Maria Pia Cinelli

Vascular Endothelial Growth Factor Stimulates Endothelial Colony Forming Cells Proliferation and Tubulogenesis by Inducing Oscillations in Intracellular Ca2+ Concentration†‡§

Endothelial progenitor cells (EPCs) home from the bone marrow to the site of tissue regeneration and sustain neovascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell‐based therapy (CBT) in regenerative medicine and provide a novel promising target in the fight against cancer. Intracellular Ca2+ signals regulate numerous endothelial functions, such as proliferation and tubulogenesis. The growth of endothelial colony forming cells (ECFCs), which are EPCs capable of acquiring a mature endothelial phenotype, is governed by store‐dependent Ca2+ entry (SOCE). This study aimed at investigating the nature and the role of VEGF‐elicited Ca2+ signals in ECFCs. VEGF induced asynchronous Ca2+ oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca2+ (0Ca2+) and SOCE inhibition with N‐(4‐[3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl]phenyl)‐4‐methyl‐1,2,3‐thiadiazole‐5‐carboxamide (BTP‐2) reduced the duration of the oscillatory signal. Blockade of phospholipase C‐γ with U73122, emptying the inositol‐1,4,5‐trisphosphate (InsP3)‐sensitive Ca2+ pools with cyclopiazonic acid (CPA), and inhibition of InsP3 receptors with 2‐APB prevented the Ca2+ response to VEGF. VEGF‐induced ECFC proliferation and tubulogenesis were inhibited by the Ca2+‐chelant, BAPTA, and BTP‐2. NF‐κB activation by VEGF was impaired by BAPTA, BTP‐2, and its selective blocker, thymoquinone. Thymoquinone, in turn, suppressed VEGF‐dependent ECFC proliferation and tubulogenesis. These data indicate that VEGF‐induced Ca2+ oscillations require the interplay between InsP3‐dependent Ca2+ release and SOCE, and promote ECFC growth and tubulogenesis by engaging NF‐κB. This novel signaling pathway might be exploited to enhance the outcome of CBT and chemotherapy. STEM CELLS 2011;29:1898–1907

Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation

We investigated the effect of hydroxytyrosol (HT), a phenolic compound from virgin olive oil, on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in J774 murine macrophages stimulated with lipopolysaccharide (LPS). Incubation of cells with LPS caused an increase in iNOS and COX-2 mRNA and protein level as well as ROS generation, which was prevented by HT. In addition, HT blocked the activation of nuclear factor-κB (NF-κB), signal transducer and activator of transcription-1α (STAT-1α) and interferon regulatory factor-1 (IRF-1). These results, showing that HT down-regulates iNOS and COX-2 gene expression by preventing NF-κB, STAT-1α and IRF-1 activation mediated through LPS-induced ROS generation, suggest that it may represent a non-toxic agent for the control of pro-inflammatory genes.

Palmitoylethanolamide reduces granuloma-induced hyperalgesia by modulation of mast cell activation in rats

The aim of this study was to obtain evidences of a possible analgesic role for palmitoylethanolamide (PEA) in chronic granulomatous inflammation sustained by mast cell (MC) activation in rats at 96 hours. PEA (200-400-800 μg/mL), locally administered at time 0, reduced in a concentration-dependent manner the expression and release of NGF in comparison with saline-treated controls. PEA prevented nerve formation and sprouting, as shown by histological analysis, reduced mechanical allodynia, evaluated by Von Frey filaments, and inhibited dorsal root ganglia activation. These results were supported by the evidence that MCs in granuloma were mainly degranulated and closely localized near nerve fibres and PEA significantly reduced MC degranulation and nerves fibre formation. These findings are the first evidence that PEA, by the modulation of MC activation, controls pain perception in an animal model of chronic inflammation, suggesting its potential use for the treatment of all those painful conditions in which MC activation is an initial key step.

Enhanced Expression of Stim, Orai, and TRPC Transcripts and Proteins in Endothelial Progenitor Cells Isolated from Patients with Primary Myelofibrosis.

Background An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1. Methodology/Principal Findings We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2–3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective. Conclusions Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.

Ca2+-dependent nitric oxide release in the injured endothelium of excised rat aorta: a promising mechanism applying in vascular prosthetic devices in aging patients

BackgroundNitric oxide is key to endothelial regeneration, but it is still unknown whether endothelial cell (EC) loss results in an increase in NO levels at the wound edge. We have already shown that endothelial damage induces a long-lasting Ca2+ entry into surviving cells though connexin hemichannels (CxHcs) uncoupled from their counterparts on ruptured cells. The physiological outcome of injury-induced Ca2+ inflow is, however, unknown.MethodsIn this study, we sought to determine whether and how endothelial scraping induces NO production (NOP) in the endothelium of excised rat aorta by exploiting the NO-sensitive fluorochrome, DAF-FM diacetate and the Ca2+-sensitive fluorescent dye, Fura-2/AM.ResultsWe demonstrated that injury-induced NOP at the lesion site is prevented in presence of the endothelial NO synthase inhibitor, L-NAME, and in absence of extracellular Ca2+. Unlike ATP-dependent NO liberation, the NO response to injury is insensitive to BTP-2, which selectively blocks store-operated Ca2+ inflow. However, injury-induced NOP is significantly reduced by classic gap junction blockers, and by connexin mimetic peptides specifically targeting Cx37Hcs, Cx40HCs, and Cx43Hcs. Moreover, disruption of caveolar integrity prevents injury-elicited NO signaling, but not the accompanying Ca2+ response.ConclusionsThe data presented provide the first evidence that endothelial scraping stimulates NO synthesis at the wound edge, which might both exert an immediate anti-thrombotic and anti-inflammatory action and promote the subsequent re-endothelialization.

