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Cardiovascular Research 2000 47(2):384-393; doi:10.1016/S0008-6363(00)00111-5
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Copyright © 2000, European Society of Cardiology

Evidence for modulation of genes involved in vascular adaptation by prolonged exposure of endothelial cells to shear stress

Mauro Bongrazio*, Clemens Baumann, Andreas Zakrzewicz, Axel R Pries and Peter Gaehtgens

Department of Physiology, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany

* Corresponding author. Tel.: +49-30-8445-1635; fax: +49-30-8445-1634 mbon{at}zedat.fu-berlin.de

Received 13 January 2000; accepted 19 April 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Shear stress is known to modulate gene expression. However, the molecular link between blood flow and long-time vessel adaptation is still unclear. In this study, the variations of gene expression by prolonged shear stress exposure was investigated in order to identify genes possibly involved in flow dependent vascular adaptation. Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress (6 dyn/cm2; 24 h) and analyzed by differential display (DDRT-PCR). Flow-modulation of differentially expressed genes by different exposure times (4, 24, 48 h) and in human cardiac microvascular endothelial cells (HCMECs) (24 h exposure) was analyzed by RT-PCR and northern blotting. Results: DDRT-PCR analysis displayed 13 down- and 20 up-regulated products in response to flow. Four known genes were identified: Angiopoietin-2, a protein reported to reduce vessel stability, was progressively (4–48 h) down-regulated by shear stress. The induction of the anti-angiogenic metalloproteinase METH-1 was maximal after 4 h exposure and sustained over the time (24–48 h). Growth arrest-specific mRNA 3 (gas3) and calpactin 1 light chain (p11) were up-regulated only by prolonged exposure (24–48 h). Analysis of the expression of angiopoietin-2, METH-1, gas3, and p11 in shear stress exposed (24 h) HCMECs showed modulation patterns comparable to those observed in HUVECs. Conclusion: Since angiopoietin-2 and METH-1 are known to be involved in vessel regression/stabilization, the reported modulation of these genes by prolonged shear stress exposure strongly suggests their participation in flow-dependent vascular adaptation.

KEYWORDS Blood flow; Endothelial function; Gene expression; Remodeling


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Growing evidence suggests a key role of shear stress in the regulation of many endothelial functions [1–4]. Exposure of endothelial cells to shear stress induces a rapid response including hyperpolarization, elevation of intracellular Ca2+, and increase of nitric oxide release (for review see [5]). This first phase is followed by a more delayed molecular response involving the modulation of gene expression and subsequent protein synthesis [1–4]. Shear stress regulated genes could serve as a link between flow conditions and long-time vessel adaptation. Among the known shear stress inducible genes, growth factors, for instance PDGF-A, PDGF-B, TGF-β1 [6,7], could be involved in structural vessel adaptation. However, it is unlikely that these molecules are sufficient to control the whole process because of the large variety of hemodynamic and metabolic situations entailing the adaptation of vascular networks [8]. For example, capillary growth is observed downstream of chronic vessel occlusions [9] and during chronic vasodilation with increased blood flow [10]. Furthermore, adaptation of vascular networks to functional demands needs capillary sprouting (angiogenesis), vessel regression, and vascular remodelling. Therefore, it is likely that the number of shear-regulated genes contributing to flow-dependent vessel adaptation is greater than reported to date.

To detect additional shear stress regulated genes possibly involved in adaptation of vascular networks, human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress for 24 h and their gene expression analyzed by differential display (DDRT-PCR) [11]. Flow-dependent differential gene expression was additionally investigated in human cardiac microvascular endothelial cells (HCMECs)


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell cultures
HUVECs were isolated as previously described [12]. Briefly, umbilical veins were rinsed with Hank's solution to remove remaining blood, filled with 0.2% collagenase type II (Biochrom, Germany), and incubated at 37°C for 15 min. Endothelial cells were washed out of the vein by two rinses with Hank's solution, pelleted, resuspended, and grown on fibronectin (10 µg/ml) coated culture flasks in a humidified incubator at 37°C with 5% CO2. MCDB 131 (500 ml, Biochrom) was used as culture medium, supplemented with fetal calf serum (25 ml), endothelial growth supplement/heparin (2 ml), hEGF (5 µg), hydrocortisone (500 µg), and gentamicinsulfate (25 mg) all purchased as SupplementPack MV® (PromoCell, Germany). Purity of endothelial cells was controlled by positive staining for von Willebrand factor and negative staining for smooth muscle actin. HUVECs were grown to confluency, harvested from culture flasks, and seeded on Primaria® culture dishes (100 mm in diameter; Becton Dickinson, France). These first-passage HUVECs were used for shear stress experiments 1 day after confluency.

