Elsevier

Journal of Biotechnology

Volume 133, Issue 2, 20 January 2008, Pages 239-244
Journal of Biotechnology

Differential gene responses in endothelial cells exposed to a combination of shear stress and cyclic stretch

https://doi.org/10.1016/j.jbiotec.2007.08.009Get rights and content

Abstract

We developed a compliant tube-type flow-loading apparatus that allows simultaneous application of physiological levels of shear stress and cyclic stretch to cultured cells and examined gene responses to a combination of the two forces. Human umbilical vein endothelial cells were exposed to shear stress and/or cyclic stretch for 24 h, and changes in the mRNA levels of endothelin-1 (ET-1), a potent vasoconstrictor, and endothelial nitric oxide synthase (eNOS), which catalyzes the production of a potent vasodilator, NO, were determined by reverse transcriptase/PCR. Cyclic stretch (10%, 1 Hz) alone increased ET-1 mRNA levels approximately 1.6-fold, but had no effect on eNOS mRNA levels. A shear stress of 7 dynes/cm2 and 15 dynes/cm2 alone decreased ET-1 mRNA levels to around 83% and 61%, respectively, of the basal level, but increased the eNOS mRNA level to around 2.2-fold and 3.2-fold, respectively. When cyclic stretch and shear stress were applied simultaneously, ET-1 mRNA levels did not change significantly, but the eNOS mRNA level increased to a level equivalent to the increase in response to shear stress alone. These results indicate that the response of endothelial genes to shear stress or cyclic stretch depends on whether the two forces are applied separately or together.

Introduction

Vascular endothelial cells (ECs) are constantly subjected to mechanical stresses in the form of the shear stress generated by blood flow and the cyclic stretch produced by pulsatile changes in blood pressure. A number of studies have indicated that ECs alter their morphology and functions in response to these hemodynamic forces (Ando et al., 2000, Pradhan and Sumpio, 2004). It is also now apparent that when EC functions are modulated by hemodynamic forces, the expression of genes related to EC functions usually changes (Ando et al., 1999). For example, shear stress regulates the expression of genes encoding various vasoactive mediators, including nitric oxide synthase (eNOS) (Nishida et al., 1992, Noris et al., 1995, Uematsu et al., 1995), C-type natriuretic peptide (Okahara et al., 1995, Chun et al., 1997), adrenomedullin (Chun et al., 1997), angiotensin-converting enzyme (Rieder et al., 1997), and endothelin-1 (ET-1) (Yoshizumi et al., 1989, Sharefkin et al., 1991, Malek and Izumo, 1992), all of which are involved in the constriction or relaxation of vascular smooth muscle, and hemodynamic-force-mediated production of these mediators plays an important role in the control of vessel tone and blood pressure in vivo.

Thus far the effects of hemodynamic forces on cultured ECs have been studied using apparatuses that allow application of either fluid shear stress or cyclic stretch to the cells (Bussolari et al., 1982, Banes et al., 1985, Stathopoulos and Hellums, 1985), and such studies have provided a large amount of valuable information on the effect of each of them. However, since little is known about the combinatorial effect of shear stress and cyclic stretch, some research groups have developed a system in which ECs cultured on the inner surface of an elastic tube can be exposed to both shear stress and stretch at the same time (Benbrahim et al., 1994, Moore et al., 1994).

In this study we developed an original compliant tube-type device and examined the combined effects of shear stress and stretch on gene expression of ET-1 and eNOS in human umbilical vein ECs (HUVECs).

Section snippets

Cell culture

HUVECs were isolated from human umbilical veins by collagenase treatment and grown in a 1% gelatin-coated flask in M199 containing 15% FBS (Hyclone Laboratories), 2 mmol/L l-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin, 50 μg/mL heparin, and 30 μg/mL endothelial cell growth factor (Becton and Dickinson). When they reached confluence, the cells were routinely passaged by trypsinization in a 0.05% trypsin/2 mmol/L EDTA solution. A suspension of HUVECs (4th–10th subculture, 105 cells/cm2) was

Results and discussion

HUVECs were exposed to shear stress (15 dynes/cm2) and stretch (10%) for 24 h in the silicon-tube-type flow-loading apparatus. The cells remained viable, with no signs of injury or desquamation, throughout the experiments. A dye exclusion test with trypan blue showed no increase in dead cells after exposure to shear stress and/or stretch (data not shown). The cells showed morphological changes in response to shear stress and cyclic stretch, becoming elongated and aligned parallel to the direction

Conclusion

The results of this study demonstrated that our original silicone-tube-type flow-loading system is useful for applying shear stress and cyclic stretch simultaneously to cultured ECs, thereby mimicking conditions in vivo where both blood flow and blood pressure constantly act on vascular endothelium. The system enabled us to observe the responses of endothelial genes to combinations of these two hemodynamic forces, and the results showed that endothelial gene responses to shear stress and cyclic

Acknowledgments

We thank J. Kuwana, Fuji Systems Co. Ltd. (Tokyo) for providing the silicone elastomer, and T. Tanaka for advice in developing the system. This work was partly supported by Grants-in-Aid for Scientific Research and for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science and Culture, and a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare.

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