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Am J Physiol Heart Circ Physiol 290: H2498-H2508, 2006. First published January 13, 2006; doi:10.1152/ajpheart.00828.2005
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Heterogeneous response of microvascular endothelial cells to shear stress

D. Hong,1 D. Jaron,1 D. G. Buerk,2 and K. A. Barbee1

1School of Biomedical Engineering, Science and Health Systems, Drexel University; and 2Departments of Physiology and Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 4 August 2005 ; accepted in final form 5 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.

calcium wave; adenosine 5'-triphosphate; heterogeneity

ENDOTHELIAL CELLS (ECs) cover the inner surface of blood vessels and are constantly exposed to shear stress due to blood flow. It is well established that ECs are sensitive to changes in shear stress. Intracellular signals generated in response to flow elicit a cascade of responses that involve cytoskeletal organization, remodeling of cellular morphology, and changes in gene expression (2, 13). One of the early responses to shear stress is the elevation of intracellular Ca2+ concentration ([Ca2+]i). Ca2+ is an essential trigger to successive physiological and biochemical reactions, such as the production of nitric oxide and the activation of Src tyrosine kinase and C kinase (13).

The cellular and molecular pathways mediating mechanotransduction in ECs have not been resolved. Several mechanisms have been proposed, including the following points. First, shear stress is sensed by integrins on the cell surface, transmitting forces through cytoskeleton, thus activating signal transduction through stretch-activated cation channels (13). Second, shear stress may cause ECs to synthesize and release ATP. The ATP concentration at the cell surface increases by overcoming the local effects of degradative enzymes, thereby mediating the Ca2+ signaling (16, 43, 44). Third, shear stress induces an efflux of K+ and membrane hyperpolarization, increasing the driving force for Ca2+ entry (33). Fourth, flow-sensitive Cl– currents play a role in modulating Ca2+ influx by altering the membrane potential (3, 30). Finally, components of the glycocalyx, of which heparan sulfates are the most abundant, function as signal transduction molecules (17). Shear stress may activate several pathways simultaneously; however, the relationships and possible interactions among the various pathways have not been determined.

ATP has been shown to be released from large vessel ECs in response to numerous stimuli, such as shear stress and vasoactive agonists, including ATP itself (7, 8, 43, 44). The released ATP can activate purinergic receptors and stimulate the formation of inositol(1,4,5)-trisphosphate [Ins(1,4,5)P3], which binds to Ins(1,4,5)P3 receptors and triggers Ca2+ release from intracellular stores (15). Thus endogenously released ATP can act as an extracellular messenger, regulating the flow-induced calcium response in large vessels. However, the possible role of ATP signaling in the endothelial transduction of shear stress-induced calcium responses in the microvascular ECs is not yet established.

The vascular system is a complex network of vessels connecting the heart with diverse organs and tissues to maintain their homeostasis in response to physiological changes. The ECs that line the vessel lumens play an integral role in the regional specialization of vascular structure and physiology. Whereas all ECs share certain common properties, phenotypic heterogeneity is observed between ECs from large vessels and microvascular ECs (10). Studies have shown longitudinal differences in endothelial functions between conduit and resistance vessels (18, 21, 42). In large vessels, shear stress-induced Ca2+ is related to vessel dilation. Either removal of extracellular calcium or chelation of intracellular calcium reduces the shear stress-dependent nitric oxide (NO) production in aortic ECs (38, 45). However, calcium appears to be less important in shear stress-induced dilations of smaller arteries and arterioles (42). The microcirculation is the site where many important biological substances are transported between the blood and surrounding tissue. In contrast to transport processes at the organ or whole body level, transport in the microcirculation is known to be highly heterogeneous, both in space and time. The heterogeneous calcium response in capillaries has been shown to account for the distribution of localized leakage sites for macromolecules in microvessels (34). Calcium waves/oscillations have also been observed in arterioles and capillaries (40, 47), which serve to coordinate and regulate the activities of cell groups.

