Sphingosine-1-phosphate Maintains Normal Vascular Permeability by Preserving Endothelial Surface Glycocalyx in Intact Microvessels

Abstract

Objective: S1P was found to protect the ESG by inhibiting MMP activity-dependent shedding of ESG in cultured endothelial cell studies. We aimed to further test that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels.

Methods: We quantified the ESG in post-capillary venules of rat mesentery and measured the vascular permeability to albumin in the presence and absence of 1 μM S1P. We also measured permeability to albumin in the presence of MMP inhibitors and compared the measured permeability with those predicted by a transport model for the inter-endothelial cleft.

Results: We found that in the absence of S1P, the fluorescence intensity of the FITC-anti-HS-labeled ESG was ~10% of that in the presence of S1P, whereas the measured permeability to albumin was ~6.5-fold of that in the presence of S1P. Similar results were observed with MMP inhibition. The predictions by the mathematical model further confirmed that S1P maintains microvascular permeability by preserving ESG.

Conclusions: Our results show that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels, consistent with parallel observation in cultured endothelial monolayers.

Keywords: heparan sulfate; in situ immunostaining; matrix metalloproteinase; rat mesentery; transport model for the inter-endothelial cleft.

Figure 1

Schematic showing the single vessel cannulation and perfusion for directly injecting FITC-anti-HS into a post-capillary venule via a theta (θ) micropipette. Upper figure shows the pre-treatment with or without 1μM S1P in 1%BSA Ringer for 20 min via a single lumen micropipette under 37°C superfusate. After pretreatment, the single lumen micropipette was changed to a θ micropipette and the superfusate temperature was gradually decreased to 4 °C. The washout (blocking) solution was first perfused into the vessel through one lumen of the θ micropipette. Then the perfusion was switched to another lumen (Dye) to perfuse FITC-anti-HS for immunolabeling the ESG of the microvessel for 2.5h. After labeling, the perfusion was back to the washout solution to wash away the free FITC-anti-HS.

Figure 2

Confocal images of the FITC-anti-HS labeled ESG in a post-capillary venule. Image at the mid-plane of the vessel (A), 3D reconstruction (B) and cross-sectional views along the vessel axis (C).

Figure 3

(A) Images of fluorescently labeled heparan sulfate in a post-capillary venule pretreated with 1 μM S1P (left) and a vessel without S1P (right). (B) Comparison of the intensity of the fluorescently labeled heparan sulfate in 4 vessels with S1P and that in 3 vessels without S1P. * p < 0.001.

Figure 4

Permeability to BSA under various treatments: S1P, BSA only, generic MMP inhibitor GM 6001, specific inhibitors to MMP-9 and -13, and negative control GM 6001 NC. *p < 0.001 compared to that under S1P treatment. n: number of vessels in each group.

Figure 5

(A) A three dimensional view of the model geometry for the interendothelial cleft in the wall of a rat mesenteric post-capillary venule (revised from Fu et al., 1994). A junction strand with periodic large breaks of width 2d lies parallel to the luminal front of the cleft. The distance between adjacent large break is 2D. There is a continuous small slit also in the junction strand. An endothelial surface glycocalyx layer (ESG) of thickness L_f is at the cleft entrance; L, the total depth of the cleft; and 2B, the width of the cleft. (B)-(D) model predictions for the effect of changing structural components of the interendothelial cleft on P to BSA, P to sodium fluorescein (NaFl) and hydraulic conductivity Lp. (B): increasing the cleft width 2B. (C): increasing the junction break width 2d. (D): increasing the number of junction breaks (decreasing 2D). P_S1P, Lp_S1P, B_S1P, d_S1P, D_S1P are control values in the presence of S1P.

Figure 6

Comparison of the model predictions for P to BSA, P to sodium fluorescein and hydraulic conductivity Lp by removing ESG in the absence of S1P and those measured from the experiments. Values are mean ± SE. P_S1P, Lp_S1P, L_fS1P are control values in the presence of S1P.