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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Vasc Surg. 2010 Nov 23;53(3):764–773. doi: 10.1016/j.jvs.2010.09.018

Prolonged Mechanical Stretch is Associated with Upregulation of Hypoxia-Inducible Factors and Reduced Contraction in Rat Inferior Vena Cava

Chung S Lim 1,2, Xiaoying Qiao 2, Ossama M Reslan 2, Yin Xia 2, Joseph D Raffetto 2,3, Ewa Paleolog 1, Alun H Davies 1, Raouf A Khalil 2
PMCID: PMC3058747  NIHMSID: NIHMS236113  PMID: 21106323

Abstract

Background

Decreased venous tone and vein wall dilation may contribute to varicose vein formation. We have shown that prolonged vein wall stretch is associated with upregulation of matrix metalloproteases (MMPs) and decreased contraction. Because hypoxia-inducible factors (HIFs) expression also increases with mechanical stretch, this study tested whether upregulation of HIFs is an intermediary mechanism linking prolonged vein wall stretch to the changes in MMP expression and venous contraction.

Methods

Segments of rat inferior vena cava (IVC) were suspended in tissue bath under 0.5g basal tension for 1hr, and a control contraction to phenylephrine (PHE, 10−5M) and KCl (96 mM) was elicited. The veins were then exposed to prolonged 18hr tension at 0.5g, 2g, 2g+HIF inhibitor (U-0126 10−5M, 17-DMAG 10−5M, or echinomycin 10−6M), or 2g+DMOG 10−4M, a prolyl-hydroxylase inhibitor which stabilizes HIF, and the fold change in PHE and KCl contraction was compared to the control contraction at 0.5g tension for 1hr. Vein tissue homogenates were analyzed for HIF-1α, HIF-2α, MMP-2 and MMP-9 mRNA and protein amount using real-time RT-PCR and Western blots.

Results

Compared to control IVC contraction at 0.5g tension for 1hr, the PHE and KCl contraction after prolonged 0.5g tension was 2.0±0.35 and 1.1±0.06, respectively. Vein contraction to PHE and KCl after prolonged 2g tension was significantly reduced (0.87±0.13 and 0.72±0.05, respectively). PHE-induced contraction was restored in IVC exposed to prolonged 2g tension plus the HIF inhibitor U0126 (1.38±0.15) or echinomycin (1.99±0.40). U0126 and echinomycin also restored KCl-induced contraction in IVC exposed to prolonged 2g tension (1.14±0.05 and 1.11±0.15, respectively). Treatment with DMOG further reduced PHE- and KCl-induced contraction in veins subjected to prolonged 2g tension (0.47±0.06 and 0.57±0.01, respectively). HIF-1α and HIF-2α mRNA were overexpressed in IVC exposed to prolonged 2g tension, and the overexpression was reversed in by U0126. The overexpression of HIF-1α and HIF-2α in stretched IVC was associated with increased MMP-2 and MMP-9 mRNA. The protein amount of HIF-1α, HIF-2α, MMP-2 and MMP-9 was also increased in IVC exposed to prolonged 2g wall tension

Conclusion

Prolonged increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α, increased MMP-2 and MMP-9 expression and reduced venous contraction in rat IVC. Together with our report that MMP-2 and MMP-9 inhibit IVC contraction, the data suggest that increased vein wall tension induces HIF overexpression, and causes an increase in MMPs expression and reduction of venous contraction, leading to progressive venous dilation and varicose vein formation.

