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1.
Peptides ; 29(11): 2013-23, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18692535

RESUMO

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.


Assuntos
Adrenomedulina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Inibidores da Angiogênese/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Inativação Gênica , Humanos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores
2.
Front Physiol ; 9: 1087, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30154729

RESUMO

Oxidative stress and mitochondrial dysfunction play a crucial role in the pathophysiology of muscular dystrophies. We previously reported that the mitochondrial enzyme monoamine oxidase (MAO) is a relevant source of reactive oxygen species (ROS) not only in murine models of muscular dystrophy, in which it directly contributes to contractile impairment, but also in muscle cells from collagen VI-deficient patients. Here, we now assessed the efficacy of a novel MAO-B inhibitor, safinamide, using in vivo and in vitro models of Duchenne muscular dystrophy (DMD). Specifically, we found that administration of safinamide in 3-month-old mdx mice reduced myofiber damage and oxidative stress and improved muscle functionality. In vitro studies with myogenic cultures from mdx mice and DMD patients showed that even cultured dystrophic myoblasts were more susceptible to oxidative stress than matching cells from healthy donors. Indeed, upon exposure to the MAO substrate tyramine or to hydrogen peroxide, DMD muscle cells displayed a rise in ROS levels and a consequent mitochondrial depolarization. Remarkably, both phenotypes normalized when cultures were treated with safinamide. Given that safinamide is already in clinical use for neurological disorders, our findings could pave the way toward a promising translation into clinical trials for DMD patients as a classic case of drug repurposing.

3.
Regul Pept ; 172(1-3): 16-22, 2011 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21871928

RESUMO

Urotensin-II (U-II) is an endogenous peptide recently characterized as a "nonclassic" pro-angiogenic cytokine. In fact, human vascular endothelial cells express the U-II receptor and exhibit a strong in vitro angiogenic response to the peptide, which was specifically triggered by the binding of U-II to its receptor and involved the activation of ERK1/2 and PI3K/Akt signaling pathways. Moreover, available studies, designed to investigate the pro-angiogenic effect quite shortly following U-II stimulation, suggested that the angiogenic action of the peptide was direct and not associated with an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. In the present study, the expression of three pro-angiogenic factors, namely VEGF, endothelin-1, and adrenomedullin, was studied in human umbilical vein endothelial cells (HUVEC) for longer times of U-II stimulation. RT-PCR and Western blot indicated that in HUVEC, exposed for at least 24h to U-II, the expression of the three angiogenic molecules was significantly increased at both mRNA and protein level, opening the possibility that U-II, not only could exert a direct stimulation of an angiogenic phenotype in endothelial cells quite shortly following exposure to the peptide, but could also further enhance the process indirectly by inducing in the cells a delayed production of other pro-angiogenic factors. Interestingly, a preliminary analysis of the time course of the in vitro capillary-like pattern formation was consistent with this view, suggesting a two phase temporal dynamics of the process.


Assuntos
Indutores da Angiogênese/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Urotensinas/farmacologia , Células Cultivadas , Humanos , Imunoensaio , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Int J Mol Med ; 26(2): 289-94, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20596610

RESUMO

Intermedin (IMD) is a recently discovered peptide closely related to adrenomedullin. Its principal physiological activity is its role in the regulation of the cardiovascular system, where it exerts a potent hypotensive effect. In addition, data were recently provided showing that this peptide is able to exert a clearcut pro-angiogenic effect both in vitro and in vivo. IMD acts through the non-selective interaction with receptor complexes formed by the dimerization of calcitonin-like receptor (CLR) with the receptor activity-modifying proteins RAMP1, 2 or 3. Thus, in the present study, the role of CLR/RAMP complexes in mediating the pro-angiogenic effect induced by IMD on human umbilical vein endothelial cells (HUVECs) cultured on Matrigel was examined. Real-time PCR demonstrated the expression of IMD, CLR/RAMP1 and CLR/RAMP2 (but not CLR/RAMP3) mRNA in HUVECs. IMD exerted a significant in vitro angiogenic action, specifically triggered by the binding of the peptide to CLR/RAMP complexes. Both CLR/RAMP1 and CLR/RAMP2 appeared to mediate the pro-angiogenic effect, which was associated with a significant increase of vascular endothelial growth factor (VEGF) mRNA expression 18 h following IMD administration, indicating that the observed pro-angiogenic effects are related, at least in part, to an increased synthesis of this growth factor promoted by the peptide. Western blot analysis, however, showed a significant increase of VEGF receptor-2 phosphorylation as early as 5 min following IMD administration, indicating that IMD induces a pro-angiogenic response in human vascular endothelial cells not only via CLR/RAMP-induced release of VEGF, but also during signal initiation and propagation by transactivating the VEGF receptor-2 machinery.


