Rk1, a Ginsenoside, Is a New Blocker of Vascular Leakage Acting through Actin Structure Remodeling

<div><p>Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage.</p> </div

Rk1, but not Rg1 or Rb1, inhibits VEGF-induced retinal endothelial permeability.

<p>(<b>A</b>) HRECs were plated onto a Transwellfilter. After reaching confluence, HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 1 h. HRECs were cultured with 50 µl (0.8 µCi/ml) of [³H] sucrose (1 µCi/µl) added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min with a liquid scintillation counter. (<b>B</b>) The structure of gensenosides. (<b>C</b>) HRECs were plated onto a Transwell filter. After reaching confluence, HRECs were pretreated for 40 min with or without Rb1, Rg1, and Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 1 h. HRECs were cultured with 50 µl (0.8 µCi/ml) of [³H] sucrose (1 µCi/µl) added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min with a liquid scintillation counter. All data are presented as the mean ± S.E. from three different experiments performed in duplicate. **, <i>P</i> < 0.01 <i>vs</i>. VEGF alone.</p

AGE induces retinal endothelial permeability <i>via</i> expression of VEGF.

<p>(<b>A</b>, <b>B</b>) HRECs were stimulated with 5 µM AGE-BSA for the indicated times. Total mRNAs were isolated and RT-PCR was performed with specific primers for human VEGF. GAPDH served as an internal control. (B) Ratio of VEGF mRNA to GAPDH mRNA. (<b>C</b>) HRECs were plated onto a Transwell filter. After reaching confluence, HRECs were pretreated for 30 min with or without 1C11 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h. HRECs were cultured with 50 µl (0.8 µCi/ml) of [³H] sucrose (1 µCi/µl) added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min with a liquid scintillation counter. Data are the mean ± S.E. **, <i>p</i> < 0.01 <i>vs</i>. AGE alone. (<b>D</b>) Confluent HRECs were incubated for 30 min with or without 10 µg/ml 1C11 and stimulated with 5 µM AGE-BSA for 10 h. Translocations of TJ proteins were assessed, as described in “Experimental Procedures”. The Triton X-100-insoluble and soluble fractions were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with anti-ZO-1, anti-ZO-2, and anti-occludin antibodies. Blots are representative of three independent experiments. (<b>E</b>) Densitometric analyses are presented as the relative ratio of tight junction proteins in membrane and cytosol and quantified with NIH ImageJ software. Data are the mean ± S.E. **, <i>p</i> < 0.01 <i>vs</i>. control. ^##, <i>p</i> < 0.01 <i>vs</i>. AGE alone.</p

Rk1 induces cortactin phosphorylation.

<p>(<b>A</b>, <b>B</b>) Confluent HRECs were treated with Rk1 (0.1, 1, 5, or 10 µg/ml) for the indicated times. Cell lysates were subject to affinity precipitation using anti-cortactin antibody. Total cortactin indicates total amount of active and inactive cortactin in the HRECs. Western blot was performed using an antibody specific for phospho-tyrosine (PY) and cortactin (Cort). (<b>C</b>) Confluent HRECs were treated with Rk1 (10 µg/ml) for 30 min. The cells were then fixed and stained with Rhodamine Phalloidin and anti-cortactin antibody. Arrows indicate cortactin. (<b>D</b>) Confluent HRECs were pretreated for 1 h with or without VEGF (20 ng/ml) prior to stimulation with Rk1 (0.5, 1, 5, or 10 µg/ml) for 1 h. The cells were then fixed and processed for immunofluorescence staining using Rhodamine Phalloidin. The samples were viewed by fluorescence microscopy.</p

Rk1 inhibits vascular leakages of retina by VEGF.

<p>(<b>A</b>) Mice were injected with 50 ng VEGF alone or in combination with 15 µg Rk1 into the vitreous of one eye and with vehicle in the contra lateral eye. At indicated times after injection, 150µl FITC-Dextran (30 mg/ml in sterile PBS) was injected into the left ventricle. The retinas were then viewed using fluorescence microscopy. Yellow arrowheads indicate region of vascular permeability. All data are presented as the mean ± S.E. from three different experiments performed in duplicate. (<b>B</b>) Quantification of FITC-dextran leakage by a multi-gauge software (Fuji). Data are the mean ± S.E.</p

Rk1 inhibits thrombin or histamine-induced retinal endothelial permeability.

