The facilitators and barriers to improving functional activity and wellbeing in people with dementia: a qualitative study from the process evaluation of Promoting Activity, Independence and Stability in Early Dementia (PrAISED).

BACKGROUND: The PRomoting Activity, Independence and Stability in Early Dementia (PrAISED) study delivered an exercise and functional activity programme to participants living with dementia. A Randomised Controlled Trial showed no measurable benefits in activities of daily living, physical activity or quality of life. OBJECTIVE: To explore participants' responses to PrAISED and explain why an intervention that might be expected to have produced measurable health gains did not do so. METHODS: A process evaluation using qualitative methods, comprising interviews and researcher notes. SETTING: Data were collected in participants' homes or remotely by telephone or videoconferencing. SAMPLE: A total of 88 interviews were conducted with 44 participants living with dementia (n = 32 intervention group; n = 12 control group) and 39 caregivers. A total of 69 interviews were conducted with 26 therapists. RESULTS: Participants valued the intervention as proactively addressing health issues that were of concern to them, and as a source of social contact, interaction, information and advice. Facilitators to achieving positive outcomes included perceiving progress towards desired goals, positive expectations, therapists' skills and rapport with participants, and caregiver support. Barriers included: cognitive impairment, which prevented independent engagement and carry-over between sessions; chronic physical health problems and intercurrent acute illness and injury; 'tapering' (progressively infrequent supervision intended to help develop habits and independent activity); and the COVID-19 pandemic. CONCLUSIONS: Self-directed interventions may not be appropriate in the context of dementia, even in the mild stages of the condition. Dementia-specific factors affected outcomes including caregiver support, rapport with therapists, availability of supervision, motivational factors and the limitations of remote delivery. The effects of cognitive impairment, multimorbidity and frailty overwhelmed any positive impact of the intervention. Maintenance of functional ability is valued, but in the face of inevitable progression of disease, other less tangible outcomes become important, challenging how we frame 'health gain' and trial outcomes.

Promoting Activity, Independence, and Stability in Early Dementia and mild cognitive impairment (PrAISED): randomised controlled trial.

OBJECTIVE: To determine the effectiveness of an exercise and functional activity therapy intervention in adults with early dementia or mild cognitive impairment compared with usual care. DESIGN: Randomised controlled trial. SETTING: Participants' homes and communities at five sites in the United Kingdom. PARTICIPANTS: 365 adults with early dementia or mild cognitive impairment who were living at home, and family members or carers. INTERVENTION: The intervention, Promoting activity, Independence, and Stability in Early Dementia and mild cognitive impairment (PrAISED), was a specially designed, dementia specific, rehabilitation programme focusing on strength, balance, physical activity, and performance of activities of daily living, which was tailored and progressive and addressed risk and the psychological needs of people with dementia. Up to 50 therapy sessions were provided over 12 months. The control group received usual care plus a falls risk assessment. Procedures were adapted during the covid-19 pandemic. MAIN OUTCOME MEASURES: The primary outcome was score on the carer (informant) reported disability assessment for dementia scale 12 months after randomisation. Secondary outcomes were self-reported activities of daily living, physical activity, quality of life, balance, functional mobility, fear of falling, frailty, cognition, mood, carer strain, service use at 12 months, and falls between months 4 and 15. RESULTS: 365 patient participants were randomised, 183 to intervention and 182 to control. The median age of participants was 80 years (range 65-95), median Montreal cognitive assessment score was 20 out of 30 (range 13-26), and 58% (n=210) were men. Intervention participants received a median of 31 therapy sessions (interquartile range 22-40) and reported completing a mean 121 minutes of PrAISED exercise each week. Primary outcome data were available for 149 intervention and 141 control participants. Scores on the disability assessment for dementia scale did not differ between groups: adjusted mean difference -1.3, 95% confidence interval -5.2 to 2.6; Cohen's d effect size -0.06, 95% confidence interval -0.26 to 0.15; P=0.51). Upper 95% confidence intervals excluded small to moderate effects on any of the range of outcome measures. Between months 4 and 15 the intervention group experienced 79 falls and the control group 200 falls (adjusted incidence rate ratio 0.78, 95% confidence interval 0.5 to 1.3; P=0.3). CONCLUSION: The intensive PrAISED programme of exercise and functional activity training did not improve activities of daily living, physical activity, or quality of life; reduce falls; or improve any other secondary health status outcomes, despite good uptake. Future research should consider alternative approaches to maintaining ability and wellbeing in people with dementia. TRIAL REGISTRATION: ISRCTN Registry ISRCTN15320670.

