effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro

Effects of Shear Stress on the Growth Kinetics of Human Aortic Smooth Muscle Cells In Vitro

Maria Papadaki,’ Larry V. Mclntire,’” and Suzanne G. Eskin2 ‘Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering, Rice University, Houston, Texas 7725 1; and Cell Biology Division, Texas Biotechnology Corporation, Houston, Texas 77030

Received May 5, 1995/Accepted December 19, 1995

After cardiovascular intervention, smooth muscle cells (SMC) are directly exposed to blood flow and thus their behavior might be affected by fluid hemodynamic forces. The aim of this study was to determine the effect of fluid shear stress on the growth rate of SMC. Human aortic smooth muscle cells (hASMC) were seeded on fibronectin-coated glass slides and were exposed to different levels of shear stress using parallel plate flow chambers. After 24 h, cell numbers in the stationary and sheared cultures were measured by a Coulter counter. Results demonstrated that increasing shear stress significantly reduces the proliferation rate of hASMC ( P < 0.05). Comparable lactate dehydrogenase levels in the media of stationary and flow cultures provided evidence that the reduction of cell number was not due to cell injury. Proliferating cell nuclear antigen (PCNA) immunofluorescence studies indicated that the cell cultures were not growth arrested 24 h after exposure to shear stress, and that the differences in PCNA staining between stationary control and flow cultures were comparable to the cell counts. CC’ 1996 John Wiley & Sons, Inc. Key words: human aortic smooth muscle cells shear stress restenosis growth rate PCNA

INTRODUCTION

Percutaneous transluminal coronary angioplasty (PTCA) has become a widely used treatment for vascular proliferative diseases. Despite the initially high suc- cess rates of the procedure, 30% to 50% of the treated arteries restenose over a period of 3 to 6 month^.^,'^.^" Restenosis occurs with similar frequencies in other types of cardiovascular interventions, and it is believed to represent a “response to injury” of the blood vessel to the trauma . y928-3”

A number of studies indicate the importance of smooth muscle cells (SMC) in the development of the restenotic lesion. Due to the release of biologically ac- tive mediators at the injury site, medial SMC proliferate, migrate to the intima, and secrete abundant quantities of extracellular matrix protein^.'^,^^*^^ In the normal artery, SMC are found in a contractile and differentiated phenotype, while those found in the restenotic lesions appear to be in a synthetic state. The synthetic phenotype

* To whom all correspondence should be addressed.

is characterized by loss of the contractile proteins and great increase in the synthetic and proliferative ca-

Upon endothelial denudation, many medial SMC are directly exposed to blood flow and it has been recently hypothesized that their healing response might be mod- ulated by the local hemodynamic e n ~ i r o n m e n t . ’ ~ , ~ ~ In vivo experiments to date have demonstrated that alterations in the local blood flow pattern, in addition to systemic risk factors, play a role in determining the rate of atherogenesis and vein graft thickening. In those studies, the effect of flow on intimal hyperplasia was examined in the presence of an intact endothelium and thus the endothelial cells were considered as a “flow sensor” and as a signal transduction system for flow f o r ~ e ~ . ~ ~ , ~ ~ , ~ ~ , ~ ~ , ~ ~ To date, few investigators have examined the SMC response to blood flow under controlled wall shear stress condition^.^',^^

The purpose of this in vitro study was to investigate possible correlations of flow-induced wall shear stress levels with the growth kinetics of vascular SMC. Cul- tured human aortic SMC were exposed to different physiological levels of laminar shear stress for a 24-h period, using parallel plate flow chambers in a recirculat- ing flow ~ O O P . ~ ~ ~

paCity.3,12,13,33

MATERIALS AND METHODS

Cell Isolation-Cell Culture

Human abdominal aortic tissue was collected from a 9- year-old kidney transplant donor (Hermann Hospital). A 3.8 X 2.5 cm2 segment was dissected and then placed in a 100-mm Petri dish in culture medium, containing Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 20% fetal bovine serum (FBS) (Hyclone Laboratories), 2 mmol L-glutamine, 200 U/mL penicil- lin, and 100 pg/mL streptomycin (Gibco BRL) (complete medium). Adventitial connective tissue was removed and the tissue was cut into pieces averaging 3 mm3, which were incubated at 37°C in complete me- d i ~ m . ~ , * ~ Human aortic smooth muscle cells (hASMC)