<p>(<b>A</b>) HRECs were plated onto a Transwellfilter. After reaching confluence, HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with Thrombin (10 units/ml) or Histamine (100 µM) for 1 h. [³H] sucrose permeability assay was performed. Data are the mean ± S.E. **, <i>P</i> < 0.01 <i>vs</i>. Thrombin or Histamine alone. (<b><i>B</i></b>) Confluent HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with Thrombin (10 units/ml) or Histamine (100 µM) for 1 h. The cells were then fixed and stained with anti-ZO-1 antibody. Arrowheads indicate disruption of ZO-1 proteins. (<b>C</b>) ZO-1 protein intensity at cell borders was analyzed and quantified with NIH ImageJ software. Data are the mean ± S.E. **, <i>P</i> < 0.01 <i>vs</i>. Control. ^##, <i>p</i> < 0.01 <i>vs</i>. Thrombin or Histamine alone.</p

Rk1 promotes cortical actin ring formation in HRECs.

<p>(<b>A</b>) Confluent HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with 20 ng/ml VEGF (1 h) or 5 µM AGE-BSA (10 h). (<b>B</b>) Confluent HRECs were treated with Rk1 (10 µg/ml) for the indicated times. The cells were then fixed and processed for immunofluorescence staining using Rhodamine Phalloidin. Arrowheads show junctional actin, arrows point to thin bundles. (<b>C</b>) Cortical actin intensity was analyzed and quantified with NIH ImageJ software. (<b>D</b>) Global levels of G-actin and F-actin pools were quantified by immunofluorescence in a time course after treatment of Rk1 (10 µg/ml). HRECs were stained with DNAse1 (G-actin) and phalloidin (F-actin) and the whole optical field quantified for each fluorophore. Values are expressed as percentage of total actin. Data are the mean ± S.E.</p

Rk1 effectively inhibits localization of TJ proteins disrupted by VEGF treatment.

<p>(<b>A</b>) Confluent HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 1 h. The cells were then fixed and stained with anti-ZO-1, anti-ZO-2, and anti-occludin antibodies. Arrowheads indicate disruption of tight junction proteins. (<b>B</b>) Tight junction protein intensity at cell borders was analyzed and quantified with NIH ImageJ software. Data are the mean ± S.E.</p

The effect of Rk1 on retinal vascular leakage of a DM model.

<p>(<b>A</b>) Diabetic mice were injected with Rk1 (1, 5, 10, 15µg) into the vitreous of one eye and with vehicle in the contra lateral eye. At 24 h after injection, 150 µl FITC-Dextran (30 mg/ml in sterile PBS) was injected into the left ventricle. The retinas were then viewed by fluorescence microscopy. Yellow arrowheads indicate region of vascular permeability. (<b>B</b>) Quantification of FITC-dextran leakage by a multi-gauge software (Fuji). Data are the mean ± S.E.</p

Rk1 induces phosphorylation of MLC and co-localization of pMLC with the cortical actin ring.

<p>(<b>A</b>) Confluent HRECs were treated with Rk1 (10 µg/ml) for 1h. The cells were then fixed and processed for immunofluorescence staining using Rhodamine Phalloidin and anti-pMLC (S19) antibodies. The samples were viewed by fluorescence microscopy. Arrowheads show junctional actin, arrows point to thin bundles and localization of pMLC. (<b>B</b>) Co-localization intensity of pMLC and junctional actin was analyzed and quantified with NIH ImageJ software. (<b>C</b>, <b>D</b>) Confluent HRECs were treated with Rk1 (10 µg/ml) for indicated times and stimulated with Rk1 (0.1, 1, 5, or 10 µg/ml) for 15 min. Western blot was performed using an antibody specific for phospho-MLC (S19). Blot was reprobed with an anti-actin antibody to verify equal loading of protein in each. Densitometric analyses are presented as the relative ratio of p-MLC and Actin and quantified with NIH ImageJ software. Data are the mean ± S.E. **, <i>p</i> < 0.01 <i>vs</i>. Control.</p