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Lust et al. Vessel Plus 2021;5:7 I http://dx.doi.org/10.20517/2574-1209.2020.52 Page 3 of 14

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.

Stimulation of HUVEC by biomechanical stretch
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.

DNA microarray
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 in vitro transcription. Quantification of cRNA was performed
by spectrophotometric analysis with an A260/A280 ratio of 1.9 to 2.1. Fragmentation of the cRNA was
achieved using a defined protocol from Affymetrix. For gene expression profiling, labeled and fragmented
cRNA was hybridized to Affymetrix Hugene-2_0-st microarrays with an incubation of 16 h at 45 °C.
Affymetrix fluidics station 450 was used to wash the microarrays and scanning was performed with
Affymetrix Genechip scanner 3000. A custom CDF version 20 with Entrez-based gene definitions was
used to annotate the arrays. Raw fluorescence intensity values were normalized by applying quantile
normalization. Differential gene expression analysis was performed with one-way analysis of variance
(ANOVA) using the software package JMP10 Genomics version 6 from SAS (SAS Institute). A false positive
rate of alpha = 0.05 with FDR correction was taken as the level of significance. The raw and normalized
data are deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/; accession
No. GSE121557).

Perfusion of isolated mouse veins
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% CO , and the veins were cultured for 1 h, (equilibration)
2
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.