This work was supported by NIH grants (R01 EY028100, EY024963, and EY031765) to JC. ZW was supported by a Knights Templar Eye Foundation Career Starter Grant.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston Children&#39;s Hospital for the generation of light microscopy and EM images (Figure 3). Protocols for the in vivo studies can be obtained from Wang et al.24. All experiments involving human retinal microvascular endothelial cells (HRMECs) were approved by the Institutional Biosafety Committee (IBC) at Boston Children&#39;s Hospital.
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BRB plays an essential role in retinal health and disease. In vitro techniques assessing vascular permeability have proven to be crucial tools in studies concerning barrier (BRB/BBB) development and function. The procedure described here could be utilized to study the molecular mechanisms underlying EC transcytosis or evaluate related molecular modulators affecting BRB permeability. In vitro EC transcytosis assays have multiple advantages over in vivo assays or techniques used for evaluating va...

The human retina is one of the highest energy-demanding tissues in the body. Proper functioning of the neural retina requires an efficient supply of oxygen and nutrients along with a restricted flux of other potentially harmful molecules to protect the retinal environment, which is mediated via the blood-retinal barrier (BRB)1. Similar to the blood-brain barrier (BBB) in the central nervous system, the BRB acts as a selective barrier in the eye, regulating the movement of ions, water, amino acids, and sugar in and out of the retina. BRB also maintains retinal homeostasis and its immune privilege by preventing exposure to circulatory factors such as immune cells, antibodies, and harmful pathogens2. BRB dysfunction contributes to the pathophysiology of several vascular eye diseases, such as diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity (ROP), retinal vein occlusion, and uveitis, resulting in vasogenic edema and subsequent vision loss3,4,5.
The BRB consists of two separate barriers for two distinct ocular vascular networks, respectively: the retinal vasculature and the fenestrated choriocapillaris beneath the retina. The inner BRB (iBRB) is primarily composed of retinal microvascular endothelial cells (RMECs) lining the retinal microvasculature, which nourishes the inner retinal neuronal layers. On the other hand, the retinal pigment epithelium forms the major component of the outer BRB, which lies between the neurosensory retina and choriocapillaris2. For the iBRB, molecular transport across RMECs takes place through both paracellular and transcellular routes (Figure 1). The high degree of substance selectivity across the iBRB relies upon (i) the presence of junctional protein complexes that restrict paracellular transport between adjacent endothelial cells (ECs), and (ii) low expression levels of caveolae mediators, transporters, and receptors within the endothelial cells that maintain low rates of transcellular transport1,6,7,8. Junctional complexes regulating paracellular flux are composed of tight junctions (claudins, occludins), adherens junctions (VE cadherins), and gap junctions (connexins), allowing the passage of water and small water-soluble compounds. While small lipophilic molecules passively diffuse across the interior of RMECs, the movement of larger lipophilic and hydrophilic molecules is regulated by ATP-driven trans-endothelial pathways including vesicular transport and membrane transporters5,9.
Vesicular transcytosis may be categorized as caveolin-mediated caveolar transcytosis, clathrin-dependent (and receptor-mediated) transcytosis, and clathrin-independent macropinocytosis (Figure 2). These vesicular transport processes involve different-sized vesicles, with macropinosomes being the largest (ranging from 200-500 nm) and caveolae being the smallest (averaging 50-100 nm), while clathrin-coated vesicles range from 70-150 nm10. Caveolae are flask-shaped lipid-rich plasma membrane invaginations with a protein coat, primarily composed of caveolin-1 that binds lipid membrane cholesterol and other structural and signaling proteins via their caveolin-scaffolding domain11. Caveolins work together with peripherally attached cavin to promote caveolae stabilization at the plasma membrane12. Caveolar membranes also may carry receptors for other molecules such as insulin, albumin, and circulating lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) to assist their movement across endothelial cells13. During development, the formation of functional BRB depends on the suppression of EC transcytosis8. Mature retinal endothelium, hence, has relatively low levels of caveolae, caveolin-1, and albumin receptors with respect to other endothelial cells under physiological conditions, contributing to its barrier properties4,9.
Because iBRB breakdown is a major hallmark of many pathological eye conditions, it is essential to develop methods to assess retinal vascular permeability in vivo and in vitro. These methods help provide probable insights into the mechanisms of compromised BRB integrity and assess the efficacy of potential therapeutic targets. Current in vivo imaging or quantitative vascular leakage assays typically employ fluorescent (sodium fluorescein and dextran), colorimetric (Evans Blue dye and horseradish peroxidase [HRP] substrate), or radioactive tracers14 to detect extravasation from the vasculature into surrounding retinal tissues with microscope imaging or in isolated tissue lysate. An ideal tracer for quantifying vascular integrity should be inert and large enough to freely permeate compromised vessels while confined within healthy and intact capillaries. Methods employing sodium fluorescein or fluorescein isothiocyanate-conjugated dextran (FITC-dextran) in live fundus fluorescein