HCMECs were isolated as described previously [13]. Briefly, tissue segments (~25 g) obtained from explanted human hearts were perfused for 30 min with Krebs–Henseleit buffer, containing 0.074% collagenase II (Sigma, Germany), 0.012% dispase (5000 U/ml, Collaborative Research, USA), 0.012% trypsin (1:250, Serva, Germany), and 0.27% bovine serum albumin (fraction V, 7.5%, Sigma). After further 20 min incubation in the enzyme solution, capillary fragments were cleared from undigested tissue by filtration through a nylon net (200 µm, Recker, Germany) and seeded on gelatin coated culture flasks. Further purification of endothelial cells was achieved with paramagnetic beads (Dynal, Germany) linked to the Ulex europaeus-I lectin (UEA-I, Sigma). Endothelial cells coupled to the beads were separated using a magnetic field. The cells were cultured with medium 199 containing fetal calf serum (20%), streptomycin (100 µg/ml, Sigma), penicillin (100 U/ml, Sigma), and endothelial cell growth factor (ECGF, 10 ng/ml, Boehringer Mannheim, Germany) in 5% CO2 at 37°C. HCMECs (passage 1–2) were seeded on Petri dishes and used for shear stress experiments 1 day after confluency.

2.2 Shear-stress experiments
To generate shear stress, a rotating cone was inserted into a 100-mm Petri dish, which served as stationary baseplate. The cone-and-plate arrangement provides uniform wall shear stress ({tau}w) over the entire cross-sectional area according to the formula

Formula
where {omega} (rad·s–1) is the angular velocity, {alpha} (rad) the cone angle and {eta} (dyn·s·cm–2) the fluid viscosity. With a medium viscosity of 0.0075 dyn·s·cm–2 at 37°C, a cone angle of 1.0°, and a constant angular velocity of 14.0 (rad·s–1) (rotational speed: 134 rpm), the parameter R, which indicates the relation between centrifugal forces and viscous forces [14], is <1 indicating the presence of laminar flow conditions. The rotational speed was measured continuously by a light emitter/reflection sensor operating in the infrared range (Siemens, Cupertino, CA, USA). The stage carrying the petri dish was elevated against a stopping face preadjusted to ensure that the tip of the cone slightly touched the petri dish. The cone-and-plate apparatus was maintained in a humidified incubator at 37°C with 5% CO2–95% air. Endothelial cells cultivated in petri dishes were placed in the cone-and-plate apparatus and subjected to shear for different times (4, 24, 48 h for HUVECs; 24 h for HCMECs), while control samples were placed under a non-rotating cone for the same time. At the end of the experiment, cells were washed twice with PBS (without Ca2+/Mg2+) and harvested by trypsinization. After centrifugation, pellets were stored in liquid nitrogen until RNA isolation.

2.3 RNA preparation
Total RNA was isolated using the RNeasy Mini kit (Qiagen, Germany). For DDRT-PCR analysis, RNA was treated with RNase-free DNase I (10 U/µl, Boehringer Mannheim) for 20 min at 37°C. DNase buffer contains 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 10 U/µl RNase Inhibitor (Ambion, USA). DNase I was denatured at 94°C for 5 min and RNA was cleaned up using the RNeasy Mini kit (Qiagen).

2.4 Primers
Primers are listed in Table 1. For DDRT-PCR analysis, three different one-base anchored oligo-dT primers (LH-T11-A, LH-T11-C, LH-T11-G) were used for cDNA synthesis and combined with 24 long (18–23 nt) arbitrary primers (l-AP1–l-AP24) for subsequent PCR. Semi-quantitative RT-PCR for bands 1/K, 41/A, 8/Q, 9/B, 19/H, 48/J, 27/O was performed using specific primers picked from the sequenced products (PRIMER3 program). For bands 17/F and 49/G, both identifying h-METH-1 [15], the primers (MET-l and MET-r) were picked from a distinct region of the known mRNA sequence (Acc. No.: AF06152; target nt 1961–2240). Primers for human glyceraldehyde phosphate dehydrogenase (GAPDH; Acc. No.: X01677 [GenBank] ) amplify a 977-bp cDNA region from nt 73 to 1051. For h-C-type natriuretic peptide (CNP; Acc. No.: D90337 [GenBank] ) and h-endothelin-1 (ET-1; Acc. No.: Y00749 [GenBank] ) target regions are nt 977–1145, nt 473–782, and nt 559–791, respectively.


