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 Ca(2+) signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca(2+) -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 Ca(2+) 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 Ca(2+) signals uniquely in presence of extracellular Ca(2+). GSK- and PMA-induced Ca(2+) 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, La(3+) and Gd(3+) dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels.

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.

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 Ca(2+) 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 Ca(2+) entry (SOCE). This study aimed at investigating the nature and the role of VEGF-elicited Ca(2+) signals in ECFCs. VEGF induced asynchronous Ca(2+) oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca(2+) (0Ca(2+)) 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 (InsP(3))-sensitive Ca(2+) pools with cyclopiazonic acid (CPA), and inhibition of InsP(3) receptors with 2-APB prevented the Ca(2+) response to VEGF. VEGF-induced ECFC proliferation and tubulogenesis were inhibited by the Ca(2+)-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 Ca(2+) oscillations require the interplay between InsP(3)-dependent Ca(2+) 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.

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.

Oligonucleotide decoy to NF-kappaB 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.

NF-kappaB blockade upregulates Bax, TSP-1, and TSP-2 expression in rat granulation tissue.

Several diseases are characterized by chronic inflammation, a condition frequently associated with angiogenesis and fibrogenesis that account for the development of granulation tissue. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) is a crucial modulator of intracellular prosurvival signaling pathways and is implicated in the pathogenesis of inflammatory process. In this study, we have investigated the role of NF-kappaB in the angiogenic and fibrogenic response induced by lambda-carrageenin in a rat model of chronic inflammation at 1, 3, and 5 days. The subcutaneous implant of lambda-carrageenin-soaked sponges in rat induced a time-related increase of granulation tissue formation accompanied by intense neovascularization. These lambda-carrageenin-induced changes were significantly reduced by coinjection of wild-type oligodeoxynucleotide (WT ODN) decoy to NF-kappaB. Molecular, morphological, and ultrastructural analysis performed on whole granulation tissue demonstrated: (1) inhibition of NF-kappaB/DNA binding activity; (2) downregulation of cyclooxygenase-2, matrix metalloproteinase-9, tumor necrosis factor-alpha, and vascular endothelial growth factor; (3) upregulation of thrombospondin (TSP)-1 at 1 day and TSP-2 at 5 days; and (4) increase in Bax to Bcl-2 ratio. Our findings show that the blockade of NF-kappaB activation by WT ODN decoy prevents the development of granulation tissue induced by lambda-carrageenin-soaked sponge implant upregulating Bax as well as TSP-1 and TSP-2 expression.

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 lambda-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-alpha). The results indicate that adelmidrol, given locally, may represent a potential therapeutic tool in controlling chronic inflammation.

Lycopene, quercetin and tyrosol prevent macrophage activation induced by gliadin and IFN-gamma.

Oxidative stress plays an important role in inflammatory process of celiac disease. We have studied the effect of the lycopene, quercetin and tyrosol natural antioxidants on the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression in RAW 264.7 macrophages stimulated by gliadin in association with IFN-gamma. The IFN-gamma plus gliadin combination treatment was capable of enhancing iNOS and COX-2 gene expression and nuclear factor-kappaB (NF-kappaB), interferon regulatory factor-1 (IRF-1) and signal transducer and activator of transcription-1alpha (STAT-1alpha) activation induced by reactive oxygen species generation at 24 h. Lycopene, quercetin and tyrosol inhibited all these effects. The results here reported suggest that these compounds may represent non toxic agents for the control of pro-inflammatory genes involved in celiac disease.

Local administration of WIN 55,212-2 reduces chronic granuloma-associated angiogenesis in rat by inhibiting NF-kappaB 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-kappaB) 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 lambda-carrageenin-soaked sponge implant in rat. Our results show that local administration of WIN 55,212-2, a CB(1)/CB(2) agonist, given daily or at time of implantation significantly decreased weight and neo-angiogenesis in granuloma tissue and inhibited nuclear factor-kappa B (NF-kappaB)/DNA binding that was associated with a reduced inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor alpha (TNF-alpha), and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein expression. Also, arachidonyl-2-chloroethylamide (ACEA), a CB(1) selective agonist, and JWH-015, a CB(2) selective agonist, exhibited the same effects that were reversed by SR141716-A and SR144528, respectively, CB(1) and CB(2) selective antagonists. These results indicate that CBs given locally may represent a potential therapeutic tool in controlling chronic inflammation avoiding psychotropic effects.

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-kappaB (NF-kappaB), signal transducer and activator of transcription-1alpha (STAT-1alpha) and interferon regulatory factor-1 (IRF-1). These results, showing that HT down-regulates iNOS and COX-2 gene expression by preventing NF-kappaB, STAT-1alpha 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.

Nuclear factor-kappaB 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.