Chronic pain in people living with dementia: challenges to recognising and managing pain, and personalising intervention by phenotype.

Pain is common in people with dementia, and pain can exacerbate the behavioural and psychological symptoms of dementia. Effective pain management is challenging, not least in people with dementia. Impairments of cognition, communication and abstract thought can make communicating pain unreliable or impossible. It is unclear which biopsychosocial interventions for pain management are effective in people with dementia, and which interventions for behavioural and psychological symptoms of dementia are effective in people with pain. The result is that drugs, physical therapies and psychological therapies might be either underused or overused. People with dementia and pain could be helped by assessment processes that characterise an individual's pain experience and dementia behaviours in a mechanistic manner, phenotyping. Chronic pain management has moved from a 'one size fits all' approach, towards personalised medicine, where interventions recommended for an individual depend upon the key mechanisms underlying their pain, and the relative values they place on benefits and adverse effects. Mechanistic phenotyping through careful personalised evaluation would define the mechanisms driving pain and dementia behaviours in an individual, enabling the formulation of a personalised intervention strategy. Central pain processing mechanisms are particularly likely to be important in people with pain and dementia, and interventions to accommodate and address these may be particularly helpful, not only to relieve pain but also the symptoms of dementia.

A Qualitative Study on the Experiences of Therapists Delivering the Promoting Activity, Independence and Stability in Early Dementia (PrAISED) Intervention During the COVID-19 Pandemic.

BACKGROUND: The Promoting Activity, Independence and Stability in Early Dementia (PrAISED) intervention is a programme of physical activity and exercise designed to maintain participation in activities of daily living, mobility, and quality of life for people living with dementia. During the COVID-19 pandemic first national lockdown in England, the PrAISED physiotherapists, occupational therapists, and rehabilitation support workers adapted to delivering the intervention remotely via telephone or video conferencing. OBJECTIVE: The aim of this study was to explore therapists' experience of delivering the PrAISED intervention during the COVID-19 pandemic and derive implications for clinical practice. METHODS: Qualitative semi-structured interviews were conducted with 16 therapists using purposive sampling. Thematic analysis was used to analyze the transcripts. RESULTS: Therapists reported a change in the relationship between themselves, the person with dementia and the caregiver, with an increased reliance on the caregiver and a loss of autonomy for the person living with dementia. There was concern that this would increase the burden on the caregiver. The therapists reported using creativity to adapt to different modes of delivery. They felt their sessions were mostly focused on providing social and emotional support, and that assessing, progressing, and tailoring the intervention was difficult. CONCLUSION: It is possible to deliver some elements of a physical intervention using remote delivery, but a dual modal approach including remote and face-to-face delivery would optimize treatment efficacy. Educational support would be required to enable people living with dementia and their caregivers to overcome barriers relating to digital literacy.

Association of pain and risk of falls in community-dwelling adults: a prospective study in the Survey of Health, Ageing and Retirement in Europe (SHARE).

PURPOSE: To investigate the longitudinal associations between pain and falls risks in adults. METHODS: Prospective cohort study on data from 40,636 community-dwelling adults ≥ 50 years assessed in Wave 5 and 6 in the Survey of Health, Ageing and Retirement in Europe (SHARE). Socio-demographic and clinical information was collected at baseline (Wave 5). At 2-year follow-up (Wave 6), falls in the previous 6 months were recorded. The longitudinal associations between pain intensity, number of pain sites and pain in specific anatomic sites, respectively, and falls risk were analysed by binary logistic regression models; odds ratios (95% confidence intervals) were calculated. All analyses were adjusted for socio-demographic and clinical factors and stratified by sex. RESULTS: Mean age was 65.8 years (standard deviation 9.3; range 50-103); 22,486 (55.3%) participants were women. At follow-up, 2805 (6.9%) participants reported fall(s) in the previous 6 months. After adjustment, participants with moderate and severe pain at baseline had an increased falls risk at follow-up of 1.35 (1.21-1.51) and 1.52 (1.31-1.75), respectively, compared to those without pain (both p < 0.001); mild pain was not associated with falls risk. Associations between pain intensity and falls risk were greater at younger age (p for interaction < 0.001). Among participants with pain, pain in ≥ 2 sites or all over (multisite pain) was associated with an increased falls risk of 1.29 (1.14-1.45) compared to pain in one site (p < 0.001). CONCLUSIONS: Moderate, severe and multisite pain were associated with an increased risk of subsequent falls in adults.

