Recombinant Tissue Plasminogen Activator (r-tPA) Induces In-Vitro Human Neutrophil Migration via Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1).

Thrombolysis is the gold standard treatment for acute ischemic stroke. Besides its fibrinolytic role, recombinant tissue plasminogen activator (r-tPA) holds several non-fibrinolytic functions. Here, we investigated the potential role of r-tPA on human primary neutrophil migration in vitro. By means of modified Boyden chamber migration assay and checkerboard analysis we showed a dose-dependent chemotactic effect of r-TPA with a maximum effect reached by 0.03 mg/mL (0.003-1 mg/mL). Pre-incubation with MAP kinases inhibitors allowed the identification of PI3K/Akt, but not ERK1/2 as the intracellular pathway mediating the observed effects. Furthermore, by means of real-time PCR, immunocytochemistry and cytofluorimetry we demonstrated that the r-tPA receptor low density lipoprotein receptor-related protein 1 (LRP-1) is synthetized and expressed by neutrophils in response to r-tPA and TNF-α. Inhibition of LRP-1 by receptor-associated protein (RAP), prevented r-tPA-mediated F-actin polymerization, migration and signal through Akt but not ERK1/2. Lastly, also neutrophil degranulation in response to r-tPA seems to be mediated by LRP-1 under adhesion conditions. In conclusion, we show that r-tPA induces neutrophil chemotaxis through LRP-1/Akt pathway. Blunting r-tPA-mediated neutrophil activation might be beneficial as an adjuvant therapy to thrombolysis in this setting.

Indoxyl Sulfate Induces Renal Fibroblast Activation through a Targetable Heat Shock Protein 90-Dependent Pathway.

Indoxyl sulfate (IS) accumulation occurs early during chronic kidney disease (CKD) progression and contributes to renal dysfunction by inducing fibrosis, inflammation, oxidative stress, and tissue remodeling. Renal toxicity of high IS concentrations (250 µM) has been widely explored, particularly in resident tubular and glomerular cells, while the effect of a moderate IS increase on kidneys is still mostly unknown. To define the effects of IS accumulation on renal fibroblasts, we first analyzed kidneys of C57BL/6 mice receiving IS (0.1%) in drinking water for 12 weeks. As a next step, we treated renal fibroblasts (NRK-49F) with IS (20 µM) with or without the HSP90 inhibitor 17-AAG (1 µM). In mouse kidneys, IS increased the collagen deposition and HSP90 and α-SMA expression (immunohistochemistry) in interstitial fibroblasts and caused tubular necrosis (histological H&E and picrosirius red staining). In NRK-49F cells, IS induced MCP1, TGF-ß, collagen I, α-SMA, and HSP90 gene/protein expression and Smad2/3 pathway activation. IS had no effects on fibroblast proliferation and ROS production. 17-AAG counteracted IS-induced MCP1, TGF-ß, collagen I, and α-SMA expression and Smad2/3 phosphorylation. Our study demonstrates that the IS increase promotes renal fibroblast activation by a HSP90-dependent pathway and indicates HSP90 inhibition as a potential strategy to restrain IS-induced kidney inflammation and fibrosis in CKD.

Causes and impact on survival of underuse of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in heart failure.

Guidelines recommend angiotensin-converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) for treatment of heart failure with reduced ejection fraction (HFrEF), but these medications are underprescribed in clinical practice. We reviewed the records of HF patients receiving a first visit in a tertiary outpatient clinic from January 1st 2004 to May 31st 2015, and selected those with a serum creatinine concentration (sCr) available at both the first and last visit and < 3.5 mg/dL at baseline, and a left ventricular ejection fraction (LVEF) < 50% at the first visit. Of 570 eligible patients, 92 (16.1%) never received ACEi/ARB. Compared to ACEi/ARB users, never-users were older, more often women, had higher sCr and lower systolic blood pressure, were less commonly on beta-blocker, and had more frequently anemia. Current or prior cancer also tended to be more common in ACEi/ARB never-users. ACEi/ARB users displayed an improvement in LVEF by ≥ 10% of the baseline value more often than ACEi/ARB never-users (33.7% vs. 20.7%, respectively, P = 0.01), whereas no difference in percent variation of sCr levels was found between the two groups (8.2% vs. 3.1%, respectively; P = 0.13). Over a median follow-up of 56 months (range 1-137 months), 215 (37.7%) patients died. After multiple adjustments, ACEi/ARB never-use was associated with an almost twofold increased risk of all-cause mortality (HR 1.97, 95%CI 1.39-2.80). ACEi/ARB underuse in HFrEF is a standing issue with dramatic prognostic consequences. Efforts are needed to eliminate perceived contraindications to these drugs and ensure their implementation in real-life cardiology.

