The clinical outcome of cardiovascular diseases as myocardial infarction and stroke are generally caused by rupture of an atherosclerotic plaque. However, the actual cause of a plaque to rupture is not yet established. Interestingly, pathology studies have shown an increased presence of the mast cell, an important inflammatory effector cell in allergy and host defense, in (peri)vascular tissue during plaque progression, which may point towards a causal role for mast cells. Very recent data in mouse models show that mast cells and derived mediators indeed can profoundly impact plaque progression, plaque stability and acute cardiovascular syndromes such as vascular aneurysm or myocardial infarction. In this review, we discuss recent evidence on the role of mast cells in the progression of cardiovascular disorders and give insight in the therapeutic potential of modulation of mast cell function in these processes to improve the resilience of a plaque to rupture. Mast celobulins -6. Mast lobulins , 6. MastAcute cardiovascular syndromes, such as myocardial infarction and vascular aneurysm formation, are still a major cause of death on the Western society. Atherosclerosis, one of the main cardiovascular disorders causing the acute cardiovascular events, is considered a chronic inflammatory disease, involving many inflammatory cells such as macrophages and T-lymphocytes , 8. At lin vitro and in vivo models that have recently given more insight into the mechanisms by which mast cells exert their pathogenic effects. Finally, the therapeutic potential of modulation of mast cell function will be evaluated.In this review, we will outline present knowledge on the contribution of mast cells to cardiovascular diseases in general. Particular emphasis will be on the pathobiologies with an inflammatory component such as the development and progression of atherosclerosis, as well as to myocardial infarction and vascular aneurysm formation. Furthermore, we will discuss 2.Human pathology studies have provided ample evidence for the presence of a variety of immune and inflammatory cell types in diseased tissue and these studies have been essential in identifying the major cellular players in the progression of disease. Mast cells can easily be detected in tissue sections through a variety of mast cell staining protocols that are available at present. Toluidin Blue as well as a Chloro-Acetate Esterase (CAE) staining both stain the granulae of mast cells , while i-/- [-/- [-/- mice [Animal models have greatly facilitated to understand the molecular mechanisms involved in cardiovascular diseases, be it atherosclerosis, ischemia/reperfusion injury, or aneurysm formation. In atherosclerosis, e.g. high fat diet fed rabbits and genetically modified hyperlipidemic mice, such as the ApoE-/- , 13, LDL-/- [-/-  or the A-/- [-/-  have all-/- [-/- , 17. Lik-/- [-/- , peri-ao-/- [-/- , arteria-/- [-/-  or Angio-/- mice . d mouse has a comparable phenotype as the W/Wv mouse, however in this strain bone marrow transfer does not rescue the phenotype, suggesting that in these mice environmental factors needed to induce mast cell differentiation and maturation are absent [sh) inversion mutation. Unlike the previous two strains, mast cell deficiency is not accompanied by anemia and sterility [-sh/W-sh) mice can also be repopulated with mast cells by injection of either bone marrow cells or cultured bone marrow derived mast cells (BMMCs), rendering this mouse a very interesting animal for studying mast cell biology in vivo. Mast cell deficient animals were reported already in 1978, when Kitamura and colleagues first described W/Wv mice that were virtually devoid of tissue and skin mast cells . These me absent , 24. A me absent . More reterility . These Kin vitro as well as in vivo studies [in vitro and in vivo models investigating the role of mast cells and mast cell constituents in cardiovascular disorders will be described.Another approach in mast cell research, albeit not frequently pursued in cardiovascular disease models, involves systemic or focal mast cell activation in animals. To date, most research has been conducted in disease models of asthma, allergy and arthritis. Mast cells can be activated in various ways, of which IgE stimulation and hapten sensitization/challenge are widely known , 28. Com studies . In the 3.Already in 1953, Constantinides reported that mast cells might be involved in the susceptibility to experimental atherosclerosis . In thisMast cell constituents such as histamine, prostaglandin D2 and leukotrienes play a role in atherogenesis and its clinical manifestations by affecting endothelial cell function and vasoreactivity. This hypothesis is strengthened by the recent finding that increased mast cell numbers in the adventitia of coronary arteries associate with vasospasms . Chymaseet al. described the presence of CCR3 positive mast cells in plaques of atherosclerotic arteries, while the primary ligand of CCR3, eotaxin, was demonstrated to be highly expressed by activated vascular smooth muscle cells [Although mast cells were on one hand demonstrated to inhibit macrophage induced oxidation of Low Density Lipoprotein (LDL) , 39, masle cells , suggestvia integrins and adhesion molecules like intercellular adhesion molecule (ICAM)-1 [Foci of mast cell accumulation were detected in the intima at later stages of plaque formation, mainly near the shoulder region and in areas of early calcification of the plaque -50. As d(ICAM)-1 . In addi(ICAM)-1 , 53.Intimal mast cells have been demonstrated to contain tryptase, and approximately 40% of the cells contained chymase as well . Both main vitro, which could translate in reduced plaque stability [It is not completely clear whether or not adventitial mast cells are capable of modulating intimal processes and instrumental in plaque rupture, although most evidence points in that direction. Pathology studies so far suggest that adventitial mast cells indeed contribute to plaque destabilization and plaque rupture, as mentioned above by release of mast cell proteases chymase and tryptase that can directly degrade matrix molecules themselves but also by activation of MMPs and by conversion of AngI into the proinflammatory, vasoactive AngII . Secondltability , 70.In vitro studies showed endothelial proliferation in response to mitogenic factors. Conceivably, VEGF secreted by mast cells [A third mechanism through which mast cells can contribute to plaque progression and destabilization is by enhancing neovascularization of the plaque. Indeed, in neovascularized areas of human coronary atheromas mast cells were demonstrated to colocalize with intraplaque microvessels . In vitrst cells  could rest cells , suggestst cells .et al. used the mast cell deficient Kit(W-sh/W-sh) mice, which had been backcrossed with the atherosclerotic LDLr-/- mouse [-/- mice was hampered compared to control LDLr-/- mice. Importantly, lipid deposition, macrophage content and plaque apoptosis were reduced, while collagen content and fibrous cap development were increased in these mice, reflective of a more stable phenotype of mast cell deficient plaques. Repopulation of these mice with wild-type BMMCs completely reversed the beneficial effect of mast cell absence. Adoptive transfer of IL-6-/- or IFN↓-/- BMMCs did however not affect lesion progression, implying that mast cell-derived IL-6 and IFN↓ are the prime factors in mast cell dependent plaque initiation. This elegant study conclusively demonstrates the contribution of mast cells to the progression of atherosclerosis, however left unanswered whether mast cells modulate the atherogenic response focally or alter the immune status of the mice, in particular as intimal mast cells are rather scarce in mouse plaques. Only recently, experimental proof was provided in support of a role of mast cells in atherosclerotic plaque development and progression. Sun /- mouse . Atherog-/- mice to stimulate flow-induced atherosclerotic plaque formation [In a parallel study by us, we have pursued a different strategy to address the direct role of vascular mast cells. We have applied slightly constrictive perivascular silastic collars at the carotid arteries of apoEormation . After tormation . SurprisN-acetyllactosamine-binding lectin family expressed amongst others by mast cells [-/-/apoE-/- showed an impaired atherogenic response as compared to apoE-/- mice, while plaques of galectin-3-/-/apoE-/- mice had a lower number of perivascular inflammatory infiltrates and mast cells than those of apoE-/- mice [Mast cell function in cardiovascular disease was recently shown to be in part dependent on galectin-3, which is a member of st cells . Galecti-/- mice .The triggers that activate mast cells during lesion progression and plaque rupture are still subject to investigation. As described above, mast cells can be activated by modified LDL particles which concurs with the notion that bioactive phospholipid components might activate mast cells , 44, whi4.Aortic valve stenosis (AS) shares many features with atherosclerosis, including infiltration of inflammatory cells such as macrophages and T-lymphocytes, accumulation of oxidized lipids and extensive tissue remodeling , 84. Ana5.et al. did not detect any differences in tryptase levels between patients with acute myocardial infarction, with unstable angina pectoris or controls that did not suffer from ischemic cardiovascular diseases [et al. [Myocardial infarction occurs mainly following rupture of an atherosclerotic plaque, leading to thrombus formation and vessel occlusion. The heart is a mast cell-rich organ and serum IgE levels were found to be significantly higher in the patients with unstable angina and acute myocardial compared to stable angina pectoris and controls, indicating that IgE might play a role in the pathogenesis of unstable angina pectoris and acute myocardial infarction . Whole bdiseases . These d [et al. . On the  [et al. . In fact [et al. .in vitro [In a guinea pig model of ischemia/reperfusion, histamine was demonstrated to be released after an ischemic period . Mast cein vitro  and in sin vitro . TreatmeIn general, mast cells have been demonstrated to accumulate in the heart after MI, while the exact role of mast cells after the MI however has not completely been elucidated. Mast cells might on one hand enhance the injury by the release of matrix-degrading enzymes such as chymase and tryptase and of pro-inflammatory cytokines and chemokines resulting in accumulation of inflammatory leukocytes in the infarcted area. Also, mast cell activation may aggravate the injury by the induction of cardiomyocyte apoptosis. As a rich source of VEGF and bFGF however, mast cells may on the other hand contribute to the wound healing response and collateral formation after ischemia. Further research will contribute to our understanding of the actual role of mast cells in MI.6.et al. showed a more pronounced presence of mast cells in arteries of the circle of Willis of patients that died of subarachnoid hemorrhage (SAH) after aneurysm rupture, although the number of patients included in this study was very limited. Besides a single case study of a patient that died of coronary artery dissection in which the perivascular tissue was demonstrated to contain a large eosinophil and mast cell infiltrate [et al. [et al. [et al. [Aortic aneurysm development is a chronic degenerative condition, which is characterized by weakening and dilation of the aortic wall that results in a life-threatening risk of rupture and is associated with the development of atherosclerosis . The firfiltrate , hardly  [et al.  conclusi [et al. , 106. Th [et al.  who demo [et al. , which w [et al.  found anIn vivo studies on the role of mast cell biology in aneurysm formation have been focussed on the mast cell protease chymase as an AngII producing enzyme. Chymase was indeed demonstrated to be involved in aneurysm development in an animal model where AAA was induced by application of elastase onto the abdominal aorta of hamsters that were treated with the specific chymase inhibitor NK3201 [r NK3201 . The aorr NK3201 . Collectet al. studied the role of mast cells itself rather than a specific mast cell constituent in an animal model of abdominal aortic aneurysm (AAA) formation, in which AAA was induced by elastase perfusion in mast cell deficient Kit(W-sh/W-sh) mice and syngenic control mice [-sh/W-sh) mice with BMMCs from wild-type or TNFα-/- mice caused susceptibility to AAA formation to be recovered while IL-6-/- or IFNγ-/- BMMCs were ineffective.Recently, Sun rol mice . In this2 application, which resulted in a decreased diameter of abdominal aorta compared to the aortic diameter of wild-type rats [Similarly, AAA were induced in mast cell-deficient mutant Ws/Ws rats by peri-aortic CaClype rats . In the In conclusion, these studies demonstrate that mast cells may not only participate in AAA pathogenesis by inducing aortic SMC apoptosis and by releasing proteases like chymase and tryptase, but also have a role in the inflammatory response in aneurysm development.7.1)). Mast cells promote lipid accumulation, matrix degradation, apoptosis, increase leukocyte influx into the plaque and enhance microvascular leakage, all resulting in a sharply increased risk of intraplaque hemorrhage, plaque destabilization and rupture. Second, mast cells contribute importantly to aneurysm formation by the release of chymase, while also mast cell IL-6 and IFNγ were seen to be involved. A role of mast cells in the response after acute myocardial infarction seems plausible but still awaits experimental support. Intervention studies in cardiovascular disease models so far (Table 1) have revealed a number of promising leads that target pathogenic mast cells per se (mast cell stabilizers) or mast cell constituents such as chymase, tryptase, histamine, IFNγ and IL6, which may have high therapeutic potential in the prevention of plaque rupture, aneurysm formation and possibly other cardiovascular disorders.Culminating evidence from pathology and experimental studies points to a key role for mast cells not only in a range of immune diseases but also in various cardiovascular disorders. Perivascular as well as intimal mast cells were seen to contribute substantially to the pathogenesis of atherosclerosis and plaque destabilization (Fig. (


Cognitive deficits and personality pathology are prevalent in opiate dependence, even during periods of remission, and likely contribute to relapse. Understanding the relationship between the two in vulnerable, opiate-addicted patients may contribute to the design of better treatment and relapse prevention strategies.The Millon Multiaxial Clinical Inventory (MCMI) and a series of neuropsychological tests were administered to three subject groups: 29 subjects receiving methadone maintenance treatment (MM), 27 subjects in protracted abstinence from methadone maintenance treatment (PA), and 29 healthy non-dependent comparison subjects. Relationships between MCMI scores, neuropsychological test results, and measures of substance use and treatment were examined using bivariate correlation and regression analysis.MCMI scores were greater in subjects with a history of opiate dependence than in comparison subjects. A significant negative correlation between MCMI scores and neuropsychological test performance was identified in all subjects. MCMI scores were stronger predictors of neuropsychological test performance than measures of drug use.Formerly methadone-treated opiate dependent individuals in protracted opiate abstinence demonstrate a strong relationship between personality pathology and cognitive deficits. The cause of these deficits is unclear and most likely multi-factorial. This finding may be important in understanding and interpreting neuropsychological testing deficiencies in opiate-dependent subjects. Cognitive impairment is prevalent in opiate dependence, even during periods of remission, and may be associated with poor addiction treatment outcomes -7. HowevBecause personality disorders are the most prevalent comorbid psychiatric condition in opiate dependence , and areSeveral reports have used the Millon Clinical Multiaxial Inventory (MCMI) to document increased prevalence of particular types of personality pathology in opiate dependence -25. CraiIn fact, to date, we are aware of no research on the relationship of personality pathology and neuropsychological functioning in opiate-addicted individuals, though an association between neuropsychological deficits and personality pathology, unrelated to substance abuse, has been documented ,18,20,28Thus, because both cognitive impairment and personality disorders are prevalent and associated with poor treatment outcomes in opiate addicted individuals, understanding the relationship between these pathologies may broaden our understanding of both impairments in these patients, as well as aid in designing more effective addiction treatments. We therefore sought to examine this relationship in two groups of treated opiate dependent individuals: those receiving methadone maintenance and those previously maintained with methadone and currently in protracted opiate abstinence. The subjects were tested using well-accepted measures of neuropsychological performance and personality pathology, and the relationships of these variables were examined using correlation and linear regression. We hypothesize that 1) personality pathology will correlate with neuropsychological test performance; 2) that the effect of drug use on neuropsychological test performance will be greater than the effect of personality pathology on neurological test performance; and 3) these relationships will be similar for both subjects currently receiving methadone and subjects who are opiate-abstinent.Eighty-five subjects  were selected for inclusion in one of three groups based on their drug use history: 1) Opiate-dependent subjects receiving methadone-maintenance therapy (MM); 2) Opiate dependent subjects previously detoxified from methadone maintenance therapy and in a state of protracted opiate abstinence (PA); and 3) comparison subjects without a history of opiate-dependence (C) , current alcoholic intake of more than 15 drinks  per week, history of head trauma, cardiovascular, pulmonary, other systemic, or neurological disease, and HIV seropositivity. Neither moderate use of caffeine, (<600 mg of caffeine per day), nor occasional marijuana use (≤ 1 marijuana cigarette/month and urine toxicology negative for THC) were exclusionary.Initial screening was performed in a telephone interview. Subjects meeting initial inclusion/exclusion criteria and who continued to express interest in participating were invited for a face-to-face interview, consisting of the Structured Clinical Interview for DSM (SCID-I), conducted by trained personnel . All subCognitive performance was determined by a small battery of tests, designed to assess aspects of cognitive functioning most frequently cited as disrupted in the addiction literature, assess neuropsychological function in multiple domains, and be quick and efficient. These were:The WAIS-R Vocabulary Test [• ary Test : A test The Stroop Color-Word Test [• ord Test ,32: A teThe Controlled Oral Word Association (COWA) [• n (COWA) -35: A teThe Benton Visual Retention Test (BVRT): A test of visual memory, and visual construction. Subjects are presented an image for 10 seconds and then asked to draw it from memory. The numbers of total correct, total errors, right, and left-sided errors are recorded [• recorded -38. OnlySubstance use history was determined by:Substance Use Inventory (SUI) [• ry (SUI) : A questPersonality pathology was measured by:Millon Clinical Multiaxial Inventory-II (MCMI) [• I (MCMI) : This 17I (MCMI) , we assiInitial analyses compared groups on demographic and clinical measures (e.g. years of heroin abuse) with ANOVA's for continuous variables and Chi-Square analyses for categorical variables.r. Methadone-maintained subjects, PA subjects, and non-dependent comparison subjects were examined individually.The scores for personality pathology  were compared across groups using MANOVA followed by Tukey's HSD pair-wise comparisons. The relationship between the MCMI scores and neuropsychological test performance were examined using bivariate correlation and regression. In order to reduce the number of multiple comparisons, the scores for the WAIS-vocabulary, COWA, Stroop CW, and Benton Correct tests were transformed to Z-scores and then combined to form a normalized composite neuropsychological performance score. The COWA total correct score was age- and education-corrected prior to normalization according to the methods of Gladsjo et al . This noIn order to try and control for any confounding effect of drug use history on neuropsychological test performance, we used multiple regression to regress the composite neuropsychological performance scores on combinations of the MCMI composite score and drug use history variables. Because the non-dependent comparison group did not have similar drug use history data, the regression analysis was carried out for the MM and PA groups only. Scatterplots of the variables composite neuropsychological performance score, composite MCMI score, and drug use history were constructed to confirm a linear relationship between the variables (not shown). The associations between independent and dependent variables are presented by means of standardized linear regression coefficients (β).2 analysis. Significant group differences were identified for the variables age and education: by Tukey's HSD paired comparisons, PA subjects were older than both non-dependent comparison and MM subjects, and the non-dependent comparison subjects had more years of education than both of the drug dependent groups. Therefore, age and years of education were entered as covariates in the MANCOVA comparing neuropsychological and personality data across groups.In all, there were 29 Methadone Maintained (MM), 27 Protracted Abstinent (PA), and 28 non-dependent comparison subjects who completed all evaluations. The three subject groups did not differ with respect to gender or ethnicity by χThe MM and PA groups did not differ significantly on any measure of substance use history  or substance treatment history . The difference between the MM and PA groups on mean age of first opiate use approached, but did not achieve significance. Protracted abstinence subjects were opiate-free an average of 10.73 months, with a range of 6–24 months. The demographic data is presented in Table p < 0.001). With age and years of education entered as covariates, this difference remained significant (Wilk's λ = .754 (Multivariate F = 2.78); df = 6, 110; p < 0.015). The results of the univariate F tests with age and years of education entered as covariates demonstrated significant differences in group means for the composite MCMI score, the MCMI Cluster B score, and Cluster C scores. The difference across group means of MCMI Cluster A score was marginally significant ; df = 6, 116; p < 0.001). Similar results were observed when correlating the three MCMI cluster scores with the normalized composite neuropsychological score. This negative correlation was