact (n = 30), and an unwillingness or inability to travel to a study site (n = 10). Based on the phone screening, 36 veterans were eligible for the study. Twenty-four veterans were enrolled, (recruitment yield = 5.4%) and twelve were not enrolled for a variety of reasons. CONCLUSIONS: Our experience with a population-based sampling method for recruitment of recent combat veterans illustrates the challenges encountered, particularly contacting and screening potential participants. The screening and enrollment data will help guide recruitment for future studies using population-based methods.

Integration of Clinical and Research Neuroimaging to Understand Traumatic Brain Injury in the Veteran Population.

There is a complex relationship between posttraumatic stress disorder and traumatic brain injury. To understand and treat these conditions, it is necessary to apply an integrated physical and mental health care approach to postdeployment care.

Ceramide 1-phosphate stimulates macrophage proliferation through activation of the PI3-kinase/PKB, JNK and ERK1/2 pathways.

Ceramide 1-phosphate (C1P) was first shown to be mitogenic for fibroblasts, but the mechanisms whereby it stimulated cell proliferation have remained largely unknown. Here we demonstrate that C1P stimulates DNA synthesis and cell division in murine bone marrow-derived macrophages. C1P caused rapid phosphorylation of protein kinase B (PKB, also known as Akt), a downstream target of phosphatidylinositol 3-kinase (PI3-K). Selective inhibition of PI3-K blocked both DNA synthesis and cell growth. C1P induced phosphorylation of GSK-3beta, which is a major target of PKB, and this effect was also abolished by inhibition of PI3-K. In addition, C1P upregulated the expression of cyclin D1 and c-Myc, two major targets of GSK-3beta, which are important regulators of cell proliferation. C1P stimulated the activity of NF-kappaB, and inhibitors of this transcription factor completely blocked macrophage proliferation. Lastly, C1P induced phosphorylation of the mitogen activated protein kinases (MAPK) extracellularly regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). Inhibition of ERK1/2 and JNK also blocked C1P-induced macrophage proliferation. It can be concluded that C1P stimulates macrophage proliferation through activation of the PI3-K/PKB, ERK and JNK pathways, and that GSK-3beta, c-Myc, cyclin D1, and NF-kappaB are important downstream effectors in this action.

Reduction of protein synthesis and statin-induced cardiomyocyte cell death.

The objective of this study was to determine whether an HMG Co A reductase inhibitor (statin) reduces protein synthesis in cardiomyocytes and whether this action maybe an underlying mechanism for statin-induced cell death. Cardiomyocytes from embryonic chick heart were maintained in culture. Cells exposed to lovastatin for 4 h showed a concentration dependent reduction in protein synthesis as assessed by [3H] leucine incorporation and [35S] methionine incorporation. Compared to control, lovastatin 100 microM, which produced a 25% increase in cell death, induced a three-fold reduction in methionine incorporation. [35S] methionine autoradiography showed little (new) protein synthesis at concentrations of lovastatin of 70 microM or higher; an effect that was not limited to specific proteins. Cardiomyocytes treated with lovastatin showed morphologic changes in the nucleoli consistent with insufficient protein synthesis. These cardiomyocytes manifested cell death under conditions of reduced protein synthesis. Interruption of protein synthesis with cycloheximide, a ribosomal RNA transcription inhibitor or reduction in protein substrate availability by lowering the media concentration of fetal calf serum was associated with a concentration-dependent reductions in cell viability. Importantly, stimulation of protein synthesis by higher concentrations of fetal calf serum limited lovastatin-induced cell death. These data suggest that statin-induced inhibition of protein synthesis is an underlying mechanism for statin-induced cell death.

The association between RhoB and caspase-2: changes with lovastatin-induced apoptosis.

