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Am J Physiol Heart Circ Physiol 290: H240-H247, 2006. First published August 19, 2005; doi:10.1152/ajpheart.01191.2004
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Inositol trisphosphate receptor calcium release is required for cerebral artery smooth muscle cell proliferation

M. Keith Wilkerson, Thomas J. Heppner, Adrian D. Bonev, and Mark T. Nelson

Department of Pharmacology, University of Vermont, Burlington, Vermont 05405

Submitted 30 November 2004 ; accepted in final form 15 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular damage signals smooth muscle cells to proliferate, often exacerbating existing pathologies. Although the role of changes in "global" Ca2+ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role of specific Ca2+ signals in determining VSM phenotype remains relatively unexplored. Earlier work with cultured VSM cells suggests that inositol 1,4,5-trisphosphate receptor (IP3R) expression and sarcoplasmic reticulum (SR) Ca2+ release may be linked to VSM cell proliferation in native tissue. Thus we hypothesized that SR Ca2+ release through IP3Rs in the form of discrete transient signals is necessary for VSM cell proliferation. To investigate this hypothesis, we used mouse cerebral arteries to design an organ culture system that permitted examination of Ca2+ dynamics in native tissue. Explanted arteries were cultured in normal medium with 10% FBS, and appearance of individual VSM cells migrating from explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca2+ waves in myocytes in the arteries that were blocked by the IP3R antagonist 2-aminoethoxydiphenylborate (2-APB). Inhibition of IP3R opening (via 100 µM 2-APB, 10 µM xestospongin C, or 25 µM U-73122) dramatically reduced outgrowth cell number compared with untreated or ryanodine-treated (10 µM) arteries. Consistent with this finding, 2-APB inhibited cell proliferation, as measured by reduced proliferating cell nuclear antigen immunostaining within 48 h of culture but did not inhibit cell migration. These results indicate that activation of IP3R Ca2+ release is required for VSM cell proliferation in these arteries.

calcium signaling; explant culture; calcium channels; sarcoplasmic reticulum

INTRACELLULAR CALCIUM, an essential regulator of vascular smooth muscle (VSM) function, controls numerous aspects of smooth muscle cell physiology, including contraction and proliferation (27). Ca2+ signaling in smooth muscle can exhibit a number of unique signatures. Changes in Ca2+ entry through L-type voltage-dependent Ca2+ channels (VDCCL) in the plasma membrane lead to graded changes in average ("global") Ca2+ throughout the cell's cytoplasm. Opening of ryanodine-sensitive, Ca2+-sensitive Ca2+ release channels [ryanodine receptors (RyRs)] in sarcoplasmic reticulum (SR) membrane can cause local Ca2+ transients ("Ca2+ sparks"), which activate large-conductance, Ca2+-sensitive K+ (BK) channels to close VDCCLs through membrane potential hyperpolarization (5). Vasoconstrictors can cause intracellular Ca2+ waves through activation of phospholipase C (PLC), production of inositol 1,4,5-trisphosphate (IP3), and activation of IP3 receptors (IP3Rs) in SR membrane (3). These Ca2+ signals differentially regulate ion channels such as the BK channel, contraction through myosin light chain kinase, and activation of Ca2+-dependent transcription factors such as cAMP response element binding protein and nuclear factor of activated T cells (NFAT) (4, 5).

The roles of IP3Rs, VDCCLs, and RyRs in regulating VSM phenotype are unclear. VSM cells undergo phenotype modulation in response to vascular damage, exhibiting the "synthetic" phenotype distinguished by the downregulation of contractile smooth muscle cell differentiation markers (25). The initiation for phenotype change is dependent on the passage of VSM cells through various cell cycle checkpoints that require specifically timed alterations in the levels of cytosolic Ca2+ concentration ([Ca2+]cyt) (3). Several studies have suggested that Ca2+-dependent cell cycle events require the release of Ca2+ from intracellular stores (8, 29, 34). The implication is that a proliferation-inducing agonist causes an increase in [Ca2+]cyt by signaling the release of Ca2+ from the SR (3, 36). In the short term, a change in Ca2+ signaling may be sufficient to stimulate VSM cells to revert to the proliferating/synthetic phenotype, whereas longer-term changes in expression of IP3R, VDCCL, and RyR may reflect cellular adaptations to the new Ca2+ signaling environment. For example, evidence from in vitro culture studies shows that IP3R expression is maintained (31, 32, 36), whereas VDCCL function (13, 18) and RyR expression (32) are lost, on conversion to the proliferating/synthetic phenotype.

