Independent application of pressure and shear stress on endothelial cells in controlled bioreactors

Federico Vozzi1, Francesca Bianchi2, Arti Ahluwalia2, Claudio Domenici1

1)Biomimetic Materials and Tissue Engineering Laboratory, CNR Institute of Clinical Physiology, Via G. Moruzzi 1, Pisa, Italy 2)Research Center “E. Piaggio”, University of Pisa, Via Diotisalvi 2, Pisa, Italy

Abstract

The principal physical forces exerted on the blood vessel wall by the passage of intra-luminal

blood are pressure and shear. In order to analyze their independent effects, these two stresses were

applied to cultured cells by means of two different bioreactors: the Pressure Controlled Bioreactor

(PCB) and the Laminar Flow Bioreactor (LFB), in which controlled levels of pressure and shear

stress can be respectively generated.

Using the bioreactor systems, Endothelin-1 and Nitric Oxide release from human umbilical vein

endothelial cells (HUVEC) were measured in varying conditions of shear stress and pressure. As

result, a decrease of Endothelin-1 production from the cells cultured in both bioreactors, with

respect to the controls, was observed, whereas Nitric Oxide synthesis was up-regulated only in the

presence of shear stress but not modulated by hydrostatic pressure.

These results show that the two hemodynamic forces acting on blood vessels affect endothelial

cell function in different ways, and that both should be considered when planning in vitro

experiments in the presence of flow.

1 Introduction

The most basic physical requirements for maintaining mammalian cells in culture are a pH value

of 7.2-7.4 and a temperature of 37 °C. For decades the backbone of in vitro cell culture has been

the incubator: an oven in which cell cultures can grow in an appropriate pH and temperature

controlled environment. In the last few years, owing to the failure of cell culture to predict in vivo

tissue responses, the issue of the environment discrepancy problem has been raised. The

prevailing view is that the classic in vitro culture environment does not adequately replicate the

physical forces and chemical concentration gradients present in vivo. As a result of this, several

alternative culture systems (bioreactors) have been developed in order to create dynamic cell

culture devices, which allow the physiological conditions of an organism to be simulated [1,2,3].

The goal of an effective bioreactor is to influence and control biological reactions and functions.

To accomplish this, in the design of the instrument, two critical aspects must be taken into

account. One of them is related to the suitable physical parameters (like geometrical dimensions,

flow rate, etc.) required to obtain the desired macro-kinetic environment. The other area of major

importance in bioreactor design involves the appropriate control of parameters which influence

biomolecular reactions including: controlled temperature, optimum pH-value, sufficient substrate

(usually a carbon source such as sugars), O2 and CO2 control, which can influence cell growth and

metabolite production. In addition, to be able to control these parameters, the bioreactor must be

capable of maintaining an appropriate cell phenotype for extended periods of time and eliminate

or reduce contamination by unwanted organisms. Among the different types of cell culture

bioreactors described in the literature, many are used in cardiovascular studies, such as laminar

flow bioreactor [4], perfusion bioreactor [5], multi-cue bioreactor [6] etc. While several groups

have performed in depth studies of endothelial cell function as a function of flow [7,8], most of

the recent systems reported are directed towards tissue engineering [9−12]. On the other hand

very little work has been done on the influence of pressure on endothelial cell structure and

function [13,14], often using in vitro systems as vessel simulators addressed to generate a

pulsatile flow, in combination with stretch, capable of simultaneously inducing shear stress and

pressure [15,14], or focusing on the characterization of the apparatus with respect to its

biomechanical and hemodynamic properties [16].

Endothelial cells cover the lumen of blood vessels and play an essential role in the regulation of

vessel homeostasis and of flow and blood pressure in the circulatory system [17,18]. The

endothelium is a dynamic system which is able to respond to different physical and chemical

conditions through the production of many vasoactive substances such as: Endothelins,

Prostaglandin and Nitric Oxide. These molecules act on the smooth muscle cells that form the

vessel side and together play a critical role in coordinating and maintaining vascular function.

Since its discovery, a great deal of effort has been devoted to understanding the role of Endothelin

(ET), a family of peptides involved in cardiovascular homeostasis, and their potential contribution

to many pathological processes [19,20]. The main vascular isoform is Endothelin-1 (ET-1), a 21

amino acid peptide [21]. In vivo studies have shown that ET-1 causes concentration dependent

arterial and venous constrictions and that it is more potent as a venoconstrictor [22]. The ET's

counter molecule is Nitric Oxide (NO), a potent vasodilator that acts on smooth muscle cells.

Moreover, it is demonstrated that the Endothelin-NO system participates in numerous physio-

pathological conditions, including hypertension, atherosclerosis and coronary artery disease [23].