Because cytoskeletal actin is regulated, in part, by Rho, and because Rho and caspases are involved in apoptosis, we sought to determine whether there was an association between RhoB and caspase-2. A RhoB-caspase-2 association was consistently demonstrated in neonatal mouse cardiomyocytes with Western Blotting, either after immunoprecipitation with RhoB followed by immunoblotting with caspase-2, or in reciprocal experiments after immuno precipitation with caspase-2 and immunoblotting with RhoB (n = 14). Although the RhoB-caspase-2 complex was constitutively present, the link between RhoB and caspase-2 may be operative in apoptosis because the HMG-CoA reductase inhibitor lovastatin increased the RhoB-caspase complex, especially in the nuclear fraction of the cell, with a peak occurrence 2 h after treatment. This association was unaffected by the caspase-2 inhibitor zVDVAD. Lovastatin produced apoptosis that was accompanied by an activation of caspase-2, as demonstrated by its immunohistochemistry and by the fact that the caspase-2 inhibitor zVDVAD reduced lovastatin-induced apoptosis. Lovastatin induced dramatic changes in cell morphology and a reduction in F-actin. Immunoblotting for actin suggests that lovastatin does not induce a degradation of the actin molecule, but rather affects filamentous F-actin. Caspase-2 inhibition with zVDVAD reduced lovastatin-induced alteration in cytoskeletal F-actin. The Rho inhibitor, Clostridium difficile toxin B, blunted the ability of lovastatin to induce apoptosis. In summary, these data show a previously unrecognized association between RhoB and caspase-2 in the cytosolic and nuclear fractions, which has ramifications for processes regulated by RhoB and caspase-2, including apoptosis.

Ceramide activates a mitochondrial p38 mitogen-activated protein kinase: a potential mechanism for loss of mitochondrial transmembrane potential and apoptosis.

This study examined the impact of ceramide, an intracellular mediator of apoptosis, on the mitochondria to test the hypothesis that ceramide utilized p38 MAPK in the mitochondria to alter mitochondrial potential and induce apoptosis. The capacity of ceramide to adversely affect mitochondria was demonstrated by the significant loss of mitochondrial potential (DeltaPsim), indicated by a J-aggregate fluorescent probe, after embryonic chick cardiomyocytes were treated with the cell permeable ceramide analogue C2-ceramide. p38 MAPK was identified in the mitochondrial fraction of the cell and p38 MAPK phosphorylation in this mitochondrial fraction of the cell occurred with ceramide treatment. In addition, SAPK phosphorylation and a decrease in ERK phosphorylation occurred in whole cell lysates after ceramide treatment. The p38 MAPK inhibitor SB 202190 but not the MEK inhibitor PD 98059 significantly inhibited ceramide-induced apoptosis and loss of DeltaPsim. These data suggest that p38 MAPK is present in the mitochondria and its activation by ceramide indicates local signaling more directly coupled to the mitochondrial pathway in apoptosis.

Ceramide-1-phosphate promotes cell survival through activation of the phosphatidylinositol 3-kinase/protein kinase B pathway.

In this report, we show for the first time that ceramide-1-phosphate (C1P) stimulates the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) pathway, which is a major mechanism whereby growth factors promote cell survival. Also, C1P induced IkappaB phosphorylation, and enhanced the DNA binding activity of the transcription factor NF-kappaB. Apoptotic macrophages showed a marked reduction of Bcl-X(L) levels, and this was prevented by C1P. These findings suggest that C1P blocks apoptosis, at least in part, by stimulating the PI3-K/PKB/NF-kappaB pathway and maintaining the production of antiapoptotic Bcl-X(L). Based on these and our previous observations, we propose a working model for C1P in which inhibition of acid sphingomyelinase and the subsequent decrease in ceramide levels would allow cell signaling through stimulation of the PI3-K/PKB pathway to promote cell survival.

Cytoskeletal actin degradation induced by lovastatin in cardiomyocytes is mediated through caspase-2.