Given the potential role of different, modulated Ca2+ signals in differential regulation of proliferation, we hypothesized that an alteration in intracellular Ca2+ signaling is involved in eliciting VSM cells to enter the cell cycle and begin proliferation. One shortcoming of previous experiments utilizing multipassaged VSM cells and established VSM cell lines is that each subsequent generation of cells progressively loses expression of key proteins that characterize the differentiated phenotype. To establish a model that more closely resembles the cellular dynamics of in vivo VSM cell migration and proliferation, we used explanted mouse cerebral arteries in organ culture to design an in vitro system. This approach permits tracking of VSM cell proliferation and migration and allows for simultaneous, high-resolution examination of VSM Ca2+ dynamics in the intact artery and in migratory cells. Our results suggest that an elevated frequency of IP3R-mediated Ca2+ waves is required for VSM cell proliferation and, furthermore, that RyR-mediated Ca2+ release inhibits this proliferation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drugs and solutions. Physiological saline solution [PSS; in mmol/l: 118.5 NaCl, 4.7 KCl, 1.2 MgCl2, 1.18 KH2PO4, 0.026 EDTA, 24 NaHCO3, 11 glucose, and 2 CaCl2 (pH 7.4)] was continuously bubbled with 95% O2-5% CO2 and heated to 37°C. Culture medium consisted of DMEM (Mediatech, Herndon, VA), penicillin-streptomycin (100 U and 100 µg/ml, respectively; Invitrogen, Carlsbad, CA), and 4 mM L-glutamate (Mediatech) and was supplemented with FBS (Invitrogen) as indicated. HEPES buffer solution contained (in mmol/l) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH. Caffeine, diltiazem, and thapsigargin were obtained from Sigma-Aldrich (St. Louis, MO); 2-aminethoxydiphenylborate (2-APB), xestospongin C, and U-73122 from Calbiochem (EMD Biosciences, San Diego, CA); and ryanodine from LC Laboratories (Woburn, MA).

Tissue preparation. Adult male C57BL/6 mice (25–31 g; Charles River Laboratories) were euthanized with an overdose of pentobarbital solution (200 mg/kg ip) and then thoracotomized in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1985) and as approved by the Institutional Animal Use and Care Committee of the University of Vermont. Posterior 70- to 120-µm diameter cerebral and cerebellar arteries were removed from the brain and placed in oxygenated, ice-cold PSS. After the connective tissue was removed, intact segments of the arteries were slipped over a glass cannula as previously described (16) for same-day [Ca2+]cyt measurements or cut into segments for culture.

Explant cultures. Arterial segments were cut into 500-µm lengths and transferred to two-well chambered coverslips (Lab-Tek, Nalge Nunc, Rochester, NY) containing culture medium supplemented with 10% or 0.1% FBS and drugs as indicated. The cultures were maintained in a water-jacketed cell incubator at 37°C under 5% CO2 in air. Culture medium and drugs were replaced daily. To stimulate outgrowth of VSM cells, arteries on glass coverslips were exposed to culture medium with 10% FBS (30). Typically, individual VSM cells migrating from the explanted arterial segments (outgrowth cells) were observed by 8 h of exposure to 10% FBS. Outgrowth cell numbers were obtained by averaging the mean daily counts from three 512-µm2 fields per cultured artery segment. Only VSM cells with nuclei clearly separated from the explanted artery segment were counted as outgrowth.

