Mecanismos de tumorogénesis hipofisaria: Estudio de factores angiogénicos y de proliferación / Mechanisms of Pituitary Tumorigenesis: Study of Angiogenic and Proliferation factors

Introducción y Objetivos: La angiogénesis es un proceso fundamental en el desarrollo tumoral. Sin embargo, se han encontrado discrepancias en el patrón angiogénico de los tumores hipofisarios. Nos propusimos estudiar la expresión de VEGF y FGF2 y su importancia en la vascularización de los adenomas hipofisarios, cuantificar los vasos con los marcadores CD31 y CD34 y determinar el índice de proliferación con PCNA y Ki67. Materiales y Métodos: Se estudiaron 76 macroadenomas hipofisarios que fueron intervenidos quirúrgicamente. Los adenomas se clasificaron según su secreción hormonal. A partir de cortes histológicos se realizó inmunohistoquímica para los marcadores de endotelio CD31 y CD34; y Ki-67 para estudio de proliferación celular. Por western blot se midieron VEGF, CD31 y PCNA. Se efectuaron comparaciones con glándulas normales. Resultados: El nivel de expresión de VEGF, hallado en todas las muestras analizadas, resultó mayor en los prolactinomas resistentes respecto a los demás tipos de adenomas hipofisarios. Esta proteína localizó en las células endoteliales de los vasos como así también en citoplasmas y núcleos de células tumorales. El 56 % de las muestras resultaron positivas para FGF2, mostrando localización citoplasmática y en matriz extracelular. Obtuvimos una fuerte correlación positiva entre VEGF y CD31 en las muestras tumorales, sin encontrar correlación lineal entre PCNA y VEGF, ni Ki-67 y VEGF en las muestras estudiadas. El área vascular resultó mayor en los tejidos normales que en los tumores utilizando CD34 como marcador de vasos. Conclusión: La importancia del estudio de la angiogénesis en los adenomas hipofisarios radica en la necesidad de hallar marcadores moleculares que predigan el comportamiento tumoral. Pudimos demostrar la expresión de los factores angiogénicos VEGF y FGF2 en estos adenomas, y la existencia de correlación lineal entre VEGF y CD31. Nuestros resultados son indicativos de existencia de angiogénesis en los adenomas hipofisarios por lo que su bloqueo podría plantearse como una estrategia alternativa para los casos resistentes a las terapias convencionales.

Introduction and objectives: Angiogenesis is an essential process in tumor development. Nevertheless, discrepancies in the angiogenic pattern of pituitary tumors, in terms of hormonal phenotype, size or invasiveness have been found. Our aim was to study the expression of VEGF and FGF2 growth factors, and their importance in the vascularization of pituitary adenomas. We also quantified blood vessels with the endothelial cell markers CD31 and CD34 determining the vascular area, and the proliferation rate through PCNA and Ki67 index. Materials and Methods: We studied 76 pituitary macroadenomas that were surgically resected in the period between 2006 and 2010 from a total of 276 patients with this pathology. Adenomas were classified into prolactinomas (PRL), somatotropinomas (GH), corticotropinomas (ACTH), non-functioning (NF) and plurihormonal (Ph) according to their hormonal secretion. Samples were collected in formalin, embedded in paraffin, and immunohistochemistry was performed from histological sections for endothelial markers CD31 and CD34; and for Ki-67 to study cell proliferation. VEGF, CD31 and PCNA were measured by Western blot. We compared results with normal glands (N=6). Results: VEGF expression levels, found in all of the samples analyzed, were higher in resistant prolactinomas than in other pituitary adenomas. This protein was detected in endothelial cells of blood vessels and in tumor cells cytoplasms and nuclei. Fifty-six percent of samples were positive for FGF2, the other potent angiogenic factor studied, showing cytoplasmatic and extracellular matrix localization. We obtained a strong positive correlation between VEGF and CD31 in tumor samples, but we did not find lineal correlation between PCNA and VEGF, or between Ki-67 and VEGF in the samples studied. The vascular area was higher in normal tissues than in tumors when CD34 was used as endothelial cell marker. Conclussion: The importance of studying angiogenesis in pituitary adenomas lies in the need to find molecular markers that can predict tumor behavior. We could demonstrate the expression of VEGF and FGF2, two potent angiogenic factors, and the existence of linear correlation between VEGF and CD31. Our results are indicative of the existence of angiogenesis in pituitary adenomas; therefore the blockage of angiogenesis might be proposed as an alternative strategy for cases of resistance to standard therapy.

