Sandra Canosa

Enhanced Susceptibility to Endotoxic Shock and Impaired STAT3 Signaling in CD31-Deficient Mice

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), an adhesion molecule expressed on hematopoietic and endothelial cells, mediates apoptosis, cell proliferation, and migration and maintains endothelial integrity in addition to its roles as a modulator of lymphocyte and platelet signaling and facilitator of neutrophil transmigration. Recent data suggest that CD31 functions as a scaffolding protein to regulate phosphorylation of the signal transducers and activators of transcription (STAT) family of signaling molecules, particularly STAT3 and STAT5. STAT3 regulates the acute phase response to innate immune stimuli such as lipopolysaccharide (LPS) and promotes recovery from LPS-induced septic shock. Here we demonstrate that CD31-deficient mice have reduced survival during endotoxic LPS-induced shock. As compared to wild-type controls, CD31-deficient mice showed enhanced vascular permeability; increased apoptotic cell death in liver, kidney, and spleen; and elevated levels of serum tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFNgamma), MCP-1, MCP-5, sTNRF, and IL-6. In response to LPS in vivo and in vitro, splenocytes and endothelial cells from knockout mice had reduced levels of phosphorylated STAT3. These results suggest that CD31 is necessary for maintenance of endothelial integrity and prevention of apoptosis during septic shock and for STAT3-mediated acute phase responses that promote survival during septic shock.

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1–negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1–positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.

Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho

Platelet endothelial cell adhesion molecule‐1 (PECAM‐1), an immunoglobulin family vascular adhesion molecule, is involved in endothelial cell migration and angiogenesis (1, 2). We found that endothelial cells lacking PECAM‐1 exhibit increased single cell motility and extension formation but poor wound healing migration, reminiscent of cells in which Rho activity has been suppressed by overexpressing a GTPase‐activating protein (3). The ability of PECAM‐1 to restore wound healing migration to PECAM‐1‐deficient cells was independent of its extracellular domain or signaling via its immunoreceptor tyrosine‐based inhibitory motif. PECAM‐1‐deficient endothelial cells had a selective defect in RhoGTP loading, and inhibition of Rho activity mimicked the PECAM‐1‐deficient phenotype of increased chemokinetic single cell motility at the expense of coordinated wound healing migration. The wound healing advantage of PECAM‐1‐positive endothelial cells was not only Rho mediated but pertussis toxin inhibitable, characteristic of migration mediated by heterotrimeric G‐protein‐linked seven‐transmembrane receptor signaling such as signaling in response to the serum sphingolipid sphingosine‐1‐phosphate (S1P) (4, 5). Indeed, we found that the wound healing defect of PECAM‐1 null endothelial cells is minimized in sphingolipid‐depleted media; moreover, PECAM‐1 null endothelial cells fail to increase their migration in response to S1P. We have also found that PECAM‐1 localizes to rafts and that in its absence heterotrimeric G‐protein components are differentially recruited to rafts, providing a potential mechanism for PECAM‐1‐mediated coordination of S1P signaling. PECAM‐1 may thus support the effective S1P/RhoGTP signaling required for wound healing endothelial migration by allowing for the spatially directed, coordinated activation of Galpha signaling pathways.—Gratzinger, D., Canosa, S., Engelhardt, B., Maori, J. A. Platelet endothelial cell adhesion molecule‐1 modulates endothelial cell motility through the small G‐protein Rho, FASEB J. 17, 1458–1469 (2003)

PECAM-1 promotes β-catenin accumulation and stimulates endothelial cell proliferation

Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine phosphorylated beta-catenin and modulates beta-catenin localization [J. Immunol. 158 (7) (1997) 3408; J. Cell Sci. 112 (Pt 18) (1999) 3005]. To elucidate functional consequences of this interaction, we studied endothelial cells from PECAM-1 knockout animals and compared them to PECAM-1 expressing endothelial cells [Mol. Biol. Cell 11 (9) (2000) 3109]. We noted an increase in the expression of beta-catenin protein in PECAM-1 expressing endothelial cells. Further, by immunofluorescence, beta-catenin localized to the cell membrane as well as to the nucleus in PECAM-1 positive endothelial cells, whereas cells not expressing PECAM-1 stained for beta-catenin only at the membrane. Additionally, we demonstrate that PECAM-1 lacking the majority of the cytoplasmic domain promotes significantly less accumulation of transcriptionally active beta-catenin than full-length PECAM-1. Finally, we note an increased proliferative rate in the PECAM-1 reconstituted cells compared to the endothelial cells lacking PECAM-1. Taken together, our data suggest that PECAM-1, an adhesion molecule, affects cell proliferation via accumulation of transcriptionally active beta-catenin.

CD44 regulates vascular endothelial barrier integrity via a PECAM-1 dependent mechanism.

Vascular integrity is a critical parameter in normal growth and development. Loss of appropriate vascular barrier function is present in various immune- and injury-mediated pathological conditions. CD44 is an adhesion molecule expressed by multiple cell types, including endothelial cells (EC). The goal of the present study was to examine how loss of CD44 affected vascular permeability. Using C57BL/6 WT and CD44-KO mice, we found no significant permeability to Evan’s Blue in either strain at baseline. However, there was significantly increased histamine-induced permeability in CD44-deficient mice compared to WT counterparts. Similar results were observed in vitro, where CD44-deficient endothelial monolayers were also impermeable to 40kD-FITC dextran in the absence of vasoactive challenge, but exhibited enhanced and prolonged permeability following histamine. However, CD44-KO monolayers have reduced baseline barrier strength by electrical resistance, which correlated with increased permeability, at baseline, to smaller molecular weight 4-kD FITC-dextran, suggesting weakly formed endothelial junctions. The CD44-KO EC displayed several characteristics consistent with impaired barrier function/dysfunctional EC junctions, including differential expression, phosphorylation, and localization of endothelial junction proteins, increased matrix metalloprotease expression, and altered cellular morphology. Reduced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression by CD44-KO EC in vivo and in vitro was also observed. Reconstitution of murine CD44 or PECAM-1 restored these defects to near WT status, suggesting CD44 regulates vascular permeability and integrity through a PECAM-1 dependent mechanism.

GSK-3β: a signaling pathway node modulating neural stem cell and endothelial cell interactions

The neurogenic areas of the brain are highly organized structures in which there is dynamic reciprocal modulation of neural stem cells (NSC) and microvascular endothelial cells (BEC) resulting in control of neural stem cell and vascular proliferation, survival and differentiation throughout the life of the individual. Select molecules such as GSK-3β, functioning as signaling nodes, and their downstream signaling components including HIF-1α, HIF-2α and β-catenin participate in regulating and orchestrating the diverse responses involved in this complex process. In this report we demonstrate GSK-3β’s role as a signaling node in two mouse strains (C57BL/6, which have been found to respond to and recover from a hypoxic insult from P3 to P11 poorly and CD-1, which have been found to respond to and recover from a hypoxic insult from P3 to P11 well both in vivo and in vitro) which mimic the wide range of responsiveness to hypoxic insult observed in the very low birth weight premature infant population. Differences in levels of neural stem cell and microvascular endothelial cell GSK-3β activation, β-catenin serine phosphorylation, HIF-1α and 2α, BDNF, SDF-1 and VEGF, β-III-tubulin and cleaved notch-1 expression in C57BL/6 and CD-1 subventricular zone tissues, and cultured NSC and BEC were noted. Specifically, CD1 pups, SVZ tissues and isolated NSC and BEC exhibit less GSK-3β and β-catenin serine phoslphorylation and greater HIF-1α and 2α, BDNF, SDF-1 and VEGF, β-III-tubulin and cleaved notch-1 expression compared to C57BL/6. Correlating with these changes were differences of several neural stem cell and microvascular endothelial cell behaviors including proliferation, apoptosis, migration and differentiation with CD1 NSC exhibiting greater proliferation and migration and decreased apoptosis and differentiation and CD1 BEC exhibiting greater angiogenesis. Further, upon treatment with nanomolar concentrations of a GSK-3β inhibitor (SB412682), C57 NSC and BEC behaviors could be brought to CD1 levels, consistent with the concept of GSK-3β functioning as a multifunctional signaling pathway node, modulating several behaviors in these cells. Lastly, the therapeutic potential of targeting GSK-3β is discussed.

Modeling the neurovascular niche: unbiased transcriptome analysis of the murine subventricular zone in response to hypoxic insult.

Premature infants often experience chronic hypoxia, resulting in cognitive & motor neurodevelopmental handicaps. These sometimes devastating handicaps are thought to be caused by compromised neural precursor cell (NPC) repair/recovery resulting in variable central nervous system (CNS) repair/recovery. We have identified differential responses of two mouse strains (C57BL/6 & CD1) to chronic hypoxia that span the range of responsiveness noted in the premature human population. We previously correlated several CNS tissue and cellular behaviors with the different behavioral parameters manifested by these two strains. In this report, we use unbiased array technology to interrogate the transcriptome of the subventricular zone (SVZ) in these strains. Our results illustrate differences in mRNA expression in the SVZ of both C57BL/6 and CD1 mice following hypoxia as well as differences between C57BL/6 and CD1 SVZ under both normoxic and hypoxic conditions. Differences in expression were found in gene sets associated with Sox10-mediated neural functions that explain, in part, the differential cognitive and motor responsiveness to hypoxic insult. This may shed additional light on our understanding of the variable responses noted in the human premature infant population and facilitate early intervention approaches. Further interrogation of the differentially expressed gene sets will provide a more complete understanding of the differential responses to, and recovery from, hypoxic insult allowing for more informed modeling of the ranges of disease severity observed in the very premature human population.

MMP-2: A modulator of neuronal precursor activity and cognitive and motor behaviors

Abstract Matrix Metalloproteinase2, (MMP2, gelatinase A) is a zinc‐containing enzyme with a broad substrate specificity including components of the extracellular matrix, cell surface molecules and a wide range bioactive molecules. MMP2 is known to play important roles in a variety of signaling pathways and processes in a wide range of cell types and tissues. In this report we elucidate the effects of the absence of MMP2 in Neural Precursor Cells (NPC) derived from C57BL/6 MMP2 KO mice and in primary and secondary neurosphere formation. We observed smaller neurosphere numbers and sizes, decreased NPC numbers, PCNA expression, DNA and Akt activation in MMP2 KO NPC compared to WT NPC. We also found decreased neurosphere formation and NPC migration outward from adherent neurospheres, decreased CXCR4 and nestin expression and increased GFAP and neuro‐filament expression in MMP2 KO NPC compared to Wt NPC. MMP2 KO mice were found to exhibit increased anxiety manifested in open field activity assays compared to Wt mice. MMP2 KO mice also exhibited differences in motor activities manifested by decreased balance and endurance during Rota‐rod testing. These studies illustrate an important role of MMP2 in cognitive and motor behaviors and confirm its importance in NPC activities crucial to brain development, growth and response to and recovery from injury.