Arterial hypertension in cancer: The elephant in the room.

The great therapeutical success achieved by oncology is counterbalanced by growing evidences of cardiovascular (CV) toxicity due to many antineoplastic treatments. Cardiac adverse events may cause premature discontinuation of effective oncologic treatments or occur as late events undermining the oncologic success. Arterial hypertension is both the most common comorbidity in cancer patients and a frequent adverse effect of anticancer therapies. A pre-existing hypertension is known to increase the risk of other cardiac adverse events due to oncologic treatments, in particular heart failure. Moreover, as a strict association between cancer and CV diseases has emerged over the recent years, various analyses have shown a direct relationship between hypertension and cancer incidence and mortality. Finally, many antineoplastic treatments may cause a rise in blood pressure (BP) values, particularly the novel anti VEGF agents, this possibly compromising efficacy of chemotherapy. Aim of this review is to revise the topic and the many aspects linking arterial hypertension and cancer, and to provide a comprehensive and practical guide of the current treatment approaches.

An increase in myocardial 18-fluorodeoxyglucose uptake is associated with left ventricular ejection fraction decline in Hodgkin lymphoma patients treated with anthracycline.

BACKGROUND: Doxorubicin (DOX)-based chemotherapy for Hodgkin lymphoma (HL) yields excellent disease-free survival, but poses a substantial risk of subsequent left ventricular (LV) dysfunction and heart failure, typically with delayed onset. At the cellular level, this cardiotoxicity includes deranged cardiac glucose metabolism. METHODS: By reviewing the hospital records from January 2008 through December 2016, we selected HL patients meeting the following criteria: ≥ 18 year-old; first-line DOX-containing chemotherapy; no diabetes and apparent cardiovascular disease; 18-fluoro-deoxyglucose positron emission tomography (18FDG-PET) scans before treatment (PETSTAGING), after 2 cycles (PETINTERIM) and at the end of treatment (PETEOT); at least one echocardiography ≥ 6 months after chemotherapy completion (ECHOPOST). We then evaluated the changes in LV 18FDG standardized uptake values (SUV) during the course of DOX therapy, and the relationship between LV-SUV and LV ejection fraction (LVEF), as calculated from the LV diameters in the echocardiography reports with the Teicholz formula. RESULTS: Forty-three patients (35 ± 13 year-old, 58% males) were included in the study, with 26 (60%) also having a baseline echocardiography available (ECHOPRE). LV-SUV gradually increased from PETSTAGING (log-transformed mean 0.20 ± 0.27) to PETINTERIM (0.27 ± 0.35) to PETEOT (0.30 ± 0.41; P for trend < 0.001). ECHOPOST was performed 22 ± 17 months after DOX chemotherapy. Mean LVEF was normal (68.8 ± 10.3%) and only three subjects (7%) faced a drop below the upper normal limit of 53%. However, when patients were categorized by median LV-SUV, LVEF at ECHOPOST resulted significantly lower in those with LV-SUV above than below the median value at both PETINTERIM (65.5 ± 11.8% vs. 71.9 ± 7.8%, P = 0.04) and PETEOT (65.6 ± 12.2% vs. 72.2 ± 7.0%, P = 0.04). This was also the case when only patients with ECHOPRE and ECHOPOST were considered (LVEF at ECHOPOST 64.7 ± 8.9% vs. 73.4 ± 7.6%, P = 0.01 and 64.6 ± 9.3% vs. 73.5 ± 7.0%, P = 0.01 for those with LV-SUV above vs. below the median at PETINTERIM and PETEOT, respectively). Furthermore, the difference between LVEF at ECHOPRE and ECHOPOST was inversely correlated with LV-SUV at PETEOT (P < 0.01, R2 = - 0.30). CONCLUSIONS: DOX-containing chemotherapy causes an increase in cardiac 18FDG uptake, which is associated with a decline in LVEF. Future studies are warranted to understand the molecular basis and the potential clinical implications of this observation.