Keywords: matrix metalloproteinases, smooth muscle, vein, varicose vein

INTRODUCTION

Varicose veins is a common disorder characterized by excessively dilated and tortuous veins.1, 2 The cause of varicose veins is unclear; however, valvular dysfunction, venous hypertension and vein dilation are common features.3 Although valvular incompetence may precede vein dilation, duplex ultrasonographic studies often demonstrate the opposite.3 Thus, increased hydrostatic venous pressure and vein dilation may play a role in the initiation and progression of varicose veins.1, 3

Changes in the composition of extracellular matrix and alterations of connective tissue and elastin may contribute to the vein wall weakness and dilation.1, 3 Matrix metalloproteases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix.4 The expression/activity of MMP-1, -2, -3, -9, -12, and -13, and their endogenous tissue inhibitors TIMP-1 and -3 are upregulated in varicose veins.3, 5 Our previous experiments on rat inferior vena cava (IVC) have shown that increases in vein wall stretch are associated with reduced contraction and overexpression of MMP-2 and MMP-9.6 Also, MMP-2 and MMP-9 induce relaxation of rat IVC, possibly through vascular smooth muscle (VSM) hyperpolarization and activation of Ca2+-dependent K+ channel (BKCa).6 These studies suggested that MMPs may play a role in the early stages of venous dilation secondary to venous hypertension.7 However, the upstream mechanisms linking the increases in vein wall tension to MMPs expression and venous dilation are unclear.

Hypoxia-inducible factors (HIFs) are nuclear transcriptional factors which regulate genes involved in oxygen homeostasis.8 HIF is a heterodimeric protein consisting of a labile α-subunit and constitutively expressed β-subunit.8 Three HIF-α isotypes (HIF-1α, -2α, -3α) have been identified. Under normoxia, HIF-1α and HIF-2α are hydroxylated by prolyl hydroxylases domain (PHD) that uses O2 and α-ketoglutarate as substrates, and facilitates the ubiquitination of HIFs and their marking for proteosomal degradation. Also, factor-inhibiting-HIF (FIH) binds to HIF-α and negatively regulates its transactivation function by hydroxylating asparagine residue. During hypoxia, the oxygen-dependent hydroxylation activity of PHD and FIH is suppressed, hence increasing HIF-α stabilization and transactivation. HIF-α translocates into the nucleus to form a dimer with HIF-β, which binds to hypoxia-responsive element (HRE) in the target genes activating their transcription.9–11 HIF-regulated genes include those involved in extracellular matrix metabolism (MMPs and TIMPs), vascular tone, cell survival and apoptosis, glucose transportation, angiogenesis, erythropoiesis and oxygen delivery.8, 11–13

In addition to the regulation of HIF by oxygen, hormones, cytokines, metallic ions and mechanical stretch may induce HIF expression.8, 14 HIF-1α and HIF-2α mRNA and protein are increased in skeletal muscle fibers exposed to stretch.15, 16 Also, HIF-1α is overexpressed in rat cardiac and aortic VSM cells exposed to mechanical stretch.17, 18 While regulation of oxygen-dependent HIF occurs at the level of protein stabilization, the induction of HIF by mechanical stretch occurs at the level of gene transcription and translation and likely involves phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK)8, 15

In search of the upstream mechanisms involved in the regulation of MMPs expression and the reduced contraction in veins under prolonged stretch, this study aimed to test the hypothesis that increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α, leading to increases in specific MMP expression and decreased venous contraction. We used rat IVC to test: 1) whether prolonged increases in vein wall tension are associated with increased expression of HIF-1α and HIF-2α; 2) whether the increased HIF expression in stretched veins is associated with increased MMP expression and decreased venous contraction; and 3) whether blockade of the expression or actions of HIFs prevents MMP overexpression and improves contraction in stretched veins.

METHODS

Solutions and drugs

Krebs solution contained (in mM): NaCl 120, KCl 5.9, NaHCO3 25, NaH2PO4 1.2, dextrose 11.5, CaCl2 2.5, MgCl2 1.2, bubbled with 95% O2 5% CO2 at pH 7.4. Membrane depolarization by high KCl induces VSM contraction by stimulating Ca2+ entry from the extracellular space.19 High KCl (96 mM) depolarizing solution was prepared as Krebs with equimolar substitution of NaCl with KCl. The α-adrenergic agonist phenylephrine (PHE) 10−5M (Sigma) was also used to stimulate IVC contraction. Tissue culture medium was used to incubate the veins overnight and was composed of Minimum Essential Medium (MEM) supplemented with penicillin, streptomycin and amphotericin B (Gibco/Invitrogen, Grand Island, NY). Drugs used to inhibit the expression/activity of HIF were U-0126 10−5M (Cayman, Ann Arbor, MI), 17-DMAG 10−5M (17-[2-(dimethylamino)ethyl]amino-17-desmethoxygeldanamycin) and echinomycin 10−6M (Alexis, San Diego, CA). Dimethyloxallyl glycine (DMOG, 10−4M, Cayman) was used to inhibit hydroxylation and protein degradation of HIF by PHD and FIH.

