3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability.

Next-generation Trop-2-targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti-Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site-mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2-expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10-12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10-8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2-expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2-targeted therapy.

Kinetics of Circulating Extracellular Vesicles Over the 24-Hour Dosing Interval After Low-Dose Aspirin Administration in Patients at Cardiovascular Risk.

Extracellular vesicles (EVs) are small vesicles deriving from all cell types during cell activation, involved in transcellular communication, and regarded as predictors of vascular damage and of cardiovascular events. We tested the hypothesis that, in patients on chronic low-dose aspirin treatment for cardiovascular prevention, aspirin may affect the release of EVs within the 24-hour interval. We enrolled 84 patients, mostly at high or very high cardiovascular risk, on chronic low-dose aspirin treatment. The numbers of circulating EVs (cEVs) and annexinV+ cEVs (total, platelet-derived, endothelial-derived, and leucocyte-derived) were assessed immediately before, and after 10 and 24 hours of a witnessed aspirin administration. Platelet cyclooxygenase 1 (COX-1) recovery was characterized by measuring serum thromboxane B2 (sTXB2 ) at the same timepoints. Nine healthy participants were also enrolled. In patients, daily aspirin administration acutely inhibited after 10 hours following aspirin administrations the release of cEVs (total and leukocyte-derived) and annexinV+ cEVs (total, platelet-derived, endothelial-derived, and leukocyte-derived), with a rapid recovery at 24 hours. The inhibition after 10 hours suggests a COX-1-dependent mechanism. Interestingly, the slope of platelet-derived and of annexinV+ platelet-derived cEVs were both directly related to sTXB2 slope and COX-1 messenger RNA, raising the hypothesis that vice versa, cEVs may affect the rate of COX-1 recovery and the subsequent duration of aspirin effect. In healthy participants, no circadian difference was observed, except for leukocyte-derived cEVs. Our findings suggest a previously unappreciated effect of aspirin on the kinetics of a subset of cEVs possibly contributing to the cardioprotective effects of this drug.

Reduced platelet glycoprotein Ibα shedding accelerates thrombopoiesis and COX-1 recovery: implications for aspirin dosing regimen.

Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.

Study of pulsed electrostatic field (PESF) in the perfusion of peripheral tissues: Microangiopathy, nutrition and quality of perceiver life.

Microangiopathy compromises the structural and functional integrity of organs and tissues in patients with type II diabetes mellitus (T2DM) negatively affecting the perceived quality of life. Nitric oxide (NO) is a multifunctional signalling molecule, acting as a vasodilator, neurotransmitter, and modulator of inflammatory processes. Patients with type II diabetes mellitus and chronic kidney disease, controlled from glycaemic status, were treated or not with pulsed electrostatic field (PESF) cycles to evaluate effect on the perfusion of peripheral tissues. Everyone was monitored for the metabolic profile, and we tested circulating NO with a commercial enzyme immunoassay kit. In addition, we tested the perceived quality of life of patients before/after a PESF cycle using a questionnaire. Patients treated with PESF were improved circulating NO levels, significant changes in systolic and diastolic blood pressure, heart rate and were more homogeneous for their metabolic profile. The questionnaire showed also a marked improvement in the perceived quality of life. The use of pulsed electrostatic fields has allowed us to observe an improvement in the metabolic, psychological, and clinical profile in patients with T2DM and chronic kidney disease whose pathological profile is strongly compromised.

Effects of liraglutide vs. lifestyle changes on soluble suppression of tumorigenesis-2 (sST2) and galectin-3 in obese subjects with prediabetes or type 2 diabetes after comparable weight loss.