Despite the recognition of regional differences in endothelium, in vitro studies of the responses of ECs to flow have focused almost exclusively on ECs from large vessels. A recent report indicated that NO concentrations in tissues cannot be accurately predicted when blood vessels are considered in isolation (20). NO production from venules and capillaries contributes significantly to the NO concentration in the vicinity of arterioles, which are usually the primary focus of experimental investigations because of their important role in the distribution of blood to tissues. NO release is highly controlled and usually preceded by elevation of intracellular calcium; therefore, it is important to investigate the calcium response to shear stress in microvascular ECs. The typical response to the initiation of shear stress in cultured aortic ECs is a rapid mobilization of intracellular calcium in a dose-dependent fashion (5, 41), although more complex behaviors (e.g., calcium oscillations) have been observed. However, little has been done to investigate whether the nature of the response or the mechanisms of mechanotransduction are similar in microvascular cells. In the present study, we have characterized the responses of microvascular ECs to shear stress in terms of their calcium signaling. We used ECs isolated from the adrenal medulla, a highly vascularized tissue containing primarily microvessels. We compared these cells with cells derived from the aorta and found differences in both the spatial and temporal characteristics of the calcium responses. Shear stress elicited calcium waves that originated from one or several cells and propagated to neighboring cells in rat adrenomedulary ECs (RAMECs), whereas the response in bovine aortic ECs (BAECs) was rapid and synchronous. We further investigated the mechanisms for this novel pattern of response and cell-cell communication in RAMECs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. BAECs were obtained from Dr. Keith Gooch's laboratory (University of Pennsylvania), and RAMECs were provided by Dr. Peter Lelkes (Drexel University). RAMECs were isolated from the adrenal medulla. During isolation, large vessels, including any sinusoidal capillaries, were carefully removed by dissection. Thus the isolated cells were primarily continuous capillary endothelium (24). ECs were cultured in Dulbecco's modified Eagle's medium, (Mediatech Cellgro), supplemented with 10% fetal bovine serum (Sigma) and 2 mmol/l L-glutamine (Mediatech Cellgro). The cultures were studied as confluent monolayers of polygonal cells.

Ca2+ measurement. Fluo 3 was used as the Ca2+ indicator. When compared with Fura-2, the higher dissociation constant (Kd) value reduces the buffering of the [Ca2+]i signal. In addition, the longer excitation wavelength avoids the cell damage by exposure to UV, and its large optical signal provides very good signal-to-noise ratio (28). ECs were loaded in the dark with Fluo 3 by incubation with 5 µmol/l Fluo 3-acetoxymethyl ester (Molecular Probes) in Dulbecco's phosphate-buffered saline (DPBS) (Sigma) at pH 7.4 for 40 min at room temperature. Cell fluorescence was monitored and recorded.

Device and mechanical stimulation. A custom-built cell-shearing device based on a cone and plate configuration was mounted on the microscope stage and used to apply precise mechanical loading conditions to the ECs (6). For each experiment, a volume of 1.2 ml DPBS (containing no exogenous ATP or other stimulatory agents) was added to the well to fill the gap between the cone and plate. For the mechanical loading period, ECs were monitored for about 30 s under static conditions before the onset of shear stress to establish basal levels of calcium. The shear stress was then increased linearly to 5, 10, 20, or 30 dyn/cm2 over 0.1 s and maintained at a steady level for 5 min. In addition, RAMECs were exposed to 60 dyn/cm2 shear stress to account for the fact that shear stress in the microcirculation can be significantly larger than in large vessels (2).

ATP assay. ATP release from ECs was determined using the luciferin-luciferase assay (Sigma). The samples (100 µl) collected from cells exposed to shear stress after 5 min or sham controls were pipetted into the wells of a microplate. The plate was placed in a luminescence counter (1450 MicroBeta Jet, PerkinElmer) and processed by injection of 50 µl ATP assay mix dilution buffer (20-fold dilution). ATP concentrations were calculated from a calibration curve constructed by using ATP standards.

Immunocytochemical staining. Cells were fixed with 4% paraformaldehyde in DPBS for 20 min. Nonspecific binding sites were blocked by incubation with blocking solution (10% normal donkey serum in DPBS) for 30 min. Cells were then incubated at room temperature with primary antibody (anti-P2Y2 receptor; Calbiochem), diluted in 1% donkey serum in DPBS to a final concentration of 3 µg/ml for 1.5 h and secondary antibody (anti-rabbit Alexa 488), diluted in 1% donkey serum in DPBS to a final concentration of 1:1,000 for 1 h in the dark. Control experiments were performed identically except for the omission of the primary antibody.

Chemicals and reagents. Suramin (a nonspecific purinergic receptor blocker), apyrase, DPBS, nucleotides ATP, ATP bioluminescent assay kit, and 1-heptanol were purchased from Sigma. Suramin, apyrase, heptanol, and ATP were directly dissolved at their final concentration in DPBS. Normal donkey serum was purchased from Jackson Immuno Research (West Grove, PA). The Fluo3-acetoxymethyl ester and anti-rabbit Alexa 488 were obtained from Molecular Probes.

Image analysis. To evaluate calcium dynamics, individual cells were outlined manually, and the average fluorescence intensity in each selected cell was calculated by using Axon Imaging Workbench. To control for cell-to-cell variations in dye loading, all fluorescence measurements were expressed as a ratio (R) of fluorescence intensity (F) to the basal fluorescence intensity (F0). A fluorescence ratio value >1.2 was selected as the criterion for cell activation because under control conditions, R fluctuated about the control level (R = 1) with a standard deviation of 0.19.