Stress, in particular mechanical pressure and stretch, induces ET release in vivo [24]. A few

in vitro studies relating flow, pressure and pulsatility to ET and NO production have been

reported. Walshe et al. [25] described a pulsatile flow system in which a co-culture of endothelial

and smooth muscle cells showed an increase of both NO and ET-1 with flow. Dai et al. [26]

reported on a venous flow simulator in which endothelial cells up-regulated NO synthesis with the

flow, while there are no reported differences in ET-1 production. In Tsukurov et al. [27], the

combined pressurized flow and cyclic strain down-regulated ET-1 production after 48 h, but not at

24 h, whereas the results for NO production were not clear. Braddon et al. [28] used a parallel

flow chamber, where endothelial cells were cultured in tissue culture plastic, or a feeder layer of

fibroblasts, or on woven polymer. The results of their study showed that NO production increases

with flow. In general, it is unclear as to whether ET-1 and NO production is pressure or flow

dependent, particularly when high flow rates are used, or if they depend on factors such as

substrate deformation. In some reports it is unclear as to what forces are actually being imposed

on the cells, and which stimulus actually causes the up- or down-regulation of these substances.

This is because high wall shear stresses usually require the application of high pressure gradients

across a sample. In fact, most commercial laminar flow systems consist of very narrow chambers;

to obtain physiological wall shear stress values in the chambers it is necessary to apply high

pressure gradients which means generating pressure differences of up to 10 mmHg or more

between inlet and outlet. Thus cells are stimulated by both shear stress and pressure.

In order to obtain more information on cell response to acute pressure and shear stress stimuli, we

report here a study carried out using two new cell-culture bioreactors, which form part of an array

of systems developed at the Research Centre “E. Piaggio”, University of Pisa [29]. While the

basic fluid and environmental control system is identical for each reactor, the cell culture chamber

can be designed and configured according to experimental requirements. The Pressure Controlled

Bioreactor (PCB) and the Laminar Flow Bioreactor (LFB), were tested by evaluating the effect of

pressure and low shear stress on human umbilical vein endothelial cell cultures, through

measurement of ET-1 and NO levels. Using these two systems, and applying the two forces in

independent well-controlled experiments we show that it is possible to determine endothelial cell

response to hydrostatic pressure and to different levels of shear stress unequivocally.

2 Materials and methods

2.1 Cell culture

HUVEC (human umbilical vein endothelial cells) were isolated from fresh human umbilical cord

veinsby enzymatic treatment with collagenase. After pooling from from multiple donors, they

were used between passages 2 to 8. The cells were cultured as already described by Jaffe et al.

[30]. In particular, endothelial cells were cultured in 75 cm2 flasks, pre-coated with 0.1% gelatin

Type B from bovine skin (Sigma, Milan, Italy) in M199 medium, supplemented with 10% of fetal

bovine serum (FBS), 2 mM of glutamine solution, 1 U/ml of penicillin, 1 µg/ml of streptomycin

(all these products from Lonza Bioscience, Basel, Switzerland), 10 ng/ml of human epidermal

growth factor (hEGF), and 0.5 µg/ml of hydrocortisone (both from Sigma, Milan, Italy). The cells

were maintained at a density of 4-7.5 ·105 cells/ml by sub-culturing every 4 to 5 days in a 1:3

dilution in fresh medium. Cells were placed in a humidified incubator with 5% CO2.

The investigation conforms with the principles outlined in the Declaration of Helsinki. The study

was approved by the local ethical committee.

2.2 The Bioreactors

In this study, the Pressure Controlled Bioreactor (PCB) and Laminar Flow Bioreactor (LFB) were

used to test the release of vasoactive factors to pressure variations and different shear stresses.

Both devices have a common structure, which includes different units: a cell culture chamber, an

electronic system for pH-value, flow, pressure and temperature control, electro-valves and

pressure regulators, heating system and control software for real-time monitoring of the

experiments.

The PCB can modulate pressure values on different kinds of cells by means of a control unit

(comprising electro-valves and a pressure regulator) interfaced with a data acquisition system

which allows us to modify, control and record two different parameters: the pH-value of the

medium and the pressure inside the chamber. The bioreactor is a double walled cylindrical glass

culture chamber (outer diameter 90 mm, inner diameter 70 mm, height 26 mm), which was

specifically designed and constructed for this application. Glass is easily sterilizable, and above

all can withstand high pressures. The lid has four orifices, one for medium sampling, one for the

pH sensor (Hamilton, Bonaduz, Switzerland), one for the pressure sensor and regulator (RS,

Cinisello Balsamo, Italy) and the last as a pinch valve (World Precision Instruments, Sarasota, FL,