also evident when examining PA subjects only . When analyzing either the non-dependent comparison group or the MM groups alone, the correlation of MCMI scores and the normalized composite neuropsychological scores was not statistically significant . The addition of any of the variables of drug use history  to the model resulted in a decrement of the coefficient of determination (r2) for each model tested. In all models tested, the β score for the composite MCMI score was greater than the β score of any of the variables of drug use history. In fact, none of the drug abuse variables were significantly related to the composite neuropsychological performance score (See Table We used linear regression to examine the functional relationship of MCMI cluster scores and drug use history variables to the composite neuropsychological performance score. When all subjects were included in the analysis, the regression of the composite MCMI cluster score on the composite neuropsychological performance score was significant (R = -0.572; Since the correlation between MCMI scores and neuropsychological performance was present only in the PA group, we performed a secondary analysis of the correlation of MCMI Cluster scores with the specific neuropsychological tests WAIS-R Vocabulary subscores, COWA Total subscore, Stroop CW subscore, and BVRT Total Correct subscore for the PA subjects only Table . The resIn agreement with our first hypothesis, we observed a significant correlation of MCMI scores and impaired neuropsychological test performance in a group of subjects with a history of opiate-dependence treated with methadone. Contrary to our second hypothesis, the regression models indicate that personality pathology explains a greater amount of the variance in neuropsychological test performance than do any of the variables of drug use history. Lastly, the relationship of personality pathology and neuropsychological performance is significant for the abstinent former opiate addicts, but not for the methadone-maintained group. We suggest that these results are best explained by postulating a multi-factorial origin to the neuropsychological deficits associated with opiate dependence, as opposed to thinking that drug use alone causes such deficits. To our knowledge, this report is the first to identify a link between personality pathology and the measurement of neuropsychological impairment in opiate addiction.Our findings are consistent with previous studies that demonstrated significantly increased MCMI scores in opiate-dependent subjects compared to healthy controls -25. AddiThe absence of significant findings in MM subjects is unexpected and contrary to our third hypothesis. One possible explanation for the group differences in statistical significance in the correlation of MCMI scores and neuropsychological test performance is differences between groups in MCMI scores and/or neuropsychological test performance. We have previously reported comparable degrees of personality pathology and cognitive impairment in both MM and PA subjects, with some minor variations – PA subjects tend to have more Cluster A pathology and worse visuospatial recall ,23. It iWe have used linear regression to examine the functional relationships of drug use history and character pathology on neuropsychological performance. In every regression model tested, the composite MCMI score accounted for a greater portion of the variance in neuropsychological performance than did any of the variables of drug use history. As a measure of the relative importance of predictor variables, the regression models suggest that drug use history has less impact on neuropsychological test performance than does personality pathology. In fact, none of the drug use history variables were any better than random chance at accounting for variance in neuropsychological scores.Because the neuropsychological variables assess different cognitive processes, it is potentially important to distinguish which, if any, of them is most responsible for the observed relationship between neuropsychological function and personality. Interestingly, for the PA subjects only, strong negative correlations of the WAIS-R Vocabulary subscore were demonstrated with each of the MCMI Clusters scores, indicating that greater personality pathology of any sort is associated with worsened verbal function. The WAIS-R Vocabulary subscore is a test of verbal function , and prePersonality disorders are thought to be enduring and stable over time . Some auCognitive deficits associated with opiate dependence have traditionally been explained as arising due to drug use itself. However, based on the results reported here, we suggest that it may be more helpful to posit a multi-factorial origin to opiate-associated cognitive impairment. A variety of factors may result in neuropsychological deficits; including opiate abuse, additional concomitant illicit substance abuse, adulterants added to illicit substances, overdoses and head trauma, lifestyle and nutritional factors, infections, educational and occupational attainment, genetic and familial factors, and early-childhood experiences. It is also possible that the neuropsychological performance deficits identified in opiate-dependent subjects, like the enduring and stable personality disorders often associated with opiate dependence, may be a manifestation of a pre-existing condition that persists through drug use, abuse, and addiction treatment.Insofar as possible, we have attempted to limit confounding influences on our results. We have recruited the subjects with a history of opiate-dependence from a residential treatment setting, thereby providing for a standardization of nutrition and activities, along with frequent testing for illicit substance use. We have included only MM subjects who have experienced a significant period of methadone treatment, and PA subjects with a prolonged period of drug abstinence. Nonetheless, our results must be interpreted in light of the study limitations. Firstly, this cross-sectional study can only identify associations, and does not support causal interpretations. Because entrance into abstinence treatment was entirely voluntary, it is possible that the observed differences between MM and PA groups reflect a selection bias rather than an actual effect of methadone. We have not controlled for additional past illicit drug use in our sample, and this may impact our outcome measures in unexpected ways. We have used a simple battery of cognitive tests, which limits the scope of our findings. Additionally, unmeasured factors, such as substance-related lifestyle and familial variables, may have influenced our results.Methadone has been used for years as a substitution treatment for opiate addiction . DespiteCurrent drug addiction treatment focuses on abstinence and/or harm reduction. The underlying assumption is that any existing neuropsychological and/or cognitive deficits will resolve with cessation of opiate use. If, as we have argued, neurocognitive difficulties do not correlate with opiate use, addiction treatment alone will not ameliorate impairments. Most studies show worsened treatment outcomes in addicts suffering from neurocognitive impairments ,7,57-59.Subjects with a history of opiate dependence demonstrate greater levels of personality pathology than do non-dependent comparison subjects. Personality pathology is highly associated with neuropsychological performance deficits in all groups studied. The cause of this deficit is poorly understood and most likely multi-factorial. Neuropsychological deficits may not resolve with cessation of opiate abuse. Given the persistence of both personality pathology and cognitive impairment in recovering opiate addicts, cognitive remediation may help to protect against relapse.The authors declare that they have no competing interests.IG and EL designed the study and wrote the protocol. JP, ED, and DE participated in data analysis, interpretation, and manuscript writing. LC participated in study design and data analysis. MS participated in data collection. All authors read and approved the final manuscript.


Transposable elements (TEs) comprise a large fraction of mammalian genomes. A number of these elements are actively jumping in our genomes today. As a consequence, these insertions provide a source of genetic variation and, in rare cases, these events cause mutations that lead to disease. Yet, the extent to which these elements impact their host genomes is not completely understood. This review will summarize our current understanding of the mechanisms underlying transposon regulation and the contribution of TE insertions to genetic diversity in the germline and in somatic cells. Finally, traditional methods and emerging technologies for identifying transposon insertions will be considered. In the 60 years since Barbara McClintock first discovered transposable elements (TEs), it has become increasingly recognized that these mobile sequences are important components of mammalian genomes and not merely 'junk DNA'. We now appreciate that these elements modify gene structure and alter gene expression. Through their mobilization, transposons reshuffle sequences, promote ectopic rearrangements and create novel genes. In rare cases, TE insertions which cause mutations and lead to diseases both in humans and in mice have also been documented. However, we are at the very earliest stages of understanding how mobile element insertions influence specific phenotypes and to the extent to which they contribute to genetic diversity and human disease.TEs are categorized into two major classes based on their distinct mechanisms of transposition. DNA transposons, referred to as Class II elements, mobilize by a 'cut-and-paste' mechanism in which the transposon is excised from a donor site before inserting into a new genomic location. These elements are relatively inactive in mammals, although one notable exception is a piggyBac element recently identified to be active in bats  encoded proteins for their mobilization [Of all mobile element families, only the retrotransposons remain actively mobile in the human and primate genomes and serve as ongoing sources of genetic variation by generating new transposon insertions. LINEs (long interspersed nucleotide elements) represent the most abundant autonomous retrotransposons in humans, accounting for approximately 18% of human DNA. Non-autonomous elements such as SINEs (short interspersed nucleotide elements) and SVAs [hybrid SINE-R-VNTR (variable number of tandem repeat)-lization ,6-9. TogAlus) have been documented in humans [in vitro, providing strong support for the idea that the V(D)J recombination machinery evolved from transposable elements [It is both impressive and puzzling that almost half of our genome is composed of these repeat sequences. Evolutionary paradigms dictate that useless elements and harmful TE insertions events should be selected against, while beneficial insertions should gain a selective advantage and thus be retained. Indeed, the most successful transposons have co-evolved with their hosts. Most transposable element insertions are expected to have few consequences for the host genome and, therefore, have little to no impact on gene function . Rarely,n humans . Less frn humans ,12,13. Pelements -16.In this review, we examine mechanisms of transposon regulation and discuss how TE insertions account for genetic diversity in the germline and in somatic cells. Traditional methods and recently developed technologies for identifying these insertions will also be considered.de novo insertions are transmitted