The following parameters characterizing the [Ca2+]i response to the flow or to agonist were analyzed: 1) fraction of activated cells (R > 1.2) in the observed field; 2) the amplitude of [Ca2+]i peak in activated cells as indicated by the peak fluorescence ratio; 3) number of calcium waves initiated in the first 5 min after the initiation of shear stress; and 4) number of activated cells in each calcium wave propagation group.

Statistical analysis. For each experimental condition, a minimum of three runs were analyzed. Relationships between groups were compared using ANOVA followed by Tukey's test for significance between treatment groups. Results were expressed as means ± SE. P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Shear stress-induced calcium waves in RAMECs. BAECs were exposed to shear stresses of 5, 10, 20, and 30 dyn/cm2, and changes in [Ca2+]i were monitored. Shear stress elicited a rapid and synchronous rise in [Ca2+]i (>90% BAECs were actived when exposed to shear stress of 20 dyn/cm2). The magnitude of the [Ca2+]i change increased as a function of shear stress (Fig. 1, A and B). The calcium rise began 1–3 s after shear stress initiation and reached its peak amplitude around 15 s. It then decreased to near baseline levels (Fig. 1C). These results are consistent with previous reports (5, 41). In contrast, shear stress induced spatially heterogeneous calcium responses in RAMECs, originating from one or several cells and propagating to neighboring cells (Fig. 2A) . The propagation radius varied widely in the series of experiments, with activated groups ranging in size from 2 to 15 cells. During the propagation, the amplitude of the [Ca2+]i peak did not decline as a function of the distance away from the initiating cells (Fig. 2, B and C). In contrast to the synchronous response of BAECs, the calcium waves in RAMECs were initiated at different times and different locations. For example, in the experiment shown in Fig. 2, 6 s after shear stress initiation, calcium waves were observed in cell group 1, and the cells exhibited calcium oscillations (Fig. 2B). Calcium waves were triggered in cells group 2 158 s later. Other small groups of calcium responses were also observed in the interim but not indicated in Fig. 2. There was no consistent pattern in the calcium wave initiation or propagation.


Figure 1
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  		Fig. 1. Intracellular Ca2+ concentration ([Ca2+]i) response to shear stress in bovine aortic endothelial cells (BAECs). A: sequential images of Fluo3 fluorescence taken from BAECs at various times after onset of shear stress ({tau} = 20 dyn/cm2). Shear stress elicited a rapid, synchronous, and relatively homogeneous calcium change in BAECs. Raw data reflect variations in dye concentration, basal [Ca2+]i level, and cell morphology. Relative changes in [Ca2+]i are indicated by the fluorescence ratio, which is insensitive to these variations. B: dose dependence of peak calcium amplitude on the magnitude of shear stress (P < 0.01). C: time course of the fluorescence ratio response of BAECs subject to different levels of shear stress. Each trace shown is the average of 5–6 independent experiments. Large triangle indicates time of flow initiation. For clarity, no error bars were shown. However, maximum SEs occurred at the peak of the calcium amplitude and is shown by error bars in B.

 

Figure 2
Figure 2
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  		Fig. 2. Shear stress-induced calcium wave in rat adrenomedullary endothelial cells (RAMECs). A: single frames of data are shown at various times after onset of shear stress. Calcium wave was observed at cell groups 1 and 2 at 16 and 172 s, respectively, after shear stress initiation. In both groups, calcium waves started from one or several cells then propagated to neighboring cells seconds later. B: time course of normalized fluorescence amplitude of the cells indicated in group 1. Ca2+ peak amplitude did not diminish during the calcium wave propagation. C: time course of normalized fluorescence amplitude of the cells indicated in group 2.

 
The calcium response time of BAECs and RAMECs is shown in Fig. 3. For BAECs, shear stress induced synchronous calcium responses, and the response reached peak amplitude within 15 s. In contrast, RAMECs responded to shear stress at different times, and the response times occurred mostly in the first 3 min. Furthermore, the average magnitude of the response did not depend on the time of the occurrence.


Figure 3
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  		Fig. 3. Scatter plots of peak calcium amplitude vs. time. Cells were exposed to 20 dyn/cm2 shear stress. Twenty responding cells were selected at random from a single experiment, which was representative of 6 similar experiments.

