Transcriptional activation of endothelial cells by TGFβ coincides with acute microvascular plasticity following focal spinal cord ischaemia/reperfusion injury

Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFβ (transforming growth factor β) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFβ-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFβ-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFβ as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI

Extracellular matrix alterations in metastatic processes

The extracellular matrix (ECM) is a complex network of extracellular-secreted macromolecules, such as collagen, enzymes and glycoproteins, whose main functions deal with structural scaffolding and biochemical support of cells and tissues. ECM homeostasis is essential for organ development and functioning under physiological conditions, while its sustained modification or dysregulation can result in pathological conditions. During cancer progression, epithelial tumor cells may undergo epithelial-to-mesenchymal transition (EMT), a morphological and functional remodeling, that deeply alters tumor cell features, leading to loss of epithelial markers (i.e., E-cadherin), changes in cell polarity and intercellular junctions and increase of mesenchymal markers (i.e., N-cadherin, fibronectin and vimentin). This process enhances cancer cell detachment from the original tumor mass and invasiveness, which are necessary for metastasis onset, thus allowing cancer cells to enter the bloodstream or lymphatic flow and colonize distant sites. The mechanisms that lead to development of metastases in specific sites are still largely obscure but modifications occurring in target tissue ECM are being intensively studied. Matrix metalloproteases and several adhesion receptors, among which integrins play a key role, are involved in metastasis-linked ECM modifications. In addition, cells involved in the metastatic niche formation, like cancer associated fibroblasts (CAF) and tumor associated macrophages (TAM), have been found to play crucial roles in ECM alterations aimed at promoting cancer cells adhesion and growth. In this review we focus on molecular mechanisms of ECM modifications occurring during cancer progression and metastatic dissemination to distant sites, with special attention to lung, liver and bone. Moreover, the functional role of cells forming the tumor niche will also be reviewed in light of the most recent findings

In vitro glioblastoma models: A journey into the third dimension

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, with an average survival time of about one year from initial diagnosis. In the attempt to overcome the com-plexity and drawbacks associated with in vivo GBM models, together with the need of developing systems dedicated to screen new potential drugs, considerable efforts have been devoted to the implementation of reliable and affordable in vitro GBM models. Recent findings on GBM molecular features, revealing a high heterogeneity between GBM cells and also between other non‐tumor cells belonging to the tumoral niche, have stressed the limitations of the classical 2D cell culture systems. Recently, several novel and innovative 3D cell cultures models for GBM have been proposed and implemented. In this review, we first describe the different populations and their functional role of GBM and niche non‐tumor cells that could be used in 3D models. An overview of the current available 3D in vitro systems for modeling GBM, together with their major weaknesses and strengths, is presented. Lastly, we discuss the impact of groundbreaking technologies, such as bioprinting and multi‐omics single cell analysis, on the future implementation of 3D in vitro GBM models

Membrane localization of N-acylphosphatidylethanolamine in central neurons: studies with exogenous phospholipases.

We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [3H]gamma-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A2 (PLA2) (Apis mellifera) and PLC (B. cereus) mobilized [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that approximately 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE

Membrane localization of N-acylphosphatidylethanolamine in central neurons: studies with exogenous phospholipases.

We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [3H]gamma-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A2 (PLA2) (Apis mellifera) and PLC (B. cereus) mobilized [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that approximately 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE

VALUTAZIONE CLINICA E TERAPEUTICA DELLA ROTTURA PREMATURA DELLE MEMBRANE PRIMA DELLA 28^ SETTIMANA GESTAZIONALE. OUTCOME NEONATALE

Abbiamo preso in considerazione 25 gravide con rottura prematura delle membrane fra la 15.ma e la 28.ma settimana gestazionale. E' stato condotto sul campione in esame un accurato monitoraggio materno fetale insieme ad una terapia antibiotica, tocolitica e induzione della maturita' polmonare. 12 dei 25 casi da noi selezionati hanno avuto un esito neonatale favorevole e sono attualmente seguiti dal Centro di Patologia Neonatale dell'Universita' di Padova. Questo risultato confortante giustifica la nostra condotta terapeutica sempre nell'interno di migliorare l'outcome neonatale