Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells
© Beese et al; licensee BioMed Central Ltd. 2010
Received: 30 November 2009
Accepted: 7 September 2010
Published: 7 September 2010
Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer in vitro. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC) which are widely used as model for endothelial cell-related studies.
We here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP) and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na) and for longer incubation periods 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin.
pCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible in vitro model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins
Endothelial cells line the surface of all vascular or lymphatic vessels and are connected by intercellular junctions consisting of tight junctions (zonula occludens), adherens junctions and gap junctions . These junctional complexes control a variety of cellular mechanisms like adhesion, paracellular transport or signaling events . In contrast to most epithelial cells that are characterized by a highly regulated organization of cellular junctions interendothelial junctions are far from being static structures. In fact, both adherens junctions and tight junctions seem to be intermingled throughout the cell-cell contact areas . Tight junctions consist of several transmembrane or membrane-associated proteins including the membrane spanning claudins (CLDN), occludin (OCLN) and junctional adhesion molecules (JAM) . Tricellulin (TRIC) represents another tight junction-associated integral membrane protein that is localized at tricellular junctions in epithelial cells and is described to regulate paracellular permeability [4, 5].
These proteins are connected to the actin cytoskeleton via adaptor proteins like the zonula occludens (ZO) proteins ZO-1, ZO-2, ZO-3, cingulin, AF6 or 7H6 [6–9]. The main constituents of endothelial adherens junctions are vascular endothelial cadherin (VE-cadherin) and catenins that link adherens junctions to the cytoskeleton . Recently, a study published by Taddei and co-workers demonstrated the importance of adherens junctions in controlling proper tight junction formation in endothelial cells in vitro .
A common problem of working with primary endothelial cells is the rapid loss of many of their endothelial characteristics as soon as they are cultivated in vitro. This de-differentiation results from the missing interaction between endothelial cells and the specific microenvironment that is necessary for endothelial specifity . As a consequence, the use of primary endothelial cells for studying endothelial junctions comprises some limitations, e.g. fragmentary assembly of tight junctions, increased paracellular permeability or low transelectrical resistance. Therefore, many efforts have been done to overcome these limitations. For instance, co-cultures of microvascular endothelial cells and vascular smooth muscle cells, pericytes or astrocytes, or conditioned supernatant of these cells, proved useful in restoring endothelial barrier properties [13–15]. Also activation of cyclic 3',5'-adenosine monophosphate (cAMP)-dependent protein kinase (PKA) by cAMP or addition of dexamethasone or hydrocortisone have been shown to successfully improve endothelial junction architecture [16–19].
A convenient and easy to obtain source for primary human endothelial cells are umbilical cords, and human umbilical vein endothelial cells (HUVEC) are widely used for studying different endothelial cell-related questions [20–22]. Although HUVEC do not possess the specific barrier properties found in highly impermeable microvascular beds like the blood-brain barrier, they are suitable for studying architecture and formation of endothelial intercellular junctions. Nevertheless, it is necessary to induce formation of intercellular junctions in HUVEC as these cells, even when grown in tight monolayer, display intercellular gaps and discontinuous junction strands.
The aim of the present study was to compare and validate different cAMP analogues regarding their effect on expression of junction-associated proteins and on formation of continuous tight junction strands as well as on paracellular permeability in freshly isolated HUVEC.
Morphology and cellular integrity of HUVEC stimulated with different cAMP derivates
Cellular localization and distribution of junction-associated proteins
cAMP derivates influence formation and dissociation of tight junctions in a calcium switch model
Expression of tight junction-associated proteins
Effects of cAMP derivates on tight junctions are partly dependent on PKA
Effect of cAMP derivates on expression of TRIC
We then performed immunostainings for TRIC. Concordant with the low expression level immunostainings for TRIC are almost not visible in untreated HUVEC (data not shown). Stimulation with pCPT-cAMP and 8-Br-cAMP/Na for 24 h induced a very faint membrane staining of TRIC whereas after 72 h TRIC localization is clearly visible along bicellular junctions (Figure 7B). However, we were not able to see TRIC membrane staining in HUVEC treated with cAMP although this compound induced an increase in TRIC mRNA expression. Of interest, different to epithelial cells TRIC could not be localized at tricellular junctions in HUVEC.