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  		Table 1 Primer sequences

 
2.5 Differential display
The original DDRT-PCR protocol [11] was modified by adoption of longer primers, improved cycling strategy, and silver staining detection in order to reduce the incidence of false positives [16–18]. In detail: total RNA (2 µg) was heated at 70°C for 8 min in the presence of 62.5 pmol of LH-T11-A, LH-T11-C or LH-T11-G and quickly chilled on ice. After addition of first strand buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 µM dNTP, final concentrations) the samples were incubated for 10 min at 25°C. SuperScript II (200 U, Life Technologies, USA) was added to a total volume of 25 µl, incubated at 42°C for 1 h, and inactivated at 70°C for 10 min. cDNA was then diluted by addition of 75 µl H2O and 4-µl aliquots were used as template for successive PCR. Samples were denatured (94°C for 3 min), quickly chilled on ice, and 6 µl of PCR reaction mix were added. Final concentrations in the reactions were: 75 mM Tris–HCl, pH 8.8, 20 mM (NH4)2SO4, 0.01% (v/v) Tween 20, 1.5 mM MgCl2, 200 µM dNTP, 1 µM of the corresponding LH-T11-X (X=A, C, G), 1 µM of arbitrary primer (l-AP1-l-AP24), 0.5 U ThermoprimePlus (Advanced Biotechnology, UK). Cycling parameters were set as follows: three cycles at low stringency (94°C for 1.5 min; 40°C for 2 min; 72°C for 1.5 min) and 30 cycles at high stringency (94°C for 1.5 min; 55°C for 1 min; 72°C for 1.5 min) conditions. Four microliters of PCR reactions were resolved on non-denaturing polyacrylamide gels (4.5%) containing 15% (w/v) urea. Gels were run at 200 V constant voltage in a 0.6x TBE buffer system. After electrophoresis, amplification products were detected by silver staining [19].

2.6 Reamplification and sequencing of differentially expressed bands
Differentially expressed bands were scraped using a sterile pipette tip and gel slices were boiled in 50 µl H2O for 20 min. Aliquots (15 µl) of supernatants were used as template for PCR reamplification in 75 mM Tris–HCl, pH 8.8, 20 mM (NH4)2SO4, 0.01% (v/v) Tween 20, 2 mM MgCl2, 200 µM dNTP, 0.5 µM of the corresponding primers, 1.5 U ThermoprimePlus (Advanced Biotechnology). Cycling conditions were: 94°C for 2 min; 35 cycles at 94°C for 45 s, 55°C for 45 s, 72°C for 1 min; 72°C for 5 min final extension. Successfully amplified products were gel purified in 1.5% TAE agarose gel and recovered using GeneElute Minus EtBr spin columns (Supelco, USA). DNA was subjected to cycle sequencing (BigDye terminator cycle sequencing, Perkin-Elmer, USA) using the primers used for DDRT-PCR. Analysis for sequence homology to known genes was performed using the BLAST program.

2.7 Semi-quantitative RT-PCR
Total RNA (2 µg) and oligo-dT12–18 (2 µg, Pharmacia, Sweden) were heated at 70°C for 8 min and quickly chilled on ice. Reaction buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 µM dNTP) and 200 U SuperScript II (Life Technologies) were added in a final volume of 25 µl. Reactions were incubated at 42°C for 1 h followed by inactivation at 70°C for 10 min. cDNA was diluted to a final volume of 100 µl and aliquots (10 µl) were used for PCR amplification. Cycling parameters were optimized to remain in the exponential phase of amplification. PCR mix contains: 75 mM Tris–HCl, pH 8.8, 20 mM (NH4)2SO4, 0.01% (v/v) Tween 20, 1.5 mM MgCl2, 200 µM dNTP, 1.0 U ThermoprimePlus (Advanced Biotechnology), and 1 µM of both primers. For GAPDH primer concentration was 20 nM. GAPDH was used to normalize the results and amplified in independent reactions. PCR products were separated on agarose gels and stained with ethidium bromide. For quantification experiments, gels were subjected to image analysis (ONE-Dscan, Scanalytics, USA). Optical density ratio (ODR) indicates the ratio between the OD of the band of interest and that of GAPDH. Results are expressed as mean±S.D. n refers to the number of umbilical cords used for isolation of endothelial cells. Statistical significance was determined by Student's t-test. Significance was assumed at a value of P<0.05.

