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Effect of flow on gene regulation in smooth muscle cells and macromolecular transport across endothelial cell monolayers.


Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, fibrinolysis, platelet aggregation, vessel tone and growth, and leukocyte traffic; and they form the principal barrier to transport of substances between the blood and the surrounding tissue space. The permeability of endothelial cells changes with environmental stimuli; shear stress, in particular, applied either in vivo or in vitro, induces changes in protein expression and secretion of vasoactive factors by endothelial cells (Nollert et al., 1991; McIntire, 1994; Papadaki and Eskin, 1997). The ability to study the effects of shear on the macromolecular permeability of the cerebral vasculature is particularly important, since in no other place is the barrier function of the endothelium more important than in the brain. The endothelial cells of this organ have developed special barrier properties that keep the cerebral system from experiencing any drastic change in composition; together with glial cells, they form the blood brain barrier (BBB). We have studied the effect of flow on bovine BBB using flow chambers and tissue culture systems.

Recent modeling studies indicate that, not only the endothelium, but also the underlying smooth muscle cells (SMC) in the vasculature are exposed to significant shear stresses that arise from interstitial flow driven by transmural pressure gradients (Wang and Tarbell, 1995). In response to vascular injury, the medial SMCs of arteries proliferate and migrate to the intima (Schwartz, 1993). Moreover, it has been hypothesized that the SMC are directly exposed to blood flow when the integrity of the endothelial monolayer is disrupted, and that their healing behavior is then modulated by the local hemodynamic environment (Kohler et al., 1991; Kohler and Jawien, 1992). Presumably, however, the effects of this environment on the SMC are not only mediated by flow, but also by small messenger molecules whose rate of production may be modulated by flow.

Nitric oxide (NO) is such a molecule; among its diverse biological functions are vasorelaxation, reduction of platelet aggregability, inhibition of adhesion of inflammatory molecules in the vascular wall, and cytostatic or cytotoxic actions in various cell types (Sessa, 1994; Koprowski and Maeda, 1995). To date, three major subtypes of nitric oxide synthase (NOS) have been identified. One subtype is the inducible NOS (NOS II), which is regulated at the transcriptional level and produces high levels of NO for extended periods. NO II is present in macrophages, SMC, and endothelial cells upon stimulation with cytokines. The other two isoforms are constitutively expressed and normally produce low levels of NO; they are termed NOS I (in neuronal, epithelial cells) and NOS III (in endothelial cells, cardiac myocytes, and skeletal muscle) (Koprowski and Maeda, 1995). Recent findings indicate that mechanical deformation of the endothelium by shear stress or by cyclic stretching increases NOS III mRNA, protein and enzymatic activity (Sessa, 1994). We have investigated the effects of fluid shear stress on the growth kinetics of cultured human aortic SMC (hASMC) and on NO released by these cells.

Materials and Methods

Cell culture

Brain microvessel endothelial cells (BMECs) were isolated by a two-step enzymatic process. Briefly, a fresh bovine brain was obtained from a local slaughterhouse, and the isolation was begun within 18 h post-mortem. The gray matter was separated from the white matter, collected, blended (Tekmar Instrument Co. stomacher), and digested with 0.5% dispase (Boehringer-Mannheim) for 3 h at 37 [degrees] C. The solution was then centrifuged on a dextran (Sigma) gradient, washed, and redigested with 1 mg/ml collagenase/dispase (Boehringer-Mannheim) for 5 h at 37 [degrees] C. The microvascular endothelial cells were separated from the other cells by centrifugation (1000 x g) on a preformed 50% Percoll (Sigma) gradient. The second layer (containing the cells) was removed, washed, and frozen in liquid nitrogen for later use. After thawing, the isolated BMECs were grown on surfaces that were treated with both type I rat tail collagen and human fibronectin. The culture media contained MEM/F-12 (Sigma) supplemented with 10% plasma derived horse serum (Hyclone), 0.1 mg/ml penicillin G/streptomycin (Gibco), 2.5 mg/ml fungizone (Gibco), and 0.1 mg/ml heparin (Sigma).

A hASMC line initiated with cells from the abdominal aorta of a 9-year-old kidney transplant donor was used in all the experiments performed in this study (Papadaki et al., 1996); the culture medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 2 mmol L-glutamine, 200 U/ml penicillin, and 100 [[micro]gram]/ml streptomycin. Phenol-red-free DMEM was used in the nitrite experiments to prevent color interference with the fluorometric assay. hASMC (P2-P10) were plated at a subconfluent density of 2.5 x [10.sup.4] cells/[cm.sup.2], on fibronectin-coated glass slides (75 x 38 mm). Twenty-four hours after seeding, hASMC were exposed to physiological levels of venous and arterial laminar stress (5 to 25 dyn/[cm.sup.2]) in parallel plate flow chambers connected to recirculating flow loops (Papadaki et al., 1996). The experiments were run in a humidified room at 37 [degrees] C and the system was gassed with 5% C[O.sub.2]. For the growth studies, the flow experiments were carried out for 24 h. At the end of each experiment, the cells were removed from the slide with the 0.05% typsin-EDTA, and the number of cells was determined with a Coulter Counter.