through the germline to subsequent generations. Indeed, successful metazoan transposons often show germline-restricted expression. As TEs pose a significant threat to genome integrity, uncontrolled activation of these elements would imperil both the host and the element. It appears that, as a consequence, metazoan genomes have evolved sophisticated mechanisms to limit the mobilization of these elements.Expansion of mobile elements occurs when Alu, and mouse IAP  elements [in vitro is modest [DNA methylation is, perhaps, the most well understood mechanism involved in the regulation of TEs in the germline of plants, fungi and mammals -20. Cytoelements . Intereselements ,25. RNA elements , althougs modest ,28.Drosophila, piRNAs are enriched in sequences containing retrotransposons and other repetitive elements. Disruption of Piwi proteins results in the reduction in piRNA abundance and transposon derepression [Drosophila and zebrafish directly implicated Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome [Drosophila germline against potentially sterilizing transposition events [Recently, a novel form of mobile element control has emerged that involves small RNAs in germ cells . At the pression ,31. A see genome -34. Thesn events ,35.Mili and Miwi2) result in the loss of TE methylation in the testes, transposon derepression and meiotic arrest during spermatogenesis [nuage [et al. demonstrated that loss of Mael leads to germ cell degeneration (at the same point in meiosis as Mili and Miwi2 mutants) and male sterility in mice [nuage , this structure is likely where the piRNA pathway defends the germline genome from the invasion of unchecked transposable elements.Mutations in two mouse Piwi orthologues  and causing malformations with craniosynostosis categorized as Apert syndrome [Autosomal transposon insertions leading to human disease have also been described. These tend to phenocopy otherwise autosomal dominant diseases caused by mutation of the transposon target locus. Examples include an intronic omatosis  and a smsyndrome ,51.de novo insertions are likely to be passed on as phenotypically silent repeats, it is well established that transposon insertions are relevant to human clinical genetics and can have severe phenotypic consequences in rare cases [de novo insertions uncommonly underlie human disease.Thus, while most re cases ,53. TherThere is a widely accepted belief that truly 'selfish' genetic elements must mobilize selectively in the germline or during early development in order to guarantee their evolutionary success. However, recent evidence from several laboratories challenges this notion. Belancio and colleagues reported that both full-length and processed L1 transcripts are detected in human somatic tissues as well as in transformed cells . Kubo anGage and colleagues reported that L1 retrotransposition occurs in cultured mouse neuronal progenitor cells and in a mouse model harboring a human L1 element . Based onuage. These cytoplasmic structures contain enzymes involved in RNA turnover, including members of the RNA-induced silencing complex. L1 RNA and ORF1 have been shown to accumulate in stress granules, which associate with P bodies in somatic cells [The extent to which TE insertions may generate diversity in somatic cells remains largely unexplored. It remains unclear why transposons do not hop more often in somatic cells. One possibility is that a transposon defense pathway present in somatic cells has yet to be discovered. One potential candidate involved in somatic TE repression might be the P body (processing body), the somatic equivalent of the germline-specific structure ic cells . It is tAPC) was described [A hallmark of neoplastic proliferation is the accumulation of somatic genetic changes. Many types of cancer involve recurrent karyotypic abnormalities or other forms of genomic instability. The roles that mobile elements may play in these processes have been largely speculative. In humans, constitutionally integrated transposons have fairly well established roles as substrates in non-allelic homologous recombinations; but do they also potentiate oncogenesis by somatic expression of, for example, genotoxic L1-encoded proteins? Beyond this, are they capable of completing retrotransposition in such a way as to inactivate key tumor suppressor genes? In rare cases, they do appear to do the latter. For example, LINE-1 retransposition was shown to be an important step in the development of a colon cancer when a tumor-specific exonic insertion in adenomatosis polyposis coli  DNA transposon system has been successfully used to identify novel cancer genes in tissues that could not be previously analysed by slow transforming retroviruses ,70. Recede novo somatic insertions requires directed strategies in order to identify younger, potentially active, transposon copies. Methods for identifying this complement of novel TE insertions have been described and are under rapid development as genomic methodologies continue to avail themselves  , a randoIn silico mining of the human genome draft [Alu, LINE-1 and SVA elements have differentially amplified in humans and chimpanzees. Building on the foundation of the human reference genome, relatively new concerted efforts are underway which may harness sequencing methods to provide insights into structural variation. Paired-end mapping of size selected DNA fragments represents a large-scale approach to identify sizable variants in the genome. For example, using this method with fragments cloned into fosmids, it is possible to detect large insertions and deletions (indels) embedded in repetitive DNA [Analyses of genomic sequencing data have since contributed significantly to our understanding of polymorphic retroelements in humans, which will presumably accelerate with the ongoing exponential increases in available data. me draft ,79, the me draft  and compme draft -83. Thestive DNA ,85. Becktive DNA . MoreoveTechnologic developments in sequencing methods and microarray platforms are expanding methods for high throughput TE discovery in the post-genomic era. Several laboratories recently published targeted methods for recovering TE insertion sites that, in combination with high resolution microarrays or deep sequencing, allow researchers to catalogue novel transposition events on a genome-wide basis ,87-89. FAlus in humans, although it is postulated that the actual number is significantly higher due to ongoing transposition and individual TE polymorphisms. In an effort to detect the degree of genetic variation that is caused by transposable elements, Bennett and colleagues [de novo transposon insertions. They estimated that human populations harbour an average estimated 2000 common transposon insertion polymorphisms. In general, these results are consistent with several other studies regarding Alu element polymorphisms [To what extent do mobile elements contribute to human genetic diversity? This is a complex question, which is just beginning to be explored in greater depth. Sequencing of the human genome revealed that individual genomes typically exhibit 0.1% variation . Most oflleagues  analysedorphisms  and L1-Horphisms ,78,91,92in vitro retrotransposition assays suggest only a small number are highly active and have accounted for the majority of de novo insertions [In an attempt to identify the number of active polymorphic L1 elements in the human genome, Brouha and colleagues  identifisertions .et al. [Alus, L1 elements and SVAs are found in an individual diploid genome. Transposon-mediated deletions were also detected. The Jorde laboratory recently demonstrated that the presence of a fixed Alu insertion is predictive of an elevated local recombination rate, which may further contribute to non-allelic recombination events [Recently, several groups have focused their efforts on determining what fraction of structural variants (SVs) in the human genome is due to TE sequences. Korbel and colleagues  employedet al.  analysedn events . Indeed,Our understanding of the basic biology of TEs has expanded dramatically in the 60 years since their initial discovery. Yet, there are still many open questions awaiting further study. For example, the mechanisms of transposon regulation and mobilization in the germline and somatic cells have not been fully elucidated. If we appreciate where, when and how these processes occur, we will ultimately better understand the impact of these elements on host genomes and the extent to which they contribute to diversity.Although major advances have been made in the identification of transposon insertions in humans, we are at the very earliest stages of recognizing the full implications of these findings. It is clear that TE insertions provide a rich source of inter-individual genetic variation. With the continued optimization of technologies that are able to identify all transposon insertions, we will undoubtedly gain a better understanding of the extent of TE diversity in individual genomes, in human populations and in disease states.APOBEC: apolipoprotein B messenger RNA editing enzyme; Catalytic polypeptide; ATLAS: amplification typing of L1 active subfamilies; IAP: intracisternal A particle; indels: insertions and deletions; LINE: long interspersed nucleotide element; LTR: long terminal repeat; MAEL: MAELSTROM; PCR: polymerase chain reaction; PIRNA: piwi-interacting RNA; SB DNA: Sleeping Beauty DNA; SINE: short interspersed nucleotide element; SV: structured variant; TE: transposable element.The authors declare that they have no competing interests.KAO and KHB wrote the review. Both authors approved the final manuscript.


Preoperative staging of gastric cancer is difficult. Several molecular markers associated with initiation and progression of cancer seem promising for obtaining preoperative prognostic information. To investigate whether these markers are indicative especially for the presence of lymph node metastases in patients with gastric cancer, we have examined primary tumour specimens from 105 patients with primary adenocarcinoma of the stomach entered in a surgical trial. In this trial, lymph node status was determined by strictly quality-controlled lymph node dissection and examination. The selected markers were growth regulators , metastasis-suppressor gene product (nm23), adhesion molecules  and urokinase-type plasminogen activator (u-PA). Also, the amount of eosinophilic and lymphocytic infiltrates available post-operatively was analysed with respect to its prognostic value for lymph node status. Moreover, the association of these parameters with survival and disease-free period (DFP) was evaluated. Of all molecular markers investigated, only Rb expression had a significant association with the presence of lymph node metastasis in both univariate and multivariate analysis. For curative resectability, a significant association was found with Rb and E-cadherin expression, while in multivariate analysis Rb and myc were selected as the combination with additional independent prognostic value, and E-cadherin had no additional independent value. For overall survival in univariate analysis, the amount of both eosinophilic and lymphocytic infiltrates and Rb and myc expression were of significant prognostic value. Only the amount of lymphocytic infiltrate had a prognostic significance for DFP. In stepwise multivariate analysis, TNM stage (I + II) and marked lymphocytic infiltrate were associated with better overall survival and longer DFP. We conclude that, if these results are confirmed in a larger series of patients, molecular markers can provide useful prognostic information.