2.8 Northern analysis
A T7 RNA polymerase promoter was ligated to the PCR products (Lig’nScribe, Ambion, USA) and biotin labelled antisense RNA probes were generated using the North2South kit (Pierce, USA). Total RNA (10–20 µg/lane) was resolved on 1.2% agarose gels containing 2.2 M formaldehyde. Blotting onto positively charged nylon membrane (Nytran SuPerCharge, Schleicher & Schuell, Germany) was performed by downward capillary transfer using 10xSSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) as transfer buffer. Blots were prehybridized for 60 min at 68°C in UltraHyb hybridization solution (Ambion). Hybridization was performed at 68°C (16–24 h) in UltraHyb containing 10–20 ng/ml of biotin-labelled probes. Two washes (68°Cx30 min) were performed in 0.1xSSC, 0.1% (w/v) SDS. Hybridizing probes were detected by chemiluminescence using CDP-Star (Tropix, USA) as substrate.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
To verify the validity of our experimental model for shear stress dependent gene modulation, we investigated the expression of C-type natriuretic peptide (CNP) and endothelin-1 (ET-1) using RT-PCR (Fig. 1). These genes have already been shown to be modulated in shear stress exposed endothelial cells [20,21]. Our results confirmed the previously reported expression pattern for CNP and ET-1 showing a shear stress dependent up- (CNP) and down-regulation (ET-1) of their expression.


Figure 1
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  		Fig. 1 RT-PCR analysis of known shear stress modulated genes in untreated (U) and shear stress (S) exposed (6 dyn/cm2; 24 h) HUVECs. Amplification was performed at 60°C annealing temperature for different cycle numbers. PCR products were resolved on 2% agarose gels and stained with ethidium bromide. GAPDH was used as control.

 
In order to screen flow regulated gene expression, untreated and shear stress (6 dyn/cm2; 24 h) exposed HUVECs were analyzed using a modified DDRT-PCR protocol. A representative fingerprint is shown in Fig. 2. The combination of three one-base anchored oligo-dT primers (LH-T11-A/C/G) and 24 arbitrary primers (l-AP1–l-AP24) displayed 33 products (100–900 bp), which were reproducibly (two independent reactions) affected by shear stress exposure (Table 2). Thirteen bands were down-regulated and twenty up-regulated by shear stress. Sixteen bands were successfully sequenced and analyzed for homology to known sequences (BLAST). Eight human genes were identified: Angiopoietin-2 (Acc. No.: AF004327 [GenBank] ), KIAA800 protein (Acc. No.: X15414 [GenBank] ), aldose reductase (Acc. No.: X15414 [GenBank] ), METH-1 (Acc. No.: AF06152), growth arrest-specific mRNA 3 (gas3) (Acc. No.: L03203 [GenBank] ), and p11 protein (calpactin 1 light chain) (Acc. No.: M81457 [GenBank] ). Band 11/P presents a moderate homology (61–82%) to regions containing L1 repeat elements. The remaining sequenced products revealed no significant homology to known genes.


Figure 2
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  		Fig. 2 Representative RNA fingerprints obtained by DDRT-PCR analysis of HUVECs. Each pair of lanes results from amplification of cDNA from untreated (U) or shear stress (S) exposed (6 dyn/cm2; 24 h) cells using the indicated primer combinations. Amplification products were separated on non-denaturing polyacrylamide (4.5%) gels and detected by silver staining. Arrows indicate two shear stress regulated PCR fragments.

 

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  		Table 2 Results of DDRT-PCR analysis for untreated and shear stress exposed (6 dyn/cm2; 24 h) HUVECs

 
In order to validate data obtained by DDRT-PCR, detected products were further analyzed by semi-quantitative RT-PCR. Of the nine tested products, six were confirmed as shear stress modulated (Fig. 3a and b). Five plots are shown since two bands (17/F and 49/G) mapped the same gene (METH-1) and were simultaneously analyzed using primers specific for this mRNA. Two products (41/A, 9/B) were down-regulated in response to prolonged shear stress exposure. mRNA for angiopoietin-2, identified by band 41/A, was markedly reduced after application of shear stress. In contrast, the expression of band 9/B was only slightly down-regulated by shear stress exposure. Three genes, corresponding to four differentially expressed DDRT-PCR sequences, were up-regulated in shear stress-treated cells. This effect was particularly pronounced for METH-1, detected by band 17/F and 49/G in DDRT-PCR screening. The expression of this mRNA was barely detectable in untreated HUVECs, but highly induced in response to shear stress exposure. Similarly, the intensity of band 19/H (gas3) was strongly increased in shear stressed cells compared to cells cultured in static conditions. Band 48/J, corresponding to the light chain (p11) of calpactin 1, was moderately expressed in untreated HUVECs and induced after shear stress exposure. Three DDRT-PCR products (bands 1/K, 8/Q, 27/O) could not be confirmed as differentially expressed (data not shown).


