Effect of Aminaphtone on in vitro vascular permeability and capillary–like maintenance

Aminaphtone (AMNA), a naphtohydrochinone used in the treatment of capillary disorders, may affect oedema in chronic venous insufficiency (CVI). Aim of study is to investigate the effect of AMNA on vascular endothelial permeability in vitro and its effects on three-dimensional capillary-like structures formed by Human Umbilical Vein Endothelial Cells (HUVECs). HUVECs were treated with 50 ng/ml VEGF for 2h and AMNA for 6h. Permeability assay, VE-cadherin expression and Matrigel assay were performed. VEGF-induced permeability was significantly decreased by AMNA in a range concentration of 1-20 μg/ml. AMNA restored VE-cadherin expression. Finally, 6h pretreatment with AMNA significantly preserved capillary-like structures formed by HUVECs on Matrigel up to 48 h compared to untreated cells. AMNA significantly protects endothelium permeability and stabilises endothelial cells organised in capillary-like structures, modulating VE-cadherin expression. These data might explain the clinical benefict of AMNA on CVI

Effects of lack of microRNA-34 on the neural circuitry underlying the stress response and anxiety

Stress-related psychiatric disorders, including anxiety, are complex diseases that have genetic, and environmental causes. Stressful experiences increase the release of prefrontal amygdala neurotransmitters, a response that is relevant to cognitive, emotional, and behavioral coping. Moreover, exposure to stress elicits anxiety-like behavior and dendritic remodeling in the amygdala. Members of the miR-34 family have been suggested to regulate synaptic plasticity and neurotransmission processes, which mediate stress-related disorders. Using mice that harbored targeted deletions of all 3 members of the miR-34-family (miR-34-TKO), we evaluated acute stress-induced basolateral amygdala (BLA)-GABAergic and medial prefrontal cortex (mpFC) aminergic outflow by intracerebral in vivo microdialysis. Moreover, we also examined fear conditioning/extinction, stress-induced anxiety, and dendritic remodeling in the BLA of stress-exposed TKO mice. We found that TKO mice showed resilience to stress-induced anxiety and facilitation in fear extinction. Accordingly, no significant increase was evident in aminergic prefrontal or amygdala GABA release, and no significant acute stress-induced amygdalar dendritic remodeling was observed in TKO mice. Differential GRM7, 5-HT2C, and CRFR1 mRNA expressionwas noted in the mpFC and BLA between TKO andWT mice. Our data demonstrate that the miR-34 has a critical function in regulating the behavioral and neurochemical response to acute stress and in inducing stress-related amygdala neuroplasticity

Prevention of Excessive Endothelin-1 Release in Sclerotherapy: In Vitro and In Vivo Studies.

Abstract BACKGROUND The foam sclerotherapy technique has become one of the most commonly used treatments for superficial venous insufficiency. Despite excellent results, few visual/neurologic disturbances have been recently reported; their pathogenesis is still debated but a correlation with endothelin-1 (ET-1) release from the treated vein has been proposed. OBJECTIVE The purpose of this work was to evaluate the ET-1 release after sclerotherapy and to investigate the effects of the anti-endothelin drug aminaphtone. METHODS AND MATERIALS As in vitro sclerotherapy model, an endothelial cell culture, mimicking vascular endothelium, was pretreated with aminaphtone and exposed to detergents. Cell survival and ET-1 release were measured. In in vivo experiments, 45 rats, fed with different aminaphtone-rich diets, were subjected to sclerotherapy, and the systemic ET-1 was measured. RESULTS A minaphtone cell exposure caused a statistically significant reduction in ET-1 release, both before and after in vitro sclerotherapy. Rats fed with aminaphtone showed a trend toward reduced mortality and a significant decrease of ET-1 release after sclerotherapy. CONCLUSION This is the first study in which an anti-endothelin agent was able to cause a significant reduction of ET-1 release during sclerotherapy. Although clinical studies are required, these findings might advocate the use of anti-endothelin agents in prophylaxis of neurologic or visual disturbances after sclerotherapy

La terapia chirurgica della malattia diverticolare in fase acuta. Nostra esperienza

The treatment of the acute diverticolitis is still a stimulating and complex problem sustained by several anatomopatological and clinical factors and the possibility of different therapeutic options, being the operative mortality among 5% and 45%. With the modern technologies it is possible to follow the evolution of the illness so to perform more appropriate therapeutic plan. From 1997 to 2007 we have observed 278 patients with acute diverticolitis. In 219 (78,7%) patients the inflammatory and sub-occlusive condition has been faced with medical therapy, with resolution of the disease in 170 (61%) cases. In 49 (17,6%) patients we have gotten the resolution of the inflammatory disease, but not of the sub-occlusion and therefore we liked to submit them to surgical treatment in election. In 1 case we have found a colovesical fistula. A total of 59 (21,2%) patients with signs of acute abdomen have been submitted to surgery in urgency, within the 24 hours from the hospitalization. We have performed a primary resection with anastomosis and without stomia in all the patients, except in 3 cases in which we have done the Hartmann procedure for the cheap general conditions. We have not recorded intra and postoperative mortality and only in 3 cases we have had a leakage, that has not needed a surgical treatment. In 9 cases we observed infection of the wound, treated with antibiotic therapy. In our experience, performing a surgical procedure, without derivative stomia and manual anastomosis, it seems to be the fittest and less expensive procedure, also in situation of emergency-urgency, without increase of mortality and morbility