Mecanismos de tumorogénesis hipofisaria: Estudio de factores angiogénicos y de proliferación / Mechanisms of Pituitary Tumorigenesis: Study of Angiogenic and Proliferation factors

Introducción y Objetivos: La angiogénesis es un proceso fundamental en el desarrollo tumoral. Sin embargo, se han encontrado discrepancias en el patrón angiogénico de los tumores hipofisarios. Nos propusimos estudiar la expresión de VEGF y FGF2 y su importancia en la vascularización de los adenomas hipofisarios, cuantificar los vasos con los marcadores CD31 y CD34 y determinar el índice de proliferación con PCNA y Ki67. Materiales y Métodos: Se estudiaron 76 macroadenomas hipofisarios que fueron intervenidos quirúrgicamente. Los adenomas se clasificaron según su secreción hormonal. A partir de cortes histológicos se realizó inmunohistoquímica para los marcadores de endotelio CD31 y CD34; y Ki-67 para estudio de proliferación celular. Por western blot se midieron VEGF, CD31 y PCNA. Se efectuaron comparaciones con glándulas normales. Resultados: El nivel de expresión de VEGF, hallado en todas las muestras analizadas, resultó mayor en los prolactinomas resistentes respecto a los demás tipos de adenomas hipofisarios. Esta proteína localizó en las células endoteliales de los vasos como así también en citoplasmas y núcleos de células tumorales. El 56 % de las muestras resultaron positivas para FGF2, mostrando localización citoplasmática y en matriz extracelular. Obtuvimos una fuerte correlación positiva entre VEGF y CD31 en las muestras tumorales, sin encontrar correlación lineal entre PCNA y VEGF, ni Ki-67 y VEGF en las muestras estudiadas. El área vascular resultó mayor en los tejidos normales que en los tumores utilizando CD34 como marcador de vasos. Conclusión: La importancia del estudio de la angiogénesis en los adenomas hipofisarios radica en la necesidad de hallar marcadores moleculares que predigan el comportamiento tumoral. Pudimos demostrar la expresión de los factores angiogénicos VEGF y FGF2 en estos adenomas, y la existencia de correlación lineal entre VEGF y CD31. Nuestros resultados son indicativos de existencia de angiogénesis en los adenomas hipofisarios por lo que su bloqueo podría plantearse como una estrategia alternativa para los casos resistentes a las terapias convencionales.(AU)

Introduction and objectives: Angiogenesis is an essential process in tumor development. Nevertheless, discrepancies in the angiogenic pattern of pituitary tumors, in terms of hormonal phenotype, size or invasiveness have been found. Our aim was to study the expression of VEGF and FGF2 growth factors, and their importance in the vascularization of pituitary adenomas. We also quantified blood vessels with the endothelial cell markers CD31 and CD34 determining the vascular area, and the proliferation rate through PCNA and Ki67 index. Materials and Methods: We studied 76 pituitary macroadenomas that were surgically resected in the period between 2006 and 2010 from a total of 276 patients with this pathology. Adenomas were classified into prolactinomas (PRL), somatotropinomas (GH), corticotropinomas (ACTH), non-functioning (NF) and plurihormonal (Ph) according to their hormonal secretion. Samples were collected in formalin, embedded in paraffin, and immunohistochemistry was performed from histological sections for endothelial markers CD31 and CD34; and for Ki-67 to study cell proliferation. VEGF, CD31 and PCNA were measured by Western blot. We compared results with normal glands (N=6). Results: VEGF expression levels, found in all of the samples analyzed, were higher in resistant prolactinomas than in other pituitary adenomas. This protein was detected in endothelial cells of blood vessels and in tumor cells cytoplasms and nuclei. Fifty-six percent of samples were positive for FGF2, the other potent angiogenic factor studied, showing cytoplasmatic and extracellular matrix localization. We obtained a strong positive correlation between VEGF and CD31 in tumor samples, but we did not find lineal correlation between PCNA and VEGF, or between Ki-67 and VEGF in the samples studied. The vascular area was higher in normal tissues than in tumors when CD34 was used as endothelial cell marker. Conclussion: The importance of studying angiogenesis in pituitary adenomas lies in the need to find molecular markers that can predict tumor behavior. We could demonstrate the expression of VEGF and FGF2, two potent angiogenic factors, and the existence of linear correlation between VEGF and CD31. Our results are indicative of the existence of angiogenesis in pituitary adenomas; therefore the blockage of angiogenesis might be proposed as an alternative strategy for cases of resistance to standard therapy.(AU)

