Factors influencing older women's decision-making related to treatment of operable breast cancer: A qualitative systematic review.

OBJECTIVE: There is variation in practice in the treatment of older women with breast cancer. International guidelines highlight the importance of patient autonomy in treatment decision-making. The aim of this study is to identify factors which influence decision-making in older women with operable breast cancer, which will enable us to further understand how to support these patients. METHODS: Systematic review in accordance with the PRISMA guidelines was performed to identify factors which influence treatment decision-making in older women with operable breast cancer. Medline, Web of Science and SCOPUS were searched. RESULTS: The search yielded 5840 results; 13 articles met the inclusion criteria and reported on a total of 1118 women. Thematic analysis identified three key themes in which decision-making factors could be categorised. These were healthcare-related factors, patient-related factors and impact of treatment. Healthcare-related factors included communication with clinicians and provision of information. Patient-related factors were age, pre-existing knowledge, preconceptions of breast cancer and treatment, decision-making style and co-morbidities. The impact of treatment considerations included body image and effect on quality of life. Decision-making style was frequently reported; older women did not demonstrate one preferred style. CONCLUSIONS: The findings have highlighted the complex interplay of factors which influence how older women make breast cancer treatment-decisions. Clinicians should have an awareness of the factors highlighted to maximise their ability to provide support and personalised care to older women with breast cancer whilst treatment decisions are made.

The impact of the cytoplasmic ubiquitin ligase TNFAIP3 gene variation on transcription factor NF-κB activation in acute kidney injury.

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.

Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

Blocking thrombospondin-1 signaling via CD47 mitigates renal interstitial fibrosis.

Acute kidney injury triggers a complex cascade of molecular responses that can culminate in maladaptive repair and fibrosis. We have previously reported that the matrix protein thrombospondin-1 (TSP1), binding its high affinity its receptor CD47, promotes acute kidney injury. However, the role of this pathway in promoting fibrosis is less clear. Hypothesizing that limiting TSP1-CD47 signaling is protective against fibrosis, we interrogated this pathway in a mouse model of chronic ischemic kidney injury. Plasma and renal parenchymal expression of TSP1 in patients with chronic kidney disease was also assessed. We found that CD47-/- mice or wild-type mice treated with a CD47 blocking antibody showed clear amelioration of fibrotic histological changes compared to control animals. Wild-type mice showed upregulated TSP1 and pro-fibrotic markers which were significantly abrogated in CD47-/- and antibody-treated cohorts. Renal tubular epithelial cells isolated from WT mice showed robust upregulation of pro-fibrotic markers following hypoxic stress or exogenous TSP1, which was mitigated in CD47-/- cells. Patient sera showed a proportionate correlation between TSP1 levels and worsening glomerular filtration rate. Immunohistochemistry of human kidney tissue demonstrated tubular and glomerular matrix localization of TSP1 expression in patients with CKD. These data suggest that renal tubular epithelial cells contribute to fibrosis by activating TSP1-CD47 signaling, and point to CD47 as a potential target to limit fibrosis following ischemic injury.

Deficiency in SIRP-α cytoplasmic recruitment confers protection from acute kidney injury.