Figure 3
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  		Fig. 3 Analysis of differential expression by semi-quantitative RT-PCR. Amplification conditions (cycle number, annealing temperature) were: 41/A=25, 58°C; 9/B=24, 62°C; METH-1=25, 62°C; 19/H=24, 63°C; 48/J=17, 58°C; GAPDH=26, 60°C. (a) Representative agarose gel of amplified PCR products. U=untreated; S=shear stress exposed (6 dyn/cm2; 24 h). (b) Quantification of ethidium bromide stained PCR products. Data (mean±S.D.; untreated n=8, sheared n=6) are expressed as optical density ratio (ODBAND/ODGAPDH). *, P<0.01 and **, P<0.05 vs. untreated.

 
The expression of angiopoietin-2, METH-1, gas3, and p11 was further analyzed by northern blot in shear exposed (24 h) HUVECs and HCMECs (Fig. 4). Results confirmed the flow-dependent modulation of mRNAs corresponding to the expected size for each gene and showed comparable expression patterns in the two endothelial cell types.


Figure 4
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  		Fig. 4 Northern blot analysis for angiopoietin-2, METH-1, gas3, and p11 in different endothelial cell types. Total RNA from HUVECs (20 µg) and HCMECs (10 µg) was hybridized with biotinylated antisense RNA probes and detected by chemiluminescence. U=untreated, S=shear stress exposed (6 dyn/cm2; 24 h). Molecular weights of the corresponding mRNAs are shown. GAPDH and 28 S were used as control for RNA loading.

 
In order to investigate the variations of angiopoietin-2, METH-1, gas3, and p11 expression by different exposure times, HUVECs were exposed for up to 48 h to shear stress and analyzed by RT-PCR (Fig. 5). The expression of angiopoietin-2 was significantly reduced after 4 h exposure and further down-regulated at 24–48 h. The up-regulation of METH-1 was maximal after 4 h shear stress treatment and sustained over the time. The expression of gas3 and p11 was not modified after 4 h shear stress exposure but significantly up-regulated by prolonged (24–48 h) exposure. No significant time-dependent variation of gene expression was observed in untreated HUVECs (data not shown).


Figure 5
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  		Fig. 5 Time-dependent modulation of angiopoietin-2, METH-1, gas3, and p11 by shear stress exposure. HUVECs were maintained in static conditions (0 h) or exposed for the indicated times to laminar shear stress (6 dyn/cm2). Semi-quantitative RT-PCR was performed as described in Fig. 3. PCR products were separated on agarose gels and stained with ethidium bromide. Optical density was measured and normalized using GAPDH as control. Data (mean±S.D.) are expressed as optical density ratio (ODBAND/ODGAPDH). For 0, 4, 24 and 48 h data, n was 14, 6, 8 and 6, respectively. *, P<0.01 and **, P<0.05 vs. 0 h; {dagger}, P<0.01; {ddagger}, P<0.05.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study revealed 33 mRNA species modulated by prolonged exposure of HUVECs to laminar wall shear stress. Four known human genes were shown for the first time to be shear stress modulated under these conditions: angiopoietin-2, METH-1, gas3, and calpactin 1 light chain.

In this study we identified a number of previously unknown shear stress modulated genes. In our approach we do not screen the entire mRNA pool, thus additional flow regulated genes should be expected by further analysis. None of the shear stress modulated genes described in previous studies were detected in the present report. Since by a random screening approach like DDRT-PCR [11] the probability to detect a specific differentially expressed gene is inversely proportional to the total number of modulated genes the pool of shear stress modulated mRNAs should be relatively large. Taken together, our observations agree with previous report [22] indicating that the number of shear stress modulated genes is probably much larger than the so far reported.