A systematic literature review and meta-analysis on digital health interventions for people living with dementia and Mild Cognitive Impairment.

OBJECTIVES: Digital health interventions enable services to support people living with dementia and Mild Cognitive Impairment (MCI) remotely. This literature review gathers evidence on the effectiveness of digital health interventions on physical, cognitive, behavioural and psychological outcomes, and Activities of Daily Living in people living with dementia and MCI. METHODS/DESIGN: Searches, using nine databases, were run in November 2021. Two authors carried out study selection/appraisal using the Critical Appraisal Skills Programme checklist. Study characteristics were extracted through the Cochrane handbook for systematic reviews of interventions data extraction form. Data on digital health interventions were extracted through the template for intervention description and replication (TIDieR) checklist and guide. Intervention effectiveness was determined through effect sizes. Meta-analyses were performed to pool data on intervention effectiveness. RESULTS: Twenty studies were included in the review, with a diverse range of interventions, modes of delivery, activities, duration, length, frequency, and intensity. Compared to controls, the interventions produced a moderate effect on cognitive abilities (SMD = 0.36; 95% CI = -0.03 to 0.76; I2  = 61%), and a negative moderate effect on basic ADLs (SMD = -0.40; 95% CI = -0.86 to 0.05; I2  = 69%). Stepping exergames generated the largest effect sizes on physical and cognitive abilities. Supervised training produced larger effect sizes than unsupervised interventions. CONCLUSION: Supervised intervention delivery is linked to greatest benefits. A mix of remote and face-to-face delivery could maximise benefits and optimise costs. Accessibility, acceptability and sustainability of digital interventions for end-users must be pre-requisites for the development of future successful services.

Tele-Rehabilitation for People with Dementia during the COVID-19 Pandemic: A Case-Study from England.

Introduction: The Promoting Activity, Independence and Stability in Early Dementia (PrAISED) is delivering an exercise programme for people with dementia. The Lincolnshire partnership National Health Service (NHS) foundation Trust successfully delivered PrAISED through a video-calling platform during the Coronavirus Disease 2019 (COVID-19) pandemic. METHODS: This qualitative case-study aimed to identify participants that video delivery worked for, to highlight its benefits and its challenges. Interviews were conducted between May and August 2020 with five participants with dementia and their caregivers (n = 10), as well as five therapists from the Lincolnshire partnership NHS foundation Trust. The interviews were analysed through thematic analysis. RESULTS: Video delivery worked best when participants had a supporting caregiver and when therapists showed enthusiasm and had an established rapport with the client. Benefits included time efficiency of sessions, enhancing participants' motivation, caregivers' dementia awareness, and therapists' creativity. Limitations included users' poor IT skills and resources. DISCUSSION: The COVID-19 pandemic required innovative ways of delivering rehabilitation. This study supports that people with dementia can use tele-rehabilitation, but success is reliant on having a caregiver and an enthusiastic and known therapist.

Protocol for the process evaluation of the Promoting Activity, Independence and Stability in Early Dementia (PrAISED), following changes required by the COVID-19 pandemic.

INTRODUCTION: The Promoting Activity, Independence and Stability in Early Dementia (PrAISED) randomised controlled trial (RCT) is evaluating a home-based, face-to-face, individually tailored, activity and exercise programme for people living with dementia. Social distancing requirements following the COVID-19 pandemic necessitated rapid changes to intervention delivery. METHODS AND ANALYSIS: A mixed-methods process evaluation will investigate how the changes were implemented and the impact that these have on participants' experience. An implementation study will investigate how the intervention was delivered during the pandemic. A study on the mechanisms of impact and context will investigate how these changes were experienced by the PrAISED participants, their carers and the therapists delivering the intervention. The study will commence in May 2020. ETHICS AND DISSEMINATION: The PrAISED RCT and process evaluation have received ethical approval number 18/YH/0059. The PrAISED process evaluation will enable us to understand how distancing and isolation affected participants, their activity and exercise routines and whether the therapy programme could be continued with remote support. This will be valuable both in explaining trial results and also contribute to understanding and designing new ways of delivering home-based services and rehabilitation interventions for people with dementia and their carers. TRIAL REGISTRATION NUMBER: ISRCTN15320670; Pre-results.