Acute kidney injury (AKI) remains an important source of progressive chronic kidney injury. Loss of renal blood flow with subsequent restoration, termed ischemia reperfusion (IR), is a common cause of AKI. The cell surface receptor signal regulatory protein α (SIRP-α) is expressed on macrophages and limits inflammation and phagocytosis. SIRP-α has recently been found to have wider cell-based expression and play a role in renal IR. We have explored this in a genetic model of deficient SIRP-α signaling. Mice lacking SIRP-α cytoplasmic signaling (SIRP-αmut) and wild-type (WT) littermate controls underwent renal ischemia and reperfusion. Chimeric mice transplanted with WT or SIRP-αmut bone marrow were similarly challenged following engraftment. Molecular and immunohistochemical analysis of renal function, tissue damage, and key molecular targets was performed. SIRP-αmut mice were protected from renal IR compared with WT animals, demonstrating improved serum creatinine, less histologic damage, reduced proinflammatory cytokine production, and diminished production of reactive oxygen species (ROS). Resistance to renal IR in SIRP-αmut occurred alongside down-regulation of CD47 and thrombospondin-1, which are known to exert SIRP-α crosstalk and also promote IR. In chimeric mice, lack of SIRP-α signaling conferred protection to IR regardless of the genotype of circulating cells. Renal tubular epithelial cells from SIRP-αmut mice produced fewer ROS and proinflammatory cytokines in vitro. These results identify parenchymal SIRP-α as an independent driver of IR-mediated AKI and a potential therapeutic target.-Ghimire, K., Chiba, T., Minhas, N., Meijles, D. N., Lu, B., O'Connell, P., Rogers, N. M. Deficiency in SIRP-α cytoplasmic recruitment confers protection from acute kidney injury.

Activated protein C binds directly to Tie2: possible beneficial effects on endothelial barrier function.

Activated protein C (APC) is a natural anticoagulant with strong anti-inflammatory, anti-apoptotic, and barrier stabilizing properties. These cytoprotective properties of APC are thought to be exerted through its pathway involving the binding of APC to endothelial protein C receptor and cleavage of protease-activated receptors. In this study, we found that APC enhanced endothelial barrier integrity via a novel pathway, by binding directly to and activating Tie2, a transmembrane endothelial tyrosine kinase receptor. Binding assays demonstrated that APC competed with the only known ligands of Tie2, the angiopoietins (Angs). APC bound directly to Tie2 (Kd ~3 nM), with markedly stronger binding affinity than Ang2. After binding, APC rapidly activated Tie2 to enhance endothelial barrier function as shown by Evan's blue dye transfer across confluent cell monolayers and in vivo studies. Blocking Tie2 restricted endothelial barrier integrity. This study highlights a novel mechanism by which APC binds directly to Tie2 to enhance endothelial barrier integrity, which helps to explain APC's protective effects in vascular leakage-related pathologies.

Neuropathic pain following traumatic spinal cord injury: Models, measurement, and mechanisms.

Neuropathic pain following spinal cord injury (SCI) is notoriously difficult to treat and is a high priority for many in the SCI population. Resolving this issue requires animal models fidelic to the clinical situation in terms of injury mechanism and pain phenotype. This Review discusses the means by which neuropathic pain has been induced and measured in experimental SCI and compares these with human outcomes, showing that there is a substantial disconnection between experimental investigations and clinical findings in a number of features. Clinical injury level is predominantly cervical, whereas injury in the laboratory is modeled mainly at the thoracic cord. Neuropathic pain is primarily spontaneous or tonic in people with SCI (with a relatively smaller incidence of allodynia), but measures of evoked responses (to thermal and mechanical stimuli) are almost exclusively used in animals. There is even the question of whether pain per se has been under investigation in most experimental SCI studies rather than simply enhanced reflex activity with no affective component. This Review also summarizes some of the problems related to clinical assessment of neuropathic pain and how advanced imaging techniques may circumvent a lack of patient/clinician objectivity and discusses possible etiologies of neuropathic pain following SCI based on evidence from both clinical studies and animal models, with examples of cellular and molecular changes drawn from the entire neuraxis from primary afferent terminals to cortical sensory and affective centers. © 2016 Wiley Periodicals, Inc.

Endogenous MMP-9 and not MMP-2 promotes rheumatoid synovial fibroblast survival, inflammation and cartilage degradation.