USA). Hot water from a thermostat flows through the glass jacket in order to maintain a

temperature of 37 °C. A steel frame holds the entire device together; Figure 1A shows the PCB

pressure and pH control circuit while Figure 2 reports the assembled bioreactor complete with

sensors and valves. Both the pH-value and pressure are controlled by the introduction of an

air/CO2 blend through the pressure regulator on the chamber cap. The PBC works under constant

over pressure, and the desired pressure is obtained either by changing the setting of the pinch

valve, or by changing the input pressure. The pH-value is controlled by regulating the amounts of

CO2 or air input into the chamber according to a feedback algorithm based on the output of the pH

meter. The signals are sent to a PC acquisition card. The pressure and pH in the chamber are

controlled by software developed in Visual Basic. Before starting, the system must be calibrated

introducing a constant pressure in the outlet from the electro-valve and verifying that no air losses

are present. After the calibration and the setting of the desired pressure and pH-value values, the

experiment can start. The pressure and pH control algorithms and electronics are described fully

in Mazzei et al. [29].

In the LFB system, a peristaltic pump and a fluid reservoir or mixing chamber is added to the

circuit to generate different rates of shear stress, while the pressure control circuit is bypassed.

The mixing chamber serves to oxygenate media, and in particular, to reduce pressure fluctuations

from the pump, the chamber is positioned between the pump and the LBF inlet. In this way it acts

as a pressure capacitor, smoothing any fluctuations from the pump. Graphs showing the minimal

pressure fluctuations using this configuration are reported in the supplementary data. The chamber

is fabricated in polydimethylsiloxane (PDMS, Sylgard 184®) (Dow Corning, Midland, MI, USA),

a biocompatible silicone polymer, through milli-molding [29]. The cell chamber, shown in

Figure 2A, is 160 mm long, 20 mm wide and 1 mm high, with a 150 µm deep rectangular

insertion for glass or plastic coverslips. Its particular shape was obtained after an accurate

modeling analysis performed with finite element software for simulation of fluid dynamic flow

(COMSOL Multiphysics, Stockholm, Sweden) in order to obtain a central region with a well-

defined and uniform wall shear stress. We used the single phase incompressible fluid flow regime

described by the Navier Stokes equations, imposing a fluid with the same density (1 g/cm3) and

viscosity as cell culture medium (1.4 mPa·s) at 37°C. No slip boundary conditions were imposed

at the walls and zero pressure at the outlet. The 3D model was solved parametrically and in steady

state conditions using different flows at the inlet boundary ranging from 1 to 20 ml/min. In the

central cell culture region the flow is fully developed and wall shear stress, τ , ranged from 0.07

to 1.4 dynes/cm2. In Figure 2B the simulation of velocity field in the LFB cell culture chamber is

reported for the xy plane at a flow rate of 15 ml/min, showing a uniform velocity and hence shear

stress in the central zone, where the maximum fluid velocity is 2.5 cm/s. At this flow rate the

pressure difference across the bioreactor is of the order of a few tenths of mmHg, as calculated

from the Finite Element Method (FEM) model (Figure 2C), and thus negligible. Further figures

(uniform wall shear stress and velocity profiles) can be found in the supplementary data.

Therefore with this geometry we can simulate different regions of the cardiovascular system

simply by adjusting flow rates. In theory a wide range of flow rates (laminar flow is ensured up to

240 mL/min) and shear stresses can be generated in the system, but the limiting factor is the

pressure difference generated by high flow rates which can give rise to leakage. The maximum

velocity is 50 mL/min, with a wall shear stress in the cell culture region of about 3.5 dynes/cm2.

In this work low flow rates were used to avoid significant pressure gradients across the bioreactor,

which could give rise to hydrostatic pressure effects.

2.3 Pressure variation experiments

When at confluence, HUVEC were seeded on glass slides (dimensions 50 mm x 25 mm),

purposely washed with ethanol 96% and sterilized under UV light for 15-20 min each side. The

slides were subsequently washed with sterile PBS to eliminate traces of ethanol. Successively, the

glass slides were treated with gelatin in the incubator for 15-20 min to improve cell adhesion.

After removal of the surplus gelatin, slides were seeded with the cell suspension (6400 cells/cm2

equivalent to 80000 cells/glass slide). About 48 hours after seeding, the experiments with the

bioreactor, whose components were previously sterilized with H2O2 gas plasma, were started.

Once assembled, the mixing chamber was filled with 10 ml of complete medium for each set of

experiments (3 slides for each). One slide was placed in the PCB which was filled with 10 mL of

complete medium and closed, ready for the experiments.