 
The number of calcium waves initiated and the average number of cells in each activated group under shear stress were significantly greater than in unsheared controls (Fig. 4A) but did not depend on the shear stress magnitude. Control cells exhibited occasional spontaneous [Ca2+]i oscillations, but there was no propagation of [Ca2+]i waves to neighboring cells. The amplitude of the responses was not dependent on shear stress nor were they statistically different from controls (Fig. 4B).


Figure 4
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  		Fig. 4. Intercellular calcium wave features of RAMECs subjected to different levels of shear stress. A: number of [Ca2+]i waves initiated within 5 min of the onset of shear stress and average number of activated cells in each group were greater than controls (P < 0.05) but not dependent of the level of shear stress (P > 0.1). In static controls (no shear stress), occasional spontaneous [Ca2+]i oscillations were observed. B: magnitude of [Ca2+]i changes was not dependent on level of shear stress. In controls, the amplitude of spontaneous [Ca2+]i oscillations was not statistically different from the magnitude of calcium changes under varying shear stress; however, there was no propagation of [Ca2+]i waves to neighboring cells, therefore, the variance of cell group number is zero.

 
Release of endogenous ATP. The application of the media collected from cells exposed to shear to unsheared cells immediately activated calcium response in a small portion of RAMECs, which suggests release of vasoactive substances from the cells induced by shear stress. In the sham controls, the concentration of ATP in the collected medium was very low (RAMEC: 16.3 ± nmol/l, BAEC: 18.9 ± 4 nmol/l). The stimulation of the cells with a shear stress of 20 dyn/cm2 for 5 min led to an increase in extracellular ATP concentration in both BAECs (102.3 ± 23 nmol/l) and RAMECs (141.2 ± 40 nmol/l) (Fig. 5). It is important to note that these final concentration values depend on the volume of fluid in the experimental chamber. Thus, to compare values from different experimental models, the amount of ATP per cell is the relevant datum. Shear stress stimulated BAECs and RAMECs to produce 13.5 and 18.6 pmol ATP per million cells per minute, respectively.


Figure 5
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  		Fig. 5. ATP release from endothelial cells under conditions of shear stress. Cells were subjected to a shear stress 20 dyn/cm2 or sham control for 5 min, and aliquots of bathing buffer were collected and assayed for ATP concentration. *P < 0.005; #P < 0.05 compared with control.

 
Effect of suramin, apyrase, and 1-heptanol on the shear stress-induced calcium waves. To investigate whether the initiation and propagation of calcium waves in response to shear stress was mediated by ATP, cells were pretreated with suramin, a nonspecific P2 receptor blocker, for at least 10 min before the shear experiments. Suramin treatment significantly inhibited both the initiation and propagation of calcium waves (Fig. 6A). However, the presence of suramin did not change the magnitude of [Ca2+]i peak amplitude in the responding cells (Fig. 6B). Suramin pretreatment significantly reduced the magnitude of calcium responses to shear stress in BAECs. As a negative control, suramin was added to both BAECs and RAMECs, and it did not lead to calcium mobilization in either of them.


Figure 6
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  		Fig. 6. Effect of suramin, 1-heptanol, and apyrase on flow induced [Ca2+]i waves in RAMECs (n = 10 for untreated, n = 8 for suramin pretreatment, n = 10 for apyrase pretreatment, n = 3 for 1-heptanol pretreatment. *P < 0.01 compared with untreated; #P < 0.01 compared with untreated) A: suramin and apyrase pretreatment significantly decreased both the initiation and propagation of shear stress-induced Ca2+ wave, but 1-heptanol pretreatment did not inhibit the shear stress-induced Ca2+ wave. B: neither suramin, apyrase, nor 1-heptanol pretreatment affected peak [Ca2+]i amplitude in responding cells.

 
To rule out the nonspecific effects of suramin, we also investigated the dependence of the calcium signaling on released ATP by conducting the experiments in the presence of apyrase, which degrades extracellular ATP. The application of 10 U/ml apyrase resulted in an inhibition similar to the suramin-pretreated group. (Fig. 6, A and B)

To test the possibility that spreading of the calcium from the initial responding cells to adjacent cells was due to gap junctional communication, we pretreated RAMECs for 15 min with 3 mmol/l 1-heptanol, which inhibits gap junctions. Inhibition of gap junctional communication had no effect on the shear stress-induced calcium waves (Fig. 6, A and B).

