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Mucopolysaccharide polysulfate promotes microvascular stabilization and barrier integrity of dermal microvascular endothelial cells via activation of the angiopoietin-1/Tie2 pathway
1 Shiori Fujiwara-Sumiyoshi and Yuhki Ueda are equal first authors.
Shiori Fujiwara-Sumiyoshi
Correspondence
Corresponding author at: Drug Development Research Laboratories, Kyoto R&D Center, Maruho Co., Ltd., 93 Chudoji Awatacho, Shimogyo-ku, Kyoto, 600-8815, Japan.
MPS increased the production of Ang-1 by HPC and the production of PDGF-BB by HDMEC.
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MPS directly activates the Ang-1/Tie2 signaling pathway by increasing the expression and phosphorylation of Tie2 in HDMEC.
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MPS enhanced the vascular barrier function in HDMEC by increasing the expression of claudin-5.
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The intradermal injection of MPS prevented VEGF-induced increase in vascular permeability in mouse skin.
Abstract
Background
Mucopolysaccharide polysulfate (MPS) is a heparinoid and MPS-containing formulations are widely used as moisturizers for dry skin and to treat peripheral vascular insufficiency. Although MPS has therapeutic effects in skin diseases with microvascular abnormalities, the effects of MPS on microvascular function remain incompletely understood.
Objective
The aim of this study was to evaluate the functional activities of MPS on human pericytes (HPC) and human dermal microvascular endothelial cells (HDMEC) in vitro, and on microvascular permeability of the skin.
Methods
The protein expression of angiopoietin (Ang)-1 in HPC, and platelet-derived growth factor-BB (PDGF-BB) and phosphorylated tyrosine-protein kinase receptor 2 (Tie2) in HDMEC were measured in the presence or absence of MPS. The vascular barrier was evaluated by the expressions of claudin-5 and vascular endothelial (VE)-cadherin, and transendothelial electrical resistance (TEER).
Results
In HPC, MPS dose-dependently enhanced Ang-1 secretion, which activated Tie2 in HDMEC. In HDMEC, MPS significantly increased the production of PDGF-BB, which is important for the recruitment of HPC to the vascular endothelium, and significantly increased the phosphorylation of Tie2, which results in the activation of the Ang-1/Tie2 signaling . MPS significantly increased the expression of tight junction protein claudin-5 and TEER in the HDMEC. Moreover, the intradermal injection of MPS prevented vascular endothelial growth factor-induced increase in vascular permeability in mouse skin.
Conclusion
We found that MPS promoted microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. These results suggest that MPS might improve microvascular abnormalities in various diseases accompanied by disturbances in Ang-1/Tie2 signaling.
Skin homeostasis is maintained by supplying the dermis and epidermis with fluid, nutrients, and oxygen via dermal microvessels. Previous studies reported that aging of the skin disturbs the vascular architecture by decreasing dermal capillary density and function, which leads to vascular hyperpermeability [
]. Dermal microvessels comprise an endothelial cell layer surrounded by pericytes; however, the number of pericyte-covered dermal microvessels is reduced with age [
]. Moreover, microvascular abnormalities have been described in acute and chronic diseases such as ultraviolet B-induced erythema, chronic urticaria, angioedema, atopic dermatitis, psoriasis, systemic sclerosis, and diabetic retinopathy. The alteration of microvessels is thought to contribute to the pathogenesis of these diseases [
]. Endothelial cells release platelet-derived growth factor BB (PDGF-BB), which binds to its receptor PDGFR-β on pericytes to recruit them to the vascular endothelium [
]. Pericytes produce angiopoietin (Ang)-1, which stabilizes the microvasculature by activating the receptor tyrosine-protein kinase receptor 2 (Tie2) on endothelial cells [
Stable expression of angiopoietin-1 and other markers by cultured pericytes: phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo.
]. Overexpressing Ang-1 in dermal microvessels or the systemic adenoviral production of Ang-1 in mice markedly reduced the vascular leakage induced by the intradermal injection of vascular endothelial growth factor (VEGF) [
]. TJs comprise several adhesive proteins including occludin, claudins, and junctional adhesion molecules. In the TJs of human dermal microvascular endothelial cells, claudin-5, but not occludin or other claudins, was mainly expressed and formed the intercellular barriers in dermal microvessels [
]. An engineered Ang-1 variant attenuated the hyperglycemia-induced reduced expressions of claudin-5, occludin, zonula occludens (ZO)-1, and vascular endothelial (VE)-cadherin in human retinal microvascular endothelial cells (HRMEC), which results in the recovery of HRMEC barrier function [
]. Thus, Ang-1 up-regulates the expressions of the TJ proteins claudin-5, occludin, and ZO-1 as well as the AJ protein VE-cadherin in HRMEC.
