An in vitro assay reveals a role for the diaphragm 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 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 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 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 and micro- Fig. 1. In vitro system of fenestra formation. (A) bEND5 cells untreated (Left) tubules in induced bEND5 cells (Figs. 2 B and C and 6). The PV-1 or induced with 2.5 ␮M latrunculin A for3h(Right) were examined by patches appeared to be surrounded by a microtubule border, similar whole-mount TEM. (B) Numerous fenestrae in the plasma membrane of to that reported in fenestrated liver endothelial cells (30). induced bEND5 cells were confirmed by SEM analysis. (C) Thin section cut To determine whether the PV-1 patches observed with LM were along the plane of a monolayer of induced bEND5 cells revealed that fenestrae indeed sieve plates, we performed immunoelectron microscopy and are organized in a sieve plate (arrow). A section plane within the cytoplasm correlative light–electron microscopy studies. First, direct immu- revealed electron-dense material surrounding the pore (arrowheads). (D) TEM nogold labeling of induced cells examined by SEM showed most of scraped monolayers revealed fenestrae both en face (arrowheads) and in PV-1 labeling in induced bEND5 cells to localize to the center of cross-sectional view (arrows). Arrows in the magnification point to fenestral each fenestra (Fig. 3A). In contrast, the general plasma membrane diaphragms. (E) Time-course of fenestra formation after induction with two different microfilament disruption agents. Mean values of two independent marker platelet endothelial cell adhesion molecule (PECAM) was experiments with standard deviations are shown. (Scale bars: 500 nm; mag- distributed across the entire plasma membrane (data not shown). nification in D, 100 nm.) Additionally, by using fluoronanogold to perform LM and electron microscopy on the same samples, we confirmed that PV-1 patches, which were identified by fluorescence LM, corresponded to gold- thelioma cell line. Whole-mount transmission electron microscopy labeled fenestrae when the same cell was examined by TEM (TEM) and scanning electron microscopy (SEM) showed that (Fig. 3B). untreated bEND5 cells had few fenestrae on their plasma mem- Finally, we used image analysis to quantify sieve plate area after brane (Fig. 1A). Upon application of latrunculin A, most of the induction. We defined sieve plates as areas positive for PV-1 and peripheral plasma membrane of bEND5 cells became perforated negative for microtubules. Next, we defined the total area of cells with hundreds of fenestrae that were organized in sieve plates and by using the cell-surface marker PECAM. By measuring the sum of were highly ordered (Fig. 1 A and B). Examination of thin sections areas that were positive for PV-1 and negative for microtubules and of embedded monolayers and cell pellets confirmed that the by expressing this value as a fraction of the sum of areas that were structures observed upon induction were surrounded by electron- positive for PECAM we obtained an estimate of the percentage of dense material (Fig. 1C) and were indeed pores that traversed the plasma membrane that was occupied by sieve plates (Fig. 7, which entire thickness of endothelial cells (Fig. 1D). The fenestrae dis- is published as supporting information on the PNAS web site). played consistent pore diameters of Ϸ60 nm, diaphragms, linear arrangement, and equidistant spacing of 100–120 nm from one Cytoskeletal Remodeling Is Essential for Fenestra Formation. Micro- fenestral center to the next (Fig. 1 A–D). filament disruption agents have previously been shown to increase To quantify the extent of fenestra formation, we applied stere- fenestra abundance by 3- to 4-fold in cultured fenestrated liver ology to randomly sampled SEM and TEM images. Quantification endothelium, leading researchers to suggest a role for the actin confirmed that fenestra formation was rapid, with large numbers in modulating fenestra density (31, 32). However, our occurring as early as 20 min after induction (Fig. 1E). Maximum observations of a potent de novo induction of the fenestrated fenestra induction was Ϸ100-fold, reaching levels of 3.5–5.0 phenotype in the bEND5 cell model suggest that cytoskeletal fenestrae per squared micrometer after3hoflatrunculin A disassembly may be a basic prerequisite for fenestra formation. We

Ioannidou et al. PNAS ͉ November 7, 2006 ͉ vol. 103 ͉ no. 45 ͉ 16771 Downloaded by guest on September 24, 2021 Fig. 3. A LM assay reveals the spatial and temporal relationship between actin rearrangements and fenestra induction. (A) Immunogold labeling for PV-1 by SEM revealed 10-nm gold particles at the center of individual fenestrae. (Scale bar: 500 nm.) (B) Correlative microscopy on induced bEND5 cells using fluoronanogold and LM (Left) confirmed that PV-1-positive patches, identified by epifluorescence microscopy (magnification), corre- sponded to gold-labeled sieve plates when the same cell was visualized by whole-mount TEM (Right). (Scale bars: Left,10␮m; magnification, 4 ␮m; Right,1␮m.) (C) bEND5 cells induced with 2.5 ␮M latrunculin A were labeled for PV-1 and filamentous actin and visualized by confocal microscopy. (Left) Immediately after (0 h) latrunculin A induction. (Center) Stress fibers were present throughout the cytoplasm immediately after (0 h) latrunculin A induction. (Right) Washout after3hofinduction resulted in reassembly of stress fibers and disappearance of sieve plates. (Scale bar: 10 ␮m.) (D) Quan- tification of sieve plates in a time-course of induction with 2.5 ␮M latrunculin A using the LM assay (compare with Fig. 1E). Mean values of two independent experiments with standard deviations are shown.

