L-citrulline prevents alveolar and vascular derangement in a rat model of moderate hyperoxia-induced lung injury.

BACKGROUND: Moderate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of L-arginine to L-citrulline in endothelial cells. We investigated whether administering L-citrulline by raising the serum levels of L-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury. METHODS: Newborn rats were exposed to FiO(2) = 0.6 or room air for 14 days to induce lung derangement and then were administered L-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed. RESULTS: Serum L-arginine rose in the L-citr + hyperoxia group (p = 0.05), as well as the Von Willebrand factor stained vessels count (p = 0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the L-citr + hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with L-citrulline under exposure to hyperoxia (p = 0.0001). Lung VEGF and eNOS increased in the L-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p = 0.003). CONCLUSIONS: We conclude that administering L: -citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups.

Urotensin-II-stimulated expression of pro-angiogenic factors in human vascular endothelial cells.

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.

Synthesis, in vitro and in vivo preliminary evaluation of anti-angiogenic properties of some pyrroloazaflavones.

This work investigated the in vitro and in vivo anti-angiogenic activity of some pyrroloazaflavones, exactly 2-phenyl-1H-pyrrolo[2,3-h]quinolin-4(7H)ones, with vinblastine as reference compound. Growth inhibitory activity, migration, and capillary-like structures formation were determined in human umbilical vein endothelial cell cultures, and Matrigel plug assay was carried out to evaluate in vivo effects on angiogenesis. Collectively, our results indicate that some pyrroloazaflavone derivatives, at non-cytotoxic concentrations and like vinblastine are able: (i) to exert in vitro anti-angiogenic activity and (ii) to counteract in vitro and in vivo the pro-angiogenic effects of fibroblast growth factor-2 (FGF-2).

Involvement of vascular endothelial growth factor signaling in CLR/RAMP1 and CLR/RAMP2-mediated pro-angiogenic effect of intermedin on human vascular endothelial cells.

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.

The pro-angiogenic activity of urotensin-II on human vascular endothelial cells involves ERK1/2 and PI3K signaling pathways.

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.

Mathematical modeling of the capillary-like pattern generated by adrenomedullin-treated human vascular endothelial cells in vitro.

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.

Pro-angiogenic activity of Urotensin-II on different human vascular endothelial cell populations.

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.

In vitro and in vivo proposal of an artificial esophagus.

Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. This study evaluates the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of homologous esophageal acellular matrix and autologous smooth muscle cells (SMCs). Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker and expressed bFGF as protein, showing angiogenic activity in vivo on the chick embryo chorioallantoic membrane (CAM). Moreover, they supported cell adhesion, and inasmuch as just after 24 h from seeding, the scaffold appeared completely covered by SMCs. To verify the biocompatibility of our constructs, defects created in the porcine esophageal wall were covered using homologous acellular matrices with and without cultures of autologous SMCs. At 3 week from surgery, the patches composed of only acellular matrices showed a more severe inflammatory response and were negative for alpha-smooth muscle actin immunostaining. In contrast, the cell-matrix implants presented ingrowth of SMCs, showing an early organization into small fascicules. Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous SMCs may represent a promising tissue-engineering approach for the repair of esophageal injuries.