Biotechnology and Bioengineering, Vol. 50, Pp. 555-561 (1996) 0 1996 John Wiley & Sons, Inc. CCC 0006-3592/96/050555-07

migrated from the explants several days later. Cultures were identified as SMC by positive staining with a monoclonal antibody to alpha-actin (Sigma Chemicals) and by the hill-and-valley pattern displayed at confluency. hASMC from passages Pz to P5 were plated subcon- fluently, at a seeding density of 2.5 X lo4 cells/cm2, on glass slides 75 X 38 mm (Fisherbrand) coated with 1 pg/ cm2 fibronectin (Collaborative Biomedical Products), in complete medium.

Exposure of hASMC to Shear Stress

Twenty-four hours after seeding, SMC cultures were exposed to well-defined levels of shear stress (5 to 25 dyn/cm2) in parallel plate flow chamber systems. The surface area exposed to shear stress was 15 cm'. Flow was driven through the chamber by a hydrostatic pressure head created by the vertical distance between the upper and the lower reservoir. The apparatus was as- sembled in a laminar flow hood, filled with 20 mL of complete medium, and run in a 37°C room.'.* A humidified mixture of 95% air and 5% C02 was gassed near the upper reservoir to maintain the pH at 7.3. All shear stress experiments were performed for 24 h.

Growth Studies

At the end of the experiment, cells from both control and flow cultures were scraped from the periphery of the slides using a flow chamber template to normalize culture surface area. Cells in the center of the slide were removed by 5-min incubation with 0.05% trypsin- EDTA (Gibco BRL). Cell number was measured by a Coulter counter with three replicates at each time point.

Light photomicrographs (Olympus IMT-2 inverted research microscope) were obtained at the beginning and at the end of each experiment.

Lactate Dehydrogenase Assay

To control for possible damage and removal of cells by shear stress, the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) into the culture medium was monitored by a LDH-L reagent kit (Ciba-Corning). Media samples were collected at the end of each experiment from both flow and control cultures and stored at -20°C. To ensure complete cell lysis, media were sonicated with a sonic dismembrator (Fisher Scientific). One hundred fifty microliters of the sonicated media samples were added to 1 mL of LDH-L reagent and the UV absorbance of the solution was measured at 340 nm by spectrophotometer (System 2600, Gilford Instruments Laboratories, Oberlin, OH). Lactate dehydrogenase activity in units (U/L) was calculated (Ciba- Corning reaction kit manual). To evaluate the sensitivity of the method, LDH measurements were taken after lysis of the entire cell population in both stationary

control and flow cultures. Furthermore, the background values of LDH were calculated, which represented the LDH activity present in the serum of media samples that were never exposed to cells. The background value varied substantially with different media preparations, mainly due to differences in serum batches. For each of the LDH values presented in the Results section, the respective background LDH values have been subtracted to give a clear picture of SMC-related LDH in the sample. Control experiments to investigate the effects of the flow system on LDH stability in cell-free media showed no difference in LDH activity between stationary culture (15.6 2 0.54 U/L) and flow culture (15.0 2 0.2 U/L) after 24 h.

PCNA lmmunofluorescence

To detect differences in hASMC proliferative state after 24 h of flow, cells from stationary cultures and from cultures exposed to 25 dyn/cm2 shear stress were fixed with cold methanol for 5 min in -20"C.2338 The methanol fixation was performed to account only for the insoluble form of the enzyme that is associated with the sites of ongoing DNA synthesis. After three washes with Dulbecco's phosphate-buffered saline (PBS) (Gibco BRL), the cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature. To reduce the amount of reagents used, cloning rings were positioned within the shear stressed area of each slide. After three washes with PBS, the areas within the rings were pre- treated with 10% normal goat serum (Sigma Chemicals) for 1 h at room temperature, to block nonspecific binding of the antibodies. Then the areas within the rings were covered with 60 p L of mouse anti-PLNA (PCNA, PC10) monoclonal antibody (Santa Cruz Biotechnol- ogy) (1:50 in 3% bovine serum albumin [BSA]/PBS), and incubated overnight at 4°C. The next day the areas inside the cloning rings were washed extensively with PBS, and then incubated with 60 p L of goat rhodamine- conjugated anti-mouse immunoglobulin antibody (1 : 100 in 3% BSA/PBS) (Calbiochem). After 1-h incubation at room temperature, the slides were washed three times with PBS and then mounted with Moviol. All the incubation steps were performed in a humidified chamber. The PCNA staining was visualized at 560 nm with a 20X fluorescence objective on a Olympus Vanox (AH-3) microscope.