fenestrae in bEND5 cells (Fig. 4). Interestingly, although VEGF did not cause global actin disruption (as observed with latrunculin A), small patches of VEGF-induced sieve plates formed between actin (Fig. 4A). When actin cytoskeletal rearrangements were blocked by incubating cells with the F-actin-stabilizing drug phalloidin before induction with VEGF, sieve plates did not form (Fig. 4). Therefore, we conclude that actin microfilament remod- eling is sufficient and necessary for fenestra induction in our in vitro model system.

Fig. 2. The diaphragm protein, PV-1, segregates from caveolae to sieve Knockdown of PV-1 Disrupts the Morphology and Organization of plates. (A) In untreated bEND5 cells, most of the staining for PV-1 and Fenestrae. PV-1 expression alone is not sufficient for fenestra caveolin-1 was colocalized, indicating that PV-1 was present predominantly in formation, because it is found in the diaphragms of caveolae in caveolae (arrowheads). Upon induction with latrunculin A, PV-1 relocalized to many endothelia that do not have fenestrae (25). However, the discrete patches in the cell periphery (arrows), which did not stain for caveo- induction of fenestrae in our system led to a dramatic and rapid lin-1. (B and C) In untreated bEND5 cells, PV-1 was present on the plasma redistribution of PV-1 into newly formed fenestrae. Thus, we sought membrane-covering organelle (DiOC6) and microtubule-rich (tubulin) re- to examine the importance of PV-1 and, hence, the importance of gions. The discrete patches of PV-1 that formed in the cell periphery upon induction excluded organelles and microtubules (arrows). (Scale bar: 10 ␮m.) the diaphragm in fenestra formation. By using an siRNA specific for PV-1, we found that PV-1 mRNA and protein levels were reduced by 90% and 70%, assessed the relationship between actin rearrangements and sieve respectively, compared with levels in cells treated with a non- plate formation upon latrunculin A induction with LM. After targeting siRNA against luciferase (Fig. 8, which is published as exposure to latrunculin A, the number of actin stress fibers de- supporting information on the PNAS web site). To determine creased, whereas the fraction of plasma membrane occupied by the extent of PV-1 knockdown in individual cells, we immuno- sieve plates increased, both with similar kinetics (Fig. 3C). At all labeled siRNA-treated bEND5 cells for PV-1. We noted that time points, stress fiber remnants and sieve plates occupied separate PV-1 immunoreactivity was greatly reduced in Ϸ70% of cells in and distinct cellular compartments (Fig. 3C). Furthermore, wash- the PV-1 siRNA condition, with the remaining 30% of the cell out of latrunculin A led to a rapid assembly of actin stress fibers population being minimally affected (data not shown). The throughout the cytoplasm and a concomitant decrease in sieve overall morphology of bEND5 cells as well as the uniform plates (Fig. 3 C and D). coverage of the plasma membrane by PECAM was unaffected by To investigate a causal role for actin depolymerization in fenestra PV-1 knockdown (Fig. 8). In addition, knockdown of PV-1 did formation, we used a more physiological stimulus for fenestra not affect the distribution of caveolin-1 at perinuclear areas, a induction. We found that VEGF produced Ϸ4-fold induction of pattern characteristic of caveolae (Fig. 8), consistent with a

16772 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603501103 Ioannidou et al. Downloaded by guest on September 24, 2021 Fig. 4. Actin rearrangements are necessary for fenestra induction by VEGF. (A) bEND5 cells untreated (Left), induced with 100 ng͞ml VEGF 164 for 4 h (Center), or induced with VEGF in the presence of phalloidin (Right), were analyzed by epifluorescence microscopy. In VEGF-induced cells, small sieve plates (arrows) were apparent between rearranged stress fibers. In cells pretreated with labeled phalloidin, sieve plate formation was prevented. (Scale bars: Right,10␮m; magnification in Center,4␮m.) (B) Quantification of sieve plates using the LM assay (P Ͻ 0.05; one-way ANOVA with post hoc Bonferroni test). CELL BIOLOGY