The Impact of a Dementia-Friendly Exercise Class on People Living with Dementia: A Mixed-Methods Study.

Exercise has multiple benefits for people living with dementia. A programme of group exercise classes for people with dementia and their family carers has been established in a University sports centre. This study aims to explore the impact of this programme on participants with dementia and their carers. A mixed-methods design including a prospective, repeated measures cohort study followed by focus groups was employed. Physiological and cognitive outcome measures were repeated at baseline and three months in a cohort of people with dementia attending a group exercise class. Focus groups on the participants' experiences and their perceptions of the impact of the exercise class on their lives were then conducted. The results were analysed and mapped on a model, to illustrate the components that most likely promote participation. Sixteen participants (n = 8 with dementia, and n = 8 carers) were recruited, and completed both baseline and follow up assessments. Positive mean differences were found in physical activity (4.44), loneliness (1.75), mood (1.33) and cognition (1.13). Ten participants were included in the focus groups, which found that accessibility of the exercise venue, opportunities for socialisation and staff who were experienced working with people living with dementia were key to participants reporting benefits. The four key themes from the focus group data were synthesised to produce a model outlining the components that might generate a positive impact of the exercise classes and promote participation. Exercise classes for people with dementia can be delivered with success in novel environments such as University sports centres. There is some indication of improvement over a short period of time. The model derived from this study will inform strategies to promote attendance at dementia-friendly exercise classes.

C3d-positive donor-specific antibodies have a role in pretransplant risk stratification of cross-match-positive HLA-incompatible renal transplantation: United Kingdom multicentre study.

Anti-HLA-antibody characteristics aid to risk-stratify patients and improve long-term renal graft outcomes. Complement activation by donor-specific antibody (DSA) is an important characteristic that may determine renal allograft outcome. There is heterogeneity in graft outcomes within the moderate to high immunological risk cases (cross-match-positive). We explored the role of C3d-positive DSAs in sub-stratification of cross-match-positive cases and relate to the graft outcomes. We investigated 139 cross-match-positive living-donor renal transplant recipients from four transplant centres in the United Kingdom. C3d assay was performed on serum samples obtained at pretreatment (predesensitization) and Day 14 post-transplant. C3d-positive DSAs were found in 52 (37%) patients at pretreatment and in 37 (27%) patients at Day 14 post-transplant. Median follow-up of patients was 48 months (IQR 20.47-77.57). In the multivariable analysis, pretreatment C3d-positive DSA was independently associated with reduced overall graft survival, the hazard ratio of 3.29 (95% CI 1.37-7.86). The relative risk of death-censored five-year graft failure was 2.83 (95% CI 1.56-5.13). Patients with both pretreatment and Day 14 C3d-positive DSAs had the worst five-year graft survival at 45.5% compared with 87.2% in both pretreatment and Day 14 C3d-negative DSA patients with the relative risk of death-censored five-year graft failure was 4.26 (95% CI 1.79, 10.09). In this multicentre study, we have demonstrated for the first time the utility of C3d analysis as a distinctive biomarker to sub-stratify the risk of poor graft outcome in cross-match-positive living-donor renal transplantation.

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells.

Recent evidence suggests that mammary cells expressing R-spondin receptor and Wnt pathway regulator Lgr5, regarded as a stem cell marker in multiple tissues, might represent mammary stem cells (MaSCs). Whether L gr5 marks a multipotent subpopulation of Lin-CD24low/medCD49fhigh MaSCs remains controversial. To some extent the differing results reflect different assays used to assess properties of stemness, including lineage tracing in vivo, mammosphere culture, and mammary fat pad transplantation assays. To address this issue directly, we isolated Lgr5+ cells from mammary glands of Lgr5-lacZ mice and established organoids based on principles adapted from studies of Wnt-driven Lgr5+ cell populations in other organs. Mammary organoids were grown from single Lgr5+ mammary cells in Matrigel, the substratum of choice for intestinal organoids, and in a growth factor cocktail containing EGF, Wnt3a and R-spondin, designed to optimally activate the endogenous Wnt signaling program of stem cells. Colonies derived from single Lgr5+ cells manifest at least four distinct cell populations: Lgr5+ and Lgr5- basal cells and c-Kit+ and c-Kit- luminal cells that spontaneously organize into a ductal structure with basal cells around the periphery and luminal cells lining an interior cavity, reminiscent of normal mammary duct structure. Lgr5+ cell-derived organoids were sustainable during prolonged passaging. In contrast, although Lgr5- cells expand into primary colonies, colony-forming efficiency immediately dissipated upon passaging. Furthermore, reproductive hormones induce epithelial cell proliferation resulting in marked increases in lumen diameter accompanied by squamous transdifferentiation. We propose this estrogen-responsive, self-organizing duct-like structure derived from single murine Lgr5+ mammary cells represents a "mini-breast" organoid.