Immunofluorescence. Arteries were fixed on the chambered coverslips with 4% formaldehyde in PSS (pH 7.4). After permeabilization and block of nonspecific binding sites with goat serum, arteries were incubated in a solution of primary antibody diluted in 5% goat serum-phosphate-buffered saline overnight at 4°C. The following primary antibodies were used: a rat monoclonal antibody against Ki-67 (1:25 dilution; DakoCytomation, Carpinteria, CA) with a mouse monoclonal antibody to smooth muscle {alpha}-actin (1:10 dilution; Research Diagnostics, Flanders, NJ); a mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA, 1:500 dilution; BD Biosciences PharMingen, San Diego, CA); or a mouse monoclonal antibody against smooth muscle myosin heavy chain (smMHC) isoforms SM1 and SM2 (1:100 dilution; Sigma-Aldrich). After several washes in 0.1 M phosphate-buffered saline, the secondary antibodies Alexa Fluor 568 goat anti-mouse (1:200 dilution; Molecular Probes) and/or Alexa Fluor 647 goat anti-rat (1:200 dilution; Molecular Probes) were applied for 2 h at room temperature. Nuclei were stained with the fluorescent nucleic acid dye Sytox (1:3,000 dilution; Molecular Probes). After several washes, the chamber sides were removed from the coverslip, which was then inverted and mounted (Citiflour, Leicester, UK) onto glass slides. Outgrowth cells and explanted arterial segments were visualized using a laser confocal scanning microscope (model MRC 1000, Bio-Rad Laboratories, Hercules, CA). Specificity of immune staining was confirmed by the absence of fluorescence in arteries incubated with primary or secondary antibodies alone. For scoring of PCNA- and Ki-67-positive cells, multiple fields for each cultured artery segment were imaged and counted under double-blind conditions with the aid of Metamorph software (Universal Imaging, Downington, PA).

Confocal Ca2+ imaging. Transient Ca2+ signals were examined in the wall of intact artery segments or in VSM outgrowth cells of artery segments cultured for 5 days. Both preparations were incubated with the Ca2+-sensitive fluorescent dye fluo 4-AM (10 µM) and Pluronic acid (2.5 µg/ml; Molecular Probes) dissolved in HEPES buffer for 60 min at 21–23°C (intact arteries) or culture medium with 0.1% FBS in a CO2 incubator for 30 min at 37°C (VSM outgrowth cells). After loading, a krypton-argon laser at 480 nm was used to excite the fluo 4, and the emitted light was detected at >500-nm wavelengths with a laser scanning confocal microscope (OZ, Noran Instruments) using Prairie Technologies (Middleton, WI) software. Images (103 x 106 µm, 512 x 480 pixels, ~10–15 smooth muscle cells) were acquired every 33.33 ms (30 images/s) for 10 or 20 s (intact arteries) or every 66.67 ms (15 images/s) for 20 s (VSM outgrowth cells). During initial experiments with VSM outgrowth cells of artery segment cultures, we observed long transient Ca2+ events (>10 s) and greater sensitivity of these cells to laser exposure, necessitating the lower acquisition frequency and increased length of recordings. Image analysis was performed with custom software written in our laboratory (A. D. Bonev). Ca2+ events were detected in individual VSM cells offline by measuring an increase in fractional fluorescence [i.e., fluorescence relative to baseline (F/F0)] ≥1.3, a value that could be distinguished above background noise. Baseline fluorescence (F0) was determined by averaging 50 images with no activity. Ca2+ events were classified as sparks or waves by examination of three rectangular regions of interest (1.5 x 1.5 µm) located in the middle and at each end of the cell. Each region was separated by ~15–20 µm. Changes in fluorescence during the recorded file (10–20 s) were measured in each of the three regions. Spatially distinct, quick (<100-ms half time of decay) peaks in fluorescence above threshold were counted as Ca2+ sparks. Ca2+ waves are defined as propagated increases in fluorescence above threshold for ≥1 s in at least two regions of interest (covering at least one-half of the length of the cell).

Ratiometric Ca2+ measurements. Changes in VSM [Ca2+]cyt were examined in the wall of intact pial arteries. Artery segments were loaded with the ratiometric Ca2+-sensitive fluorescent dye fura 2-AM (2 µM; Molecular Probes) and Pluronic acid (2.5 µg/ml) dissolved in HEPES buffer for 60 min, as previously described (17). Fluorescence was measured using a photomultiplier system (IonOptix, Milton, MA) attached to a Nikon Diaphot microscope (x40 Fluor lens) in which background-corrected ratios of 510-nm emission were obtained at a sampling rate of 15 Hz from arteries alternately excited at 340 and 380 nm. Ratios were measured from background-corrected images of the 510-nm emission. Arterial wall VSM [Ca2+]cyt was calculated using the following equation (11)

where Kd is apparent dissociation constant and Rmin and Rmax are ratios of emission signals under Ca2+-free and Ca2+-saturated conditions, respectively.

