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An in Vitro Assay Reveals a Role for the Diaphragm Protein PV-1 in Endothelial Fenestra Morphogenesis

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An in Vitro Assay Reveals a Role for the Diaphragm Protein PV-1 in Endothelial Fenestra Morphogenesis An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis Sofia Ioannidou*†, Katrin Deinhardt†‡, Jadwiga Miotla†, John Bradley*, Eunice Cheung*, Steven Samuelsson*, Yin-Shan Ng*, and David T. Shima*†§ *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) Fenestrae are small pores in the endothelium of renal glomerular, VEGF to certain tissues in vivo results in fenestra induction (19), gastrointestinal, and endocrine gland capillaries and are involved and genetic ablation of components in the VEGF signaling pathway in the bidirectional exchange of molecules between blood and (20–22) or antagonism of its receptor (23, 24) lead to a loss of the tissues. Although decades of studies have characterized fenestrae fenestrated phenotype. However, the low levels of fenestra induc- at the ultrastructural level, little is known on the mechanisms by tion in vitro obtained with VEGF [mean of Ͻ1 fenestra per cell which fenestrae form. We present the development of an in vitro (15)], have precluded in depth mechanistic studies. assay in which rapid and abundant fenestra induction enables a The only known component of fenestrae is PV-1, a type II detailed study of their biogenesis. Through the use of agents that membrane glycoprotein first discovered in caveolae (25) and later stabilize or disassemble actin microfilaments, we show that actin recognized to be a component of the diaphragm of endothelial cell microfilament remodeling is part of fenestra biogenesis in this caveolae, fenestrae, and transendothelial channels (26). PV-1 is model. Furthermore, by using a loss-of-function approach, we present in vascular beds containing diaphragmed fenestrae but is show that the diaphragm protein PV-1 is necessary for fenestral absent from fenestrated endothelia of the adult liver sinusoids and pore architecture and the ordered arrangement of fenestrae in kidney glomerulus, which are devoid of diaphragms (2, 26, 27). sieve plates. Together, these data provide insight into the cell PV-1 has been proposed to associate in multiple coiled-coil ho- biology of fenestra formation and open up the future study of the modimers to form the fibrils of the diaphragm (28) and was recently fenestra to a combined morphological and biochemical analysis. shown to be necessary and sufficient for the formation of dia- phragms (29). However, a role for PV-1 in fenestra formation and actin filaments ͉ sieve plates ͉ VEGF ͉ vascular permeability function has not been addressed. To gain insight into the cellular and molecular events required for egulated vascular permeability is essential for normal circula- fenestra formation we developed an in vitro assay in which fenestrae Rtory function and tissue homeostasis. Throughout the vascular can be induced at densities that approach those seen in vivo, thereby network, endothelial cells employ a number of mechanisms to facilitating cell biological studies. By establishing and applying control permeability. One such mechanism is the formation of quantitative ultrastructural and light microscopy (LM) methods, we pore-like structures called fenestrae, which are implicated in the discovered that actin microfilament depolymerization is a prereq- permeability of water, solutes, and small macromolecules. uisite for fenestra formation in our model. Furthermore, through a Fenestrae occur at sites of high blood–tissue exchange, such as the loss-of-function study using siRNA, we demonstrated that the kidney glomerulus, the gastrointestinal tract, endocrine glands, liver diaphragm protein PV-1 is required for normal fenestra biogenesis. sinusoids, and the choriocapillaris of the eye (1–3). In addition, In particular, we found that PV-1 defines the dimensions of fenestrae have been documented in vessels during cancer and individual fenestrae and specifies their precise arrangement within diabetic retinopathy (4–6), pathologies associated with uncon- a sieve plate. trolled permeability and edema. Results Ultrastructural studies have determined that fenestrae traverse the entire thickness of endothelial cells in attenuated regions as thin Development of an in Vitro Assay for the Study of Fenestrae. We as 40 nm, forming a pore Ϸ60–70 nm in diameter (7, 8). In most screened a large panel of endothelial cells for their response to fenestrated vascular beds, the pore of fenestrae is dissected into 5- single factors and combinations of factors previously reported to to 6-nm openings by a diaphragm composed of radially arranged induce