OBJECTIVE: The aim of this study was to investigate the effect of endogenous matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) on the invasive characteristics of RA synovial fibroblasts. METHODS: Synovial fibroblasts isolated from patients with RA or OA were treated with MMP small interfering RNA (siRNA), inhibitors and recombinant proteins or TNF-α, with or without cartilage explants. Cell viability and proliferation were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and 5-bromo-2-deoxyuridine (BrdU) proliferation assays, respectively; apoptosis by an in situ cell death detection kit; migration and invasion by CytoSelect invasion assay, scratch migration and collagen gel assays; cartilage degradation by 1,9-dimethylmethylene blue assay; and inflammatory mediators and MMPs by ELISA, western blot and zymography. RESULTS: MMP-2 was expressed by both OA and RA synovial fibroblasts, whereas only RA synovial fibroblasts expressed MMP-9. Suppressing MMP-2 or MMP-9 reduced RA synovial fibroblast proliferation equally. However, MMP-9 siRNA had greater effects compared with MMP-2 siRNA on promoting apoptosis and suppressing RA synovial fibroblast viability, migration and invasion. Suppression/inhibition of MMP-9 also decreased the production of IL-1ß, IL-6, IL-8 and TNF-α, inactivated nuclear factor κB (NF-κB), extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and suppressed RA synovial fibroblast-mediated cartilage degradation. In contrast, suppression/inhibition of MMP-2 stimulated TNF-α and IL-17 secretion and activated NF-κB, while recombinant MMP-2 (rMMP-2) inactivated NF-κB and suppressed RA synovial fibroblast-mediated cartilage degradation. Results using specific inhibitors and rMMPs provided supportive evidence for the siRNA results. CONCLUSION: Endogenous MMP-2 or MMP-9 contribute to RA synovial fibroblast survival, proliferation, migration and invasion, with MMP-9 having more potent effects. Additionally, MMP-9 stimulates RA synovial fibroblast-mediated inflammation and degradation of cartilage, whereas MMP-2 inhibits these parameters. Overall, our data indicate that MMP-9 derived from RA synovial fibroblasts may directly contribute to joint destruction in RA.

Thrombospondin-1 Drives Cardiac Remodeling in Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) face a high risk of cardiovascular disease. Previous studies reported that endogenous thrombospondin 1 (TSP1) involves right ventricular remodeling and dysfunction. Here we show that a murine model of CKD increased myocardial TSP1 expression and produced left ventricular hypertrophy, fibrosis, and dysfunction. TSP1 knockout mice were protected from these features. In vitro, indoxyl sulfate is driving deleterious changes in cardiomyocyte through the TSP1. In patients with CKD, TSP1 and aryl hydrocarbon receptor were both differentially expressed in the myocardium. Our findings summon large clinical studies to confirm the translational role of TSP1 in patients with CKD.

Activated protein C inhibits proliferation and tumor necrosis factor α-stimulated activation of p38, c-Jun NH2-terminal kinase (JNK) and Akt in rheumatoid synovial fibroblasts.

Synovial fibroblast proliferation is a hallmark of the invasive pannus in the rheumatoid joint. Activated protein C (APC) is a natural anticoagulant that exerts antiinflammatory and cyto-protective effects in various diseases via endothelial protein C receptor (EPCR) and proteinase-activated receptor (PAR)-mediated pathways. In this study, we investigated the effect and the underlying cellular signaling mechanisms of APC on proliferation of human rheumatoid synovial fibroblasts (RSFs). We found that APC stimulated proliferation of mouse dermal fibroblasts (MDFs) and normal human dermal fibroblasts (HDFs) by up to 60%, but robustly downregulated proliferation of RSFs. APC induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) and enhanced expression of p21 and p27 in a dose-dependent manner in RSFs. The latter effect was inhibited by pre-treatment with the ERK inhibitors PD98059 and U0126 but not by p38 inhibitor SB203580. In addition, APC significantly downregulated tumor necrosis factor (TNF)α-stimulated cell proliferation and activation of p38, c-Jun NH2-terminal kinase (JNK) and Akt in RSFs. These results provide the first evidence that APC selectively inhibits proliferation and the inflammatory signaling pathways of RSFs. Thus, APC may reduce synovial hyperplasia and pannus invasion in rheumatoid arthritis.

Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function.