At the end of every experiment, Rmin and Rmax were determined from ionomycin-treated arteries, and {beta} was determined. A Kd of 282 nM Ca2+ was used for fura 2 (17). Experimental protocols were started after an additional 20-min equilibration period to allow intracellular deesterification of fura 2-AM.

Migration assay. Arterial explant cultures were placed in an open chamber with atmospheric and temperature controls. Conditions were maintained at 37°C in 5% CO2 in air for 6–8 h. After a 30-min adjustment period, images were obtained and processed using a Deltavision RT system (Applied Precision, Issaquah, WA) at 10-min intervals. After 3–4 h, culture medium + 10% FBS was replaced with fresh medium + 10% FBS with or without drug. Migration distances for individual cells were calculated by tracking movement of nuclei over time.

Statistics. Values are means ± SE where applicable. Unless otherwise noted, n is the number of explants. For all except Ca2+ imaging data, significance was tested at the 95% (P < 0.05) confidence level by one-way ANOVA followed by appropriate comparisons (Tukey's procedure or Kruskal-Wallis ANOVA on ranks). Transient and whole cell Ca2+ imaging data were tested using one-way repeated-measures ANOVA (Holm-Sidak method).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Outgrowth cells from arterial segments are VSM cells. Immunofluorescence staining was performed on a subset of cultured arterial segments to confirm that outgrowth cells were VSM in origin and actively proliferating (28). All cells were positive for the smooth muscle markers smooth muscle {alpha}-actin and smMHC (data not shown). Ten percent of cells within the explanted artery segments and 54% of VSM outgrowth cells (individual VSM cells migrating from the explanted artery segments) were positive for the proliferation indicator Ki-67 (Fig. 1).



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  		Fig. 1. Outgrowth cells are proliferating vascular smooth muscle (VSM) cells. A: overlay of smooth muscle {alpha}-actin (blue) and Ki-67 (red) in cerebral arteries cultured for 5 days. Nuclear counterstain Sytox green was also used. Yellow arrows, Ki-67-positive nuclei; white dotted line, explanted artery edge. Magnification x20. B: Ki-67 stain only, from A. Light blue dotted lines, outgrowth cells. Note Ki-67-positive nuclei in artery wall. C: overlay image of VSM outgrowth cells from 5-day artery cultures. Inset: secondary antibody control. Magnification x40. Scale bars, 50 µm.

 
Serum induces Ca2+ waves in arteries. Activation of IP3Rs by vasoconstrictors leads to a characteristic increase in Ca2+ waves in arterial myocytes (14). We therefore examined the effects of serum on Ca2+ signals in intact cerebral arteries (Fig. 2). In the absence of serum, arteries exhibited primarily Ca2+ sparks and few waves (15). Exposure to FBS (10%) for 10 min induced an increase in the number of Ca2+ waves. These FBS-elicited Ca2+ waves diminished after 10 min of treatment with 2-APB [an IP3R blocker (21)] or U-73122 [a PLC inhibitor (12)], suggesting that Ca2+ waves resulted from activation of IP3Rs (Table 1, supplemental movies 1a and 1b). [Supplemental data (movies 1a, 1b, 2, and 3) for this article may be found at http://ajpheart.physiology.org/cgi/content/full/01191.2004.DC1.] FBS contains numerous mitogens, which stimulate PLC through G protein-coupled receptors (e.g., catecholamines, endothelin-1, and thrombin) or by activation of protein tyrosine kinases (e.g., platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor) (2, 26). These data imply that FBS stimulates the production of IP3 through receptor-mediated PLC cleavage of phosphatidylinositol 4,5-bisphosphate to IP3 and diacylglycerol (4). In contrast to the substantial effects of FBS on Ca2+ waves, the frequency of Ca2+ sparks in PSS was not significantly altered by FBS or the further addition of 2-APB or U-73122 (Table 1). Addition of either 2-APB or U-73122 alone failed to significantly affect the frequency of Ca2+ sparks or Ca2+ waves (Table 1).



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  		Fig. 2. Spontaneous Ca2+ sparks and waves in cerebral arteries. Left: gray-scale averaged image of representative artery with individual smooth muscle cells aligned from top left to bottom right. Scale bar, 20 µm. Right: Ca2+ transient event activity recorded sequentially after 20 min of exposure to physiological saline solution (PSS), 10 min of exposure to 10% FBS, and 10 min of exposure to 100 µM 2-aminoethoxydiphenylborate (2-APB) + 10% FBS. Colored traces correspond to representative 1.5 x 1.5 µm regions of interest in individual cells. Ca2+ transients are indicated as sparks (*) or waves (^). Note change in predominant Ca2+ events within a region of interest, from sparks to waves, after FBS and cessation of waves after 2-APB. Images were acquired every 33.33 ms during 10-s scans. F/F0, fractional fluorescence.

