The Effects of Silencing PTX3 on the Proteome of Human Endothelial Cells.

The human long pentraxin PTX3 has complex regulatory roles at the crossroad of innate immunity, inflammation, and tissue repair. PTX3 can be produced by various cell types, including vascular endothelial cells (ECs), in response to pro-inflammatory cytokines or bacterial molecules. PTX3 has also been involved in the regulation of cardiovascular biology, even if ambiguous results have been so far provided in both preclinical and clinical research. In this study, we compared the proteomic profiles of human ECs (human umbilical vein ECs, HUVECs), focusing on differentially expressed proteins between the control and PTX3-silenced ECs. We identified 19 proteins that were more abundant in the proteome of control ECs and 23 proteins that were more expressed in PTX3-silenced cells. Among the latter, proteins with multifunctional roles in angiogenesis, oxidative stress, and inflammation were found, and were further validated by assessing their mRNAs with RT-qPCR. Nevertheless, the knock down of PTX3 did not affect in vitro angiogenesis. On the contrary, the lack of the protein induced an increase in pro-inflammatory markers and a shift to the more oxidative profile of PTX3-deficient ECs. Altogether, our results support the idea of a protective function for PTX3 in the control of endothelial homeostasis, and more generally, in cardiovascular biology.

Sex-dependent differences in the secretome of human endothelial cells.

BACKGROUND: Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and "omics" approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms. METHODS: We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. RESULTS: Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression. CONCLUSIONS: Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

Inhibition of Chloride Intracellular Channel 1 (CLIC1) as Biguanide Class-Effect to Impair Human Glioblastoma Stem Cell Viability.

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC50 values significantly lower than metformin, (range 0.054-0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanide-structured molecules, characterized by high antitumor efficacy and safe toxicological profile.

Fatty acids rather than hormones restore in vitro angiogenesis in human male and female endothelial cells cultured in charcoal-stripped serum.

Charcoal-stripped serum (CSS) is a well-accepted method to model effects of sex hormones in cell cultures. We have recently shown that human endothelial cells (ECs) fail to growth and to undergo in vitro angiogenesis when cultured in CSS. However, the mechanism(s) underlying the CSS-induced impairment of in vitro EC properties are still unknown. In addition, whether there is any sexual dimorphism in the CSS-induced EC phenotype remains to be determined. Here, by independently studying human male and female ECs, we found that CSS inhibited both male and female EC growth and in vitro angiogenesis, with a more pronounced effect on male EC sprouting. Reconstitution of CSS with 17-ß estradiol, dihydrotestosterone, or the lipophilic thyroid hormone did not restore EC functions in both sexes. On the contrary, supplementation with palmitic acid or the acetyl-CoA precursor acetate significantly rescued the CSS-induced inhibition of growth and sprouting in both male and female ECs. We can conclude that the loss of metabolic precursors (e.g., fatty acids) rather than of hormones is involved in the impairment of in vitro proliferative and angiogenic properties of male and female ECs cultured with CSS.

Hormone-deprived serum impairs angiogenic properties in human endothelial cells regardless of estrogens.

AIMS: In vitro studies on hormone biological activities are commonly performed on cells cultured in nominally hormone-free media consisting of phenol-red-free media supplemented with charcoal-stripped (CS) serum. These media are largely used in almost all cell types, including endothelial cells (ECs). METHODS: Cell number and metabolic activity were measured with standard methods. Angiogenesis was evaluated in a three-dimensional spheroid sprouting assay. RESULTS: When we compared human umbilical vein ECs (HUVECs) cultured in standard conditions (199 medium supplemented with normal serum) with HUVECs grown in the hormone-free medium (phenol-red-free 199 medium supplemented with CS serum), we found that cells stop to grow in the absence of hormones. Notably, neither 17-ß2 estradiol nor dihydrotestosterone reversed this inhibition. Moreover, the presence of the CS serum was sufficient to abrogate the ability of HUVECs to sprout in a three-dimensional spheroid assay, thus affecting a functional property of ECs. CONCLUSIONS: Our results suggest that one or possibly more substances removed by stripping procedure from serum and different from sex hormones are crucial for the maintenance of in vitro ECs distinctive properties. Therefore, caution should be used when ECs are studied in media containing the CS serum.