Endothelial cells are normally exposed to pressures ranging from 2-5 mmHg up to 140 mmHg,

although in cases of acute hypertension pressures can go up to 240 mmHg. In this work the value

of 0 mmHg (with respect to atmospheric pressure), identified as P0, was used to simulate normal

conditions (controls), while a “representative” physiological pressure (about 70 mmHg, identified

as P70) was used to stimulate the in vitro conditioned cells. Besides an increase in pressure, there

is also a slight increase in oxygen solubility. The Henry constant for oxygen is 1.3.10-6 M/mmHg,

thus the increase in oxygen concentration at 830 mmHg is only about 8%. Since the Km

(Micheles Menten constant for mammalian cells, 1 µM) is much lower than the predicted oxygen

concentration (around 0.2 mM), we can assume that the additional 70 mmHg in the physiological

pressure experiment does not contribute significantly to increase oxygen consumption, and the

only stimulus being applied is hydrostatic pressure. Samples were cultured in the bioreactor filled

with fresh medium and placed for 1 hour in the incubator at P0 (as controls), 1 hour at 70 mmHg

(P70) in the pressure bioreactor and, finally, for another hour at P0 in the incubator, to verify cell

recovery. In all cases, the entire volume of medium was collected for the biological tests (ET-1

and NO), and refreshed every hour. At the end of each experiment, cells were analyzed under a

microscope (Olympus IX-81, Olympus Italia, Segrate, Italy) in order to observe structural changes

and for counting using trypan blue exclusion. The experiment was replicated five times.

2.4 Shear stress experiments

For this set of experiments, cells at confluence were seeded on glass slides (dimensions 17 mm x

32 mm x 0.15 mm) with a cell suspension density of 6400 cells/cm2 (equivalent to about 35000

cells/glass slide), treated as previously described. About 48 hours after seeding, the experiments

started with the bioreactor. Once assembled, the mixing chamber was filled with 10 ml of fresh

complete medium M199 and sensors were connected for the control of air/CO2 inlet, thermostatic

system and peristaltic pump (Masterflex L/S, Cole-Palmer, Vernon Hills, IL, USA).

For ET-1 and NO quantization, the experiments were conducted at flow rates of 0 (control), 1.5,

5, and 15 ml/min (for 1 hour each one), corresponding to wall shear stress values of 0, 0.105,

0.35, and 1.05 dynes/cm2 respectively. It should be noted that these are fairly low shear stresses in

comparison to those typically encountered in vivo, with values varying from a few to tens of

dynes/cm2 as reported in literature [31, 32].

If high flow rates are used, then the pressure gradient generated across the slide in the LFB could

give rise to hydrostatic pressure induced effects on the cells, and it is impossible to determine the

effects of the two stimuli unambiguously.

At the end of each experiment samples were analyzed for ET-1 and NO, and cells were stained

with Coomassie brilliant blue stain or trypan blue for micrographs and counting. In order to

quantify the structural re-organization of cells after mechanical stress, the eccentricity values of

cells at the end of every experiment were also analyzed. The cell shapes were approximated to an

ellipse using image processing software [33] and the eccentricity was calculated applying the

formula:

⎟⎟⎠

⎞⎜⎜⎝

⎛−= 2

2

1abe

where e is the eccentricity, a and b are major (longest) axis and minor (shortest perpendicular to a)

axis, respectively. Three experiments were performed for each flow rate and the eccentricity values

were averaged over 60-100 cells in 5 different locations on each slide.

2.5 Biological assays

ET-1 and NO concentrations were assayed in both pressure studies and shear stress experiments.

The concentration of Endothelin-1 was assayed in medium samples by enzymatic immunoassay

with an ELISA kit (IBL America, Minneapolis MN, USA) based on a double-antibody sandwich

technique. Nitric Oxide concentrations were evaluated using a commercial colorimetric assay

(CaymanChem, Ann Arbor, MI, USA) based on the Griess reaction [34]. Fresh medium did not

contain detectable amounts of either compound.

2.6 Statistical tests

Statistical analysis was performed using the Student’s t-test. A p value of less than 0.05 was

considered statistically significant. Since different cell numbers were used in the LFB and PCB all

results are expressed as relative concentrations, with respect to controls.

3 Results

3.1 Pressure variation In each set of experiments, HUVEC cells at confluence on glass slides were subjected to pressure

cycles between high pressure (P70 = 70 mmHg higher with respect to control conditions P0) in the

PCB bioreactor chamber, and control conditions P0 = 0 mmHg, in a standard incubator. The ET-1

and NO concentrations after 1 hour of exposure at the different levels of pressure were evaluated.

The results of ET-1 dosage, shown in Figure 3A, demonstrated that HUVEC react to the pressure

imposed by down-regulating ET-1 production in comparison with control conditions. Moreover

after the second hour at P0, ET-1 levels were restored indicating that the down-regulation is

reversible. On the other hand, NO levels were not changed significantly between P0 and P70

(Figure 3B), and neither was cell morphology. The cells retained their typical cobblestone

appearance throughout the experiments, with no detectable changes in viable cell numbers.