BACKGROUND: Soluble suppression of tumorigenesis-2 (sST2) and galectin (Gal)-3 are two biomarkers related to inflammation, metabolic disturbances and to myocardial fibrosis that characterize several cardiac pathological conditions. Increased circulating levels of these molecules have been associated with risk of cardiovascular death. Treatment with liraglutide, a glucagon-like peptide 1 analog, is associated with weight loss, improved glycemic control, and reduced cardiovascular risk. We wanted to assess (I) potential differences between subjects with prediabetes or type 2 diabetes mellitus (T2DM) and healthy controls in sST2 and Gal-3 circulating levels, and their relationship with glycemic control and markers of beta cell function and myocardial injury; (II) whether liraglutide treatment modulates these markers in subjects with prediabetes or early T2DM independently of weight loss; (III) whether baseline levels of any of these two molecules may predict the response to liraglutide treatment. METHODS: Forty metformin-treated obese subjects (BMI ≥ 30) with prediabetes [impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) or both (n = 23)] or newly diagnosed T2DM (n = 17), were randomized to liraglutide or lifestyle counseling until achieving a comparable weight loss (7% of initial body weight). Thirteen subjects were enrolled as healthy controls for baseline sST2 and Gal-3 levels. RESULTS: Baseline sST2 levels were comparable between controls and obese patients (p = 0.79) whereas Gal-3 levels were significantly higher in patients as compared to controls (p < 0.001). Liraglutide treatment, but not weight loss achieved by lifestyle counseling, decreased plasma sST2 levels (- 9%, beta = - 14.9, standard deviation 6.9, p = 0.037) while Gal-3 levels did not change. A reduction in serum hs-Troponin I was observed after intervention, due to a 19% (p = 0.29) increase in the lifestyle arm, and a 25% decrease (p = 0.033) in the liraglutide arm (between-group difference p = 0.083). Lower baseline Gal-3 levels predicted a better improvement in beta cell function after liraglutide treatment. CONCLUSIONS: Liraglutide-induced reduction in sST2 and possibly hs-TnI suggests that in obese patients with prediabetes or early T2DM this drug may have a positive effect on (cardiac) fibrosis, whereas plasma level of Gal-3 before liraglutide initiation may predict response to the drug in terms of beta cell function improvement. Trial registration Eudract: 2013-001356-36.

Trop-2, Na+/K+ ATPase, CD9, PKCα, cofilin assemble a membrane signaling super-complex that drives colorectal cancer growth and invasion.

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca2+ signal. We reveal here that Trop-2 binds the cell membrane Na+/K+-ATPase, and that clustering of Trop-2 induces an intracellular Ca2+ rise followed by membrane translocation of PKCα, which in turn phosphorylates the Trop-2 cytoplasmic tail. This feed-forward signaling is promoted by the binding of Trop-2 to the PKCα membrane-anchor CD9. CRISPR-based inactivation of CD9 in CRC cells shows that CD9 is required by Trop-2 for recruiting PKCα and cofilin-1 to the cell membrane. This induces malignant progression through proteolytic cleavage of E-cadherin, remodeling of the ß-actin cytoskeleton, and activation of Akt and ERK. The interaction between Trop-2 and CD9 was validated in vivo in murine models of CRC growth and invasion. Overexpression of the components of this Trop-2-driven super-complex significantly worsened disease-free and overall survival of CRC patients, supporting a pivotal relevance in CRC malignant progression. Our findings demonstrate a previously unsuspected layer of cancer growth regulation, which is dormant in normal tissues, and is activated by Trop-2 in cancer cells.

Insulin resistance and NAFLD may influence memory performance in obese patients with prediabetes or newly-diagnosed type 2 diabetes.

BACKGROUND AND AIMS: Diabetes has consistently been shown to increase risk for cognitive decline. Cognitive deficits may occur at the very earliest stages of diabetes. We sought to estimate the determinants of memory function in a group of middle-aged obese subjects with prediabetes or newly-diagnosed type 2 diabetes mellitus. METHODS AND RESULTS: Sixty-two obese patients in treatment with metformin-with prediabetes (n = 41) or newly diagnosed T2DM (n = 21), were studied. Short- and long-term memory function was assessed through a neuropsychological assessment consisting of two tests and a composite domain z score was calculated. Cardiometabolic variables, such as abdominal MRI quantification of subcutaneous (SAT) and visceral (VAT) adipose tissue content, and of intra-hepatocellular lipid content, as well as insulin sensitivity (Matsuda Index, HOMA-IR) and beta cell performance (Beta Index), by multiple sampling, 8-point oral glucose tolerance test, were also evaluated. Age, non-alcoholic fatty liver disease (NAFLD), and lnHOMA-IR together explained 18% (R square) of the variance in memory domain. Including NAFLD increased the explained variance by 8% and including lnHOMA-IR by 9.1%, whereas the contribution of age and other factors was negligible. CONCLUSION: Preventing and managing insulin resistance in precocious and possibly earlier stages of diabetes might provide benefit in slowering down future cognitive decline.