Silencing of Eps8 inhibits in vitro angiogenesis.

AIMS: Eps8 is an actin-binding protein which has been proposed as a regulator of cancer cell motility and invasion. However, nothing much is known about its contribution to the invasive properties of endothelial cells (ECs), and more generally to angiogenesis. MAIN METHODS: Expression and silencing of Eps8 were evaluated by western blot analysis. The effect of Eps8 silencing on cell number and VEGF-induced signaling was tested with standard methods. Migration was evaluated by scratch wound assay and morphogenesis with 2-dimensional (2-D) tube formation and 3-dimensional (3-D) sprouting assays. Actin cytoskeleton was visualized by immunofluorescence. KEY FINDINGS: We found that silencing of Eps8 profoundly affected the ability of human ECs to migrate and to undergo tube formation and sprouting in 2-D and 3-D in vitro assays, respectively. Notably, capillary-like outgrowth was strictly depending on Eps8 expression also in human tumor-derived ECs. SIGNIFICANCE: Our data demonstrate for the first time the involvement of Eps8 in the morphological processes required for in vitro angiogenesis, and suggest that this protein might represent a common target for the design of new anticancer drugs, acting at the same time on both tumor and endothelial cells.

Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells.

BACKGROUND: Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs). METHODS: Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting. RESULTS: Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother's weight may exert a different influence on male and female babies. CONCLUSIONS: The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.

Silencing of Eps8 blocks migration and invasion in human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most malignant human primary brain tumor, and its infiltrative nature represents the leading cause for the failure of therapies and tumor recurrences. It is therefore crucial the knowledge of the molecular mechanisms underlying GBM invasion to identify novel therapeutic targets to limit motility. In this study, we evaluated the role of Epidermal growth factor receptor Pathway Substrate 8 (Eps8), a crucial regulator of the actin cytoskeleton dynamics accompanying cell motility and invasion, in GBM migration and invasiveness. We found that silencing of the protein by small interfering RNAs (siRNAs) abrogated the migratory and invasive capacity of three different human GBM cell lines both in 2-dimensional (2-D) and 3-dimensional (3-D) in vitro assays. The inhibitory effect on invasion was maintained independently by the migration mode utilized by the cells in our 3-D model, and was accompanied by an impaired formation of actin-based cytoskeletal protrusive structures. Our data propose Eps8 as a key molecule involved in the control of the intrinsic invasive behavior of GBM cells, and suggest that this protein might represent a useful target for the design of new drugs for the treatment of these tumors.

Chronic deficiency of nitric oxide affects hypoxia inducible factor-1α (HIF-1α) stability and migration in human endothelial cells.

BACKGROUND: Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated. RESULTS: Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N(G)-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O(2) consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells. CONCLUSION: Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.

Oxytocin stimulates in vitro angiogenesis via a Pyk-2/Src-dependent mechanism.

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.

Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells.

Nitric oxide (NO) generated by endothelial NO synthase (eNOS) is a key regulator of endothelial cell (EC) migration. Whereas the effects of acute NO generation are generally stimulatory, the role of chronic basal NO release has not been explored so far. Here, we addressed this question both in HeLa and in human endothelial cells. In stably transfected HeLa cells, inducibly expressing eNOS, expression of the enzyme per se blunted the phosphorylation of Akt/PKB in response to serum and strongly inhibited chemotaxis, an effect partially blocked by eNOS- and soluble guanylyl cyclase (sGC) inhibitors. Likewise, long-term pre-treatment of non-transfected HeLa cells with nanomolar concentrations of an NO donor inhibited subsequent migration, an effect blocked by sGC inhibition and mimicked by a cGMP analog. Finally, EC migration was stimulated by chronic pre-treatment with an eNOS inhibitor. Thus, in addition to its well-known stimulatory role, eNOS attenuates migration through basal long-term NO release.

A molecular dynamics study of human endostatin and its synthetic fragments with antiangiogenic properties.