Systemic Correlates of White Adipose Tissue Inflammation in Early-Stage Breast Cancer.

PURPOSE: Obesity, insulin resistance, and elevated levels of circulating proinflammatory mediators are associated with poorer prognosis in early-stage breast cancer. To investigate whether white adipose tissue (WAT) inflammation represents a potential unifying mechanism, we examined the relationship between breast WAT inflammation and the metabolic syndrome and its prognostic importance. EXPERIMENTAL DESIGN: WAT inflammation was defined by the presence of dead/dying adipocytes surrounded by macrophages forming crown-like structures (CLS) of the breast. Two independent groups were examined in cross-sectional (cohort 1) and retrospective (cohort 2) studies. Cohort 1 included 100 women undergoing mastectomy for breast cancer risk reduction (n = 10) or treatment (n = 90). Metabolic syndrome-associated circulating factors were compared by CLS-B status. The association between CLS of the breast and the metabolic syndrome was validated in cohort 2, which included 127 women who developed metastatic breast cancer. Distant recurrence-free survival (dRFS) was compared by CLS-B status. RESULTS: In cohorts 1 and 2, breast WAT inflammation was detected in 52 of 100 (52%) and 52 of 127 (41%) patients, respectively. Patients with breast WAT inflammation had elevated insulin, glucose, leptin, triglycerides, C-reactive protein, and IL6 and lower high-density lipoprotein cholesterol and adiponectin (P < 0.05) in cohort 1. In cohort 2, breast WAT inflammation was associated with hyperlipidemia, hypertension, and diabetes (P < 0.05). Compared with patients without breast WAT inflammation, the adjusted HR for dRFS was 1.83 (95% CI, 1.07-3.13) for patients with inflammation. CONCLUSIONS: WAT inflammation, a clinically occult process, helps to explain the relationship between metabolic syndrome and worse breast cancer prognosis. Clin Cancer Res; 22(9); 2283-9. ©2015 AACR.

Molecular pathways: adipose inflammation as a mediator of obesity-associated cancer.

The increasing rate of obesity worldwide is predicted to be associated with a surge in diseases. Notably, obesity has been linked to approximately 20% of cancer cases in the United States; obesity is associated with both increased risk and worse outcomes after diagnosis. Altered levels of circulating factors are strongly implicated, including insulin, insulin-like growth factor 1, leptin, adiponectin, and interleukin-6 (IL-6). In addition, increasing attention has focused on the consequences of local adipose inflammation. Inflammatory foci characterized by crown-like structures consisting of dead adipocytes encircled by macrophages occur in white adipose depots, including the breast tissue, of most overweight and obese women. Saturated fatty acids, released as a consequence of obesity-associated lipolysis, induce macrophage activation via Toll-like receptor 4, thereby stimulating NF-κB signaling. This, in turn, activates transcription of proinflammatory genes including COX-2, IL-6, IL-1ß, and TNFα. Elevated levels of proinflammatory mediators cause both local and systemic effects. Of particular relevance with regard to breast cancer is increased transcription of the CYP19 gene encoding aromatase, the rate-limiting enzyme for estrogen synthesis. Notably, this obesity-inflammation-aromatase axis provides a plausible explanation for increased rates of postmenopausal, hormone receptor-positive breast cancer associated with obesity and hence may offer targets for interventions to attenuate risk or improve prognosis. Potential approaches include weight reduction, exercise, and suppression of obesity-driven signaling pathways using pharmaceutical or dietary agents. A key future goal is to identify biomarkers that accurately report adipose inflammation, both for identification of at-risk individuals and to assess the efficacy of interventions. Clin Cancer Res; 19(22); 6074-83. ©2013 AACR.

Genetic deletion of microsomal prostaglandin E synthase-1 suppresses mouse mammary tumor growth and angiogenesis.

