11 research outputs found
ACVRL1 (activin A receptor type II-like 1)
Activin A receptor, type II-like kinase 1 (ALK1 is a serine-threonine kinase) predominantly expressed on endothelial cells surface. Mutations in its ACVRL1 encoding gene (12q11-14) cause type 2 Hereditary Haemorrhagic Telangiectasia (HHT2), an autosomal dominant multisystem vascular dysplasia. Its involvement in cancer neoangiogenesis has lead to the recent development of novel anti-cancer drugs, which are now in clinical trials
Pharmacological characterization of 50-kHz ultrasonic vocalizations in rats: comparison of the effects of different psychoactive drugs and relevance in drug-induced reward
Significant evidence suggests that ultrasonic vocalizations (USVs) may index the emotional state in rats, and 50-kHz USVs have been proposed as a tool to investigate the rewarding properties of drugs. Apart from the evidence on some psychostimulants, little is known about the effects of other drugs with rewarding properties on emission of 50-kHz USVs. To further elucidate the neuropharmacology of 50-kHz USVs and their relevance in drug-induced reward, this study characterized the effects of different drugs possessing rewarding properties on 50-kHz USVs in adult male rats. Rats received the acute administration of 3,4-methylenedioxymethamphetamine (MDMA, 5-15 mg/kg, i.p.), methylphenidate (2.5-10 mg/kg, i.p.), morphine (1-5 mg/kg, s.c.), or nicotine (0.1-0.4 mg/kg, s.c.). The number and acoustic features of 50-kHz USVs and their subtypes were then measured. As a comparison, additional rats received the acute administration of amphetamine (2 mg/kg, i.p.), which strongly stimulates the emission of 50-kHz USVs. Methylphenidate, similar to amphetamine, increased the total number of 50-kHz USVs emitted by rats, and also modified their acoustic features. Conversely, MDMA, morphine, and nicotine did not elevate the total number of 50-kHz USVs. However, these drugs modified the acoustic features of 50-kHz USVs, as well as the number and acoustic features of specific subtypes of vocalizations. This study demonstrates that major differences exist in the effects of psychoactive drugs on 50-kHz USVs in rats. These findings provide a better understanding of psychoactive properties of drugs with rewarding properties and usefulness of 50-kHz USVs in assessment of these properties
ACVRL1 (activin A receptor type II-like 1)
Activin A receptor, type II-like kinase 1 (ALK1 is a
serine-threonine kinase) predominantly expressed
on endothelial cells surface. Mutations in its
ACVRL1 encoding gene (12q11-14) cause type 2
Hereditary Haemorrhagic Telangiectasia (HHT2),
an autosomal dominant multisystem vascular
dysplasia. Its involvement in cancer
neoangiogenesis has lead to the recent development
of novel anti-cancer drugs, which are now in
clinical trials
Differential effects of intravenous R,S-(+/-)-3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and its S(+)- and R(-)-enantiomers on dopamine transmission and extracellular signal regulated kinase phosphorylation (pERK) in the rat nucleus accumbens shell and core
R,S(+/-)-3,4-methylenedioxymethamphetamine (R,S(+/-)-MDMA, 'Ecstasy') is known to stimulate dopamine (DA) transmission in the nucleus accumbens (NAc). In order to investigate the post-synaptic correlates of pre-synaptic changes in DA transmission and their relationship with MDMA enantiomers, we studied the effects of R,S(+/-)-MDMA, S(+)-MDMA, and R(-)-MDMA on extracellular DA and phosphorylated extracellular signal regulated kinase (pERK) in the NAc shell and core. Male Sprague-Dawley rats, implanted with a catheter in the femoral vein and vertical concentric dialysis probes in the NAc shell and core, were administered i.v. saline, R,S(+/-)-MDMA, S(+)-MDMA, or R(-)-MDMA. Extracellular DA was monitored by in vivo microdialysis with HPLC. Intravenous R,S(+/-)-MDMA (0.64, 1, and 2 mg/kg) increased dialysate DA, preferentially in the shell, in a dose-related manner. S(+)-MDMA exerted similar effects but at lower doses than R,S(+/-)-MDMA, while R(-)-MDMA (1 and 2 mg/kg) failed to affect dialysate DA. R,S(+/-)- and S(+)-MDMA but not R(-)-MDMA increased ERK phosphorylation (expressed as density/neuron and number of pERK-positive neurons/area) in both subdivisions of the NAc. The administration of the D1 receptor antagonist, SCH 39166, prevented the increase in pERK elicited by R,S(+/-)-MDMA and S(+)-MDMA, while the D2/3 receptor antagonist, raclopride, increased pERK in the NAc core per se but failed to affect the R,S(+/-)-MDMA-elicited stimulation of pERK. The present results provide evidence that the DA stimulant effects of racemic MDMA are accounted for by the S(+)-enantiomer and that pERK may represent a post-synaptic correlate of the stimulant effect of R,S(+/-)-MDMA on D1-dependent DA transmission
Synthesis and characterization of new phthalhydrazothiazole derivatives: A preliminary investigation on their activity against hepatocellular carcinoma
The synthesis of new 2-(4-substituted thiazol-2-yl)-2,3-dihydrophthalazine-1,4-diones, 2-(4-oxo-4,5-dihydro-thiazol-2-yl)-2,3-dihydrophthalazine-1,4-diones, and 2-(5-arylidene-4-oxo-4,5-dihydrothiazol-2-yl)-2,3-dihy-drophthalazine-1, 4-diones is reported. The introduction of different substituents on the phthalazine, the thiazole and the thiazolinone has been studied. The new compounds have been characterized and evaluated for their antiproliferative activity against hepatocellular carcinoma, one of the most lethal tumors. The activity shown by some of these compounds towards liver tumor cells is encouraging
Functional analysis of a novel ENG variant in a patient with hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH) identifies a new SP1 binding site
Objectives: To investigate the role of a novel intronic ENG variant
found in a Patient with a PAH diagnosis followed by the identification
of typical HHT clinical signs. The pathogenic role of this variant was
demonstrated.