previous study that showed intact caveola morphology after PV-1 siRNA treatment (29). We used whole-mount TEM to assess the formation of fenestrae in cells upon PV-1 knockdown (Fig. 5A). Interestingly, we found no Fig. 5. Reduction of PV-1 disrupts the morphology of fenestrae. (A) Variable difference in total sieve plate areas between the luciferase and PV-1 fenestra diameters and spacing after PV-1 knockdown. (Left) The luciferase siRNA conditions. Furthermore, quantitative analysis of fenestra siRNA condition. (Right) The PV-1 siRNA condition. The magnifications show density within sieve plates revealed no significant difference be- the recordings of fenestra centers (yellow dots) that were used to determine tween the two conditions, although a trend toward a lower overall fenestra spacing. (Scale bars: 500 nm; magnification, 250 nm.) (B) Fenestra density within sieve plates was not significantly different between luciferase density was observed in the PV-1 siRNA condition (Fig. 5B). and PV-1 siRNA conditions. (C) Distances between fenestrae in the PV-1 However, the appearance and order of fenestrae within the sieve condition were significantly smaller or greater than in the luciferase siRNA plates of cells in the PV-1 siRNA condition was significantly condition (P Ͻ 0.01; see also Fig. 9, which is published as supporting informa- different from that of controls (Fig. 5 C–E). The spacing between tion on the PNAS web site). (D) Fenestra diameters showed a wider distribu- fenestrae in the PV-1 siRNA condition was highly variable, being tion in the PV-1 versus the luciferase siRNA condition but were not signifi- both shorter and longer than the average distance of 100–120 nm cantly different. The discontinuous y axis simplifies comparisons between in the luciferase siRNA condition (Fig. 5C). The diameters of 75% conditions within one graph. (E) Fenestra diameters within the PV-1 siRNA condition were significantly greater in fenestrae without diaphragms versus of fenestrae in the PV-1 siRNA condition fell in the range of 60–80 Ͻ nm, similar to all fenestrae in the luciferase siRNA condition. fenestrae with diaphragms (P 0.01). However, 25% of fenestrae in the PV-1 siRNA condition showed diameters as small as 20 nm and as large as 400 nm (Fig. 5D). vivo in terms of morphology, abundance, and higher-order orga- Importantly, diaphragms were still evident in a population of nization. Our model system confers a number of advantages over fenestrae in the PV-1 siRNA condition, a partial phenotype that is other approaches: (i) Fenestrae are formed in a transformed predictable because we observed a reduction rather than a com- endothelial cell line that can be maintained for numerous passages; plete elimination of PV-1 protein. To examine the correlation (ii) fenestra induction occurs within minutes rather than days; (iii) between the observed morphological irregularities and a reduction fenestrae are abundant; (iv) the fold-induction is large and is in PV-1 levels, we determined the pore size of fenestrae as a function of the presence or absence of a diaphragm. Quantitative suitable for studies using comparative biochemical methods and assessment of whole-mount TEM images from the PV-1 siRNA LM; and (v) the model system is amenable to gain- and loss-of- condition revealed that fenestrae that were devoid of diaphragms function approaches. had enlarged and variable diameters, whereas fenestrae that dis- In developing a system to study fenestrae, we found that numer- played regular or smaller-than-usual diameters contained a dia- ous endothelial cell lines and primary cell cultures failed to induce phragm (Fig. 5E). Taken together, our observations strongly sug- fenestrae upon treatment with a variety of stimuli. At present, we gest that PV-1 is required during fenestra biogenesis to define the do not know what predisposes the bEND5 brain endothelioma cell dimensions of fenestrae as well as their organization within sieve line to respond so robustly to latrunculin A. Although our system plates. should prove useful in gaining insight into fenestra morphology and biogenesis, care must be taken when extrapolating these data to Discussion fenestra regulation in vivo, because we observed only a low response In this study we have described an in vitro culture system that to VEGF, a physiological stimulus for fenestra formation. Under- produces fenestrae similar to those observed in capillary beds in standing the predisposing factors behind the robust response to