Trop-2 induces ADAM10-mediated cleavage of E-cadherin and drives EMT-less metastasis in colon cancer.

We recently reported that activation of Trop-2 through its cleavage at R87-T88 by ADAM10 underlies Trop-2-driven progression of colon cancer. However, the mechanism of action and pathological impact of Trop-2 in metastatic diffusion remain unexplored. Through searches for molecular determinants of cancer metastasis, we identified TROP2 as unique in its up-regulation across independent colon cancer metastasis models. Overexpression of wild-type Trop-2 in KM12SM human colon cancer cells increased liver metastasis rates in vivo in immunosuppressed mice. Metastatic growth was further enhanced by a tail-less, activated ΔcytoTrop-2 mutant, indicating the Trop-2 tail as a pivotal inhibitory signaling element. In primary tumors and metastases, transcriptome analysis showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 is through alternative mechanisms. Trop-2 can tightly interact with ADAM10. Here, Trop-2 bound E-cadherin and stimulated ADAM10-mediated proteolytic cleavage of E-cadherin intracellular domain. This induced detachment of E-cadherin from ß-actin, and loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of ß-catenin signaling, which were further enhanced by the ΔcytoTrop-2 mutant. This Trop-2/E-cadherin/ß-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival. In patients with colon cancer, activation of this Trop-2-centered program led to significantly reduced relapse-free and overall survival, indicating a major impact on progression to metastatic disease. Recently, the anti-Trop-2 mAb Sacituzumab govitecan-hziy was shown to be active against metastatic breast cancer. Our findings define the key relevance of Trop-2 as a target in metastatic colon cancer.

Endogenous PCSK9 may influence circulating CD45neg/CD34bright and CD45neg/CD34bright/CD146neg cells in patients with type 2 diabetes mellitus.

Protease proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of LDL cholesterol clearance and has been associated with cardiovascular risk. PCSK9 inhibitors increase in vivo circulating endothelial progenitor cells (EPCs), a subtype of immature cells involved in ongoing endothelial repair. We hypothesized that the effect of PCSK9 on vascular homeostasis may be mediated by EPCs in patients with or without type 2 diabetes mellitus (T2DM). Eighty-two patients (45 with, 37 without T2DM) at high cardiovascular risk were enrolled in this observational study. Statin treatment was associated with higher circulating levels of PCSK9 in patients with and without T2DM (p < 0.001 and p = 0.036) and with reduced CD45neg/CD34bright (total EPC compartment) (p = 0.016) and CD45neg/CD34bright/CD146neg (early EPC) (p = 0.040) only among patients with T2DM. In the whole group of patients, statin treatment was the only independent predictor of low number of CD45neg/CD34bright (ß = - 0.230; p = 0.038, adjusted R2 = 0.041). Among T2DM patients, PCSK9 circulating levels were inversely related and predicted both the number of CD45neg/CD34bright (ß = - 0.438; p = 0.003, adjusted R2 = 0.173), and CD45neg/CD34bright/CD146neg (ß = - 0.458; p = 0.002, adjusted R2 = 0.191) independently of age, gender, BMI and statin treatment. In high-risk T2DM patients, high endogenous levels of PCSK9 may have a detrimental effect on EPCs by reducing the endothelial repair and worsening the progression of atherothrombosis.

Sex-Specific Association of Endogenous PCSK9 With Memory Function in Elderly Subjects at High Cardiovascular Risk.

Background: Growing evidence indicates that cognitive decline and cardiovascular diseases (CVDs) share common vascular risk factors. Protease proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with CV disease risk and has been also involved in neuronal differentiation. Aim: Evaluate whether in patients at high CV risk cognitive function is related to PCSK9 levels. Methods. One hundred sixty-six patients (67 female) were enrolled. A detailed neuropsychological (NP) assessment was performed. PCSK9 levels were measured with ELISA. Results: Men had significantly higher short-term memory, executive function, and praxic and mental representation skills, as reflected by Forward Digit Span (FDS) (p = 0.005), Trail Making Test-A (TMT-A) (p = 0.047), Clock Drawing Test (CDT) (0.016). Endogenous PCSK9 levels were higher in female (p = 0.005). On linear regression analysis PCSK9 predicts short term memory only in females (Beta = 0.408, p = 0.001), with an interaction between PCSK9 and gender (p = 0.004 for interaction PCSK9 by sex). The association of PCSK9 with FDS in female was partially mediated by waist circumference (mediation effect 8.5%). Conclusions: In patients at high CV risk short term memory was directly related to PCSK9 levels only in women, revealing the relevance of sex in this relationship. The association of PCSK9 with memory function may be mediated, at least in part, by waist circumference.

Liraglutide improves memory in obese patients with prediabetes or early type 2 diabetes: a randomized, controlled study.

BACKGROUND/OBJECTIVES: Diabetic subjects are at increased risk of subtle cognitive impairment since the disease early stages and of dementia later in life. In animal models, glucagon-like peptide-1 receptor agonizts (GLP1-RAs) have been shown to exert neuroprotective effects, expecially in the memory domain. We assessed whether treatment with a GLP1-RA might affect cognitive functions in type 2 diabetic subjects independently on the weight loss it might induce. SUBJECTS/METHODS: Forty metformin-treated obese subjects with prediabetes or newly diagnosed type 2 diabetes mellitus, received liraglutide (1.8 mg/d) (n = 20) or lifestyle counseling (dietary intervention and exercise training) (n = 20) until achieving a modest and comparable weight loss (-7% of initial body weight). INTERVENTIONS/METHODS: A detailed neuropsychological assessment before and after weight loss was completed in 16 patients per arm, who were administered a total of seven psychological tests, thus assessing three composite domain z-scores for attention, memory, and executive control. RESULTS: After comparable weight loss and superimposable glycemic control and insulin sensitivity, a significant increase in short term memory (mean Digit Span Z score from -0.06 to 0.80, p = 0.024) and memory composite z-score (mean memory z-score from -0.67 to 0.032, p = 0.0065) was observed in the liraglutide exposed subjects (between group p = 0.041 and p = 0.033, respectively). CONCLUSIONS: Liraglutide might slow down memory function decline in diabetic patients in early, and possibly preclinical stages of the disease.

In vivo thromboxane-dependent platelet activation is persistently enhanced in subjects with impaired glucose tolerance.

BACKGROUND: Impaired glucose tolerance (IGT) is associated with increased cardiovascular morbidity and mortality. Enhanced thromboxane (TX)-dependent platelet activation plays a pivotal role in atherothrombosis and characterizes type 2 diabetes mellitus (DM). Whether this also pertains to IGT is currently unknown. We investigated whether TXA2 -dependent platelet activation, as reflected by 11-dehydro-TXB2 (TXM) urinary excretion, is comparably abnormal in IGT as in DM, is persistent over long-term follow-up, changes as a function of metabolic disease progression, and is influenced by food intake. METHODS: We prospectively investigated subjects with IGT (n = 48) and two control groups with DM diagnosed either less than 12 months (n = 60) or 12 months or more (n = 58). RESULTS: Baseline TXM excretion was comparable between subjects with IGT and DM, with no evidence of a circadian variation. During a 36-month follow-up, urinary TXM excretion was stable over time in the DM groups, while tended to increase in subjects with IGT. Increasing urinary TXM excretion over time was observed in the subjects who progressed to diabetes vs nonprogressors. CONCLUSIONS: We conclude that TXA2 -dependent platelet activation was at least as high in IGT as in patients with DM and further increased over time, especially in those who progressed to overt diabetes.

Thromboxane-Dependent Platelet Activation in Obese Subjects with Prediabetes or Early Type 2 Diabetes: Effects of Liraglutide- or Lifestyle Changes-Induced Weight Loss.

Thromboxane (TX)-dependent platelet activation and lipid peroxidation, as reflected in vivo by the urinary excretion of 11-dehydro-TXB2 and 8-iso-prostaglandin (PG)F2α, play a key role in atherothrombosis in obesity and type 2 diabetes mellitus (T2DM) since the earlier stages. Thirty-five metformin-treated obese subjects with prediabetes or newly-diagnosed T2DM were randomized to the glucagon-like peptide receptor agonist (GLP-RA) liraglutide (1.8 mg/day) or lifestyle counseling until achieving a comparable weight loss (-7% of initial body weight), to assess whether changes in subcutaneous (SAT) and visceral (VAT) adipose tissue distribution (MRI), insulin sensitivity (Matsuda Index) and beta-cell performance (multiple sampling OGTT beta-index), with either intervention, might affect TX-dependent platelet activation, lipid peroxidation and inflammation. At baseline, Ln-8-iso-PGF2α (Beta = 0.31, p = 0.0088), glycosylated hemoglobin (HbA1c) (Beta = 2.64, p = 0.0011) Ln-TNF-α (Beta = 0.58, p = 0.0075) and SAT (Beta = 0.14, p = 0.044) were significant independent predictors of 11-dehydro-TXB2. After achievement of the weight loss target, a comparable reduction in U-11-dehydro-TXB2 (between-group p = 0.679) and 8-iso-PGF-2α (p = 0.985) was observed in both arms in parallel with a comparable improvement in glycemic control, insulin sensitivity, SAT, high-sensitivity C-reactive protein (hs-CRP). In obese patients with initial impairment of glucose metabolism, the extent of platelet activation is related to systemic inflammation, isoprostane formation and degree of glycemic control and abdominal SAT. Successful weight loss, achieved with either lifestyle changes or an incretin-based therapy, is associated with a significant reduction in lipid peroxidation and platelet activation.

Establishment and long-term culture of human cystic fibrosis endothelial cells.

Endothelial cell (EC) dysfunction has been reported in cystic fibrosis (CF) patients. Thus, the availability of CF EC is paramount to uncover mechanisms of endothelial dysfunction in CF. Using collagenase digestion, we isolated cells from small fragments of pulmonary artery dissected from non-CF lobes or explanted CF lungs. These cells were a heterogeneous mixture, containing variable percentages of EC. To obtain virtually pure pulmonary artery endothelial cells (PAEC), we developed an easy, inexpensive, and reliable method, based on the differential adhesion time of pulmonary artery cells collected after collagenase digestion. With this method, we obtained up to 95% pure non-CF and CF-PAEC. Moreover, we also succeed at immortalizing both PAEC and CF-PAEC, which remained viable and with unchanged phenotype and proliferation rate over the 30th passage. These cells recapitulated cystic fibrosis transmembrane conductance regulator expression and functions of the parental cells. Thus, we isolated for the first time endothelial cells from CF patients, providing a valuable tool to define the emerging role of EC in CF lung and vascular disease.

Oxidant stress as a major determinant of platelet activation in invasive breast cancer.

The hypothesis that increased oxidative stress in breast cancer (BC) patients could induce enhanced lipid peroxidation, which, in turn, would contribute to platelet activation and poor clinical outcome is attractive. To address this issue, we investigated pre-surgical urinary 8-iso-prostaglandin (PG)F2α (marker of in vivo oxidative stress) and 11-dehydro-thromboxane (TX)B2 (marker of in vivo platelet activation) levels in patients with primary BC (n = 115) compared with control women paired for comorbidities and their association with patients' metabolic profile and clinical prognostic factors. The results obtained showed that presurgical urinary excretion of both biomarkers was enhanced in BC patients compared to controls and was associated with patients' estrogen receptor (ER) expression, but not HER2 status or Ki67 proliferation index. Accordingly, both urinary biomarkers were increased in patients with luminal-like BC molecular subtypes compared with triple negative or HER2-enriched tumors. ER status was an independent predictor of 8-iso-PGF2α urinary levels, which, in turn, significantly predicted 11-dehydro-TXB2 urinary levels together with disease stage and ER status. The prognostic value of 11-dehydro-TXB2 was then evaluated showing a significant correlation with BC pathological response to neoadjuvant treatment. Furthermore, among relapsing patients, those with elevated urinary biomarker levels were more likely to develop distant metastasis rather than local recurrence. In conclusion, we may speculate that enhanced oxidative stress due to estrogen-related mechanisms might cause a condition of persistent platelet activation capable of sustaining BC growth and progression through the release of bioactive stored molecules, ultimately contributing to tumor invasiveness. Further studies specifically addressing this hypothesis are presently needed.

Increased circulating resistin is associated with insulin resistance, oxidative stress and platelet activation in type 2 diabetes mellitus.