Association between cavernous angioma and cerebral glioma. Report of two cases and literature review of so-called angiogliomas.

The association between vascular malformations and cerebral gliomas is unusual. While the association between cavernous angioma with gliomatous lesions is even more rare, it is considered by certain authors to be a particular pathological entity termed angioglioma. The authors report on two cases of association of a cavernous angioma with a ganglioglioma and an oligodendroglioma respectively. Subsequent review of the literature on the so-called angiogliomas was conducted. In the author's opinion, the entity of angiogliomas represents a general spectrum of angiomatous neoplasms that include gliomatous tumors, in the majority low-grade gliomas, associated with a major vascular component.

Relationship between ADIPOQ gene, circulating high molecular weight adiponectin and albuminuria in individuals with normal kidney function: evidence from a family-based study.

AIMS/HYPOTHESIS: Insulin resistance is associated with reduced serum adiponectin and increased albuminuria levels. Thus, one would anticipate an inverse relationship between circulating adiponectin and albuminuria. However, several studies have described a 'paradoxical' elevation of serum adiponectin in patients with elevated albuminuria. These findings may have been confounded by the presence of diseases and related treatments known to affect circulating adiponectin and albuminuria. We therefore studied the relationship between circulating adiponectin and albuminuria in the absence of such confounders. METHODS: To this purpose, the relationship between adiponectin isoforms and albumin:creatinine ratio (ACR) was investigated in a family-based sample of 634 non-diabetic untreated white individuals with normal kidney function. We also investigated whether the two variables share a common genetic background and addressed the specific role of the gene encoding adiponectin on that background by genotyping several ADIPOQ single nucleotide polymorphisms (SNPs). RESULTS: ACR was directly associated with high molecular weight (HMW) adiponectin isoform (p = 0.024). The two variables shared some genetic correlation (ρ(g) = 0.38, p = 0.04). ADIPOQ promoter SNP rs17300539 was associated with HMW adiponectin (p = 4.8 × 10(-5)) and ACR (p =0.0027). The genetic correlation between HMW adiponectin and ACR was no longer significant when SNP rs17300539 was added to the model, thus reinforcing the role of this SNP in determining both traits. CONCLUSIONS/INTERPRETATION: Our study shows a positive, independent correlation between HWM adiponectin and ACR. ADIPOQ variability is associated with HMW adiponectin and ACR, and explains some of the common genetic background shared by these traits, thus suggesting that ADIPOQ and HMW adiponectin modulate albuminuria levels.

Circulating high molecular weight adiponectin isoform is heritable and shares a common genetic background with insulin resistance in nondiabetic White Caucasians from Italy: evidence from a family-based study.

