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

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 [Figure 2A]. However, nanoscale protein analyses performed by
applying capillary electrophoresis, indicated a pressure-induced increase in COX-2, but not COX-1 protein
abundance in venous cells [Figure 2B-G]. In contrast, the expression of the corresponding genes Ptgs1/2
was not significantly altered under these conditions [Supplement 3]. Corresponding experiments with
diclofenac-treated veins showed a decline in the protein level of COX-2, but not of COX-1 [Supplement 4].

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 [Supplement 5], a marker of
proteolytic capacity of venous cells. Exposure to 16 mmHg intraluminal pressure led to an increased level
of ERK1/2 phosphorylation [Figure 3A] and an abundance of active MMP-2 in the venous wall [Figure 3B].
Diclofenac treatment diminished these effects [Figure 3A and B].

Diclofenac inhibits varicose-like venous remodeling in mice
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, in vivo. To
test this, we employed a mouse model, in which varicose-like venous remodeling can be mimicked by
occlusion of a single vein in the mouse auricle to increase the filling pressure in the connected local venous
network [5,17-20] . Diclofenac was applied transdermally by utilizing liposome-based diclofenac formulation
(Voltaren® spray), and venous remodeling was determined by comparing the diameter of veins immediately
and four days after the venous occlusion.

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
[Figure 4A]. Immunofluorescence-based techniques indicated a generally low COX-2 protein abundance
in the unaffected auricle veins [Figure 4B and C], but it was predominantly localized in endothelial cells of
remodeling vessels [arrows, Figure 4D and E]. Further analyses detected the proliferation marker, PCNA,
the protease MMP-2, and COX-2 in cells of enlarged (remodeled) veins [Figures 5A-C], thus confirming
earlier observations [5,17-20] . These markers were barely detectable in vessels from auricles treated with
diclofenac [Figures 5D-F].

DISCUSSION
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 [21,22] , although COX-1 is also detected in murine vascular smooth
[23]
muscle cells in vivo . In contrast, induction of vascular COX-2/PTGS-2 expression is usually associated
[24]
[23]
[25]
with inflammatory responses , atherosclerosis , or responses to injury ; but it may additionally be
[26]
constitutively expressed in distinct organs such as the kidney .