An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis

doi:10.1073/pnas.0603501103

An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis

*Eyetech Research Center, OSI Eyetech, 35 Hartwell Avenue, Lexington, MA 02420; and
^†Endothelial Cell Biology Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Edited by Judah Folkman, Harvard Medical School, Boston, MA, and approved September 25, 2006 (received for review May 15, 2006)

Abstract

Fenestrae are small pores in the endothelium of renal glomerular, gastrointestinal, and endocrine gland capillaries and are involved in the bidirectional exchange of molecules between blood and tissues. Although decades of studies have characterized fenestrae at the ultrastructural level, little is known on the mechanisms by which fenestrae form. We present the development of an in vitro assay in which rapid and abundant fenestra induction enables a detailed study of their biogenesis. Through the use of agents that stabilize or disassemble actin microfilaments, we show that actin microfilament remodeling is part of fenestra biogenesis in this model. Furthermore, by using a loss-of-function approach, we show that the diaphragm protein PV-1 is necessary for fenestral pore architecture and the ordered arrangement of fenestrae in sieve plates. Together, these data provide insight into the cell biology of fenestra formation and open up the future study of the fenestra to a combined morphological and biochemical analysis.

Footnotes

^§To whom correspondence should be addressed. E-mail: davesfenestra{at}yahoo.com
Author contributions: S.I., K.D., J.M., Y.-S.N., and D.T.S. designed research; S.I., K.D., J.M., and E.C. performed research; S.I., K.D., J.M., J.B., E.C., S.S., and D.T.S. analyzed data; and S.I., J.B., and D.T.S. wrote the paper.
↵ ^‡Present address: Molecular Neuropathobiology Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations:

TEM,

transmission electron microscopy;

LM,

light microscopy;

PECAM,

platelet endothelial cell adhesion molecule;

PFA,

paraformaldehyde