The objective of this study was to test the hypothesis that cytoskeletal actin fragmentation is mediated through caspase-2, specifically examining the ability of a caspase-2 inhibitor to interfere with actin fragmentation, in comparison with a caspase-3 inhibitor. Cardiomyocytes were cultured from embryonic chick heart. The fine structural element of cellular F-actin was visualized by staining cardiomyocytes with NBD-phallacidin. Lovastatin induced a dramatic and concentration-dependent loss of intact F-actin. The selectivity of this effect of lovastatin was demonstrated by the absence of similar changes in F-actin when cardiomyocytes were treated with the apoptotic stimulus palmitate, the metabolism of which produces acetyl CoA, the early substrate of cholesterol synthesis, through the mevalonate pathway. FACS analysis of NBD-phallacidin-stained cells was used to quantify the amount of F-actin loss. Actin fragmentation produced by lovastatin was operative through a caspase-2 pathway, as the caspase-2 inhibitor, z-VDVAD-fmk, significantly blocked lovastatin-induced changes in F-actin, but the caspase-3 inhibitor, Ac-DEVD-CHO, did not. Interruption of the mevalonate pathway was in part responsible for lovastatin's action, as the downstream metabolite mevalonate partially reversed the effect of lovastatin on actin fragmentation. These data indicate a previously unrecognized link between cytoskeletal actin and caspase-2.

Reduction of palmitate-induced cardiac apoptosis by fenofibrate.

The objective of this study was to test the hypothesis that a strategy based on alteration of lipid metabolism would moderate the cellular toxicity of the C16:0 saturated fatty acid-palmitate. Cardiomyocytes from neonatal mice and embryonic chicks were treated with palmitate and both oncotic and apoptotic death were observed. Fenofibrate pretreatment, 1 microM, 24 h prior to palmitate, significantly (p < 0.05) reduced palmitate-induced apoptosis. In contrast, fenofibrate had no significant effect on palmitate-induced apoptosis when fenofibrate treatment was concomitant with palmitate. The protective effect of fenofibrate was restricted to the apoptotic population. The more potent and specific PPARalpha agonist WY 14643, 1 microM, also reduced palmitate-induced apoptosis but to a smaller extent than fenofibrate. The long pretreatment time, 24 h, was necessary to show fenofibrate's effect on apoptosis, suggesting an increase in gene transcription and protein expression. Indeed, fenofibrate increased PPARalpha expression that was mainly demonstrated in the nucleus. These data suggest a novel approach to the reduction of cardiac apoptosis by the chronic treatment with the PPARalpha agonist fenofibrate.

Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages.

It was reported previously that ceramide-1-phosphate (Cer-1-P) is mitogenic for fibroblasts (Gómez-Muñoz, A., P. A. Duffy, A. Martin, L. O'Brien, H-S. Byun, R. Bittman, and D. N. Brindley. 1995. Mol. Pharmacol. 47: 883-889; Gómez-Muñoz, A., L. M. Frago, L. Alvarez, and I. Varela-Nieto. 1997. Biochem. J. 325: 435-440). We now show that Cer-1-P prevents cell death in bone-marrow-derived macrophages (BMDMs) after withdrawal of macrophage colony-stimulating factor (M-CSF). Removal of M-CSF is known to induce apoptosis in these cells. Cer-1-P blocked activation of the caspase-9/caspase-3 pathway and prevented DNA fragmentation, indicating that the enhancement of cell survival was due to inhibition of apoptosis. M-CSF deprivation resulted in activation of acid sphingomyelinase (A-SMase), increased ceramide levels, and a decrease in intracellular Cer-1-P. Exogenously added Cer-1-P inhibited A-SMase in intact BMDMs at concentrations that also prevented apoptosis. Cer-1-P also inhibited A-SMase in cell homogenates, suggesting a possible direct physical interaction of Cer-1-P with the enzyme. In conclusion, these data demonstrate that Cer-1-P blocks apoptosis in BMDMs through inhibition of A-SMase, thereby reducing ceramide generation. This adds a new dimension to the understanding of the metabolic interrelationship of ceramides and Cer-1-P, and shows how altering the balance of intracellular levels of these mediators can affect cell survival.