Ioannidou et al. PNAS ͉ November 7, 2006 ͉ vol. 103 ͉ no. 45 ͉ 16773 Downloaded by guest on September 24, 2021 latrunculin A and the low response to VEGF in bEND5 cells will disassembly after latrunculin A treatment also was gradual, whereas help in the future characterization of the signaling cascades and actin filament reassembly after stimulus removal was more rapid, subcellular changes controlling fenestra biogenesis. consistent with the mode of action of latrunculin A, an inhibitor of Absolute numbers of Ϸ5.0 fenestrae per squared micrometer and actin (43). a relative induction of 100-fold favorably compare to previous in Our data indicate that expression of PV-1, the only recognized vitro studies where adrenal cortex endothelial cells or human constituent of fenestrae, is not sufficient for their formation, umbilical vein endothelial cells induced with VEGF, phorbol esters, consistent with previous studies of PV-1 overexpression in endo- or retinoic acid attained maximal levels of only 0.187 fenestrae per thelial cells (29). However, the dysmorphic appearance of fenestrae squared micrometer, with peak levels requiring days of treatment and disorganized sieve plates after PV-1 knockdown by siRNA and yielding a relatively low fold-induction (11–15). Fenestra den- suggested that PV-1 is required for the morphology of fenestrae. sities of Ϸ9.0 per squared micrometer were reported for liver Furthermore, we observed a reduction of diaphragms in fenestrae endothelial cells treated with swinholide A (33), but these outcomes after PV-1 knockdown, consistent with previous reports showing represented only a Ϸ3-fold induction and were restricted to already that PV-1 is the major constituent of caveolar and fenestral fenestrated, freshly isolated, cells (34). Our estimated density of diaphragms (29). Analysis restricted to fenestrae without dia- fenestrae in sieve plate areas (30 fenestrae per squared micrometer) phragms revealed an increase in fenestra diameter and in the is in agreement with the value reported for kidney capillaries (8). variability of these diameters, further supporting a role for PV-1 in Although the development of a fenestra biogenesis model was fenestra morphogenesis. focused on obtaining the maximum amount of fenestrae, our model Observations on the fenestrated capillary beds of the kidney has provided some insight into the cell biology of fenestra forma- glomerulus and liver sinusoids indicated a periodic presence of tion. The potent effect of microfilament disassembly on fenestra diaphragms during vascular development, despite the absence of a induction in bEND5 cells and the spatial and temporal correlation diaphragm in the adult tissues (refs. 27 and 44 and unpublished between stress fiber disassembly and sieve plate formation high- observation). Taken together, these observations and our siRNA lighted the importance of actin microfilaments. Whereas previous data are suggestive of a requirement for PV-1 during fenestra studies had addressed the role of actin disassembly in sustaining or formation but not maintenance. PV-1 is unlikely to play a role in the modulating the number of pores in fenestrated liver endothelial fusion of the apical and basal plasma membranes, given that cells (31–33, 35) and isolated kidney glomeruli (36), we demon- fenestral pores, albeit devoid of diaphragms, were evident in the strated a role for actin disassembly in de novo fenestra formation. PV-1 knockdown condition. Plausible functions of PV-1 include Our observations of a rearrangement of large actin filaments during constraining the fenestral opening by oligomerizing with other fenestra induction by the physiological stimulus VEGF and the PV-1 molecules at opposite sides of the pore or regulating the prevention of such rearrangements and fenestra induction by the spacing between fenestrae through interactions with intracellular F-actin-stabilizing drug phalloidin further supported a role for actin scaffolds. remodeling in fenestra formation. Actin remodeling in response to The in vitro model for fenestra formation and the initial charac- VEGF was not as dramatic as the complete stress fiber disassembly terization described here should provide a platform and conceptual seen with latrunculin A; however, in light of the reported low framework to enable further studies of fenestra biogenesis to stress-fiber content of capillaries (37), it is likely to reflect more characterize fenestra components and their function, to help probe closely the subcellular changes in vivo. Furthermore, a recent study the function of fenestrae in vivo, and to eventually modulate the showed that an antagonist of the actin regulator Rac, blocked barrier properties of the endothelium for therapeutic purposes. VEGF-mediated induction of fenestrae in the corneal micropocket assay (38). Together with evidence for a modulation of actin Materials and Methods polymerization by VEGF signaling in other systems (39), these data Antibodies and Reagents. The following primary antibodies were suggest that actin remodeling may be part of VEGF-driven fenestra used for immunocytochemistry: rat anti-PV-1 (MECA-32; Devel- biogenesis in vivo. opmental Studies Hybridoma Bank, Iowa City, IA), rabbit anti- We hypothesize that actin stress fibers are a structural barrier, PV-1 (prepared against the 12 C-terminal amino acids), mouse the disruption or remodeling of which is required to bring the anti-caveolin 1 (BD Biosciences, Franklin Lakes, NJ), mouse apical and basal plasma membranes of the cell in close proximity anti-tubulin (Sigma, St. Louis, MO), rat anti-PECAM (MEC13.3; for fusion. A parallel can be drawn with organelle membrane BD Biosciences), allophycocyanin-conjugated rat anti-PECAM fusion, where persistence of a surrounding actin coat acts as a (MEC13.3; BD Biosciences). Rhodamine phalloidin (Molecular physical barrier that prevents the recruitment of tethering factors Probes, Eugene, OR) and DiOC6 (Molecular Probes) were used to and, in turn, the close apposition of membranes (40, 41). visualize F-actin and intracellular organelles, respectively. All stan- Furthermore, the absence of stress fibers from fenestrated areas dard chemicals were obtained from Sigma unless otherwise appears to be required for their maintenance, given that washout indicated. of latrunculin A was followed by a reassembly of actin stress fibers and the disappearance of fenestrae. The reversal in cellular Cell Culture, siRNA Transfection, and Fenestra Induction. The bEND5 attenuation suggested by the increase in actin stress fibers and endothelioma cell line (45) was kindly provided by Urban Deutsch the redistribution of organelles to the cell periphery is likely and Britta Engelhardt (University of Bern, Bern, Switzerland). similar to the increase in thickness observed in vivo during bEND5 cells were maintained in high-glucose DMEM (Invitrogen, fenestra disappearance after VEGF inhibition (42) or preven- Carlsbad, CA) containing 10% FBS and antibiotics. Cells were tion of fenestra formation by a Rac antagonist (38). transfected with Oligofectamine (Invitrogen) in culture medium The timing of fenestra appearance and disappearance in our lacking FBS and antibiotics. Predesigned siRNA against PV-1 (ID model and the dynamic and reversible nature of the relationship 85339; Ambion, Austin, TX) and luciferase negative control siRNA between actin microfilaments and fenestrae are suggestive of a (Ambion) were used at a final concentration of 5 nM. PV-1 plasticity of endothelial cells. Although the low response to VEGF knockdown and cell phenotypes were assessed 72 h later. in our model precludes direct comparisons to the situation in vivo, Twenty-four hours before fenestra induction, glass coverslips, a similar endothelial plasticity has been reported in studies using formvar grids, or culture dishes were coated with 1% gelatin in PBS VEGF and VEGF inhibitors to induce or remove fenestrae (19, 42). and bEND5 cells were seeded at a density equivalent to 1.5 ϫ 106 Furthermore, the gradual appearance of fenestrae in our model, in cells per 100-mm dish. Cells were induced with either 10 ␮M contrast to their rapid disappearance upon stimulus removal, likely cytochalasin B for2h(Sigma), 2.5 ␮M latrunculin A for 3 h reflects the kinetics of actin filament polymerization. Actin filament (Molecular Probes), or 100 ng͞ml murine VEGF 164 for 4 h

16774 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603501103 Ioannidou et al. Downloaded by guest on September 24, 2021 (Peprotech, Rocky Hill, NJ). For actin stabilization studies, cells (70–100 nm) were stained with uranyl acetate and Reynolds were preincubated with 80 nM rhodamine phalloidin for 30 min stain for 2 min and dried. (Molecular Probes). Morphometrics. We randomly captured 20–30 SEM images per Immunocytochemistry. Cells on coverslips were fixed with either time-point at a magnification of ϫ10,000. A grid with 150 points methanol at Ϫ20°C for 8 min or 4% paraformaldehyde (PFA) for of intersection was placed on each image, and the number of grid 15 min. PFA-fixed cells were permeabilized in PBT (0.1% Triton points falling on any cellular structure versus the number of grid X-100͞PBS). Before incubation with primary and secondary points falling on sieve plates were counted. The density of antibodies (30 min each at room temperature), cells were fenestrae was estimated by multiplying the fraction of total cell blocked in 10% goat serum͞0.2% fish skin gelatin in PBS for 15 membrane area that was occupied by sieve plates with the min. Fluorescent images were captured with an LSM510 laser average density of fenestrae within 10 representative sieve plates scanning confocal microscope (Zeiss, Go¨ttingen, Germany) and (30 fenestrae per squared micrometer of sieve plate area). a DMRA2 epifluorescence microscope (Leica Microsystems, For sieve plate quantification by LM, seven images were Wetzlar, Germany). randomly acquired per coverslip from preparations triple stained Immunolabeling for SEM was performed on live cells at 4°C, for PV-1, tubulin, and PECAM. The total cell area was calcu- followed by fixation in 1% PFA͞3% glutaraldehyde (Electron lated by measuring the PECAM-positive area with Openlab Microscopy Sciences, Hatfield, PA) in cacodylate buffer (0.1 M software (Improvision, Lexington, MA). The area occupied by sodium cacodylate, pH 7.4). For whole-mount TEM immuno- sieve plates was calculated by measuring the PV-1-positive, labeling, cells were fixed in 4% PFA͞0.5% glutaraldehyde in 0.1 tubulin-negative area. Sieve plate area per cell was estimated as M phosphate buffer for 15 min, labeled, and postfixed with 2.5% the percentage of the total PECAM-positive area that was glutaraldehyde. positive for PV-1 and negative for tubulin (Fig. 7). For the siRNA phenotype quantification, 36 whole-mount Electron Microscopy. For SEM, cells on coverslips were fixed in TEM images per condition were randomly captured at a mag- ϫ 2% PFA (EM grade; Electron Microscopy Sciences)͞2.5% glu- nification of 25,000. Only images containing sieve plates taraldehyde in cacodylate buffer, dehydrated, dried in hexam- (20–30 images per condition) were included in the analysis (see ethyldisalazane, gold-coated, and examined under a JSM-6700 also Supporting Materials and Methods, which is published as field emission SEM (JEOL, Tokyo, Japan) in Backscatter mode. supporting information on the PNAS web site). Statistical analysis was performed by using one-way ANOVA CELL BIOLOGY Immunogold labeling was visualized in Backscatter mode. Cells Ͻ grown on formvar grids were fixed in 1.25% glutaraldehyde and with a post hoc Bonferroni test for which P 0.05 was 2.5% PFA in cacodylate buffer, postfixed, dehydrated, dried as considered a statistically significant difference. for SEM and examined under a JEOL 1010 TEM. For TEM thin sections, intact monolayers or pellets of monolayers that had We thank Amy Snodgrass, Peter Munro, Kirsty Roberts, Rose Watson, and Steve Gschmeissner for technical assistance; Vladimir Mastyugin for been scraped and centrifuged at 1,000 ϫ g for 10 min were fixed ͞ help in designing siRNA transfections; and members of the Endothelial in 2.5% glutaraldehyde 2% PFA in cacodylate buffer for 20 min. Cell Biology Laboratory and the Eyetech Research Center for useful After osmium fixation and dehydration, specimens were infil- discussions. This work was supported by Cancer Research UK and OSI trated with resin and polymerized overnight at 60°C. Sections Eyetech.

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