Serial Troponin for Early Detection of Nivolumab Cardiotoxicity in Advanced Non-Small Cell Lung Cancer Patients.

BACKGROUND: Rare cases of severe myocarditis are reported during treatment with nivolumab. Troponin, a biomarker of cardiac damage, is a key component of the diagnostic workup of many cardiac disorders, including myocarditis. This study investigates the role of troponin to assess cardiac involvement during nivolumab therapy for non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: We evaluated 59 NSCLC patients, analyzing serum samples collected within a translational research study. Troponin above the upper normal limit (0.046 ng/mL) was defined as Tn+, whereas normal but detectable troponin (0.015-0.045) was defined as Tndet. Troponin alterations were interpreted on the grounds of the following elements: peak values and time curve, cardiac comorbidities, signs and symptoms coincident to troponin elevation, ECG, echocardiography, and disease progression. RESULTS: No patient had cardiovascular events. Among 362 available blood samples, Tn+ (max 0.317 ng/mL) was found in 13 determinations belonging to 6 patients. Seven other patients had isolated Tndet. In five patients, Tn+ was attributed to cardiac comorbidities, disease progression, or worsening clinical status. One patient without cardiac history and in good clinical condition had a sustained troponin increase-soon after the start of therapy-and after careful evaluation of all relevant elements, it was interpreted as a marker of nivolumab-related subclinical myocarditis. CONCLUSION: Tn+ may occur in NSCLC patients treated with nivolumab, but in most cases it does not indicate nivolumab cardiotoxicity. In some cases, however, a careful interpretation of troponin alteration, especially at the beginning of therapy, enables identification of subclinical myocarditis, thus allowing early cardiac treatment. IMPLICATIONS FOR PRACTICE: Myocarditis is a rare but serious adverse event of immune checkpoint blockade with nivolumab, which needs to be recognized as soon as possible. This article suggests that troponin, a user-friendly biomarker of myocardial cytotoxicity, might be useful for early detection of immune-mediated myocarditis. However, because troponin abnormalities might also be related to a number of conditions capable of causing myocardial oxygen demand-supply mismatch, a careful cardiac assessment should be performed in non-small cell lung cancer patients in order to properly interpret any troponin increase. According to the available evidence, monitoring troponin during the first weeks of treatment can be considered reasonable.

Ivabradine in Cancer Treatment-Related Left Ventricular Dysfunction.

BACKGROUND: Patients developing cancer treatment-related left ventricular dysfunction (CTrLVD) require a prompt therapy. Hypotension, dizziness, and fatigue often limit the use of angiotensin-converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARB), and ß-blockers (BB) in cancer patients who may already be afflicted by these symptoms. Ivabradine is a heart rate-lowering drug that does not cause hypotension and may be used in heart failure with reduced left ventricular ejection fraction (LVEF). OBJECTIVE: The aim of this paper was to investigate the role of ivabradine to treat CTrLVD. METHODS: A retrospective analysis in a cohort of 30 patients with CTrLVD (LVEF < 50%) receiving ivabradine on top of the maximal tolerated dose of ACEi/ARB and BB was performed. We evaluated cardiovascular treatment, oncologic treatment, LVEF, functional class (New York Heart Association [NYHA]), and fatigue during the study period. RESULTS: Ivabradine was initially started at the dose of 2.5 mg/b.i.d. in most patients and then carefully titrated. Hypotension (70%) and fatigue (77%) were the main causes limiting the treatment with ACEi/ARB and BB. After a mean follow-up of 6.5 months, LVEF increased from 45.1% (SD = 6.4) to 53.2% (SD = 3.9; p < 0.001). When patients were analyzed according to the type of cancer therapy, no difference in LVEF changes across the groups was found. NYHA class ameliorated in 11 patients, while fatigue improved in 8 patients. No serious cardiovascular side effects were reported. CONCLUSIONS: The ability to improve symptoms and LVEF in unfit cancer patients makes ivabradine a reasonable pharmacological tool for treating CTrLVD.

