Edited by: Per Hellstrand, Lund University, Sweden
Reviewed by: Holger Nilsson, University of Gothenburg, Sweden; Stephanie Lehoux, McGill University, Canada
This article was submitted to Vascular Physiology, a section of the journal Frontiers in Physiology
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Development of spider veins is caused by the remodeling of veins located in the upper dermis and promoted by risk factors such as obesity or pregnancy that chronically increase venous pressure. We have repeatedly shown that the pressure-induced increase in biomechanical wall stress is sufficient to evoke the formation of enlarged corkscrew-like superficial veins in mice. Subsequent experimental approaches revealed that interference with endothelial- and/or smooth muscle cell (SMC) activation counteracts this remodeling process. Here, we investigate whether the herbal agent glycyrrhetinic acid (GA) is a suitable candidate for that purpose given its anti-proliferative as well as anti-oxidative properties. While basic abilities of cultured venous SMCs such as migration and proliferation were not influenced by GA, it inhibited proliferation but not angiogenic sprouting of human venous endothelial cells (ECs). Further analyses of biomechanically stimulated ECs revealed that GA inhibits the DNA binding capacity of the mechanosensitive transcription factor activator protein-1 (AP-1) which, however, had only a minor impact on the endothelial transcriptome. Nevertheless, by decreasing gelatinase activity in ECs or mouse veins exposed to biomechanical stress, GA diminished a crucial cellular response in the context of venous remodeling. In line with the observed inhibitory effects, local transdermal application of GA attenuated pressure-mediated enlargement of veins in the mouse auricle. In summary, our data identifies GA as an inhibitor of EC proliferation, gelatinase activity and venous remodeling. It may thus have the capacity to attenuate spider vein formation and remodeling in humans.
Pathologic remodeling of veins often results in significant morbidity and discomfort for the patient while remodeling of smaller, i.e., superficial veins results in formation of spider veins or telangiectasias that often cause a cosmetic nuisance/discontent. Spider veins comprise dilated, intradermal vessels less than 1 mm in diameter (venous telangiectasias) or between 1 and 3 mm in diameter (reticular veins) and often resemble varicosities albeit on a much smaller scale (Partsch,
A number of risk factors have been associated with the development of spider veins, including prolonged sitting or standing, pregnancy, being overweight, wearing tight undergarments or clothes which suggest an underlying increase in the hydrostatic venous filling pressure associated with the deep veins in the lower extremities (Sisto et al.,
Here, we attempted to attenuate activation of venous endothelial and smooth muscle cells (SMCs) and enlargement of superficial veins by utilizing bioactive compounds frequently utilized in skin care products. From among them, we selected glycyrrhetinic acid (GA)—the predominant lipophilic bioactive compound extracted from the root of licorice—due to its broad range of beneficial properties comprising anti-inflammatory, anti-oxidative and anti-allergic effects(Chang et al.,
The anti-Ki67 antibody was obtained from abcam (Cambridge, UK; ab16667). The DQ-gelatin (EnzChek gelatinase assay kit) was obtained from ThermoFisher Scientific (Pittsburgh, PA, USA: E12055).
Isolation of human umbilical vein endothelial and SMCs (HUVEC/HUVSMC) was approved from the Local Ethical Committee (document number 336/2005, Heidelberg Germany) and conformed to the principles outlined in the Declaration of World Medical Association declaration of Helsinki (
HUVEC: Umbilical veins were flushed with Hank's buffer solution to remove residual blood. The veins were filled with 10 ml of dispase solution (3.1 g/l) and incubated for 30 min at 37°C. Veins were then flushed with 40 ml of M199 medium resulting in a cell-containing media suspension. Both M199 and dispase solutions were centrifuged at 160 × g for 5 min and the cell pellet was re-suspended in M199 media (Sigma-Aldrich, Germany) supplemented with EC growth supplement (PromoCell, Germany, C-39215), 5% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin and 0.25 μg/ml Fungizone® antimycotic. The EC phenotype of these cells was confirmed by positive immunofluorescence for CD31 endothelial marker and assessment of a cobble-stone like morphology. The cells were routinely cultured on standard plates pre-coated with 2% (w/v) gelatin at 37°C, 5% CO2. Only cells subcultured up to passage 4 were utilized for all subsequent experiments.