Methods: We analysed all coding exons of ENG, ACVRL1 and
BMPR2 by Sanger sequencing and mlPA. We expressed the ENG
variant in vitro and evaluated protein levels by western blotting. Then,
we confirmed the results by qRT-PCR on an RNA sample of the
Patient. We used in silico tools to evaluate the presence of putative
transcription factor binding-site, changed in the variant. EMSA
analyses were performed to validate the involvement of the transcription
factor Sp1.
Results: We identified the ENG novel variant c.1852 + 42 C[T in a
Patient with both PAH and HHT. No other disease-causing mutation was
found. We proved by western blotting and qRT-PCR that the variant
significantly reduced ENG expression. In silico analyses predicted that the
variant changes a putative binding-site for the transcription factor Sp1,
already demonstrated as involved in ENG expression. By EMSA, we
observed that nuclear extract proteins of human fibroblasts bind with
different affinity wild-type and mutated oligonucleotides.
Conclusion: We identified a novel Sp1 binding-site in ENG intron14.
We demonstrated the pathogenic role of ENG c.1852 + 42 C[T
mutation, which impairs this Sp1 binding-site reducing the transcription
level of the gene. These results stress the importance of
joining genetic findings to functional studies, in order to understand
the role of novel variants of uncertain significance in the disease
pathogenesis
Genetic characteristics of the HHT Italian population: the experience of the three Italian Reference Centres
Objectives: To describe the disease-causing mutations distribution of
ENG and ACVRL1 in the HHT Italian population.
Methods: Patients were recruited for the clinical screening and the
diagnosis in the three main HHT Italian reference centres of Pavia,
Crema and Bari. Mutation analyses were performed by DHPLC and/
or Sanger sequencing of ENG and ACVRL1 coding exons. A dedicated
mlPA kit (Salsa mlPA Probe mix P093-C2, HHT/PPH1; MRCHolland)
was used to detect large deletions and duplications.Results: From 2000 up to October 2016, we collected about 2000
samples from more than 500 families. Until now, we have studied 450
families; a disease-causing mutation was identified in 390 families
and about 68% of them have HHT type 2. We found 240 different
mutations spread among the whole coding sequence and flanking
introns of both genes with the exception of ACVRL1 exon 2 and ENG
exons 11 and 15, where no mutation was detected. Noteworthy, about
47% of Patients have a mutation in exons 3, 7 or 8 of ACVRL1. If we
consider the geographical origin of the families, a founder effect is
suggested for at least 5 mutations.
Conclusion: We confirm the peculiar genetic characteristic of the HHT
Italian population with a higher incidence of HHT2. In particular, an
unexplained 22% of cases carry a mutation in ACVRL1 exon 3. In
addition, a founder effect is to be supposed in some geographic regions
Circulating microRNAs In Hereditary Hemorrhagic Telangiectasia: Preliminary Results Identify Significant Differences Among Patients
Objectives: We are investigating the role of circulating microRNAs
(miRNAs) in HHT as potential disease biomarkers. The main goal is
to define an HHT-related miRNAs signature. Particular attention has
been paid on miRNAs-genotype and miRNAs-phenotype correlations.
Here we present the preliminary results of this study.
Methods: We performed a circulating miRNAs profiling in 15 subjects:
5 HHT1, 5 HHT2 Patients, and 5 controls, age and gender matched.
miRNAs profile was analysed by qPCR, using serum/plasma microRNA
PCR Panel (I + II), V4.M (Exiqon). Each panel contains 752 LNA™
primer sets of human miRNAs, including different controls. Statistical
analyses were performed using parametric and non-parametric methods.
miRNAs with a p value\0.05 were considered statistically significant
and underwent enrichment analysis.