angiography (FFA) or isolated retinal flat mounts are widely used for quantitating retinal extravasation in vivo or ex vivo. FITC-dextran has the advantage of being available in different molecular weights ranging from 4-70 kDa for size-selective studies15,16,17. FITC-albumin (~68 kDa) is an alternative large-sized protein tracer of biological relevance for vascular leakage studies18. Evans Blue dye, injected intracardially19, retro-orbitally, or through the tail vein20, also relies on its binding with endogenous albumin to form a large molecule that can be quantified by mostly spectrophotometric detection or, less commonly, fluorescence microscopy in flat mounts20,21. These quantitative or light imaging methodologies, however, often do not distinguish paracellular transport from trans-endothelial transport. For the specific analysis of transcytosis with ultrastructural visualization of transcytosed vesicles, tracer molecules such as HRP are typically used to locate transcytosed vesicles within endothelial cells that can be observed under an electron microscope22,23,24 (Figure 3A-C).
The development and use of in vitro iBRB models to evaluate the endothelial cell permeability could provide robust and high throughput assessment to complement in vivo experiments and aid the investigation of molecular regulators of vascular leakage. Commonly used assays to assess the paracellular transport and integrity of tight junctions include trans-endothelial electrical resistance (TEER), a measure of ionic conductance (Figure 4)2,25, and in vitro vascular leakage assay using small molecular weight fluorescent tracers26. In addition, transferrin-based transcytosis assays modeling BBB have been utilized to explore clathrin-dependent transcytosis27. Despite this, assays to evaluate BRB and, more specifically, retinal EC caveolar transcytosis in vitro are limited.
In this study, we describe an EC transcytosis assay using human retinal microvascular endothelial cells (HRMECs) as an in vitro model to determine iBRB permeability and EC transcytosis. This assay relies on the ability of HRMECs to transport transferrin or HRP via the receptor-mediated or caveolae-dependent transcytosis pathways, respectively (Figure 2). HRMECs cultured to full confluency in the apical chamber (i.e., filter insert) were incubated with fluorescent-conjugated transferrin (Cy3-Tf) or HRP to measure the fluorescence intensity corresponding to the levels of transferrin or HRP transferred to the bottom chamber through EC transcytosis solely. Confluency of the cell monolayer can be confirmed by measuring TEER, indicating the tight junction integrity25. To demonstrate the TEER and transcytosis assay technique, known molecular modulators of vascular permeability and EC transcytosis were used, including vascular endothelial growth factor (VEGF)28 and those in Wnt signaling (Wnt ligands: Wnt3a and Norrin)29.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biological Safety Cabinet&#160;</td>
			<td>Thermo Electron Corporation, Thermo Fisher Scientific</td>
			<td>1286</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture petridish&#160;</td>
			<td>Nest Biotechnology</td>
			<td>704001</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL)</td>
			<td>Corning Inc.</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 mL)</td>
			<td>Denville Scientific Inc.</td>
			<td>C1062-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanine 3-human Transferrin&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>AB_2337082</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Basal Medium-2 (EBM-2)</td>
			<td>Lonza Bioscience</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium-2 (EGM-2) SingleQuots supplements</td>
			<td>Lonza Bioscience</td>
			<td>CC-4176</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM Millicell Electrical Resistance System-2 (ERS-2)</td>
			<td>Millipore</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Lonza Bioscience</td>
			<td>CC-4102B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer (2-chip)</td>
			<td>Bulldog Bio</td>
			<td>DHC-N002</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish Peroxidase (HRP)</td>
			<td>Sigma-Aldrich</td>
			<td>P8250</td>
			<td></td>
		</tr>
		<tr>
			<td>Human retinal microvascular endothelial cells (HRMEC)</td>
			<td>Cell Systems</td>
			<td>ACBRI 181</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Electron Corporation, Thermo Fisher Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr>
			<td>L cells (for Control-conditioned medium)</td>
			<td>ATCC</td>
			<td>CRL-2648</td>
			<td></td>
		</tr>
		<tr>
			<td>L Wnt-3A cells (for Wnt3A-conditioned medium)</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimode Plate Reader</td>
			<td>EnSight, PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) buffer (1x)</td>
			<td>GIBCO</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantaBlu Fluorogenic Peroxidase Substrate kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>15169</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human Norrin (rhNorrin)</td>
			<td>R&#38;D Systems</td>
			<td>3014-NR</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human Vascular endothelial growth factor (rhVEGF)</td>
			<td>R&#38;D Systems</td>
			<td>293-VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.22 &#181;m)</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell inserts (6.5 mm transwell, 0.4 &#181;m pore polyester membrane insert)</td>
			<td>Corning Inc.</td>
			<td>CLS3470-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%) (1x)</td>
			<td>GIBCO</td>
			<td>25-200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Precision, Thermo Fisher Scientific</td>
			<td>51221060</td>
			<td></td>
		</tr>
		<tr>
			<td>XAV939 (Wnt/&#946;-catenin Inhibitor)</td>
			<td>Selleckchem</td>
			<td>S1180</td>
			<td></td>
		</tr>
	</tbody>
</table>