HUVSMC: The umbilical veins were flushed with D-PBS buffer to remove residual blood. The tunica intima, the inner most layer of the vein was denuded of the ECs and the tunica media layer was cut into small pieces (~0.3 mm × 0.3 mm) which were spread around in a 6 cm cell culture Petri dish. The venous fragments were covered gently with 15% FCS, DMEM media making sure not to dislodge the attached vein segments. Approximately 2 weeks later the cell outgrowths were trypsinized and centrifuged for 5 min at 160 × g. The pellet was re-suspened in 15% FCS, DMEM media supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin and 0.25 μg/ml Fungizone® antimycotic mix and transferred to T-75 cell culture flask. The cells were routinely cultured on standard tissue culture plates at 37°C, 5% CO2 and cells cultured up to passage 5 were utilized for all subsequent experiments.
Viability of HUVECs and HUVSMCs cultured with increasing GA concentrations for 6 and 24 h was assessed with a PresoBlue cell Viability Reagent (Invitrogen, Frederick, U.S.) according to the manufacturer's instructions. Fluorescence readings were taken at 544 nm/590 nm excitation and emission wavelength respectively which correspond directly to the amount of viable cells.
To expose HUVECs to biomechanical stretch, cells were cultured on BioFlex Collagen type I 6-well plates (Flexcell, Hillsborough, NC, USA) pre-coated with Geltrex® (basement membrane surrogate, 1:10 in cell media) for 1 h at 37°C. One day prior to stretch, the endothelial supplement content of the media was reduced to half and diluted in the M199 media supplemented with 12.5% FCS, 25 U/ml penicillin, 25 μg/ml streptomycin and 0.125 μg/ml Fungizone® antimycotic. Cell monolayers were treated with GA dissolved in DMSO at 20 μM or 40 μM final concentration or equivalent volume of DMSO vehicle control. Cyclic stretch was applied 1.5 h later using a microprocessor controlled vacuum pump (FX-3000 FlexerCell Strain Unit, Flexcell, Hillsborough, NC) with 15% cyclic elastomer elongation at frequency of 0.5 Hz. Cyclic, as opposed to static, elongation is needed to prevent the cells from evading the biomechanical stimulus through rearranging their focal contacts. To expose HUVSMCs to biomechanical stretch, cells were cultured on BioFlex Collagen type I 6-well plates (Flexcell, NC, USA) in 15% FCS, DMEM media. One day prior to stretch the cell media was exchanged to pure DMEM media in order to stabilize HUVSMCs phenotype in the absence of serum. Cell monolayers were treated with GA (1 h at 5 μM final concentration) or an equivalent volume of DMSO vehicle control. Cyclic stretch was applied using a microprocessor controlled vacuum pump (FX-5000 FlexerCell Strain Unit, Flexcell, NC) with 15% cyclic elastomer elongation at frequency of 0.5 Hz.
HUVECs and HUVSMCs were fixed with methanol for 15 min at 4°C, air-dried and blocked with Casein/BSA block buffer (0.25% Casein, 0.1% BSA, 50 mM Tris, pH 7.6) for 30 min. Determination of gelatinase activity was performed by incubating methanol-fixed cells with fluorescein-conjugated DQ-gelatin (EnzChek gelatinase assay kit; Molecular Probes/Invitrogen, Leiden, Netherlands) for 1 h at 37°C as per manufacturer's instructions. Nuclei were visualized by counterstaining with DAPI (2 μg/mL, diluted in PBS) for 10 min and the cells were then washed and mounted with Mowiol 4-88 (Fluka). Fluorescence intensity was recorded using a fluorescence microscope IX83 (Olympus) and quantified by using the Cell∧R software (Olympus, Hamburg, Germany) analyzing at 4–5 different regions of the specimen per experimental group. Exposure times during digital imaging were kept constant.