Human endostatin is one of the better characterized endogenous angiogenesis inhibitors, and its ability to modulate vascularization of tumours could be of great therapeutic interest. These properties are not exclusive to the full-length protein, but are shared by some of its synthetic fragments. A number of research groups have partitioned human endostatin in different peptides and have investigated their activity, in order to collect a body of experimental data which could be important in shedding new light on their structure-activity relationships. It was also reported that a small active fragment can become inactive when contained in a larger fragment, revealing an apparent discrepancy in the experimental results. Very few studies have been devoted to the computational analysis of these systems and to the rationalization of their properties using molecular modelling. Through molecular dynamics simulations of human endostatin and of four synthetic fragments, we have been able to rationalize the experimental findings. In particular, we have identified a pattern consisting of six amino acids, namely R-R(G)-A-D-R-A, which appears to be an active epitope if it is properly exposed to the solvent. Interestingly, this pattern can be already present in sequential order in the primary structure, or it can be generated by the spatial approach of two groups of residues, far apart in the primary structure, as an effect of the peptide folding. Comparing the structural features and the time evolution of all the simulated peptides we provide a coherent explanation of their activity or inactivity.

Expression studies in gliomas and glial cells do not support a tumor suppressor role for LGI1.

Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.

Anti-migratory and anti-invasive effect of somatostatin in human neuroblastoma cells: involvement of Rac and MAP kinase activity.

Cell motility and invasion are crucial events for the spread of cancer and, consequently, the metastatic process. Platelet-derived growth factor (PDGF) is not only capable of stimulating the proliferation of SH-SY5Y human neuroblastoma cells, but also their migration and invasion through an extracellular matrix barrier. Experiments using wortmannin and PD98059, specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and of the mitogen-activated protein kinases (ERK 1 and 2) signaling, respectively, show that the activation of both pathways is required for the PDGF-induced cell motility responses. We have previously shown that somatostatin inhibits cell division and ERK 1/2 and Ras activity in SH-SY5Y cells. We report here that it is also capable of potently and effectively inhibiting their PDGF-stimulated migration and invasion. The inhibitory effect of somatostatin is sensitive to pertussis toxin. Although somatostatin does not affect PI3-K, it inhibits ERK 1/2 and the small G-protein Rac activation and ruffle formation induced by PDGF. These results indicate that somatostatin can be considered an anti-migratory and anti-invasive agent that acts by inhibiting ERK 1/2 signaling and the PI3-K pathway via the inhibition of Rac in SHSY5Y cells.

Avaliação do desempenho de um teste sorológico rápido utilizando quatro antígenos para o diagnóstico de tuberculose pulmonar / Evaluation of a rapid serologic test with four antigens for the pulmonary tuberculosis diagnosis

Introdução: Os testes sorológicos não fazem parte da rotina da investigação diagnóstica da tuberculose pulmonar. Métodos: Um teste imunocromatográfico (ICT-TB) utilizando quatro diferentes antígenos purificados para a pesquisa de anticorpo IgG foi avaliado em 268 amostras de soro de 69 casos de tuberculose pulmonar, 41 de doença não tuberculosa, 12 de sequela de tuberculose, 107 controles saudáveis e 39 contatos domiciliares de tuberculose pulmonar. Resultados: A sensibilidade foi de 54% e a especificidade 86%. Entre os contatos domiciliares o ICT-TB foi positivo em 10%. Conclusão: concluímos que a sensibilidade e a especificidade do teste sorológico ICT-TB conferiu a ele pouco valor para o diagnóstico de tuberculose pulmonar na nossa amostra.

Introduction: Serologic tests are not routinely used for the diagnostic investigation of pulmonary tuberculosis. Methods: An immunochromatographic test (ICT-TB) using four different purified antigens against an IgG antibody was evaluated in 268 serum samples of 69 patients with pulmonary tuberculosis, 41 with a nontuberculous disease, 12 subjects with healed pulmonary tuberculosis and 107 healthy volunteers and 39 close contacts of pulmonary tuberculosis patients. Results: Sensitivity was 54% and specificity 86%. Among close contacts, ICT-TB was positive in 10%. Conclusion: We concluded that sensitivity and specificity of ICT-TB test made it a poor diagnostic tool for pulmonary tuberculosis diagnosis in our sample.