Eulalia de la Torre

Arginine metabolic pathways involved in the modulation of tumor‐induced angiogenesis by macrophages

Neovascularization, an essential step for tumor progression and metastasis development, can be modulated by the presence of macrophages (Mps) in the tumor microenvironment. The ability of Mps to regulate the angiogenicity of the LMM3 tumor cell line was studied. Peritoneal Mps from LMM3 tumor‐bearing mice (TMps) potentiate in vivo LMM3 angiogenicity. These results were confirmed by CD31 immunoblotting assays. The activity of TMps depended on nitric oxide synthase (NOS) and arginase (A) activity. By immunoblotting we evidenced that AI and AII isoforms were up‐regulated in TMps while the inducible and neuronal NOS isoforms were highly expressed in normal Mps. TMps might positively modulate tumor growth by stimulating angiogenic cascade mainly through polyamine synthesis.

Activation of muscarinic cholinergic receptors induces MCF-7 cells proliferation and angiogenesis by stimulating nitric oxide synthase activity

Muscarinic acetylcholine receptors (mAChR) are members of the G-protein coupled receptor family. These receptors play key physiological roles and changes in their expression and/or function are involved in several diseases. We had previously demonstrated that mAChR expression is up regulated in three different cell lines derived from distinct murine mammary adenocarcinomas that spontaneously arose in BALB/c female mice, in comparison with normal murine mammary cells. Stimulation of mAChR with the muscarinic agonist carbachol (CARB) potentiated different steps of tumor progression. We here evidence that similarly to previous results obtained in mice, human breast tumor homogenates over expressed mAChR in comparison with normal breast tissue. Thus, to test the muscarinic actions on human breast adenocarcinoma cells we investigate the effect of CARB on MCF-7 cells proliferation and neovascular response. Particularly we observe that: CARB stimulates tumor cells proliferation, being 10-9 M the maximal effective dose for the muscarinic agonist. This action was due to M3 and M1 receptors activation being nitric oxide synthase (NOS) its effector enzyme via phospholipase C and protein kinase C signaling pathway. NOS1 and NOS3 isoforms are expressed in MCF-7 cells and its activation by CARB triggers nitric oxide synthesis and vascular endothelial growth factor expression increasing blood vessels formation induced by mammary tumor cells in vivo. We can conclude that non-neuronal cholinergic system activation stimulates MCF-7 tumor cells growth and neovascular response promoting tumor progression.

Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice.

IntroductionThe role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR).MethodsPeritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 × 105 cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E2 (PGE2) production, respectively.ResultsTMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M1 and M3 receptor antagonists, and partly by the M2 receptor antagonist methoctramine. M1 receptor activation by carbachol in TMps triggers neovascularization through arginase products because Nω-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE2 is responsible for the promotion of TMps angiogenic activity by M3 receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol significantly increased VEGF expression in TMps, and this effect was totally reversed by methoctramine and pirenzepine. Arginase and COX inhibitors partly decreased VEGF derived from TMps.ConclusionTMps themselves induce a potent angiogenic response that is augmented by carbachol action. mAchR activation triggers arginine metabolism, PGE2 synthesis and VEGF production, promoting neovascularization.

Different mechanisms lead to the angiogenic process induced by three adenocarcinoma cell lines

Neoangiogenesis is essential for tumor and metastasis growth, but this complex process does not follow the same activation pathway, at least in tumor cell lines originated from different murine mammary adenocarcinomas. LMM3 cells were the most potent to stimulate new blood vessel formation. This response was significantly reduced by preincubating cells with indomethacin and NS-398, non-selective cyclooxygenase (COX) and COX-2 selective inhibitors, respectively. COX-1 and COX-2 isoenzymes were both highly expressed in LMM3 cells, and we observed that indomethacin was more effective than NS-398 to inhibit prostaglandin E2(PGE2) synthesis. In addition, nitric oxide synthase (NOS) inhibitors, Nωmonomethyl l-arginine and aminoguanidine, also reduced LMM3-induced angiogenesis and nitric oxide (NO) synthesis as well. NOS2 > NOS3 proteins and arginase II isoform were detected in LMM3 cells by Western blot. The latter enzyme was also involved in the LMM3 neovascular response, since the arginase inhibitor, Nω hydroxy l-arginine reduced the angiogenic cascade. On the other hand, parental LM3 cells were able to stimulate neovascularization via COX-1 and arginase products since only indomethacin and Nω hydroxy l-arginine, which diminished PGE2 and urea synthesis, respectively, also reduced angiogenesis. In turn, LM2 cells angiogenic response could be due in fact to PGE2-induced VEGF liberation that stimulated neoangiogenesis at very low levels of NO.