[Ca2+]i response to ATP in RAMECs. That suramin inhibited calcium wave initiation and propagation suggests that ATP release and autocrine/paracrine signaling are involved in the shear stress-induced [Ca2+]i response in RAMECs. To further investigate the heterogeneity of the response and the role of ATP, we measured calcium responses of the cells to exogeneous ATP stimulation. The application of ATP to RAMECs elicited a rapid [Ca2+]i response that was spatially and temporally heterogeneous (Fig. 7). ATP induced an immediate [Ca2+]i response in some individual cells. The [Ca2+]i increase was then observed in adjacent cells, and the magnitude of the [Ca2+]i transient did not decline as the [Ca2+]i changes propagated away from the initial response. Another characteristic of the RAMEC response to ATP was the large variability in the calcium oscillations even for contiguous cells (Fig. 7, B and C). ATP could initiate calcium oscillations that persisted for the full duration of the observation (around 5 min) (cell 1) or oscillations that had only 1 or 2 transients (cell 2 and 3). In some of the cells, [Ca2+]i decreased to baseline without any oscillations (cell 4). The long-lasting oscillations were usually observed in the initially responding cells.


Figure 7
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  		Fig. 7. A: 10 µM ATP evoked synchronous [Ca2+]i transients in BAECs and heterogeneous [Ca2+]i responses in RAMECs. B: application of ATP to RAMECs elicited a rapid [Ca2+]i response that was spatially and temporally heterogeneous. Addition of ATP immediately induced a [Ca2+]i response in a subset of individual cells. [Ca2+]i increase then spread to adjacent cells. C: temporal characteristics of the [Ca2+]i changes elicited by ATP depended on the cell's position within the group. Initially responding cells exhibited sustained oscillations, whereas more distant cells had a single transient.

 
The fraction of activated cells increased as a function of ATP concentration (Fig. 8). Even 100 µmol/l ATP evoked a response in only ~70% of the cells. Low doses of ATP evoked [Ca2+]i increases just in individual cells. Higher ATP concentrations induced [Ca2+]i transients that propagated to adjacent cells. The magnitude of the rise in [Ca2+]i elicited by ATP was not dependent on the concentration in the range of 0.1–100 µmol/l (Fig. 8B). The peak amplitude of [Ca2+]i was reduced only at a level of 0.01 µmol/l ATP. The rise time and return to basal level were not affected by ATP concentration level (Fig. 8C).


Figure 8
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  		Fig. 8. Dose-dependent effect of ATP on [Ca2+]i response in RAMECs and effect of suramin pretreatment (n = 7–8 for each group). A: percentage of activated cells increased as a function of the ATP concentration (+P < 0.001 compared with 0.01 µM, *P < 0.001 compared with 0.1 µM, {ddagger}P < 0.005 compared with 1 µM), and suramin pretreatment significantly reduced fraction of activated cells (#P < 0.01). B: [Ca2+]i amplitude was not dependent on ATP concentration in the range of 0.1–100 µmol/l. Also, suramin pretreatment did not significantly affect peak [Ca2+]i amplitude in activated cells. C: normalized fluorescence amplitude of activated cells over time. Each trace shown is average of 7~8 independent experiments. With suramin treatment, there were no activated cells at ATP concentration of 0.01–1 µmol/l.

 
When two different doses of ATP (1 and 100 µmol/l) were applied sequentially to the same cells, 100 µmol/l ATP activated more cells than 1 µmol/l. The same cells that responded to 1 µmol/l ATP (Fig. 9, left) were also all activated by 100 µmol/l ATP (Fig. 9, right) with similar Ca2+ peak amplitude.


Figure 9
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  		Fig. 9. Ca2+ response of RAMECs to sequential application of 1 (left) and 100 µmol/l ATP (right) separated by 60 s. ATP at 100 µmol/l activated more cells than 1 µmol/l; however, all the cells that responded to 1 µmol/l ATP also responded to 100 µmol/l with similar Ca2+ peak amplitude (indicated by white outline).

 
[Ca2+]i response to ATP in BAEC. Parallel experiments were conducted using BAECs. BAECs responded to ATP in a dose-dependent manner. At the lowest concentrations of ATP (0.01 and 0.1 µmol/l), a very small fraction of cells was activated with low [Ca2+]i amplitude. ATP evoked rapid, simultaneous, and homogeneous [Ca2+]i transients in all the cells (Fig. 7A). Higher doses of ATP evoked higher peak amplitude and longer duration of [Ca2+]i response elevation (Fig. 10).


Figure 10
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  		Fig. 10. Time course of normalized intracellular [Ca2+]i response of BAECs subject to different concentrations of ATP. For BAECs, both magnitude and duration of response were dependent on ATP concentration. Each trace shown is average of 6~8 independent experiments. Large triangle indicates addition of ATP.

