Claudio Molinari

1α,25-dihydroxycholecalciferol induces nitric oxide production in cultured endothelial cells

Background: Recently, 1α,25-dihydroxycholecalciferol (vitD) has received increasing interest for its effects on many tissues and organs other than bone. A number of experimental studies have shown that vitD may have an important role in modifying risk for cardiovascular disease. Aims: This study was planned to test the effects of vitD on endothelial nitric oxide (NO) production and to study the intracellular pathways leading to NO release. Methods: In human umbilical vein endothelial cells (HUVEC) cultures the effects of vitD on NO production and p38, Akt, ERK and eNOS phosphorylations were examined in absence or in presence of the NO synthase inhibitor L-NAME and protein kinases specific inhibitors SB203580, wortmannin and UO126. Results: VitD caused a concentration-dependent increase in NO production. The maximum effect was observed at a concentration of 1 nM and the optimal time of stimulation was 1 min. Effects induced by vitD were abolished by L-NAME and by pre-treatment with protein kinases inhibitors. To verify the effective involvement of vitD receptor (VDR) in the action mechanism of vitD, experiments were repeated in presence of the specific VDR ligands ZK159222 and ZK191784. Conclusions: The results of this study demonstrate that vitD can induce a significant increase in endothelial NO production. VitD interaction with VDR caused the phosphorylation of p38, AKT and ERK leading to eNOS activation.

The pattern of c-Fos immunoreactivity in the hindbrain of the rat following stomach distension

It has been previously shown that the walls of the stomach contain vagal and splanchnic afferents, connected to low and high threshold (LT and HT) gastric receptors, that convey physiological and noxious information to areas of the hindbrain involved mainly in the control of gastrointestinal function. Because distension of the stomach also reflexly increases the sympathetic drive to the cardiovascular system, the present study was planned to examine the pattern of activation of all nuclei encountered throughout the hindbrain in response to gastric distension. In anaesthetized rats, the stimulus was controlled by employing different transmural pressures and frequencies of distension, and c-Fos immunohistochemistry was used to characterize neuronal activation. Low intensity stimulation induced c-Fos expression in the cranial part of nucleus of solitary tract (NTS), the nucleus ambiguus (NA), the lateral reticular area (LRt) and the ventrolateral medulla (RVL/CVL). At low frequency of stimulation c-Fos positive nuclei (p.n.) were found in NTS only. At high frequency of stimulation an increase in c-Fos immunoreactivity was found. High intensity stimulation induced c-Fos expression in area postrema (AP), the lateral vestibular nucleus (LVe) and the caudal part of the NTS. At low frequency, only the number of c-Fos p.n. was increased. Increasing the frequency of stimulation induced c-Fos expression in further nuclei such as the parabrachial nucleus (PBN), the inferior olive subnuclei (IOn), the oral part of spinal trigeminal nucleus (Sp5O) and locus coeruleus (LC). At higher frequencies c-Fos immunoreactivity decreased in NTS and LRt, disappeared in VLM and increased in NA. Thus stomach distension activated several neuronal excitatory and inhibitory circuits that are involved in the control of gastrointestinal function as well as in cardiovascular, respiratory and pain regulation. The differences in c-Fos immunoreactivity induced by changing the distension patterns suggested interactions between groups of vagal and splanchnic afferents.

1α,25-Dihydroxycholecalciferol (Vitamin D3) induces NO-dependent endothelial cell proliferation and migration in a three-dimensional matrix.

Background/Aims: The 1α,25-dihydroxycholecalciferol (Vit. D) induces eNOS dependent nitric oxide (NO) production in human umbilical vein endothelial cells (HUVEC). To our knowledge, there are no reports directly relating Vit. D induced NO production to proliferation and/or migration in endothelial cells (EC). The aim of this study was to evaluate whether Vit. D addition to porcine EC could affect their proliferation and/or migration in a three-dimensional matrix via NO production. Materials and Methods: Porcine aortic endothelial cells (PAE) were used to evaluate Vit. D effects on cell proliferation and migration in a three-dimensional matrix. Results: Vit. D induced NO production in PAE cells. Moreover, it induced a significant increase in cellular proliferation and migration in a three-dimensional matrix. These effects were NO dependent, as inhibiting eNOS activity by L-NAME PAE migration was abrogated. This effect was strictly related to MMP-2 expression and apparently dependent on Vit. D and NO production. Conclusions: Vit. D can promote both endothelial cells proliferation and migration in a three-dimensional matrix via NO-dependent mechanisms. These findings cast new light on the role of Vit. D in the angiogenic process, suggesting new applications for Vit. D in such fields as tissue repair and wound healing.