Immunoglobulin G from Breast Cancer Patients in Stage I Stimulates Muscarinic Acetylcholine Receptors in MCF7 Cells and Induces Proliferation. Participation of Nitric Oxide Synthase-Derived Nitric Oxide

IntroductionMuscarinic acetylcholine receptors (mAChR) belong to the G-protein-coupled receptor family and are extensively expressed in most cells in mammals. We had reported the expression of mAChR in murine and human breast tumors.MethodsThe presence of antibodies in the sera of patients with different tumors directed against self-proteins has been recently described. In this work, we investigated the presence of autoantibodies against mAChR in the sera of breast cancer patients in stage I (T1N0Mx-IgG). IgG purification was performed by affinity chromatography in protein G-agarose. We also studied the ability of these antibodies to modulate the proliferation of MCF-7 breast tumor cells by the MTS colorimetric assay. The ability of T1N0Mx-IgG to stimulate muscarinic signaling pathway via nitric oxide synthase was tested by Griess reaction.ResultsWe demonstrated M3 and M4 receptors expression in MCF-7 cells. T1N0Mx-IgG promotes cell proliferation, mimicking the action of the muscarinic agonist carbachol. This effect was preferentially due to M3 receptor activation in tumor cells via phospholipase C-induced nitric oxide liberation by calcium-dependent nitric oxide synthases. IgG from control patients was unable to produce this effect.DiscussionIgG from patients with breast cancer in early stages could be promoting tumor progression by muscarinic activation, and its presence could be determining the prognosis of this illness.

Macrophages derived from septic mice modulate nitric oxide synthase and angiogenic mediators in the heart

Macrophages (Mps) can exert the defense against invading pathogens. During sepsis, bacterial lipopolisaccharide (LPS) activates the production of inflammatory mediators by Mps. Nitric oxide synthase (NOS) derived‐nitric oxide (NO) is one of them. Besides, Mps may produce pro‐angiogenic molecules such as vascular endothelial growth factor‐A (VEGF‐A) and metalloproteinases (MMPs). The mechanisms involved in the cardiac neovascular response by Mps during sepsis are not completely known. We investigated the ability of LPS‐treated Mps from septic mice to modulate the behavior of cardiac cells as producers of NO and angiogenic molecules. In vivo LPS treatment (0.1 mg/mouse) increased NO production more than fourfold and induced de novo NOS2 expression in Mps. Immunoblotting assays also showed an induction in VEGF‐A and MMP‐9 expression in lysates obtained from LPS‐treated Mps, and MMP‐9 activity was detected by zymography in cell supernatants. LPS‐activated Mps co‐cultured with normal heart induced the expression of CD31 and VEGF‐A in heart homogenates and increased MMP‐9 activity in the supernatants. By immunohistochemistry, we detected new blood vessel formation in hearts cultured with LPS treated Mps. When LPS‐stimulated Mps were co‐cultured with isolated cardiomyocytes in a transwell assay, the expression of NOS2, VEGF‐A and MMP‐9 was induced in cardiac cells. In addition, MMP‐9 activity was up‐regulated in the supernatant of cardiomyocytes. The latter was due to NOS2 induction in Mps from in vivo LPS‐treated mice. In conclusion LPS‐treated Mps are inducers of inflammatory/angiogenic mediators in cardiac cells, which could be triggering neovascularization, as an attempt to improve cardiac performance in sepsis. J. Cell. Physiol. 228: 1584–1593, 2013.

Parasympathetic Modulation of Local Acute Inflammation in Murine Submandibular Glands

The parasympathetic nervous system controls submandibular glands (SMG) functions in physiological and pathological conditions via muscarinic acetylcholine receptors (mAchR). We had previously demonstrated that IFNγ and carbachol stimulate amylase secretion in normal murine SMG by mAchR activation. While the cytokine action depended on nitric oxide synthase activation, the effect of the agonist was mediated by prostaglandin E2 (PGE2) production. Both IFNγ and carbachol triggered IFNγ secretion in SMG. We here show that during local acute inflammation (LAI) induced by intraglandular injection of bacterial endotoxin, lypopolisaccharide (LPS), amylase secretion is decreased in comparison to control glands. We also observed that the muscarinic agonist carbachol stimulates in a dose-dependent manner amylase activity by M2 and M3 mAchR activation. Moreover, cyclooxygenase-2 (COX-2) activation and subsequent PGE2 liberation, in a nitric oxide independent manner, seem to be involved in M3 and M2 receptor activation by carbachol. In contrast, the addition of exogenous IFNγ or carbachol inhibits the cytokine liberation in LAI glands.