OBJECTIVE: Reduced circulating adiponectin levels contribute to the aetiology of insulin resistance. Adiponectin circulates in three different isoforms: high molecular weight (HMW), medium molecular weight (MMW) and low molecular weight (LMW) isoforms. The genetics of adiponectin isoforms is mostly unknown. Our aim was to investigate whether and to which extent circulating adiponectin isoforms are heritable and whether they share common genetic backgrounds with insulin resistance-related traits. METHODS: In a family-based sample of 640 nondiabetic White Caucasians from Italy, serum adiponectin isoforms concentrations were measured by ELISA. Three single nucleotide polymorphisms (SNPs) in the ADIPOQ gene previously reported to affect total adiponectin levels (rs17300539, rs1501299 and rs677395) were genotyped. The heritability of adiponectin isoform levels was assessed by variance component analysis. A linear mixed effects model was used to test the association between SNPs and adiponectin isoforms. Bivariate analyses were conducted to study genetic correlations between adiponectin isoforms levels and other insulin resistance-related traits. RESULTS: All isoforms were highly heritable (h(2) = 0.60-0.80, P = 1.0 x 10(-13)-1.0 x 10(-23)). SNPs rs17300539, rs1501299 and rs6773957 explained a significant proportion of HMW variance (2-9%, P = 1.0 x 10(-3)-1.0 x 10(-5)). In a multiple-SNP model, only rs17300539 and rs1501299 remained associated with HMW adiponectin (P = 3.0 x 10(-4) and 2.0 x 10(-2)). Significant genetic correlations (P = 1.0 x 10(-2)-1.0 x 10(-5)) were observed between HMW adiponectin and fasting insulin, homeostasis model assessment of insulin resistance, HDL cholesterol and the metabolic syndrome score. Only rs1501299 partly accounted for these genetic correlations. CONCLUSION: Circulating levels of adiponectin isoforms are highly heritable. The genetic control of HMW adiponectin is shared in part with insulin resistance-related traits and involves, but is not limited to, the ADIPOQ locus.

DNA polymorphisms associated with Hb D-Los Angeles [beta 121(GH4)Glu-->Gln] in southern Italy.

We detected Hb D-Los Angeles [beta 121(GH4)Glu-->Gln], the most common hemoglobin variant after Hb S and Hb Lepore-Boston, in six unrelated families in Southern Italy. Ten patients were studied; eight patients were heterozygotes and two were compound heterozygotes for the hemoglobin variant and the beta-thalassemia codon 39 (C-->T) nonsense mutation. The beta-globin gene sequence was characterized by polymerase chain reaction direct sequencing; restriction fragment length polymorphisms were defined by Southern blot analysis. The gene variant, due to the GAA-->CAA substitution at codon 121, was found in association with the 5' subhaplotype [+ - - - -] and the beta-globin gene framework 1; in addition, it was found to be associated with the absence of Ava II/phi beta and Xmn I/5'G gamma, and with the presence of Hpa I/3' beta. This restriction fragment length polymorphism haplotype is common in the Mediterranean area as well as in other populations. The findings are equally compatible with an independent origin in the Mediterranean area or with origin in Asia and subsequent spread to Italy.

Origin heterogeneity of Hb Lepore-Boston gene in Italy.

Forty-three hybrid delta-beta-globin genes were characterized by DNA sequence analysis and associated RFLP haplotypes in 40 families from Abruzzo and Campania, which are on the east and west coast of Italy, respectively. All the genes had the delta-globin sequence up to the exon 2 codon 87 and had the beta-globin sequence from IVS-2-8; between these two ends, they had 58 bp in common with the delta- and beta-globin genes. Thus, they were all of the Lepore-Boston type. A chromosomal background heterogeneity was present among the mutant genes. In fact, they were all associated with (+ - - - -) 5' subhaplotype, but 23/31 from Campania were associated with (+ +) 3' subhaplotype, whereas 12/12 genes from Abruzzo and 8/31 from Campania were associated with (+ -). DNA sequencing of homozygous subjects showed that (+ +) 3' subhaplotype was associated, at IVS-2-74, with G, while (+ -) was associated with T; that is they were associated with the beta-globin gene sequence of frameworks 1 and 2, respectively. The molecular characteristics of this heterogeneity, as well as its geographical patterns in the eastern and western regions of Italy, represent strong evidence for the recurrent and multicentric origins of the mutation.