fenestrae in vitro. The factors tested included extracellular fibrils that converge in a central knob (9). Within an endothelial cell, matrix, growth factors such as VEGF, actin microfilament depo- fenestrae are organized in clusters referred to as ‘‘sieve plates,’’ lymerizing agents, and phorbol 12-myristate 13-acetate. Most cell lines and primary cell cultures examined demonstrated very few being arranged with equidistant spacing, often forming linear or fenestrae or no fenestrae at all under basal or induced conditions two-dimensional arrays (8, 10). Despite the extensive morpholog- (data not shown). However, the greatest and most reproducible ical characterization of fenestrae, their composition and biogenesis fenestra induction occurred with the application of the actin remain poorly understood. The small size of fenestrae, together depolymerizing agent latrunculin A to the bEND5 mouse endo- with a lack of specific markers for these structures, has rendered their study dependent on ultrastructural methods, which are tech- nically demanding and provide only limited descriptive information. Author contributions: S.I., K.D., J.M., Y.-S.N., and D.T.S. designed research; S.I., K.D., J.M., Attempts at analyzing fenestrae in vitro, an approach that facilitates 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., experimental manipulation and observation, have been limited by J.B., and D.T.S. wrote the paper. the rapid dedifferentiation and loss of the fenestrated phenotype in The authors declare no conflict of interest. culture. Moreover, attempts at de novo induction of the fenestrated This article is a PNAS direct submission. phenotype in cultured endothelial cells have resulted in low yields Abbreviations: TEM, transmission electron microscopy; LM, light microscopy; PECAM, that have limited the application of cell biological analyses (11–15). platelet endothelial cell adhesion molecule; PFA, paraformaldehyde. VEGF is a prime candidate for induction of fenestrae in vivo. ‡Present address: Molecular Neuropathobiology Laboratory, Cancer Research UK, 44 Lin- VEGF is a potent angiogenic and permeability mediator that is coln’s Inn Fields, London WC2A 3PX, United Kingdom. expressed in epithelia adjacent to fenestrated vascular beds (16, 17) §To whom correspondence should be addressed. E-mail: [email protected]. and fenestrated neovasculature (6, 18). Ectopic administration of © 2006 by The National Academy of Sciences of the USA 16770–16775 ͉ PNAS ͉ November 7, 2006 ͉ vol. 103 ͉ no. 45 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603501103 Downloaded by guest on September 24, 2021 treatment (Fig. 1E). Because VEGF is a potent inducer of fenestrae, we wondered whether the effects of latrunculin A were mediated by VEGF. However, a VEGF function-blocking antibody (recognizing all VEGF isoforms) did not affect latrunculin A induction of fenestrae, indicating that fenestrae form as a direct result of actin depolymerization (data not shown). Furthermore, cytochalasin B, an actin-depolymerizing agent with a mode of action different than latrunculin A, also produced large numbers of fenestrae (Fig. 1E). Together, these data reveal a close relationship between actin depolymerization and fenestra induction. A LM Assay for the Study of Fenestrae in Vitro. We next developed an alternative approach for the characterization of fenestrae that did not require technically demanding and laborious ultrastructural analysis. Because the diaphragm protein PV-1 is the only known component of fenestrae and because almost all fenestrae in our system were spanned by a diaphragm (Fig. 1D, magnification), we reasoned that PV-1 would be enriched in fenestrated areas in vitro. Furthermore, we hypothesized that fenestrated areas in our model would not stain for cytoskeletal elements and organelles, because sieve plates are only 40 nm thick and exclude such structures. Double immunolabeling revealed that, in untreated bEND5 cells, PV-1 colocalized with caveolin-1 on the plasma membrane in a pattern that is characteristic for caveolae (Fig. 2A) (29). DiOC6 and tubulin, markers for membrane-bound organelles and microtu- bules, respectively, were distributed widely throughout the cell and significantly overlapped with the localization of PV-1 and caveo- lin-1 in untreated bEND5 cells (Fig. 2 B and C). In contrast, double CELL BIOLOGY immunolabeling of induced bEND5 cells for PV-1 and caveolin-1 showed PV-1 concentrated in discrete patches at the cell periphery (Fig. 2A; see also Fig. 6, which is published as supporting infor- mation on the PNAS web site). Furthermore, labeling for PV-1, in combination with either the dye DiOC6 or an antibody against tubulin, showed that PV-1 patches excluded organelles
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