HUVECs from three different donors were stimulated with GA (40 μM) or DMSO as solvent control and exposed to biomechanical stretch for 6 h. RNA was isolated and processed for DNA microarray analysis according to manufacturers' instructions: Gene expression profiling was performed using the GeneChip® Human Genome Array from Affymetrix. After RNA isolation RNA was purified using the RNA Clean-Up and Concentration Micro Kit. cDNA synthesis was performed using the SuperScript Choice System according to the recommendations of the manufacturer. Using ENZO BioArray HighYield RNA Transcript Labeling Kit biotin-labeled cRNA was produced. Standard protocol from Affymetrix was used for the
HUVEC spheroids of defined cell number were generated as described previously (Heiss et al.,
HUVSMCs were suspended in growth medium containing 0.25% (w/v) methylcellulose and seeded onto non-adherent round bottom 96-well plates overnight allowing for the formation of a single, well-defined a round spheroids which were suspended in collagen gels as described earlier (Pfisterer et al.,
HUVECs/HUVSMCs were treated with various concentrations of GA or equivalent volume of the DMSO vehicle control at ~30% confluency and allowed to grow for an additional 24 h. Light microscopy images from the same areas of the well were obtained immediately after pre-treatment as well as 24 h and the total number of cells in each field of view was quantified with the help of ImageJ software (NIH, Bethesda, MD, USA). The cell number was converted into doubling time using the following formula:
TissueGnostics/TissueQuest microscopy and software were utilized to simultaneously automatically assess the number of Ki67-positive cells. In brief, HUVECs were fixed with methanol for 15 min at 4°C, air-dried and blocked with Casein/BSA block buffer for 30 min, incubated with the anti-Ki67 antibody overnight, washed with PBS and nuclei were visualized by DAPI staining, automatically detected and defined as region of interest (ROI). ROI-associated ki67 fluorescence was automatically detected and expressed as percentage fluorescence-positive nuclei with fluorescence signal above background using TissueFAXS microscopy (TissueGnostics AG, Austria) and TissueQuest (TissueGnostics AG, Austria) software.
HUVECs were pre-treated with GA (40 μM) or DMSO vehicle control subject to 6 h of biomechanical stretch as described in the previous section. Preparation of nuclear extracts from the cultured cells and subsequent non-denaturing 4% polyacrylamide gel electrophoresis was carried out as described previously (Krzesz et al.,
The impact of GA on the activity of gelatinases was determined by measuring the MMP2 or MMP9-mediated turnover of the gelatinase-specific substrate BML-P125-9090. In brief, GA or an equivalent volume of solvent (DMSO) were mixed with recombinant human MMP2 or MMP9 (90 mU/μl) and incubated for 30 min at 37°C. After adding the substrate (2 mM), absorption of the produced chromogen was acquired at A412 every minute according to manufacturer's instructions (MMP2/9 colorimetric drug discovery kits, Enzo Life Sciences, USA).
All animal studies were approved by the Regional Council Karlsruhe and carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Ligation of mouse auricle veins was performed as described earlier (North and Sanders,
Animals were sacrificed and the facial and saphenous veins (~1 cm long segments, diameter: 50–100 μm) were extracted and inserted into a perfusion chamber (Culture Myograph, DMT, Copenhagen, Denmark). The chambers were placed in an incubator at 37°C and 5% CO2, and the free end of blood vessel segments were tied off with a suture. This allowed for an increase in intraluminal hydrostatic pressure of the vessels upon continuous stretch distension at a transmural pressure difference of 4 or 16 mmHg without the application of flow (varicosis-inducing conditions: ΔP: 16 mmHg; physiological (control) condition: ΔP: 4 mmHg). The vessels were continuously stretch-distended for 18 h in Panserin 401 media (PAN-Biotech, Aidenbach, Germany) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin and 0.25 μg/ml Fungizone® antimycotic in the presence of GA (10 μM) or the equivalent volume of DMSO vehicle control. After perfusion, vessel segments were fixed in Dent's fixative (4°C for 24 h; 80% methanol, 20% DMSO) and subsequently processed for whole-mount immunofluorescence analyses.