Alpha alpha alpha alpha anti-3.7 type II: a new alpha-globin gene rearrangement suggesting that the alpha-globin gene duplication could be caused by intrachromosomal recombination.

We report here a new human alpha-globin gene rearrangement carrying the two normal, alpha 2 and alpha 1, and two hybrid, alpha 1/alpha 2, globin genes in the order 5'-alpha 2-alpha 1/alpha 2-alpha 1/alpha 2-alpha 1-3'. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the beta-thalassaemia IVS 1 nt 1 G----A mutation caused thalassaemia intermedia. We also report a case of an alpha alpha alpha-globin gene rearrangement in the twin of one of the alpha alpha alpha alpha-globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an alpha alpha alpha alpha-anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the alpha-globin gene rearrangements could be of the intrachromosomal rather than the interchromosomal type.

Hb City of Hope [beta 69(E13)Gly----Ser] in Italy: association of the gene with haplotype IX.

Hb City of Hope [beta 69(E13)Gly----Ser] was detected by reversed phase high performance liquid chromatography in an asymptomatic carrier from Naples, Southern Italy. The amino acid substitution, identified by fast atom bombardment mass spectrometry, was due to a TGG----TGA substitution as assessed by DNA sequencing. Analysis of the chromosomal background indicates that the globin gene cluster containing the mutant gene has most probably been rearranged by a recombination event, since the mutation was associated with restriction fragment length polymorphism haplotype IX, instead of haplotype I, as previously reported.

Hemoglobin Neapolis, beta 126(H4)Val----Gly: a novel beta-chain variant associated with a mild beta-thalassemia phenotype and displaying anomalous stability features.

A novel beta-chain, beta 126(H4)Val----Gly, electrophoretically silent, was detected by reverse-phase high performance liquid chromatography in three unrelated families from Naples (Southern Italy) and accounted for about 30% of the total beta-chains. The amino acid substitution was detected by HPLC fingerprint. The eight heterozygous patients showed hematologic and biosynthetic alterations of mild beta-thalassemia type. The hemoglobin variant showed abnormal stability features. It was unstable in the heat stability and isopropanol precipitation tests, but did not cause a hemolytic syndrome in vivo and was stable in a time-course experiment of biosynthesis in vitro. DNA polymerase chain reaction direct sequencing of the mutated gene from 135 nt upstream of the cap site to 106 nt downstream of the polyadenylation site showed only the beta 126 GTG----GGG mutation, which was confirmed in the other patients by allele-specific oligonucleotide hybridization. The mutation was found to be associated with a type II beta-globin framework and restriction fragment length polymorphism haplotype V. The novel variant was named hemoglobin Neapolis.

(Alpha)alpha 5.3: a novel alpha(+)-thalassemia deletion with the breakpoints in the alpha 2-globin gene and in close proximity to an Alu family repeat between the psi alpha 2- and psi alpha 1-globin genes.

A novel 5.3-kb deletion of the alpha-globin gene cluster was observed in a family from Naples, Southern Italy. It removes the 5' end of the alpha 2-globin gene, causing an alpha (+)-thalassemia defect. Because of the presence of the residual 3' end of the alpha 2-globin gene, we indicated this new haplotype with the symbol (alpha)alpha 5.3. The 5' breakpoint, the first to be reported in the intergene region of the psi alpha 2- and psi alpha 1-globin genes, is located 822 bp upstream of the cap site of the psi alpha 1-gene and about 150 bp upstream of a 300-nt Alu family member. The 3' breakpoint is located in the IVS-1 nt 58 of the alpha 2-globin gene. The 5.3-kb deleted fragment shows particular characteristics: it contains four Alu sequences having long regions 80% complementary and the 5'-GGCC-3' short repeat at both ends. The sequences spanning across the breakpoints on the same strand and containing this repeat on their 3' and 5' ends, respectively, are 17 of 25 base complementary. These particular features led us to assume the formation of a multistem-loop due to the intrastrand interaction between the complementary regions as intermediate to the deletion. The unusual localization of the 5' breakpoint suggests that even the intergene region of the psi alpha 2- and psi alpha 1-globin genes may function as a deletion target.