endothelial cell transcytosis assay as an in vitro model to evaluate inner blood-retinal barrier permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

EM images of retinal vascular endothelium show transcytotic vesicular transport and caveolar vesicles in endothelial cells in vivo. 
EC transcytosis can be visualized in vivo within retinal cross-sections with dark brown precipitate reflecting HRP-containing blood vessels under a light microscope (Figure 3A) and as electron-dense precipitate indicative of HRP-containing transcytotic vesicles (Figur...

Watch this Scientific Journal Video about Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability at JoVE.com

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

This protocol provides a useful cell-based model for studying the function and permeability of selective vascular barriers such as blood-retinal or blood-brain barrier with a specific focus on endothelial transcytosis. This assay is easy to set up and use requiring no live animals. It can be utilized for investigating various molecular regulators of endothelial cell permeability and transcytosis affecting inner blood-retinal barrier integrity.This assay can provide insights into vascular biology and eye and brain research, allowing the better understanding of molecule mechanisms underlying BRB BBB control and exploring potential drug delivery across the barriers. To begin, prepare the cell culture filter inserts for seeding human retinal microvascular endothelial cells or HRMECs, by coating each insert with 200 microliters of 0.1%gelatin solution for 30 minutes under a laminar flow hood. Ensure the solution covers the entire bottom surface of the filter insert.Take a Petri dish with cultured HRMECs incubated at 37 degrees Celsius and 5%carbon dioxide, and aspirate the growth media. Gently rinse the cells twice with 10 milliliters of 1X PBS under the laminar flow hood to get rid of the potential floating or dead cells. Dissociate the cells with 0.5 to 1 milliliter of 0.25%trypsin-EDTA solution and place the Petri dish in the incubator at 37 degrees Celsius and 5%carbon dioxide for 5 minutes.Quench the trypsin activity by adding 4.5 to 9 milliliters of growth media and transfer the cell suspension into a 15-milliliter tube using a 10-milliliter pipette. Spin the cells at 200 times g for 5 minutes at room temperature. Carefully remove the supernatant and resuspend the pellet in 3 milliliters of growth media.Count the number of cells using a manual hemocytometer or an automated cell counter and seed at a density of 40, 000 cells per filter insert. The volume of cell suspension for each insert is 250 microliters. Aspirate the coating solution from the wells containing permeable inserts and transfer 250 microliters of the cell suspension per insert, then add only medium to one of the inserts, which will be used as a blank control for transendothelial electrical resistance, or TEER, measurement.Simultaneously, also add 750 microliters of medium per well in the basolateral chambers. Keep the 24-well plate with permeable inserts in the incubator at 37 degrees Celsius and 5%carbon dioxide for 7 to 12 days until the cultured cells become fully confluent and the desired TEER value of around 20 ohms per square centimeter is achieved. Before TEER measurements, place the cell-containing 24-well plate in the laminar flow hood at room temperature for 15 to 20 minutes for temperature equilibration.To measure the TEER for HRMECs, using an epithelial volt/ohm meter electrical resistance system, connect the electrode to the meter, and equilibrate the electrode by first soaking it in 70%ethanol for 15 to 20 minutes and then immersing it briefly in the cell culture EGM growth medium. Perform TEER measurement by carefully immersing the electrode, such that the shorter tip is in the insert and the longer tip touches the bottom of the well. Measure the resistance across the blank control first, then for each insert, measure TEER in triplicates.Upon reaching confluency with TEER values around 20 ohm square centimeters, serum-deprive the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide using 0.5%FBS in EBM in both the chambers prior to treatment with the ligand. Serum-reduced EBM was used throughout the assay. Incubate the cells using the serum-reduced