Exposure to hypoxic-ischemic insults during the neonatal or perinatal developmental periods produces various forms of pathology. Injuries that occur in response to these events often manifest as severe cognitive and/or motor disturbances over time. Due to difficulties regarding the early diagnosis and treatment of hypoxic-ischemic injury, there is a growing need for effective therapies that can be delivered at delayed time points. Much of the research into mechanisms of neural injury has focused on molecular targets associated with excitotoxicity and free oxygen radicals. Despite repeated success in animal models, these compounds have failed to show efficacy in clinical trials. Increasing evidence indicates that hypoxic-ischemic injury in the neonate is progressive, and the resulting neuropathies are linked to the activation of neuroinflammatory processes that occur in response to the initial wave of cell death. Understanding this latter response, therefore, will be critical in the development of novel therapies to block the progression of the injury. In this review, we summarize emerging concepts from rodent models concerning the regulation of various cytokines, chemokines, and matrix metalloproteinases in response to ischemia, and the various ways in which the delayed neuroinflammatory response may contribute to the progressive nature of neonatal hypoxic-ischemic injury in rat. Finally, we discuss data that supports the potential to target these neuroinflammatory signals at clinically relevant time points. Due to improvements in medical care over past decades, increasing numbers of premature and low birth weight infants survive the neonatal period. Not surprisingly, there has been a rise in the incidence of diseases associated with prematurity, including neuropathies. Advances in diagnostic methods for these neuropathies and general knowledge of the cellular and molecular consequences have provided insight into potential causes of these disorders. Hypoxia-ischemia (H-I) is thought to be a major cause of perinatal brain injury, producing lesions of variable severity including focal necrotic cell death, diffuse white-matter injury, and cystic or cavitary infarction. It is estimated that nearly 40% of premature infants may suffer from either intraventricular-periventricular hemorrhage or periventricular lesions. In both cases, age of prematurity and birth weight are associated with increased risk, with younger, lighter infants at highest risk .The escalating incidence of these deficiencies and lack of effective therapies underscores the importance of research in this area. Rodent H-I models have provided strong and convincing evidence supporting the detrimental roles of oxidative stress and free radicals, blood-brain barrier (BBB) disruption, cytokine and chemokine signaling, and matrix metalloproteinase (MMP) activity after H-I. In fact, there is growing evidence demonstrating a discreet temporal injury profile whereby neural injury is exacerbated due to neuroinflammatory signaling from activated microglia and peripheral macrophages. This review highlights the roles of inflammatory cytokines, chemokines, and MMPs after ischemia, and the precise manner in which these proteins may mediate perinatal H-I injury in rat brain.In the developing brain, H-I results in a biphasic injury profile. Many of the initial events leading to progressive damage have been identified. The ischemic brain relies heavily on anaerobic glycolysis, which is energetically unfavorable . Energy Thus, the early response to H-I is characterized, in large part, by neuronal necrosis resulting from excitotoxic damage. While much of this injury is irreversible, the progressive nature of this pathology offers potential for intervention at delayed time points when neuroinflammatory mediators modulate the neural response to insult.The contributions of excitotoxicity and free radical production to the early ischemic response are clear, yet the selective targeting of these mechanisms has not led to significant functional improvements in clinical trials. Importantly, a second wave of cell death occurs in response to ischemic insult and is mediated by the neuroinflammatory response to injury. Accumulating evidence suggests that targeting delayed neuroinflammatory mechanisms may be a promising avenue for therapeutic intervention.The immune response in brain is complex and highly regulated by a host of different cell types and signaling pathways. Resident microglia are among the first to become activated . These cregulated upon activation, normal T cell expressed and secreted) both 18 hrs and 72 hrs after H-I compared to non-transgenic controls. These effects were also associated with reduced leukocyte infiltration [in vitro and in vivo methodologies also suggests that MCP-1 is a key mediator of the microglial chemotactic response to neonatal hypoxia. In these experiments, hypoxia resulted in a robust increase in activated microglia in the periventricular white matter, a region of selective vulnerability. Additionally, intracranial injection of MCP-1 induced a robust migratory response of CD11b-positive microglia, and hypoxic primary microglial cultures from rat neonates showed increased expression of MCP-1 [Cytokines seated on cell surfaces are activated and subsequently released into the extracellular milieu, where they serve as important mediators of apoptotic cell death. Following ischemic insult, activated microglia upregulate the expression of various cytokines and chemokines -12. In pltration , suggestof MCP-1 . Other dIn contrast to these findings, evidence supporting a neuroprotective role of microglial activation and cytokine signaling has raised issues as to the precise role of this signaling in various injury models. For example, experiments using primary cultures from rat showed that while cultured microglia upregulated TNF-α and IL-1β only in response to mild hypoxia-induced neuronal death, these same activated microglia were protective when added to separately cultured neurons. Furthermore, microglia co-cultured with media from severely compromised neurons released neurotrophic factors . SimilarAlthough the precise mechanisms have yet to be elucidated, data from numerous studies suggests that MMPs are instrumental in the production and maintenance of a proinflammatory microenvironment . CollectIn the developing brain, microglia mediate the neuroinflammatory response through a variety of mechanisms . In the In vivo experiments revealed MMP-9 expression that was localized to neutrophils and endothelial cells, and showed elevated MMP-2 expression in astrocytic endfeet [ endfeet . The lat endfeet . There i endfeet ,21,28.in vivo.Though the relative contributions of the resident and peripheral immune responses have not been elucidated, a recent study showed that resident microglia were associated with neural injury resulting from oxygen glucose deprivation in organotypic hippocampal slices from rat neonates. In these experiments, CD11b – positive microglia expressed MMP-9 after insult, and increased microglial MMP-9 expression occurred concomitantly with enhanced neurodegeneration and gelatinolytic activity . Thus, iAccumulating evidence suggests that gelatinases may contribute to the inflammatory response through "sheddase" activity, the process by which MMPs cleave proinflammatory cytokines attached to cell surfaces. This proteolytic processing activates and releases these molecules into the extracellular milieu to exert a wide range of effects depending upon the specific state of the microenvironment. MMPs have previously been shown to process both TNF-α  and IL-1In addition to their roles in activating cytokines and processing basement membrane proteins, MMPs are efficient at cleaving lecticans . LecticaAlthough MMP activation is often associated with proinflammatory processes, data from numerous studies demonstrates the ability of MMPs to alter cell growth, signaling and migration in a neuroprotective fashion . For exain vivo ischemic models linking MMP activity to growth factors, the aforementioned studies illustrate the distinct ways in which MMPs modulate numerous cellular processes and may, in fact, provide protection depending upon the temporal activation profile and the overall conditions of the microenvironment.Other evidence suggests that gelatinase MMPs can act on growth factor receptors to alter receptor turnover. In experiments that used a cervical carcinoma cell/mixed lymphocyte co-culture system to study the effects of immunosuppression, MMP-9 was shown to cleave interleukin-2 receptor alpha on T-lymphocytes . AdditioTo date, the majority of research into neonatal H-I has emphasized the contributions of glutamate excitotoxicity and free radical production to the resulting neuropathology . ConsistData from previous experiments lends support to the notion that selective targeting of MMP-2 and MMP-9 should be considered in developing novel therapeutics. MMP-9 knockout mice showed improved outcomes after cerebral ischemia using either the Vannucci model that mimics neonatal H-I ,59 or thin vitro and in vivo [Minocycline is another compound that has shown great promise as a therapeutic to combat neonatal H-I. Neuroprotective effects have been shown previously ,63, yet  in vivo . Indeed, in vivo , furtherDespite these encouraging data, it will be critical to proceed with caution when developing novel therapeutics. A hallmark of the neonatal brain is the unique potential for plasticity. Although advantageous from a treatment perspective, it is also likely that administration of exogenous compounds could alter critical developmental processes and/or mechanisms associated with neuroplasticity. It is now known that neurogenesis is prominent after ischemic insult in the neonatal rat brain. The Levison laboratory found that neural progenitors from the subventricular zone (SVZ), and possibly glial progenitors, migrate to and populate columnar regions of the neocortex that show prominent cell death in response to H-I . AnotherNeonatal hypoxic-ischemic injury is progressive and leads to debilitating neuropathies later in development. Difficulties in diagnosis and treatment underscore the need for novel therapeutics that can be administered at delayed time points. The initial injury results from energy failure and cytotoxicity, yet the delayed neuroinflammatory response is a more viable option for treatment in the clinical setting. As accumulating evidence from rodent models suggests that gelatin-degrading MMPs mediate the neuroinflammatory response to H-I in the developing brain, detailed investigations into the concerted actions of proinflammatory cytokines, chemokines, and MMPs Figure  may provThe authors declare that they have no competing interests.CL drafted and edited the manuscript. KP assisted in the editing of the manuscript. All authors read and approved the final manuscript.