Activated protein C (APC) is an anticoagulant, approved as a treatment for severe sepsis, that can prevent apoptosis, inflammation, and vascular leakage. The aim of this study was to investigate whether APC protects endothelial barrier function through the angiopoietin (Ang)/Tie2 axis. APC significantly up-regulated gene and protein expression of Tie2 and Ang1 in a dose (0.01-10 microg/ml)- and time (0.5-24 h)-dependent manner in human umbilical vein endothelial cells (HUVECs). Interestingly, it markedly inhibited Ang2 with an IC(50) of approximately 0.1 microg/ml. HUVEC permeability, measured using Evans blue dye transfer, was significantly reduced in the presence of APC, and, in concordance, the tight junction associated protein zona occludens (ZO)-1 was up-regulated and localized peripherally around cells, compared with controls. Smooth muscle cell migration toward APC-stimulated HUVECs was elevated compared with unstimulated cells. Blocking antibodies and small interfering (si) RNA treatment, compared with isotype (IgG) or scrambled siRNA controls, showed that APC requires 3 receptors, the endothelial protein C receptor, protease-activated receptor 1, and Tie2 to perform all these barrier stabilization functions. In summary, this study demonstrates that APC has novel effects on the Ang/Tie2 axis, which enhance endothelial barrier function and are likely to contribute to its therapeutic effect in sepsis and other diseases associated with vascular leakage.-Minhas, N., Xue, M., Fukudome, K., Jackson, C. J. Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function.

Endogenous protein C is essential for the functional integrity of human endothelial cells.

Circulating protein C (PC) plays a vital role as an anti-coagulant and anti-inflammatory mediator. We show here that human endothelial cells produce PC that acts through novel mediators to enhance their own functional integrity. When endogenous PC or its receptor, endothelial protein C receptor (EPCR), was suppressed by small interfering (si) RNA, human umbilical cord endothelial cell (HUVEC) proliferation was decreased and apoptosis elevated. Interestingly, PC or EPCR siRNA significantly increased HUVEC permeability, which is likely via reduction of the angiopoietin (Ang)1/Ang2 ratio and inhibition of the peripheral localization of the tight junction protein, zona occludins-1. In addition, PC or EPCR siRNA inhibited type IV collagen and matrix metalloproteinase-2, providing the first evidence that PC contributes to vascular basement membrane formation. These newly described actions of endogenous PC act to stabilize endothelial cells and enhance barrier function, to potentially promote the functional integrity of blood vessels.

Repurposing of metformin and colchicine reveals differential modulation of acute and chronic kidney injury.

Acute kidney injury (AKI) is a major health problem affecting millions of patients globally. There is no effective treatment for AKI and new therapies are urgently needed. Novel drug development, testing and progression to clinical trials is overwhelmingly expensive. Drug repurposing is a more cost-effective measure. We identified 2 commonly used drugs (colchicine and metformin) that alter inflammatory cell function and signalling pathways characteristic of AKI, and tested them in models of acute and chronic kidney injury to assess therapeutic benefit. We assessed the renoprotective effects of colchicine or metformin in C57BL/6 mice challenged with renal ischemia reperfusion injury (IRI), treated before or after injury. All animals underwent analysis of renal function and biomolecular phenotyping at 24 h, 48 h and 4 weeks after injury. Murine renal tubular epithelial cells were studied in response to in vitro mimics of IRI. Pre-emptive treatment with colchicine or metformin protected against AKI, with lower serum creatinine, improved histological changes and decreased TUNEL staining. Pro-inflammatory cytokine profile and multiple markers of oxidative stress were not substantially different between groups. Metformin augmented expression of multiple autophagic proteins which was reversed by the addition of hydroxychloroquine. Colchicine led to an increase in inflammatory cells within the renal parenchyma. Chronic exposure after acute injury to either therapeutic agent in the context of reduced renal mass did not mitigate the development of fibrosis, with colchicine significantly worsening an ischemic phenotype. These data indicate that colchicine and metformin affect acute and chronic kidney injury differently. This has significant implications for potential drug repurposing, as baseline renal disease must be considered when selecting medication.