Nuclear Factor-κB Regulates Inflammatory Cell Apoptosis and Phagocytosis in Rat Carrageenin-Sponge Implant Model

In the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-kappaB in a model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-kappaB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-kappaB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-beta1 and decrease of tumor necrosis factor-alpha as well as nitrite/nitrate production. Our results, showing that blockade of NF-kappaB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process.

Local administration of WIN 55,212-2 reduces chronic granuloma-associated angiogenesis in rat by inhibiting NF-κB activation

Chronic inflammation is often associated with granuloma formation that is a hallmark of many human diseases. The transcription factor nuclear factor-kappa B (NF-κB) plays a central role in this process by regulating the expression of several pro-inflammatory genes. Cannabinoids (CBs) from Cannabis sativa L. exert a large number of biological effects including anti-inflammatory and anti-angiogenic effects. In this study, we investigated the role of CBs on granuloma formation induced by λ-carrageenin-soaked sponge implant in rat. Our results show that local administration of WIN 55,212-2, a CB1/CB2 agonist, given daily or at time of implantation significantly decreased weight and neo-angiogenesis in granuloma tissue and inhibited nuclear factor-kappa B (NF-κB)/DNA binding that was associated with a reduced inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein expression. Also, arachidonyl-2-chloroethylamide (ACEA), a CB1 selective agonist, and JWH-015, a CB2 selective agonist, exhibited the same effects that were reversed by SR141716-A and SR144528, respectively, CB1 and CB2 selective antagonists. These results indicate that CBs given locally may represent a potential therapeutic tool in controlling chronic inflammation avoiding psychotropic effects.

Adelmidrol, a palmitoylethanolamide analogue, reduces chronic inflammation in a carrageenin-granuloma model in rats

Palmitoylethanolamide (PEA) and some of its analogues have shown great efficacy in the treatment of pain and inflammation. Adelmidrol – the International Nonproprietary Name (INN) of the di‐amide derivative of azelaic acid – is one of these analogues. The anti‐inflammatory and analgesic effects of PEA and adelmidrol are hypothesized to be mediated, at least in part, by mast cell down‐modulation. Mast cell mediators released at early stage of the inflammatory process drive the inflammatory reaction to chronicity as it happens in X‐carrageenin‐induced granulomatous tissue formation. In the present study, the choice of testing adelmidrol depends upon the physicochemical properties of the compound, i.e. the amphipatic feature, that make it more easily soluble than PEA. In this study, we investigated the effect of adelmidrol on granuloma formation induced by λ‐carrageenin‐soaked sponge implant in rats. Our results show that the local administration of the compound under study significantly decreases weight and neo‐angiogenesis in granulomatous tissue. The anti‐inflammatory effect was due to the modulation of mast cells degranulation, as shown by histological analysis and by the inhibition of the release of several pro‐inflammatory and pro‐angiogenic enzymes (e.g. iNOS, chymase and metalloproteinase MMP‐9), and mediators (e.g. nitric oxide and TNF‐α). The results indicate that adelmidrol, given locally, may represent a potential therapeutic tool in controlling chronic inflammation.

A functional transient receptor potential vanilloid 4 (TRPV4) channel is expressed in human endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca2+ signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca2+‐permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store‐operated Ca2+ entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca2+ signals uniquely in presence of extracellular Ca2+. GSK‐ and PMA‐induced Ca2+ elevations were inhibited by RN‐1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor‐enriched culture medium. Conversely, SOCE inhibition with BTP‐2, La3+ and Gd3+ dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels. J. Cell. Physiol. 230: 95–104, 2015.

Oligonucleotide decoy to NF-κB slowly released from PLGA microspheres reduces chronic inflammation in rat

Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the immune and inflammatory process. Previously, we demonstrated that NF-kappaB activation can be significantly inhibited by a double stranded oligodeoxynucleotide (ODN). Nevertheless, the therapeutic use of ODN requires a delivery system able to improve poor crossing of cell membranes and rapid in vivo enzymatic degradation. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres can increase ODN stability in biological environment and release the encapsulated drug in long time frames. Here, we used a decoy ODN against NF-kappaB and we investigated its effect, when administered in naked form or when delivered by PLGA microspheres, in a rat model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges caused leukocyte infiltration and formation of granulation tissue which were inhibited up to 15 days by co-injection of microspheres releasing decoy ODN whereas naked decoy ODN showed this effect only up to 5 days. Molecular analysis performed on granulation tissue demonstrated an inhibition of NF-kappaB activation correlated to a decrease of tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) expression. Our results suggest that microspheres could be an useful tool to improve pharmacokinetics of decoy ODN and may represent a strategy to inhibit NF-kappaB activation in chronic inflammation.