3.2 Shear stress

The experiments carried out using the LFB highlighted the high sensitivity of endothelial cells to

flow. Even at shear stress values much lower than those typical of the in vivo environment, there

were significant variations in ET-1 and NO concentrations, as well as structural modifications

induced by flow. As reported in Figure 4A, shear stress induces down-regulation of ET-1 with

respect to static conditions. This result is in accordance with some literature reports [35,36]. In

particular, the decrease of ET-1 production has been related to a reduction of synthesis of mRNA

for ET-1 in HUVEC [37]. NO concentrations on the other hand increased with shear stress

(Figure 4B), as generally reported in the literature [38,39].

Flow also induced structural reorganization of endothelial cells, even at the low stresses used

here. Endothelial cells, which are known to respond strongly to flow [40], changed their

morphology becoming more elongated and aligning with the direction of flow. This well-known

phenomenon which has been ascribed to the reorganization of F-actin and microtubule networks,

is thought to be activated through shear stress sensitive mechanoreceptors. As can be observed in

the micrographs of Figure 5, HUVEC exposed to different levels of shear stress are elongated

along flow lines, and this effect increases with the increasing shear stress.

In order to compare the eccentricity data obtained using the LFB with eccentricity values of

endothelial cells in vivo, e, was calculated using the data reported in Kim et al. [41].

Interestingly, as shown in Figure 6, we observed that even at very low values of shear stress

(0.35 dynes/cm2) eccentricity is quite similar to that obtained for endothelial cells exposed to

higher shear stress in vivo, demonstrating again that these cells are extremely sensitive to flow.

This morphological effect was not observed in the PCB, indicating that tangential flow rather than

hydrostatic pressure is responsible for these observed cytoskeletal changes.

4 Discussion

Endothelial cells are known to undergo significant changes, such as increased membrane

permeability and expression of adhesion factors, under flow. At present there is some debate as to

whether fluid mechanical forces directly influence cell response to flow through mechano-

sensitive receptors, or if the increased mass transport brought about by convection is actually

responsible for most of the flow dependent phenomena observed in endothelial cells. Recently

Vandrangi et al. showed that even in the absence of mechano-sensitivity, mass-transfer limited

systems show shear and viscosity dependent behavior [42]. However mechanical stimulation due

to pressure variations have not been considered in this debate, probably due to the experimental

difficulties in decoupling hydrostatic pressure and viscous flow. In fact, as mentioned in the

introduction, the effect of pressure on HUVEC Endothelin and NO release has been little

investigated in the literature with respect to that of shear-stress. Here we show that the two stimuli

induce different response profiles in endothelial cells. As most reports do not observe any

morphological changes at shear stresses less than about 2 dyne/cm2, probably the most striking

result is the change in morphology and NO downregulation in the presence of flow. Quite likely

not only shear but also mass transfer effects, play a role in these cell responses. On the other hand

Endothelin−1 is stimulated by both shear and pressure, indicating that its pathway could indeed be

force, rather than transport modulated.

These results indicate that our system can be useful even for in vitro experiments simulating

pathological conditions. In fact, it has been widely confirmed that low shear stress contributes to

atherosclerosis development, giving rise to lipid accumulation and intimal thickening in blood

vessels [43]. Therefore these results, although preliminary, could open new important

perspectives regarding the differential role of pressure and flow in the study of cardiovascular

diseases [8].

5 Conclusion

The bioreactors described in this work, with the two different configurations, allow the action of

pressure and shear stress to be assessed independently. The experiments were performed using

the two bioreactor chambers, in which steps of hydrostatic pressure and different levels of shear

stress were respectively applied. Both the application of hydrostatic pressure and flow induced

shear stress resulted in a decrease in ET-1 production, with respect to the controls in static

conditions in an incubator. NO production and cell morphology were unaffected by pressure, but

highly sensitive to shear stress. Cells aligned with the direction of flow even at very low flow

rates, while their typical cobblestone morphology was unchanged by pressure.

We show here how the two principal hemodynamic forces which normally act on blood vessels,

affect endothelial cell function. This is the first report in which the two forces are considered

separately and provides an important advancement in the understanding of physiological and

pathological processes in vascular remodeling and adaptation in health and disease.

The authors declare no commercial or financial conflict of interest.

6 References

[1] Barron, V., Lyons, E., Stenson-Cox, C., McHugh, P.E. et al., Bioreactors for cardiovascular

cell and tissue growth: A review. Ann. Biomed. Eng. 2003, 31, 1017-1030.

[2] Morsi, Y.S., Yang, W.W., Owida, A., Wong, C.S., Development of a novel pulsatile

bioreactor for tissue culture. J.  Artif.  Organs  2007,  10,  109-­‐114.

[3] Jaasma, M.J., Plunkett, N.A., O’Brien, F.J., Design and validation of a dynamic flow

perfusion bioreactor for use with compliant tissue engineering scaffolds. J. Biotechnol. 2008, 133,

490-496.

