Emanuela Fiumana

Antiproliferative effect of sildenafil on human pulmonary artery smooth muscle cells

AbstractPulmonary arterial hypertension (PAH) is characterized by vasoconstriction and by obstructive changes of the pulmonary vasculature including smooth muscle cell proliferation which leads to medial hypertrophy and subsequent luminal narrowing. Sildenafil, an orally active inhibitor of cGMP phosphodiesterase–type–5, exerts pulmonary vasodilator activity in PAH patients. We evaluated the effects of sildenafil on growth of cultured human pulmonary artery smooth muscle cells (PASMC). The results indicate that sildenafil reduced DNA synthesis stimulated by PDGF and dose dependently inhibited PASMC proliferation. These effects were paralleled by a progressive increase in cGMP content, followed by an accumulation of cAMP. The treatment with 8–bromo–cGMP or dibutyryl–cAMP mimicked all the effects of sildenafil. On the other hand, treatment of PASMC with inhibitors of cGMP–dependent protein kinase (PKG) or cAMP–dependent protein kinase (PKA) reversed the antiproliferative effect of sildenafil. In addition, sildenafil inhibited the phosphorylation of ERK, a converging point for several pathways leading to cell proliferation. This effect was partially reduced by PKG inhibition and completely abolished by PKA inhibition.We conclude that sildenafil exerts an antiproliferative effect on human PASMC that is mediated by an interaction between the cGMP–PKG and the cAMP–PKA activated pathways, leading to inhibition of PDGF–mediated activation of the ERK.

Restored perfusion and reduced inflammation in the infarcted heart after grafting stem cells with a hyaluronan-based scaffold

The aim of this study is to investigate the blood perfusion and the inflammatory response of the myocardial infarct area after transplanting a hyaluronan‐based scaffold (HYAFF®11) with bone marrow mesenchymal stem cells (MSCs). Nine‐week‐old female pigs were subjected to a permanent left anterior descending coronary artery ligation for 4 weeks. According to the kind of the graft, the swine subjected to myocardial infarction were divided into the HYAFF®11, MSCs, HYAFF®11/MSCs and untreated groups. The animals were killed 8 weeks after coronary ligation. Scar perfusion, evaluated by Contrast Enhanced Ultrasound echography, was doubled in the HYAFF®11/MSCs group and was comparable with the perfusion of the healthy, non‐infarcted hearts. The inflammation score of the MSCs and HYAFF®11/MSCs groups was near null, revealing the role of the grafted MSCs in attenuating the cell infiltration, but not the foreign reaction strictly localized around the fibres of the scaffold. Apart from the inflammatory response, the native tissue positively interacted with the HYAFF®11/MSCs construct modifying the extracellular matrix with a reduced presence of collagene and increased amount of proteoglycans. The border‐zone cardiomyocytes also reacted favourably to the graft as a lower degree of cellular damage was found. This study demonstrates that the transplantation in the myocardial infarct area of autologous MSCs supported by a hyaluronan‐based scaffold restores blood perfusion and almost completely abolishes the inflammatory process following an infarction. These beneficial effects are superior to those obtained after grafting only the scaffold or MSCs, suggesting that a synergic action was achieved using the cell‐integrated polymer construct.

Enhanced engraftment and repairing ability of human adipose-derived stem cells, conveyed by pharmacologically active microcarriers continuously releasing HGF and IGF-1, in healing myocardial infarction in rats.