Chitosan-Based Nanoparticles Containing Cherry Extract from Prunus avium L. to Improve the Resistance of Endothelial Cells to Oxidative Stress

Cherries are known for their nutraceutical properties, in particular for their antioxidant ability due to their polyphenol content, which causes a reduction of cardiovascular disease (CVD) risk factors. However, once ingested these molecules are degraded in the Gastrointestinal (GI) tract before reaching the blood, which is the action site. The object of the present work is to evaluate the ability of cherry extract (CE), encapsulated in nanoparticles (NPs) based on different chitosan (Ch) derivatives, to promote a protective effect of human umbilical vein endothelial cells (HUVECs) involved in vascular dysfunction against oxidative stress. CE-loaded NPs based on quaternary ammonium chitosan (NP1) and an S-protected thiolated derivative thereof (NP2) were prepared. The mean particle size (NP1 344.9 ± 17.8, NP2 339.9 ± 68.2 nm), the polydispersity index, the encapsulation efficiency (NP1 78.4 ± 4.5, NP2 79.8 ± 0.6%), and the zeta potential (NP1 14.8 ± 0.3, NP2 15.8 ± 0.5 mV) did not appear to be significantly different. Both NP types improved the CE apparent permeation parameters with respect to the control. Conversely, CE-loaded NP2 protected HUVECs from oxidative stress and reduced reactive oxygen species (ROS) production more than CE-loaded NP1 and free CE. In addition to promoting HUVEC resistance, NP2 could be a useful tool to overcome the problem of cherry seasonality

RENAL DENERVATION RAPIDLY RESTORES CIRCULATING PROGENITOR CELLS IN PATIENTS AFFECTED BY RESISTANT HYPERTENSION

Objective: To investigate whether blood pressure (BP) lowering after renal sympathetic denervation (RSD) affects CD34+ cell number in drug-resistant hypertension (R-HTN). Design and method: We enrolled 11 patients with R-HTN, already treated with at least 6 antihypertensive drugs, including a diuretic, at full dosages; patients with offi ce BP of > 160 mmHg (>150 mmHg for type 2 diabetes) were considered eligible for the procedure. Adherence to drug treatment was accurately checked by patient’s general practitioners. Mean age was 61 ± 7.9 years; M: F 8:5. We measured clinic (sphygmomanometer) and ambulatory (Tonoport V GEHealthcare) BP, and heart rate (HR; electrocardiogram), at baseline and 30 days after RSD procedure (Symplicity; Medtronic). 24 h BP recordings and home BP protocols were consulted in addition to offi ce BP measurements at the hospital before enrollment. Results: At T0: SBP: 179.1 ± 9.3mmHg; DBP: 101.2 ± 5.5 mmHg; HR 79.9 ± 9.4; CD34+ cells: 1.66 ± 0.51. At T1 SBP values were reduced on the average of 40.2 mmHg (138.9 ± 7.3; –22.5%, p < 0.001) DBP of 18 mmHg (83.2 ± 3.2; –17.7%, p < 0.001), and HR of 10.4 bpm (67.3 ± 6.0; -17.7%, p < 0.005), and CD34+cell number increased on an average of 0.34 cells /microL (2.0 ± 0.51; +21.2%, p < 0.001). Conclusions: RSD rapidly restores CD34+cell number in patients affected by true R-HTN; if these results will be confi rmed on a larger scale, they could provide new insights about CD34+ cells and pathophysiological aspects of arterial hypertension

Effect of platelet lysate on human cells involved in different phases of wound healing

Background Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Methodology/Principal Findings Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p&lt;0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. Conclusion/Significance These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing

Fibrin gel: a new scaffold for cardiovascular applications

Aims: Peripheral blood endothelial progenitor cells (EPC) are promising therapies for irreversible myocardial damage, heart failure and peripheral ischemia disease. Natural biopolymers as fibrin are appealing in tissue engineering, because fibrin is biocompatible and bioresorbable. In vitro studies indicate that fibrin can support the growth migration and proliferation of several cells types. Up to date numerous studies have proved the potential of fibrin based injectable cell delivery systems. No studies are available with fibrin as scaffold for EPC. The goal of this study was to investigate if fibrin is a suitable matrix for EPC culture as compared with fibronectin and if different concentrations of fibrinogen (Fb) and thrombin (Th) can influence fibrin structure and EPC behaviour. Methods: Fibrin (Kedrion S.p.a. Lucca, Italy) was prepared mixing Fb (final 4.5-9-18-36 mg/ml) and Th (final 6-12.5-25-50 U/ml). The scaffolds were maintained for 1 hour at 37?C, 5% CO2 before cell seeding. The ultrastructure of fibrin was investigated by scanning electron microscopy (SEM), cryogenic SEM (CRYO-SEM) and atomic force microscopy (AFM) that allow the hydratating analysis of the sample, to evaluate fibre diameter and density. EPC were obtained from peripheral blood of healthy donors and cultured for 1 week on fibrin at the concentration of 1x106 cell/ml in endothelial growth medium. EPC seeded on fibronectin were used as control. Metabolic cell activity on the different scaffolds was assessed after 7 and 14 days by WST1 while cell viability by confocal microscopy (Calcein AM incorporation). Results: Fibrin polymerization rate ranged between 17 and 68 seconds and increased at higher Fb or Th concentrations. Both AFM and SEM analysis revealed a nanometric fibrous structure, with a decrease in fiber diameter with higher fibrinogen concentrations (4.5 mg/ml: 166?4 nm. vs. 36 mg/ml: 119?3 nm, p<0.005, n=5). Different concentrations of Th didn\u27t affect fibre diameter and density. CRYO-SEM suggested a reticulate structure with mesh-size up to 10?m. WST1 assay showed that EPC metabolic activity was better with lower fibrinogen concentrations (4.5 mg/ml: 0.890?0.134 a.u. vs. 36 mg/ml 0.234?0.046 a.u., p<0.05, n=5), while Th had no significant effect. Calcein staining demonstrated that EPC were viable at 14 days and even organised in cluster. Conclusions: Fibrin combines important properties of an ideal biological scaffold, like the nanometric structure, important for the growth and migration of cells. Fibrin is also an ideal scaffold for EPC but the ratio between fibrinogen and thrombin is important for cell viability

Correction to: MicroRNA-34 Contributes to the Stress-related Behavior and Affects 5-HT Prefrontal/GABA Amygdalar System through Regulation of Corticotropin-releasing Factor Receptor 1

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Development of a new technology for 3-D nanostructured scaffolds with potential cardiovascular applications

Aims The in situ release and maintaining of cells to promote revascularization is a new goal of cardiovascular therapy. Endothelial progenitor cells (EPC) may contribute to the process of vascular repair. Medical devices realized according to tissue engineering are composed by a cellular component and by an artificial component, usually made of a biocompatible polymer. Scaffolds may be coated with bio-polymers like fibrin to enhance cell adhesion and growth. Aim of this study was to realize nanocomposite 3D scaffolds composed by a synthetic polymer coated with fibrin to support EPC growth and to promote in vivo angiogenesis. Methods 3D PEtU-PDMS scaffolds were studied in vitro for their biocompatibility (viability and proliferation tests; citokine release). In vivo biocompatibility was studied by intramuscular implant in a rabbit model. The scaffolds were fabricated by spray-phase inversion technique. 25U/mL thrombin was sprayed during the fabrication process. The composite scaffold was then incubated o.n. at 37?C with 18mg/mL fibrinogen. The scaffold morphology was analysed by stereo-microscopy and by scanning electron microscopy (SEM). EPC obtained from peripheral blood were cultured for 1 week on the scaffolds at the concentration of 1x106 cell/ml. Fibronectin coating was used as a control. Cell viability was assessed by confocal laser (Calcein-AM incorporation). To test in vivo angiogenesis, EPC-seeded scaffolds were subcutaneously implanted into the back of rats for 14 days. After harvesting, the scaffolds were examined histologically and immunohistochemically to evaluate inflammatory response and neovascularization. Results In vitro and in vivo biocompatibility data demonstrated absence of any citotoxic effect, immunocompatibility and a slight inflammatory reaction without any sign of encapsulation and implant rejection. Morphological analyses showed an homogeneus fibrin coating of the scaffolds, tightly bound and interconnected to the PEtU-PDMS surface. SEM showed the presence of a well organized layer of fibres in a nm scale (mean diameter ~140nm). Cell viability and phenotype were not affected when EPC were seeded on PEtU-PDMS/fibrin scaffolds. The histological observation of explanted scaffolds revealed a slightly inflammatory response and a significant increased numbers of neovessels in tissues surrounding the EPC-seeded scaffold as compared to the scaffold without cells. Conclusions Our data suggest that PEtU-PDMS/fibrin scaffold obtained with a new spray manufacturing technology can support in vitro EPC growth and promote in vivo neovascularisation. Further studies are currently under way in an ischemic hindlimb rat model