Protective effects of 1α,25-Dihydroxyvitamin D3 on cultured neural cells exposed to catalytic iron.

Recent studies have postulated a role for vitamin D and its receptor on cerebral function, and anti‐inflammatory, immunomodulatory and neuroprotective effects have been described; vitamin D can inhibit proinflammatory cytokines and nitric oxide synthesis during various neurodegenerative insults, and may be considered as a potential drug for the treatment of these disorders. In addition, iron is crucial for neuronal development and neurotransmitter production in the brain, but its accumulation as catalytic form (Fe3+) impairs brain function and causes the dysregulation of iron metabolism leading to tissue damage due to the formation of toxic free radicals (ROS). This research was planned to study the role of vitamin D to prevent iron damage in neuroblastoma BE(2)M17 cells. Mechanisms involved in neurodegeneration, including cell viability, ROS production, and the most common intracellular pathways were studied. Pretreatment with calcitriol (the active form of vitamin D) reduced cellular injury induced by exposure to catalytic iron.

NO-dependent proliferation and migration induced by Vitamin D in HUVEC.

Recently, Vitamin D (Vit. D) has gained importance in cellular functions of a wide range of extraskeletal organs and target tissues, other than bone. In particular, Vit. D has displayed important beneficial effects in the cardiovascular system. Although little is known about the mechanism by which this response is exerted, a Vit. D-induced eNOS-dependent nitric oxide (NO) production in endothelial cells (EC) has been reported. The aim of this study was to evaluate whether Vit. D administration could affect human EC proliferation and/or migration through NO production. For this purpose, HUVEC (human umbilical vein endothelial cells) were used to evaluate Vit. D effects on cell proliferation and migration in a 3D matrix. Experiments were also performed in the presence of the specific VDR ligand ZK159222 and eNOS inhibitor L-NAME. This study demonstrated that Vit. D can promote both HUVEC proliferation and migration in a 3D matrix. These effects were NO dependent, since HUVEC proliferation and migration were abrogated along with Vit. D induced MMP-2 expression by inhibiting eNOS activity by L-NAME. These findings support the role of Vit. D in the angiogenic process, suggesting new applications for Vit. D in tissue repair and wound healing.

Protective effects of vitamin D 3 on fimbrial cells exposed to catalytic iron damage

BackgroundRecently, vitamin D3 (1alpha, 25-dihydroxyvitamin D) has shown its capability to take part in many extraskeletal functions and its serum levels have been related to patient survival rate and malignancy of many types of neoplasms, including ovarian cancers. Catalytic iron is a free circulating form of iron that is able to generate reactive oxygen species and consequently to promote a number of cellular and tissutal dysfunctions including tumorigenesis. In fertile women an important source of catalytic iron is derived from retrograde menstruation. Epithelial secretory cells from fimbriae of fallopian tubes are greatly exposed to catalytic iron derived from menstrual reflux and so represent the site of origin for most serous ovarian cancers.The aim of this study was to assess whether vitamin D3 can play a role in counteracting catalytic iron-induced oxidative stress in cells from fimbriae of fallopian tubes.MethodsThe cells, isolated from women undergoing isteroannessiectomy, were treated with catalytic iron 50-75-100xa0mM and vitamin D3 at a concentration ranging from 0.01 to 10 nM to study cell viability, radical oxygen species production, p53, pan-Ras, Ki67 and c-Myc protein expressions through Western Blot, and immunocytochemistry or immunofluorescence analysis.ResultsThe pre-treatment with vitamin D3 1 nM showed its beneficial effects that consists in a significant decrease in ROS production. In addition a novel finding is represented by the demonstration that pre-treatment with vitamin D3 is also able to significantly counteract tumoral biomarkers activation, such as p53, pan-Ras, Ki67 and c-Myc, and consequently the catalytic iron-induced cellular injury.ConclusionsThis study demonstrates for the first time that vitamin D3 plays an important role in preventing catalytic iron-dependent oxidative stress in cultured fimbrial cells. These results support the hypothesis that vitamin D3 could counteract carcinogenic changes induced by catalytic iron.