Disturbance of the Ang-1/Tie2 signaling pathway is thought to contribute to the pathogenesis of diseases such as systemic sclerosis, diabetic retinopathy, age-related macular degeneration, and carcinoma [
]. A previous report showed that compared with healthy controls, the dermal microvessels of patients with systemic sclerosis abundantly expressed Ang-2, but not Ang-1, and Tie2 expression on microvessels was markedly decreased [
]. Thus, abnormalities of the Ang-1/Tie2 signaling pathway cause microvascular dysfunction, leading to Raynaud’s phenomenon, capillary collapse, and tissue ischemia.
Hirudoid® cream/soft ointment/lotion/foam is a prescription drug in Japan and is usually used topically to treat patients with thrombophlebitis, pain, and inflammatory disease accompanied by vascular insufficiency, chilblains, hypertrophic scars, keloid, keratodermia tylodes palmaris progressiva, and asteatosis. The active ingredient of Hirudoid® is a heparinoid, mucopolysaccharide polysulfate (MPS), which contains polysulfated chondroitin sulfate (ChS), a sulfated glycosaminoglycan (GAG) containing repeating units of N-acetyl-D-galactosamine and D-glucuronic acid [
]. Conversely, non-sulfated GAGs, such as hyaluronic acid (HA), comprise repeating units of N-acetyl-D-glucosamine and D-glucuronic acid. Although the percutaneous application of MPS products has therapeutic effects in skin diseases with microvascular abnormalities, the specific effects of MPS on dermal microvascular integrity and function have not been fully elucidated. In this study, we evaluated the functional activities of MPS on human pericytes and human dermal microvascular endothelial cells (HDMEC) in vitro and on microvascular permeability in mouse skin. Furthermore, to clarify whether the effect of MPS on dermal microvascular function in vitro associate with its sulfation degree, we studied the effect of other GAGs such as ChS and HA as well.
2. Materials and methods
2.1 Test drugs and reference drugs
MPS and ChS were produced in-house by Maruho Co., Ltd. (Kyoto, Japan). HA (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan, Cat# 9004-61-9), Ang-1 and VEGF (R&D Systems, Inc., MN, USA, Cat# 923-AN-025, 493-MV-005) were obtained commercially.
2.2 Cell culture
Human placenta-derived pericytes (HPC, Cat# C-12980) (PromoCell GmbH, Heidelberg, Germany) were seeded in Pericyte Growth Medium with Pericyte Growth Medium Supplement Mix (PromoCell GmbH, Cat# C-28041) and cultured at 37 °C in 5% CO2 and 95% air. HDMEC (PromoCell GmbH, Cat# C-12212) were seeded in Endothelial Cell Growth Medium MV2 with Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell GmbH, Cat# C-22121) and cultured using dishes coated with collagen I (Corning Incorporated, NY, USA, Cat# 354236) at 37 °C in 5% CO2 and 95% air. Both cell types were used within four passages for experiments.
2.3 Animals and ethics
Female 6-week-old hairless mice (Hos: HR-1) were purchased from Hoshino Laboratory Animals (Ibaraki, Japan) and housed in a specific pathogen-free animal facility under conditions of controlled temperature (23 ± 3 °C), relative humidity (50 ± 20 %), and lighting (lights for 12 h, 7:00 to 19:00). They were given a laboratory diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and ultraviolet sterile water ad libitum. All animal experimental procedures were approved by the Ethics Committee for Animal Experiments of Maruho Co., Ltd., and conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals at Maruho Co., Ltd.