 

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  		Table 1. Effects of FBS and IP3R antagonists on Ca2+ waves and sparks in cultured cerebral arteries

 
FBS treatment could potentially increase whole cell cytosolic (i.e., global) Ca2+, which has been previously associated with the initiation of VSM cell proliferation (4). To dissect the effects of global Ca2+ elevations and Ca2+ waves, fura 2-loaded cerebral arteries were exposed to FBS and then to 2-APB, xestospongin C, U-73122, and diltiazem. FBS induced a sustained increase in [Ca2+]cyt, which was not decreased by subsequent addition of 2-APB, xestospongin C, or U-73122 (Fig. 3). The specific inhibitor of VDCCL, diltiazem, attenuated FBS-induced increases in [Ca2+]cyt. The elevated fluorescent signal caused by FBS-induced increases in global Ca2+ may overwhelm more subtle drug effects on [Ca2+]cyt; therefore, a subset of arteries were exposed to 2-APB, U-73122, or diltiazem in the absence of FBS. Neither 2-APB nor U-73122 decreased [Ca2+]cyt: 88.8 ± 9.2 and 112.9 ± 24.0 nM Ca2+ with PSS and 100 µM 2-APB, respectively (n = 4, P = 0.39), and 94.3 ± 9.0 and 85.9 ± 10.0 nM Ca2+ with PSS and 25 µM U-73122, respectively (n = 4, P = 0.52). However, diltiazem reduced [Ca2+]cyt: 93.7 ± 14.3 and 60.2 ± 12.9 nM Ca2+ with PSS and 100 µM diltiazem, respectively (n = 4, P = 0.003). These results reveal that FBS-induced Ca2+ waves do not contribute significantly to the global Ca2+ signal and that FBS increases global Ca2+ independently of Ca2+ waves and IP3.



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  		Fig. 3. FBS elevates arterial wall Ca2+ in cerebral arteries. A: original trace of cytosolic Ca2+ concentration ([Ca2+]cyt) in an artery during exposure to PSS and after sequential addition of 10% FBS and 100 µM 2-APB. Gaps in trace correspond to pauses in excitation during perfusate changes. B: summary graph. Each grouping represents mean peak [Ca2+]cyt measurements in arteries exposed sequentially to PSS, 10% FBS, and drug (2-APB, U-73122, xestospongin c, or diltiazem) + 10% FBS. Addition of 10% FBS increased [Ca2+]cyt. Only subsequent addition of 100 µM diltiazem abrogated FBS-induced [Ca2+]cyt elevations. *Significantly less than 10% FBS alone (P < 0.05).

 
VSM cell outgrowth is dependent on the SR Ca2+ store. To confirm that the increases in Ca2+ waves reflected release of SR Ca2+, we cultured artery segments in medium with 10% FBS or medium with 10% FBS + thapsigargin and tracked the effect on VSM cell proliferation over 5 days. Thapsigargin inhibits SR Ca2+ reuptake, leading to SR Ca2+ store depletion (19). Thapsigargin drastically inhibited VSM cell outgrowth to a level similar to that observed in 0.1% (low) FBS (Fig. 4). These data indicate that SR Ca2+ stores are necessary for VSM cell outgrowth from cultured arteries.



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  		Fig. 4. Depletion of sarcoplasmic reticulum Ca2+ store inhibits VSM cell outgrowth. All experiments were performed in culture medium + 10% FBS + diltiazem or thapsigargin or in culture medium + 0.1% FBS. Top: representative images. Scale bar, 100 µm. Bottom: summary graph. Cell numbers were obtained by averaging mean daily counts from three 512-µm2 fields per artery culture. {dagger}Significantly less than control (P < 0.05).

 
It is also possible that extracellular Ca2+ entry, through VDCCL, may have an impact on proliferation. To examine this possibility, we treated a subset of explants from the start of culture though day 5 with diltiazem, a very specific VDCCL inhibitor (Fig. 4). Although diltiazem reduced FBS-induced elevations in global Ca2+ (Table 1), it did not affect outgrowth, suggesting that extracellular Ca2+ entry through VDCCLs is not an absolute requirement for VSM cell outgrowth.

