Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction: Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition. Methods: Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients. Results: In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r(2) = 0.33 and r(2) = 0.34 respectively, p < 0.0015). In patients with a low platelet count (<150 x 10(9) platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r(2) = 0.33 and r(2) = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05). Conclusions: There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count. (C) 2009 Elsevier Ltd. All rights reserved.

Collagen is reduced and disrupted in human aneurysms and dissections of ascending aorta

In ascending aorta aneurysms, there is an enlargement of the whole vessel, whereas aortic dissections (ADs) are characterized by the cleavage of the wall into 2 sheets at the external half. We searched if alterations in collagen could be related to these diseases. Sections of aortas from 14 case patients with acute dissections, 10 case patients with aneurysms, and 9 control subjects were stained with picrosirius. Slides were analyzed under polarized microscopy to evaluate the structure of collagen fibers. The proportion of collagen was calculated in each half of the medial layer by color detection in a computerized image analysis system. Collagen appearance under polarized light was consistent with collagenolysis. The mean collagen proportions at the inner and outer halves, respectively, were 0.50 +/- 0.13 and 0.40 +/- 0.08 in the control group, 0.20 +/- 0.10 and 0.18 +/- 0.12 in the AD group, and 0.33 +/- 0.12 and 0.19 +/- 0.12 in the aneurysm group. The AD (P < .01) and control (P = .04) groups had less collagen at the external half, no difference was found in the aneurysm group (P = .71). In both halves, there was less collagen in the case patients than in the control subjects (all P < .01), but at the internal half, the decrease was significantly greater in the case patients with aneurysms than in those with dissections (P = .03; at the external half, P = .99). Aortic dissections and aneurysms show a decrease in collagen content that could be related to a weakness of the wall underlying the diseases, but the locations of the decrease differ: in dissections, it is situated mostly at the external portion of the media (site of cleavage), whereas in aneurysms, it is more diffuse, consistent with the global enlargement. (c) 2008 Elsevier Inc. All rights reserved.

Sequential Changes of Longitudinal and Radial Myocardial Deformation Indices in the Healthy Neonate Heart

Background: Significant hemodynamic changes, including preload and afterload modifications, occur during the transition from the fetal to the neonatal environment. The ductus arteriosus closes, pulmonary vascular resistance decreases, and pulmonary blood flow increases. Strain rate (SR) and strain (e) have been proposed as ultrasound indices for quantifying regional wall deformation. This study was designed to determine if these indices can detect variations in regional deformation between early and late neonatal periods. Methods: Data were obtained from 30 healthy neonates (15 male). The initial study was performed at a mean age of 20.1614 hours (exam 1) and the second at 31.962.9 days (exam 2). Apical and parasternal views were used to quantify regional left ventricular (LV) and right ventricular (RV) longitudinal and radial SR and e, and systolic, early, and late diastolic values were calculated from these curves. A paired-samples t test was performed comparing the two groups. Results: Compared with exam 1, LV radial deformation showed significant reductions in peak systolic e in the basal and mid segments (51615% vs 4669%, P < .01). LV longitudinal deformation behaved similarly, showing significant peak systolic e reductions in all measured segments. Systolic SR showed reductions only in the basal and apical segments of the lateral wall and in the mid portion of the inferior wall (-1.9 +/- 0.5 vs -1.7 +/- 0.3 s(-1) and -1.9 +/- 0.4 vs -1.7 +/- 0.2 s(-1), respectively, P = .03). RV longitudinal free and inferior wall systolic SR and e values were significantly higher in exam 2. Conclusions: LV peak systolic e decreases in exam 2 were possibly due to afterload increase and preload decrease. The lower RV initial deformation indices could be attributed to increased afterload caused by physiologic pulmonary hypertension or immature RV contractile properties. SR seemed to be a more robust index than e and less influenced by preload and afterload hemodynamic alteration. (J Am Soc Echocardiogr 2010;23:294-300.)

