Varicose veins frequently develop in the veins of lower extremities including the superficial, deep or perforating veins[
Recently, MMP activity and collagen content have been linked to the level of prostaglandin E2 (PGE2), a cyclooxygenase (COX)-derived prostanoid, in human varicose veins[
Herein, we hypothesized that the activity of COX-1/2 promotes remodeling of the venous wall via hypertensive venous pressure levels and chronically elevated venous wall stress. Consequently, this study aimed to investigate the effect of the COX-1/2 inhibitor, diclofenac, on pressure-induced responses of veins and venous remodeling by employing
The procedure for isolating human umbilical vein endothelial cells (HUVECs) was carried out with the approval from the Local Ethical Committee (document number 336/2005, Heidelberg, Germany) and conformed to the principles outlined in the Declaration of Helsinki (1997). Isolation of HUVECs from the umbilical cords of newborns was carried out upon parental consent. HUVECs were cultured in EASY medium (PeloBiotech, Germany) supplemented with endothelial cell growth supplement and 5% fetal calf serum (FCS). For proliferation analyses, HUVECs were cultured in EASY medium supplemented with 15% FCS. Human umbilical smooth muscle cells (huSMCs) were purchased from Provitro AG (Germany) and cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 15% FCS. For proliferation analyses, huSMCs were cultured in DMEM supplemented with 5% FCS.
Cells were cultured on BioFlex® Collagen type I 6-well plates (Flexcell, North Carolina, USA) pre-coated with Geltrex® (basement membrane surrogate, 1:10 in cell media; ThermoFisher Scientific, Massachusetts, USA) for 1 h at 37 °C. One day prior to the stretch, the endothelial cell supplement content of the media was reduced to half and diluted in M199 media supplemented with 12.5% FCS, 25 U/ml penicillin, 25 μg/ml streptomycin and 0.125 μg/ml Fungizone® antimycotic. Cyclic stretch was applied using a microprocessor controlled vacuum pump (FX-5000 FlexerCell® strain unit, Flexcell®, North Carolina, USA) with 15% cyclic elastomer elongation at a frequency of 0.5 Hz. Cyclic elongation, as opposed to static, is needed to prevent the cells from evading the biomechanical stimulus through rearranging their focal contacts. Cells were stretched at 85%-95% confluency.
HUVECs from three different donors were exposed to biomechanical stretch for 6 h or cultured under static conditions (control). RNA was isolated and processed for DNA microarray analysis according to the manufacturers’ instructions: Gene expression profiling was performed using the GeneChip® Human Genome Array from Affymetrix (Thermo Fisher Scientific Inc). 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
Adult NMRI mice (age: 12-20 weeks, outbred strain) were euthanized and branches of the mesenteric veins were excised, cannulated (with ligated open ends), and inserted into a perfusion chamber (Culture Myograph, DMT, Copenhagen, Denmark), which contained serum-free medium (Panserin 401, PanBiotechTM) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin, and 0.25 μg/ml Fungizone® antimycotic in the presence of diclofenac (2 µg/ml) or an equivalent volume of a control solvent. The chambers were incubated at 37 °C with 5% CO2, and the veins were cultured for 1 h, (equilibration) followed by exposure to 4 mmHg (control) or 16 mmHg (hypertension) pressure for additional 5 h (occluded at one end). This translates into a fourfold increase in venous wall stress. Vessel segments were processed for whole-mount immunofluorescence analyses. For RNA and protein extraction, several vessel segments of the same experimental group (vessels from one mouse) were pooled.
RNA from murine veins was isolated utilizing the RNeasy® Micro Kit 50 (Qiagen, Cat No./ID: 74004), according to the manufacturer’s instructions. Subsequently, cDNA was synthesized using the Sensiscript Reverse Transcription Kit (205213, Qiagen), and quantitative real-time RT-PCR for the target sequences was performed in the Rotor-Gene Q (Qiagen) using the LightCycler® 480 SYBR Green I Master Mix (Roche, Mannheim).
Summary of PCR primer pairs
Gene name | Sequence |
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5’-GAAGCTGCCAAAGCCTTAGA-3’
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5’-ATGAGTCGAAGGAGTCTCTCG-3’
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5’-TGAGCAACTATTCCAAACCAGC-3’
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Fluorescence was monitored (excitation at 470 nm and emission at 530 nm) at the end of the annealing phase. Threshold cycle (Ct) was set within the exponential phase of the PCR. Quantification of the PCR product was done by using the ΔΔCt method. Amplification of the ribosomal protein, S12 (S12) cDNA, served as an internal standard.