The marker molecules for the permeability experiments were fluoroisothiocynate (FITC) dextran (Pharmacia for all molecular weights except 2 million). The 70 and 2000 kD probes were dialyzed extensively before use. The dextrans were also tested for purity on thin-layer chromatography (70% chloroform; 25% methanol and 5% acetic acid, on silica gel plates, as recommended by Molecular Probes). Permeability was measured in a modified parallel plate flow chamber according to methodology described earlier (Nollert et al., 1991; McIntire, 1994; Casnocha et al., 1989; Wagner et al., 1997).

Nitrate assay

Samples of the conditioned media samples were collected at different times, and nitrite, as an index of nitric oxide production, was measured with a quantitative fluorometric assay (Misko et al., 1993, Papadaki et al., 1998). This assay is based on the reaction of nitrite with an acid form of 2,3-diaminonaphthalene to form the highly fluorescent product 1-(H)-naphthotriazole. The intensity of the fluorescent product was maximized by the addition of 2.8 N NaOH, and the signal was measured with a fluorescent 96-well plate reader, with excitation of 365 nm and emission read at 460 nm.

Western blotting

NOS protein was detected in total cell lysates. Cells were harvested from both the control and flow cultures in 150 [[micro]liter] of lysis buffer (0.5% SDS, 50 mM Tris/Cl, pH 7.4, leupeptin 1 mg/ml, pepstatin 1 mg/ml, 0.1 M phenylmethylsufonyl fluoride). Cell homogenates were centrifuged for 20 min at 14,000 x g at 4 [degrees] C to remove insoluble material, and the viscosity of the supernatant was then reduced by several passages through a 26-gauge needle. Protein concentration was measured in a small aliquot of sample with the micro BCA method. The samples were further diluted, at a ratio of 3:1 in a 4x sample buffer (0.2 mM TrisCl, pH 6.8, 4% SDS, 40% glycerol, 0.4% bromophenol blue, 10% [Beta]-mercaptoethanol) and boiled for 5 min. Equal amounts of protein were loaded in a 7.5% SDS-polyacrylamide minigel and electrophoresed at a constant current of 15 mA for 2 hours. The separated proteins were transferred to nitrocellulose membranes, and the blots were incubated for 1 h with 5% nonfat dry milk in Dulbecco's phosphate buffered saline (PBS) and 0.05% Tween-20, (PBS-T) to block nonspecific binding of the antibody. The membranes were incubated overnight with primary monoclonal and polyclonal antibodies against all isoforms of NOS protein (Transduction Laboratories); the antibodies were diluted 1:500 in PBS-T. Blots were washed (PBS-T x 5) and then incubated for 1 h with a donkey anti-rabbit secondary antibody conjugated to horseradish peroxidase and used at a dilution 1:1000 in PBS-T. Nitric oxide synthase immunoreactivity was detected by the enhanced chemiluminescence (ECL) method, followed by autoradiography.
Table I

Increase in permeability over baseline(a)

           1 dyne/[cm.sup.2]      10 dyne/[cm.sup.2]
MW KDa     18 h     30 h(b)        18 h     30 h(b)

2000        76       4              34        2.3
70          20       0.8            11       -0.4
4            2       1.2             3.4      1.4(*)

a Increases shown are multiples of baseline values.

b Not significantly larger than baseline values, except (*).



The permeability of BMECs under shear stresses of 1 dyne/[cm.sup.2] or 10 dyne/[cm.sup.2] rose rapidly at first, became maximal between 10 and 18 h, and had largely recovered by 30 h (Table I).