 
Effect of suramin pretreatment on the [Ca2+]i response to ATP. In these experiments, we confirmed that suramin treatment can block the calcium response to ATP in RAMECs. Incubation with 400 µmol/l suramin resulted in a complete inhibition of [Ca2+]i in response to ATP stimulation up to 1 µmol/l. Higher concentrations of ATP induced an increasing percentage of activated cells, but suramin pretreatment caused a 77% inhibition in 10 µmol/l ATP and 22% in 100 µmol/l ATP activation. Whereas suramin significantly reduced the number of responding cells to ATP stimulation, the amplitude of [Ca2+]i peak in the activated cells was not affected (Fig. 8). Suramin significantly reduced the [Ca2+]i peak amplitude to 1 µM ATP in BAECs.

P2Y2 receptor staining. The expression of P2Y2 receptors on RAMECs and BAECs was compared by immunofluorescence (Fig. 11). P2Y2 receptor immunoreactivity was clearly present on the BAECs, and the mean fluorescence intensity was significantly higher than in RAMECs. The immunolabeling of P2Y2 receptors in RAMECs also showed a nonuniform pattern of expression. Strong immunoreaction was found in a few cells that had bright fluorescence over the whole cell membrane. Lighter binding was present in some cells that expressed faint fluorescence. Unlabeled or very weakly labeled cells were also present with very dim fluorescence (Fig. 11).


Figure 11
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  		Fig. 11. Expression of P2Y2 receptor on RAMECs and BAECs. P2Y2 receptor immunoreactivity was clearly present on BAECs and mean fluorescence intensity was significantly higher than RAMECs. Immunoreactivity for P2Y2 receptor in RAMECs was heterogeneous with a few cells staining brightly and little or no staining in surrounding cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study demonstrate for the first time that BAECs and RAMECs respond differently to shear stress. In BAECs, shear stress elicited a rapid and transient rise in [Ca2+]i that was synchronous across the monolayer. Furthermore, the magnitude of the [Ca2+]i change was dependent on the level of shear stress, consistent with previous reports (5, 41). In contrast to the response in aortic ECs, RAMECs responded to shear stress with [Ca2+]i transients that were spatially heterogeneous and asynchronous. Responding cells initiated a wavelike propagation to neighboring cells of the [Ca2+]i transients of which their magnitude was independent of the shear stress level. Our results with BAECs are also similar to previous findings using rat aortic ECs (22, 45), suggesting that the characteristics of the response are not species dependent.

We showed that both BAECs and RAMECs release ATP in response to shear stress. Spatially uniform, exogenously applied ATP produced a spatially heterogeneous calcium response in RAMECs that was similar to the shear stress response. The initiation and the propagation of calcium waves in RAMECs, induced either by shear stress or exogenous ATP, were significantly suppressed by suramin, a purinergic receptor blocker. Furthermore, immunocytochemical staining indicated nonuniform expression of P2Y2 receptors in RAMECs. Based on our observations, we propose a mechanism for the spatial heterogeneity of the shear stress response of RAMECs in which shear stress causes the release of ATP, which then elicits calcium responses from a subset of cells that are differentially sensitive to ATP. Additional ATP is then released from those cells, creating a locally elevated concentration, sufficient to activate the calcium response in neighboring cells.

It has previously been shown that shear stress induces ATP release from large vessel ECs (9, 43, 44, 46). Our data provide for the first time evidence that ATP is also released from microvascular ECs in response to shear stress. Meanwhile, our data strongly suggest that ATP mediates both the initiation and propagation of calcium waves in response to shear stress via purinergic receptor activation. Preincubation with a purinergic receptor blocker or enzymatic degradation of extracellular ATP significantly inhibited the calcium wave initiation and propagation. Furthermore, when unsheared RAMEC cells were exposed to the media collected from cells that were exposed to shear stress, a calcium response was immediately activated in a small portion of the unsheared cells. It should, however, be mentioned that suramin is relatively nonselective. It may disturb other cellular pathways in addition to its effect on purinergic receptors. Caution is needed in interpreting the data generated from the use of suramin. Nevertheless, the data from the apyrase experiment provide confirmation that ATP release during the shear stress is required for the calcium response.

Our study indicates that ATP released in response to shear stress mediates mechanotransduction in microvascular ECs. The physiological roles of ATP in the microcirculation have also been pointed out by other previous studies. With regard to shear stress, Liu et al. (23) showed a synergistic effect of ATP and flow on dilation that was attenuated by application of purinergic antagonists in isolated rat small mesenteric arteries; however, they did not investigate the response to flow in the absence of exogenous ATP.

The potential role of shear stress-stimulated ATP release in the control of microvascular perfusion is further suggested by the following studies. McCullough et al. (26) reported that application of micromolar amounts of ATP into first- and second-order arterioles resulted in a significant conducted vasodilation in the same arteriole as far as 1,750 µm upstream from the site of application. Furthermore, Collins et al. (11) showed that intraluminal application of ATP in a collecting venule resulted in an increase in arteriolar diameter and red blood cell flux in capillaries. Thus it appears that ATP is capable of stimulating vasodilatory responses in microvascular networks that propagate well beyond the point of application (upstream and downstream). Therefore, flow-induced ATP release by capillary ECs, as observed in our experiments, may serve a role in coordinating the microvascular response to meet the perfusion requirements of the tissue.