IP3Rs play an important role in VSM cell outgrowth. Two types of SR Ca2+ release channels are present in VSM: IP3Rs and RyRs. Thapsigargin depletion of SR Ca2+ stores affects Ca2+ release through both receptor types. We first focused on IP3Rs for several reasons: 1) exposure of arteries to 10% FBS induced significant changes in Ca2+ waves; 2) 10% FBS most likely stimulates VSM cell proliferation via receptor-mediated PLC activation; and 3) evidence from studies using traditional cell culture lines suggests that IP3R expression is preserved during dedifferentiation (32, 36). Consequently, to determine whether FBS stimulates VSM cell outgrowth by signaling through the PLC pathway, we utilized an inhibitor (U-73122) of PLC-{beta} and PLC-{gamma} (12). U-73122 significantly inhibited VSM cell outgrowth (Fig. 5), indicating PLC dependence.



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  		Fig. 5. Blockers of inositol 1,4,5-trisphosphate receptors (IP3Rs) and phospholipase C inhibit VSM cell outgrowth. All experiments were performed in culture medium + 10% FBS + ryanodine, 2-APB, xestospongin C, or U-73122. Top: representative images. Scale bar, 100 µm. Bottom: summary graph. Outgrowth cell numbers were obtained by averaging daily counts of three 512-µm2 fields per artery culture. *Significantly greater than control (P < 0.05). {dagger}Significantly less than control (P < 0.05).

 
To explore further the role of IP3Rs, the effects of two inhibitors of IP3Rs, 2-APB and xestospongin C, were tested on VSM cell outgrowth. VSM cell outgrowth was effectively inhibited by both drugs (Fig. 5). Although 2-APB has other effects (7, 21, 22), 2-APB and xestospongin C block IP3Rs (7, 21, 22). Taken together with the inhibitory effects of thapsigargin and U-73122, these data show that VSM cell outgrowth is dependent on SR Ca2+ release through IP3Rs.

Smooth muscle SR contains another Ca2+ release channel, the RyR, which is activated by cytoplasmic Ca2+. Ryanodine, which selectively inhibits Ca2+ release from SR RyRs, failed to prevent VSM cell outgrowth (Fig. 5). In fact, ryanodine increased the number of VSM outgrowth cells over 5 days. This suggests that RyR activity may block VSM cell outgrowth, a role in opposition to that of IP3Rs.

IP3R activity is necessary for VSM cell proliferation. Reductions in VSM cell outgrowth from cultured arteries by IP3R blockers could be attributed to inhibition of cell proliferation and/or cell migration. To examine cell proliferation, arteries were treated with 2-APB for up to 48 h and then immunostained for the proliferation indicator protein PCNA (red) and counterstained with Sytox to identify nuclei. IP3R blockade with 2-APB dramatically reduced the number of PCNA-positive nuclei in the walls of the explanted arteries and inhibited VSM cell outgrowth (Fig. 6). These data suggest that IP3R-dependent proliferation is responsible, at least in part, for our observations of VSM cell outgrowth.



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  		Fig. 6. 2-APB inhibits VSM cell proliferation. Top: representative images of artery cultures fixed at 24 h and immunostained for proliferating cell nuclear antigen (PCNA, red). Experiments were performed in culture medium + 10% FBS or with 2-APB. Nuclei were counterstained with Sytox green. Dotted lines, explanted artery edge. Scale bar, 50 µm. Bottom: summary graph. Cell numbers were obtained by averaging mean daily counts from three 512-µm2 fields within each artery culture. *Significantly less than 10% FBS (P < 0.05).

 
To test whether IP3R blockade via 2-APB affects the ability of VSM outgrowth cells to migrate, we used phase-contrast imaging of day 5 artery cultures. This allowed for observation of the active migration of VSM outgrowth cells under control conditions (culture medium + 10% FBS) and immediately after application of 2-APB. Migration rates were unaffected by 2-APB over a 160-min period (Fig. 7; see supplemental Movie 2). Therefore, it appears that IP3R signaling is required for proliferation of VSM cell outgrowth, rather than migration.