Smooth muscle cell metabolism

Our results demonstrated that fluid shear stress decreased the proliferation rate of hASMC [ILLUSTRATION FOR FIGURE 1 OMITTED]. The cell number at high shear-stress levels ([greater than] 17.5 [dyn.sup.a]/[cm.sup.2]) was significantly lower than at low levels of shear stress ([greater than] 15 [dyn.sup.a]/[cm.sup.2]). Furthermore, at all shear-stress levels tested, the growth rate was reduced relative to stationary control cultures (Papadaki et al., 1996). The flow-related reduction in the cell number was not due to cell injury, as demonstrated by the equality of lactate dehydrogenase (LDH) activity in the conditioned media of control and sheared cultures (results not shown). The LDH concentration in the medium of the stationary cell (3.5 [+ or -] 0.8 U/L) was not different from those in the media containing cells at all different shear stress levels used (2.4 [+ or -] 1.5 U/L) (Papadaki et al., 1996). In addition, indirect immunofluorescence for the proliferating cell nuclear antigen (PCNA) provided further evidence that, in the range of shear stresses used and for the time course of these experiments, the growth of the cell population was not arrested. Control cultures had more PCNA-positive nuclei (37% [+ or -] 6%) than cultures exposed to flow, and this difference was essentially the same as that seen for cell density (35% [+ or -] 6%). This comparison suggests that the reduction in the cell number observed with shear stress is due to slower movement of cells through the cell cycle (Papadaki et al., 1996).

The effects of flow on nitrite production by hASMC are shown on Figure 2. Shear stress significantly increased nitrite levels in the conditioned media, whereas the levels present in the stationary control cells were almost undetectable (Papadaki et al., 1998). Cumulative nitrite production in conditioned media increased with the duration of flow exposure and with shear. However, nitrite production rates were inversely correlated with time. An initial burst in nitrite production rate, detected as early as I h after flow exposure, was followed by a gradual decrease with time. Similar experiments with endothelial cells have shown similar trends in nitrite production rates (Kuchan and Frangos, 1994). This result was the first indication that the NOS isoform responsible for shear-induced NO production is not NOS II; i.e., activation of NOS II results in sustained nitrite production rates for extended periods (up to 72 h after addition of stimulant). Treatment with 100/[[micro]molar] [N.sup.G]-amino-L-arginine (L-NAA) for 30 min before and during exposure to shear stress (25 dynes/[cm.sup.2]) completely abolished the flow-induced release of nitrite without affecting release from stationary cultures. L-NAA is a potent amino-substituted NOS inhibitor (Kuchan and Frangos, 1994). The complete inhibition of the nitrite signal in the presence of inhibitor provided evidence that the NO production in response to shear stress comes from the enzymatic reaction of L-arginine to L-citrulline, and is not a result of cell debris.

To identify the NOS isoform involved, cultures were incubated with 1 [[micro]molar] dexamethasone (DM) 24 h before and during exposure to shear stress (Papadaki et al., 1998). DM is asteroid that blocks transcription of NOS II by interfering with the binding of transcription factors to the promoter region of the gene (Rees et al., 1990). Dexamethasone had no effect on the nitrite levels in either the controls or the flow cultures. This result provided further evidence that NOS II plays no role in flow-induced nitrite production by hASMC. Monoclonal or polyclonal antibodies against NOS II showed no immunoreactivity with Western blot analysis [ILLUSTRATION FOR FIGURE 3 OMITTED], which verified that the inducible isoform was not present, either in control or sheared hASMC cultures. On the other hand, polyclonal antibodies against the constitutively expressed isoform of neuronal NOS (NOS I) gave specific products in all stationary control and flow samples [ILLUSTRATION FOR FIGURE 3 OMITTED]. The intensities of the control and shear NOS I bands are identical at 6 h, indicating that cultured hASMC express a constitutive NOS I protein whose enzymatic activity, rather than the amount of protein, is modulated by shear stress. Endothelial NOS (NOS III) was found in neither control nor sheared hASMC [ILLUSTRATION FOR FIGURE 3 OMITTED].

Summary and Conclusions

BMECs in static culture have a swirling spindoidal morphology. After exposure to flow for 10-18 h, BMECs appeared rounded with no preferred orientation. Further application of 10 dynes/[cm.sup.2], but not 1 dyne/[cm.sup.2], induced the cells to elongate in the direction of flow (data not shown). We demonstrated that BMECs initially respond to either of 1 dyne/[cm.sup.2] or 10 dynes/[cm.sup.2] shear stress with a dramatically increased macromolecular permeability. Maximum permeabilities were obtained between 10 and 18 h in the shear field for both shear rates, and these timepoints corresponded to the most rounded morphology. Continued application of the shear field led to a partial recovery in the permeability of the cerebral endothelial cells to macromolecules. The initial increase in permeability and the recovery was most dramatic for the higher molecular weight dextran markers.

