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Circulation Research. 1997;81:328-337

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(Circulation Research. 1997;81:328-337.)
© 1997 American Heart Association, Inc.

Articles

Effects of Changes in Blood Flow Rate on Cell Death and Cell Proliferation in Carotid Arteries of Immature Rabbits

Aesim Cho, Lylieth Mitchell, Donna Koopmans, , B. Lowell Langille

From the Banting and Best Diabetes Centre, The Toronto Hospital Research Institute, and Department of Pathology, University of Toronto (Canada).

Correspondence to B. Lowell Langille, PhD, The Toronto Hospital Research Institute, CCRW 1-836, 200 Elizabeth St, Toronto, ON M5G 2C4, Canada. E-mail lowell.langille{at}utoronto.ca


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Spontaneous and experimental changes in arterial blood flow rates affect tissue accumulation in developing arteries. To examine whether cell proliferation and/or cell death are affected by alterations in blood flow, we ligated the left external carotid artery of 3-week-old rabbits, which reduces left common carotid blood flow by 71%. In control arteries and after 2 days of flow reduction, agarose gel electrophoresis of DNA extracted from all carotid arteries resolved multiple low molecular weight bands characteristic of apoptosis; however, DNA fragmentation in arteries carrying reduced blood flow was 2.5-fold higher than that of control arteries. The effect of reduced blood flow on cell death subsequently waned but remained significant at 7 days. Cell death in carotid arteries was also detected by in vivo uptake of propidium iodide, a DNA-binding fluorescent dye that labels the nuclei of nonviable cells. Both smooth muscle and endothelial cells exhibited large and statistically significant increases in labeling index in the flow-reduced artery. Propidium iodide–labeled cells were cleared from the vessel wall within 1 to 4 hours of labeling, and nuclear staining displayed condensation (clumping) of chromatin in all labeled cells at later time points. This time course and nuclear morphology and the rapid clearance of labeled cells are consistent with death via apoptosis. Many propidium iodide–positive cells did not display chromatin condensation immediately after labeling; however, this was also true of cultured endothelial cells that were driven into apoptosis with sphingomyelinase treatment and then double-labeled with propidium iodide and the apoptosis marker annexin V. We infer that propidium iodide can label apoptotic vascular cells before these cells display chromatin condensation that is detectable with fluorescence labeling of DNA. Replication rates of smooth muscle and endothelial cells, determined by 5-bromo-2'-deoxyuridine uptake, were inhibited by >75% with decreased blood flow. The inhibition of proliferation was unabated after 7 days of reduced flow. These findings indicate that the coordinated regulation of cell death and cell proliferation, in response to changes in arterial blood flow rates, contributes to arterial remodeling during development.


Key Words: blood flow • cell death • apoptosis • cell proliferation • arterial remodeling


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The development of large arteries involves continuous remodeling of wall tissues that adjusts vessel diameter in accord with prevailing blood flow rates and adjusts vessel wall thickness in accord with prevailing blood pressures.1 This remodeling is achieved, at least in part, by a direct sensitivity of vascular tissues to local hemodynamic stresses, since experimental changes in hemodynamic conditions affect subsequent arterial structure.2 3 4 5 Effects of arterial pressure on the growth of arterial wall thickness have been well characterized, largely because of the relevance of these effects to the pathogenesis of hypertension.2 The effects of blood flow on arterial development have been characterized less extensively; however, studies during the past decade have shown that experimental changes in blood flow rates affect the accumulation of medial tissue mass,3 including arterial cells and extracellular matrix,4 and that they also induce reorganization of matrix, particularly elastin.5

The effects of changes in blood flow on arterial cell accumulation4 6 are generally assumed to be mediated by modulation of cell proliferation rates. However, we recently demonstrated that apoptosis of both endothelial and smooth muscle cells in arteries contributes to perinatal arterial development.7 Furthermore, after birth, the incidence of apoptosis increased greatly in the abdominal aorta, which experiences a 95% reduction in blood flow at birth,8 and in the intra-abdominal umbilical arteries, which experience total cessation of blood flow at birth.7 These findings support the hypothesis that changes in blood flow rates affect growth of cell populations through effects on vascular cell apoptosis. However, these observations did not establish whether apoptosis in these arteries was due to decreased blood flow rate or to the profound changes in endocrine function,9 10 11 12 13 blood gas status,14 15 or blood pressure15 that occur at birth. In this regard, partial pressure of oxygen,16 17 glucocorticoid levels,18 19 and tissue stretch20 have been implicated in the induction of apoptosis in other systems, and all of these factors change in arteries in the perinatal period.15 21 Furthermore, our previous work has not assessed the role of apoptosis in developmental arterial remodeling outside of the perinatal period.