During the S phase of the cell cycle the amount of PCNA is increased two- to threefold in normal replicating cells and had granular distribution in the cell nuclei.36 The brightly stained nuclei were counted as cells in S phase of the cell cycle. To determine the percentage of cells in the S phase, the brightly stained nuclei in a given field were counted using fluorescence microscopy and the total number of cells in the same field were measured using phase optics. For each slide, three different fields were measured twice.

556 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 50, NO. 5, JUNE 5, 1996

Statistics

Two-tailed Student’s t-tests for paired samples were used to compare means of cell number and percent of PCNA-positive nuclei from the stationary and flow cultures. Differences in the cell number between all the shear stress levels used were evaluated using one-way analysis of variance (ANOVA) followed by Fisher’s least-squares difference. Differences were considered significant when P < 0.05. Results are expressed as mean 2 SD.

3.5 1

RESULTS

Our studies demonstrated that fluid shear stress reduced the growth rate of hASMC (Fig. 1). At all shear stresses, the cell number in the stationary control cultures was significantly higher than the cultures exposed to flow for 24 h (e.g.. the stationary control culture cell number was 5.18 ? 36 X lo4 celldcm’, whereas at 5 dyn/cm’ the cell number was 4.29 2 0.30 X lo4 cells/cm2). Further- more, increasing shear stress reduced the replication rate of hASMC. There were 17.3 t 5.7% fewer cells in experiments exposed to shear stress of 5 dynkm’ compared to the respective stationary control values, while at 25 dynkm’ the cell numbers were 35 ? 6.58% less than controls. These differences between 5 dyn/cm2 and 25 dyn/cm2 shear stress levels were significant (Fig. 1). In all the experiments, the number of cells at the onset of flow was equal to the seeding density as confirmed by cell counts.

The LDH assay, which serves as an indirect measure of cell injury and detachment, provided evidence that the flow-related reduction in the cell number was not

Control

[ ] Flow

0

2.5

5 2 z

1.5

1

0.5

0 5 1 1 1 5 17.5 21.5

Shear stress dynlcm2

Figure 1. Effects of shear stress on the growth rate

2 5

f cultured hASMC. N indicates the number of cells per sq. centimeter at the end of a 24-h experiment, while No indicates the number of cells per sq. centimeter at the beginning of an experiment. Asterisks indicate significant difference from the respective stationary culture cell number; double asterisks indicate significant difference from the cell number at 5 , 11, and 15 dyn/cm* shear stress.

due to shear-stress-induced cell damage or death. As shown in Figure 2, no significant difference was observed in the LDH activity of conditioned media from cultures at any shear stress employed. For example, corrected LDH values for hASMC sheared at 25 dyn/ cm2 were 2.35 -C 1.5 U/L, while LDH for stationary hASMC controls was 3.5 2 0.89 U/L, after the respective baseline LDH levels of serum were subtracted. From these experiments, when the stationary control cells were lysed, LDH activity was 30.62 ? 4.9 U/L (after subtracting the respective baseline value). The average baseline LDH level of serum in media samples that were never exposed to cell was 21.92 ? 8.65 U/L. From these data, it was calculated that one unit change in LDH represents 1600 cells/cm’; and that, in this experimental design, the LDH assay can detect as low as 3.3% cell death.

Light photomicrographs taken at the beginning and the end of each experiment supported the above findings (Fig. 3). Figure 3a shows an hASMC culture 24 h after seeding, at the beginning of an experiment, while Figure 3b shows a typical stationary culture at the end of a 24-h experiment (48 h after seeding). Figure 3c and d show hASMC after a 24-h exposure to different levels of shear stress, 5 and 25 dydcm’, respectively. Under the conditions used, hASMC remained viable, and there were no morphological alternations evident at this magnification. hASMC were long and thin, growing in a monolayer, and they formed swirling patterns. No cell orientation in the direction of flow was observed during the time course of the experiments and for the levels of shear stress used, which is in contrast to results re- ported for bovine endothelial cell^.^^'^

10

el LDH in control medid

3 r LDH in flow media .

