Abstract
Engagement of platelet endothelial cell adhesion molecule 1 (PECAM, PECAM-1, CD31) on the leukocyte pseudopod with PECAM at the endothelial cell border initiates transendothelial migration (TEM, diapedesis). We show, using fluorescence lifetime imaging microscopy (FLIM), that physical traction on endothelial PECAM during TEM initiated the endothelial signaling pathway. In this role, endothelial PECAM acted as part of a mechanotransduction complex with VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2), and this predicted that VEGFR2 was required for efficient TEM. We show that TEM required both VEGFR2 and the ability of its Y1175 to be phosphorylated, but not VEGF or VEGFR2 endogenous kinase activity. Using inducible endothelial-specific VEGFR2-deficient mice, we show in three mouse models of inflammation that the absence of endothelial VEGFR2 significantly (by ≥75%) reduced neutrophil extravasation by selectively blocking diapedesis. These findings provide a more complete understanding of the process of transmigration and identify several potential anti-inflammatory targets.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2311-2324.e6 |
| Journal | Immunity |
| Volume | 56 |
| Issue number | 10 |
| DOIs | |
| State | Published - Oct 10 2023 |
Funding
We would like to thank Clifford D. Carpenter and Lily A. Miller for assistance with experiments and editing the manuscript. We are grateful to Martin Schwartz (Yale Medical School) for the PECAM tension sensor constructs. We thank Ralf Adams (Max Planck Institute) for the Cdh5-CreERT2 mice and Susan Quaggin (Northwestern) for the VEGFR2flox/flox mice. Expert technical guidance for FLIM was provided by the Northwestern University Center for Advanced Microscopy, and DNA sequencing was performed by the NUSeq Core Facility, both generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We would also like to thank the Sidney & Bess Eisenberg Memorial Fund for partially covering publication costs. This work was supported by NIH F31 HL131355 (to N.S.R.); Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship and F30 HL134202 (to P.J.D.); and R01 HL064774, R01 HL046849, and R35 HL155652 (to W.A.M.), T.F. D.P.S. E.W.W. A.M.G. and W.A.M. conceived, designed, and performed experiments; interpreted data, and wrote the manuscript. M.E.H. A.L.T. and J.A.Y. performed and analyzed experiments. P.J.D. and N.S.R. analyzed raw intravital microscopy data. W.A.M. oversaw the project and secured funding. All authors read and approved the manuscript. The authors declare no competing interests. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as living with a disability. We would like to thank Clifford D. Carpenter and Lily A. Miller for assistance with experiments and editing the manuscript. We are grateful to Martin Schwartz (Yale Medical School) for the PECAM tension sensor constructs. We thank Ralf Adams (Max Planck Institute) for the Cdh5-CreERT2 mice and Susan Quaggin (Northwestern) for the VEGFR2 flox/flox mice. Expert technical guidance for FLIM was provided by the Northwestern University Center for Advanced Microscopy, and DNA sequencing was performed by the NUSeq Core Facility, both generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We would also like to thank the Sidney & Bess Eisenberg Memorial Fund for partially covering publication costs. This work was supported by NIH F31 HL131355 (to N.S.R.); Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship and F30 HL134202 (to P.J.D.); and R01 HL064774 , R01 HL046849 , and R35 HL155652 (to W.A.M.)
Keywords
- PECAM
- VEGFR2
- endothelial cell
- inflammation
- inflammatory signaling
- leukocyte
- mechanotransduction
- monocyte
- neutrophil
- transmigration
ASJC Scopus subject areas
- Immunology and Allergy
- Immunology
- Infectious Diseases