2.4 Protein extraction and immunoblotting
At the end of cell culture, the cells were harvested and sonicated with radioimmunoprecipitation assay buffer (Nacalai Tesque, Inc., Kyoto, Japan, Cat# 08714-04) containing phosphatase inhibitors (Thermo Fisher Scientific Inc., MA, USA, Cat# 78440). After protein concentrations in the cell lysates were adjusted to the same level, the samples were subjected to SDS-PAGE using a 5%–20% polyacrylamide gel. Proteins were transferred to a PVDF membrane using the Transfer-Blot Turbo Transfer Pack (Bio-Rad Laboratories, Inc., CA, USA, Cat# 1704156), and then the membrane was blocked with Blocking One-P (Nacalai Tesque, Cat# 05999-84) or 5% diluted skim milk solution. Subsequently, the membranes were incubated at 4 °C overnight with the following antibodies: anti-phosphorylated Tie2 antibody, anti-β-actin antibody (Cell Signaling Technology, Inc., MA, USA, Cat# 4221, 3700), anti-Tie2 antibody (Sigma-Aldrich Co. LLC, Cat# 05-584), anti-claudin-5 antibody, or anti-VE-cadherin antibody (Thermo Fisher Scientific Inc, Cat# 35-2500, 14-1449-82). Then, the secondary antibody, anti-rabbit IgG HRP-linked antibody or anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, Inc, Cat# 7074, 7076) was added and the membrane was incubated at room temperature for 1 h. Protein bands were detected by chemiluminescence.
2.5 ELISA and Bio-Plex
Ang-1 and Ang-2 in culture supernatants were measured using the Human Angiopoietin-1 Quantikine ELISA Kit and the Human Angiopoietin-2 Quantikine ELISA Kit (R&D Systems, Inc, Cat# DANG10, DANG20), respectively. PDGF-BB and VEGF-A were measured using Bio-Plex Human Cytokine Assay Kits (Bio-Rad Laboratories, Inc.).
2.6 Transendothelial electrical resistance (TEER)
An Electrical Cell-Substrate Impedance Sensor (ECIS) ZΘ System (Applied Biophysics, NY, USA) was used for barrier function measurements as previously described [
The importance of multifrequency impedance sensing of endothelial barrier formation using ECIS technology for the generation of a strong and durable paracellular barrier.
Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.
]. HDMEC were seeded into each well of a 96W20idf ECIS plate pre-coated with collagen I (Corning Incorporated, Cat# 354236) and 10 mM l-cysteine. TEER was measured at 4 kHz. Cells were allowed to attach to the well overnight and form a confluent monolayer. The medium was changed 24 h after cell seeding. At 72 h after cell seeding, MPS or 500 ng/mL Ang-1 (positive control), was added. The ratio of change of the TEER value was calculated by dividing the TEER value at each measurement point (84, 96, 108, and 120 h after cell seeding) by the TEER value at 72 h.
2.7 Intradermal miles assay
To investigate the effects of MPS on vascular permeability, the intradermal Miles assay was performed as described previously [
]. First, 100 μL of physiological saline solution containing 1% Evans blue dye (Nacalai Tesque, Inc, Cat# 09158-74) was injected into the tail vein of hairless mice. Thirty minutes later, 20 μL of PBS (negative control) (Sigma-Aldrich Co. LLC, Cat# D5796-500ML), VEGF, a mixture of VEGF and Ang-1, or a mixture of VEGF and MPS was administered intradermally to the back of mice. Twenty minutes later, skin at the administration site was collected. Deionized formamide (Gel Company Inc., CA, USA, Cat# DFM-025) was added to the collected skin, and extraction was performed at 55 °C overnight. The amount of leaked Evans blue dye was calculated using the absorbance (620 nm) of the formamide extract.
2.8 Statistical analysis
Data are shown as the mean ± standard error (S.E.). Statistical analysis was performed using EXSUS software (Ver.8.1) (CAC EXICARE Corporation, Tokyo, Japan) with a statistical significance level of less than 5% (P < 0.05, two-tailed test). Student’s t-test or Aspin-Welch’s t-test were used to test differences between two groups. Dunnett’s multiple comparison test was performed for differences between more than three groups.
3. Results
3.1 The effect of MPS on the production of Ang-1 by HPC
To evaluate the effect of MPS on the production of Ang-1, HPC were cultured for 48 h in the presence of MPS, ChS, or HA. Compared with growth medium alone, MPS (>1 μg/mL) significantly increased the production of Ang-1 by HPC, which was concentration dependent (Fig. 1a). However, ChS or HA, which are sulfated or non-sulfated GAGs, did not affect the production of Ang-1, even at 10 μg/mL (Fig. 1b).
Fig. 1Effects of MPS on the protein expression of Ang-1 in HPC, and Ang-2 and PDGF-BB in HDMEC.