Changes in paracellular permeability
The aim of the present study was to evaluate whether primary macrovascular endothelial cells isolated from human umbilical cords prove useful as a cell culture model to study tight junction assembly. The decision to choose HUVEC for these assays was based on the fact that umbilical cords are easy to obtain and the subsequent isolation of the endothelial cells out of the vein is well described and does not require specific technical skills [21, 23]. We also decided to concentrate on human cells as a broad variety of appropriate antibodies directed against human tight and adherens junction-associated proteins are available.
Although endothelial cells apparently form dense monolayer in vitro microscopic analysis revealed intercellular gaps and clefts as well as discontinuous junction strands  demonstrating that single monocultures of endothelial cells are not sufficient to induce proper cellular junction formation. Therefore, many approaches were undertaken to overcome these limitations by co-cultivating endothelial cells with "helper cells" like astrocytes or pericytes or with their appropriate conditioned cell culture medium. Under these conditions cerebral microvascular endothelial cells succeeded in building blood-brain barrier like monolayer with enhanced transelectrical resistance and decreased paracellular permeability [15, 25–27].
Another approach takes advantage of the junction-protecting effect of the second messenger cAMP. Beneath its ability to modulate PKA activity cAMP directly influences Epac, a guanine nucleotide exchange factor for Rap1 that modulates actin reorganization and distribution of adherens and tight junction-associated proteins [28, 29].
We here focused on the cAMP-approach and described the effects of different cAMP analogues on formation of cellular junctions and changes in paracellular permeability in HUVEC. Although the effect of cAMP on assembly of tight junctions in endothelial cells is well known a characterization of the cellular and molecular events that are modulated by different cAMP derivates is necessary as endothelial cells from macro- or microvascular beds may differ in their response to cAMP. Moreover, evidence exists that different chemically modified cAMP analogues modulate specific cellular responses. Recently, a study by Sand and colleagues demonstrated that 8-CPT-conjugated but not bromine-conjugated cAMP analogues act as competitive thromboxane receptor antagonists .
We here demonstrated that, although all cAMP derivates improved formation of continuous junction strands in HUVEC, they differ regarding their cell compatibility and kinetics of junction assembly. cAMP, 8-Br-cAMP and its sodium salt 8-Br-cAMP/Na induced formation of continuous tight junction strands as soon as 24 h after addition. Nevertheless, longer incubation periods led to a disassembly of the junction strands suggesting that these compounds proved useful for solely short-termed experiments. Moreover, they did not show any significant effect on improving paracellular permeability of HUVEC monolayer and, more importantly, cAMP and 8-Br-cAMP seemed to induce extensive vacuolization when exposed for longer time periods. pCPT-cAMP, on the other hand, displayed its best effects on HUVEC when added for longer time periods (≥72h) and generated the most robust phenotype as shown by calcium switch experiments. Moreover, pCPT-cAMP is the only compound that actually induced an improvement in barrier properties. Nevertheless, all four compounds reduced thrombin-induced increases in paracellular permeability to a certain degree and partly diminished breakdown of junctions in response to calcium depletion. These results suggest that cAMP and its derivates exhibit rather protective properties towards barrier breakdown than improving existing barrier properties.
For us, it was interesting to note that the compounds tested here had different effects on transcript expression of tight junction-associated proteins in HUVEC. All four cAMP derivates induced enhanced synthesis of CLDN5 mRNA after 24 h but only in pCPT-cAMP and 8-Br-cAMP/Na-treated cells mRNA level of CLDN5 remained elevated in the next 48 h. CLDN5 is one of the main endothelial tight junction proteins and its expression correlates with formation of tight junctions and endothelial barrier properties [31–33]. On the other hand, substances known to disrupt endothelial barrier properties down-regulate CLDN5 transcript amounts . Concordantly, the expression profiles of CLDN5 mRNA correlated quite well with the assembly of proper tight junction strands in HUVEC treated with the different cAMP derivates. Expression of OCLN mRNA followed a slightly different pattern. Although pCPT-cAMP and 8-Br-cAMP/Na showed the greatest effect on induction of OCLN after 24 h at the end of the experiment mRNA level did not differ compared to untreated cells. Surprisingly, the remaining two cAMP derivates did not induce any significant changes in OCLN expression. We therefore performed western blots for CLDN5 and OCLN. Whereas the protein and mRNA data for CLDN were consistent expression of OCLN was increased after stimulation with all four cAMP derivates. This discrepancy might be explained by the fact that changes in mRNA expression do not necessarily induce altered protein synthesis and that in case of doubt the protein analysis provided the most reliable data.