Advanced Oxidation Protein Products-Modified Albumin Induces Differentiation of RAW264.7 Macrophages into Dendritic-Like Cells Which Is Modulated by Cell Surface Thiols.

Local accumulation of Advanced Oxidation Protein Products (AOPP) induces pro-inflammatory and pro-fibrotic processes in kidneys and is an independent predictor of renal fibrosis and of rapid decline of eGFR in patients with chronic kidney disease (CKD). In addition to kidney damage, circulating AOPP may be regarded as mediators of systemic oxidative stress and, in this capacity, they might play a role in the progression of atherosclerotic damage of arterial walls. Atherosclerosis is a chronic inflammatory disease that involves activation of innate and adaptive immunity. Dendritic cells (DCs) are key cells in this process, due to their role in antigen presentation, inflammation resolution and T cell activation. AOPP consist in oxidative modifications of proteins (such as albumin and fibrinogen) that mainly occur through myeloperoxidase (MPO)-derived hypochlorite (HOCl). HOCl modified proteins have been found in atherosclerotic lesions. The oxidizing environment and the shifts in cellular redox equilibrium trigger inflammation, activate immune cells and induce immune responses. Thus, surface thiol groups contribute to the regulation of immune functions. The aims of this work are: (1) to evaluate whether AOPP-proteins induce activation and differentiation of mature macrophages into dendritic cells in vitro; and (2) to define the role of cell surface thiol groups and of free radicals in this process. AOPP-proteins were prepared by in vitro incubation of human serum albumin (HSA) with HOCl. Mouse macrophage-like RAW264.7 were treated with various concentrations of AOPP-HSA with or without the antioxidant N-acetyl cysteine (NAC). Following 48 h of HSA-AOPP treatment, RAW264.7 morphological changes were evaluated by microscopic observation, while markers of dendritic lineage and activation (CD40, CD86, and MHC class II) and allogeneic T cell proliferation were evaluated by flow cytometry. Cell surface thiols were measured by AlexaFluor-maleimide binding, and ROS production was assessed as DCF fluorescence by flow cytometry. HSA-AOPP induced the differentiation of RAW264.7 cells into a dendritic-like phenotype, as shown by morphological changes, by increased CD40, CD86 and MHC class II surface expression and by induction of T cell proliferation. The cell surface thiols dose dependently decreased following HSA-AOPP treatment, while ROS production increased. NAC pre-treatment enhanced the amount of cell surface thiols and prevented their reduction due to treatment with AOPP. Both ROS production and RAW264.7 differentiation into DC-like cells induced by HSA-AOPP were reduced by NAC. Our results highlight that oxidized plasma proteins modulate specific immune responses of macrophages through a process involving changes in the thiol redox equilibrium. We suggest that this mechanism may play a role in determining the rapid progression of the atherosclerotic process observed in CKD patients.

Moderate Increase of Indoxyl Sulfate Promotes Monocyte Transition into Profibrotic Macrophages.