HPC were cultured in the presence or absence of MPS (a), ChS, or HA (b) for 48 h and human Ang-1 levels in the culture medium were measured by ELISA.
HDMECs were cultured in the presence or absence of MPS (c and e), ChS, or HA (d and f) for 48 h. Human Ang-2 and PDGF-BB levels in the culture medium were measured by ELISA or Bio-Plex, respectively. Each column represents the mean ± S.E. (n = 3 or 4). *: P < 0.05, **: P < 0.01 by Dunnett’s multiple comparison test compared with growth medium alone (–).
Furthermore, we examined the effect of MPS, ChS and HA on the production of Ang-2 by HDMEC. However, all of them had no significant effect on the Ang-2 production (Fig. 1c and d).
3.2 The effect of MPS on the production of PDGF-BB by HDMEC
The effect of MPS on the production of PDGF-BB, which is important for the recruitment and migration of HPC to the vascular endothelium, was investigated. The amount of PDGF-BB produced by HDMEC cultured with MPS at 10 μg/mL was significantly increased compared with HDMEC cultured with growth medium alone (Fig. 1e). However, HA or ChS had no clear effect on the production of PDGF-BB (Fig. 1f). Thus, MPS increased the production of Ang-1 by HPC and the production of PDGF-BB by HDMEC.
3.3 The effect of MPS on the expression and phosphorylation of Tie2 in HDMEC
Next, we used HDMEC to study the effect of MPS on the expression and phosphorylation of Tie2, the receptor for Ang-1. The results showed that MPS at 0.1 μg/mL and 1 μg/mL increased the amount of Tie2 (Fig. 2a) and significantly increased the phosphorylation of Tie2 in HDMEC compared with the medium alone (Fig. 2b and c). Furthermore, MPS and Ang-1 slightly increased the relative ratio of phosphorylated Tie2 to total Tie2 protein (Fig. 2d). These results suggested that MPS directly activates the Ang-1/Tie2 signaling pathway by increasing the expression and phosphorylation of Tie2 in HDMEC.
Fig. 2Effects of MPS on the protein expression of Tie2 and phosphorylated Tie2 in HDMEC.
HDMEC were cultured in the presence or absence of MPS or recombinant Ang-1 protein (500 ng/mL) for 10 min. The expressions of Tie2 and phosphorylated Tie2 were detected by western blotting (c), and the relative expressions of Tie2 (a) and phosphorylated Tie2 (b) to β-actin used as a loading control were determined by the image analysis of western blotting. The relative ratio of phosphorylated Tie2 to total Tie2 protein was determined by densitometric analysis of western blotting (d). Each column represents the mean ± S.E. (n = 4). *: P < 0.05 by Dunnett’s multiple comparison test compared with growth medium alone (–).
3.4 The effect of MPS on the TEER in HDMEC monolayers
Because the activation of the Ang-1/Tie2 signaling pathway plays an important role in the barrier function of HDMEC, the effect of MPS on the barrier function of HDMEC monolayers was investigated over time using TEER as an indicator of barrier function. TEER reached a steady state at 72 h after HDMEC seeding (data not shown). With the addition of Ang-1 (500 ng/mL), the ratio of change in the TEER of HDMEC was significantly increased at 108 and 120 h after the start of HDMEC seeding compared with growth medium alone. Similarly, MPS at 1 and 10 μg/mL significantly increased the TEER of HDMEC in a concentration-dependent manner (Fig. 3a). At 120 h after the start of HDMEC seeding, MPS at 1 and 10 μg/mL increased TEER levels in HDMEC compared with Ang-1 (Fig. 3b); however, no effects of HA or ChS were observed on the TEER of HDMEC (Supplementary data 1).
Fig. 3Effect of MPS on the TEER in HDMEC monolayers.
(a) TEER was measured for 120 h post-HDMEC seeding. MPS (0.1, 1 or 10 μg/mL) or Ang-1 (500 ng/mL) was added at 72 h post-HDMEC seeding (black line = Non-treated, white line = Ang-1, red and orange line = the concentration of each MPS). Relative resistance of each point represents the mean ± S.E. (n = 3). (b) Concentration-dependent effects of MPS or Ang-1 on TEER at 120 h post-HDMEC seeding, that is, after 48 h-treatment with MPS or Ang-1. Each column represents the mean ± S.E. (n = 3). For MPS, **: P < 0.01 by Dunnett’s multiple comparison test compared with growth medium alone (Medium). For Ang-1, *: P < 0.05, **: P < 0.01 by F-test followed by Student’s t-test compared with growth medium alone (Medium).
3.5 The effects of MPS on claudin-5 and VE-cadherin in HDMEC
To clarify the mechanisms of vascular barrier function enhancement by MPS, we measured the expressions of claudin-5, a TJ protein, and VE-cadherin, an AJ protein, both of which constitute the vascular barrier. MPS at 1 and 10 μg/mL significantly increased the amount of claudin-5 expressed in HDMEC in a concentration-dependent manner (Fig. 4a and b) but not ChS or HA (Supplementary data 2). There was no effect of MPS (Fig. 4c), ChS, or HA (Supplementary data 2) on VE-cadherin expression. These results indicate MPS enhanced the vascular barrier function in HDMEC by increasing the expression of claudin-5.
Fig. 4Effects of MPS on the protein expressions of claudin-5 and VE-cadherin in HDMEC.
HDMEC were cultured in the presence or absence of MPS for 48 h. (a) Expressions of claudin-5 and VE-cadherin were detected by immunoblotting. β-actin was used as a loading control. The effects of MPS (0.1, 1 and 10 μg/mL) on the relative expressions of claudin-5 (b) and VE-cadherin (c) were calculated by the signal intensity of β-actin protein bands. Each column represents the mean ± S.E. (n = 4). *: P < 0.05, **: P < 0.01 by Dunnett’s multiple comparison test compared with growth medium alone (–).
3.6 The effect of MPS on vascular permeability in vivo
Because our findings in this study suggested that MPS strengthens vascular barrier function, we evaluated the effect of MPS on vascular permeability induced by VEGF in vivo using the Miles assay.
Compared with the intradermal administration of PBS, VEGF (50 ng/site) significantly increased the amount of leaked dye, an index of vascular permeability, in mice (Fig. 5). The amount of leaked dye induced by Ang-1 (500 ng/site) mixed with VEGF tended to be smaller than that induced by VEGF alone (Fig. 5a). Moreover, MPS (10 and 20 μg/site) significantly and dose-dependently suppressed the VEGF-induced increase in vascular permeability in mice (Fig. 5a). Image data show that 20 μg/site of MPS markedly suppressed dye leakage in the skin induced by VEGF (Fig. 5b). These findings indicated that MPS stabilized the vascular structure and had a stronger suppressive effect on vascular leakage compared with Ang-1.
Fig. 5Inhibitory effect of MPS on VEGF-induced vascular permeabilityin vivo.
Effect of MPS on vascular permeability was assessed using the Miles assay. Mice were treated with an intravenous injection of 100 μL 1% Evans blue solution into the tail vein 30 min prior to the intradermal injection of PBS control, VEGF alone (50 ng/site), VEGF (50 ng/site) + MPS (2.5, 5, 10, or 20 μg/site), or VEGF (50 ng/site) + Ang-1 (500 ng/site). (a) Evans blue dye extravasation to the adjacent tissue was quantified (n = 16 per group, 2 tissues were sampled from 8 mice; 1 sample per administration site). Each column represents the mean ± S.E. (b) The images show the back of mice under each condition. **: P < 0.01 by Dunnett’s multiple comparison test compared with VEGF (50 ng/site) (VEGF+, MPS-), ††: P < 0.01 by F-test followed by Aspin-Welch’s t-test, compared with PBS (VEGF-, MPS-).
In the present study, we demonstrated that MPS increased the production of Ang-1 in HPC (Fig. 1a) and PDGF-BB in HDMEC (Fig. 1e) in a dose-dependent manner. Furthermore, MPS increased the expression of Tie2 in HDMEC, which results in the increased phosphorylation of Tie2 (Fig. 2). Conversely, the other mucopolysaccharides ChS and HA did not affect Ang-1 expression in HPC (Fig. 1b) or PDGF-BB expression in HDMEC (Fig. 1f). These results suggest that MPS potentiates interactions between dermal microvascular endothelial cells and pericytes to enhance microvascular stabilization via activation of the Ang-1/Tie2 signaling pathway. A previous study reported that unfractionated heparin, a sulfated GAG similar to ChS, suppressed the lipopolysaccharide-induced disruption of human pulmonary microvascular endothelial barrier function by upregulating Ang-1 and Tie2 mRNAs, and ZO-1 protein, which is associated with the cytoskeleton and provides junctional stability possibly by activating the Ang-1/Tie2 signaling pathway [
]. Thus, the effects of GAGs on activation of the Ang-1/Tie2 pathway may vary according to the type of GAG or cell. We speculate that the polysulfation of mucopolysaccharides is important for activation of the Ang-1/Tie2 signaling pathway in HDMEC because MPS, like heparin, has a higher content of sulfation motifs compared with HA and ChS. The negatively-charged sulfated motifs in MPS promote electrostatic interactions with various proteins including ligands and cell surface receptors. However, the different mechanism of action between MPS and other GAGs remains to be defined.
We also showed that MPS (Fig. 3), but not HA and ChS (Supplementary data 1), dose-dependently potentiated the TEER of HDMEC monolayers, an index of endothelial barrier tightness. Furthermore, treatment of HDMEC with MPS dose-dependently and significantly enhanced the expression of claudin-5. Conversely, MPS did not affect the expression of VE-cadherin in HDMEC (Fig. 4). Recent reports indicated that the expression levels of claudin-5, but not VE-cadherin, in HDMEC were directly proportional to TEER and barrier strength, whereas the extent of claudin-5 knockdown by shRNAs correlated with the decrease in TEER [
]. Thus, claudin-5 is an essential TJ protein that maintains HDMEC barrier function. In addition, activation of Ang-1/Tie2 signaling promoted lymphatic integrity by increasing the expressions of the tight junction proteins claudin-5 and ZO-1 [
]. These findings suggest that the MPS-induced enhancement of claudin-5 expression and TEER in HDMEC is associated with the activation of the Ang-1/Tie2 signaling pathway.
To elucidate whether MPS affects the Ang-1/Tie-2 signaling-associated response in vivo, we investigated the effect of MPS on VEGF-induced vascular leakage in hairless mice. MPS dose-dependently inhibited the VEGF-induced vascular leakage of Evans blue dye in the skin of mice (Fig. 5). Previous reports showed that VEGF-induced acute vascular leakage was inhibited by a combination of Ang-1 and VEGF, which activated Tie2 [
]. Ang-1 has been reported to suppress the disruption of the endothelial barrier by preventing Src activation, which is a critical component of the VEGF pathway that induces vascular permeability [
]. The activation of the Src-dependent intracellular signaling pathway leads to the phosphorylation and internalization of VE-cadherin, and eventually to the disassembly of cell-cell junctions, which enhances vascular permeability [
]. Therefore, the inhibitory effect of MPS on VEGF-induced acute vascular leakage might be exerted by preventing the activation of Src-dependent intracellular signaling via Ang-1/Tie2 signaling as well as Ang-1.
Whereas there is a limitation for this in vivo study in which the inhibitory effect of MPS on VEGF-induced acute vascular leakage has not been proved to depend on Ang-1/Tie2 signaling, we need to elucidate whether MPS activates Ang-1/Tie2 signaling in vivo, leading to the increase in claudin-5 expression related to microvascular barrier function. Moreover, to clarify the involvement of the Ang-1/Tie2 signaling pathway in the in vivo action of MPS, it would be necessary to analyze the actions of MPS on microvascular barrier functional disorder in not only the acute model used in this study but also a chronic model such as systemic sclerosis model [
]. In future studies, we will resolve the above problems.
In conclusion, we found that MPS promoted dermal microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. This suggests that MPS-containing products might be useful to treat age-related alterations of microvessels and microvascular abnormalities in diseases such as systemic sclerosis, diabetic retinopathy, and carcinoma accompanied by disturbances of the Ang-1/Tie2 signaling pathway.
Funding sources
This study was supported by Maruho Co., Ltd.
Declaration of Competing Interest
The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. This study was funded by Maruho Co., Ltd.
Acknowledgements
We thank Yusuke Kumagai and Azusa Miwa (Maruho. Co., Ltd) for their technical assistance. This study was fully supported by Maruho. Co., Ltd.
Appendix A. Supplementary data
The following are Supplementary data to this article:
Stable expression of angiopoietin-1 and other markers by cultured pericytes: phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo.
The importance of multifrequency impedance sensing of endothelial barrier formation using ECIS technology for the generation of a strong and durable paracellular barrier.
Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.
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