Blood perfusion of an area of ~20 × 20 mm containing a spider vein (left leg, knee level) was made from one healthy female volunteer using the PeriCam PSI blood perfusion imager placed at a 10 cm distance perpendicular to the skin (image resolution: 20 μm/pixel; Camera resolution: 752 × 580 pixels). This system is based on the Laser Speckle Contrast Analysis (LASCA) technology which allows for visualization of blood perfusion (Perfusion Units, PU) of the superficial organs such as skin. Informed (written) consent in accordance with the Declaration of Helsinki has been obtained from the volunteer for imaging of a spider vein as well as online open-access publication of the information/images.
As normality tests of small samples (<10) have little power to discriminate between Gaussian and non-gaussian populations, we made assumptions about their distribution in a given basic population by considering the variability/scatter/source of scatter of data from previous experiments assessing the same variables(Feldner et al.,
Superficial veins or venules are connected to the deeper venous plexus (Somjen,
Morphometrical and functional analyses of superficial veins. Blood perfusion of one spider vein (left leg, knee level) was made from a healthy female volunteer using the PeriCam PSI blood perfusion imager (Superficial image: top panel; Perfusion measurement: bottom panel). An increase in blood perfusion of a spider vein was observed when the volunteer actively contract muscles of the leg (II) as opposed to the low perfusion level while standing still (I). Morphology and perfusion (upon muscle contraction) of the same spider vein was assessed 19.5 months later in a follow up recording (III).
One hallmark of spider veins is their increased diameter and given that blood vessel growth almost always relies on the proliferation of ECs and SMCs, we hypothesized that cream formulations containing bioactive compounds interfering with biomechanically evoked activation of vascular cells may attenuate the development of spider veins. GA was selected due to its frequent usage in personal care products and anti-proliferative features (Huang et al.,
GA does not affect viability and migration of cultured HUVECs or HUVSMCs. HUVECs
Impact of GA on HUVEC and HUVSMC proliferation. Proliferation of HUVECs
As has been repeatedly shown by results of our group, biomechanical wall stress or stretch of venous endothelial and SMCs is sufficient to promote their activation and acts as a major determinant of venous remodeling (Feldner et al.,
GA attenuated the capacity of AP-1 binding to DNA in biomechanically stimulated HUVECs. HUVECs were treated with GA (40 μM) or DMSO control vehicle for 1.5 h and subjected to biomechanical stretch (15% cyclic elongation at 0.5 Hz) for 6 h. Binding of the mechanosensitive transcription factor AP-1 was assessed in an EMSA assay (***
Assessment of changes in the transcriptome of GA-treated HUVECs exposed to biomechanical stretch.