Animals and tissues

Male Sprague-Dawley rats (12 weeks, 250–300g, Charles River Lab, Wilmington, MA) were euthanized by inhalation of CO2. The abdominal cavity was opened, and the IVC was excised, placed in Krebs, cleaned of adventitial tissue under dissecting microscope, and portioned into four 3mm-wide rings. All procedures followed the guidelines of Harvard Animal Care and Use Committee.

Isometric contraction

Each IVC segment was suspended between two tungsten wire hooks, in water-jacketed tissue bath filled with 50mL Krebs bubbled with 95% O2 5% CO2 at 37°C. One hook was fixed to a glass rod at the bottom of the tissue bath and the other hook was connected to Grass force transducer (FT03, Astro-Med, West Warwick, RI), and the changes in isometric contraction were recorded on Grass polygraph (Model 7D, Astro-Med). O2 tension in the tissue bath was kept constant in all experiments so that any changes in HIF-1α and HIF-2α expression would be related to the changes in vein wall tension. Since HIF is known to be regulated by O2 tension, future experiments should test whether alteration of O2 tension in the tissue bath would further affect the contraction of the veins exposed to prolonged stretch.

We have previously demonstrated that in rat IVC subjected to step-wise increases in basal tension (0.0625, 0.125, 0.25, 0.5, 1, 2 and 3g) for 30min, membrane depolarization by 96 mM KCl caused tension-dependent increases in contraction that reached a maximum at 0.5g basal tension.6 Increases in tension to 1g or 2g did not show further increase in KCl contraction. When basal tension was increased to 3g, KCl contraction was reduced.6 Based on these findings, we selected 0.5g as the control basal tension that produces maximum contraction. We also selected 2g as the maximum tension that produces maximum contraction without causing excessive vein wall stretch and tissue damage.

We have estimated the venous pressure corresponding to the basal tension on IVC segments using the formula P=F/A, where P=pressure in gram force/cm2, F=force, A=area, and assuming an average IVC diameter of 1.5 mm as detected by histology. At 0.5g tension the pressure generated is 28.4 gram force/cm2 or 20.8 mmHg, and at 2g tension the pressure generated is 113.2 gram force/cm2 or 83.4 mmHg. These pressures are in accordance with the venous pressures observed in the lower extremity of humans, which could vary between 10 and 100 mmHg depending on posture and muscule contraction. Although the estimated pressure generated by 0.5g and 2g tension appeared similar to lower extremity venous pressure in humans, it is important to note that the present study was conducted on rat IVC and, therefore, any extrapolation of the findings in the rat veins to human varicose veins should be interpreted with extreme caution.

Vein segments were initially stretched under 0.5g tension for 1hr. To determine the control contraction properties, IVC segments were stimulated twice with 96 mM KCl and then with PHE 10−5M. Each control IVC contraction was followed by 3×10min washes in Krebs. The bathing solution was then changed to tissue culture medium. The IVC was then subjected to either 0.5g tension for 18hr, high 2g tension for 18hr, or 2g tension for 18hr plus the HIF modulator U-0126 10−5M, 17-DMAG 10−5M, echinomycin 10−6M or DMOG 10−4M. IVC segments were then washed in Krebs 3×10 min, and a second contraction to 96 mM KCl and PHE 10−5M was elicited. The fold change of contraction was calculated by dividing the PHE- and KCl-induced contraction after prolonged tension for 18hr by the corresponding control contraction at 0.5g tension for 1hr.