OBJECTIVE: The uremic toxin Indoxyl-3-sulphate (IS), a ligand of Aryl hydrocarbon Receptor (AhR), raises in blood during early renal dysfunction as a consequence of tubular damage, which may be present even when eGFR is normal or only moderately reduced, and promotes cardiovascular damage and monocyte-macrophage activation. We previously found that patients with abdominal aortic aneurysms (AAAs) have higher CD14+CD16+ monocyte frequency and prevalence of moderate chronic kidney disease (CKD) than age-matched control subjects. Here we aimed to evaluate the IS levels in plasma from AAA patients and to investigate in vitro the effects of IS concentrations corresponding to mild-to-moderate CKD on monocyte polarization and macrophage differentiation. METHODS: Free IS plasma levels, monocyte subsets and laboratory parameters were evaluated on blood from AAA patients and eGFR-matched controls. THP-1 monocytes, treated with IS 1, 10, 20 µM were evaluated for CD163 expression, AhR signaling and then induced to differentiate into macrophages by PMA. Their phenotype was evaluated both at the stage of semi-differentiated and fully differentiated macrophages. AAA and control sera were similarly used to treat THP-1 monocytes and the resulting macrophage phenotype was analyzed. RESULTS: IS plasma concentration correlated positively with CD14+CD16+ monocytes and was increased in AAA patients. In THP-1 cells, IS promoted CD163 expression and transition to macrophages with hallmarks of classical (IL-6, CCL2, COX2) and alternative phenotype (IL-10, PPARγ, TGF-ß, TIMP-1), via AhR/Nrf2 activation. Analogously, AAA sera induced differentiation of macrophages with enhanced IL-6, MCP1, TGF-ß, PPARγ and TIMP-1 expression. CONCLUSION: IS skews monocyte differentiation toward low-inflammatory, profibrotic macrophages and may contribute to sustain chronic inflammation and maladaptive vascular remodeling.

Prevalent cardiac, renal and cardiorenal damage in patients with advanced abdominal aortic aneurysms.

Chronic kidney disease (CKD), cardiac damage (CD) and the combination of the two are associated with increased morbidity and death in patients admitted to vascular surgery units. We assessed the prevalence of cardiac and renal damage and cardiorenal syndrome (CRS) in 563 patients with abdominal aortic aneurysms (AAA) who underwent cardiac screening before either an endovascular procedure (EVAR) or open surgery (OS) for aneurysm repair. CD was defined by ≥stage B as per the ACC/AHA classification of congestive heart failure (CHF), while CKD was defined by estimated GFR <60 mL/min/1.73 m(2) (CKD-EPI). Anemia [World Health Organization (WHO) guidelines] and iron deficiency (ID) (criteria for CHF patients) were also calculated. AAA patients were stratified into the following groups: CD, CKD, CRS or none of these conditions [no risk factors (NoRF)]. The prevalence of isolated cardiac and renal structural damage, of combined cardiorenal damage and of ID was 24.1, 15.0, 20.6 and 23.4 %, respectively. The frequency of anemia (mostly unrecognized) among the groups increased from NoRF (12.8 %)/CKD (19 %)/CD (25 %) up to CRS (38.8 %). This large-scale observational study provides clues for the increased CD/CKD risk profiles of unselected AAA patients, and underlines the need for better identification of ID/anemia and for appropriate treatment of CKD and CD before these patients undergo EVAR/OS.

Doxorubicin impairs the insulin-like growth factor-1 system and causes insulin-like growth factor-1 resistance in cardiomyocytes.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R). Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3), which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes. METHODS AND RESULTS: Besides inducing apoptosis, concentrations of doxorubicin comparable to those observed in patients after bolus infusion (0.1-1 µM) caused a progressive decrease in IGF-1R and increase in IGFBP-3 expression. Exogenous IGF-1 was capable to rescue cardiomyocytes from apoptosis triggered by 0.1 and 0.5 µM, but not 1 µM doxorubicin. The loss of response to IGF-1 was paralleled by a significant reduction in IGF-1 availability and signaling, as assessed by free hormone levels in conditioned media and Akt phosphorylation in cell lysates, respectively. Doxorubicin also dose-dependently induced p53, which is known to repress the transcription of IGF1R and induce that of IGFBP3. Pre-treatment with the p53 inhibitor, pifithrin-α, prevented apoptosis and the changes in IGF-1R and IGFBP-3 elicited by doxorubicin. The decrease in IGF-1R and increase in IGFBP-3, as well as apoptosis, were also antagonized by pre-treatment with the antioxidant agents, N-acetylcysteine, dexrazoxane, and carvedilol. CONCLUSIONS: Doxorubicin down-regulates IGF-1R and up-regulates IGFBP-3 via p53 and oxidative stress in H9c2 cells. This leads to resistance to IGF-1 that may contribute to doxorubicin-initiated apoptosis. Further studies are needed to confirm these findings in human cardiomyocytes and explore the possibility of manipulating the IGF-1 axis to protect against anthracycline cardiotoxicity.