ZBTB43 | Zinc finger and BTB domain containing 43 | ENSG00000169155 | 0.446 | 0.020 |
PHLPP2 | PH domain and leucine rich repeat protein | ENSG00000040199 | 0.424 | 0.043 |
TMOD2 | Tropomodulin 2 (neuronal) | ENSG00000128872 | 0.391 | 0.012 |
ALG1 | ALG1, chitobiosyldiphosphodolichol beta- | ENSG00000033011 | 0.383 | 0.004 |
SNX18 | Sorting nexin 18 | ENSG00000178996 | 0.339 | 0.003 |
CTSF | Cathepsin F | ENSG00000174080 | 0.338 | 0.025 |
ATG10 | Autophagy related 10 | ENSG00000152348 | 0.313 | 0.004 |
LACTBL1 | Lactamase, beta-like 1 | ENSG00000215906 | −0.338 | 0.043 |
DEFB113 | Defensin, beta 113 | ENSG00000214642 | −0.368 | 0.018 |
SNORA11B | Small nucleolar RNA, H/ACA box 11B | ENSG00000221102 | −0.379 | 0.039 |
IQCD | IQ motif containing D | ENSG00000166578 | −0.419 | 0.019 |
RNF126 | Ring finger protein 126 | ENSG00000070423 | −0.687 | 0.021 |
DDX11L9 | DEAD/H (Asp-Glu-Ala-Asp/His) box helicas | ENSG00000248472 | −0.736 | 0.050 |
Another prototypic response of ECs subject to biomechanical stress is the activation of proteases which promote degradation of extracellular matrix components as a prerequisite for further remodeling of the vessel wall (Kowalewski et al.,
GA attenuates gelatinase activity in biomechanically stimulated HUVECs or mouse veins. HUVECs were exposed to biomechanical stretch (15% cyclic elongation at 0.5 Hz) for 24 h with or without GA pretreatment (1.5 h, 20 μM) followed by quantitative analysis of gelatinase activity (
As evidenced by our findings, GA may diminish remodeling of superficial veins at least by interfering with (i) EC proliferation, (ii) AP-1 binding capacity, and (iii) the gelatinase activity. To scrutinize the relevance of these findings in the context of venous remodeling
GA attenuates formation of spider veins in the mouse auricle. Mouse auricles were treated with GA (~5 μg/ear) or DMSO (corresponding vehicle volume) applied as cream formulations before ligation and on every other day. Ligation of a central auricle vein was utilized to locally increase the venous volume load. Morphological and functional changes in the venous system was recorded by digital (
Veins are constantly exposed to biomechanical forces exerted by the movement of blood inside the vessel and depending on the magnitude and duration this may stabilize or “activate” the cells comprising the venous vessel wall (Pfisterer et al.,
While not much is known of the effects of GA on ECs and SMCs in the context of their stretch-dependent activation, GA has been shown to non-specifically block gap junctions, e.g., composed of connexin-43 in vascular SMCs (Matchkov et al.,
Surprisingly, GA appears to robustly interfere with the function of AP-1 in stretch-exposed ECs which, however, did not much affect their transcriptional profile. Other reports suggest that in TNF-α-stimulated ECs GA may also influence the activity of NFκB (Chang et al.,
Despite the ambivalent effects of GA observed so far, it robustly inhibited the capacity of both human and mouse ECs and SMCs to degrade gelatin-based matrices. This inhibitory feature may in fact attenuate the renovation of the extracellular matrix in the context of venous remodeling which is usually associated with increased activity of matrix metalloproteinases (MMP-2/9) in animal models (Pascarella et al.,
Although the mechanistic details of the GA-induced inhibitory effects remain unknown, collectively they may interfere with the capacity of biomechanically stressed ECs and partly SMCs to adequately promote the structural reorganization of a superficial vein. In fact, this study shows for the first time that GA attenuates venous remodeling
Pharmacokinetic and pharmacodynamic properties of GA have been also thoroughly examined in the past in a number of studies utilizing various
In summary, the results of this study indicate that GA has the capacity to interfere with the activation of biomechanically stressed venous ECs and inhibits attenuated remodeling of superficial veins upon topical application. Although it remains to be investigated how GA exerts its effects, it is tempting to speculate that skin care products containing this bioactive compound may decelerate the formation of spider veins.
HK, CA, and AW: Performed the experiments; MH: Contributed expertise and wrote the manuscript; HK, CS, CA, AW, and TK: Conceived the study, analyzed data and wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to acknowledge the excellent technical assistance of Yvonne Feuchter, Gudrun Scheib, and Maria Harlacher.
The Supplementary Material for this article can be found online at: