Research

Project 1

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), growth factors and neurotrophins that have been shown to be involved in the responses to hypoxia.

In recent studies we have found that a decreased expression of a particular transcription factor (Sox10) correlates with a poor response to chronic hypoxia in mouse pups, resulting in multiple neurodevelopmental deficits. Sox10 regulates oligodendrocytogenesis, differentiation and myelination, known modulators of synaptic transmission and neuronal impulse.

Currently, we have identified a small molecule (minocycline) that induces Sox10 and improves cognitive behavior in mice following hypoxic insult.

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 (Figure 1).

Figure 1. Summary of the laboratory’s work on the neurovascular niche and the response to and recovery from chronic hypoxia in the very premature newborn.

Selected references:

Boisvert, E. M., Means, R. E., Michaud, M. R., Madri, J.A., Katz, S. G., Minocycline mitigates the effect of neonatal hypoxic insult on brain organoids, Cell Death & Disease, Apr 11;10(4):325, 2019. doi: 10.1038/s41419-019-1553-x.

Madri, J.A., Modeling the Neurovascular niche: Implications for recovery from CNS injury, J. Physiol & Pharmacol., 60, Suppl 4, 95-104, 2009. http://www.jpp.krakow.pl/journal/archive/1009_s4/pdf/95_1009_s4_article.pdf

Li, Q., Michaud, M, Canosa, S, Kuo, A., Madri, J.A., GSK-3beta: a signaling pathway node modulating neural stem cell and endothelial cell interactions, Angiogenesis, 14(2): 173-185, 2011.

Li, Q, Canosa S, Michaud, M., Flynn, K., Krauthammer, M., J.A. Madri. Modeling the Neurovascular Niche: Unbiased Transcriptome Analysis of the Murine Subventricular Zone in Response to Hypoxic Insult, PLOS One, 8(10): e76265. doi:10.1371/journal.pone.0076265,2013. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076265

Li, Q., Tsuneki, M, Krauthammer, M., Couture, R., Schwartz, M., Madri, J.A., Modulation of Sox10, HIF-1alpha, Survivin and YAP by minocycline in the treatment of neurodevelopmental handicaps following hypoxic insult, Amer J. Pathol., Sep;185(9):2364-78. doi: 10.1016/j.ajpath.2015.05.016. Epub 2015 Jul 22. PMID: 26209807.

Project 2

Elucidation of the mechanisms involved in modulation of the Blood Brain Barrier and lymphocyte transmigration 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 & Tregs) and endothelial cell biology. We are also investigating CD44’s role in modulating vascular permeability and mononuclear cell transmigration in the brain microvasculature via modulation of CD31. Our recent studies illustrate a signaling pathway involving CD44, CD31, VE-cadherin and the Hippo pathway. (See Figure 2).

Figure 2. Working model for the involvement of CD44 and CD31 in the modulation of proliferation and active caspase cascades in endothelial cells.

A. In WT-BEC the cells exhibit a contact-inhibited phenotype with the cells expressing optimal levels of CD31 and VE-cadherin, low levels of Survivin, non-detectable levels of Ajuba (dashed lines), moderate levels of active caspases, reduced nuclear YAP and increased cytoplasmic P-YAP, consistent with an active Hippo pathway.
B. Compared to WT-BEC, EOMA cells exhibit decreased contact inhibition and junctional molecule expression resulting in down-regulation of the Hippo pathway with increases in Survivin, Ajuba (thick lines) and MMP expression and increased YAP nuclear translocation, suppressing caspase mediated apoptosis and increasing proliferation.
C. EOMA cells treated with YM155 (a small molecule Survivin inhibitor) or transfected with Survivin siRNA exhibit increased contact inhibition and junctional molecule expression resulting from increased CD31 and VE-cadherin expression and decreases in Survivin, Ajuba (dashed lines) and nuclear YAP expression, resulting in up-regulation of the Hippo pathway which in turn results in increased cytoplasmic P-YAP and decreased MMP2 expression and caspase mediated apoptosis. (WT-BEC cells are not affected by YM155 Treatment.)

Selected References:

Boisvert, E.M., Means, R E., Michaud, M., Thomson, J.J., Madri, J.A., Katz, S.G. A Static Self-directed Method for Generating Brain Organoids from Human Embryonic Stem Cells. J. Vis. Exp.( J Vis Exp. 2020 Mar 4;(157): 10.3791/60379, 2020. doi:10.3791/60379. PMID: 32202516.

Graesser, D., Solowiej, A., Bruckner,M., Osterweil, E., Judes, A., M., Davis, S., Ruddle, N., Engelhardt, B., Madri, J.A. Changes in Vascular Permeability and Early Onset of Experimental Autoimmune Encephalomyelitis in PECAM-1 (CD31) Deficient Mice. J. Clin Invest., 109:383-392, 2002.

Esparza, J., Kruse, M., Lee, J., Michaud, M and Madri, J.A., MMP-2 null mice exhibit an early onset and severe experimental autoimmune encephalomyelitis due to an increase in MMP-9 expression and activity. FASEB J. 18: 1682-1691, 2004.

Flynn, K., Michaud, M., J.A. Madri. CD44 Deficiency Contributes to Enhanced Experimental Autoimmune Encephalomyelitis: A Role in Immune Cells and Vascular Cells 2 of the Blood Brain Barrier3, American Journal of Pathology, 182(4):1322-1336, 2013.

Flynn, K., Michaud, M., Canosa, S., J.A. Madri. CD44 Regulates Vascular Endothelial Barrier Integrity via a PECAM-1 Dependent Mechanism, Angiogenesis, 16:689-705, 2013.

Tsuneki, M., Madri, J.A.,CD44 regulation of endothelial cell proliferation and apoptosis via modulation of CD31 and VE-cadherin expression., J Biol Chem. 2014, 289(9):5357-5370. doi: 10.1074/jbc.M113.529313. Epub 2014 Jan 14.PMID: 24425872

Tsuneki, M., Madri, J.A. Adhesion Molecule-Mediated Hippo Pathway Modulates Hemangioendothelioma Cell Behavior, Mol Cell Biol., 34(24):4485-99, 2014. doi: 10.1128/MCB.00671-14, Epub 2014 Sep 29

Tsuneki, M., Hardee, S., Michaud, M., Morotti, R, Lavik, E, Madri JA. A Hydrogel-Endothelial Cell implant Mimics Infantile Hemangioma:, Modulation by Survivin and the Hippo pathway, Laboratory Investigation, Jul;95(7):765-80. doi: 10.1038/labinvest.2015.61. Epub 2015 May 11., PMID: 25961170.

Tsuneki, M., Madri J.A., CD44 influences fibroblast behaviors via modulation of cell-cell and cell-matrix interactions, affecting Survivin and Hippo pathways, Journal of Cellular Physiology, Aug 6. doi: 10.1002/jcp.25123. [Epub ahead of print], August, 2015.

Project 3

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. (See Figure 3.)

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.

Figure 3. Summary of the laboratory’s work on elucidating the roles of PECAM-1 (CD31) as a dynamic modulator of cellular behaviors, several of which are regulated by differential phosphorylation of specific Serine and/or Tyrosine residues as well as by selected polymorphisms.

Selected references:

Ilan, N., Madri J.A., PECAM-1: Old friend, new partners, Curr. Opin. in Cell Biol., 15(5):515-24, 2003.

Wu, Y., Tworkoski, K., Michaud, M, Madri J.A., Bone Marrow Monocyte PECAM-1 Deficiency Elicits Increased Osteoclastogenesis Resulting in Trabecular Bone Loss, J. Immunol., 182(5):2672-2679, 2009.

Wu, Y., Madri J.A., Insights into Monocyte-Driven Osteoclastogenesis and Its Link with Hematopoiesis: regulatory roles of PECAM-1 and SHP-1, Critical Reviews in Immunology, 30(5): 423-432, 2010.

Flynn, K., Michaud, M., Canosa, S., J.A. Madri. CD44 Regulates Vascular Endothelial Barrier Integrity via a PECAM-1 Dependent Mechanism, Angiogenesis, 16:689-705, 2013.

Tsuneki, M., Madri, J.A. Adhesion Molecule-Mediated Hippo Pathway Modulates Hemangioendothelioma Cell Behavior, Mol Cell Biol., 34(24):4485-99, 2014. doi: 10.1128/MCB.00671-14, Epub 2014 Sep 29.