Pressure-exposed veins were lysed in RIPA buffer (pH 7.4) containing 65 mM TRIS, 154 mM NaCl, 10% NP-40, 10% sodium deoxycholate and 1 mM EDTA. Veins were centrifuged at 13,000 × g for 15 min at 4 °C. The supernatant was analyzed by applying capillary electrophoresis due to the low yield of protein. For capillary electrophoresis, the Wes system (ProteinSimple®, San Jose, California, USA) was used. Samples and reagents were prepared according to ProteinSimple® instructions. The primary antibodies for capillary electrophoresis were used using following dilutions: anti-COX-1 (NBP1-85500, Biotechne) 1:20, anti-COX-2 (AF4198-SP, Biotechne) 1:10, anti-ERK1/2 (#4695, Cell Signaling Technology) 1:50, anti-pER1/2 (#4370, Cell Signaling Technology) 1:25, anti-β-actin (ab6276, Abcam) 1:50, anti-MMP2 (NB200-193SS, Biotechne) 1:10. The images were automatically analyzed with Compass for SW software (Version 3.1.7, ProteinSimple®); β-actin and ERK1/2 (for pERK1/2 only) were utilized as references.
Vein segments were glued onto a glass slide, fixed with 4% PFA/PBS for 30 min, rinsed in PBST (PBS supplemented with 0.5% Triton X-100) and incubated with the goat anti-CD31 antibody (Biotechne, AF3628; 1:200) and the anti-alpha-SMA-Cy3 (Sigma, C6198; 1:400) for 24 h, rinsed in PBST, incubated with a fluorescein-conjugated secondary antibody (donkey anti-goat, 705-546-147 Dianova; 1:100) for 4 h, rinsed in PBST and counterstained with DAPI for visualization of the nuclei. Vessels were mounted in Mowiol 4-88 mounting media and imaged utilizing a confocal microscope (Olympus IX81, software: xcellence rt 2.0).
All animal studies were approved by the Karlsruhe Regional Council 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 previously[
For diclofenac treatment, the auricle was embalmed with 10 µl Voltaren® Spray, which contains 400 µg diclofenac-sodium in liposomes allowing for rapid penetration through the dermis, or ethanol-containing solvent (control), before, one, and three days after ligation of the vein. Four days upon ligation, mice were sacrificed and perfused with Ringer’s solution and a zinc-fixative. Mouse auricles were dissected and processed for paraffin embedding and histological examination.
MMP-2, PCNA, COX-2 and CD31 abundance were assessed as a part of the immunofluorescence detection technique performed on 4-5 µm thick paraffin sections of auricle veins by using the corresponding primary antibodies (rabbit anti-MMP2, Biotechne #NB200-193, 1:200; rabbit anti-COX2, CST #4842, 1:200; rabbit anti-PCNA, Abcam #2426, 1:500; goat anti-CD31, Biotechne #AF3628, 1:200) in combination with compatible fluorescence-labeled secondary antibodies (Dianova, 1:100) and employing standard operating procedures. Nuclei were visualized by counterstaining with DAPI.
Results are expressed as means ± SD. Differences between two matched experimental groups were analyzed by unpaired Student’s
Sustained increase in venous intraluminal pressure and consequent increase in circumferential wall tension, may ultimately result in an elevated level of cellular stretch. In order to first assess the various transcriptional targets whose expression may be affected in venous cells under these conditions, HUVECs were exposed to biomechanical stretch for 6 h and processed for RNA extraction. Subsequent gene expression profiling [
Transcriptional profile of stretch-stimulated HUVECs (A); responses of HUVECs (B); and huSMCs (C) to prostaglandin E2. HUVECs from three different donors were subjected to biomechanical stretch (6 h). mRNA was isolated and processed for microarray-based analysis. Table A shows selected genes which were affected by biomechanical stretch (
PTGS2 encoding COX-2 was identified as another significantly up-regulated transcript in stretch-stimulated HUVEC
In order to better elucidate the role of COX in varicose development, we aimed to investigate its functional relevance in the activation of biomechanically stressed venous cells. To this end, we developed an experimental model that allows exposing mouse veins to elevated pressure levels while being treated with diclofenac-an inhibitor of COX-1 and COX-2 activity. Venous wall stress was elevated by increasing the pressure from 4 (physiological pressure level) to 16 mmHg (mimics venous hypertension), which by itself, had no major impact on the architecture and integrity of the venous wall as evidenced by whole-mount immunofluorescence analyses
Pressure stimulation of isolated mouse veins. Isolated branches of the mesenteric vein of male NMRI mice were exposed to pressure levels of 4 or 16 mmHg, respectively. Afterwards, vessel segments were processed for whole mount immunofluorescence staining, scale bars: 0.1 mm (A) to detect CD31 (endothelial cell marker), αSMA (smooth muscle cell marker), and DAPI (visualization of the nuclei). Protein samples generated from these blood vessels were pooled and analyzed by automated capillary electrophoresis/immunodetection [required due to the low yield of protein; specified antigens were detected by antibodies and corresponding signals (B, D, F) and were automatically evaluated by their size (kDa) and intensity (area under the curve, C, E, G)]. Signals specific for COX-1 (B and C) and COX-2 (D and E) were detected. b-actin served as a loading reference (F and G).