Intramyocardial transplantation of fibroblasts expressing vascular endothelial growth factor attenuates cardiac dysfunction

In this study, we analyzed whether transplantation of cardiac fibroblasts (CFs) expressing vascular endothelial growth factor (VEGF) mitigates cardiac dysfunction after myocardial infarction (MI) in rats. First, we observed that the transgene expression lasts longer (45 vs 7 days) when fibroblasts are used as vectors compared with myoblasts. In a preventive protocol, induction of cardiac neovascularization accompanied by reduction in myocardial scar area was observed when cell transplantation was performed 1 week before ischemia/reperfusion and the animals analyzed 3 weeks later. Finally, the therapeutic efficacy of this approach was tested injecting cells in a fibrin biopolymer, to increase cardiac retention, 24 h post-MI. After 4 weeks, an increase in neovascularization and a decrease in myocardial collagen were observed only in rats that received cells expressing VEGF. Basal indirect or direct hemodynamic measurements showed no differences among the groups whereas under pharmacological stress, only the group that received cells expressing VEGF showed a significant reduction in end-diastolic pressure and improvement in stroke volume and cardiac work. These results indicate that transplantation of CFs expressing VEGF using fibrin biopolymer induces neovascularization and attenuates left ventricle fibrosis and cardiac dysfunction in ischemic heart. Gene Therapy (2010) 17, 305-314; doi:10.1038/gt.2009.146; published online 10 December 2009

Cardiovascular prevention in HIV patients: Results from a successful intervention program

Objectives: To analyze the effect of a prevention program oil the estimated cardiovascular risk calculated by three risk scores. Methods: We prospectively evaluated 87 HIV+ patients with elevated cardiovascular risk estimation. Framirigham (FIRS), PROCAM and National Cholesterol Education Program (ATP-III) were applied. Cardiovascular risk was defined as elevated if >10%. All patients received non-pharmacological (diet, exercise, smoking cessation) and, when appropriate, pharmacological therapy. Results: Mean age was 52 years, 92% were male, 39.1% were smokers, 70.1% had hypertension, 18.4% had diabetes. All patients were under HAART, 56.3% were receiving protease inhibitors (131). After 6 months, intervention was associated to significant changes oil triglycerides (298 242 and 206 +/- 135 mg/dL, p<0.05), total-cholesterol (224 +/- 47 and 189 +/- 38 mg/dL, p<0.001). LDL-cholesterol (129 +/- 44 and 109 +/- 30 mg/dL,p<0.001). Frequencies of patients with elevated cardiac risk before and 6 months after intervention were 92% x 27.6% (p < 0.0001), 80.5% x 50.6% (p < 0.0002), and 25.3% x 14.9% (p = 0.12), for FIRS, ATP III and PROCAM, respectively. Conclusions: An intervention Program focused on reduction of traditional risk factors was able to decrease the frequency of patients with HIV infection and elevated cardiovascular risk estimation. FIRS showed greater sensitivity than the other scores. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Refractory Angina Cell Therapy (ReACT) Involving Autologous Bone Marrow Cells in Patients Without Left Ventricular Dysfunction: A Possible Role for Monocytes

Autologous bone marrow mononuclear cell (BMMC) transplantation has emerged as a potential therapeutic option for refractory angina patients. Previous studies have shown conflicting myocardium reperfusion results. The present study evaluated safety and efficacy of CellPraxis Refractory Angina Cell Therapy Protocol (ReACT). in which a specific BMMC formulation was administered as the sole therapy for these patients. The phase I/IIa noncontrolled, open label. clinical trial, involved eight patients with refractory angina and viable ischemic myocardium, without left ventricular dysfunction and who were not suitable for conventional myocardial revascularization. ReACT is a surgical procedure involving a single series of multiple injections (40-90 injections, 0.2 ml each) into ischemic areas of the left ventricle. Primary endpoints were Canadian Cardiovascular Society Angina Classification (CCSAC) improvement at 18 months follow-up and myocardium ischemic area reduction (assessed by scintigraphic analysis) at 12 months follow-up, in correlation with a specific BMMC formulation. Almost all patients presented progressive improvement in angina classification beginning 3 months (p = 0.008) postprocedure which was sustained at 18 months follow-up (p = 0.004), as well as objective myocardium ischemic area reduction at 12 months (decrease of 84.4%, p < 0.004). A positive correlation was found between monocyte concentration and CCSAC improvement (r = -0.759, p < 0.05). Improvement in CCSAC, followed by correlated reduction in scintigraphic myocardium ischemic area, strongly suggests neoangiogenesis as the main stem cell action mechanism. The significant correlation between number of monocytes and improvement strongly supports a cell-related effect of ReACT. ReACT appeared safe and effective.