We have previously demonstrated that prolonged increases in rat IVC wall tension for 24 hr was associated with decreased contraction and increased expression of MMP-2 and MMP-9.6 In the present study we used a slightly shorter 18 hr period of stretch to avoid possible negative feedback mechanisms that could downregulate HIF-α.20 Previous studies on hypoxia regulation of HIF-α used a duration of about 16 hr to avoid the negative feedback mechanisms that may complicate the findings.21 Examples of negative feedback mechanisms that can be activated by hypoxia include the generation of an antisense RNA to HIF-1α (aHIF),22 production of CBP/p300-Interacting-Transactivator-with-ED-rich-tail-2 (CITED2) that can bind and block p300 recruitment required for HIF transcriptional activity,20, 23 and induction of PHDs transcription which negatively regulate HIF expression.20

Real-Time RT-PCR

RNA was isolated from IVC using an RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA). Total RNA (1μg) was used for reverse transcription to synthesize single-strand cDNA in 15–33μl reaction mixture following the First-Strand cDNA Synthesis Kit (Amersham Biosciences, Pittsburgh, PA). 2μl of the cDNA dilution (1:5 for HIF-1α, HIF-2α, MMP-2, MMP-9 and 1:25 for α-actin) of the reverse transcription product was applied to 20μl RT-PCR mixture. Quantification of gene expression was performed using a real-time RT-PCR machine (Mx4000, Multiplex Quantitative PCR System, Stratagene, La Jolla, CA), published oligonucleotide primers for HIF-1α24, HIF-2α24, MMP-225, and MMP-925 (Integrated DNA Technologies, Coralville, IA) and iQ-SYBR-Green Supermix (Bio-Rad, Hercules, CA). α-Actin primer was included in the RT-PCR as an internal standard to normalize the results.

The following primers were used:

HIF-1α forward 5′-AAGAAACCGCCTATGACGTG-3′
reverse 5′-CCACCTCTTTTTGCAAGCAT-3′
HIF-2α forward 5′-CCCCAGGGGATGCTATTATT-3′
reverse 5′-GGCGAAGAGCTTCTCGATTA-3′
MMP-2 forward 5′-CATCGCTGCACCATCGCCCATCATC-3′
reverse 5′-CCCAGGGTCCACAGCTCATCATCATCAAAG-3′
MMP-9 forward 5′-GAAGACTTGCCGCGAGACCTGATCGATG-3′
reverse 5′-GCACCAGCGATAACCATCCGAGCGAC-3′
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PCR was carried out with 1 cycle for 10min at 95°C and then 40–45 cycles of 30sec of denaturation at 95°C, 45sec of annealing at 56°C, and 30sec of extension at 72°C followed by 1min of final extension at 95°C. The number of PCR cycles varied according to the expression level of the target gene. The relative gene expression was calculated by comparison of cycle thresholds with the housekeeping gene α-actin.26

Western blots

IVC segments were homogenized in 2mL homogenization buffer at 4°C, centrifuged at 10,000g for 2min, and the supernatant was collected and protein concentration was determined. Tissue homogenate was subjected to electrophoresis on 8% SDS polyacrylamide gel then transferred electrophoretically to nitrocellulose membranes (Bio-Rad). The membranes were incubated in 5% dried non-fat milk in phosphate buffer solution (PBS)-Tween buffer for 1hr, then in the antibody solution containing HIF-1α [1:1000], HIF-2α [1:500] (Novus, Littleton, CO), MMP-2 [1:500] or MMP-9 [1:500] rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 24hr. The internal control actin was detected using monoclonal anti-smooth muscle α-actin antibody (1:500000, Sigma). The membranes were washed in PBS-Tween then incubated in horseradish peroxidase-conjugated secondary antibody (1:1000) for 1.5hr. The membrane blots were washed with PBS-Tween, and visualized with ECL Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Piscataway, NJ) and the reactive bands corresponding to HIF and MMP were analyzed by optical densitometry and ImageJ software (NIH). The densitometry values represented the pixel intensity normalized to α-actin to correct for loading.6

Statistical analysis

The data were presented as means±SEM, with n=number of experiments. The data were first analyzed using ANOVA. When a statistical difference was observed, the data were further analyzed using Student’s t-test for unpaired data for comparison of two means (Graphpad Prism 5.00, Graphpad Software, San Diego, CA). Differences were considered statistically significant when P<0.05.