[4] Thompson, C.A., Colon-Hernandez, P., Pomerantseva, I., MacNeil, B.D. et al., A novel

pulsatile, laminar flow bioreactor for the development of tissue-engineered vascular structures.

Tissue Eng. 2002, 8, 1083-1088.

[5] Williams. C., Wick, T.M., Perfusion bioreactor for small diameter tissue-engineered arteries.

Tissue Eng. 2004, 10, 930-941.

[6] McCulloch, A.D., Harris, A.B., Sarraf, C.E., Eastwood, M., New multi-cue bioreactor for

tissue engineering of tubular cardiovascular samples under physiological conditions. Tissue Eng.

2004, 10, 565-573.

[7] Dewey, C.F. Jr., Effects of fluid flow on living vascular cells. J. Biomech. Eng. 1984, 106,

31-35.

[8] Li, Y.-S.J., Haga, J.H., Chien, S., Molecular basis of the effects of shear stress on vascular

endothelial cells. J. Biomech. 2005, 38, 1949-1971.

[9] Martin, I., Wendt, D., Heberer, M., The role of bioreactors in tissue engineering. Trends

Biotechnol. 2004, 22, 80-86.

[10] Poertner, R., Nagel-Heyer, S., Goepfert, C., Adamietz, P. et al.., Bioreactor design for tissue

engineering. J. Biosci. Bioeng. 2005, 100, 235-245.

[11] Chen, H.-C., Hu, Y.-C., Bioreactors for tissue engineering. Biotechnol. Lett. 2006, 28, 1415-

1423.

[12] Vinci, B., Cavallone, D., Vozzi, G., Domenici, C. et al., In vitro liver model using

microfabricated scaffolds in a modular bioreactor. Biotechnol. J. 2010, 5, 232-241.

[13] Dai, G., Tsukurov, O., Orkin, R.W., Abbott, W.M. et al., An in vitro cell culture system to

study the influence of external pneumatic compression on endothelial function. J. Vasc. Surg.

2000, 32, 977-897.

[14] Estrada, R., Giridharan, G.A., Nguyen, M.D., Roussel, T.J. et al., Endothelial cell culture

model for replication of physiological profiles of pressure, flow, stretch, and shear stress in vitro.

Anal. Chem. 2011, 83, 3170-3177.

[15] Peng, X., Recchia, F.A., Byrne, B.J., Wittstein, I.S. et al., In vitro system to study realistic

pulsatile flow and stretch signaling in cultured vascular cells. Am. J. Physiol. Cell Physiol. 2000,

279, C797-C805.

[16] Nakadate, H., Hirose, Y., Sekizuka, E., Minamitani, H., A new in vitro pulsatile perfusion

system that mimics physiological transmural pressure and shear stress in any size of in vivo

vessel. J. Biomech. Sci. Eng. 2008, 3, 25-37.

[17] Bilsel, A.S., Moini, H., Tetik, E., Aksungar, F. et al., 17β-estradiol modulates endothelin-1

expression and release in human endothelial cells. Cardiovasc. Res. 2000, 46, 579-584.

[18] Korff, T., Kimmina, S., Martiny-Baron, G., Augustin, H.G., Blood vessel maturation in a 3-

dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates

endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 2001, 15, 447-457.

[19] Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y. et al., A novel potent vasoconstrictor

peptide produced by vascular endothelial cells. Nature 1988, 332(6163), 411-415.

[20] Lüscher, T.F., Oemar, B.S., Boulanger, C.M., Hahn, A.W., Molecular and cellular biology

of endothelin and its receptors-Part I. J. Hypertens. 1993, 11, 7-11.

[21] Rubanyi, G.M., Polokoff, M.A., Endothelins: molecular biology, biochemistry,

pharmacology, physiology, and pathophysiology. Pharmacol. Rev. 1994, 46, 325-415.

[22] Johnson, R.J., Fink, G.D., Watts, S.W., Galligan, J.J., Endothelin receptor function in

mesenteric veins from deoxycorticosterone acetate salt-hypertensive rats. J. Hypertens. 2002, 20,

587-589.

[23] Schiffrin, E.L., Vascular endothelin in hypertension. Vascul. Pharmacol. 2005, 43, 19-29.

[24] Hasdai, D., Holmes, D.R. Jr., Garratt, K.N., Edwards, W.D. et al., Mechanical pressure and

stretch release endothelin-1 from human atherosclerotic coronary arteries in vivo. Circulation

1997, 95, 357-362.

[25] Walshe, T.E., Ferguson, G., Connell, P., O’Brien, C. et al., Pulsatile flow increases the

expression of eNOS, ET-1, and Prostacyclin in a novel in vitro coculture model of the retinal

vasculature. Invest. Ophthalmol. Vis. Sci. 2005, 46, 375-382.