Resistin is an adipokine that promotes inflammation and insulin resistance by targeting several cells including platelets. We hypothesised that in type 2 diabetes (T2DM), resistin may foster in vivo oxidative stress, thromboxane-dependent platelet activation and platelet-derived inflammatory proteins release, key determinants of atherothrombosis. A cross-sectional comparison of circulating resistin, sCD40L, as a marker of platelet-mediated inflammation, asymmetric dimethylarginine (ADMA), endothelial dysfunction marker, Dickkopf (DKK)-1, reflecting the inflammatory interaction between platelets and endothelial cells, and urinary 8-iso-PGF2α and 11-dehydro-TxB2, reflecting in vivo lipid peroxidation and platelet activation, respectively, was performed between 79 T2DM patients and 30 healthy subjects. Furthermore, we investigated the effects of the α-glucosidase inhibitor acarbose and the PPARγ agonist rosiglitazone, targeting hyperglycaemia or insulin resistance, versus placebo, in 28 and 18 T2DM subjects, respectively. Age- and gender-adjusted serum resistin levels were significantly higher in patients than in controls. HOMA (ß=0.266, p=0.017) and 11-dehydro-TXB2 (ß=0.354, p=0.002) independently predicted resistin levels. A 20-week treatment with acarbose was associated with significant reductions (p=0.001) in serum resistin, DKK-1, urinary 11-dehydro-TXB2 and 8-iso-PGF2α with direct correlations between the change in serum resistin and in other variables. A 24-week rosiglitazone treatment on top of metformin was associated with significant decreases in resistin, DKK-1, 11-dehydro-TXB2 and 8-iso-PGF2α, in parallel with HOMA decrease. In conclusion, resistin, antagonising insulin action in part through PPARγ activation, may favour insulin resistance and enhance oxidative stress, endothelial dysfunction and platelet activation. The adipokine-platelet interactions may be involved in platelet insulin resistance and their consequent pro-aggregatory phenotype in this setting.

Oxidative stress and thromboxane-dependent platelet activation in inflammatory bowel disease: effects of anti-TNF-α treatment.

Patients with inflammatory bowel disease (IBD) are at higher risk of venous thromboembolism and coronary artery disease despite having a lower burden of traditional risk factors. Platelets from IBD patients release more soluble CD40 ligand (CD40L), and this has been implicated in IBD platelet hyper-activation. We here measured the urinary F2-isoprostane 8-iso-prostaglandin (PG)2α (8-iso-PGF2α), urinary 11-dehydro-thromboxane (TX) B2 (11-dehydro-TXB2) and plasma CD40L in IBD patients, and explored the in vitro action of anti-tumour necrosis factor (TNF)-α antibody infliximab on IBD differentiating megakaryocytes. Urinary and blood samples were collected from 124 IBD patients and 37 healthy subjects. Thirteen IBD patients were also evaluated before and after 6-week infliximab treatment. The in vitro effect of infliximab on patient-derived megakaryocytes was evaluated by immunoflorescence microscopy and by flow cytometry. IBD patients had significantly (p<0.0001) higher urinary 8-iso-PGF2α and 11-dehydro-TXB2 as well as plasma CD40L levels than controls, with active IBD patients displaying higher urinary and plasma values when compared to inactive patients in remission. A 6-week treatment with infliximab was associated with a significant reduction of the urinary excretion of 8-iso-PGF2α and 11-dehydro-TXB2 (p=0.008) and plasma CD40L (p=0.001). Infliximab induced significantly rescued pro-platelet formation by megakaryocytes derived from IBD patients but not from healthy controls. Our findings provide evidence for enhanced in vivo TX-dependent platelet activation and lipid peroxidation in IBD patients. Anti-TNF-α therapy with infliximab down-regulates in vivo isoprostane generation and TX biosynthesis in responder IBD patients. Further studies are needed to clarify the implication of infliximab induced-proplatelet formation from IBD megakaryocytes.

Oxidative Stress Modulation Through Habitual Physical Activity.