Indoxyl sulfate: a candidate target for the prevention and treatment of cardiovascular disease in chronic kidney disease.

The awareness that chronic kidney disease (CKD) is a condition of dramatically increased cardiovascular risk has prompted an intense research activity, aimed at identifying factors that are specifically involved in the development of cardiovascular complications of CKD and that can be delayed or reduced by novel pharmacological approaches. This may be the case with indoxyl sulfate (IS). IS is an endogenous molecule derived from indole, a product of protein metabolism by intestinal bacteria, which acts via the aryl hydrocarbon receptor (AhR). IS accumulates early in CKD and exerts proinflammatory and other detrimental effects on the cardiovascular system, in particular promoting atherosclerosis and atherosclerosis-related arterial remodeling. Furthermore, IS also contributes to renal damage, thereby fueling a vicious circle. Dialysis is poorly effective in removing IS, but its levels can be lowered by preventing the bacterial generation of indole or by absorbing this latter within the intestine. More intriguing, although still theoretical, is the possibility of inhibiting the action of IS at the cell level, by antagonizing the binding to AhR or IS intracellular signaling. Therefore, IS targeting might become an option for reducing the cardiovascular burden of CKD.

Adipose tissue immune response: novel triggers and consequences for chronic inflammatory conditions.

Adipose tissue inflammation mediates the association between excessive body fat accumulation and several chronic inflammatory diseases. A high prevalence of obesity-associated adipose tissue inflammation was observed not only in patients with cardiovascular conditions but also in patients with inflammatory bowel diseases, abdominal aortic aneurysm, or cardiorenal syndrome. In addition to excessive caloric intake, other triggers promote visceral adipose tissue inflammation followed by chronic, low-grade systemic inflammation. The infiltration and accumulation of immune cells in the inflamed and hypertrophied adipose tissue promote the production of inflammatory cytokines, contributing to target organ damages. This comorbidity seems to delimit subgroups of individuals with systemic adipose tissue inflammation and more severe chronic inflammatory diseases that are refractory to conventional treatment. This review highlights the association between adipose tissue immune response and the pathophysiology of visceral adiposity-related chronic inflammatory diseases, while suggesting several new therapeutic strategies.

Role of bone mineral density in the inverse relationship between body size and aortic calcification: results from the Baltimore Longitudinal Study of Aging.

OBJECTIVE: There is a J-shaped relationship between body mass index (BMI) and cardiovascular outcomes in elderly patients (obesity paradox). Whether low BMI correlates with aortic calcification (AC) and whether this association is accounted for by bone demineralization is uncertain. METHODS: Presence of AC was evaluated in 687 community-dwelling individuals (49% male, mean age 67 ± 13 years) using CT images of the thoracic, upper and lower abdominal aorta, and scored from 0 to 3 according to number of sites that showed any calcification. Whole-body bone mineral density (BMD) was evaluated by dual-energy X-ray absorptiometry. Predictors of AC were assessed by logistic regression, and the role of BMD using mediation analysis. RESULTS: Age and cardiovascular risk factors were positively associated while both BMI (r = -0.11, p < 0.01) and BMD (r = -0.17, p < 0.0001) were negatively associated with AC severity. In multivariate models, lower BMI (OR 0.96, 95%CI 0.92-0.99, p = 0.01), older age, higher systolic blood pressure, use of lipid-lowering drugs and smoking were independent predictors of AC. A nonlinear relationship between BMI and AC was noticed (p = 0.03), with decreased AC severity among overweight participants. After adjusting for BMD, the coefficient relating BMI to AC was reduced by 14% and was no longer significant, whereas BMD remained negatively associated with AC (OR 0.82, 95%CI 0.069-0.96, p = 0.01), with a trend for a stronger relationship in older participants. CONCLUSION: Low BMI is associated with increased AC, possibly through calcium mobilization from bone, resulting in low BMD. Prevention of weight loss and bone demineralization with aging may help reducing AC.