Organizational leaders in business and medicine have been experiencing a similar dilemma: how to ensure that their organizational members are adopting work innovations in a timely fashion. Organizational leaders in healthcare have attempted to resolve this dilemma by offering specific solutions, such as evidence-based medicine (EBM), but organizations are still not systematically adopting evidence-based practice innovations as rapidly as expected by policy-makers (the knowing-doing gap problem). Some business leaders have adopted a systems-based perspective, called the learning organization (LO), to address a similar dilemma. Three years ago, the Society of General Internal Medicine's Evidence-based Medicine Task Force began an inquiry to integrate the EBM and LO concepts into one model to address the knowing-doing gap problem.During the model development process, the authors searched several databases for relevant LO frameworks and their related concepts by using a broad search strategy. To identify the key LO frameworks and consolidate them into one model, the authors used consensus-based decision-making and a narrative thematic synthesis guided by several qualitative criteria. The authors subjected the model to external, independent review and improved upon its design with this feedback.The authors found seven LO frameworks particularly relevant to evidence-based practice innovations in organizations. The authors describe their interpretations of these frameworks for healthcare organizations, the process they used to integrate the LO frameworks with EBM principles, and the resulting Evidence in the Learning Organization (ELO) model. They also provide a health organization scenario to illustrate ELO concepts in application.The authors intend, by sharing the LO frameworks and the ELO model, to help organizations identify their capacities to learn and share knowledge about evidence-based practice innovations. The ELO model will need further validation and improvement through its use in organizational settings and applied health services research. You are the new Chief of Quality in a university health center and are studying the inadequate utilization of an evidence-based guideline designed to reduce the number of missed diagnoses and improve the early treatment of sepsis. Your predecessor implemented the guideline with standard implementation strategies, including training providers using the guideline's results, using timely chart audits with individual/unit reports, periodic detailing by an infection expert, identifying and engaging local opinion leaders and integrating reminders into the electronic patient record. These strategies improved the sepsis outcomes, but there is an obvious need to improve early diagnoses and antibiotic utilization. From the audit reports, you note that most units are underperforming on the practice standards. You are mystified how further to improve this situation.Research evidence about decision errors of omission, from diagnostic errors to the underutilization of medications, suggests that these errors may be due, in part, to unnecessary delays in knowledge translation and result in needless human suffering ,2. Some To address a similar dilemma, successful business organizations respond to market changes by efficiently implementing practice innovations that incorporate market-generated information and make organizational changes to enhance utilization of this knowledge; such organizations are called "Learning Organizations" (LO) . In the Because of the similarity of the knowing-doing gap problems in medicine and business, some of the LO solutions learned in business organizations might be applied to the healthcare organizational setting. Indeed, some researchers admit that there are gaps in their knowledge about how healthcare decision-makers use knowledge contextually and adapt their decisions to new information -11, and Over three years, a multidisciplinary team of scholars completed a review of LO frameworks, interpreted them for healthcare organizational contexts, and consolidated them into a cohesive model. We describe the LO frameworks and the consolidated model, called Evidence in the Learning Organization (ELO), to provide healthcare leaders, managers, and their employees a practical framework for understanding how their organizations learn and build contextually-based knowledge gained through evidence-based decision making. This model should help organizational leaders and implementation experts identify their organizational flaws with learning and sharing this knowledge and, therefore, identify targets for interventions.The ELO team consisted of six members of the Society of General Internal Medicine's EBM Task Force who have experience in EBM, medical education, healthcare management, public health, clinical research and quality improvement.Our first task was to identify the scope of the LO and related literature within and outside of medical databases; thus, our aim was to get a large sample of the concepts by using a broad search strategy. We searched several databases using each database's unique thesaurus and field codes. We identified the following database/strategy combinations most useful:- In PubMed: "organizational learning [EXPLODE]," "organizational culture [EXPLODE]," "organizational innovation [EXPLODE]," "organizational culture [MeSH] AND evidence-based medicine [MeSH]," "evidence-based medicine [MeSH] AND organizational innovation [MeSH]," "informatics [MESH] AND organizational Innovation [MeSH]", and "models, organization [MeSH] AND evidence-based medicine [MeSH]."- In Business Source Primer: "organizational learning [EXPLODE]," "organizational learning [TX] AND Knowledge Management [TX]," "organizational learning [TX] AND organizational change [TX]," "organizational learning [TX] AND culture [TX]," and "organizational learning [TX] AND organizational behavior [TX] AND knowledge management [TX]."- In Sociological Abstracts: "organizational learning [KEY WORD]," "organizational culture AND organizational learning [SUT]," and "organizational culture [SUT] AND organizational development [SUT]."- In SocINDEX: "organizational learning [EXPLODE]," "organizational learning [SU] AND corporate culture [SU]," and "organizational change [SU] AND corporate culture [SU]."We used several of the search strategies described above with the ERIC database but were unable to identify any useful references.The Learning Organization, back to its inception (1994). We also identified key texts from both academia and consultative business practitioners by cross-referencing these texts from resources identified in the search. We engaged several experts in various fields  to identify key works in organization leadership and management. We continued to review the literature until we consistently failed to identify new theories, frameworks, models, or concepts related to the LO principles (information saturation).In addition to the database searches, we searched the indices for one journal, Because we were searching for the best theoretical LO frameworks and their constituent concepts to support the EBM and LO integration, we used consensus-based decision-making to select the relevant frameworks. We identified several qualitative criteria to assist our decisions: how seminal a framework was in establishing a new line of inquiry; how frequently a framework was cross-referenced in the literature; how representative a framework was for healthcare organizational applications; how well empiric research supported a framework; how well a framework fit with other frameworks, and how well a framework appealed to team members' intuition and experience. We reviewed all frameworks and their concepts independently and later, during a retreat, built consensus around our final choices. During the retreat, each ELO team member described his/her reasoning for choosing specific frameworks and concepts using the criteria. The other team members would subject these arguments to critical discourse and offer opportunities for rebuttal. The discourse continued until all team members could agree on which frameworks and constituent concepts to include in the review.To consolidate the selected frameworks into one model, we used a narrative thematic synthesis, rather than a quantitative systematic review. For this consolidation phase, we identified a series of candidate ELO models and subjected them to the same consensus process described above. After reaching consensus on the best candidate model, we subjected it to a second review process by engaging leaders in medical management, quality control, medical sociology and health systems scholarship to review the model for clarity, representativeness, and inclusiveness. We updated the model with the feedback from these experts.From our literature review, we developed a glossary to guide our discourse Appendix 1) and identified seven key LO frameworks that best informed healthcare contexts  learning if work processes are adequate to implement current strategies , 2) learning if the current organizational assumptions about strategy effectiveness are valid , and 3) studying the effectiveness of organizational learning structures and processes  [The first three frameworks in Table earning) ,43."4i" framework  extends the description of loop learning processes across three organizational levels: members sense and capitalize upon organizational learning opportunities (intuiting to interpreting); teams, using the results of these learning processes, discuss and create a collective understanding for needed changes (interpreting to integrating); and organizations capture and share this new knowledge to benefit all its members  [Although Argyris and Schön's seminal loop learning framework provides a general understanding of organizational learning, it needs clarification on how it flows across different organizational levels. Crossan's alizing) . Once thalizing) . Thus, iWe can illustrate the loop-learning and 4i frameworks by describing a hypothetical initiative designed to improve Prostate Specific Antigen (PSA) testing decisions for prostate cancer screening. An organization can discover, through routine audit mechanisms, that providers' documentation show unacceptably low frequencies of orders for PSA tests. After implementing changes to improve work processes, managers determine, through further audit and reporting cycles, if the changes result in improved compliance with the practice standard. When organizations delegate to teams the responsibility for deliberating (interpreting) upon the team members' learning opportunities (intuiting) and forming recommendations about the needed process changes (integrating), the organization is adept at practicing single-loop learning at the team level. Single-loop learning provides organizations opportunities to learn more about the practical limitations of specific policies and how working teams can help provide feedback about these limitations.Despite adapting adequate processes of care, teams may find through the same intuiting/interpreting/integrating processes that their patients are declining PSA tests at a high frequency because their patients desire more knowledge, discussion time, and decision options for all the complicated prostate screening issues, including their costs and consequences. In this example of double-loop learning, the organization should rethink its priorities and adapt its strategies accordingly, such as offering providers more time to discuss PSA decisions with patients, providing patients more education on screening decisions or resetting their benchmark targets to reflect more realistically on their patients' preferences. Finally, organizations may discover that they have inadequate structures or processes to facilitate sharing this new knowledge; for example, organizations may discover that no one is tasked to filter and reformulate knowledge gained from single- and double-loop learning at the management or committee level (deutero-loop learning). To be successful at single-, double- and deutero-loop learning, organizations must have adequate resources and processes to capture loop learning and flexibility to adapt policies when the assumptions underlying these policies are proven incorrect.Decision-Execution Cycles framework describes more specifically how organizational members use the intuiting process as an opportunity for loop learning by testing recommendations from new  or old practice policies, called knowledge claims, with patient care decisions. In this process, members compare the expected outcomes of their decisions to the actual ones  and form epistemic gaps, or "a-ha" moments, as learning moments about specific knowledge claims [Firestone and McElroy's e claims . The knoe claims .In their analysis of the Partners HealthCare case study, Firestone and McElroy described how physicians in Partners HealthCare, when provided with electronic prescribing alerts integrated with the electronic patient record, improved compliance with prescribing standards by challenging the physicians to rethink the assumptions of their decisions . Becauseorganizational knowledge creation framework helps clarify the team learning (interpreting/integrating) process by describing how "know-how" knowledge, called tacit knowledge, is continually built, used, and reformulated [Sometimes external knowledge claims need to be translated into working knowledge through