In the present study, we tested whether experimental changes in blood flow rates in developing arteries cause changes in the incidence of cell death and/or cell replication. Well-established techniques for manipulating blood flow rate in rabbit carotid arteries4 were exploited using young (3-week-old) animals, and we assessed subsequent cell proliferation and cell death in these arteries. Cell death was assessed by detection of DNA degradation into oligonucleosomes, a hallmark of apoptosis, and by in vivo labeling with propidium iodide. All of the immature carotid arteries, regardless of flow conditions, exhibited apoptosis of both smooth muscle and endothelial cells. Apoptosis was upregulated for both smooth muscle and endothelial cells, and cell proliferation was downregulated for both cell types when blood flow rates were reduced experimentally. Furthermore, at low flow rates, cell death rates transiently exceeded proliferation rates. These findings support the hypothesis that signals derived from local blood flow control the growth of cell populations in developing arteries by influencing both cell proliferation and cell death.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Three-week-old male New Zealand White rabbits, weighing 389±13 g, were used in the present study. All experiments were conducted in accord with guidelines approved by the Canadian Council of Animal Care.

Experimental Reduction of Blood Flow
Rabbits were anesthetized with intramuscular injections of 0.08 mL/kg xylazine (20 mg/mL) and 0.72 mL/kg ketamine hydrochloride (100 mg/mL). The proximal left external carotid artery was exposed by a midline incision over the larynx and ligated just distal to the origin of the internal carotid artery using a 5-0 silk suture. Alternatively, the common carotid artery was ligated just distal to the thyroid artery to produce a more severe flow reduction. The incision was closed in layers, and the animals were allowed to recover. In sham-operated rabbits, the suture was tied loosely around the left external carotid artery or the distal left common carotid artery.

Hemodynamic Assessments
Maximum induction of apoptosis by low flow occurred 2 days after flow reduction (see "Results"). Therefore, hemodynamic function was assessed in unmanipulated animals (n=6) and in animals 2 days after common carotid ligation (n=6), external carotid ligation (n=5), or sham surgeries (n=7). The animals were anesthetized as described above. To measure arterial pressure, a PE-90 catheter coupled to a fluid-filled pressure transducer (Statham P23 XL) was introduced into the femoral artery via a skin incision and advanced retrogradely into the lower abdominal aorta. Next, both carotid arteries were exposed via a midline incision in the neck, and 2- to 3-mm ultrasonic transit time flow transducers were mounted on each vessel and coupled to a flowmeter (Transonics model T206). Pressures and flows were recorded on a four-channel chart recorder (Gould 2400S). Low-pass filters on the flowmeter (for flows) or the chart-recorder amplifiers (for pressure) were set to record mean flow; however, the filters were switched frequently to record pulsatile flow (cutoff frequency was set to 100 Hz) for brief periods to ensure that high-quality phasic signals were being recorded. Mean pressures and flows recorded between 20 and 30 minutes after the start of hemodynamic assessments were used for statistical analysis.

Morphometry
We assessed whether flow reduction significantly affected vessel morphology. Seven days after left external carotid ligation, animals were killed with 0.2 mL intravenous injection of T-61 containing 200 mg/mL N[2-m-methoxyphenyl)-2-ethylbutyl-(1)]-2-hydroxybutyramide, 50 mg/mL 4,4'-methylene-bis(cyclohexyltrimethylammonium iodide), and 5 mg/mL tetracaine HCl (Hoechst) and then perfusion-fixed with 3% paraformaldehyde in phosphate buffer at a perfusion pressure of 65 mm Hg, ie, at approximately systolic pressure for these animals. Short segments of the midregion of the common carotid artery were embedded in paraffin, and 7-µm cross sections were then stained with hematoxylin and eosin. Cross sections were viewed with a Nikon Labophot microscope, and images were transferred to an image analysis system (C{bullet}Imaging, Compix) using a video camera interface. Image analysis included computation of internal vessel circumference and medial cross-sectional area (area between the internal and external elastic lamina). Arterial diameters were computed by dividing circumferences by {pi}. In practice, the medial area was equivalent to the intimal-medial area, since the intima consisted of only endothelium on a basal lamina. In addition, medial smooth muscle cell nuclei per cross section were counted to assess medial cellularity.