One of the main cause of ineffective cell therapy in repairing the damaged heart is the poor yield of grafted cells. To overcome this drawback, rats with 4-week-old myocardial infarction (MI) were injected in the border zone with human adipose-derived stem cells (ADSCs) conveyed by poly(lactic-co-glycolic acid) microcarriers (PAMs) releasing hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) (GFsPAMs). According to treatments, animals were subdivided into different groups: MI_ADSC, MI_ADSC/PAM, MI_GFsPAM, MI_ADSC/GFsPAM, and untreated MI_V. Two weeks after injection, a 31% increase in ADSC engraftment was observed in MI_ADSC/PAM compared with MI_ADSC (p < 0.05). A further ADSC retention was obtained in MI_ADSC/GFsPAM with respect to MI_ADSC (106%, p < 0.05) and MI_ADSC/PAM (57%, p < 0.05). A 130% higher density of blood vessels of medium size was present in MI_ADSC/GFsPAM compared with MI_ADSC (p < 0.01). MI_ADSC/GFsPAM also improved, albeit slightly, left ventricular remodeling and hemodynamics with respect to the other groups. Notably, ADSCs and/or PAMs, with or without HGF/IGF-1, trended to induce arrhythmias in electrically driven, Langendorff-perfused, hearts of all groups. Thus, PAMs releasing HGF/IGF-1 markedly increase ADSC engraftment 2 weeks after injection and stimulate healing in chronically infarcted myocardium, but attention should be paid to potentially negative electrophysiological consequences.

Comparison between Stem Cells Harvested from Wet and Dry Lipoaspirates

Adipose-derived stem cells (ASC) are usually isolated from lipoaspirates, but it is not known if the anesthetic solution injected into adipose tissue affects cell yield and functions. Two different samples were drawn from the abdominal region of female subjects. In the first, a physiological solution containing lidocaine/adrenaline was injected (wet liposuction, WL), while in the contralateral area, the sample was collected without injecting any solution (dry liposuction, DL). The aspirates were processed to investigate the yield of the stromal-vascular fraction (SVF) cells and ASC frequency, growth rate, apoptosis, and differentiation potential. The solid dried mass of fresh WL isolates was lower than that of DL isolates (p < 0.01) due to the presence, in the former, of a liquid solution. As a consequence, the amount of WL-SVF cells was 18.7% lower than those obtained from DL (p < 0.01); this difference was also observed under culture conditions. In addition, the number of colony-forming unit-fibroblasts (CFU-Fs) obtained from 1 × 103 SVF cells was 25.5% lower in WL-aspirates than DL-aspirates (p < 0.05) owing, at least in part, to the observed presence of apparent diffusion coefficient in the liquid solution of the WL isolates. After WL and DL, no differences were observed in ASC growth rate, apoptosis, or differentiation potential toward adipogenic, osteogenic, and endothelial cell lineages. In conclusion, WL yields about 40% fewer ASC than DL due to the combined effect of tissue dilution and the reduced frequency of ASC in the SVF. The main biological features of ASC are suitable for cell-based therapies.

Polyamine depletion inhibits etoposide-induced NF-κB activation in transformed mouse fibroblasts

Summary.In a previous research, we have shown that adequate levels of polyamines are required in transformed mouse fibroblasts for the correlated activations of MAPK subtypes (ERK and JNK) and caspases induced by etoposide and leading to apoptosis. We report now that the treatment of fibroblasts with etoposide also elicited a progressive and sustained increase of NF-κB activation. The DNA binding activity of p65 NF-κB subunit was increased up to approximately 4-fold and was accompanied by enhancement of p65 phosphorylation. A two days pre-treatment of fibroblasts with α-difluoromethylornithine (DFMO), which caused polyamine depletion, provoked a slight activating effect when given alone, but markedly inhibited the etoposide-induced increases in p65 DNA binding and phosphorylation. The NF-κB inhibiting effect of DFMO was prevented by the addition of exogenous putrescine, which restored the intracellular content of polyamines. Selective inhibitors of the etoposide-stimulated MAPK subtypes also reduced NF-κB activation. Moreover, pharmacological NF-κB inhibition reduced the increase in caspase activity and cell death elicited by etoposide, suggesting that NF-κB is involved in signaling to apoptosis. The results of the present study, together with our previous findings, suggest that polyamines play a permissive role in the pathways triggered by etoposide and leading to cell death of fibroblasts, by supporting the activation of MAPKs, NF-κB and caspases.