Tuesday, December 9, 2014

Research of Josephine Lok, MD

Josephine Lok, MD, Physician, Assistant in Pediatrics, Pediatric Critical Care Medicine, Massachusetts General Hospital for Children; Assistant Professor in Pediatrics, Harvard Medical School.

The microvascular endothelium is instrumental in many processes in the brain, including oxygen delivery, barrier function, and response to inflammation.  After brain trauma, microvasculature disturbances lead to increased permeability, vasoconstriction, and capillary occlusion, events which amplify the primary insult.  An understanding of these endothelial responses to injury is important in the investigation of neuroprotective strategies during brain injury.  This topic forms one major area of my research. 

I am also interested in neuregulin1 (NRG1) signaling in microvascular endothelial cells, in particular its effect on endothelial permeability.  NRG1 is an endogenous growth factor which is important in the embryonic development and postnatal functioning of the heart and the nervous system.  Within the central nervous system, NRG1’s actions, mediated through 4 erbB receptors, have been implicated in diverse neuronal processes including synaptic transmission, myelination, astrocyte differentiation, embryonic migration of interneurons, and survival of subsets of neurons and astrocytes.  NRG1 is also thought to play a role in the pathological mechanisms of schizophrenia.  In the field of cardiology, NRG1 is being investigated in clinical trials for the treatment of chronic heart failure.  NRG1 is expressed as multiple isoforms, enabling many different functions.  The isoform utilized in these studies is NRG1-β, currently the most widely studied NRG1 isoform in brain research.

Working under the mentorship of Dr. Eng Lo, my research focuses on three topics - 1. alterations in endothelial permeability, leukocyte adhesion, and thrombogenicity during cytokine-induced injury;  2. the effect of neuregulin-1 (NRG1) on these endothelial responses; 3. the neuroprotective potential of NRG1.  These investigations are performed in cell culture models of cytokine –induced injury and oxygen/glucose deprivation, and in an in-vivo model of cortical contusion in mice.  Relevant outcome parameters in endothelial permeability, neutrophil adhesion, and endogenous NRG1/erbB signaling are examined. In the in-vivo experiments, additional outcomes in motor and cognitive performance are examined.   The effect of exogenous NRG1-β administration on these outcomes is also being investigated. 

Initial experiments consisted of determining whether NRG1/erbB signaling is present and whether it performs significant functions in brain microvascular endothelial cells.  Although NRG1 function had been studied in neurons, glia, and endothelial cells of non-CNS origin, its role in brain microvascular endothelial cells had not been well defined.   Our work provided evidence that NRG1 and erbB receptors are present in brain microvascular endothelial cells, and identified endothelial permeability as a target of NRG1-β signaling.  In in-vitro studies, NRG1-β reduced endothelial hyper-permeability induced by IL-1β.  In parallel in-vivo experiments using a controlled cortical impact (CCI) model of brain trauma in mice, NRG1-β treated mice had a reduction in acute blood brain barrier (BBB) permeability compared to vehicle-treated mice.  Additionally, mice that received NRG1-β after TBI had improved cognitive outcomes compared to PBS-treated controls. 

We also examined endothelial adhesion molecule expression and neutrophil adhesion to endothelial cells after IL-1β exposure.  As expected, IL-1β increased adhesion molecule expression and neutrophil adhesion to endothelial cells.  Co-incubation with NRG1-β reduced the expression of adhesion molecules VCAM-1 and e-selectin in brain microvascular endothelial cells that have been activated by IL-1β, and ameliorated the IL-1β induced increase in neutrophil adhesion.  In the context of neuro-inflammation, these actions of NRG1 may be significant as an excessive inflammatory response can be damaging to surviving cells after injury.

Neurological outcome after brain trauma depends on the interplay between the different types of cells in the brain.  We are therefore exploring related topics in pericyte/endothelial interactions and in the survival of sub-populations of neurons in culture during oxygen-glucose deprivation.

In summary, our data have provided evidence that NRG1-β signaling decreases IL-1β induced increases in endothelial permeability, adhesion molecule expression, and neutrophil adhesion. Additionally, intravenous administration of NRG1- β improves cognitive outcome in mice after TBI.    Current experiments aim are directed at investigation of pathways through which NRG1 interacts with brain microvascular endothelial cells and how these interactions may improve outcome after TBI.

References

  1. Lok J, Wang H, Murata Y, Zhu H, Qin T, Whalen MJ, Lo EH.  Effect of Neuregulin-1 on histopathological and functional outcome after controlled cortical impact in mice.  J Neurotrauma 2007; 24:1817-1822.
  2. Lok J, Sardi SP, Guo S, Besancon E, Ha Duy M, Rosell A, Kim WJ,  Corfas G, Lo EH.  Neuregulin-1 signaling in brain endothelial cells.  JCBFM 2009 Jan;29(1):39-43. Epub Aug 27 2008.
  3. Lok J, Zhao S, Leung W, Seo JH, Navaratna D, Wang X, Whalen MJ, Lo EH.  Neuregulin-1 effects on endothelial and blood-brain-barrier permeability after experimental injury.  Transl Stroke Res. 2012 Jul 1;3(S1):S119-S124. Epub 2012 Apr 13.
  4. Guo S, Zhou Y, Xing C, Lok J, Som AT, Ning M, Ji X, Lo EH.  The vasculome of the mouse brain.  PLoS One. 2012;7 (12).
  5. Guo S, Lok J, Liu Y, Hayakawa K, Leung W, Xing C, Ji X, Lo EH. Assays to examine endothelial cell migration, tube formation, and gene expression profiles. Methods Mol Biol. 2014;1135:393-402
  6. Wu L, Walas S, Leung W, Sykes DB, Wu J, Lo EH, Lok J. Neuregulin1-β Decreases IL-1β-Induced Neutrophil Adhesion to Human Brain Microvascular Endothelial Cells. Transl Stroke Res. 2014 May 28.

Figure 1

Josephine Lok, MD: Figure 1

Figure 1. Effects of  NRG1-β  on barrier integrity.  (A) Trans-Endothelial Electrical Resistance (TEER) was measured continuously in primary human brain microvascular endothelial cells (BMEC) with increasing concentrations of NRG1: 10ng/ml, 30 ng/ml, 100ng/ml, 300 ng/ml, 600 ng/ml and 1200 ng/ml.  After baseline steady-state TEER was achieved, the various concentrations of NRG1 were introduced at time = 0.  The resistance was measured at 4000 Hz in 15 min intervals for the duration of the time shown. (B) TEER (measured as above) from BMECs exposed to IL1β (100ng/ml) or IL1β (100ng/ml) added together with NRG1.  TEER values are represented as the change (from baseline) in the average normalized TEER shown with the positive SEM (N= 3).  (C) Evaluation of BMEC viability after exposure to IL-1β (100ng/ml) alone or with either NRG1 at 100ng/ml or 300ng/ml.  After formation of BMEC monolayers, the indicated experimental condition was added for 18 hr.  The viability of the BMECs were evaluated by MTT assay (left axis) or LDH (right axis).  For the MTT results, the data is represented as the percent in cell viability when compared to the untreated control (shown as the mean + SEM).  For LDH, the results are expressed as the percentage of LDH release (shown as the mean + SEM).  This set of experiments was done in collaboration with Dr. Servio Ramirez at Temple University.

Figure 2.  NRG1-β reduces the IL-1β- induced increase in neutrophil adhesion to human brain endothelial cells

In PBS-treated BMECs, an average of 14 (14 +/- 6.5) neutrophils were adherent to the endothelial monolayer in each high-powered (10x) field. In IL-1β – treated endothelial cells, the number of adherent neutrophils increased to 35 (35+/- 12.9), a 2.5 fold increase (p<0.05). In BMECs treated with IL-1β and co-incubated with NRG1-β (100ng/ml or 12.5nM) NRG1-β, the number of adherent neutrophils decreased to the baseline level of 14 (14.6 +/- 4.7; p<0.05 when compared to BMECs treated with IL-1β and co-incubated with PBS). In endothelial cells treated with IL-1β and 300ng/ml (37.5nM) NRG1-β, the number of adherent neutrophils was 9.9 (9.9 +/- 4.0), again a significant decrease from that in cells treated with IL-1β without NRG1-β (p<0.05).

Josephine Lok, MD: Figure 2

Figure 3

Figure 3.  Effect of NRG1 on probe trial performance after CCI.  Vehicle-treated and NRG1- β treated mice were subjected to the MWM training and testing paradigm, starting on post-injury day14 after CCI.  After 5 training sessions, retention and recall of spatial memory was evaluated in the probe trial, in which the amount of time that each mouse spent in the target quadrant was recorded. (The target quadrant is the quadrant of the swim tank in which a previously hidden platform was located, and which the mice learned to associate with the platform over the course of the training sessions.)   NRG1-β treated mice had an improved performance in the probe trial, as evidenced by their preference for the target quadrant during the trial. 

Josephine Lok, MD: Figure 3

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