(n=5)

11 15 17.5 21.5 25

Shear stress dyn/cm2

Figure 2. Effects of shear stress on the adhesion of hASMC on the surface. Lactate dehydrogenase enzyme concentration in the media of stationary and flow cultures in units (U/L). Each LDH value was corrected for the respective LDH concentration in serum, in media that were never exposed to cells. Total cell lysis would correspond to approximately 30 U/L.

PAPADAKI, MclNTIRE, AND ESKIN: FLOW REGULATION OF GROWTH KINETICS OF HASMC 557

Figure 3. Light photomicrographs of representative fields of hASMC. (a) Stationary culture 24 h after seeding (at onset of flow). (b) Cells in a stationary culture at the end of a 24-h experiment (48 h after seeding). Cells exposed to shear stress for 24 h at (c) 5 dynicm' and (d) 25 dyn/cm2. The arrow indicates the direction of flow in the parallel plate flow chamber. (Top to bottom).

Immunofluorescence analysis for PCNA confirmed that the cells were actively replicating and they were not growth arrested after 24-h flow exposure at 25 dynl cm2 (Fig. 4). Both in the stationary (Fig. 4a) and in the sheared cultures (Fig. 4c) there were brightly stained nuclei, which indicated cells in the S phase of the cell cycle.36 Figure 4b and d are the phase contrast photomicrographs of 4a and 4c, respectively. The percentage of positively stained cells for PCNA was 28.4 2 7% and 17.7 -t 4% in the control and flow cultures, respectively (n=3). There was a significant difference in the PCNA staining between the stationary and shear-stressed cultures (37 -t 6%, P < 0.05, n=3), which was comparable to the differences seen in the cell density measurements.

DISCUSSION

This study has demonstrated that alternations in shear stress significantly affect the growth rate of hASMC in vitro. The proliferation rate of hASMC decreases with increasing shear stress and this reduction is not due to flow-induced cell injury, as indicated by similar LDH activities in the media of stationary and sheared cultures.

Intimal thickening is an important result of vascular proliferative diseases and may be the major cause of failure of autogenous vein grafts in coronary bypass operations and restenosis following angioplasty proce-

dures. It has previously been observed that mechanical forces are involved in the hyperproliferative response of the vascular wall. In particular, regions of low wall shear stress have been correlated with increased rate of atherosclerosis progression,'0,20,2' while studies on vascular grafts indicated that low blood flow is correlated with the development of neointimal h y p e r p l a ~ i a . ' ~ . ~ ~ . ~ ~ In most of the previously mentioned models, the effects of shear stress on the healing response of the vasculature have been studied in the presence of an intact endothelium.

Little is known about the relationship between fluid hemodynamic forces and abnormal SMC behavior after endothelial denudation accompanying cardiovascular interventions. In the few in vivo studies published, investigators have implied that because of the absence of the endothelial monolayer the luminal SMC were directly affected by alterations in the blood fl0w."3'~ Specifically, experiments after balloon catheter injury of the rat carotid artery demonstrated that increased blood flow was associated with decreased neointimal thickening. It was also suggested that luminal SMC can affect the arterial wall structure by modifying the function of underlying SMC.

The results from our in vitro model seem to correlate well with the above findings. We found a 24% difference in the cell number between human SMC cultures ex-


Figure 4. (a, c) Immunofluorescence staining patterns of PCNA in hASMC in a stationary culture, and hASMC exposed to 25 dyn/cm2 shear stress, respectively, at the end of a 24-h experiment. (b, d) Phase contrast photomicrographs of the (a) and (c) fields, respectively. The arrow indicates the same human aortic smooth muscle cell with bright nuclei that was scored as a S-phase cell. Original magnification: 2 0 ~ .

posed to 5 and 25 dyn/cm* shear stress levels for 24 h. This indicates that shear-stress differences within the physiological range may have significant effects on SMC replication rate. The work of Sterpetti et al. on bovine SMC showed similar trends in the growth rate of SMC under flow condition^."^'^ Their cultures were growth arrested when exposed to shear stress above 3 dyn/cm2, while our results showed that, although increased shear stress reduced the proliferation of SMC, the cell population was actively replicating, even at 25 dyn/cm*. These differences may be due to differences in cell source (human vs. bovine). Other in vitro studies, in which bovine aortic endothelial cells (BAEC) were exposed to shear stress, showed that shear stress levels less than 5 dyn/cm2 had no effect on the proliferation rate of BAEC; whereas shear stress levels above 15 dyn/cm* considerably decreased the BAEC replication rate.''