The cyclooxygenase/prostaglandin (COX/PG) signaling pathway is of central importance in inflammation and neoplasia. COX inhibitors are widely used for analgesia and also have demonstrated activity for cancer prophylaxis. However, cardiovascular toxicity associated with this drug class diminishes their clinical utility and motivates the development of safer approaches both for pain relief and cancer prevention. The terminal synthase microsomal PGE synthase-1 (mPGES-1) has attracted considerable attention as a potential target. Overexpression of mPGES-1 has been observed in both colorectal and breast cancers, and gene knockout and overexpression approaches have established a role for mPGES-1 in gastrointestinal carcinogenesis. Here we evaluate the contribution of mPGES-1 to mammary tumorigenesis using a gene knockout approach. Mice deficient in mPGES-1 were crossed with a strain in which breast cancer is driven by overexpression of human epidermal growth factor receptor 2 (HER2/neu). Loss of mPGES-1 was associated with a substantial reduction in intramammary PGE2 levels, aromatase activity, and angiogenesis in mammary glands from HER2/neu transgenic mice. Consistent with these findings, we observed a significant reduction in multiplicity of tumors ≥1mm in diameter, suggesting that mPGES-1 contributes to mammary tumor growth. Our data identify mPGES-1 as a potential anti-breast cancer target.

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.

BACKGROUND: We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects. METHODS: RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes. RESULTS: We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67. CONCLUSION: Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.

Cyclooxygenase-2-derived prostacyclin regulates arterial thrombus formation by suppressing tissue factor in a sirtuin-1-dependent-manner.

BACKGROUND: Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood. METHODS AND RESULTS: We found that ferric chloride-induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-δ antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-δ agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice. CONCLUSIONS: Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-δ pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors.

Pioglitazone, a PPARγ agonist, suppresses CYP19 transcription: evidence for involvement of 15-hydroxyprostaglandin dehydrogenase and BRCA1.

Estrogen synthesis is catalyzed by cytochrome P450 aromatase, which is encoded by the CYP19 gene. In obese postmenopausal women, increased aromatase activity in white adipose tissue is believed to contribute to hormone-dependent breast cancer. Prostaglandin E(2) (PGE(2)) stimulates the cAMP→protein kinase A (PKA) pathway leading to increased CYP19 transcription and elevated aromatase activity in inflamed white adipose tissue. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) plays a major role in the catabolism of PGE(2). Here, we investigated the mechanism by which pioglitazone, a ligand of the nuclear receptor PPARγ suppressed aromatase expression. Treatment of human preadipocytes with pioglitazone suppressed Snail, a repressive transcription factor, resulting in elevated levels of 15-PGDH and reduced levels of PGE(2) in the culture medium. Pioglitazone also inhibited cAMP→PKA signaling leading to reduced interaction between phosphorylated cAMP responsive element-binding protein, p300, and CYP19 I.3/II promoter. BRCA1, a repressor of CYP19 transcription, was induced by pioglitazone. Consistent with these in vitro findings, treatment of mice with pioglitazone activated PPARγ, induced 15-PGDH and BRCA1 while suppressing aromatase levels in the mammary gland. Collectively, these results indicate that the activation of PPARγ induces BRCA1 and suppresses the PGE(2)→cAMP→PKA axis leading to reduced levels of aromatase. PPARγ agonists may have a role in reducing the risk of hormone-dependent breast cancer in obese postmenopausal women.

Activation status of Wnt/ß-catenin signaling in normal and neoplastic breast tissues: relationship to HER2/neu expression in human and mouse.

Wnt/ß-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/ß-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma in situ (DCIS), and invasive carcinomas. Cores were scored for membranous ß-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic ß-catenin, a hallmark of Wnt/ß-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated ß-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas (45%, P<0.001), consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (P = 0.04) and estrogen receptor-negative status (P = 0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic ß-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic ß-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic ß-catenin in node-positive carcinomas (P = 0.02). Furthermore, cytoplasmic ß-catenin was detected in HER2/neu-induced mouse mammary tumors. The Axin2(NLSlacZ) mouse strain, a previously validated reporter of mammary Wnt/ß-catenin signaling, was utilized to define in vivo transcriptional consequences of HER2/neu-induced ß-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/neu, Axin2(NLSlacZ) mice, highlighted by robust ß-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/neu mice. Our study thus provides provocative evidence for Wnt/ß-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia.