Carmen Capobianco

An intimate interplay between precocious, migrating pericytes and endothelial cells governs human fetal brain angiogenesis

In order to better understand the process of angiogenesis in the developing human brain, we have examined the spatial relationship and relative contributions of endothelial cells and pericytes, the two primary cell types involved in vessel growth, together with their relation with the vascular basement membrane. Pericytes were immunolocalized through use of the specific markers nerve/glial antigen 2 (NG2) proteoglycan, endosialin (CD248) and the platelet-derived growth factor receptor β (PDGFR-β), while endothelial cells were identified by the pan-endothelial marker CD31 and the blood brain barrier (BBB)-specific markers claudin-5 and glucose transporter isoform 1 (GLUT-1). The quantitative analysis demonstrates that microvessels of the fetal human telencephalon are characterized by a continuous layer of activated/angiogenic NG2 pericytes, which tightly invest endothelial cells and participate in the earliest stages of vessel growth. Immunolabelling with anti-active matrix metalloproteinase-2 (aMMP-2) and anti-collagen type IV antibodies revealed that aMMP-2 producing endothelial cells and pericytes are both associated with the vascular basement membrane during vessel sprouting. Detailed localization of the two vascular cell types during angiogenesis suggests that growing microvessels of the human telencephalon are formed by a pericyte-driven angiogenic process in which the endothelial cells are preceded and guided by migrating pericytes during organization of the growing vessel wall.

Ischemia-Reperfusion Induces Glomerular and Tubular Activation of Proinflammatory and Antiapoptotic Pathways: Differential Modulation by Rapamycin

Ischemia-reperfusion (I-R) injury in transplanted kidney, a key pathogenic event of delayed graft function (DGF), is characterized by tubular cell apoptosis and interstitial inflammation. Akt-mammalian target of rapamycin-S6k and NF-kappaB-inducing kinase (NIK)-NF-kappaB axis are the two main signaling pathways regulating cell survival and inflammation. Rapamycin, an immunosuppressive drug inhibiting the Akt axis, is associated with a prolonged DGF. The aim of this study was to evaluate Akt and NF-kappaB axis activation in patients who had DGF and received or not rapamycin and in a pig model of I-R and the role of coagulation priming in this setting. In graft biopsies from patients who were not receiving rapamycin, phosphorylated Akt increased in proximal tubular, interstitial, and mesangial cells with a clear nuclear translocation. The same pattern of activation was observed for S6k and NIK. However, in rapamycin-treated patients, a significant reduction of S6k but not Akt and NIK activation was observed. A time-dependent activation of phosphatidylinositol 3-kinase, Akt, S6k, and NIK was observed in the experimental model with the same pattern reported for transplant recipients who did not receive rapamycin. Extensive interstitial and glomerular fibrin deposition was observed both in pig kidneys upon reperfusion and in DGF human biopsies. It is interesting that the activation of both Akt and NIK-NF-kappaB pathways was induced by thrombin in cultured proximal tubular cells. In conclusion, the data suggest that (1) coagulation may play a pathogenic role in I-R injury; (2) the Akt axis is activated after I-R, and its inhibition may explain the prolonged DGF observed in rapamycin-treated patients; and (3) NIK activation in I-R and DGF represents a proinflammatory, rapamycin-insensitive signal, potentially leading to progressive graft injury.

Fetal blood-brain barrier P-glycoprotein contributes to brain protection during human development.

During brain development and blood-brain barrier (BBB) differentiation the expression of P-glycoprotein (P-gp) may complement the protective function of the placental barrier against xenobiotic substances. To establish an immunohistochemical procedure for P-gp detection, different anti-P-gp monoclonal antibodies were first tested on a fibrosarcoma cell line and colonic carcinoma tissue. The protocol was then tested on adult human brains as a BBB-P-gp tissue-specific control and for double labeling with anti-P-gp and the astroglia marker glial fibrillary acidic protein (GFAP). The protocol was then used to analyze the expression and localization of P-gp in human fetuses during cerebral cortex formation. At the earliest examined stage, 12 weeks of gestation (wg), P-gp was detectable as diffuse cytoplasmic labeling of the endothelial cells lining the primary cortex microvessels. At 18 wg, a punctate P-gp staining pattern was detected on cortex and subcortical vessels and on their side branches. At 22 wg, P-gp staining was linear and concentrated on endothelial cell membranes. In all examined ages, GFAP-positive radial glial cells and astrocytes did not stain for P-gp, even at their perivascular processes, whereas faint P-gp labeling was seen on vimentin-reactive radial glia at the earliest examined fetal age. At midgestation, P-gp colocalized with caveolin-pY14 on the abluminal endothelial cell membrane. These results demonstrate that P-gp is expressed early during human cerebral cortical microvessel development, and suggest that at midgestation there may be efflux activity that is regulated by interactions with the caveolar endothelial cell compartment.

Increase of Proliferating Renal Progenitor Cells in Acute Tubular Necrosis Underlying Delayed Graft Function

Background. Delayed graft function (DGF) is associated with acute tubular necrosis. In this setting, surviving tubular cells may proliferate and replace injured cells. CD133+Pax-2+cells may play a role in the regeneration of tubular damage. The aim of this study was to demonstrate the presence of these cells in human kidneys before transplantation and in grafts with DGF. Methods. Ten normal kidneys (group 1) and pretransplant biopsy of 25 deceased donors (group 2) were examined. The latter group included 10 kidneys with early graft function (2A) and 15 with DGF (2B). Group 2B patients received a second biopsy during DGF (2C). CD133, Pax-2, and Ki-67 protein expression was investigated by confocal microscopy. Results. CD133+Pax-2+ and CD133−Pax-2+cells were present within the Bowman’s capsule and proximal tubules in all groups except group 2B. Number of CD133+Pax-2+ and CD133−Pax-2+cells at tubular level was similar in groups 1 and 2A. Within group 2B we observed a striking reduction in both cell types. There was a significant increase of both cell populations within group 2C, compared with group 2B. CD133+Pax-2+ and CD133−Pax-2+cell number in group 2 correlated inversely with cold ischemia time. Pax-2+Ki-67+cells were absent from group 1 and 2B samples, and increased significantly in groups 2A and 2C. Proliferating CD133+ cells increased significantly in group 2C. Conclusions. Our data suggest that regenerative response in posttransplant acute tubular necrosis, underlying DGF, is characterized by an increase in proliferating renal progenitor/stem cells CD133+Pax-2+ and CD133−Pax-2+ cells involved in repairing tubular damage.

CD40L Proinflammatory and Profibrotic Effects on Proximal Tubular Epithelial Cells: Role of NF-κB and Lyn

Chronic allograft nephropathy (CAN) is the main cause of renal graft loss, but its pathogenic mechanisms are still unclear. Immune system activation has been suggested as a key event in the development of CAN. CD40 is a co-stimulatory protein whose expression is upregulated in proximal tubular epithelial cells (PTEC) in acute rejection. This receptor interacts with CD40L, expressed by activated T cells. CD40L induces the production by PTEC of different proinflammatory cytokines, but very little is known of its profibrotic effects. The aim of this study was to investigate the effect of CD40/CD40L interaction on PTEC expression of plasminogen activator inhibitor-1 (PAI-1), a powerful profibrotic mediator, and monocyte chemoattractant protein-1 (MCP-1), a proinflammatory cytokine, and to investigate the signaling pathways that lead to these effects. Soluble CD40L induced a time-dependent increase in both PAI-1 and MCP-1 gene expression and protein production in PTEC. CD40 cross-linking on PTEC caused TNF-R-associated factors 2 and 6 membrane translocation. This event led to NF-kappaB activation, through the NF-kappaB-inducing kinase, and to a significant increase in the phosphorylation of lyn, a src-related tyrosine kinase. Lyn, upon phosphorylation, became strictly associated with caveolin-1, a scaffolding protein enriched in caveolae. Lyn inhibition did not have any effect on CD40L-induced NF-kappaB activation and MCP-1 expression but abolished PAI-1 induction. On the contrary, NF-kappaB inhibition significantly reduced only MCP-1 expression. In conclusion, CD40L could play a key role in the pathogenesis of CAN through PAI-1 induction. CD40L profibrotic and proinflammatory effects are mediated by different signaling pathways, suggesting that drugs that inhibit inflammation may not be equally effective in reducing fibrosis.

HIF activation and VEGF overexpression are coupled with ZO-1 up-phosphorylation in the brain of dystrophic mdx mouse.

In Duchenne muscular dystrophy (DMD) metabolic and structural alterations of the central nervous system are described. Here, we investigated in the brain of 10 mdx mice and in five control ones, the expression of hypoxia inducible factor‐1α (HIF‐1α) and we correlated it with the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) and of the endothelial tight junction proteins zonula occludens‐1 (ZO‐1) and claudin‐1. Results showed an activation of mRNA HIF‐1α by reverse transcription polymerase chain reaction (RT‐PCR) and a strong HIF1‐α labeling of perivascular glial cells and cortical neurons by immunohistochemistry, in mdx mouse. Moreover, overexpression of VEGF and VEGFR‐2, respectively, in neurons and in endothelial cells coupled with changes to endothelial ZO‐1 and claudin‐1 expression in the latter were detected by immunoblotting and immunohistochemistry, in the mdx brain. Furthermore, by immunoprecipitation, an up‐phosphorylation of ZO‐1 was demonstrated in mdx endothelial cells in parallel with the reduction in ZO‐1 protein content. These data suggest that the activation of HIF‐1α in the brain of dystrophic mice coupled with VEGF and VEGFR‐2 up‐regulation and ZO‐1 and claudin‐1 rearrangement might contribute to both blood–brain barrier opening and increased angiogenesis.