Mechanisms of modulation of brain microvascular endothelial cells function by thrombin

Abstract

Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic Ca²⁺ concentration in RBMVEC via Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and Ca²⁺ influx from extracellular space. Thrombin increases nitric oxide production; the effect is abolished by inhibition of the nitric oxide synthase or by antagonism of PAR1 receptors. In addition, thrombin increases mitochondrial and cytosolic reactive oxygen species production via PAR1-dependent mechanisms. Immunocytochemistry studies indicate that thrombin increases F-actin stress fibers, and disrupts the tight junctions. Thrombin increased the RBMVEC permeability assessed by a fluorescent flux assay. Taken together, our results indicate multiple mechanisms by which thrombin modulates the function of RBMVEC and may contribute to the blood-brain barrier dysfunction.

Keywords: Blood-brain barrier; Calcium signaling; Cerebral microvasculature; Protease-activated receptor 1.

Figure 1. Thrombin increases cytosolic Ca²⁺ concentration, [Ca²⁺]_i , in RBMVEC

A, Examples of fura-2 AM fluorescence ratio (F₃₄₀/F₃₈₀) in RBMVEC before (basal) and after treatment with thrombin (0.5 u/ml), or thrombin (0.5 u/ml) in cells pretreated with the PAR-1 antagonist, FR-171113 (1 μM). Cold colors represent low ratios and hot colors represent high ratio (scale 0–2). B, Representative examples of [Ca²⁺]_i increases produced by thrombin (0.1u/ml, 0.5 u/ml and 1 u/ml) and thrombin (0.5 u/ml) in the presence of FR-171113 (1 μM). Thrombin induced a fast and transient increase in [Ca²⁺]_i whose amplitude was dose-dependent; the response to thrombin was abolished by FR-171113. C, Comparison of the amplitude of [Ca²⁺]_i produced by each concentration of thrombin tested and by thrombin (0.5 u/ml) in the presence of FR-171113 (1 μM). P < 0.05 as compared to the response to the other concentrations of thrombin tested (*), or to the response produced by thrombin 0.5 u/ml (**).

Figure 2. Thrombin releases Ca²⁺ from endoplasmic reticulum

A, Examples of increases in [Ca²⁺]_i produced by thrombin in Ca²⁺-free HBSS, in the absence and presence of inhibitors of lysosomal and endoplasmic reticulum Ca²⁺ stores. Disruption of lysosomal Ca²⁺ stores with bafilomycin A1 (Baf, 1 μM, 1 h), did not affect the response to thrombin. Inhibition of ryanodine receptors with ryanodine (Ry, 1 μM, 1 h) reduced the response to thrombin, and blockade of IP₃ receptors with xestospongin C (XeC, 10 μM, 15 min) and 2-APB (100 μM, 15 min) abolished the response to thrombin. B, Comparison of the amplitude of Ca²⁺ responses produced by thrombin in each of the conditions mentioned. P < 0.05 as compared to the response to thrombin in Ca²⁺-free HBSS (*), or in the presence of ryanodine (**).

Figure 3. Thrombin increases nitric oxide (NO) production in RBMVEC

A, Examples of increases in DAF-FM diacetate fluorescence ratio (F/F₀), as a measure of NO level, produced by thrombin (0.5 u/ml) in the absence and presence of L-NAME and of PAR-1 antagonist, FR-171113 (1 μM). The effect of FR-171113 (1 μM) alone is also illustrated. B, Comparison of increases in Δ DAF-FM ratio in each of the conditions mentioned; L-NAME and FR-171113 abolished the response produced by thrombin. P < 0.05 as compared to the basal level (*), or to the response produced by thrombin (**).

Figure 4. Thrombin increases mitochondrial superoxide in RBMVEC

A, Examples of increases in MitoSOX Red fluorescence ratio (F/F0), as a measure of mitochondrial superoxide produced by thrombin (0.5 u/ml) in the absence and presence of the PAR-1 antagonist, FR-171113 (1 μM) or by FR-171113 (1 μM) alone. B, Comparison of increases in Δ MitoSOX Red fluorescence ratio produced by thrombin alone or in the presence of FR-171113. The PAR-1 antagonist abolished the response produced by thrombin. P < 0.05 as compared to the response produced by thrombin (*). P < 0.05 as compared to the basal level (*), or to the response produced by thrombin (**).

Figure 5. Thrombin increases cytosolic ROS in RBMVEC

A, Examples of increases in CM-D₂-DCFDA fluorescence ratio (F/F0), as a measure of ROS level, produced by thrombin (0.5 u/ml), FR-171113 (1μM) and thrombin in the presence of FR-171113(1 μM). B, Comparison of increases in Δ CM-D₂-DCFDA ratio produced by thrombin alone, FR-171113 alone or thrombin in the presence of FR-171113. The PAR-1 antagonist while did not have a significant effect by itself, abolished the response produced by thrombin. P < 0.05 as compared to the basal level (*), or to the response produced by thrombin (**).

Figure 6. Morphological changes induced by thrombin in RBMVEC

A, Distribution of F-actin (red), a component of cytoskeleton, and ZO-1 (green), a component of tight junctions, in RBMVEC in control cells, cells treated with thrombin (0.5 u/ml) or thrombin (0.5 u/ml) and FR-171113 (1 μM). Treatment with thrombin increased F-actin stress fiber formation, produced a reduction in ZO-1 staining, indicating cytoskeletal rearrangement and disruption of tight junctions; in addition, intercellular gaps, indicated by arrows, became visible in the endothelial monolayer. Pretreatment with the PAR1 antagonist prevented the changes produced by thrombin. Cellular nuclei were stained with DAPI. B, Thrombin increased the permeability of RBMVEC monolayers assessed using the FITC-dextran flux assay *P < 0.05 as compared to control.

Figure 7. Proposed mechanism of thrombin effects in RBMVEC

Thrombin acting on PAR1, produces Ca²⁺ release from endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptors (IP₃) receptors, and ryanodine receptors (RyR). Depletion of ER Ca²⁺ store leads to Ca²⁺ influx (store-operated Ca²⁺ entry, SOCE). The increase in [Ca²⁺]_i promotes NO formation, increase mitochondrion-derived superoxide (mROS) and cytosolic ROS (cytoROS) levels and determines cytoskeletal changes (increase in F-actin stress fibers formation) and disruption of tight junctions, leading to increased permeability and barrier dysfunction. Abbreviations: PIP₂ phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C.