medium in the apical chamber with fluorescent cyanine-3-tag to transferrin ligand for 60 minutes at 37 degrees Celsius.After washing the monolayer apically and basolaterally 4 times with the serum-reduced medium, add fresh medium to the filter inserts containing the cells and transfer the inserts to fresh wells of the 24-well plate containing prewarmed serum-reduced medium. Incubate the cells for another 90 minutes in the incubator and then collect the medium from the basolateral chamber. Record the fluorescence intensity of the solution from the basolateral chamber using a fluorescence detector.Upon reaching confluency with TEER values around 20 ohm square centimeter, serum-deprive the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide using 0.5%FBS in EBM in both the chambers prior to treatment with the ligand. Serum-reduced EBM was used throughout the assay. Next, treat the cells in the apical chamber with the desired treatments and vehicle controls.Add 5 milligrams per milliliter of horseradish peroxidase, or HRP, to the cells, and incubate for 15 minutes at 37 degrees Celsius. Add fresh serum-reduced medium to the apical chamber and transfer the inserts to a fresh well containing prewarmed media. Incubate the monolayer for an additional 90 minutes in the incubator.After collecting medium from the basolateral chamber, add 100 microliters of HRP fluorogenic peroxidase substrate to the collected medium and incubate at room temperature for 10 minutes. Stop the reaction with 100 microliters of stop solution and detect the levels of HRP substrate reaction product in the media using a fluorescence plate reader. Before demonstrating the in vitro transcytosis assays, visualization of endothelial cell transcytosis in the retina is shown as background information.Here, the light microscope image shows an HRP-filled blood vessel lumen from a 3-month-old wild-type mouse retinal section stained with DAB. HRP was retro-orbitally injected. HRP-filled retinal vessels can be seen as dark brown precipitate under the light microscope.Transmission electron microscope ultra thin section shows HRP-filled transcytotic vessels with RMECs depicting EC transcytosis across the inner blood-retinal barrier. Here, the large vesicle potentially reflects macropinosome and a red blood cell on the luminol side. TeM image shows HRP-filled small transcytotic vesicles, likely caveola vesicles, within the RMEC.Immunohistochemistry staining of CAV-1 antibody and isolectin B4 demonstrates the localization of CAV-1, A marker of caveola vesicles in retinal blood vessels, in the 3-month-old wild-type mouse retina. TeM image of immunogold-labeled CAV-1 within the retinal endothelial cells is shown here with a magnified image of CAV-1 positive caveola vesicle. For clathrin-mediated EC transcytosis assay in HRMECs using transferrin, here, a fluorescence microscope image shows endocytosed Cy3 transferrin in red color within HRMECs stained with DAPI in blue.For caveolae-mediated EC transcytosis using HRP, Wnt signaling here is used as an example to demonstrate the assay. Wnt signaling was found recently to regulate caveolae-mediated EC transcytosis. Cells were treated with Wnt pathway activator Wnt3a-conditioned medium and recombinant Norrin, with or without Wnt signaling inhibitor XAV939 and their reactive controls.Wnt activators reduced the level of the HRP-based transcytosis, which were reversed by XAV939. Correct TEER measurement is crucial to determine monolayer integrity. It is greatly affected by various factors such as improper handling, temperature fluctuations, culture medium, culture duration, et cetera.This assay could be modified as co-culture or 3D organotypic culture systems mimicking in vivo conditions.

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4.3K visualizações.  Harvard Medical School. Este protocolo fornece um modelo celular útil para estudar a função e a permeabilidade de barreiras vasculares seletivas, como a barreira hemato-retiniana ou hematoencefálica, com foco específico na transcitose endotelial. Este ensaio é fácil de configurar e usar, não exigindo animais vivos. Pode ser utilizado para investigar vários reguladores moleculares da permeabilidade das células endoteliais e da transcitose que afetam a integridade da barreira hemato-retiniana interna....