To evaluate the cellular activity during this early phase of biomechanical stress response, we determined the levels of phosphorylation of the MAP-kinases ERK1/2 in the venous wall, a known and important indicator of growth regulating signaling cascades. Additionally, we assessed the abundance of the pro-form (~ 72 kDa) and active form (~ 62 kDa) of the matrix-metalloproteinase, MMP-2
Impact of elevated pressure levels on markers of cell activity. Isolated branches of the mesenteric vein of male NMRI mice were exposed to 4- or 16-mmHg, respectively in the presence or absence of diclofenac. Protein extracts generated from these blood vessels were analyzed by capillary electrophoresis as described above. The level of phosphorylated ERK1/2 and the abundance of MMP-2 (pro-enzyme: 72 kDa was below detection limit, active form: ~ 62 kDa) were determined; A: *
The results obtained thus far suggested that diclofenac may inhibit, or at least delay, pressure-induced or biomechanically evoked stress responses of venous cells. Consequently, we hypothesized that treatment with diclofenac will interfere with the onset of varicose-like remodeling in mammalian veins,
While a corkscrew-like morphology and a significant increase in the diameter of remodeling veins was observed under control conditions, this effect was significantly attenuated upon treatment with diclofenac
Effect of diclofenac on venous remodeling,
Influence of diclofenac-treatment on SMC activation in mouse auricle veins. Selected auricle tissue segments (4 d after vein ligation) containing remodeling veins were fixed, paraffin-embedded, cross-sectioned, and processed for immunostaining (nuclei were visualized by DAPI staining). While PCNA (proliferation marker, A, arrows), MMP-2 (B, arrows), and COX-2 (C, arrows) were detectable in cells of the remodeling veins, corresponding proteins were barely detected in diclofenac-treated auricles (arrows in D-F, scale bar: 50 µm).
Diclofenac belongs to the class of non-steroidal anti-inflammatory drugs (NSAIDs), which are broadly applied to fight fever, attenuate pain, prevent thrombosis, or limit inflammatory responses. NSAIDs specifically inhibit the activity of COX-1 and COX-2 that act as rate-limiting factors in prostanoid synthesis by generating prostaglandin H2 (PGH2) from arachidonic acid. Depending on the cell-specific availability of defined prostaglandin synthases (e.g., prostaglandin E2 synthase or prostaglandin I2 synthase), several bioactive products such as PGE2 and prostacyclin (PGI2) may be generated from PGH2. In the vascular system, recent findings suggest that COX-1 and PGI2-synthase are constitutively active in endothelial, but not vascular smooth muscle cells[
In this study, we have verified both COX-1 and COX-2 protein expression in isolated and cannulated branches of mesenteric mouse veins whose
In the vascular system, PGI2 as well as PGE2 are crucial for regulating vascular tone and inflammatory responses via specific G-protein coupled receptors located on the surface of vascular endothelial and smooth muscle cells. Indeed, PGI2 acts as the most relevant cardioprotective prostanoid[
Although supplementary corresponding analyses are lacking for venous diseases, the association of varicose vein development with accentuated
One of the limiting factors for the translational impact of our study is the utility of a mouse model, which only mimics certain aspects of venous remodeling processes in humans. As such, although occlusion- or reflux-mediated venous wall stresses induce comparable cellular responses in mice and humans[
On the molecular level however, many cellular responses in the remodeling veins are comparable between the two species, considering their proteolytic, transcriptional, or proliferative activity[
The authors would like to acknowledge Ralph Mayer and Dr. Caroline Arnold for their technical assistance in this study.
Performed experiments, analyzed the data, and revised the manuscript: Lust L, Kuk H, Kohlhaas J
Performed the microarray analysis and the statistical evaluation: Sticht C
Designed the study, analyzed the data and wrote the manuscript: Korff T
The raw and normalized data are deposited in the Gene Expression Omnibus database (
This work was supported by the Swiss Society for Phlebology.
All authors declare that there are no conflicts of interest.
Human umbilical vein endothelial cells (HUVECs) were isolated as per approval from the Local Ethical Committee (document number 336/2005, Heidelberg Germany) and conformed to the principles outlined in the Declaration of Helsinki (1997). Parental consent was obtained for isolation of HUVECs from the umbilical cords of newborns.
Not applicable
© The Author(s) 2021.