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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 loss^3,4,5.

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 transport^1,6,7,8. 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 transporters^5,9.

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 properties^4,9.

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 studies^15,16,17. 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 mounts^20,21. 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 microscope^22,23,24 (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.

Protokół

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston Children'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's Hospital.

1. Preparation of Reagents

1. Solution for coating tissue culture dish: Prepare 0.1% gelatin solution by dissolving 1 mL of gelatin stock solution (40%-50%) in 500 mL of sterile tissue culture grade 1x PBS (pH = 7.4). Filter the solution through a 0.22 µm filter. Store 0.1% gelatin solution at 2-8°C. It can be stored at the said temperature for an indefinite time in a sterile condition.
2. Growth medium: Prepare 500 mL of endothelial cell growth complete medium (EGM) by dissolving EGM supplements into the endothelial cell growth basal medium (EBM) according to the manufacturer's instructions (Table of Materials). Aliquot growth medium into 50 mL tubes and store the tubes at 4 °C. The shelf life of the growth medium is 12 months at 4 °C.
3. Trypsin solution: For 0.25% trypsin-EDTA solution, aliquot from the stock vial into 50 mL tubes and store at 4 °C (up to 2 weeks) or −20 °C (up to 24 months).

2. Culturing HRMECs

1. Initial cell culture
   1. Coat cell culture Petri dishes (100 mm diameter x 20 mm height) with 5 mL of 0.1% gelatin solution under a laminar flow hood and keep them undisturbed in the hood for 30 min at room temperature (RT) for uniform coating.
      NOTE: Gelatin coating of dishes enhances cell attachment. Alternatively, gelatin-coated T75 culture flasks can also be used.
   2. Before seeding the cells, aspirate the gelatin solution using a vacuum pump. The vacuum pressure of the aspirator used was up to 724 mmHg.
      NOTE: Aspiration must be done immediately before seeding cells to prevent drying out of the coating solution.
   3. Thaw a frozen vial of HRMECs (from storage in liquid nitrogen) either by using a water bath at 37 °C or by adding a warm growth medium into the vial. Once thawed, transfer the cell suspension to 9 mL of growth medium (assuming thawed vial of HRMECs is 1 mL).
      NOTE: The HRMECs were commercially obtained (Table of Materials). The growth medium added to the cells should always be prewarmed to 37 °C before use.
   4. Spin the cells at 200 x g for 5 min at RT and carefully aspirate the supernatant to avoid removing the cell pellet.
   5. Resuspend the cell pellet in 10 mL of growth medium and transfer the resultant suspension onto a gelatin-coated Petri dish. Keep the cells in the incubator at 37 °C and 5% CO₂. Typically, HRMECs are 70%-80% confluent after 72 h.
      NOTE: The growth medium is changed every other day.
2. Subculture of HRMECs on permeable membrane inserts
   1. Prepare cell culture filter inserts (6.5 mm inserts with 0.4 µm pore size polycarbonate membrane, placed in a 24-well plate) for seeding HRMECs by coating each insert (apical chamber) with 200 µL of 0.1% gelatin solution for 30 min under a laminar flow hood. Ensure the solution covers the entire bottom surface of the filter insert.
      NOTE: Each well has an apical and basolateral chamber separated by a porous membrane.
   2. Take out the Petri dish with cultured HRMECs (step 2.1.5.) from the incubator and aspirate the growth media. Gently rinse the cells 2x with 10 mL of 1x PBS under a laminar flow hood to get rid of potential floating/dead cells.
   3. Dissociate the cells with 0.5-1 mL of 0.25% trypsin-EDTA solution and place the Petri dish in the incubator (37 °C and 5% CO₂) for 5 min.
   4. Quench the trypsin activity by adding 4.5-9 mL of growth media and transfer the cell suspension into a 15 mL tube using a 10 mL pipette.
   5. Spin the cells at 200 x g for 5 min at RT, carefully remove the supernatant, and resuspend the pellet in 3 mL of growth media.
   6. Count the number of cells using a manual hemocytometer or an automated cell counter and seed at a density of 4 x 10⁴ cells per filter insert (i.e., 1.25 x 10⁵ cells/cm²). The volume of cell suspension for each insert is 250 µL.
   7. Aspirate the coating solution from the wells containing permeable inserts and transfer 250 µL of the cell suspension per insert (apical chamber). Add only medium (i.e., without cells) to one of the inserts, which will be used as a "blank control" for TEER measurement. Simultaneously, also add 750 µL of medium per well, in the basolateral chambers.
   8. Keep the 24-well plate with permeable inserts in the incubator (37 °C and 5% CO₂) for 7-12 days until the cultured cells become fully confluent and the desired TEER value of ~20 Ω·cm² is achieved.
   9. Change the growth medium every other day. During media change, carefully aspirate the media to minimize disruption of the cell monolayer and add 250 µL and 750 µL of fresh media per well to the apical and basolateral chambers, respectively.
      ​NOTE: Growth medium is changed for both the apical and basolateral chambers of the wells.