RESULTS

In IVC segments under 0.5g basal tension for 1hr both α-adrenergic receptor stimulation with PHE (10−5M) and membrane depolarization by KCl (96 mM) caused significant contraction (Fig. 1,2). Compared to the initial IVC contraction at 0.5g tension for 1hr, the fold change of contraction to PHE and KCl after prolonged tension at 0.5g for 18hr was 2.0±0.35 and 1.1±0.06, respectively (Fig. 1,2). In IVC exposed to prolonged 2g tension for 18hr the PHE-and KCl-induced contraction was significantly decreased (Fig. 1,2).

Figure 1.

Figure 1

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Figure 2.

Figure 2

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The effects of the HIF inhibitors U-0126 10−5M, 17-DMAG 10−5M, and echinomycin 10−6M on PHE- and KCl-induced contraction in IVC segments exposed to prolonged stretch were then examined. PHE- and KCl-induced contraction of IVC exposed to prolonged 2g tension for 18hr was restored by U0126 and echinomycin (Fig. 1,2). On the other hand, treatment with the HIF inhibitor 17-DMAG did not restore the reduced PHE- or KCl-induced contraction in IVC exposed to prolonged 2g tension for 18hr (Fig. 1,2).

The effect of HIF stabilization by DMOG on PHE- and KCl-induced contraction was also examined. Treatment of IVC exposed to prolonged 2g tension for 18hr with DMOG 10−4M further decreased PHE- and KCl-induced contraction (Fig. 1,2)..

RT-PCR analysis demonstrated expression of HIF-1α and HIF-2α mRNA in control IVC under 0.5g tension for 1hr. Small but significant increases in HIF-1α and HIF-2α mRNA were observed in IVC exposed to 0.5g tension for 18hr. In contrast, significant and robust increases in HIF-1α and HIF-2α mRNA were observed in IVC exposed to prolonged 2g tension for 18hr (Fig. 3). The increases in HIF-1α and HIF-2α mRNA in stretched veins were reversed in IVC treated with the HIF inhibitor U0126 and to a less extent with 17-DMAG or echinomycin, but not with the HIF stabilizer DMOG (Fig. 3).

Figure 3.

Figure 3

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RT-PCR also demonstrated prominent expression of MMP-2 and MMP-9 mRNA in control IVC under 0.5g tension for 1hr. In IVC exposed to 0.5g tension for 18hr, small increases in MMP-2 and MMP-9 mRNA were observed. In contrast, in IVC exposed to prolonged 2g tension for 18hr significant and robust increases in MMP-2 and MMP-9 mRNA were observed (Fig. 4). The increases in MMP-2 or MMP-9 mRNA expression in stretched veins were reversed in IVC treated with the HIF inhibitor U0126, 17-DMAG or echinomycin, but not with the HIF stabilizer DMOG (Fig. 4).

Figure 4.

Figure 4

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Western blot analysis in control IVC under 0.5g tension for 1hr revealed little immunoreaction at 100kDa corresponding to HIF-1α, but a robust band in IVC under prolonged 2g tension for 18hr (Fig. 5A). A band at 116kDa corresponding to HIF-2α was detected in control IVC under 0.5g tension for 1hr, and was significantly increased in IVC under prolonged 2g tension for 18hr (Fig. 5B). Western blots also revealed an immunoreactive band at 72kDa corresponding to pro-MMP-2 and a second band at 63kDa corresponding to active MMP-2 in IVC under 0.5g tension for 1hr, that were significantly increased in IVC under prolonged 2g tension for 18hr (Fig. 5C). In control IVC under 0.5g tension for 1hr a little immunoreaction at 92kDa corresponding to MMP-9 could be detected. In IVC exposed to prolonged 2g tension for 18hr a robust MMP-9 immunoreactive band was observed (Fig. 5D).

Figure 5.