[26] Dai, G., Tsukurov, O., Chen, M., Gertler, J.P. et al., Endothelial nitric oxide production

during in vitro simulation of external limb compression, Am. J. Physiol. Heart Circ. Physiol.

2002, 282, H2066-2075.

[27] Tsukurov, O.I., Kwolek, C.J., L'Italien, G.J., Benbrahim, A. et al., The response of adult

human saphenous vein endothelial cells to combined pressurized pulsatile flow and cyclic strain,

in vitro. Ann. Vasc. Surg. 2000, 14, 260-267.

[28] Braddon, L.G., Karoyli, D., Harrison, D.G., Nerem, R.M., Maintenance of a functional

endothelial cell monolayer on a fibroblast/polymer substrate under physiologically relevant shear

stress conditions. Tissue Eng. 2002, 8, 695-708.

[29] Mazzei, D., Vozzi, F., Cisternino, A., Vozzi, G. et al., A high-throughput bioreactor system

for simulating physiological environments, IEEE Trans. Ind. Electron. 2008, 55, 3273-3280.

[30] Jaffe, E.A., Nachman, R.L., Becker, C.G., Minick, C.R., Culture of human endothelial cells

derived from umbilical vein. Identification by morphologic and immunologic criteria. J. Clin.

Invest. 1973, 52, 2745-2756.

[31] Lipowsky, H.H., Kovalcheck, S,. Zweifach, B.W., The distribution of blood rheological

parameters in the microvasculature of cat mesentery. Circ. Res. 1978, 43, 738-749.

[32] Koutsiaris, A.G., Tachmitzi, S.V., Batis, N, Kotoula, M.G. et al., Volume flow and wall

shear stress quantification in the human conjunctival capillaries and post-capillary venules in

vivo. Biorheology 2007, 44, 375-386.

[33] Santarelli, M.F., Sani, L., Ahluwalia, A., Vozzi, G. et al., A new method for quantitative

cellular imaging on 3-D scaffolds using fluorescence microscopy. IEEE Trans. Nanobiosci. 2003,

2, 110-117.

[34] Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L. et al., Analysis of nitrate, nitrite

and [15N]-nitrate in biological fluids. Anal. Biochem. 1982, 126, 131-138.

[35] Malek, A.M., Greene, A.L., Izumo, S., Regulation of endothelin 1 gene by fluid shear stress

is transcriptionally mediated and independent of protein kinase C and cAMP. Proc. Natl. Acad.

Sci. U.S.A. 1993, 90, 5999-6003.

[36] Kuchan, M.J., Frangos, J.A., Shear stress regulates endothelin-1 release via protein kinase C

and cGMP in cultured endothelial cells. Am. J. Physiol. 1993, 264, H150-156.

[37] Sharefkin, J.B., Diamond, S.L., Eskin, S.G., McIntire, L.V. et al., Fluid flow decreases

preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human

endothelial cells. J. Vasc. Surg. 1991, 14, 1-9.

[38] Ranjan, V., Xiao, Z., Diamond, S.L., Constitutive NOS expression in cultured endothelial

cells is elevated by fluid shear stress. Am. J. Physiol. 1995, 269, H550-555.

[39] Morawietz, H., Talanow, R., Szibor, M., Rueckschloss, U. et al., Regulation of the

endothelial system by shear stress in human endothelial cells. J. Physiol. 2000, 525, 761-770.

[40] Li, Y.-S.J., Haga, J.H., Chien, S., Molecular basis of the effects of shear stress on vascular

endothelial cells. J. Biomechanics 2005, 38, 1949-1971.

[41] Kim, D.W., Gotlieb, A.I., Langille, B.L., In vivo modulation of endothelial F-actin

microfilaments by experimental alterations in shear stress. Arterioscler. Thromb. Vasc. Biol.

1989, 9, 439-445.

[42] Vandrangi, P., Sosa, M., Shyy, J.Y.-J., Rodgers, V.G.J., Flow-dependent mass transfer may

trigger endothelial signaling cascades. PLoS ONE 2012, 7, e35260.

[43] Caro, C.G., Discovery of the role of wall shear in atherosclerosis. Arterioscler. Thromb.

Vasc. Biol. 2009, 29, 158-161.

FIGURE CAPTIONS Figure 1 - Schematic of pressure and pH control circuit for the Pressure Controlled Bioreactor,

PCB (A) Assembled set up of the PCB (B).

A

B

Figure 2: Open view of the Laminar Flow Bioreactor, LFB (the white blocks are used to hold

horizontal tubing in place) (A), flow streamlines in LFB (B), and simulation of

pressure field in LFB cell culture chamber at a flow rate of 15 ml/min (C).

