Regulation of Ca2+ channel and phosphatase activities by polyamines in intestinal and vascular smooth muscle - implications for cellular growth and contractility.

Polyamines added extracellularly to intestinal and vascular smooth muscle cells cause relaxation through inhibition of Ca2+ channel activity. Intracellularly applied polyamines also affect Ca2+ channel properties. Polyamines do not readily pass over the plasma membrane because of their positive charges but in permeabilized smooth muscle preparations they have free access to the cytoplasm. In this system they increase sensitivity of the contractile machinery to Ca2+ through inhibition of myosin phosphatase activity. The magnitude of Ca2+ channel and phosphatase inhibition depends on the number of positive charges on the polyamine molecule. Polyamines have an obligatory, but yet undefined, role in regulation of cell growth and proliferation. Several groups of protein kinases, such as tyrosine and mitogen activated protein (MAP)-kinases transmit the growth signal from the plasma membrane to the cell nucleus where mitosis and protein synthesis are initiated. The data reviewed here show that polyamines may affect such signal transmission via inhibition of phosphatase activity

Inhibition of MicroRNA-125a Promotes Human Endothelial Cell Proliferation and Viability through an Antiapoptotic Mechanism.

The microRNA-125a (miR-125a) is highly expressed in endothelial cells, but its role in vascular biology is not known. Endothelial cell proliferation and viability play an important role in endothelial healing, and we hypothesize that miR-125a regulates this process. The aim of the present study was to investigate if miR-125a controls human endothelial cell proliferation, viability and endothelial healing, and to assess the mechanisms involved. We showed that overexpression of miR-125a by transfection with miR-125a mimic reduced human umbilical vein endothelial cell (HUVEC) proliferation and viability, and stimulated apoptosis as demonstrated by a miR-125a-induced increase of the proportion of annexin V-positive cells monitored by flow cytometry. Moreover, we showed that the miR-125a mimic downregulated the antiapoptotic Bcl2 protein and upregulated caspase 3, suggesting that these two proteins represent molecular targets for miR-125a. Accordingly, transfection with miR-125a inhibitor, downregulating miR-125a expression, promoted HUVEC proliferation and viability, and reduced apoptosis. Importantly, transfection with miR-125a inhibitor promoted HUVEC tube formation in Matrigel, suggesting that reduction of miR-125a has a proangiogenic effect. In conclusion, downregulation of miR-125a through local transfection with miR-125a inhibitor might be a new way to enhance endothelial cell proliferation and viability, thereby promoting the reendothelialization observed in response to intimal injury. © 2014 S. Karger AG, Basel

LL-37-induced host cell cytotoxicity depends on cellular expression of the globular C1q receptor (p33).

The human host-defense peptide LL-37 not only displays antimicrobial activity but also immune modulating properties that trigger intracellular signaling events in host cells. Since the cytolytic activity of high LL-37 concentrations affects cell viability, the function of LL-37 requires tight regulation. Eukaryotic cells therefore benefit from protective measures to prevent harmful effects of LL-37. p33, also known as globular C1q receptor, is reported to act as an LL-37 antagonist by binding the peptide thereby reducing its cytotoxic activity. In this report, we show that high levels of endogenous p33 correlate with an increased viability in human cells treated with LL-37. Sub-cellular localization analysis showed p33 distribution at the mitochondria, the plasma membrane and in the cytosol. Strikingly, cytosolic over-expression of p33 significantly antagonized detrimental effects of LL-37 on cell fitness, while the reverse effect was observed by siRNA-induced down-regulation of p33. However, modulation of p33 expression had no effect on LL-37-induced plasma membrane pore forming capacity pointing to an intracellular mechanism. A scavenging function of intracellular p33 is further supported by co-immunoprecipitation experiments, showing a direct interaction between intracellular p33 and LL-37. Thus, our findings support an important role of intracellular p33 in maintaining cell viability by counteracting LL-37-induced cytotoxicity

The Polyamine Pathway as a Potential Target for Vascular Diseases: Focus on Restenosis

Polyamines are organic polycations expressed by all living organisms, which are known to play an essential role in cell proliferation and differentiation. Recent studies revealed their involvement also in cell contractility and migration and in programmed cell death. These processes are known to contribute to restenosis, a pathophysiological process occurring in 10-20% of patients submitted to revascularization procedures. The advent of bare metal stents and of drug-eluting stents has significantly reduced but not eliminated the incidence of restenosis, which thus remains a clinically relevant problem. Despite the potential role of the polyamine pathway as a therapeutic target due to its involvement in proliferation, apoptosis and migration of vascular cells, experimental inhibition of polyamine synthesis and/or uptake has been poorly investigated in animal models of vascular disease. Here we review the current knowledge about molecular mechanisms related to polyamine functions, with particular reference to the role played by polyamines in vascular cell pathophysiology, together with experimental evidence obtained so far in animal models of (re) stenosis. We also evaluate the advantages of different routes of administration of polyamine synthesis/transport inhibitors and polyamine analogue molecules. Increasing knowledge about the molecular mechanisms and functions of polyamines is expected to shed new light on their potential role as a therapeutic target for restenosis reduction

The G protein-coupled oestrogen receptor 1 agonist G-1 disrupts endothelial cell microtubule structure in a receptor-independent manner.

