Elucidation of the roles of PECAM-1 (CD31) in vasculo- and angio-genesis, permeability, inflammation, directed migration, hemostasis and hematopoiesis and bone metabolism.We have been investigating the roles of PECAM-1, a member of the Ig superfamily of transmembrane proteins, in a variety of in vivo and in vitro models with the goal of elucidating the effects of its alternatively spliced isoforms and polymorphisms on vascular and immune cell behaviors. We have found that PECAM-1 functions as a scaffolding protein, mediating the binding and/or activation of beta- and gamma-catenin, SHP1 & 2, STAT3 & 5, src family members and syk, having a wide range of effects on endothelial cells, polymorphonuclear leukocytes, lymphocytes, megakaryocytes and mononuclear cells.
Elucidation of the mechanisms involved in lymphocyte transmigrtation through endothelial cells during inflammation.We have a long-standing interest in the roles of selected adhesion molecules, their cognate receptors and proteases in the process of directed migration of immune cells during the inflammatory process. Specifically, we have studied the transmigration of T lymphocytes into the CNS of animals that have been induced to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. We have found that the expression of a particular integrin (VLA4) on antigen-specific T lymphocytes and its engagement by an endothelial cell adhesion molecule (VCAM-1) is required for transmigration and transmigration requires the induction of specific proteases (MMP-2 and MMP-14).
Elucidation of the mechanisms involved in the recovery of neurodevelopmental handicaps incurred during and following premature birth and recovery from spinal cord injury.Very low birth weight premature infants exhibit a high incidence of hypoxic-driven neurodevelopmental handicaps including cognitive and motor deficits. Over time some children show improvement while others do not. This variability in improvement has been shown to be due to differences in individuals' neurogenic zone responses to injury. Previously, using canine, rodent and murine models as well as tissue culture models, we have demonstrated that induction of selected growth factors and neurotrophins, their receptors, extracellular matrix components and proteases are involved in the response to the hypoxic insult associated with premature birth and identified several signaling pathways involved in response to hypoxic insult.
Elucidation of the mechanisms underlying the increased incidence of congenital abnormalities (cardiovascular) in the offspring of maternal diabetics and the development of diagnostic methods to enable earlier diagnosis.We have developed both in vivo and in vitro models to elucidate the dysregulations of the signaling cascades involved in vitelline vasculo- and angio-genesis and endocardial cushion formation.
The many facets of PECAM-1 signaling: a schematic representation of PECAM-1's roles as a dynamic modulator of endothelial, hematopoietic precursor cell and immune cell junctional, cytoskeletal, adhesive, and signaling pathways based on data accrued in the laboratory.
The thick arrows and the BOLD type denote this proposal's focus (SHP-1,2 -> Syk -> osteoclastogenesis).
- Illustrates the binding of SHP-1 & -2 to differentially phosphorylated/alternatively spliced PECAM-1 (Y663 & Y686), resulting in distinct binding to PECAM-1 and substrate specificities. PECAM-1/SHP-1 & 2 interactions have been shown to modulate moesin phosphorylation, affecting directed migration of neutrophils and megakaryocytes; the tyrosine phosphorylation state of beta-catenin and FAK, vascular permeability, proliferation, apoptosis, gene expression and migration; activation state of ERK1/2, affecting STAT phosphorylation and cytokine responsiveness, in turn affecting vascular permeability, proliferation, apoptosis, gene expression and migration; & tyrosine phosphorylation state of STAT3, affecting cytokine induction.
- Illustrates the interaction of PECAM-1 with Galphai2, affecting Rho activation, cell motility and migration;
- Illustrates the modulation of MMP-2 & -9 expression by the presence of PECAM-1 on the surface of endothelial cells via induction and nuclear targeting of GATA2 and p53 transcription factors;
- Illustrates PECAM-1's interactions with PI3K that modulates Akt activity, which in turn regulates Egr-1 expression via p38 activation, leading to blunting of tissue factor induction, as well as MMP-14 and PAI-1 induction, reducing thrombosis, permeability and apoptosis in endothelial cells;
- Also illustrates PECAM-1/PI3K interactions that also regulate GSK-3beta activity via Akt phosphorylation, resulting in blunting of beta-catenin serine phsophorylation, reducing its proteosomal degradation;
- Illustrates tyrosine phosphorylated beta-catenin binding to PECAM-1, resulting in sequestration of beta-catenin, rendering it incapable of binding to VE-cahderin, affecting junction formation;
- Illustrates the binding of gamma-catenin to exon 13 of PECAM-1, dependent upon the phosphorylation state of PECAM-1 residue S673.
Upon engagement of alpha4beta1 integrin by VCAM-1 (thick black arrow), MT1-MMP (MMP-14), MMP-2 and MMP-9 induction and activation occur, facilitating mononuclear cell migration through the endothelial cell and basement membrane layers as well as cleavage of adhesion molecules, ECM components (thin blue arrows) and chemokines & cytokines (thin red arrows), affecting a wide range of epithelial, lymphocytic and endothelial cell behaviors. The presence of CD44 was found to modulate expression levels of endothelial cell PECAM-1 (CD31), VE-Cadherin and TGFbeta Receptor I and serine phosphorylation of beta-catenin (thick red arrow), affecting endothelial permeability and mononuclear cell transmigration (dashed red arrow).
Schematic summary of the laboratory's work elucidating the proteins and pathways dysregulated during the sublethal chronic hypoxia experienced in the premature newborn population.
- Over 60,000 very low birth weight premature infants are born in the USA every year having a survival of approx. 85%. Many suffer significant intellectual disability and cerebral palsy.
- This condition has been successfully modeled in mice, with the mice mimicking the morphological, biochemical and behavioral pathologies observed in the human population.
- Using this murine model and transcriptome analyses we have identified many dysregulated genes.
- We have used neural stem cells and microvascular endothelial cells isolated from murine pup brains in 2- and 3-dimensional co-culture to mimic the subventricular neurovascular niche.
- Using these co-culture models, we have elucidated several signaling pathways affected by specific gene dysregulations.
- Using this information, we have utilized selected ELISA assays and selected SNP analyses to validate our in vitro findings in our murine models and in cord blood samples of premature infants with the goal of developing a rapid diagnostic test to identify premature infants "at risk" for developing significant neurodevelopmental handicaps and ultimately to monitor future therapies developed from our mechanistic studies.