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Page 2 of 14 Lust et al. Vessel Plus 2021;5:7 I http://dx.doi.org/10.20517/2574-1209.2020.52

Conclusion: The cyclooxygenase inhibitor, diclofenac, interferes with stress-induced activation of venous cells
and attenuates venous remodeling in vivo. Additional research is warranted to investigate whether nonsteroidal
anti-inflammatory drugs interfere with the processes promoting the onset of varicose vein development and
biomechanical activation of venous cells.

Keywords: Varicose veins, endothelial cells, prostanoids, cyclooxygenase, biomechanical stress

INTRODUCTION
Varicose veins frequently develop in the veins of lower extremities including the superficial, deep or
[1]
perforating veins , reflecting the relevance of pressure gradients such as biomechanical stimuli in this
pathophysiological process. As such, prolonged standing and tight undergarments are considered as
[2,3]
primary risk factors of the disease , leading to elevated venous filling pressure and consequently, increases
in biomechanical stress to which the venous wall is exposed. Likewise, the development of pelvic venous
[4]
insufficiency and varicosities in the context of pelvic congestion syndrome may also be promoted by an
increase in venous pressure due to the partial occlusion of pelvic veins. In the long run, any congestion
of blood flow in large veins may cause chronic elevation of blood pressure in the connected venous
network and elevate venous wall tension. This biomechanical force is sufficient to promote the detrimental
and structural remodeling of the venous wall architecture in mouse models , and is thought to drive
[5,6]
[7]
comparable processes in humans . Morphologically, this results in the development of thin-walled varicose
veins, further characterized by their corkscrew-like and tortuous appearance, enlarged diameter, altered
[8]
extracellular matrix composition and a change in the activity of endothelial and smooth muscle cells . At
[9]
the molecular level, loss of the regular collagen-elastin network, changes in the collagen type I to III-ratio ,
and an elevated activity of matrix-metalloproteinases (MMPs) such as the gelatinases MMP-2 and MMP-9 [9-11]
are reported for varicose veins.

Recently, MMP activity and collagen content have been linked to the level of prostaglandin E2 (PGE2),
[12]
a cyclooxygenase (COX)-derived prostanoid, in human varicose veins . Moreover, elevated PGE2
[13]
levels as well as increased expression of the prostaglandin-endoperoxide synthase gene (PTGS1/2;
[14]
encoded by COX-1/2) have been detected previously in the development of varicosities. In this context,
biomechanical wall stress could serve as a determinant of PTGS2 expression, which is up-regulated in
[15]
biomechanically stressed venous endothelial cells , and has been associated with the remodeling of the
[16]
extracellular matrix and vascular stiffness of arteries in hypertensive rats . Although there is evidence that
demonstrates a general association between COX activity and varicose vein development, their functional
relevance for the onset of pressure-induced venous remodeling has not been investigated so far.

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 in vitro and in vivo experimental mouse models.

METHODS
HUVEC and huSMC culture
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