CD16(+) monocyte subsets are increased in large abdominal aortic aneurysms and are differentially related with circulating and cell-associated biochemical and inflammatory biomarkers.

Proinflammatory components are present in abdominal aortic aneurysm (AAA). Circulating monocytes display heterogeneity, and three subsets have been identified, based on the differential expression for CD14 and CD16 receptors: CD14(+)CD16(−), classical, CD14(+)CD16(+), intermediate and CD14(dim)CD16(+), non-classical monocytes. Increased proinflammatory CD16+ monocytes with high expression of CD143 are present in CKD patients. D-dimer is increased in AAA patients, and might contribute to the pro-inflammatory response associated to circulating monocytes. We aimed to investigate the frequency of CD14(+)CD16(+), CD14(dim)CD16(+) monocytes and monocyte CD143 expression in AAA patients, and their relationship with Ddimer, eGFR and other inflammatory parameters. Blood from 74 AAA patients and 30 healthy controls was analyzed to determine the frequency of CD14(+)CD16(+), CD14(dim)CD16(+) monocytes and the monocyte CD143 expression by means of flow-cytometry. AAA patients had expanded CD16+ subsets (CD14(+)CD16(+): 7.66 ± 0.31% vs 5.42 ± 0.27%; CD14(dim)CD16(+): 7.43 ± 0.48% vs 5.54 ± 0.38%, AAA vs controls, mean ± SE, both p < 0.05). CD14(+)CD16(+) cells were associated to D-dimer and age, and to reduced eGFR. CD14(dim)CD16(+) cells were associated to uric acid, surface CD143, and reduced count of total leukocytes and neutrophils. Within AAA patients, the two CD16(+) subsets and the monocyte CD143 expression display different relationships with D-dimer, parameters of renal function and circulating biochemical and inflammatory biomarkers.

Inhibition of doxorubicin-induced senescence by PPARδ activation agonists in cardiac muscle cells: cooperation between PPARδ and Bcl6.

Senescence and apoptosis are two distinct cellular programs that are activated in response to a variety of stresses. Low or high doses of the same stressor, i.e., the anticancer drug doxorubicin, may either induce apoptosis or senescence, respectively, in cardiac muscle cells. We have demonstrated that PPARδ, a ligand-activated transcriptional factor that controls lipid metabolism, insulin sensitivity and inflammation, is also involved in the doxorubicin-induced senescence program. This occurs through its interference with the transcriptional repressor protein B cell lymphoma-6 (Bcl6). Low doses of doxorubicin increase the expression of PPARδ that sequesters Bcl6, thus preventing it from exerting its anti-senescent effects. We also found that L-165041, a specific PPARδ activator, is highly effective in protecting cardiomyocytes from doxorubicin-induced senescence through a Bcl6 related mechanism. In fact, L-165041 increases Bcl6 expression via p38, JNK and Akt activation, and at the same time it induces the release of Bcl6 from PPARδ, thereby enabling Bcl6 to bind to its target genes. L-165041 also prevented apoptosis induced by higher doses of doxorubicin. However, while experiments performed with siRNA analysis techniques very clearly showed the weight of Bcl6 in the cellular senescence program, no role was found for Bcl6 in the anti-apoptotic effects of L-165041, thus confirming that senescence and apoptosis are two very distinct stress response cellular programs. This study increases our understanding of the molecular mechanism of anthracycline cardiotoxicity and suggests a potential role for PPARδ agonists as cardioprotective agents.