discourse prior to their use. Nonaka and Takeuchi's rmulated . Within rmulated . Team mermulated . Teams ormulated . Professrmulated .An example of the organizational knowledge creation framework comes from Gabay and Lemay's ethnographic "mindlines" study. These investigators studied how primary care doctors and nurses used external evidence-based guidelines to create "mindlines," which were internally-based, tacit guidelines that the providers socially-constructed through an iterative process . For exaorganizational culture, and these cultural influences can impact their acceptance or resistance to organizational learning [The last four frameworks in Table learning -52. For learning ,19,53-55learning ,37. If tBeyond broad organizational cultural beliefs that may inhibit decentralized learning, specific subculture conflicts may exist in organizations and cause variable adoption of evidence-based practices. For example, the subcultural values of autonomy held by physicians or pragmaticism held by nurses may be sources of resistance to change ,56. OrgaComplex Adaptive Systems (CAS) framework views organizations as a group of semi-autonomous decision-makers who try to reconcile best practice recommendations (minimum specifications) and their policies (attractors) with the complex, local influences on healthcare delivery , including the contextual barriers to decision-making and the beliefs of co-workers, management and patients [The patients -59. As tpatients . The impIn their description of an a new initiative at a United Kingdom Primary Care Trust, Rowe and Hogarth illustrated their attempt to influence more responsive public health decision-making using CAS as a guiding framework . The TruDiffusion and dissemination of innovation are two closely related perspectives that help us understand how evidence-based practice innovations are adopted passively and variably by members and teams (diffusion) or can be accelerated thorough intentional organizational strategies (dissemination) [ination) ,38,62,63ination) .A systematic review on diffusion and dissemination research from several research fields  has identified additional characteristics about readiness for innovation adoption . An indiThese multiple lines of inquiry have been fairly consistent in identifying the types of cognitive, attitudinal, cultural, relational and resource characteristics that promote readiness for innovation adoption within organizations, and many of these characteristics are reflected in earlier frameworks. One barrier to implementing this research is the lack of knowledge about how these characteristics fit together to predict adaptability by decision-makers and teams.Total Quality Management (TQM) framework views evidence-based decisions as mostly linear logic models intended to improve health outcomes by re-designing care processes and measuring success through achievement of practice standards and benchmark targets [The  targets . The TQM targets ; thus, T targets ,33,64,65 targets ,33,47.We consolidated these frameworks into the ELO model to assist organizational leaders and scholars with the task of diagnosing organizational learning and knowledge sharing flaws. The ELO model is constituted of four themes and their subthemes that represent the processes required for organizations to learn and share new knowledge more effectively. These processes are not necessarily sequential but can occur simultaneously and interactively:1) Inquiring: Are members are ready to inquire on behalf of teams/organizations to facilitate the loop learning processes?a. Acquiring: Do they possess technical skills related to locating resources and communicating feedback about this inquiry ?b. Informing: Do they possess the cognitive skills  that support evidence-based decisions?c. Transforming: Do they possess cognitive traits that facilitate behaviors for inquiry ?2) Deciding: Are members and teams utilizing effective decision processes to integrate evidence into healthcare decisions?a. Deliberating: Are they comparing and analyzing new working goals/strategies and structures/processes that will lead to better decisions ?b. Decision-taking: Are they using appropriate decision methods/tools to support better decision-making making ?c. Evaluating: Are they using adequate analytical methods  to measure outcomes of evidence-based decisions ?Relating: Are members, teams, and organizations facilitating evidence-based practices through effective organizational communication and relationships?3) a. Sharing: Do the organizational communication structures and processes facilitate sharing knowledge ?b. Cooperating: Are teams available and functioning to facilitate efficient knowledge generation and evaluation ?c. Advocating: Is there adequate and sufficient leadership with effective motivational strategies to induce organizational cultural change towards learning ?4) Interpreting: Are members and teams sensing the need for evidence-based practice innovations and explicitly describing their tacit knowledge?a. Judging: Are they properly evaluating judgments about the outcomes of decisions and needed practice changes ?b. Knowing: Are they building new models of shared understanding based upon the results of evidence-based decision-making ?c. Formulating: Are they codifying this new knowledge  for organizational consumption?To illustrate the application of this model, we return to our opening scenario to diagnose what went wrong Figure :With the ELO model in hand, you, the Quality Chief, do further investigation to discover why the guideline initiative failed. Over three days, you talk with providers from patient care units, observe their practice patterns, and review their charts. Additionally, you interview several individuals from leadership, management and hospital committees. After completing your investigation, you find several flaws that may be amenable to specific interventions:Inquiring difficulty: Some physicians have yet to learn the reminder system and seem unmotivated to learn it or information shared through the other implementation strategies; thus they lose opportunities to adapt their practices, provide feedback about practical limitations of the initiative  or to recognize when certain reminders are clinically inappropriate (flaws of acquiring and transforming). Additionally, nurses from several nursing units possess a "punch-the-clock" norm, through which team members are discouraged by influential senior staff members to challenge team leadership with ideas about improving patient care (flaw of transforming).1) Because of lost opportunities to improve decision-making processes (single-loop) and to address the quality of the clinical reminders and their limitations (double-loop), teams do not have the capacity to take advantage of these learning opportunities. Also, the committee that oversees the reminder system fails to integrate into it the practice innovations identified by teams that enhance early diagnosis and to adjust the benchmarks with feedback about inappropriate reminders (flaw of deutero-loop learning). The latter flaw has led, in some cases, to an overestimation of the number of missed diagnoses.Deciding difficulties: Because of time constraints, providers have little time to review the results of audit reports (flaws of deliberating). Also, the hospital's nursing subculture admires some physicians more than others, and many nurses defer decisions to these physicians (flaws of decision-taking). Unfortunately, these physicians are largely responsible for delayed empiric antibiotic courses and have been identified as local opinion leaders by a recent survey.2) Because the diagnosis and treatment decisions are mostly made by under-informed individuals (physicians), teams are not fully vetting decision evaluations and needed changes . Organizational leaders and management committees, unaware of these flaws, make uninformed strategic resource decisions .Relating Difficulties: Because of the physicians' inattentiveness to reminders, nurses do not provide feedback to the system about the practical limitations of certain recommendations and when reminders are inappropriate (flaw of sharing). Also, instead of using performance-based incentives, middle managers have used other more controversial tactics to motivate non-compliant physicians, including threats to modify hospital privileges (flaws of advocating). These tactics conflict directly with the autonomy beliefs held by physicians, and this conflict has led more resistance to change.3) Because the patient care teams work under these contextual constraints, there are no opportunities for meaningful team discussions about the potential advantages of the guideline, how to modify local practices to achieve its general goals, and address its contextual limitations (flaws of cooperating). Additionally, the hierarchal organizational culture inhibits senior managers delegating process decisions to teams and creating incentives at the team level . Despite its potential as a flexible knowledge repository, the current IT system is currently managed only for information exchange and, therefore, the organization cannot share new knowledge .Interpreting Difficulties: Because of impairment of individual learning and decision-making, providers are unable to generate accurate judgments about what is happening with their practices and what, if any, changes should occur. Teams cannot form and validate explicit practice recommendations based on these experiences and thus they fail to "buy-into" the initiative. Because of its resource structure and leadership, the organization is failing at knowledge management. The consequences of all these flaws, when magnified at three organizational levels, result in the failure to build and share new organizational knowledge.4) During your investigation, you do identify a unit that seems to more successful with this initiative than others. This team consists of technically  sound providers and local opinion leaders who encourage the norms of inquiry, experimentation, open dialogue, and critical debate. The collective beliefs that guide these norms are shared by most team providers (including physicians) and seem to have helped the team members in adapting to audit reports, detailing, and reminders when these measures suggest improvements are needed. This team is also somewhat successful at identifying inappropriate reminders and adapting their processes to address the initiative's local limitations , but they have not experienced a push-back from organizational leadership for variations with standard practices on these issues. The managers who oversee this unit have been adept at shielding the team from this push-back, using incentives to induce practice changes , and using a collaborative leadership style by delegating decisions about process changes to their teams (at their own peril). These managers also help share knowledge gained through team deliberations with other shifts, use creative management techniques to improve time efficiencies to support team discourse, and use their available budgets to improve the technologies (IT system) that support learning and sharing of knowledge across the unit.We need to make three points about our description of this scenario as a representative ELO model application. First, we described the scenario narrative in a linear manner, but the processes that compose the four themes interact and influence each other in a complex and iterative way . In this case, the answers for all eight questions were "yes"; therefore the Quality Chief could quickly bypass this unit to focus her efforts on more flawed units. If any of the answers to the questions are "no," then the unit or organization may have flaws in its learning competencies that could be, in part, contributing to the failures. In this case, leaders would need a broader framework, such as the ELO model, to perform a more thorough analysis.In summary, we described the LO frameworks that we believe relevant for understanding how healthcare organizations learn, create, and share knowledge about evidence-based practices and the system issues that facilitate or inhibit these learning processes. We also describe the ELO model as a cohesive framework to help organizations and implementation experts diagnose flaws in their organizational learning and knowledge management.From the implementation perspective, the ELO model's weakness may be that it only helps identify targets (monitoring the system) for organizational change, not in its ability to identify specific implementation strategies or tactics to reach this end (managing the system). Fortunately, the research about useful organizational interventions that help facilitate evidence uptake is growing, and we refer readers to a sample of this literature ,37,66-69The strengths of our model is its inclusiveness of LO topics, thoughtful consolidation, theoretical grounding, and flexibility to be applied in naturalistic organizational contexts. In the future, we aim to use this model as a framework for research on ideal ELO organizational structures and processes. For example, we intend to use this ELO model to organize and guide the inquiry into the characteristics that are expressed by excellent healthcare organizations and compare them to less successful organizations. This inquiry should both uncover these organizational features and guide refinements and improvements in the ELO model itself. The model also needs validated through its application in naturalistic organizational settings and feedback about its practical application should lead to further refinements.Some expenses related to this study were supported from an internal, unrestricted educational fund from the Society of General Internal Medicine.GEC, MCM, EAA, and WSR contributed to the literature review, identification of frameworks, model synthesis and manuscript authorship. CAU and JN contributed to the model synthesis and manuscript authorship.The 4i Model  [1. alizing) : The 4i Communities of Practice [2. Practice : SponsorComplex Adaptive Systems (CAS) [3. ms (CAS) : CAS theDecision-Execution Cycles (DEC) [4. es (DEC) : A decisInnovation Diffusion Theory and Dissemination of Innovation [5. novation ,62: DiffKnowing-doing Gap [6. oing Gap : A systeKnowledge Management [7. nagement : MethodsLearning Organization [8. Loop Learning  [9. eutero-) : OrganizOrganizational Culture [10.  