Assuntos
Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Hormônios Peptídicos/metabolismo , Receptores da Calcitonina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Biomarcadores/metabolismo , Proteína Semelhante a Receptor de Calcitonina , Células Cultivadas , Colágeno , Combinação de Medicamentos , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Laminina , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Hormônios Peptídicos/genética , Fosforilação , Proteoglicanas , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Proteína 1 Modificadora da Atividade de Receptores , Proteína 2 Modificadora da Atividade de Receptores , Proteína 3 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/antagonistas & inibidores , Receptores da Calcitonina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
Regul Pept ; 162(1-3): 26-32, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20171992

RESUMO

Human vascular endothelial cells express the urotensin-II (U-II) receptor and exhibit a strong in vitro angiogenic response to the peptide. Thus, in the present study an in vitro model, based on human umbilical vein endothelial cells (HUVEC) cultured on Matrigel, was used to characterize more in detail the signaling pathways that control the pro-angiogenic action of U-II. The activation of the U-II receptor (UT) was associated with an increase of intracellular calcium concentration. Both calcium rise and pro-angiogenic effect of the peptide can be blocked by U73122, a selective inhibitor of phospholipase-C, indicating that the signal transduction from UT mainly involves the phospholipase-C/IP(3) pathway. As far as the downstream signaling pathways are concerned, western blot analyses and experiments with specific inhibitors indicated that the U-II-induced self-organization of the cells into capillary-like structures was PKC dependent and involved the activation of the ERK1/2, but not p38-MAPK, transduction pathway. Interestingly, the pharmacological inhibition of PI3K (obtained with LY294002), hindered the capacity of U-II to induce a proangiogenic effect on HUVEC, suggesting that PI3K-dependent pathways also play a role in regulating the process.


Assuntos
Endotélio Vascular/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Urotensinas/fisiologia , Western Blotting , Células Cultivadas , Endotélio Vascular/enzimologia , Humanos
6.
Dev Dyn ; 238(8): 1951-63, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19618467

RESUMO

A recently proposed approach was used to model the self-organization into capillary-like structures of human vascular endothelial cells cultured on Matrigel. The model combines a Cellular Potts Model, considering cell adhesion, cytoskeletal rearrangement and chemotaxis, and a Partial Differential Equation model describing the release and the diffusion of a chemoattractant. The results were compared with the data from real in vitro experiments to establish the capability of the model to accurately reproduce both the spontaneous self-assembly of unstimulated cells and their self-organization in the presence of the pro-angiogenic factor adrenomedullin. The results showed that the model can accurately reproduce the self-assembly of unstimulated cells, but it failed in reproducing the adrenomedullin-induced self-organization of the cells. The extension of the model to include cell proliferation led to a good match between simulated and experimental patterns in both cases with predicted proliferation rates in agreement with the data of cell proliferation experiments.


Assuntos
Adrenomedulina/farmacologia , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Sequência de Bases , Capilares/citologia , Capilares/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno , Primers do DNA/genética , Combinação de Medicamentos , Células Endoteliais/metabolismo , Humanos , Técnicas In Vitro , Laminina , Neovascularização Fisiológica/genética , Fenótipo , Proteoglicanas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Regul Pept ; 157(1-3): 64-71, 2009 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-19362580

RESUMO

Urotensin-II (U-II), along its receptor UT, is widely expressed in the cardiovascular system, where it exerts regulatory actions under both physiological and pathological conditions. In the present study, human vascular endothelial cells (EC) from one arterious and three venous vascular beds were used to investigate in vitro their heterogeneity in terms of expression of U-II and UT and of angiogenic response to the peptide. Real-time PCR and immunocytochemistry demonstrated the expression of UT, as mRNA and protein, in all the EC populations investigated. U-II, on the contrary, was detectable only in EC from aorta and umbilical vein. U-II did not affect the proliferation rate of adult human EC, but induced a moderate proliferative effect on EC from human umbilical vein. When tested in the Matrigel assay, however, all EC exhibited a strong angiogenic response to the peptide, comparable to that of fibroblast growth factor-2 (FGF-2) and it was not associated to an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. The angiogenic effect of U-II was abolished by the UT antagonist palosuran. Overall, these data suggest that U-II, in addition to the well known role in the regulation of cardiovascular function, also exert a specific angiogenic activity.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Neovascularização Fisiológica , Urotensinas/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/citologia , Humanos , Imuno-Histoquímica , Fenótipo , Quinolinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ureia/análogos & derivados , Ureia/farmacologia , Urotensinas/antagonistas & inibidores , Urotensinas/biossíntese
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