MOESM3 of Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis
Additional file 3. A single channel, grayscale, bas-relief image of Fig. 2a, c. The digital filte... more Additional file 3. A single channel, grayscale, bas-relief image of Fig. 2a, c. The digital filters applied to pictures a and b in Fig. 2, highlight the endothelial profile (a, d), the pericyte origin of TNTs (b, e), and their excavated course (tunneling) through the dense, fetal cerebral cortex parenchyma (c, f; arrows). Scale bars a, b 25 µm; c, d 20 µm.
MOESM7 of Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis
Additional file 7. CD105 is a marker of angiogenically activated endothelial tip cells in the fet... more Additional file 7. CD105 is a marker of angiogenically activated endothelial tip cells in the fetal cerebral cortex. a–c Examples of CD105+ activated. d–f Examples of CD105+ endothelial cells, covered by CD146+ pericytes (PC). Nuclear counterstaining TOPRO-3. Scale bar a, b, d 20 µm; c, e, f 10 µm.
MOESM6 of Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis
Additional file 6. Does the large TNTs convey cell nuclei? Sequence of single optical planes from... more Additional file 6. Does the large TNTs convey cell nuclei? Sequence of single optical planes from the z-stack of Fig. 5d, shows the TNT plasma membrane and a cell nucleus inside the TNT.
Prognostic role of XTP1/DEPDC1B and SDP35/DEPDC1A in high grade soft-tissue sarcomas
Histology and histopathology, 2018
BACKGROUND The outcome of patients with metastatic soft tissue sarcoma (STS) remains unfavourable... more BACKGROUND The outcome of patients with metastatic soft tissue sarcoma (STS) remains unfavourable and new therapeutic strategies are needed. The aim of this study was to determine the role of RhoGAP, XTP1/DEPDC1B and SDP35/DEPDC1A, as possible prognostic markers, to be used to identify candidate patients for more effective and personalized therapies. MATERIALS-METHODS SDP35/DEPDC1A and XTP1/DEPDC1B transcriptional levels were evaluated by Real-Time PCR in 86 primary STS and 22 paired lung metastasis. 17 normal tissues were used as control. Protein expression was evaluated by tissue microarray, including 152 paraffin-embedded STS samples and by western blot in 22 lung metastases and paired primary STS. Non-parametric and parametric analysis were used to establish the differences in gene and protein expression and prognostic factors were tested with Kaplan Meier and Cox's regression analyses. RESULTS SDP35/DEPDC1A and XTP1/DEPDC1B gene were down-regulated in adjacent normal tissue...
The chondroitin sulfate proteoglycan 4 (CSPG4) gene encodes a transmembrane proteoglycan (PG) con... more The chondroitin sulfate proteoglycan 4 (CSPG4) gene encodes a transmembrane proteoglycan (PG) constituting the largest and most structurally complex macromolecule of the human surfaceome. Its transcript shows an extensive evolutionary conservation and, due to the elaborated intracellular processing of the translated protein, it generates an array of glycoforms with the potential to exert variant-specific functions. CSPG4-mediated molecular events are articulated through the interaction with more than 40 putative ligands and the concurrent involvement of the ectodomain and cytoplasmic tail. Alternating inside-out and outside-in signal transductions may thereby be elicited through a tight functional connection of the PG with the cytoskeleton and its regulators. The potential of CSPG4 to influence both types of signaling mechanisms is also asserted by its lateral mobility along the plasma membrane and its intersection with microdomain-restricted internalization and endocytic trafficking. Owing to the multitude of molecular interplays that CSPG4 may engage, and thanks to a differential phosphorylation of its intracellular domain accounted by crosstalking signaling pathways, the PG stands out for its unique capability to affect numerous cellular phenomena, including those purporting pathologic conditions. We discuss here the progresses made in advancing our understanding about the structural-functional bases for the ability of CSPG4 to widely impact on cell behavior, such as to highlight how its multivalency may be exploited to interfere with disease progression.
Background: Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in rec... more Background: Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in recent times as involved in cell-to-cell communication between distant cells. Nevertheless, TNT-like, long filopodial processes had already been described in the last century as connecting facing, growing microvessels during the process of cerebral cortex vascularization and collateralization. Here we have investigated the possible presence and the cellular origin of TNTs during normal brain vascularization and also in highly vascularized brain tumors. Methods: We searched for TNTs by high-resolution immunofluorescence confocal microscopy, applied to the analysis of 20-µm, thick sections from lightly fixed, unembedded samples of both developing cerebral cortex and human glioblastoma (GB), immunolabeled for endothelial, pericyte, and astrocyte markers, and vessel basal lamina molecules. Results: The results revealed the existence of pericyte-derived TNTs, labeled by proteoglycan NG2/CSPG4 and CD146. In agreement with the described heterogeneity of these nanostructures, ultra-long (> 300 µm) and very thin (< 0.8 µm) TNTs were observed to bridge the gap between the wall of distant vessels, or were detected as short (< 300 µm) bridging cables connecting a vessel sprout with its facing vessel or two apposed vessel sprouts. The pericyte origin of TNTs ex vivo in fetal cortex and GB was confirmed by in vitro analysis of brain pericytes, which were able to form and remained connected by typical TNT structures. Conclusions: None of the multiple roles described for TNTs can be excluded from a possible involvement during the processes of both normal and pathological vessel growth. A possible function, suggested by the pioneering studies made during cerebral cortex vascularization, is in cell searching and cell-to-cell recognition during the processes of vessel collateralization and vascular network formation. According to our results, it is definitely the pericyte-derived TNTs that seem to actively explore the surrounding microenvironment, searching for (site-to-site recognition), and connecting with (pericyte-to-pericyte and/or pericyte-to-endothelial cell communication), the targeted vessels. This idea implies that TNTs may have a primary role in the very early phases of both physiological and tumor angiogenesis in the brain.
In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs)... more In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood-brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knockout (NG2KO) mice and used laser confocal microscopy immunofluorescence and M. Errede and F. Girolamo contributed equally as second authors to this work. N. Kerlero de Rosbo and A. Uccelli contributed equally as last authors to this work.
NG2 is a membrane spanning proteoglycan with a large extracellular domain showing binding sites f... more NG2 is a membrane spanning proteoglycan with a large extracellular domain showing binding sites for several molecules, including extracellular matrix components and growth factors. In the developing CNS, NG2 is expressed by subpopulations of oligodendroglia precursors and additionally by microvascular pericytes. The expression of NG2 is downregulated in mature oligodendrocytes and also pericytes of stable brain vessels in healthy brains do not express the proteoglycan. Our previous studies have demonstrated that during human cerebral cortex development, 'activated' NG2pericytes form vascular cord that allow endothelial cell migration and elongation thus contributing to vascular sprouting. Braced by these first results, we have concentrated our attention on the possible involvement of NG2 and NG2-expressing cells on cell/ cell and cell/matrix relationships during vessel growth, in developing human brain at 9 and 22 weeks of gestation. Double immunolabelling has been carried out utilizing anti-NG2 antibodies directed against different antigenic sites of the core protein; namely, a rabbit anti-NG2 D2, mouse monoclonal antibodies from two different clones, F9 and D7, and a commercially available rabbit anti NG2 (CSPG4, Sigma). At the earlier developmental stage, NG2 + pericytes are already an integral part of the vessel wall and are involved, together with Glut + endothelial cells, in basal lamina collagen type IV and VI deposition. In double immunolabelling with two different NG2 antibodies (D2/F9), the staining patterns almost completely overlap and also coincide with collagen type IV/VI distributions. Later on, NG2 D2 and NG2 F9 staining patterns are largely unrelated, D2 appearing associated with both pericytes and vessel basal membrane, whereas NG2 F9 is primarily restricted to pericytes. It is known that in 'in vitro' assay almost the entire ectodomain of NG2 can be released by proteolitic shedding. These observations support the concept that both membrane-bound and unbound NG2 forms are involved in vessel growth, and that the released proteoglycan may contribute as an ECM substrate to endothelial cell migration and sprouting.
NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic ... more NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic factor, a valuable tool for ex vivo and non-invasive molecular diagnostics and, by virtue of its tight association with malignancy, a tantalizing therapeutic target in several tumour types. Although the biology behind its involvement in cancer progression needs to be better understood, implementation of NG2/CSPG4 in the routine clinical practice is attainable and has the potential to contribute to an improved individualized management of cancer patients. In this context, its polymorphic nature seems to be particularly valuable in the effort to standardize informative diagnostic procedures and consolidate forcible immunotherapeutic treatment strategies. We discuss here the underpinnings for this potential and highlight the benefits of taking advantage of the intra-tumour and inter-patient variability in the regulation of NG2/CSPG4 expression. We envision that NG2/CSPG4 may effectively be ...
176 POSTER Drug resistance in highly aggressive acute leukemias is controlled by de novo expressed NG2 proteoglycan acting via modulation of selected transporters
NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed m... more NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/ CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating between phenotype-specific and neoplastic versus non-neoplastic variants of the PG, thus opening up vistas for more selective immunotherapeutic targeting of brain tumours.
In an effort to define the biological functions of COMP, a functional genetic screen was performe... more In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 Colocalize in the Growth Plates in Vivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMPmediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth.
Journal of Neuropathology & Experimental Neurology, 2012
The pathophysiology of cerebral cortical lesions in multiple sclerosis (MS) is not understood. We... more The pathophysiology of cerebral cortical lesions in multiple sclerosis (MS) is not understood. We investigated cerebral cortex microvessels during immune-mediated demyelination in the MS model chronic murine experimental autoimmune encephalomyelitis (EAE) by immunolocalization of the endothelial cell tight junction (TJ) integral proteins claudin-5 and occludin, a structural protein of caveolae, caveolin-1, and the blood-brain barrierYspecific endothelial transporter, Glut 1. In EAE-affected mice, there were areas of extensive subpial demyelination and well-demarcated lesions that extended to deeper cortical layers. Activation of microglia and absence of perivascular inflammatory infiltrates were common in these areas. Microvascular endothelial cells showed increased expression of caveolin-1 and a coincident loss of both claudin-5 and occludin normal junctional staining patterns. At a very early disease stage, claudin-5 molecules tended to cluster and form vacuoles that were also Glut 1 positive; the initially preserved occludin pattern became diffusely cytoplasmic at more advanced stages. Possible internalization of claudin-5 on TJ dismantling was suggested by its coexpression with the autophagosomal marker MAP1LC3A. Loss of TJ integrity was confirmed by fluorescein isothiocyanateYdextran experiments that showed leakage of the tracer into the perivascular neuropil. These observations indicate that, in the cerebral cortex of EAE-affected mice, there is a microvascular disease that differentially targets claudin-5 and occludin during ongoing demyelination despite only minimal inflammation.
The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells ... more The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells (hMSCs) by simple in vitro treatments is appealing both conceptually and practically. However, whether phenotypic modulations observed after chemical manipulation of such stem cells truly represent a genuine trans-lineage differentiation remains to be established. We have re-evaluated the effects of a frequently reported biochemical approach, based on treatment with butylated hydroxyanisole and dimethylsulphoxide, to bring about such phenotypic conversion by monitoring the morphological changes induced by the treatment in real time, by analysing the expression of phenotype-specific protein markers and by assessing the modulation of transcriptome. Video time-lapse microscopy showed that conversion of mesenchymal stem cells to a neuron-like morphology could be reproduced in normal primary fibroblasts as well as mimicked by addition of drugs eliciting cytoskeletal collapse and disruption of...
MOESM3 of Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis
Additional file 3. A single channel, grayscale, bas-relief image of Fig. 2a, c. The digital filte... more Additional file 3. A single channel, grayscale, bas-relief image of Fig. 2a, c. The digital filters applied to pictures a and b in Fig. 2, highlight the endothelial profile (a, d), the pericyte origin of TNTs (b, e), and their excavated course (tunneling) through the dense, fetal cerebral cortex parenchyma (c, f; arrows). Scale bars a, b 25 µm; c, d 20 µm.
MOESM7 of Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis
Additional file 7. CD105 is a marker of angiogenically activated endothelial tip cells in the fet... more Additional file 7. CD105 is a marker of angiogenically activated endothelial tip cells in the fetal cerebral cortex. a–c Examples of CD105+ activated. d–f Examples of CD105+ endothelial cells, covered by CD146+ pericytes (PC). Nuclear counterstaining TOPRO-3. Scale bar a, b, d 20 µm; c, e, f 10 µm.
MOESM6 of Tunneling nanotubes evoke pericyte/endothelial communication during normal and tumoral angiogenesis
Additional file 6. Does the large TNTs convey cell nuclei? Sequence of single optical planes from... more Additional file 6. Does the large TNTs convey cell nuclei? Sequence of single optical planes from the z-stack of Fig. 5d, shows the TNT plasma membrane and a cell nucleus inside the TNT.
Prognostic role of XTP1/DEPDC1B and SDP35/DEPDC1A in high grade soft-tissue sarcomas
Histology and histopathology, 2018
BACKGROUND The outcome of patients with metastatic soft tissue sarcoma (STS) remains unfavourable... more BACKGROUND The outcome of patients with metastatic soft tissue sarcoma (STS) remains unfavourable and new therapeutic strategies are needed. The aim of this study was to determine the role of RhoGAP, XTP1/DEPDC1B and SDP35/DEPDC1A, as possible prognostic markers, to be used to identify candidate patients for more effective and personalized therapies. MATERIALS-METHODS SDP35/DEPDC1A and XTP1/DEPDC1B transcriptional levels were evaluated by Real-Time PCR in 86 primary STS and 22 paired lung metastasis. 17 normal tissues were used as control. Protein expression was evaluated by tissue microarray, including 152 paraffin-embedded STS samples and by western blot in 22 lung metastases and paired primary STS. Non-parametric and parametric analysis were used to establish the differences in gene and protein expression and prognostic factors were tested with Kaplan Meier and Cox's regression analyses. RESULTS SDP35/DEPDC1A and XTP1/DEPDC1B gene were down-regulated in adjacent normal tissue...
The chondroitin sulfate proteoglycan 4 (CSPG4) gene encodes a transmembrane proteoglycan (PG) con... more The chondroitin sulfate proteoglycan 4 (CSPG4) gene encodes a transmembrane proteoglycan (PG) constituting the largest and most structurally complex macromolecule of the human surfaceome. Its transcript shows an extensive evolutionary conservation and, due to the elaborated intracellular processing of the translated protein, it generates an array of glycoforms with the potential to exert variant-specific functions. CSPG4-mediated molecular events are articulated through the interaction with more than 40 putative ligands and the concurrent involvement of the ectodomain and cytoplasmic tail. Alternating inside-out and outside-in signal transductions may thereby be elicited through a tight functional connection of the PG with the cytoskeleton and its regulators. The potential of CSPG4 to influence both types of signaling mechanisms is also asserted by its lateral mobility along the plasma membrane and its intersection with microdomain-restricted internalization and endocytic trafficking. Owing to the multitude of molecular interplays that CSPG4 may engage, and thanks to a differential phosphorylation of its intracellular domain accounted by crosstalking signaling pathways, the PG stands out for its unique capability to affect numerous cellular phenomena, including those purporting pathologic conditions. We discuss here the progresses made in advancing our understanding about the structural-functional bases for the ability of CSPG4 to widely impact on cell behavior, such as to highlight how its multivalency may be exploited to interfere with disease progression.
Background: Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in rec... more Background: Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in recent times as involved in cell-to-cell communication between distant cells. Nevertheless, TNT-like, long filopodial processes had already been described in the last century as connecting facing, growing microvessels during the process of cerebral cortex vascularization and collateralization. Here we have investigated the possible presence and the cellular origin of TNTs during normal brain vascularization and also in highly vascularized brain tumors. Methods: We searched for TNTs by high-resolution immunofluorescence confocal microscopy, applied to the analysis of 20-µm, thick sections from lightly fixed, unembedded samples of both developing cerebral cortex and human glioblastoma (GB), immunolabeled for endothelial, pericyte, and astrocyte markers, and vessel basal lamina molecules. Results: The results revealed the existence of pericyte-derived TNTs, labeled by proteoglycan NG2/CSPG4 and CD146. In agreement with the described heterogeneity of these nanostructures, ultra-long (> 300 µm) and very thin (< 0.8 µm) TNTs were observed to bridge the gap between the wall of distant vessels, or were detected as short (< 300 µm) bridging cables connecting a vessel sprout with its facing vessel or two apposed vessel sprouts. The pericyte origin of TNTs ex vivo in fetal cortex and GB was confirmed by in vitro analysis of brain pericytes, which were able to form and remained connected by typical TNT structures. Conclusions: None of the multiple roles described for TNTs can be excluded from a possible involvement during the processes of both normal and pathological vessel growth. A possible function, suggested by the pioneering studies made during cerebral cortex vascularization, is in cell searching and cell-to-cell recognition during the processes of vessel collateralization and vascular network formation. According to our results, it is definitely the pericyte-derived TNTs that seem to actively explore the surrounding microenvironment, searching for (site-to-site recognition), and connecting with (pericyte-to-pericyte and/or pericyte-to-endothelial cell communication), the targeted vessels. This idea implies that TNTs may have a primary role in the very early phases of both physiological and tumor angiogenesis in the brain.
In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs)... more In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood-brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knockout (NG2KO) mice and used laser confocal microscopy immunofluorescence and M. Errede and F. Girolamo contributed equally as second authors to this work. N. Kerlero de Rosbo and A. Uccelli contributed equally as last authors to this work.
NG2 is a membrane spanning proteoglycan with a large extracellular domain showing binding sites f... more NG2 is a membrane spanning proteoglycan with a large extracellular domain showing binding sites for several molecules, including extracellular matrix components and growth factors. In the developing CNS, NG2 is expressed by subpopulations of oligodendroglia precursors and additionally by microvascular pericytes. The expression of NG2 is downregulated in mature oligodendrocytes and also pericytes of stable brain vessels in healthy brains do not express the proteoglycan. Our previous studies have demonstrated that during human cerebral cortex development, 'activated' NG2pericytes form vascular cord that allow endothelial cell migration and elongation thus contributing to vascular sprouting. Braced by these first results, we have concentrated our attention on the possible involvement of NG2 and NG2-expressing cells on cell/ cell and cell/matrix relationships during vessel growth, in developing human brain at 9 and 22 weeks of gestation. Double immunolabelling has been carried out utilizing anti-NG2 antibodies directed against different antigenic sites of the core protein; namely, a rabbit anti-NG2 D2, mouse monoclonal antibodies from two different clones, F9 and D7, and a commercially available rabbit anti NG2 (CSPG4, Sigma). At the earlier developmental stage, NG2 + pericytes are already an integral part of the vessel wall and are involved, together with Glut + endothelial cells, in basal lamina collagen type IV and VI deposition. In double immunolabelling with two different NG2 antibodies (D2/F9), the staining patterns almost completely overlap and also coincide with collagen type IV/VI distributions. Later on, NG2 D2 and NG2 F9 staining patterns are largely unrelated, D2 appearing associated with both pericytes and vessel basal membrane, whereas NG2 F9 is primarily restricted to pericytes. It is known that in 'in vitro' assay almost the entire ectodomain of NG2 can be released by proteolitic shedding. These observations support the concept that both membrane-bound and unbound NG2 forms are involved in vessel growth, and that the released proteoglycan may contribute as an ECM substrate to endothelial cell migration and sprouting.
NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic ... more NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic factor, a valuable tool for ex vivo and non-invasive molecular diagnostics and, by virtue of its tight association with malignancy, a tantalizing therapeutic target in several tumour types. Although the biology behind its involvement in cancer progression needs to be better understood, implementation of NG2/CSPG4 in the routine clinical practice is attainable and has the potential to contribute to an improved individualized management of cancer patients. In this context, its polymorphic nature seems to be particularly valuable in the effort to standardize informative diagnostic procedures and consolidate forcible immunotherapeutic treatment strategies. We discuss here the underpinnings for this potential and highlight the benefits of taking advantage of the intra-tumour and inter-patient variability in the regulation of NG2/CSPG4 expression. We envision that NG2/CSPG4 may effectively be ...
176 POSTER Drug resistance in highly aggressive acute leukemias is controlled by de novo expressed NG2 proteoglycan acting via modulation of selected transporters
NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed m... more NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/ CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating between phenotype-specific and neoplastic versus non-neoplastic variants of the PG, thus opening up vistas for more selective immunotherapeutic targeting of brain tumours.
In an effort to define the biological functions of COMP, a functional genetic screen was performe... more In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 Colocalize in the Growth Plates in Vivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMPmediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth.
Journal of Neuropathology & Experimental Neurology, 2012
The pathophysiology of cerebral cortical lesions in multiple sclerosis (MS) is not understood. We... more The pathophysiology of cerebral cortical lesions in multiple sclerosis (MS) is not understood. We investigated cerebral cortex microvessels during immune-mediated demyelination in the MS model chronic murine experimental autoimmune encephalomyelitis (EAE) by immunolocalization of the endothelial cell tight junction (TJ) integral proteins claudin-5 and occludin, a structural protein of caveolae, caveolin-1, and the blood-brain barrierYspecific endothelial transporter, Glut 1. In EAE-affected mice, there were areas of extensive subpial demyelination and well-demarcated lesions that extended to deeper cortical layers. Activation of microglia and absence of perivascular inflammatory infiltrates were common in these areas. Microvascular endothelial cells showed increased expression of caveolin-1 and a coincident loss of both claudin-5 and occludin normal junctional staining patterns. At a very early disease stage, claudin-5 molecules tended to cluster and form vacuoles that were also Glut 1 positive; the initially preserved occludin pattern became diffusely cytoplasmic at more advanced stages. Possible internalization of claudin-5 on TJ dismantling was suggested by its coexpression with the autophagosomal marker MAP1LC3A. Loss of TJ integrity was confirmed by fluorescein isothiocyanateYdextran experiments that showed leakage of the tracer into the perivascular neuropil. These observations indicate that, in the cerebral cortex of EAE-affected mice, there is a microvascular disease that differentially targets claudin-5 and occludin during ongoing demyelination despite only minimal inflammation.
The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells ... more The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells (hMSCs) by simple in vitro treatments is appealing both conceptually and practically. However, whether phenotypic modulations observed after chemical manipulation of such stem cells truly represent a genuine trans-lineage differentiation remains to be established. We have re-evaluated the effects of a frequently reported biochemical approach, based on treatment with butylated hydroxyanisole and dimethylsulphoxide, to bring about such phenotypic conversion by monitoring the morphological changes induced by the treatment in real time, by analysing the expression of phenotype-specific protein markers and by assessing the modulation of transcriptome. Video time-lapse microscopy showed that conversion of mesenchymal stem cells to a neuron-like morphology could be reproduced in normal primary fibroblasts as well as mimicked by addition of drugs eliciting cytoskeletal collapse and disruption of...
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Papers by Roberto Perris