Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier - PubMed (original) (raw)
Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier
Paul G McGuire et al. Arterioscler Thromb Vasc Biol. 2011 Dec.
Abstract
Objective: The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier.
Methods and results: Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2.
Conclusions: Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.
Figures
Figure 1
Confocal images of human retinal endothelial cells (HREC) alone (A), co-cultures of HREC and pericytes (B) and HREC after the addition of pericyte conditioned media (C). Cells are stained for smooth muscle actin (red), N-cadherin (green) and DNA (blue). The increase in N-cadherin expression seen by immunostaining is confirmed by Western blot analysis of HREC treated with pericyte conditioned media (D). The addition of pericytes (E) or pericyte conditioned media (F) to confluent monolayers of HREC increases the monolayer resistance as measured by ECIS. The addition of HREC or the Muller glial cell line MIO-M1, or their conditioned media, did not alter the monolayer resistance. Values represent the mean ± SEM from triplicate wells. For (E) values at each time point starting at 8 hours are significantly different and for (F) values at each time point starting at 6 hours are significantly different.
Figure 2
N-cad Fc disrupts pericyte-endothelial interactions. A suspension of retinal pericytes was added to a pre-established monolayer of retinal endothelial cells in the presence of control IgG Fc (0.5μg/ml) (A) or N-cadherin Fc (0.5μg/ml) (B) and incubated for 5 hours. The presence of N-cadherin Fc significantly inhibited the spreading of pericytes and the extent of contact between pericytes and endothelial cells. Cultures with N-cadherin Fc 12 hours after plating demonstrated a normal flattened morphology (C). The differences in pericyte morphology in the different conditions was confirmed by quantitation of cell area (D). Values represent the mean ± SEM from triplicate coverslips. * Significantly less than cells in the presence of control IgG Fc at 5 hours or N-cadherin Fc after 12 hours of incubation. (P<0.001). The altered morphology of pericytes in the presence of N-cadherin Fc was correlated with changes in the ability of the pericytes to increase the resistance of the endothelial monolayer (E). Values at each time point for HREC + Pericytes are significantly greater than HREC alone or HREC + Pericytes + N-cad Fc beginning at 6 hours. Values for HREC + Pericytes + N-cad Fc are significantly different from HREC alone beginning at 10 hours.
Figure 3
Stimulation of the S1P1 receptor mediates the effects of pericyte conditioned media. (A) ECIS normalized resistance in HREC alone, HREC plus pericyte conditioned media, HREC plus pericyte conditioned media in the presence of Tie-2 Fc chimera (200ng/ml) or HREC with pericyte conditioned media in the presence of anti TGF-β antibody (50ng/ml). The change in resistance was monitored for 18 hours after the addition of perictye conditioned media and the resistance values normalized to time 0. Values represent the mean ± SEM from triplicate wells. Values for pericyte CM, Tie-2 Fc and anti TGF-β are significantly greater than HREC alone at all time points beginning at 4 hours. (B) HREC monolayers incubated with pericyte CM alone or with the S1P1 antagonist VPC23019 (0.3μM), or the S1P2 antagonist JTE-013 (1μM). Values for pericyte CM, and S1P2 antagonist are significantly greater than HREC alone and S1P1 antagonist at all time points beginning at 4 hours. (C and D) Quantitation of percent change in resistance compared to basal level at time 0. * Significantly greater than HREC alone (P<0.001). ** Significantly less than pericyte CM and pericyte CM+S1P2 antogonist (P<0.05).
Figure 4
Knockdown of Sphk1 in pericytes or S1P1 in endothelial cells reduces the ability of pericytes to increase the endothelial monolayer resistance. (A) Representative Western blot for Sphk1 in untreated perictyes (lane 1), perictyes treated with control morpholinos (lane 2) or Sphk1 morpholinos (lane 3) (consistent among N=3 separate cell cultures for each treatment). (B) Conditioned media from each set of cells tested in an ECIS assay. (C) Quantitation of percent change in resistance compared to basal level at time 0. (D) Representative Western blot for S1P1 in HREC (lane 1), HREC treated with control morpholinos (lane 2) or S1P1 morpholinos (lane 3) (consistent among N=3 separate cultures for each treatment). (E) Conditioned media from untreated pericytes added to HREC with each of the treatments in an ECIS assay. (F) Quantitation of percent change in resistance compared to basal level at time 0. * Significantly greater than HREC untreated or morpholino-treated cells (P<0.0001).
Figure 5
Purified S1P increases the expression of adhesion proteins in human retinal endothelial cells. Confluent HREC monolayers were incubated for 12 hours in the presence of S1P and collected for PCR and Western blotting. Both N-cadherin mRNA (A) and VE-cadherin mRNA (D) were signficantly upregulated in S1P-treated cells in a dose-dependent manner. Representative Western blots for N-cadherin (B) and VE-cadherin (E) from HREC incubated with 10μM S1P demonstrate increased protein associated with the insoluble membrane fraction of the cells. Quantitation of band density normalized to tubulin levels indicates a significant increase in both N-cadherin (C) and VE-cadherin (F) proteins. *Significantly greater than untreated and 1mM (P<0.01).
Figure 6
The adhesion proteins N-cadherin and VE-cadherin have different functions in establishment and maintenance of the endothelial cell barrier. (A) Western blot for N-cadherin in HREC treated with N-cadherin morpholinos or control morpholinos. (B) Morpholino-treated HREC or untreated HREC were plated onto ECIS electrodes in the absence or presence of N-cadherin Fc (0.5μg/ml) or VE-cadherin Fc (0.5μg/ml) and the developing resistance was monitored. The decreased expression of N-cadherin or the addition of N-cadherin Fc had no effect on the resistance that developed over the subsequent 6 hours compared to control cells. Cells incubated with VE-cadherin Fc demonstrated significantly less resistance at all time points beginning at approximately 20 minutes after plating and continued to decrease for more than 5 hours.
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