Heart Rate Variability modifications induced by high doses of incobotulinumtoxinA and onabotulinumtoxinA in hemiplegic chronic stroke patients: A single blind randomized controlled, crossover pilot study

Background: Botulinum toxin type A is a valid and safe treatment for focal spasticity, with documented effects on both sympathetic and parasympathetic systems. Heart rate variability can provide detailed information about the control of the autonomic nervous system on cardiovascular activities. Previous studies in literature showed no significant changes in Heart Rate Variability with doses >600 U of incobotulinumtoxinA in chronic post stroke spastic patients; however, at present time, these results have not been confirmed with doses >600 U of onabotulinumtoxinA. Aim: To evaluate changes in Heart Rate Variability induced by high doses (>600 U) of incobotulinumtoxinA or onabotulinumtoxinA in spastic stroke patients over a 1‐year period. Design: single blind randomized controlled crossover study design. Setting: Rehabilitation Unit of the University Hospital in Novara. Population: 10 stroke survivors with spastic hemiplegia (Modified Ashworth Scale ≥ 2) were recruited and randomly divided in two groups (A and B). Methods: In the first part of the study, patients in Group A were injected with incobotulinumtoxinA while patients in Group B with onabotulinumtoxinA; after 6 months, a crossover intervention was performed. All patients were blinded to Botulinum toxin type A type, and performed an ECG registration in the 24 h before injection (t0) and 10 days after treatment (t1), both in the first and in the second part of the study. Functional status was evaluated with Barthel Index, Motricity Index and Functional Ambulation Category scores. Results: Heart Rate Variability analysis showed no significant changes after each Botulinum toxin type A injection in both groups at any evaluation time. Moreover, no statistically significant differences were found regarding each variable between the two groups. Conclusions: Our data show that high doses (>600 U) of incobotulinumtoxinA and onabotulinumtoxinA do not influence the cardiovascular activity of the autonomic nervous system in chronic hemiplegic spastic stroke survivors. HighlightsIncobotulinumtoxinA up to 12 units/kg was safe in terms of heart drive modifications in adult stroke hemiplegic patients.OnabotulinumtoxinA up to 12 units/kg was safe in terms of heart drive modifications in adult stroke hemiplegic patients.Multiple administrations of BoNT‐A at high doses seemed not to induce cumulative effects in adult stroke hemiplegic patients.

Gastric distension causes changes in heart rate and arterial blood pressure by affecting the crosstalk between vagal and splanchnic systems in anesthetised rats

Various hindbrain nuclei have been demonstrated to be involved in the control of the cardiovascular reflexes elicited by both non-noxious and noxious gastric distension, through parasympathetic and sympathetic activation. The different role played by the branches of autonomic nervous system in exerting these effects and their crosstalk in relation to low-/high-pressure distension rate has not been examined yet. Therefore, in the present work, monolateral and bilateral vagotomy and splanchnicotomy were performed in anesthetised rats to analyse the involvement of hindbrain nuclei in haemodynamic changes caused by gastric distension at high (80xa0mmHg) and low (15xa0mmHg) pressure. The analysis of c-Fos expression in neuronal areas involved in cardiovascular control allowed us to examine their recruitment in response to various patterns of gastric distension and the crosstalk between vagal and splanchnic systems. The results obtained show that the low-pressure (non-noxious) gastric distension increases both heart rate and arterial blood pressure. In addition, the vagus nerve and hindbrain nuclei, such as nucleus ambiguous, ventrolateral medulla and lateral reticular nucleus, appear to be primarily involved in observed responses. In particular, we have found that although vagus nerve plays a central role in exerting those cardiovascular reflex changes at low gastric distension, for its functional expression an intact splanchnic system is mandatory. Hence, the absence of splanchnic input attenuates pressor responses or turns them into depressor responses. Instead at high-pressure (noxious) gastric distension, the splanchnic nerve represents the primary component in regulating the reflex cardiovascular effects.