A

B

C

Figure 2 - Effect of pressure on endothelial cell release of ET-1 (A) and NO (B). Data correspond

to 1h at P0 , 1h at P70 and 1h again at P0. Medium was refreshed every hour, so the

values indicate the level of NO release after each hour of incubation at the pressures

indicated. The asterisk in (A) indicates a statistically significant difference with

respect to the ET-1 concentration measured at P0 at the beginning of the experiment,

p<0.01 .

A

B

Figure 3 - Effect of shear stress on ET-1 (A) and NO (B) release by endothelial cells. Cells were

placed in fresh medium at the beginning of the experiment, and assayed at the end.

The asterisk * indicates a statistically significant difference with respect to ET-1 and

NO concentration at zero shear stress with p<0.01 , while ** indicates a statistically

significant difference with p<0.05 .

A

B

Figure 4 - Micrographs of HUVEC exposed to different levels of shear stress: 0 (A), 0.105 (B),

0.35 (C), and 1.05 (D) dynes/cm2. The scale bare is 100 µm, while the arrows

indicate the flow direction.

A B

D C

Figure 5 - Cell eccentricity calculated for different levels of shear stress and compared with

in vivo value estimated from the data reported in [41]. Eccentricity values present a

statistically significant difference with respect to the control at zero shear stress,

p<0.01 .

152 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.biotechnology-journal.com www.biotecvisions.com

BiotechnologyJournal Biotechnol. J. 2014, 99, 146–154

will always have problems decoupling biochemicaleffects (i.e. enhanced mass transport) from biophysicaleffects (forces, pressures, etc.). This was very elegantlyunderlined by Vandrangi et al. [43]. The authors showedthat even in the absence of mechano-sensitivity, masstransfer-limited systems show shear- and viscosity-dependent behavior [43]. However, mechanical stimula-tion due to pressure variations has not been considered inthis debate, probably due to the experimental difficultiesin decoupling pressure and viscous flow. Additionally, anincrease in pressure may also be accompanied by anincrease in dissolved oxygen concentration through Hen-ry’s law. In the PCB, the increase in pressure results in an8% increase in oxygen concentration, which according toour calculations, does not affect oxygen consumption bycells (Section 2.3). Interestingly, although we were unableto find any studies on ET-1 and oxygen, investigations byFismen et al. [44, 45] suggest that NO synthase activityand NO production by endothelial cells are not signifi-cantly correlated with oxygen concentration. Here weshow that flow and pressure induce different responseprofiles in endothelial cells. As most reports do notobserve any morphological changes at shear stresses lessthan about 2 dyne/cm2, probably the most striking resultis the change in morphology and NO regulation in thepresence of flow. Quite likely not only shear but also masstransfer effects play a role in these cell responses. On theother hand, ET-1 is stimulated by both shear and pres-sure, indicating that its pathway could indeed be forcerather than transport modulated.

These results indicate that our system can be usefuleven for in vitro experiments simulating pathological con-ditions. In fact, it has been widely confirmed that low

shear stress contributes to atherosclerosis development,giving rise to lipid accumulation and intimal thickening inblood vessels [46]. Therefore, these results suggest newimportant perspectives regarding the differential roles ofpressure and flow in the study of cardiovascular diseases[8].

The bioreactors described in this work, with the twodifferent configurations, allow the action of pressure andshear stress to be assessed independently. The experi-ments were performed using the two bioreactor cham-bers, in which steps of hydrostatic pressure and differentlevels of shear stress were respectively applied. Both theapplication of hydrostatic pressure and flow-inducedshear stress resulted in a decrease in ET-1 production,with respect to the controls in static conditions in an incu-bator. NO production and cell morphology were unaffect-ed by pressure, but highly sensitive to shear stress. Cellsaligned with the direction of flow, even at very low flowrates, while their typical cobblestone morphology wasunchanged by pressure.

We show here how the two principal hemodynamicforces, which normally act on blood vessels, affectendothelial cell function. This is the first report in whichthe two forces are considered separately, and provides animportant advancement in the understanding of physio-logical and pathological processes in vascular remodelingand adaptation in health and disease.

The authors declare no commercial or financial conflict ofinterest.

Figure 5. Micrographs of HUVECs exposed to different levels of shearstress: (A) 0, (B) 0.105, (C) 0.35, and (D) 1.05 dynes/cm2. Stained cellswere analyzed under a microscope to quantify their elongation. The scalebar represents 100 µm; the arrows indicate the flow direction. Three exper-iments were performed for each flow rate and the eccentricity values wereevaluated in five different locations on each sample.

Figure 6. Cell eccentricity calculated for different levels of shear stress andcompared with in vivo value estimated from the data reported in [42].Eccentricity values, averaged over 60–100 cells in five different locationson each sample, present a statistically significant difference with respectto the control at zero shear stress, p<0.01. The error bars indicate stan-dard deviation.