Figure 5

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DISCUSSION

This study demonstrates that prolonged increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α in rat IVC. The upregulation of HIF-1α and HIF-2α secondary to prolonged increases in rat IVC wall tension is associated with increased MMP-2 and MMP-9 expression and reduced venous contraction.

Previous studies demonstrated an increase of MMP-2 expression in mechanically stretched skeletal muscle fibres.16 Also, our previous experiments on rat IVC have demonstrated that prolonged increases in vein wall tension are associated with reduced contraction and increased protein amount of MMP-2 and MMP-9.6 We have previously performed immunohistochemistry studies and demonstrated that MMP-2 and MMP-9 were expressed in tunica intima, media and adventitia of rat IVC. Significant increases in MMP-2 and MMP-9 immunostaining were observed in the tunica intima of IVC subjected to 2g basal tension for 24 hr as compared to tissues under 0.5g basal tension. Also, a relative increase in the MMP immunostaining in the VSM media could be observed in tissues under 2g tension for 24 hr.6 These data suggested that the endothelium and VSM cells may function as sources/targets of MMPs in rat IVC. Other studies have shown the expression of MMP-9 in VSM cells of rat aorta 27 and human saphenous vein.28 We have also shown that MMP-2 and MMP-9 cause relaxation of Ca2+-dependent contraction in rat IVC.7 Consistent with our previous reports,6 both the PHE-induced α-adrenergic receptor-mediated contraction and KCl-induced receptor-independent response were reduced in rat IVC exposed to prolonged stretch. Because membrane depolarization by KCl mainly stimulates Ca2+ entry,19 the reduced contraction in IVC under prolonged stretch is likely due to reduction in the Ca2+-dependent pathways or downstream mechanism of VSM contraction. The present observations that prolonged increases in basal tension were associated with decreased IVC contraction and increased expression of MMP-2 and MMP-9 are consistent with previous reports and further support a relationship between vein wall stretch, MMP expression and decreased venous contraction.

In search for the upstream mechanisms linking the increases in vein tension to the changes in MMP expression and reduction in venous contraction, the present study demonstrates HIF-1α and HIF-2α as potential candidates because: 1) The reduction in venous contraction associated with prolonged vein stretch was reversed by the HIF inhibitors U0126 and echinomycin. 2) The reduction in venous contraction associated with prolonged vein stretch was enhanced in the presence of the HIF stabilizer DMOG. 3) Prolonged vein wall stretch was associated with increased expression of HIF-1α and HIF-2α. 4) The increased expression of MMP-2 and MMP-9 associated with prolonged vein stretch were reversed by HIF inhibitors.

The potential involvement of HIF in the decreased venous contraction associated with prolonged vein stretch was first examined using HIF inhibitors. To circumvent potential lack of specificity, we used three HIF inhibitors with different mechanisms of action (Fig. 6). U0126, a MAPK (dual MEK1 and MEK2) inhibitor, has been shown to inhibit the increases in HIF-α mRNA and protein in response to mechanical stretch of rat skeletal muscle microvascular endothelial and aortic VSM cells.15, 18 Echinomycin inhibits HIF-1 DNA binding activity, specifically the binding of HIF to the HRE sequence on the promoter of target genes.29 In this study, U0126 and echinomycin restored contraction of rat IVC, suggesting a role of HIF in the reduced contraction associated with prolonged vein wall stretch.

Figure 6.

Figure 6

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To further examine the role of HIF in the decreased venous contraction associated with prolonged vein wall stretch, we investigated the effect of stabilizing HIF with DMOG. DMOG inhibits PHD and FIH which mediate the oxygen-dependent degradation of HIF-α protein, and thereby increases the amount/activity of HIF-α protein even in normoxia.30 The IVC segments treated with DMOG demonstrated further reduction in contraction, consistent with a role of HIF in the reduced contraction associated with prolonged vein stretch.