Soluble form of the endothelial adhesion molecule CD146 binds preferentially CD16+ monocytes.

The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16-, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62-3.87) and of CD14+/CD16+ (2.59, 1.28-4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16- (0.66, 0.47-1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16- monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.

Direct relationship between cell density and FDG uptake in asymptomatic aortic aneurysm close to surgical threshold: an in vivo and in vitro study.

PURPOSE: Conflicting results have been reported about the clinical value of fluorodeoxyglucose (FDG) imaging in predicting the risk of rupture of abdominal aortic aneurysm (AAA). The present study tests the hypothesis that FDG uptake is low in asymptomatic noninflammatory AAA due to the low cell density in aneurysmal walls. METHODS: Positron emission tomography (PET)/CT imaging was performed in 12 consecutive candidates for AAA surgical repair and in 12 age- and sex-matched controls. At intervention, aneurysmal walls were cut into three sequential blocks. Block A was frozen to cut three 5-µm slices for incubation with 2-3 MBq of FDG for 5 min. Block C was first incubated with the same tracer solution for the same time and subsequently frozen to cut three 5-µm slices. Autoradiographic images were coregistered with immunohistochemical pictures of cell density, type and DNA synthesis as assessed on block B. RESULTS: No visible uptake in abdominal aorta occurred in any patient or control subject. Immunohistochemistry documented a severe loss of wall structure, with low numbers of cells. Tracer retention directly correlated with overall cell density and with prevalence of cells synthesizing DNA. The metabolic nature of FDG uptake was confirmed by the selective effect of preliminary freezing that decreased tracer content by 90% in regions with high cell density and only by 34% in cold acellular areas. CONCLUSION: The loss of tissue structure and the marked decrease in cell density account for the low prevalence of positive findings at FDG PET imaging, at least in asymptomatic patients bearing AAAs whose diameter is close to surgical indication.

Resveratrol counteracts systemic and local inflammation involved in early abdominal aortic aneurysm development.

BACKGROUND: Monocyte activation, macrophage infiltration, vascular oxidative stress and matrix proteolysis are inflammatory key steps contributing to abdominal aortic aneurysm (AAA) development. A phenotypical and functional heterogeneity is recognizable in monocytes by the differential expression of surface molecules: CD62L- subset corresponds to activated monocytes, while CD143/ACE surface expression increases during their differentiation into macrophages. In this work, Resveratrol, which is an antioxidant polyphenol with vasoprotective properties, has been evaluated for its potential to limit aneurysm development and monocyte-dependent inflammatory response in a model of elastase-induced AAA. METHODS: Male Sprague-Dawley rats received Resveratrol (10 mg/kg/die) (Rsv group, n=15) or vehicle (ethanol) alone (Et-OH group, n=15) continuously from 7 d before until 14 d after the AAA induction with elastase; five littermates were used as untreated control group (Ctr group, n=5). At the end of treatment, CD143 and CD62L monocyte expression was analyzed by flow cytometry, serum antioxidant capacity was evaluated using the TRAP method and circulating TNFα, and MMP-9 were measured with ELISA and gel zymography, respectively. Aortas were subjected to histology and immunohistochemistry for morphological analysis, macrophage infiltration, and MMP-9, TNFα, and VEGF expression. RESULTS: Resveratrol counteracted the CD62L-monocyte subset expansion, CD143 monocyte expression, and circulating levels of MMP-9 activity and TNFα associated to AAA induction. Similarly, treatment with Resveratrol significantly attenuated AAA expansion, vessel wall macrophage infiltration and MMP-9, VEGF, and TNFα expression, compared with AAA from Et-OH group. CONCLUSIONS: Resveratrol limited the monocyte-dependent inflammatory response, macrophage differentiation and aortic lumen enlargement in elastase-induced AAA. These data suggest that Resveratrol might be tested in selected patients with small AAA to modulate the early systemic and local inflammatory response associated to AAA progression.