The G protein-coupled oestrogen receptor GPER1, also known as GPR30, has been implicated in oestrogen signalling, but the physiological importance of GPER1 is not fully understood. The GPER1 agonist G-1 has become an important tool to assess GPER1-mediated cellular effects. Here, we report that this substance, besides acting via GPER1, affects the microtubule network in endothelial cells. Treatment with G-1 (3 μM) for 24 h reduced DNA synthesis by about 60 % in mouse microvascular endothelial bEnd.3 cells. Treatment with 3 μM G-1 prevented outgrowth of primary endothelial cells from mouse aortic explants embedded in Matrigel. Treatment with G-1 (0.3-3 μM) for 24 h disrupted bEnd.3 cell and HUVEC microtubule structure in a concentration-dependent manner as assessed by laser-scanning confocal immunofluorescence microscopy. G-1-induced (3 μM) disruption of microtubule was observed also after acute (3 and 6 h) treatment and in the presence of the protein synthesis inhibitor cycloheximide. Disruption of microtubules by 3 μM G-1 was observed in aortic smooth muscle cells obtained from both GPER1 knockout and wild-type mice, suggesting that G-1 influences microtubules through a mechanism independent of GPER1. G-1 dose dependently (10-50 μM) stimulated microtubule assembly in vitro. On the other hand, microtubules appeared normal in the presence of 10-50 μM G-1 as determined by electron microscopy. We suggest that G-1-promoted endothelial cell anti-proliferation is due in part to alteration of microtubule organization through a mechanism independent of GPER1. This G-1-promoted mechanism may be used to block unwanted endothelial cell proliferation and angiogenesis such as that observed in, e.g. cancer

The G Protein-Coupled Estrogen Receptor 1 (GPER1/GPR30) Agonist G-1 Regulates Vascular Smooth Muscle Cell Ca Handling.

The G protein-coupled estrogen receptor GPER1/GPR30 is implicated in blood pressure regulation but the mechanisms are not identified. Here, we hypothesize that GPER1 controls blood pressure by regulating vascular smooth muscle cell Ca(2+) handling. Treatment with the GPER1 agonist G-1 (in the µM concentration range) acutely reduced spontaneous and synchronous Ca(2+) spike activity in A7r5 vascular smooth muscle cells expressing mRNA for GPER1. Furthermore, G-1 (1 µM) attenuated the thromboxane A2 analogue U46619-stimulated Ca(2+) spike activity but had no effect on the U46619-induced increase in the basal level of Ca(2+). The voltage-sensitive L-type Ca(2+) channel blocker nifedipine (100 nM) reduced Ca(2+) spike activity similar to G-1. Pharmacological, but not physiological, concentrations of the estrogen 17β-estradiol reduced Ca(2+) spike activity. The GPER1 antagonist G-15 blocked G-1-induced downregulation of Ca(2+) spike activity, supporting a GPER1-dependent mechanism. G-1 (1 µM) and nifedipine (100 nM) attenuated the 30-mM KCl-evoked rise in intracellular Ca(2+) concentration, suggesting that G-1 blocks inflow of Ca(2+) via voltage-sensitive Ca(2+) channels. In conclusion, we demonstrate that the GPER1 agonist G-1 regulates vascular smooth muscle cell Ca(2+) handling by lowering Ca(2+) spike activity, suggesting a role for this mechanism in GPER1-mediated control of blood pressure. © 2013 S. Karger AG, Basel

Local inhibition of ornithine decarboxylase reduces vascular stenosis in a murine model of carotid injury

Objectives: Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re) stenosis. Methods: The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd. 3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression. Results: DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd. 3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury. Conclusions: Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process. (C) 2013 Elsevier Ireland Ltd. All rights reserved

Cerebellar mutism syndrome in children with brain tumours of the posterior fossa

Background: Central nervous system tumours constitute 25% of all childhood cancers; more than half are located in the posterior fossa and surgery is usually part of therapy. One of the most disabling late effects of posterior fossa tumour surgery is the cerebellar mutism syndrome (CMS) which has been reported in up to 39% of the patients but the exact incidence is uncertain since milder cases may be unrecognized. Recovery is usually incomplete. Reported risk factors are tumour type, midline location and brainstem involvement, but the exact aetiology, surgical and other risk factors, the clinical course and strategies for prevention and treatment are yet to be determined. Methods: This observational, prospective, multicentre study will include 500 children with posterior fossa tumours. It opened late 2014 with participation from 20 Nordic and Baltic centres. From 2016, five British centres and four Dutch centres will join with a total annual accrual of 130 patients. Three other major European centres are invited to join from 2016/17. Follow-up will run for 12 months after inclusion of the last patient. All patients are treated according to local practice. Clinical data are collected through standardized online registration at pre-determined time points pre- and postoperatively. Neurological status and speech functions are examined pre- operatively and postoperatively at 1-4 weeks, 2 and 12 months. Pre- and postoperative speech samples are recorded and analysed. Imaging will be reviewed centrally. Pathology is classified according to the 2007 WHO system. Germline DNA will be collected from all patients for associations between CMS characteristics and host genome variants including pathway profiles. Discussion: Through prospective and detailed collection of information on 1) differences in incidence and clinical course of CMS for different patient and tumour characteristics, 2) standardized surgical data and their association with CMS, 3) diversities and results of other therapeutic interventions, and 4) the role of host genome variants, we aim to achieve a better understanding of risk factors for and the clinical course of CMS - with the ultimate goal of defining strategies for prevention and treatment of this severely disabling condition.Peer reviewe