CYCLIC STRAIN INFLUENCES THE EXPRESSION OF THE VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND
THE HYPOXIA INDUCIBLE FACTOR 1 ALPHA (HIF-α) IN TENDON FIBROBLASTS
1
Petersen, W; 2Varoga, D; 3 Zantop, T; 3 Hassenpflug, J; 2Mentlein, R, 2Pufe T
1
Department of Trauma-, Hand- and Reconstructive Surgery, University Hospital Münster, Germany
INTRODUCTION:
Neoangiogenesis might be involved in beneficial but also in
detrimental effects to tendon tissue. Angiogenesis is controlled by
various mitogenic, chemotactic, One of the most important angiogenic
factors is the vascular endothelial cell growth factor (VEGF). Previous
studies have shown that hypoxia and several growth factors upregulate
VEGF expression synergistically in tendon tissue. But during daily living
or sportive activities tendon tissue is subjected to repetitive loading and it
is well known that changes in mechanical environment significantly
affect the mechanical properties of tendon tissue. The aim of the current
study was to examine effect of cyclic loading on VEGF expression in
tendon cells. Since the transcription of the VEGF gene is mediated by
the hypoxia-inducible factor-1 (HIF-1) (1) we further examined the
possibility that mechanical stress might also be a factor which is able
to induce HIF-1α expression in tendon cells.
METHODS
For cell culture experiments 3T3 fibroblasts and cultures of rat
Achilles tendon cells were used. For the application of uniaxial strains,
dishes with the monolayer cell cultures were subjected to cyclic,
homogenous stretching in a custom made six station motor driven
apparatus. The eccentric motor allowed variation of stretch magnitude
(0.5%-10%) and stretch frequency (0.5 Hz to 5 Hz).The dishes were
optically clear and have a cell culture surface area of 50 mm x 23 mm.
This model has been used in previous studies (§). The dishes were then
cyclically (sinusoidally) stretched with 8% uniaxial strain at different
frequencies (0.5 Hz, 1 Hz) for 24 hours.
For ELISA, the supernatant of the cell cultures was homogenized
in 150 mM NaCl 20 mM Tris/HCl-buffer, pH 7.4; a soluble fraction
obtained by centrifugation (48 000 xg, 60 min), and aliquots (100 µl)
were analyzed by a sandwich ELISA (R&D Systems, Minneapolis, MN,
USA).
For Western blots, samples were reduced in the presence of 10 mM
dithiothreitol, proteins separated by sodium dodecylsulfate-
polyacrylamide gel electrophoresis (SDS-PAGE; 10% gels), transferred
onto nitrocellulose membranes that were blocked and incubated with
antibodies.
For RT-PCR, the following primers have been used (2.5 µl each
containing 10 pmol): selective for VEGF splice variants 5'-TGC-ACC-
CAC-GAC-AGA-AGG-GGA- -3' (sense) and 5'-TCA-CCG-CCT-TGG-
CTT-GTC-ACA-T-3' (antisense) yielding different bp fragments:
VEGF110-330bp; VEGF120-360bp; VEGF144-432bp; VEGF164-492bp;
VEGF188-564bp; VEGF205-615bp (40 cycles, annealing temperature 62°C
).
For statistical analysis of the ELISA results the unpaired Mann
Whitney U Wilcoxon test have been used. The p-level for significance
was set at 0,05.
RESULTS SECTION
Fibroblasts (3T3 fibroblasts and rat Achilles tendon fibroblasts)
cultured under normal oxygen pressure without intermittent hydrostatic
pressure released measurable amounts of VEGF into their culture
supernatants (Fig. 1).
VEGF ELISA
80
70
60
VEGF [pg/ml]
50
40
30
20
10
0 3.
no cyclic cyclic strain cyclic strain 1
strain 0,5 Hz Hz
Fig. 1: VEGF ELISA
Application of uniaxial stretch with a frequency of 0.5 Hz did not
increase the VEGF production significantly. In contrast, at a strain
frequency of 1 Hz VEGF concentrations increased significantly. There
was no significant difference in the VEGF concentrations between 3T3
fibroblasts and rat Achilles tendon fibroblasts.
These results could also be verified in Western blotting experiments
which further proved the specificity of the immunoreaction.
From all cultures, two PCR products were obtained: One with 526
bp corresponding to VEGF121 and one with 658 bp corresponding to
VEGF165. Both splice products were also obtained with other VEGF-
positive human tissues (rheumatoid synovium, gliomas) whereas a
human cartilage from the growth plate yielded the splice forms
VEGF121 and VEGF189 (not shown) illustrating that the method has
the resolution necessary to detect other forms. The strongest bands
have been observed in cells that have been stretched with a rate of 1
Hz.
∅ 0,5Hz 1 Hz
Fig. 2: RT PCR
Western blotting experiments showed that cells subjected to uniaxial
strain at 1 Hz expressed the hypoxia-inducible factor-1α (HIF-1α). A
band of 103 kDa that corresponds to HIF-1α was heavily stained in
cultures which has been subjected to uniaxial strain but not in
unstimulated control cultures.
HIF-1-α - 103 kDa kDa
control 1Hz
Fig. 3: HIF 1 alpha Western Blot
DISCUSSION
This study is the first to demonstrate that mechanical stretch
regulates expression vascular endothelial growth factor (VEGF) in
tendon cells. Evidence that VEGF is upregulated by stretch of the
ventricular wall was firstly provided by Li et al. (2). They found a
marked increase in VEGF mRNA in the heart after diastolic pressure.
The present study shows that the effect of uniaxial stretching was highly
sensitive to strain frequency since only strain rates of 1 Hz resulted in
increased VEGF levels whereas at 0.5 Hz VEGF concentrations differed
not significantly from the levels of the control cultures. It is well known
that mechanical stimulation parameter such as the frequency strongly
affect cellular reactions.
Kim et al. (1) were the first who studied the role of HIF-1α for
stress mediated VEGF induction in the rat heart. They found that HIF-
1α plays an important role in the induction of VEGF in the
nonischemic and mechanically stretched myocardium, and that this is
regulated by stretch-activated channels and the PI3K/Akt/FRAP
pathway (1). The cell culture model of the present study confirms
these in vivo findings and demonstrates that HIF-1α protein is induced
by cyclic strain in tendon cells.
3T3 fibroblasts
Rat Achilles tendon
REFERENCES
fibroblasts 1. Kim et al. (2002). Circ Res. 2002 Feb 8;90(2):E25-3
2. Li et al. (1997) J Clin Invest. 1997 Jul 1;100(1):18-24
Skutek et al. (2001) Eur J Appl Physiol. Nov;86 (1):4
AFFILIATED INSTITUTIONS FOR COAUTHORS
2) Department of Anatomy, CAU Kiel, Germany
3) Department of Orthopaedic Surgery, CAU Kiel, Germany
50th Annual Meeting of the Orthopaedic Research Society
Poster No: 0828