Double dissociation between temporal and spatial pattern processing in patients with frontal and parietal damage.

One hundred and twenty nine patients affected by a cerebral lesion confined to a single lobe, underwent a battery of tests including the "Temporal Rule Induction" (TRI) and the Raven's "Coloured Progressive Matrices" (CPM). Frontal patients scored lower than any other group on TRI and parietal patients on CPM. This contrasting pattern of performance provides strong empirical support to the hypothesis that the frontal lobe is specifically involved in tasks that require a control on temporally ordered information whereas the parietal lobe is concerned with cognitive activities that imply visuo-spatial analysis.

Contralateral and ipsilateral tactile extinction in patients with right and left focal brain damage.

Heilman has suggested that right hemisphere lesions produce neglect both in the spaces contralateral and ipsilateral to the damaged hemisphere. To test this hypothesis, we studied the incidence of contralateral and ipsilateral tactile extinctions in 44 right and 59 left brain-damaged patients, by means of a standard test of a double tactile simultaneous stimulation of symmetrical and asymmetrical parts of the two sides of the body and of ipsilateral body parts. Results did not confirm Heilman's hypothesis; while contralateral tactile extinction was more frequent and severe in right than in left brain-damaged patients, no hemispheric difference was found when the number of ipsilateral extinctions was taken into account. Furthermore, ipsilateral tactile extinction was significantly related to aspecific factors (such as age, severity of sensorimotor impairment and widespread mental deterioration), irrespective of the hemispheric side of lesion.

[Clinical aspects and mechanisms of visual-spatial neglect]. / Aspects cliniques et mécanismes de la négligence visuo-spatiale.

Several interpretations have been advanced to explain the nature of unilateral spatial neglect. According to some authors, unilateral neglect could be due to a sensory deficit or to an oculomotor disorder hampering the patient from completely exploring the half space contralateral to the damaged hemisphere. According to other authors, the unilateral neglect syndrome could result either from a mutilation of the inner representation of the outside space or from a lack of attention for stimuli falling in the half space opposite to the hemispheric locus of lesion. We have wondered whether the relationships between unilateral neglect and hemispheric lateralization could not help to understand the nature of the neglect phenomena. A retrospective analysis of data gathered in neuropsychological literature, and results of an experimental study in which we had contrasted results obtained on an "overlapping figures" task and on a visual search task and a qualitative analysis of neglect phenomena observed in right brain-damaged patients, have led to the following conclusions: a) unilateral neglect is specifically linked to right hemisphere lesions in tasks requiring extraction of information from the central part of the visual field, whereas no difference between right-sided and left-sided lesions is observed on tasks requiring a full exploration of extra-personal space; b) tasks selectively affected in right brain-damaged patients require an automatic orienting of attention toward the more peripheral parts of the stimuli, which could be partly impaired owing to an early orientation of attention toward the right side of the pattern and a subsequent difficulty to detach attention from this early focus to orient it toward parts fo the stimulus lying in the left half space.

Constructive disabilities in focal brain-damaged patients. Influence of hemispheric side, locus of lesion and coexistent mental deterioration.

The performance of 185 focal brain-damaged patients on two non-verbal constructive tasks ('Copying Drawings' and 'Copying Drawings with Landmarks') was evaluated with regard to three variables: laterality of cerebral lesion, intrahemispheric locus of lesion, and coexistence of mental deterioration. The aim of the study was to assess the relationships between each of these variables and both the incidence and severity of constructional apraxia. Different evaluations were carried out in order to partial-out the possible interference of unilateral spatial neglect. Results showed that, regardless of whether faults attributable to unilateral spatial neglect were penalized or not: right brain-damaged patients performed slightly worse and showed a higher percentage of pathologic performances than left brain-damaged patients; subjects with parietal--and particularly with right parietal--damage obtained the poorest mean scores and exhibited the highest incidence of apraxic performances; and coexistence of mental deterioration was the most relevant variable associated with global decay of constructive scores and incidence of constructional apraxia.