We have also shown that the flow-induced shear stress stimulates NO production in hASMC due to activation of a constitutively expressed NOS I enzyme. The constitutive expression of NOS I in SMC, and its concomitant activation by flow-induced shear stress, may play a regulatory role in the blood vessel wall in the absence of endothelium due to vascular injury. Shear-induced NO production from vascular SMC may inhibit excessive adhesion of platelets and other inflammatory molecules at the injury site, and may regulate the release of mitogenic factors by activated blood cells. In vascular wall homeostasis, constitutive NO production by underlying SMC, modulated due to transmural flow, may act in concert with endothelial-cell-derived NO to regulate vascular tone and maintain a non-proliferative phenotype for SMC.

Literature Cited

Casnocha, S. A., S. G. Eskin, E. R. Hall, and L. V. McIntire. 1989. Permeability of human endothelial cell monolayers: effect of vasoactive agonists and cAMP. J. Appl. Physiol. 87: 1997-2005.

Kohler, T. R., and A. Jawien. 1992. Flow affects development of intimal hyperplasia after arterial injury in rats. Arterioscler. Thromb. 12: 963-971.

Kohler, T. R., T. R. Kirkman, L. W. Krais, B. K. Ziegler, and A. W. Clowes. 1991. Increased blood flow inhibits neointimal hyperplasia in endothelialized vascular grafts. Circ. Res. 69: 1557-156.

Koprowski, H., and H. Maeda. 1995. The Role of Nitric Oxide in Physiology and Pathophysiology. Springer-Verlag, New York.

Kuchan, M. J., and J. A. Frangos. 1994. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am. J. Physiol. 35: C628-C636.

McIntire, L. V. 1994. Bioengineering and vascular biology. Ann. Biomed. Eng. 22: 2-13.

Misko, T. P., R. J. Schilling, D. Salvemini, W. M. Moore, and M. G. Currie. 1993. A fluorometric assay for the measurement of nitrite in biological samples. Anal. Biochem. 214: 1-6.

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Papadaki, M., and S. G. Eskin. 1997. Effects of fluid shear stress on gene replication in vascular cells. Biotechnol. Prog. 13: 209-221.

Papadaki, M., L. V. McIntire, and S. G. Eskin. 1996. Effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro. Biotechnol. Bioeng. 50: 555-561.

Papadaki, M., R. G. Tilton, S. G. Eskin, and L. V. McIntire. 1998. Effects of shear stress on nitric oxide production by human aortic smooth muscle cells. Am. J. Phys. 274: H616-H626.

Rees, D. D., R. M. J. Palmer, and S. Moncada. 1990. Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone. an insight into endotoxin shock. Blochem. Biophys. Res. Com. 173: 541-547.

Schwartz, R. S. 1993. Coronary Restenosis. Blackwell Scientific, Cambridge, MA.

Sessa, W. C. 1994. The nitric oxide synthase family of proteins. J. Vas. Res. 31: 131-143.

Wagner, J. E., L.V. McIntire, and P. A. Whitson. 1997. Macromolecular permeability of bovine brain endothelial cells monolayers subjected to shear stress in vitro. Am. J. Physiol. (in press).

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LUNA: Did you say that htere is a 36-fold increase for high molecular weight species and only a 20-fold increase for the low molecular weights?

MCINTIRE: Yes, 36- to 70-fold for high molecular weight species and 2- to 4-fold for low molecular weight species.

LUNA: Can you speculate on what kind of hole would be opening up that would allow the bigger molecules through, or is it some kind of transcytosis through the cell?

MCINTIRE: We think t hat there is a cytoskeletal rearrangement that leads to a change in the junction integrity. This would allow larger molecular weight species through, whereas the lower molecular weight species are going through relatively fast anyway.

FUJIWARA: Do you have any explanation for the fact that the mololayer with the round cell shape has increased permeability?

MCINTIRE: Those time points happen to coincide with the high flow. At the lower shear stress we see even bigger changes in permeability; but we don't see changes in gross cell shape because we don't see any realignment at very low stress. I assume this to mean there are variuos cytoskeletal rearrangements occurring even at the low stresses, but they don't lead to alignment in the direction of the flow, because the flow forces aren't forcing the cells to do that. I don't know why the maximum permeability occurs for that rounded shape at that particular ti me.

FUJIWARA: We are looking at the motility of cells in the monolayer, and when cells are round they move at lot more. Perhaps movement breaks the cell-cell adhesion more frequently than in the aligned area, contributing to the increased permeability.

MCINTIRE: Yes, that's possible.
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Title Annotation:The Cytoskeleton: Mechanical, Physical, and Biological Interactions; includes panel discussion
Author:McIntire, Larry V.; Wagner, John E.; Papadaki, Maria; Whitson, Peggy A.; Eskin, Suzanne G.
Publication:The Biological Bulletin
Date:Jun 1, 1998
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