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  		Fig. 7. 2-APB does not inhibit VSM cell migration. VSM outgrowth cell migration was recorded from artery cultures on day 5. Images were acquired every 10 min over 6–8 h. Left: sequential phase-contrast images showing active migration of outgrowth cells for 160 min in culture medium + 10% FBS and for 160 min after addition of 100 µM 2-APB. A single cell has been highlighted (red) to show movement over time. Blue circles and yellow arrows, measured nuclear movement of highlighted cell. Scale bar, 50 µm. Right: summary graph. Migration rates were obtained by averaging nuclear distance traveled over time per cell in cultures before (pretreatment) and after (posttreatment) replacement of culture medium + 10% FBS with fresh medium + 10% FBS (vehicle) or medium + 10% FBS and 2-APB.

 
Ca2+ transients in proliferative VSM cells. If IP3Rs are maintained, and RyRs and VDCCLs are lost, during VSM cell proliferation, then cytosolic Ca2+ dynamics should reflect this. To address this, spontaneous Ca2+ signals were recorded from individual VSM outgrowth cells from day 5 artery explant cultures in the presence of 10% FBS. Long transient Ca2+ events (>100-ms half time of decay) and propagated Ca2+ waves, but not Ca2+ sparks, were observed in these cells (Fig. 8; see supplemental Movie 3). The lack of Ca2+ spark activity in these cells supports the hypothesis that outgrowth cells have reduced RyR activity and that the spontaneous Ca2+ transients are due to agonist-induced opening of IP3Rs.



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  		Fig. 8. Propagation of spontaneous Ca2+ transients in VSM outgrowth cells. Activity was recorded from a representative VSM outgrowth cell on day 5. Images were acquired every 66.67 ms over 20 s. Top left: Ca2+ activity traces. Each trace corresponds to regions (1–5) at top right. [Ca2+]cyt peak at each region occurs in sequential order, indicating signal propagation. Top right: gray-scale averaged image. Scale bar, 20 µm. Bottom: sequential F/F0 images showing progression of a Ca2+ wave in a single cell, starting at 1 and propagating through the cell body (2 and 3) to 4 and 5.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major Ca2+ entry pathways (VDCCs, RyRs, and IP3Rs) into the smooth muscle cytoplasm mediate distinctly different Ca2+ signals and are differentially regulated (15). Activation of VDCCLs leads to a uniform (i.e., global) elevation of [Ca2+]cyt. RyR activation largely manifests as stationary, local elevation of Ca2+ (Ca2+ spark), which activates closely opposed BK channels, leading to membrane potential hyperpolarization and decreased VDCCL activity (23). Finally, IP3R stimulation is dependent on receptor-mediated PLC activation, which elevates levels of IP3, causing Ca2+ waves to propagate through smooth muscle cells (14, 15).

Our results (Fig. 2, Table 1) indicate that FBS, which induces proliferation, causes Ca2+ waves, consistent with the activation of IP3Rs. Indeed, FBS contains numerous mitogens (e.g., catecholamines, endothelin-1, thrombin, platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor) that should elevate IP3 and cause Ca2+ waves in VSM cells. We investigated the relation between serum-induced proliferation of VSM cells and serum-induced alteration in Ca2+ signaling. 2-APB prevented Ca2+ waves and VSM cell proliferation without substantially affecting [Ca2+]cyt. Because 2-APB is not a specific blocker of IP3Rs and may also affect gap junction conductance and store-operated Ca2+ entry (10), we used U-73122 to target the PLC pathway upstream from IP3Rs. Similar to 2-APB, U-73122 completely abrogated FBS-induced Ca2+ waves and proliferation without affecting [Ca2+]cyt, thus indicating that activation of the PLC pathway and IP3R-mediated Ca2+ waves are required for VSM cell proliferation. Inhibition of SR Ca2+-ATPase (SERCA) with thapsigargin also inhibited proliferation, further implicating SR Ca2+ stores in this process. Interestingly, blocking VDCCLs with diltiazem had no effect on proliferation (Fig. 4), whereas inhibition of RyRs augmented proliferation (Fig. 5). One mechanism that may account for the potentiating effect of RyR inhibition is that treatment of explanted arteries with ryanodine stimulates proliferation by elevating average [Ca2+]cyt through increased VDCCL activation (17) and, thereby, increases IP3-mediated Ca2+ release through an elevation of SR Ca2+ load. Alternatively, if Ca2+ release from RyRs and IP3Rs is dependent on a shared Ca2+ pool, it is possible that blockade of one channel type provides a greater driving force for SR Ca2+ release from the unblocked channel.