LDH measurements have been used to measure shear sensitivity of cell lysis due to hydrodynamic f o r ~ e s . * ~ , ~ ~ The similarity of LDH concentrations in the media of stationary and flow cultures provided evidence that flow in the range employed did not cause significant detachment of hASMC from the surface. In all the experiments less than 5% cell lysis was observed and there was no difference in cell death between control and shear stress cultures. Studies by Ziegler et al. demonstrated that subconfluent endothelial cells attach more firmly to the

substrate under flow condition^.^^ Assuming a similar response for SMC, this finding, together with the measured LDH levels, indicates that the reduction in cell number at high shear stress is not due to cell detachment from the slide.

PCNA is a cyclin that is synthesized in the late GI and S phase of the cell cycle, permitting DNA synthesis, and most of it is degraded at the end of the S p h a ~ e . * , ~ ~ , ~ ' Only cells with bright nuclei, with a granular distribution of the PCNA protein were scored as S-phase cells, because the cellular content of the PCNA protein substantially increases during the S phase as it is associated with the ongoing DNA synthesis. There are two forms of the PCNA protein: a soluble form that is not involved in replication and is sensitive to organic solvents, and an insoluble one which is associated with the DNA synthesis. To measure only the form of the cyclin that is a component of the DNA replication system the cells were fixed in methanol. PCNA is a highly conserved protein and most of the antibodies show cross-reactivity among species.

PCNA immunofluorescence experiments demonstrated that the hASMC population was viable and was normally proliferating after 24 h, both in stationary control cultures and under a wall shear stress of 25 dyn/ cm*. The PCNA staining, together with the cell counts, demonstrated that, in the range of shear stress used and

PAPADAKI, MclNTIRE, AND ESKIN: FLOW REGULATION OF GROWTH KINETICS OF HASMC 559

for the time course of these experiments, cells were not arrested. An interesting finding was that the differences in the percentage of PCNA-positive nuclei between the stationary control and flow-exposed cultures were es- sentially the same as the differences in the cell density. Based on these findings, with both LDH activity in the media and PCNA staining, we speculate that the reduction in cell number under flow exposure is due to slower movement through the different phases of the cell cycle, and not to increased cell death.

Flow did not appear to have any effect on the orientation of hASMC for the time of exposure and shear stress levels studied. Cells in both stationary and sheared cultures were long and thin and formed swirling patterns. In contrast, in vitro experiments with bovine endothelial cells have consistently demonstrated that the cells elongate in the direction of fl0w.~3~9'~ In those studies, less alignment was observed in cell populations that were close to confluency, indicating that cell contact might inhibit the kinetics of this phenomenon. It is possible that, because SMC do not have a history of direct flow exposure in the normal arterial wall, and because they are not contact inhibited, they sense and respond to the flow in different ways than do endothelial cells.

Our in vitro data suggest that SMC directly exposed to fluid hemodynamic forces, as would occur after endothelial denudation, respond to flow alterations by changes in their population dynamics. This may result from the modulation of synthesis and secretion of, or binding of, growth factors at the blood-vessel wall inter- face. These results also indicate that shear stress in the normal artery may be homeostatic for medial SMC. Recent modeling studies have indicated that interstitial fluid flow driven by the transmural pressure gradient across the arterial wall may impose significant shear stress on underlying SMC and may affect mass transfer of macromolecules to the SMC surface. 1335 Differences observed when comparing cell numbers in low- and high-flow groups suggest that efforts to increase blood flow across injured segments of the arterial wall may reduce intimal hyperplasia.

This work was partially supported by Texas Biotechnology Corporation, NIH Grants HL18672 and NS23326, Welch Foundation Grant C-0938, and TATP Grant 003604 (L.V.M.). Assistance from L. Sturgis and C. Kagan is ap- preciated.

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effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro

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