Culture : The shaOrganizational Knowledge [11. nowledge : The embOrganizational Knowledge Creation (OKC) [12. on (OKC) : MembersOrganizational Learning [13. Learning : KnowledTotal Quality Management TQM) [14. ent TQM) : Describ• Are providers asking questions about current practices and finding, appraising and using external knowledge to inform their practices?YES: The exemplary unit is composed of providers (nurses and doctors) who have taken their own time to bookmark evidence-based resources and updates about team-generated best practices on their workstations. They have a workable EBM knowledge and use their resources during point-of-service decisions.◦ • Are providers effectively deliberating, taking, and evaluating decisions among a viable set of decision options, decision tactics, and potential outcomes?YES: The providers from this unit rely both on the audit reports and their own experiences to provide feedback about the outcomes of their decisions and demonstrate flexibility to consider other decisions when they are required.◦ • Are providers participating in collaborative relationships and encouraging open dialogue?YES: The providers are remarkably egalitarian; they all demonstrate openness, experimentation, tolerance, and teamwork.◦ • Are providers forming sensible conclusions about the reasons for the outcomes of their decisions?YES: The providers often make useful suggestions about changes to the unit's work space, work flow, and best practices that will improve care delivery and outcomes.◦ • Are practice teams forming and functioning collaboratively?YES: Teams from different shifts are composed of all care providers and deliberate regularly using open discourse with input from all team members.◦ • Are practice teams deliberating the reasons for patient care outcomes?YES: During deliberations, the teams discuss and debate the conclusions for their failure to achieve their expected outcomes and ways to improve work space, work flow and application of knowledge to achieve the desired outcomes.◦ • Are teams explicitly sharing their recommendations about needed practice and policy changes with its members, other teams, and organizational leadership?YES: The team's author and share  these conclusions with teams from other shifts and their local managers.◦ • Are organizational leaders providing necessary resources  and effective motivational strategies to encourage organizational learning, knowledge creation, and knowledge sharing?YES: Although the broader organization is failing to support learning and knowledge management, the managers from this unit use creative time management techniques to create more time for team deliberations, use deft capital project decisions to improve their information systems, demonstrate a collaborative management style, and use thoughtful incentives to induce adaptive practices.◦


Sociodemographic characteristics, such as sex, have been shown to influence health care delivery. Acute care surgery models are effective in decreasing mortality and morbidity after emergency surgeries, but sex-based differences in delivery and outcomes have not been explored. Our objective was to explore sex associated differences in the patient characteristics and clinical outcomes of those admitted to emergency general surgery.A post-hoc analysis of 512 emergency general surgical patients admitted consecutively to two tertiary care hospitals in Alberta Canada, between April 1, 2014 and July 31, 2015. We measured associations between sex and patient demographics, pre-, intra- and post-operative delivery of care, as well as post-operative outcomes.Of those excluded from the analysis, older females were more likely to undergo conservative management compared to older men . Overall, there were no differences between sexes for time from admission to surgery, time spent in surgery, overall complication rate, mortality, hospital length of stay, or discharge disposition. Women were more likely to have a cancer diagnosis , while men were more likely to receive hernia surgery . Finally, men were more likely to have a major respiratory complication .Only two differences in peri and post-operative complications between sexes were noted, which suggests sex-based disparity in quality of care is limited once a decision has been made to operate. Future studies with larger databases are needed to corroborate our findings and investigate potential sex biases in surgical versus conservative management. Differences in health care delivery and outcome between men and women have been documented in many areas of research including sepsis , 2, myocIn the general surgery setting, studies have shown conflicting findings of sex disparities. For instance, females are less often diagnosed then men with colon cancer through screening –17; howeAcute care surgery is a relatively newer surgical specialty that encompasses trauma, critical care and emergency general surgery (EGS) such as appendectomies, cholecystectomies, gastrointestinal obstruction release or resection, perforation repair, and emergency cancer surgery. The goal of EGS is to provide surgical care within the first 24 hours of hospitalization. Few studies have examined differences in sex in the EGS setting. Two studies that both used data from the Nationwide Inpatient Sample have noted that females undergo EGS more frequently ; howeverExploring sex disparity is crucial to examining the models of surgical disease in men and women and expanding our understanding of health determinants for each sub-population. We sought to identify if there were sex differences within an EGS system for pre-, intra- and post-operative delivery of care, as well as post-operative outcomes.All screened patients for the Elder-friendly Approaches to the Surgical Environment (EASE) study  were revAll screened patients had age, sex and reason for inclusion and/or exclusion from the EASE study recorded. Trained research assistants collected pre-, intra- and post-operative details for those eligible for post-hoc analysis. Details included: patient demographics ; pre-, intra- and post-operative delivery of care ; and post-operative outcomes .Given the exploratory nature of this analysis, missing data was not imputed, nor outliers excluded. Descriptive statistics were calculated after patients were stratified by sex and compared using student’s t test, chi-squared test, Wilcoxon Rank Sum test, or a non-parametric equivalent as dictated by the data. Univariate difference between groups that had a p < 0.1 was explored using regression analysis, to identify covariate-adjusted independent associations of each variable with sex. We limited regression covariates to the a priori specified explanatory variables of sex, and sequentially included potential confounders including age, Charlson Comorbidity Index score and/or surgery type (six categories) when the outcome event rate was sufficient to support it (one independent variable per 10 patients who experienced an event of interest). Model fit was judged using the Hosmer–Lemeshow goodness-of-fit test and Area Under the Receiver Operating Characteristics curve for logistic regressions and Rn = 857), there were no statistically significant differences in EASE study exclusion rates between sex for transfers, nursing home residency, elective, trauma, palliative, and non-abdominal surgeries (p > 0.05). Older women were more likely than men to undergo conservative management as opposed to surgery .There were 3506 patients screened for the EASE study between April 1, 2014 and July 31, 2015. In older patients, where data was available  and 267 men (52%), with a sex-ratio consistent with our overall excluded cohort. There were no differences in patient demographics or admission location type (p > 0.05). Women had a significantly more frequent diagnosis of cancer when compared to men  . Unlike When gender bias was investigated at the primary care referral process, physicians themselves may be favoring the selection of men, as observed in elective knee and hip arthroplasties , 26, wheSeveral social reasons have been identified as to why women are having less aggressive surgical intervention compared to men. Women themselves may have a decreased willingness to undergo surgery , due to In non-emergent surgeries, a US nationwide retrospective data analysis in gastrointestinal surgery has shown that women tend to have lower mortality then men, and that this particularly pronounced in those 50 years or less . FemalesLimitations of our study include the over-representation of elderly patients given the nature of the EASE study, bias introduced by doing a post-hoc assessment, inability to adjust for confounding for the majority of our univariate comparisons because of low outcome event rates, as well relatively short follow-up time which was limited to hospital admission. As all non-operative patients were excluded from further data collection, we are unable to further explore important social selection biases in the selection of surgery patients. In the future, using larger administrative data bases would be ideal to further investigate these differences, although this may make specific outcomes unavailable. Our data may not be generalizable to other countries that do not have universal health care. However, even adjusting for insurance, age and diseases severity, US data from California suggests that women tend to receive less procedures , highligStrengths of our study include the relatively large sample size given the prospective study design, the wide age range including elderly, as well as the extensive data on comorbidities, intra and postoperative surgical details that was collected during study enrollment.In summary, in this prospective study of EGS patients we found that women were more likely to be treated conservatively with non-operative approaches when presenting to an acute surgical service. However, once an operative approach was decided there were no sex differences with regards to perioperative care, overall mortality, hospital length of stay or discharge disposition. This study supports that the EGS model does not have sex biases once an operative approach is chosen. However, further work is necessary to explore potential sex biases in patients who are considered for operative verses non-operative management in EGS. These biases may disadvantage women in receiving inadequate care.


Vascular calcification is associated with physiological aging; genetic diseases, such as Hutchinson-Gilford progeria syndrome and generalized arterial calcification of infancy; and various pathological conditions, such as chronic kidney disease and diabetes . VasculaPyrophosphate is a potent inhibitor of calcium-phosphate crystal formation and growth . ExtraceVascular tissue mineralization occurs when the synthesis of vascular calcification inhibitors is impaired or when the formation of calcium-phosphate crystals is enhanced, for example, by hyperphosphatemia, the main risk factor for vascular calcification . Hyperphex vivo model of phosphate-induced calcification showed that devitalized aortas calcify faster than aortas containing living cells. These results confirmed the findings of a previous in vitro study with live and dead vascular smooth muscle cells, which showed that calcification is a passive process that can be actively prevented by pyrophosphate [Despite findings showing that hyperphosphatemia triggers vascular calcification, the effects of hyperphosphatemia on extracellular pyrophosphate metabolism remain unclear. A recent study  investighosphate ,7.Finally, we showed that there are two responses to high phosphate concentration : early (in vitro and rat aortas ex vivo. Moreover, the hydrolysis of pyrophosphate was found to decrease during early stages, but increase during later stages, of hyperphosphatemia. Although overall pyrophosphate production is higher during hyperphosphatemia, it was not sufficient to block calcium-phosphate deposition.These results may explain the contradictory results showing that TNAP expression is increased or decreased during vascular smooth muscle cell calcification ,8. NeverA growing body of evidence suggests that pyrophosphate is the predominant endogenous inhibitor of vascular calcification. The results of this study, along with previous findings, suggest that induction of pyrophosphate synthesis may be an easy and effective therapeutic strategy to inhibit vascular calcification associated with aging and other pathological conditions.