Results: The overall result was the detection of 18 deregulated
miRNAs. We observed differences between: HHT Patients versus
controls; either HHT1 or HHT2 versus controls; HHT1 versus HHT2
Patients and also comparing Patients’ subgroups showing different
clinical features.
The enrichment analysis identified the top predicted target genes and
the related pathways. Among these, we highlighted different pathways
already described in association with HHT or angiogenesis.
Conclusions: We obtained a preliminary “HHT signature” for circulating
miRNAs, underlying, for the first time, differences between
the two disease subtypes and a more peculiar miRNAs profile in
HHT2. We also described miRNA-PAVMs (Pulmonary Arteriovenous
Malformations) correlations. Confirmation of these results in a
larger cohort of patients is therefore mandatory, and Patients enrolment
for the second step of this study is ongoin
Pulmonary artery systolic pressure as estimated by TTE in a group of HHT patients.
Hereditary hemorrhagic telangiectasia (HHT) or Rendu syndrome-Osler-Weber disease, is an autosomal dominant vascular dysplasia characterized by epistaxis, mucocutaneous telangiectasias and arteriovenous malformations in specific locations mainly in liver, lungs and brain. The genes involved are two: ENG responsible for HHT1 and ACVRL1 associated with HHT2. We previously reported that estimated pulmonary artery systolic pressure (ePASP) was increased in a subgroup (9/68; 13,2%) of HHT patients in whom the clinical diagnosis defined according to Curaçao criteria was confirmed by the demonstration of mutations in ACVRL1 (Olivieri et al 2006). We extended our experience by performing Doppler transthoracic echocardiography (TTE) to estimate PASP in 104 patients (58 females), previously diagnosed with HHT according to Curaçao criteria. We found 79 ACVRL1 and 25 ENG mutations, and the distribution of the mutations in both genes resembles the general distribution in our lab. While no differences were observed for a ePASP in the whole group of HHT1/HHT2 patients, a trend for higher levels in HHT2 was observed when levels of a ePASP ≥40 mmHg were considered: HHT1 43,5±1,3.; 48,8±8,8 in HHT2. As expected a highly significant correlation was found between age and ePASP and, similarly, the presence of hepatic vascular malformations is significantly associated with the levels of ePASP in both HHT1/HHT2. Although not significant, the correlation between age and ePASP was slightly different in the two groups: the mean age of HHT1 showing values ≥40 mmHg was 72,2 while in HHT2 the mean age was 63,5
Efficacy of thalidomide in the treatment of severe recurrent epistaxis in hereditary hemorrhagic teleangiectasia: a comparison between HHT1 and 2.
Hereditary hemorrhagic telangiectasia (HHT: OMIM 187300 and 600376) is an autosomal dominant vascular dysplasia that affects 1 in 5-8000. The diagnosis is clinical and based on the presence of at least three of the “Curaçao criteria”: spontaneous and recurrent epistaxis, multiple telangiectases, visceral lesions (AVMs) in lungs, liver, brain; and finally a family history. Heterozygous mutations of activine receptor-like kinase-1 (ACVRL1) gene and endoglin (ENG) gene cause HHT2 and HHT1 respectively. Recurrent severe epistaxis is the most common presentation in HHT patients (95%), frequently leading to severe anemia requiring blood transfusions. Since angiogenesis has been implicated in the pathogenesis of HHT and since thalidomide has been shown to be a potent angiogenic inhibitor in experimental models, we tried to evaluate the effectiveness of thalidomide in reducing epistaxis and in the blood transfusions requirement. By assessing parameters such as: grading of epistaxis according to the severity score by Pagella et al (Acta Otolaryngol. 2013; 133(2):174-80), transfusion requirement and hemoglobin in the blood, we report results obtained on a group of 29 HHT patients treated with thalidomide for five months. We observed differences in the response to the use of thalidomide between HHT2 and HHT1 patients; in particular the first ones showed a reduction of transfusion requirement of 45,14% compared to HHT1 patients (5,77%). These data indicate a better response in patients with mutations in ACVRL1 and then you can think of to target treatment to patients preferentially HHT2. According to data in literature, thalidomide is metabolized in small part by CYP2C19, and since it is known that specific polymorphisms in these enzymes may modulate the activity of the drug, we set out to evaluate their presence in treated patients and how they can possibly modulate the answer and the side-effects of thalidomide. The results showed that the effects in patients (5) heterozygous for the polymorphism (*1/*2) have a lower reduction of epistaxis (37%) compared to wild type (46.49%)