Lovastatin-induced cardiac toxicity involves both oncotic and apoptotic cell death with the apoptotic component blunted by both caspase-2 and caspase-3 inhibitors.

The objective of this study was to evaluate the cardiac toxicity of the HMG-CoA reductase inhibitors by testing the hypothesis that lovastatin induces apoptotic and/or oncotic cell death in the myocyte element of the heart and further that cell death is mediated through interruption of the mevalonate pathway and that apoptosis is induced through activation of caspase-2 and caspase-3. Cardiomyocytes were cultured from embryonic chick heart. Lovastatin-induced apoptosis in these cells was demonstrated by three independent techniques, namely (1) FACS analysis of low DNA content by propidium iodide (PI); (2) microscopic assessment for cellular changes of apoptosis; and (3) FACS analysis of cells stained with PI and fluorescein diacetate. Lovastatin produced a concentration-dependent increase in apoptotic cell death and 100 microM lovastatin showed over a 4-fold increase in apoptosis compared to control. Lovastatin also induced oncotic cell death, as there was a 2.5-fold increase in the amount of oncotic cell death compared to control. Lovastatin-induced apoptosis operated, in part, through the mevalonate pathway. The caspase-2 inhibitor z-VDVAD-fmk and the caspase-3 inhibitor Ac-DEVD-CHO reduced the extent of lovastatin-induced cardiac apoptosis. In contrast, lovastatin-induced oncosis was not only insensitive to these caspase-2 or -3 inhibitors but occurred through a mevalonate-independent mechanism of action. In summary, lovastatin-induced cardiotoxicity is complex and represents the sum of two distinct modes of cell death operating in part through the mevalonate pathway with the apoptotic component subject to modification by inhibitors of the initiator caspase, caspase-2, as well as the effector caspase, caspase-3.

Oxidized low density lipoprotein inhibits macrophage apoptosis by blocking ceramide generation, thereby maintaining protein kinase B activation and Bcl-XL levels.

Macrophages play a central role in the development and progression of atherosclerotic lesions. It is well known that oxidized low density lipoprotein (ox-LDL) promotes the recruitment of monocytes (which differentiate to macrophages) into the intima. We reported recently that ox-LDL blocks apoptosis in bone marrow-derived macrophages deprived of macrophage colony-stimulating factor (M-CSF) by a mechanism involving protein kinase B (PKB) (Hundal, R., Salh, B., Schrader, J., Gómez-Muñoz, A., Duronio, V., and Steinbrecher, U. (2001) J. Lipid Res. 42, 1483-1491). The aims of the present study were 1) to define the apoptotic pathway involved in the pro-survival effect of ox-LDL; 2) to determine which PKB target mediated this effect; and 3) to identify mechanisms responsible for PKB activation by ox-LDL. Apoptosis following M-CSF withdrawal was accompanied by activation of the caspase 9-caspase 3 cascade and cytochrome c release from mitochondria, but the caspase 8 pathway was unaffected. M-CSF withdrawal resulted in a marked and selective reduction in Bcl-XL protein and mRNA levels, and this decrease was prevented by ox-LDL. The ability of ox-LDL to preserve Bcl-XL levels was blocked by NFkappaB antagonists, thereby implicating IkappaB kinase as a key PKB target. M-CSF deprivation resulted in activation of acid sphingomyelinase and an increase in ceramide levels. Desipramine (a sphingomyelinase inhibitor) prevented the increase in ceramide and inhibited apoptosis after M-CSF deprivation. Ox-LDL completely blocked the increase in acid sphingomyelinase activity as well as the increase in ceramide after M-CSF deprivation. Pretreatment of macrophages with C2-ceramide reversed the effect of ox-LDL on PKB and macrophage survival. These results indicate that ox-LDL prevents apoptosis in M-CSF-deprived macrophages at least in part by inhibiting acid sphingomyelinase. This in turn prevents ceramide-induced down-regulation of PKB, the activity of which is required to maintain production of Bcl-XL.