The present study shows for the first time shear stress dependent down-regulation of angiopoietin-2. Angiopoietin-2 competes with angiopoietin-1 for binding to their common endothelial receptor Tie2 [23]. Experiments in angiopoietin transgenic mice embryos underline the role of the two proteins in regulation of vessel structure by which angiopoietin-1 is involved in preserving vessel integrity while angiopoietin-2 leads to loss of structure and matrix contact [23]. In adult human tissues, mRNA for angiopoietin-2 is exclusively expressed in ovary, placenta, and uterus [23], where physiological angiogenesis by capillary sprouting occurs. Before capillary sprouts can establish contact to other capillaries, their endothelium has to proliferate and migrate under no-flow conditions. According to the present findings, expression of angiopoietin-2 may be up-regulated in these cells due to the lack of shear forces at the endothelial surface. This interpretation is supported by the findings that angiopoietin-2 is more abundant at the front of vessel ingrowths into the early corpus luteum [23] and in degenerating vessels of aging corpora lutea [23] where the blood flow is also reduced or even absent. In contrast to angiopoietin-1, angiopoietin-2 is able to sustain VEGF-induced neovascularization [24]. In turn, VEGF increases the expression of angiopoietin-2, but not that of angiopoietin-1 [25,26]. Furthermore, it has been demonstrated that endothelial cells migrate along a chemotactic gradient of angiopoietin-1 but not angiopoietin-2 [27].

With the above-cited biological effects of angiopoietins in mind, our finding of shear stress-dependent down-regulation of angiopoietin-2 mRNA could explain vessel adaptation via capillary sprouting or vascular regression in a number of hemodynamically different situations. Poststenotic sprouting may involve up-regulation of angiopoietin-2 due to reduced blood flow combined with hypoxia-induced VEGF expression [28]. The high expression of angiopoietin-2 in no-flow capillary sprouts may establish a ‘chemotactic gradient’ of angiopoietin-1 [27] directing the sprouting vessel to establish contact with perfused vessels. Conversely, the preponderance of angiopoietin-1 effects caused by shear stress-induced down-regulation of angiopoietin-2 may explain the stabilization and maturation of vessel structures in the presence of blood flow.

In this study we describe the shear stress dependent induction of METH-1, a protein containing metalloprotease and thrombospondin domains [15]. METH-1, a human ortholog of mouse ADAMTS-1 [29], was originally identified in a human heart cDNA library [15]. The protein is secreted and proteolytically processed [15]. Functional studies showed that METH-1 was able to suppress fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited VEGF-induced angiogenesis in the chorioallantoic membrane assay [15]. Consistent with an endothelial specific response, METH-1 was shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth [15]. On the basis of this evidence, the induction of METH-1 under flow conditions observed in our study suggests a possible involvement of this protein in controlling the maintenance of vessel structure under normal flow conditions. By contrast, down-regulation of METH-1 by reduced flow, i.e. lack of endothelial growth inhibition, could serve as a prerequisite for adaptation of vascular networks.

The present study also reports the shear stress induced expression of human growth arrest-specific mRNA 3 (gas3). Gas3, originally described in human fibroblasts [30], is involved in cell cycle regulation and cell differentiation [31]. This protein is also expressed in Schwann cells where it represents a major component of the peripheral nerve myelin (PMP22) [32]. Recent reports strongly support the correlation between expression of gas genes and positive regulation of the cell cycle [33]. Thus, the up-regulation of gas3 by shear stress could be involved in the control of endothelial cell growth by altered flow conditions.

Band 48/J identified the mRNA for p11 protein [34], the light chain of calpactin 1. Its expression was markedly increased in HUVECs by shear stress. The heavy chain of calpactin 1 acts as a receptor for TNfnA-D [35], a splice variant of the matrix glycoprotein tenascin C. TNfnA-D is involved in tissue rearrangement, since it induces the loss of focal adhesions, supports the mitogenic response to growth factors, and enhances cell migration [36]. Thus, an increase of calpactin 1 expression in shear stress exposed endothelial cells could modulate shear stress induced vascular adaptation by binding of TNfnA-D. On the other hand, calpactin 1 is required for efficient regulation of exocytosis [37]. Therefore, its induction by shear stress in endothelial cells may correlate with the increased release of mediators, especially growth factors, under these conditions [2].

Taken together, the current study supports the concept that shear stress is able to modulate the expression of a much larger pool of genes than described to date. The reported shear stress dependent modulation of genes involved in vessel regression/stabilization strongly proposes their participation in flow-mediated adaptation of vascular networks.

Time for primary review 25 days.


   Acknowledgements
 
We thank Dr. M. Gräfe for providing human cardiac microvascular endothelial cells. This work was supported by Deutsche Forschungsgemeinschaft, Za 184-1-2.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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