It is well known that cAMP exerts both PKA-dependent and -independent effects. In HUVEC cAMP-induced expression of OCLN and CLDN5 is controlled by PKA whereas membrane translocation and formation of continuous strands seemed to be independent of PKA at least for pCPT-cAMP, cAMP and 8-Br-cAMP. Interestingly, these findings are in opposite to a study by Ishizaki et al. who showed that induction of CLDN5 by pCPT-cAMP in brain microvascular endothelial cells is independent of PKA . This discrepancy may be explained by the different sources of the endothelial cells; we used human macrovascular cells whereas the group of Ishizaki worked on microvascular cells isolated from porcine brain capillaries.
The intention of this study was to evaluate whether HUVEC proved to be suitable for studying endothelial tight junction formation. These primary endothelial cells express most if not all of the known endothelial adherens and tight junction-associated proteins and form proper junction strands when cultured under appropriate conditions. Nevertheless, they definitely miss some of the typical vascular endothelial characteristics like increased transelectrical resistance or highly impermeable paracellular barriers found in other endothelial cell culture models established to study e.g. blood-brain barrier properties in vitro [35, 36]. Certainly, these different properties of the cells are due to the specific vascular beds they derived from. Whereas most of the culture models used for permeability and drug transport studies utilizes cerebral microvascular endothelial cells we worked with macrovascular venous endothelial cells from the umbilical cord. Accordingly, HUVEC are definitely not an adequate model to study blood-brain barrier-related topics. But on the other hand, umbilical cords are easy to obtain, and isolation of HUVEC does not require highly specific technical skills and is not as time-consuming as, for example, isolation of microvascular endothelial cells. Therefore, they represent a suitable culture model for studying formation or disassembly of endothelial intercellular junctions and the signaling pathways that are linked to this process.
In summary, we demonstrated that for short-term experiments 8-Br-cAMP/Na and for long-term studies pCPT-cAMP have shown to be useful for formation of dense endothelial monolayer and induction of continuous tight junction strands in HUVEC. We concluded that HUVEC proved useful for studying formation and disassembly of endothelial junction architecture or characterization of junction-associated proteins. Nevertheless, their use for studying functional parameters e.g. paracellular transport or permeability seemed to be restricted under the conditions tested here.
HUVEC were isolated from umbilical cords as described earlier  with the only modification that collagenase II (Biochrom, Berlin, Germany) was used instead of chymotrypsin. Cells were maintained in EBM-2 basal medium supplemented with the EGM-2 SingleQuot kit containing growth factors, gentamycin, amphotericin-B and 2% fetal bovine serum (Lonza, Vervier, Belgium). Contaminating fibroblasts were removed using CD90-coated paramagnetic beads (Invitrogen, Karlsruhe, Germany). For all experiments HUVEC were used up to passage three. The use of HUVEC was approved by the Hannover Medical School Ethics Committee and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.
Stimulation of HUVEC with cAMP or cAMP-derivates
cAMP derivates used in this study and their final working concentration
cyclic 3',5'-adenosine monophosphate
8-bromoadenosine 3'5'-cyclic monophosphate
8-bromoadenosine 3',5'-cyclic monophosphate sodium salt
8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate sodium salt
Determination of cellular cytotoxicity or apoptosis
Cellular cytotoxicity and metabolic activity of HUVEC due to the different cAMP derivates was determined using the CytoTox-Glo Cytotoxicity and the CellTiter-Glo Luminescent Cell Viability assay (Promega, Mannheim, Germany). Activation of caspases 3 and 7 was measured using the Caspase-Glo 3/7 assay (Promega). Assays were performed according to the manufacturer's instruction.