Mitochondrial effects with ceramide-induced cardiac apoptosis are different from those of palmitate.

The objective of this study was to evaluate whether ceramide, palmitate, and inhibitors of mitochondrial electron transport chain shared similar effects on the mitochondria of intact cardiomyocytes in order to determine the likelihood that ceramide and palmitate utilize similar mitochondrial mechanisms or pathways to apoptosis. In embryonic chick cardiomyocytes, ceramide, 100 microM for 24h, induced a 42.9+/-5.8% increase in cell death assessed by the MTT assay, and a significant (P<0.01) 3.9+/-0.6-fold increase in apoptosis assessed by propidium iodide staining of permeabilized cells. Mitochondrial potential (delta psi (m)), as demonstrated microscopically and by flow cytometry of cardiomyocytes stained with a J-aggregate dye, was markedly and significantly reduced by ceramide, palmitate, and two different inhibitors of the mitochondrial electron transport chain-rotenone and antimycin A. In contrast, the effect on mitochondria as assessed by CMX-Ros oxidation was dramatically different, as palmitate, rotenone, and antimycin A each produced a reduction, while ceramide increased CMX-Ros fluorescence. Further ceramide-induced cardiomyocyte apoptosis and loss of delta psi (m) operated through a cyclosporine-insensitive pathway similar to rotenone and antimycin A but distinct from palmitate which induced apoptosis though a cyclosporine-sensitive mechanism in these cells. These data suggest that ceramide acts on the mitochondria of intact cells through a cyclosporine-insensitive mechanism likely from a combination of actions including production of mitochondrial oxidants. The discordant findings between ceramide and palmitate suggest that palmitate-induced cell death is not primarily mediated by de novo ceramide synthesis.

Discordance between the effect of modulators of calcium on staurosporine-induced apoptosis and staurosporine-induced actin degradation.

Staurosporine produced DNA fragmentation characteristic of apoptosis and a dramatic alteration of cell structure that include alterations of cytoskeletal actin and cytoplasmic condensation with vacuolization. These changes were not induced by chelerythrine, a more specific PKC inhibitor than staurosporine. The calcium chelator, BAPTA, significantly reduced staurosporine-induced DNA fragmentation but did not affect staurosporine-induced changes in cytoskeletal actin. These data suggest that DNA fragmentation and actin degradation in apoptosis, induced by staurosporine, are under different regulatory control by calcium.

Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin.

To test the hypothesis that regulation of palmitate metabolism, through carnitine palmitoyl transferase-1 (CPT-1) or through alterations of glycolysis, was involved in the pathway of palmitate-mediated cell death, cardiomyocytes were cultured from 7-day-old chick embryos. Palmitate-induced cell death, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was enhanced by carnitine, a cofactor needed for palmitate transport into mitochondria via CPT-1. Carnitine co-incubation with palmitate significantly (P < 0.01) increased the amount of apoptotic cells, assessed by propidium iodine staining and fluorescent-activated cell sorting analysis compared with treatment with either palmitate or carnitine alone. The CPT-1 inhibitor oxfenicine significantly (P < 0.05) blocked the cell death induced by the combination of palmitate and carnitine. The short-chain saturated fatty acid capric acid (100 microM), which is not likely transported by CPT-1, did not significantly affect cell viability, whereas the C18 saturated fatty acid stearic (100 microM) significantly (P < 0.01) reduced cell viability and to a similar extent as palmitate. In contrast, there was no significant alteration of palmitate-induced cell death by cotreatment with 100 nM insulin + 2 g/l glucose or 1 mM lactate, which promote ATP generation by glycolysis rather than fatty acid oxidation. Fumonisin did not alter palmitate-induced cell death or apoptosis, suggesting that the effect of palmitate was not operative through increased ceramide synthesis. These results suggest that oxidation of palmitate through CPT-1 is involved in the production of apoptosis in cardiomyocytes.