Burnstock's laboratory (46) previously quantified the ATP concentration when human umbilical vein ECs (HUVECs) were exposed to 25 dyn/cm2 shear stress by using a luciferin-luciferase assay, and results showed that the highest concentration of ATP (0.1 µmol/l) was seen after 3 min of shear. In our study, the amount of ATP released from RAMECs exposed to 20 dyn/cm2 shear stress was very similar (0.14 µmol/l). This value, however, represents the average concentration in the bulk fluid. As ATP is released from the cell monolayer under flow, the concentration in the diffusive boundary layer adjacent to the cells will be much higher than in the bulk. To estimate the concentration of ATP at the cell surface in our shearing experiments, we can find the exogenously applied ATP concentration for which suramin treatment inhibited the calcium response to a similar degree. Thus we estimate the cell surface concentration to be ~1–10 µM.

We have proposed that the spatial heterogeneity of the calcium response of RAMECs to shear stress is due to the nonuniform sensitivity to ATP. This is supported by the finding of a nonuniform pattern of expression of P2Y2 receptors and by the heterogeneous calcium response to the (spatially uniform) exogenous application of ATP. The mechanism by which shear stress elicits the release of ATP is unknown, and it is possible that heterogeneity in the sensing mechanism involved in this response also contributes to the heterogeneity of the [Ca2+]i response. Davies et al. (4, 14) proposed that heterogeneous responses to shear stress could be due to the variations in the detailed distribution of shear stresses acting on the cell surface, owing to the specific surface topography of the monolayer. While it is possible that this mechanism contributes to the heterogeneity that we observed, this explanation seems incomplete in this case. There was no dramatic difference in the morphology of the two cell types; therefore, one would expect similar heterogeneity in the BAEC response if it were determined by surface topography alone.

There are two major families of nucleotide receptors, defined based on molecular structure and signal transduction mechanisms. P2X receptors are ATP-gated, Ca2+-permeable channels, where ATP binding directly causes Ca2+ influx from extracellular space. P2Y receptors are G protein-coupled receptors and are linked to the stimulation of phospholiphase C and the generation of Ins(1,4,5)P3, which triggers Ca2+ release from intracellular stores (39). Even though Yamamoto et al. (43) demonstrated shear stress-activated Ca2+ influx into HUVECs via P2X4 purinoceptors there is evidence from several studies that supports the Ins(1,4,5)P3-mediated release from intracellular stores as the primary source of calcium in response to flow (5, 41). Prasad et al. (36) and Nollert et al. (32) showed that Ins(1,4,5)P3 production increases in responses to flow with an initial peak at 15 s, and the increase is proportional to shear stress magnitude. We evaluated the P2Y2 receptors in this study because P2Y2 are present in ECs from both the aorta (29) and adrenal medulla (25). Furthermore, the Ins(1,4,5)P3 response to ATP appears to be mediated exclusively by P2Y2 in the ECs from the adrenal medulla (1, 37). However, other purinergic receptors coexist in ECs from both aorta and adrenal medulla, and ATP is able to evoke calcium increases by activating these receptors (12, 25). Thus it is possible there is similar heterogeneity in other purinergic receptors, which could also contribute to the calcium response.

Our results suggest that the calcium wave propagation in RAMECs is due to ATP-induced ATP release. 1-Heptanol pretreatment did not inhibit the calcium waves, suggesting RAMECs did not require gap junctions to propagate calcium response or that gap junctions were not the main pathway of the intercellular calcium wave. The observation that suramin incubation reduced the spreading of calcium indicates that the calcium wave operates via ATP release. The spreading of the calcium signal when RAMECs were exposed to spatially uniform exogenous ATP suggests that a localized peak in ATP concentration due to its release by the initially responding cell is capable of stimulating neighboring cells. This is consistent with the previous finding that ATP released from ECs stimulates the release of additional ATP (8).

It has been documented that the magnitude of ATP release from large vessel ECs in response to shear stress is dependent on the level of shear stress (9, 46). If in the RAMECs, the ATP is released in proportion to shear stress, then one might expect that the number and size of the responding groups would increase with shear stress. However, as the shear rate is increased, the diffusive boundary layer will be reduced in thickness, facilitating the removal of ATP from the cell surface by convective transport. This situation has been modeled by John and Barakat (19) who found that depending on the relationship between shear stress and ATP release, the surface concentration does not necessarily increase monotonically with increasing shear stress. In our study, the number and size of the responding groups were not statistically different over the shear stress range 5–60 dyn/cm2.