Continuous positive airway pressure increases inspiratory capacity of COPD patients

Background and objective: Hyperinflation with a decrease in inspiratory capacity (IC) is a common presentation for both unstable and stable COPD patients. As CPAP can reduce inspiratory load, possibly secondary to a reduction in hyperinflation, this study examined whether CPAP would increase IC in stable COPD patients. Methods: Twenty-one stable COPD patients (nine emphysema, 12 chronic bronchitis) received a trial of CPAP for 5 min at 4, 7 and 11 cmH(2)O. Fast and slow VC (SVC) were measured before and after each CPAP trial. In patients in whom all three CPAP levels resulted in a decreased IC, an additional trial of CPAP at 2 cmH(2)O was conducted. For each patient, a `best CPAP` level was defined as the one associated with the greatest IC. This pressure was then applied for an additional 10 min followed by spirometry. Results: Following application of the `best CPAP`, the IC and SVC increased in 15 patients (nine emphysema, six chronic bronchitis). The mean change in IC was 159 mL (95% CI: 80-237 mL) and the mean change in SVC was 240 mL (95% CI: 97-386 mL). Among these patients, those with emphysema demonstrated a mean increase in IC of 216 mL (95% CI: 94-337 mL). Six patients (all with chronic bronchitis) did not demonstrate any improvement in IC. Conclusions: The best individualized CPAP can increase inspiratory capacity in patients with stable COPD, especially in those with emphysema.

Real-time and Doppler US after pediatric segmental liver transplantation

Background Accurate diagnosis of portal vein (PV) stenosis by real-time and color Doppler US (CD-US) after segmental liver transplantation in children can decrease morbidity by avoiding unnecessary biopsy, PV hypertension, thrombosis and loss of the graft. Objective To evaluate CD-US parameters for the prediction of PV stenosis after segmental liver transplantation in children. Materials and methods We retrospectively reviewed 61 CD-US examinations measuring the diameter at the PV anastomosis, velocities at the anastomosis (PV1) and in the segment proximal to the anastomosis (PV2), and the PV1/PV2 velocity ratio. The study group comprised patients with stenosis confirmed by angiography and the control group comprised patients with a good clinical outcome. Results PV stenosis was seen in 12 CD-US examinations. The mean PV diameter was smaller in the study group (2.6 mm versus 5.7 mm) and a PV diameter of < 3.5 mm was highly predictive of stenosis (sensitivity 100%, specificity 91.8%). Conclusion A PV diameter of < 3.5 mm is a highly predictive CD-US parameter for the detection of hemodynamically significant stenosis on angiography.

Endostatin- and interleukin-2-expressing retroviral bicistronic vector for gene therapy of metastatic renal cell carcinoma

Background Metastatic renal cell carcinoma (mRCC) is one of the most treatment-resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)-2 using an orthotopic metastatic RCC mouse model. Methods Balb/C-bearing Renca cells were treated with NIH/3T3-LendIRES-IL-2-SN cells. In the survival studies, mice were monitored daily until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and the number of metastatic nodules, nodule area, tumor vessels and proliferation of tumor-infiltrating Renca cells were determined. Results Inoculation of NIH/3T3-LendIRES-IL-2-SN cells resulted in an increase in ES and IL-2 levels in the treated group (p < 0.05). There was a significant decrease in lung wet weight, lung nodule area and tumor vessels in the treated group compared to the control group (p < 0.001). The proliferation of Renca cells in the bicistronic-treated group was significantly reduced compared to the control group (p < 0.05). Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice submitted to bicistronic therapy (log-rank test, p = 0.0016). Bicistronic therapy caused an increase in the infiltration of CD4, CD4 interferon (IFN)gamma-producing, CD8, CD8 IFN gamma-producing and natural killer (CD49b) cells. Conclusions Retroviral bicistronic gene transfer led to the secretion of functional ES and IL-2 that was sufficiently active to: (i) inhibit tumor angiogenesis and tumor cell proliferation and (ii) increase the infiltration of immune cells (C) Copyright. 2011 John Wiley & Sons, Ltd.

Endostatin gene therapy enhances the efficacy of IL-2 in suppressing metastatic renal cell carcinoma in mice

We investigated whether the administration of IL-2 combined with endostatin gene therapy was able to produce additive or even synergistic immunomodulatory activity in a mouse model of metastatic renal carcinoma. Renca cells were injected into the tail vein of BALB/c mice. After 24 h, the animals were randomly divided into four groups (5 mice/group). One group of mice was the control, the second group received treatment with 100,000 UI of Recombinant IL-2 (Proleukin, Chiron) twice a day, 1 day per week during 2 weeks (IL-2), the third group received treatment with a subcutaneous inoculation of 3.6 x 10(6) endostatin-producing cells, and the fourth group received both therapies (IL-2 + ES). Mice were treated for 2 weeks. In the survival studies, 10 mice/group daily, mice were monitored daily until they died. The presence of metastases led to a twofold increase in endostatin levels. Subcutaneous inoculation of NIH/3T3-LendSN cells resulted in a 2.75 and 2.78-fold increase in endostatin levels in the ES and IL-2 + ES group, respectively. At the end of the study, there was a significant decrease in lung wet weight, lung nodules area, and microvascular area (MVA) in all treated groups compared with the control group (P < 0.001). The significant difference in lung wet weight and lung nodules area between groups IL-2 and IL-2 + ES revealed a synergistic antitumor effect of the combined treatment (P < 0.05). The IL-2 + ES therapy Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice treated with the combined therapy (log-rank test, P = 0.0028). Conjugated therapy caused an increase in the infiltration of CD4, CD8 and CD49b lymphocytes. An increase in the amount of CD8 cells (P < 0.01) was observed when animals received both ES and IL-2, suggesting an additive effect of ES over IL-2 treatment. A synergistic effect of ES on the infiltration of CD4 (P < 0.001) and CD49b cells (P < 0.01) was also observed over the effect of IL-2. Here, we show that ES led to an increase in CD4 T helper cells as well as cytotoxic lymphocytes, such as NK cells and CD8 cells, within tumors of IL-2 treated mice. This means that ES plays a role in supporting the actions of T cells.

Neural activity changes to emotional stimuli in healthy individuals under chronic use of clomipramine

Previous functional magnetic resonance imaging (fMRI) studies examined neural activity responses to emotive stimuli in healthy individuals after acute/subacute administration of antidepressants. We now report the effects of repeated use of the antidepressant clomipramine on fMRI data acquired during presentation of emotion-provoking and neutral stimuli on healthy volunteers. A total of 12 volunteers were evaluated with fMRI after receiving low doses of clomipramine for 4 weeks and again after 4 weeks of washout. Fear-, happiness-, anger-provoking and neutral pictures from the International Affective Picture System (IAPS) were used. Data analysis was performed with statistical parametric mapping (P < 0.05). Paired t-test comparisons for each condition between medicated and unmedicated states showed, to negative valence paradigms, decrease in brain activity in the amygdala when participants were medicated. We also demonstrated, across both positive and negative valence paradigms, consistent decreases in brain activity in the medicated state in the anterior cingulate gyrus and insula. This is the first report of modulatory effects of repeated antidepressant use on the central representation of somatic states in response to emotions of both negative and positive valences in healthy individuals. Also, our results corroborate findings of antidepressant-induced temporolimbic activity changes to emotion-provoking stimuli obtained in studies of subjects treated acutely with such agents.

Galectin-3 plays a modulatory role in the life span and activation of murine neutrophils during early Toxoplasma gondii infection

Galectins are beta-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (N phi) from C57BL/6 wildtype (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MU assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in NO culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in N phi cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Not. number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates gondii-induced N phi toxicity as well as N phi degranulation regardless of infection. Furthermore, Gal-3 expression by N phi was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-alpha later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in N phi following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in N phi life span and activation during early T gondii infection. (C) 2009 Elsevier GmbH. All rights reserved.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Purpose We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the antihypertensive drug losartan (LOS). Results We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumorassociated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Conclusions Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGF beta 1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. (C) 2009 UICC

Concomitant Lucio Phenomenon and Erythema Nodosum in a Leprosy Patient: Clues for Their Distinct Pathogeneses

Lepromatous leprosy patients may develop necrotic lesions, usually in the context of Lucio phenomenon (LP) or severe erythema nodosum (EN). The clinical and histopathological characteristics of the necrotic manifestations of both entities may eventually be confounded. We describe a patient with lepromatous leprosy who developed, since the 4th month of her first pregnancy, recurrent necrotic lesions in lower limbs, which, at the postpartum, worsened and led to partial destruction of ears and nose. In addition, she referred painful nodes oil upper limbs since I year before pregnancy and intermittent swelling and tenderness of the ankles, which together with a right tibial and ulnar neuritis led to the diagnosis of, erythema nodosum leprosum (ENL). The histopathology of a biopsy of the upper limb (ENL) revealed a dermal-hypodermal inflammation with vasculitis and vascular lumen narrowing, whereas biopsy of the lower limb (LP) revealed small vessels with fibrin thrombi on the superficial layer of the dermis without inflammatory infiltrate and no evidence of vasculitis. Thus, besides having several different clinical features, LP and ENL result from different pathogenetic mechanisms. The histopathological and clinical features distinguishing both entities are proposed. This distinction is important because decrease in bacillary load through multidrug therapy is the main target in LP, whereas in ENL, concomitant reduction of the reaction by means of thalidomide or high-dose steroids is recommended.