BACKGROUND: Oxidative stress is involved in different pathophysiological states, such as aging, inflammatory, cardiovascular and neurodegenerative diseases, by damaging several cellular and tissue components including proteins, DNA and lipids. On the other hand, free radicals generated during physical activity are important modulators of muscle contraction, antioxidant protection, and oxidative damage repair. Indeed, ROS, generated during physical activity, are likely main mediators of antioxidant molecules upregulation, as reflected by increased glutathione reductase levels after exercise training. METHODS: The aim of this review is to summarize the main mechanisms responsible for ROS-dependent adaptations to exercise training. RESULTS: Regular moderate exercise seems to counteract oxidative stress-related detrimental changes and to promote a healthy lifestyle. Conversely, acute and strenuous exercise can generate an excess of free radicals production. Moreover, regular habitual physical activity is related to reduced risk of coronary heart disease and death, whereas vigorous exercise has been shown to favour sudden cardiac death in sedentary individuals with preexisting vascular disease. New specific markers of mitochondrial or ER dysfunction may be better clues of oxidative stress, and their application to clinical practice may help set up the optimal dose, intensity and modality of exercise training for every single subject. CONCLUSION: The relationship between exercise and oxidative stress is extremely complex, depending on the mode, intensity, and duration of exercise. These conflicting effects and outcomes may be explained by the hormesis theory, in which low doses of an agent that is detrimental at high doses, induces an adaptive beneficial effect on the cells or organism.

Trop-2 Induces Tumor Growth Through AKT and Determines Sensitivity to AKT Inhibitors.

PURPOSE: Inhibition of AKT is a key target area for personalized cancer medicine. However, predictive markers of response to AKT inhibitors are lacking. Correspondingly, the AKT-dependent chain of command for tumor growth, which will mediate AKT-dependent therapeutic responses, remains unclear. EXPERIMENTAL DESIGN: Proteomic profiling was utilized to identify nodal hubs of the Trop-2 cancer growth-driving network. Kinase-specific inhibitors were used to dissect Trop-2-dependent from Trop-2-independent pathways. In vitro assays, in vivo preclinical models, and case series of primary human breast cancers were utilized to define the mechanisms of Trop-2-driven growth and the mode of action of Trop-2-predicted AKT inhibitors. RESULTS: Trop-2 and AKT expression was shown to be tightly coordinated in human breast cancers, with virtual overlap with AKT activation profiles at T308 and S473, consistent with functional interaction in vivo AKT allosteric inhibitors were shown to only block the growth of Trop-2-expressing tumor cells, both in vitro and in preclinical models, being ineffective on Trop-2-null cells. Consistently, AKT-targeted siRNA only impacted on Trop-2-expressing cells. Lentiviral downregulation of endogenous Trop-2 abolished tumor response to AKT blockade, indicating Trop-2 as a mandatory activator of AKT. CONCLUSIONS: Our findings indicate that the expression of Trop-2 is a stringent predictor of tumor response to AKT inhibitors. They also support the identification of target-activatory pathways, as efficient predictors of response in precision cancer therapy. Clin Cancer Res; 22(16); 4197-205. ©2016 AACR.

Pentraxin 3 and Platelet Activation in Obese Patients After Gastric Banding.

BACKGROUND: Circulating pentraxin 3 (PTX3), the main regulator of the inflammatory response, rapidly increases following cardiovascular events, and low PTX3 is associated with high body mass index. METHODS AND RESULTS: We conducted a 12-month longitudinal study, to test the hypothesis that laparoscopic adjustable gastric banding (LAGB)-induced weight loss was associated with changes in platelet activation markers and PTX3. Twelve obese patients, scheduled to undergo LAGB, were enrolled at the University Obesity Center. Urinary 11-dehydro-thromboxane (Tx)B2excretion rate was measured on radioimmunoassay, and PTX3 and CD40L were determined on immunoassay. Plasma PTX3 increased by 178.8 and 214.9% (P<0.0001), respectively, 6 and 12 months after LAGB. High-sensitivity CRP decreased by 24 and 29.7% (P<0.0001), whereas CD40L decreased by 64.3 and 58.6% (P=0.002), respectively. Urinary 11-dehydro-TxB2decreased from 1,443 to 715 and 564 pg/mg creatinine, respectively 6 months and 12 months after LAGB (P<0.0001). PTX3 was inversely related to platelet activation markers, 11-dehydro-TxB2and CD40L. Moreover, multiple regression analysis on pooled data showed that plasma PTX3 was an independent predictor of urinary 11-dehydro-TxB2. CONCLUSIONS: There is an association between inflammation, platelet activation and metabolic dysfunction in obesity, and PTX3 is a key player within these circuits.