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Lust et al. Vessel Plus 2021;5:7 I http://dx.doi.org/10.20517/2574-1209.2020.52 Page 5 of 14

a full view of the murine auricle were obtained with a digital camera adjusted to a dissecting microscope
[17]
(Microscope Wild, Heerbrugg; Leica, Wetzlar, Germany), as described by North and Sanders . Images
were then morphometrically analyzed using the software Image J (NIH, Maryland, USA) by measuring
across the diameter of the remodeling vessels.

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.

Morphological analyses of tissue samples
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.

Statistical analysis
Results are expressed as means ± SD. Differences between two matched experimental groups were analyzed
by unpaired Student’s t-test with a probability value of P < 0.05 considered as statistically significant.
Differences among 3 or more experimental groups were analyzed by ANOVA, followed by Sidak’s multiple
comparisons test.

RESULTS
Biomechanical stress stimulates PTGS2 expression in human venous endothelial cells
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 [Figure 1, Supplement 1] revealed significant up-regulation of transcripts that control
inflammation (e.g., CXCL8, ESM-1, VCAM-1), growth/differentiation (KITLG/KIT, TGFB2, PDGFC,
HBEGF, GDF6) and extracellular matrix remodeling (HAS2, ADAMTS2). Down-regulated transcripts
included those associated with nitric-oxide signaling (NOS3, guanylate cyclases), redox-signaling (HMOX1,
TXNIP) and transcriptional modulation of cellular differentiation (SMAD6, KLF2, SNAI2).

PTGS2 encoding COX-2 was identified as another significantly up-regulated transcript in stretch-
[15]
stimulated HUVEC [Figure 1A], confirming results from earlier publications . COX-2 attracted our
attention as it encodes a stress-inducible enzyme that synthesizes prostaglandin H2 (PGH2), thus acting
as a precursor molecule for multiple prostanoid effectors capable of activating vascular cells. For instance,
stimulation with PGE2 spurred the proliferation of both endothelial as well as vascular smooth muscle
cells [Figure 1B and C, Supplement 2]. Moreover, COX-2 has been associated with the development of
varicose veins and is an important drug target for the non-steroidal anti-inflammatory drug class (NSAIDs).
Currently, it is still unknown whether COX activity may causally contribute to the remodeling of the
venous wall or represents a mere consequence of the cellular activation under these conditions.

Diclofenac decreases pressure-induced ERK1/2 and MMP-2 activation in mouse veins
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