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.
We are currently investigating the roles of PECAM-1 and its particular polymorphisms in modulating osteoclast behaviors utilizing bone marrow chimeras, bone marrow cultures and selected PECAM-1 SNP analysis of normal and osteoporotic human cohorts. A more complete understanding of PECAM-1 roles and the specific differences in PECAM-1 polymorphisms and alternatively spliced isoforms will allow for the potential use of PECAM-1-based reagents as diagnostics and therapeutics. (see figure 1)

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).
In follow-up studies we examined the role of MMP-2 using MMP-2 knock out mice. In these studies we found that surprisingly MMP-2 KO mice exhibited an earlier onset and a more severe clinical presentation compared to wildtype mice. We found that the absence of MMP-2 triggered a compensatory induction of MMP-9, which was responsible for the earlier onset and more severe clinical presentations.
We are currently investigating the roles of CD44 in modulating lymphocyte (TH17) and endothelial cell biology. Currently we are investigating CD44's role in modulating vascular permeability and mononuclear cell transmigration in the brain microvasculature. (see figure 2)

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.
These studies lead us to examine the neurogenic regions of the CNS, namely the subventricular zone (SVZ) and the subgranular zone (SGZ), regions of the brain in which there is coupled vasculogenesis and neurogenesis. Using standard and three-dimensional co-culture techniques employing bio-compatible, bio-degradable scaffolds and implantation techniques, we found a dynamic cross-talk between microvascular endothelial cells and neural stem cells mediated by specific growth factors and neurotrophins known to be up-regulated in response to hypoxic insult and spinal cord injury.
Currently we are investigating specific mouse strains, which mimic the wide range of responses to hypoxia observed in the human very low birth weight premature infant population. We have found these strains to exhibit significant differences in selected signaling nodes (GSK-3beta and HIF-1alpha) and growth factors and neurotrophins that have been shown to be involved in the responses to hypoxia. These studies should lead to a more complete understanding of the proteins and pathways involved and provide us with needed insights for rational drug design. (see figure 3)

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.
We have published our work using classical developmental, genetic and cell biological techniques. We have utilized a proteomic approach employing LC-MS and LC-MS-MS to acquire a global understanding of which proteins are up- and down-regulated following a hyperglycemic insult in utero. We have identified approximately 170 proteins and have validated approximately 15 of them in ELISA/Western/activity assays and in yolk sac and endocardial cushion culture models. We are currently using these proteins in ELISA/Western/activity assays to develop a protein chip that will allow for earlier and more specific diagnosis of cardiovascular defects in utero at earlier times than currently available. Testing amniotic fluid and sera samples obtained from a group of pregnant women with normal fetuses and a group of women with fetuses having cardiovascular defects has yielded promising results.
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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).

  1. 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.
Recently we have demonstrated a PECAM-1/SHP-1/Syk interaction which modulates osteoclastogenesis
  1. Illustrates the interaction of PECAM-1 with Galphai2, affecting Rho activation, cell motility and migration;
  2. 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;
  3. 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;
  4. 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;
  5. 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;
  6. Illustrates the binding of gamma-catenin to exon 13 of PECAM-1, dependent upon the phosphorylation state of PECAM-1 residue S673.
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Schematic illustrating the signaling properties ofalpha4beta1 integrin and CD44 on the cell surface.

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).

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Schematic summary of the laboratory's work elucidating the proteins and pathways dysregulated during the sublethal chronic hypoxia experienced in the premature newborn population.

  1. 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.
  2. This condition has been successfully modeled in mice, with the mice mimicking the morphological, biochemical and behavioral pathologies observed in the human population.
  3. Using this murine model and transcriptome analyses we have identified many dysregulated genes.
  4. 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.
  5. Using these co-culture models, we have elucidated several signaling pathways affected by specific gene dysregulations.
  6. 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.