To directly examine the role of HIF in the reduced contraction associated with prolonged increases in vein wall tension, we examined the effects of prolonged vein stretch on HIF expression. Studies have shown that HIF-1α and HIF-2α are expressed in vascular endothelial cells 31, 32 and VSM cells.33, 34 Also, Increased expression of HIF in response to mechanical stretch has been demonstrated in the myocardium,17 fibroblasts,35 VSM cells18 and skeletal muscle fibres.15, 16 In support of transcriptional regulation of HIF-1α by mechanical stress, Chang and colleagues reported upregulation of HIF-1α mRNA in VSM cells subjected to cyclic stretch for 4hr.18 Also, HIF-1α and HIF-2α mRNA and proteins are increased in rat capillary endothelial cells of skeletal muscle fibers exposed to prolonged mechanical stretch.15 Other studies support that the protein amount and activity of HIF are regulated by mechanical stretch.15, 18, 36 Kim and colleagues have demonstrated upregulation of HIF-1α protein in response to increased mechanical stress of the left ventricular wall by aortocaval shunt formation or intraventricular balloon expansion.17 Similar increases in HIF-1α protein have been reported in fibroblasts that were cyclically stretched for 24hr.35 Although HIF-1α and HIF-2α have been identified in vascular tissues,13, 36–38 their role in venous tissue has not been thoroughly examined. The present RT-PCR experiments demonstrate that HIF-1α and HIF-2α mRNA are expressed in rat IVC. Importantly, the HIF-1α and HIF-2α mRNA are overexpressed in rat IVC subjected to prolonged increases in wall tension, supporting an association between prolonged mechanical vein stretch and HIF-regulated pathways. Also, Western blots revealed no HIF-1α and little HIF-2α protein in IVC under control basal tension, but significant increases in IVC subjected to prolonged stretch. Thus both RT-PCR and Western blot data support the contention that prolonged vein stretch is associated with increased expression of HIF-1α and HIF-2α.

It is unlikely that the upregulation of HIF-1α and HIF-2α mRNA and protein in IVC subjected to prolonged mechanical stretch was due to external hypoxic stimuli. The culture medium in which the IVC was incubated when subjected to prolonged tension was exposed to room air. Also, the contraction measurements in IVC exposed to either control basal tension or prolonged stretch were performed in Krebs solution bubbled with oxygen. Furthermore, hypoxia usually causes stabilization of HIF protein rather than de novo mRNA expression.13, 15 The observations that the HIF inhibitor U0126 and to a less extent echinomycin inhibited the overexpression of HIF-1α and HIF-2α mRNA and prevented the reduction in IVC contraction associated with prolonged vein wall stretch are consistent with a role of HIF-1α and HIF-2α in the reduced venous contractile response during wall stretch (Fig. 6). Also, the observation that the HIF stabilizer DMOG did not reduce HIF-1α and HIF-2α mRNA expression while further reducing contraction supports the contention that HIF-1α and HIF-2α are involved in the reduced contraction associated with prolonged vein stretch.

We examined whether the regulation of venous contraction by mechanical stretch and HIF also involves MMPs. Studies have shown that the expression and activity of MMP-2 and MMP-9 are regulated by HIF.39, 40 Consistent with our previous report,6 the prolonged increases in IVC tension were associated with increased expression of MMP-2 and MMP-9 mRNA and protein. Importantly, the increased MMP-2 and MMP-9 mRNA associated with prolonged vein stretch was reversed by the HIF inhibitors U0126, 17-DMAG and echinomycin, supporting our hypothesis that overexpression of MMP-2 and MMP-9 in rat IVC subjected to prolonged mechanical stretch is regulated by HIF. Also, the rat IVC treated with DMOG during prolonged increases in vein wall tension still demonstrated upregulation of MMP-2 and MMP-9 mRNA and further reduction in IVC contraction, consistent with a potential relation between prolonged mechanical vein stretch, increased HIF expression, upregulation of MMPs and reduced venous contraction.