Calcium switch experiments
HUVEC were normally cultivated in EGM medium that contain several sources of calcium like D-calcium pantothenate, folinic acid calcium salt or calcium chloride dehydrate. To generate a calcium-free environment the cells were cultivated for 2 h in EGM medium in the presence of EDTA (3 μM) to chelate the free calcium or in PBS without any calcium but supplemented with all growth factors contained in the Lonza single quots. After 2 h medium was changed to normal EGM medium with the appropriate cAMP derivates and cells were cultivated for another 4 h before they were fixed.
RNA extraction and real-time quantitative PCR (qPCR)
Ordering information of the QuantiTect primer assays
Ordering numbers (Qiagen)
Cells were lysed in ice-cold 1 × lysis buffer (NEBiolabs, Frankfurt, Germany) containing protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics). Protein content of the samples was determined using the BCA protein assay kit (Thermo Fisher Scientific, Bonn, Germany). 50 μg of whole cell lysates was separated by SDS PAGE electrophoresis and blotted on a PVDF nylon membrane. Filters were incubated with the appropriate primary antibody followed by incubation with a HRP-conjugated secondary antibody. The bands were visualized by Western Lighting chemiluminiscence reagent (Perkin Elmer, Rodgau, Germany) and quantified by densitometry using a CCD camera and Quantity One software (Biorad Laboratories, Munich, Germany).
Cells were grown on collagen-coated glass cover slips and fixed in ice-cold acetone for 15 minutes at -20°C followed by permeabilization with ice-cold methanol for 20 min at -20°C. After washing with PBS the cells were blocked in normal donkey serum, incubated with the primary antibody for one hour followed by incubation with the appropriate secondary antibody coupled to ALEXA-488 or ALEXA-546 (Invitrogen) for an additional hour. DNA was counterstained with DAPI (Sigma Aldrich) and confocal images were taken using a Leica DM IRB microscope with a TCS SP3 AOBS scan head equipped with argon and krypton laser beams. Micrographs were obtained using a HCX PL APO 63 × 1.4 numerical aperture objective. Antibodies used in this study were a murine monoclonal anti-VE-cadherin (clone 55-7H1 from BD Pharmingen, Heidelberg, Germany; working concentration 1 ng/μl), a murine monoclonal anti-ZO-1 antibody (clone 1/ZO-1 from BD Pharmingen, working concentration 2.5 ng/μl), a murine monoclonal anti-OCLN antibody (clone 3F10 from Invitrogen, working concentration 5 ng/μL), a polyclonal goat anti-Jam-A antibody (R&D Systems, Wiesbaden, Germany, working concentration 1 ng/μl), a polyclonal anti-TRIC antibody (Invitrogen) and a rabbit anti-CLDN5 antibody (Santa Cruz Biotechniques, Heidelberg, Germany, working concentration 2 ng/μl).
Paracellular tracer flux assay
For determination of paracellular permeability HUVEC were seeded into the upper compartment of transwell devices with a pore size of 0.4 μm and cultivated for two days to reach confluence. After stimulation for 24 to 72 h with cAMP or its derivates the low molecular weight markers Lucifer yellow (LY, 457 Da, 20 μmol/L) and sodium fluorescein (Na-F, 10 μg/mL) or fluorescein (FITC)-coupled dextrans with the molecular masses of 10 kDa, 20 kDa or 70 kDa (of 200 μg/ml) were added to the upper compartment (Sigma-Alrich). In some experiments cells were pretreated with thrombin (2 U/mL, Merck, Darmstadt, Germany). After 30 minutes the concentrations of FITC-labeled dextrans in the lower compartment were measured using an ELISA reader equipped for fluorescence measurement (Tecan, Crailsheim, Germany).
Results were expressed as the mean ± SD of at least three independent experiments with cells derived from different donors. Statistical significance was calculated using Mann-Whitney-U test.
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