Original article
A novel approach to prevent endothelial hyperpermeability: The Crataegus extract WS® 1442 targets the cAMP/Rap1 pathway

https://doi.org/10.1016/j.yjmcc.2011.10.020Get rights and content

Abstract

Endothelial hyperpermeability followed by edema formation is a hallmark of many severe disorders. Effective drugs directly targeting endothelial barrier function are widely lacking. We hypothesized that the hawthorn (Crataegus spp.) extract WS® 1442, a proven multi-component drug against moderate forms of heart failure, would prevent vascular leakage by affecting endothelial barrier-regulating systems. In vivo, WS® 1442 inhibited the histamine-evoked extravasation of FITC–dextran from mouse cremaster muscle venules. In cultured human endothelial cells, WS® 1442 blocked the thrombin-induced FITC–dextran permeability. By applying biochemical and microscopic techniques, we revealed that WS® 1442 abrogates detrimental effects of thrombin on adherens junctions (vascular endothelial-cadherin), the F-actin cytoskeleton, and the contractile apparatus (myosin light chain). Mechanistically, WS® 1442 inhibited the thrombin-induced rise of intracellular calcium (ratiometric measurement), followed by an inactivation of PKC and RhoA (pulldown assay). Moreover, WS® 1442 increased endothelial cAMP levels (ELISA), which consequently activated PKA and Rap1 (pulldown assay). Utilizing pharmacological inhibitors or siRNA, we found that PKA is not involved in barrier protection, whereas Epac1, Rap1, and Rac1 play a crucial role in the WS® 1442-induced activation of cortactin, which triggers a strong cortical actin rearrangement. In summary, WS® 1442 effectively protects against endothelial barrier dysfunction in vitro and in vivo. It specifically interacts with endothelial permeability-regulating systems by blocking the Ca2 +/PKC/RhoA and activating the cAMP/Epac1/Rap1 pathway. As a proven safe herbal drug, WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases. This in-depth mechanistic work contributes to a better acceptance of this herbal remedy.

Highlights

► The Crataegus extract WS® 1442 protects against endothelial hyperpermeability in vivo and in vitro. ► WS® 1442 inhibits detrimental effects of thrombin on adherens junctions, the F-actin cytoskeleton, and the contractile apparatus. ► Mechanistically, WS® 1442 blocks the thrombin-induced barrier-disrupting calcium/PKC/RhoA signaling. ► Concurrently, WS® 1442 activates the barrier-stabilizing cAMP/Epac/Rap1 pathway. ► WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases.

Introduction

Endothelial hyperpermeability, i.e. a compromised endothelial barrier function, and the subsequent formation of edema are hallmarks of many severe disorders, such as atherosclerosis [1], asthma [2], sepsis [3], or heart failure [4]. In the last years, significant progress has been made in elucidating the mechanisms involved in endothelial barrier dysfunction. Focusing on paracellular permeability, barrier destabilization is described as the formation of minute interendothelial gaps [5]. These gaps are mainly regulated by subcellular systems that form adhesive cell–cell contacts (e.g. adherens junctions/VE-cadherin) and exhibit retraction forces (e.g. the contractile apparatus/actomyosin). The function of these systems has been extensively characterized and the signaling mechanisms governing their operating status have been analyzed in detail [5]. Nevertheless, this profound knowledge is as yet not reflected in the implementation of new drugs that counteract barrier dysfunction by tackling these signaling systems. The search for novel pharmacological approaches is an ongoing need [6].

We proposed that the herbal remedy WS® 1442 prevents endothelial hyperpermeability and investigated its action on barrier dysfunction in vitro and in vivo. WS® 1442, a standardized hawthorn (Crataegus spp.) special extract, is widely used for the treatment of congestive heart failure according to the New York Heart Association (NYHA) functional class II because of its beneficial direct actions on the heart [7], [8]. Its efficacy and safety have been verified in several double-blind randomized clinical trials [9], [10], [11]. Crataegus extracts are among the most frequently used herbal medicinal products world-wide. The acceptance of herbal remedies—whether they are approved/registered drugs or nutrition supplements—is often ideologically influenced and, moreover, their use is controversially discussed due to the extract's inherent multi-component feature (polypharmacology) and the frequently missing in-depth mechanistic analysis. We set out to address this issue by coming up with a profound explanation for the action of WS® 1442: We provide a comprehensive insight into the underlying mechanisms affected by this extract in the human endothelium.

Section snippets

Materials

The Crataegus special extract WS® 1442 was kindly provided by the company Dr. Willmar Schwabe GmbH & Co. KG (Karlsruhe, Germany). Thrombin and GGTI-298 were from Sigma-Aldrich (Taufkirchen, Germany), adenosine-3′,5′-monophosphorothioate triethylammonium salt Rp-isomer (cAMPS-Rp), 8-bromoadenosine-3′,5′-cyclic monophosphate sodium salt (8-Br-cAMP), and forskolin were from Biotrend (Cologne, Germany). 8-(4-Chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic mono-phosphate sodium salt

WS® 1442 protects endothelial barrier function in vivo and in vitro

The extravasation of the macromolecule FITC–dextran from postcapillary venules of the mouse cremaster muscle was monitored via intravital fluorescence microscopy. Histamine was used as permeability-increasing factor. WS® 1442 (100 μg, applied i.a. 30 min prior to histamine) clearly abrogated the histamine-induced dextran extravasation and maintained endothelial barrier function (Figs. 1A/B). In vitro, macromolecular permeability through an HMEC-1 monolayer was assessed. WS® 1442 (30 min

Discussion

In the present study, we introduce WS® 1442 as a novel endothelial barrier-protecting approach, both in vitro and in vivo. WS® 1442 was found to beneficially act on (1) adhesion junction stability (VE-cadherin morphology, internalization, Tyr731 phosphorylation), (2) actin stress fiber formation (F-actin distribution and content), and (3) endothelial contraction (phospho-myosin light chain distribution and content). Thus, WS® 1442 influences pivotal systems that tightly regulate endothelial

Conclusion

We have shown that the herbal drug WS® 1442 effectively protects against endothelial barrier dysfunction by its action on key determinants of endothelial permeability (adherens junctions, actin cytoskeleton, and contractile apparatus). Mechanistically, WS® 1442 inhibits the barrier-destabilizing calcium/PKC/Rho signaling and activates the barrier-stabilizing cAMP/Epac1/Rap1 pathway. Thus, our study highlights WS® 1442 as a novel valuable approach for preventing endothelial hyperpermeability and

Disclosures

None.

Acknowledgments

This work was supported by the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft) [FU 691/7-1 to RF and AMV]. The extract WS® 1442 was kindly provided by the company Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany. MF, EAW, PB, and HA performed the research and analyzed the data. GJ contributed essential reagents. FK, SZ, AMV, and RF designed the study. RF wrote the paper.

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