The mechanism of HIF regulation by mechanical stretch is unclear, but may involve PI3K and MAPK.14, 15, 18 Cell membrane ion channels, integrins, and receptor tyrosine kinases are mechano-sensitive to stretch.41 Mechanical stretch may stimulate PI3K by activating Ca2+ influx through transient receptor potential ion channels such as TRPV4.42 Also, integrins may transduce mechanical stretch to initiate signaling cascades and cause MAPK activation.43 Receptor tyrosine kinases and G protein-coupled receptors are also stimulated by biomechanical stress with subsequent activation of MAPK.44 Furthermore, mechanical stretch may increase the generation of reactive oxygen species, which activate MAPK.45, 46 U-0126 may inhibit MAPK.15, 18 The observation that the increased HIF-1α and HIF-2α mRNA expression and the reduced IVC contraction associated with prolonged stretch were reversed by U-0126 suggests a role of MAPK in the regulation of HIF by mechanical stretch.

Although 17-DMAG inhibited the overexpression of HIF-1α, HIF-2α, MMP-2 and MMP-9, it did not reverse the reduced contraction in IVC subjected to prolonged stretch. 17-DMAG is a geldanamycin-derived heat shock protein 90 (Hsp90) inhibitor which promotes HIF-α protein destabilization and degradation. Hsp90 stabilizes HIF-α by acting as a molecular chaperone that associates with HIF-α during nuclear translocation.8, 14 However, Hsp90 may affect other pathways and regulate vascular tone through nitric oxide synthase and superoxide anion.47, 48 The inhibition of Hsp90 by 17-DMAG could cause venorelaxation effects unrelated to HIF inhibition.

In conclusion, prolonged increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α, increased MMP-2 and MMP-9 expression and reduced venous contraction in rat IVC (Fig. 6). The data are consistent with the view that increased vein wall tension secondary to venous hypertension may induce HIF overexpression, and cause an increase in MMPs expression and reduction of venous contraction, leading to progressive venous dilation and varicose vein formation. We should note that HIF-1 may target other proteins such as vascular endothelial growth factor (VEGF) and could affect other processes such as cell apoptosis, glucose metabolism, pH regulation and cell cycle and the role of these responses in the changes in vein function should be examined in future studies

CLINICAL RELEVANCE.

Varicose veins is a major disease charcterized by venous dilation and tortuousity, and involves structural changes in the vein wall, including fragmentation of elastin and disorganized venous smooth muscle. Venous hypertension is often associated with varicose veins and chronic venous insufficiency. Also, MMPs are overexpressed in the varicose vein wall, and have been shown to promote venous dilation. Hypoxia-inducible factors (HIFs) also increase with mechanical stretch. The present study examined whether upregulation of HIFs is an intermediary mechanism linking the increases in vein wall tension to the increases in MMP expression and venous dilation. The results suggest that protracted elevation in venous pressure and vein wall stretch induce HIF overexpression and cause an increase in the expression of MMPs, which in turn produce venous dilation and cause further increases in venous pressure, leading to a recalcitrant cycle, and resulting in progressive venous dilation and varicose vein formation. Specific HIF inhibitors and MMP antagonists could be useful tools in disrupting the link between mechanical vein wall stretch and venous dilation.

Acknowledgments

R.A. Khalil was supported by grants from The National Heart, Lung, and Blood Institute (HL-65998 and HL-70659), and The Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD-60702). C.S. Lim was a recipient of the Royal College of Surgeons of England and Rosetrees’ Trust Research Fellowship, Simpson-Smith Travelling Scholarship, the United Kingdom Venous Forum Travelling Fellowship, European Venous Forum Pump Priming Grant and a Research Grant from the European Society for Vascular Surgery. Y. Xia was a visiting scholar from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P. R. China, and a recipient of a scholarship from the China Scholarship Council. We would like to thank Virak Mam for his assistance in the setting of the vein contraction experiments.

List of Abbreviations

Ca2+

calcium

FIH

factor-inhibiting HIF

HIF

hypoxia-inducible factor

HRE

hypoxia-responsive element

Hsp90

heat shock protein 90, mitogen-activated protein kinase

MMP

matrix metalloprotease

PHD

prolyl hydroxylases domain

PHE

phenylephrine

PI3K

phosphatidylinositol 3-kinase

VSM

vascular smooth muscle

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