# Beyond PECAM:  Mechanisms of Transendothelial Migration

> **NIH NIH R01** · NORTHWESTERN UNIVERSITY · 2020 · $430,552

## Abstract

Project Summary
 Transendothelial migration (TEM) of leukocytes is a critical step in the inflammatory response, since
most of the damage due to unwanted inflammation occurs after leukocytes cross blood vessels. TEM is
regulated by molecules such as platelet/endothelial cell adhesion molecule-1 (PECAM, CD31) and CD99
expressed on both the leukocytes and at endothelial cell (EC) borders. On EC, these molecules also reside in
an interconnected membrane reticulum of membrane called the lateral border recycling compartment (LBRC)
that is “targeted” to the site of TEM. Homophilic interactions between leukocyte PECAM and EC PECAM
control the start of diapedesis. Homophilic interactions between leukocyte CD99 and EC CD99 are important
for the completion of TEM. In this proposal, we will build on the knowledge gained in the last cycle to test the
overarching hypothesis that these leukocyte/endothelial cell interactions recruit the LBRC to promote
efficient TEM and that their distribution on the leukocyte is responsible for the sequence in which leukocyte/EC
interactions trigger signaling pathways that promote TR to facilitate TEM.
 In Aim I we will test the hypothesis that the distribution of PECAM and CD99 on the leukocyte is
responsible for the sequential regulation of TEM. Preliminary data show that during TEM, PECAM is
concentrated in the front of the leukocyte and CD99 at the rear. We suspect that this concentration is critical
to clustering PECAM and CD99 on the endothelial cell. We will test our hypothesis in vitro by switching the
cytoplasmic domains of PECAM and CD99 to reverse their distribution on the leukocyte and determine
whether this affects the order in which they regulate TEM. We will also test whether they regulate TEM
sequentially in vivo using spinning disc confocal intravital microscopy to study the process in real time.
 CD99L2 (L2) is a molecule we recently discovered to play an important role in TEM. In Aim II we will
identify how L2 functions to regulate TEM. L2 is diffusely distributed on the leukocyte surface. We will test the
hypothesis that it regulates a step between those regulated by PECAM and CD99 using the “sequential block”
assay that we have developed and published. We will identify the signaling pathways used by L2 to carry out
its function and determine whether it plays a significant role in ischemia/reperfusion (I/R) injury. We will study
response to I/R injury by intravital microscopy in the cremaster muscle circulation over hours and in a mouse
model of myocardial infarction over days.
 We recently identified the long-sought mechanism by which endothelial cell CD99 signals in TEM. It
resides in a multimolecular complex that activates protein kinase A (PKA). In Aim III we will identify the CD99
signaling pathways downstream of PKA. Based on preliminary data we hypothesize that Rac1 is a critical
downstream effector of PKA to promote targeted recycling. We will test the role of CD99 in I/R injury in the two
models a...

## Key facts

- **NIH application ID:** 9843556
- **Project number:** 5R01HL064774-18
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** William A Muller
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $430,552
- **Award type:** 5
- **Project period:** 2000-04-01 → 2021-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9843556

## Citation

> US National Institutes of Health, RePORTER application 9843556, Beyond PECAM:  Mechanisms of Transendothelial Migration (5R01HL064774-18). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9843556. Licensed CC0.

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