It is important to note that inhibition of VSM cell outgrowth by thapsigargin and IP3R blockade is due to prevention of VSM cell proliferation, rather than migration. Although our results do not rule out the possibility that IP3R-mediated Ca2+ is required to initiate migration, they do suggest that IP3R-mediated Ca2+ release is required for proliferation but not for migratory activity per se. Furthermore, if the initial event in outgrowth cell appearance was migration of VSM cells from the cultured artery, rather than proliferation within the arterial wall, then one would expect to see outgrowth at a rate at least equal to the appearance of PCNA-positive cells within the artery wall. Figure 6 shows that this is not the case: the number of PCNA-positive cells clearly exceeded the number of outgrowth cells over the first 48 h of culture. The near-complete absence of PCNA-positive cells in the 2-APB-treated arteries suggests that the primary initial event in VSM cell outgrowth is cell proliferation.

The important role of IP3Rs in VSM cell proliferation has also been demonstrated by Wang et al. (36). Utilizing the A7r5 VSM cell line, they showed that suppressing expression of the dominant IP3R subtype (type I) in VSM cells via an antisense strategy resulted in a loss of IP3-induced Ca2+ release and a failure to proliferate (24). Our findings demonstrate that SR Ca2+ released through IP3Rs, in the form of Ca2+ waves, is required for dedifferentiation of native VSM cells from the contractile to the proliferative state. This observation is consistent with a central role for SR Ca2+ in controlling VSM cell proliferation, as suggested by previous studies showing that SERCA activity is necessary for serum-induced cell growth (34) and that emptying of SR Ca2+ stores via SERCA inhibition triggers entry of cells into a quiescent state (29).

Although the exact physiological role of Ca2+ waves remains elusive, our laboratory has reported a potential role for Ca2+ wave signaling in regulating nuclear localization of the Ca2+-activated transcription factor NFAT. We found that blocking IP3Rs prevented NFATc3 nuclear localization (9). NFAT requires dephosphorylation by the Ca2+-activated en-zyme calcineurin to move into the nucleus and become transcriptionally active (1). By blocking calcineurin activity with cyclosporin A or FK-506, Wada et al. (33) found that they could inhibit the endogenous expression of smMHC in differentiated VSM cells, suggesting that the calcineurin pathway is activated during differentiation of VSM cells and is required for smMHC expression. Similarly, it is possible that the alteration in IP3R-mediated Ca2+ signaling that precedes VSM cell proliferation is linked to stimulation of proliferation through effects on Ca2+-sensitive transcriptional regulators. To this end, Lipskaia et al. (20) found that treating cultured VSM cells with very-low-density lipoproteins induces a calcineurin-dependent translocation of NFATc3 to the nucleus, which coincided with proliferation. Other Ca2+-dependent transcription factors, such as cAMP response element binding protein and Rho kinase, could also be involved (6, 35).

The main finding of the present study is that inhibition of SR Ca2+ release through IP3Rs in the form of Ca2+ waves in cerebral arteries effectively blocks VSM cell proliferation. An important novel aspect of these studies is that they were performed with native arteries. Thus the findings presented here more closely resemble the cellular dynamics of in vivo VSM cell migration and proliferation. These results are significant because of their potential application in preventing pathological VSM cell proliferation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grants HL-44455, HL-63722, and DK-53832 (to M. T. Nelson). M. K. Wilkerson received support through NIH Postdoctoral Trainee Fellowship HL-07944. Migration assays were performed in the Vermont Neuroscience COBRE facility, which receives funding from NIH National Center for Research Resources Grant P20 RR-16435.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Ammon Fager, Dr. Laura Gonzalez-Bosc, and Theresa Wellman (Department of Pharmacology, University of Vermont) and Dr. Sarah Locknar [Neuroscience Center of Biomedical Research Excellence (COBRE) Facility, University of Vermont] for technical advice and assistance. We thank Dr. David Hill-Eubanks, Paul Doetsch, and Dr. Kevin Thorneloe for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. K. Wilkerson, Dept. of Pharmacology, Univ. of Vermont College of Medicine, 89 Beaumont Ave., Burlington, VT 05405-0068 (e-mail: keith.wilkerson{at}uvm.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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