Our study did not directly identify the mechanisms for the heterogeneity in the temporal response in RAMECs. One possible cause for the delay of the response is the time course of ATP accumulation in the medium necessary to reach the threshold of individual cells with different sensitivities to ATP. Our data (Fig. 3) show that most responses occurred in the first 3 min. This is consistent with the result of Burnstock's group that the peak concentration of ATP in the collected medium occurred 3 min after the application of shear stress (9, 46). Another possible mechanism is the different time course for Ins(1,4,5)P3 accumulation induced by ATP stimulus in aortic and adrenal medullary ECs (1). In aortic cells, the Ins(1,4,5)P3 response reaches a maximum within a few seconds, followed by a rapid fall. In adrenal medullary ECs, a substantial response is observed within a few seconds, and then the Ins(1,4,5)P3 concentration continues to rise without reaching a maximum up to 5 min after stimulation (1). Ins(1,4,5)P3 acts as a messenger to link G protein-linked receptors with intracellular calcium activity. The slower time course of Ins(1,4,5)P3 accumulation to reach its threshold and then induce calcium response in RAMEC may explain the delay in the [Ca2+]i rise induced by shear stress. One of the characteristics of the response in RAMECs was the large number of responding cells with long duration calcium oscillations in contrast to the very few BAECs with oscillations. It is possible that the incidence of calcium oscillations observed in RAMECs is also due to the slower time course of Ins(1,4,5)P3 accumulation because sustained concentrations of Ins(1,4,5)P3 are known to generate intracellular calcium oscillations (31).

Another feature of the calcium signaling in RAMECs was also qualitatively different from BAECs. The RAMECs did not exhibit a graded response either to different levels of shear stress or to different concentrations of exogenous ATP. The following observations support the all-or-none response of RAMECs. First, only a fraction of RAMECs responded to ATP. Higher levels of ATP increased the fraction of activated cells but did not increase the calcium peak amplitude, suggesting a threshold in the purinergic signaling in RAMECs. Once the threshold was reached, the transduction system was maximally activated. Second, suramin pretreatment significantly reduced the fraction of activated cells, but the Ca2+ peak amplitude of activated cells remained the same. Third, in the flow experiments, suramin pretreatment inhibited the calcium initiation and propagation but had no effect on the calcium peak amplitude. Finally, when the same culture was exposed sequentially to 1 µmol/l ATP and 100 µmol/l ATP, the high concentration of ATP activated more cells than low concentration but did not change the Ca2+ peak amplitude. Furthermore, the same cells that responded to the low concentration (Fig. 9A) were also all activated by the high concentration (Fig. 9B) with similar calcium amplitude. The mechanisms of this all-or-none response remain unclear, and additional studies are needed to elucidate them.

The framework we offer for considering the differences between aortic and microvascular ECs in their response to shear stress may be a useful extension of our understanding of the diversity and regional specificity in ECs. It provides insight into the well-recognized regional variations in physiological properties that affect the vascular system. In particular, we have identified two distinct modes of signal modulation. In the aortic endothelium, the strength of the signal is modulated by the dose-dependent amplitude of the response in individual cells. In the microvascular endothelium, taken as a multicellular unit, the strength of the signal is modulated by the number and frequency of responding cells. The ramifications of this novel mode of signaling for the physiological function of microvascular networks are not clear and require further study.

Heterogeneous calcium responses in microvascular ECs have been reported in vivo and in isolated vessels (34, 35). The transient increases in [Ca2+]i can remain within localized regions (34), which correlate with sites of macromolecule leakage (27, 34) or can spread to neighboring cells as a calcium wave (47), serving to coordinate vascular functions. Our in vitro model appears to simulate many of these characteristics and has allowed us to propose a mechanism for heterogeneous cell activation and signal transmission to neighboring cells. Our hypothesis suggests that in microvessels, ATP plays a central role in the cell signaling in response to shear stress and serves to amplify and spread the effects of vasoactive agonists or shear stress beyond their initial sites of action. By this means, ATP acts as an autocrine and paracrine hormone, serving to integrate the heterogeneity of individual cell responses and coordinate vascular functions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-068164 and National Science Foundation Grant BES0301446.


   ACKNOWLEDGMENTS
 
We thank Dr. Gooch and Dr. Lelkes for providing the cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Jaron, School of Biomedical Engineering, Science, and Health Systems, Drexel Univ., Philadelphia, PA 19104 (e-mail: dov.jaron{at}drexel.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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