3. TEER measurements (Figure 4)

1. On day 14 post cell seeding (step 2.2.9.), measure the TEER for HRMECs using an epithelial volt-ohm meter (EVOM) electrical resistance (ER) system (Table of Materials) as follows.
2. Pre-charge the ER system and check the meter functionality using the STX04 test electrode. Calibrate, if required.
3. Connect the electrode to the meter and equilibrate the electrode by first soaking it in 70% ethanol for 15-20 min and then immersing it briefly in the cell culture EGM growth medium.
4. In the meantime, keep the cell-containing 24-well plate in the laminar flow hood at RT for 15-20 min for temperature equilibration.
   NOTE: TEER measurements are affected by temperature, so the equilibration step is essential.
5. For TEER measurement, add fresh growth medium to both the apical (250 µL) and basolateral (750 µL) chambers of the wells.
6. 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. For each insert, measure TEER in triplicates.
   NOTE: For rinsing the electrode in between the measurements, culture media is used. Ensure the electrode is held at a 90° angle to the bottom of the insert for stable readings.
7. Calculate electrical resistance (in Ω·cm²) across the monolayer using the formula, TEER = net resistance (Ω) x surface area of filter insert (cm²); here, net resistance is the difference between the resistance of each well (growth medium with cells) and the blank well (only growth medium).
8. Carry out further treatment of the cells only after the TEER value reaches ~20 Ω·cm². If the desired TEER level is not reached, keep the plate in the incubator and measure the TEER the following day.
   ​NOTE: HRMEC confluency can also be validated by morphological examination of cell shape (under a microscope) with typical cobblestone morphology and/or by the presence of cell junction proteins with immunohistochemistry staining separately.

4. Transcytosis assay

1. Clathrin-mediated in vitro transcytosis assay using Cy3-tagged transferrin (Figure 5)
   1. Upon reaching confluency with TEER values around 20 Ω·cm², serum deprive the cells for 24 h at 37 °C and 5% CO₂ using 0.5% FBS in EBM (serum-reduced medium) in both chambers (apical chamber: 250 µL and basolateral chamber: 750 µL) prior to treatment with the ligand. Serum-reduced EBM was used throughout the assay.
   2. Incubate the cells (using the serum-reduced medium in step 4.1.1.) in the apical chamber with fluorescent (cyanine 3)-tagged transferrin ligand (Cy3-Tf) (final concentration of 40 µg/mL) for 60 min at 37 °C.
      NOTE: Plates containing Cy3-Tf should be protected from light to avoid photobleaching of Cy3-Tf by wrapping in them aluminum foil and performing the experiment in a cell culture hood with the lights turned off.
   3. After 1 h, place the plate on ice and wash the monolayer apically and basolaterally 4x (3-5 min per wash) with the serum-reduced medium at RT to remove the free unbound Cy3-Tf.
      NOTE: Washing is essential to remove free tracer molecules and allow accurate reading of transcytosis without potential leakage from the paracellular route.
   4. Add fresh serum-reduced medium (as in step 4.1.1.) to the thoroughly washed filter inserts containing cells and transfer the inserts to fresh wells of the 24-well plate containing pre-warmed serum-reduced medium.
   5. Incubate the cells for another 90 min in the incubator (37 °C and 5% CO₂), and then collect the medium from the basolateral chamber.
   6. Record the fluorescence intensity of the solution from the basolateral chamber using a fluorescence detector. The levels of fluorescence intensity, measured as relative fluorescent units (RFU), indicate the amount of Cy3-Tf complex transcytosed across the HRMEC monolayer via clathrin-dependent transcytosis.
2. Caveolae-mediated in vitro transcytosis assay using HRP (Figure 6)
   1. Upon reaching full confluency with TEER values around 20 Ω·cm², serum deprive the cells for 24 h at 37 °C and 5% CO₂ using 0.5% FBS-containing EBM medium (serum-reduced medium) before treatment (as in step 4.1.1.). Serum-reduced EBM medium was used throughout the entire assay.
   2. Treat the cells in the apical chamber with the desired treatments and vehicle controls.
      NOTE: Here, we have used Wnt modulators as an example to demonstrate the regulation of caveolae-mediated transcytosis by the Wnt signaling pathway in HRMECs: human recombinant Norrin and Wnt inhibitor XAV939. In a typical experiment, cells were treated for 24 h in an incubator (37 °C and 5% CO₂) with the following concentrations: Norrin (125 ng/mL), Norrin (125 ng/mL) + XAV939 (10 µM), and vehicle control solution. In addition, Wnt3a-conditioned medium (produced from L Wnt-3A cells) was also used.
   3. Incubate the cells in the apical chamber with HRP (5 mg/mL) for 15 min at 37 °C.
   4. Afterward, place the 24-well plate on ice and wash the apical and basolateral chambers intensively 6x with P buffer (10 mM HEPES pH = 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl₂, 145 mM NaCl) in order to remove the free extracellular HRP.
      NOTE: Washing is essential to remove free tracer molecules and allow accurate reading of transcytosis without potential leakage from the paracellular route.
   5. Add fresh serum-reduced medium (as in step 4.1.1.) to the apical chamber and transfer the inserts to a fresh well containing pre-warmed media.
   6. Incubate the monolayer for an additional 90 min in the incubator at 37 °C and 5% CO₂ before collecting the medium from the basolateral chamber.
   7. To the collected medium, add 100 µL of HRP fluorogenic peroxidase substrate (Table of Materials) as per the manufacturer's instructions, and incubate at RT for 10 min before stopping the reaction with 100 µL of Stop solution.
   8. Detect the levels of HRP substrate reaction product in the media using a fluorescence plate reader. Measure the fluorescence intensity at 420 nm emission wavelength (with 325 nm excitation) and as relative fluorescent units (RFU). The values indicate the level of HRP transcytosed across the HRMEC layer through caveolae-mediated transcytosis.
      NOTE: The fluorescent product used here (Table of Materials) does not photobleach. Light protection against photobleaching is not needed.

Wyniki

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...

Dyskusje

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...

Materiały

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