In some cases, arteries that had been perfusion-fixed at a pressure of 100 mm Hg were used for assessments of medial area and vessel cellularity but not for internal circumferences, since the latter depends on vessel distension. In total, circumferences were measured for 7 animals, medial cross-sectional areas were measured for 11 animals, and smooth muscle cell nuclei per cross section were counted for 12 animals.

Analysis of Oligonucleosome Formation
One, 2, 4, and 7 days after blood flow reduction, rabbits were euthanized as described above. Both common carotid arteries were harvested from their origins to the carotid bifurcations and cleared of adventitia, and then DNA was extracted and analyzed for degradation into oligonucleosomes using the method of Rosl22 as previously described.7 Briefly, DNA was extracted by overnight incubation of arterial tissues in DNA lysis buffer (20 mmol/L Tris-HCl [pH 7.4], 1% SDS, 5 mmol/L EDTA, and 100 µg/mL proteinase K) at 50°C, followed by a phenol/chloroform extraction. Five micrograms of arterial DNA plus 2.5 ng of 30-bp oligonucleotide was incubated with 10 µCi of [32P]dCTP and 10 U of Klenow polymerase (Pharmacia) for 15 minutes at 30°C. The 30-bp oligonucleotide was added to control for variability in loading of wells for electrophoresis. Unincorporated nucleotides were removed using a Wizard DNA cleanup system (Promega Corp) according to the manufacturer's instructions. Radiolabeled DNA was electrophoresed on a 1.8% agarose gel and blotted onto Hybond nylon membrane, and the membrane was used to expose Kodak X-OMAR x-ray film.

The area of the blotted membrane corresponding to the 100- to 800-bp DNA fragments was cut out, and the radioactivity was measured by scintillation counting. Counts were normalized by dividing by the radioactivity of the band corresponding to 30-bp oligonucleotide. The amount of DNA fragmentation (normalized cpm) in the flow-reduced left carotid artery was expressed as a ratio of right carotid arterial DNA fragmentation. Differences in apoptosis rates between the two arteries were inferred when this ratio was significantly different from 1.

In order to obtain sufficient DNA for electrophoresis, carotid arteries from four rabbits were pooled for each sample. Six such samples were used for each time point.

In Situ Detection of Nonviable Cells
Two days after reducing left common carotid blood flow, 5 rabbits were reanesthetized by intramuscular injections of xylazine plus ketamine and then given intravenous injections of 5 µmol/kg propidium iodide (Calbiochem-Novabiochem Corp). Propidium iodide fluoresces when bound to nuclear DNA, but it is excluded from viable cells; therefore, nonviable cells were identified by uptake of the dye.7 After 15 minutes, the rabbits were killed by intravenous injections of T-61. The thoracic aorta was exposed by performing a bilateral thoracotomy, the descending aorta was cannulated retrogradely, and the carotid arteries were fixed by perfusion with 3% paraformaldehyde in phosphate buffer as described above. The left and right carotid arteries were removed, opened lengthwise, and mounted lumen side up on glass slides under coverslips in glycerol/PBS (9:1). The tissue samples were viewed under a laser scanning confocal microscope (Bio-Rad MRC 600) at x60 magnification. Endothelial cells and smooth muscle cells that did not exclude the propidium iodide (nonviable) were separately counted in each full-thickness field. Here, a full-thickness field includes the endothelial cells in one microscope field and all medial smooth muscle cells below that field. Smooth muscle cells were assayed by optical sectioning through the complete media of the vessel in 1-µm steps. For each artery, 50 full-thickness fields, 2 to 3 mm apart, were counted to determine the number of labeled cells in each carotid artery.

In arteries of 3 additional animals, all nuclei were stained in vitro by treating the vessel with 1% Triton X-100 for 5 minutes before exposure to propidium iodide. These preparations allowed us to count total cells per field and therefore to estimate the percentage of cells labeled.

Clearance of Labeled Cells From the Vessel Wall
To study the fate of cells labeled with propidium iodide in vivo, we more severely reduced blood flow in the proximal carotid artery by ligating the left common carotid just distal to the thyroid artery.23 This procedure produced higher labeling indexes than did external carotid ligation. One day after surgery, rabbits were infused with propidium iodide as previously outlined and then killed 15 (n=8), 45 (n=8), 75 (n=9), 105 (n=6), or 255 (n=6) minutes later. The descending aorta was catheterized, the carotid arteries were perfusion-fixed, the left carotid arteries were harvested and mounted on glass slides, and labeled cells were counted using a laser scanning confocal microscope as described previously.

The decline in number of labeled cells over time was a direct measure of the loss of labeled cells and was not influenced by ongoing labeling of cells that entered apoptosis after propidium iodide infusion, because propidium iodide was cleared rapidly from the circulation. When 1-mL blood samples were drawn from 3 animals at 1 minute before and 1, 5, 10, and 30 minutes after the infusion of propidium iodide, the fluorescence of plasma after the addition of calf thymus DNA became negligible by 10 minutes after infusion (data not shown).

Estimation of Daily Cell Death Rate
The percentage of cells dying per day is equal to the percentage of cells labeled at any instant in time with a marker of cell death (propidium iodide in the present study) divided by the duration of time (in days) over which dying cells can be labeled. Thus, if dying cells can take up propidium iodide for T hours, then Daily Death Rate (%)=% Cells Labeledx24÷T

T could not be measured directly in the present study; however, T is equal to the time taken for clearance of labeled cells from the vessel wall (see previous section) if it can be assumed that cells that have become incapable of excluding the dye do not regain this capacity before they are deleted from the vessel. This assumption was made (see "Discussion"), and the above equation was used to estimate daily cell death rates. Note that the calculation does not require that cells take up propidium iodide throughout the cell death pathway, only that they do so over a defined interval during this pathway. By analogy, daily cell replication rates are measured using markers of a defined portion of the cell cycle (5-bromo-2'-deoxyuridine [BrdU] or [3H]thymidine labeling of cells in the S phase).

In Vitro Assessment of Propidium Iodide–Positive Cells
Propidium iodide is a marker of cell nonviability that has previously been found to label nuclei of apoptotic cells late in the apoptotic pathway.24 25 To assess whether the staining patterns we observed were consistent with vascular cell apoptosis, we examined propidium iodide uptake by cultured endothelial cells after treatment with sphingomyelinase. Sphingomyelinase induces the hydrolysis of sphingomyelin to produce ceramide, a physiological mediator of apoptosis in many cell types, including endothelium.26 Cell surface binding of fluorescently labeled annexin V was used to identify apoptotic cells. Annexin V binds to phosphotidylserine, which is confined to the cytoplasmic leaflet of the plasma membrane in viable cells but is also found on the outer leaflet of the membrane during apoptosis.27

Confluent monolayers of porcine aortic endothelial cells (passages 3 to 8) on glass coverslips were maintained in medium 199 containing 1% penicillin-streptomycin (Gibco BRL), 1% fungizone, and 5% FBS. The cells were treated for 48 hours with 1 U/mL of sphingomyelinase (Sigma Chemical Co), and then they were exposed for 15 minutes to 50 µg/mL propidium iodide and 50 µL/mL of FITC-labeled recombinant human annexin V (annexin V-FITC, Biowhittaker). Cells were washed three times in HEPES buffer, fixed for 10 minutes with 3% paraformaldehyde in PBS, and again washed three times in HEPES buffer. The coverslips were inverted onto glass slides that had been spotted with glycerol/PBS (9:1) and viewed by confocal or conventional fluorescence microscopy. For counting frequency of apoptosis, 50 fields for each of triplicate experiments were examined using a x60 objective, and cells positive for propidium iodide, annexin V-FITC, or both were counted. Propidium iodide–positive cells that displayed normal nuclear morphology were counted separately from those exhibiting evidence of lobular fluorescence suggestive of chromatin condensation.

Measurement of Cell Proliferation Rates
Rabbits were given intramuscular injections of 30 mg/kg BrdU (Sigma) at 17, 9, and 1 hour before they were killed by intravenous injections of T-61 at 2 (n=5 rabbits) or 7 (n=4 rabbits) days after left external carotid ligation. The carotid arteries were perfusion-fixed with 3% paraformaldehyde in phosphate buffer as described above. Arteries and small intestine (a positive control for replicating cells) were harvested and immersed in fixative for 18 to 24 hours at 4°C. Segments of tissues were embedded in paraffin, and then 6-µm sections were deparaffinized in xylene and processed for BrdU immunocytochemistry. Deparaffinized and hydrated arterial sections were treated with endogenous peroxidase blocker (Biomeda) for 5 minutes at 37°C and then subjected to pepsin digestion for 5 minutes at 40°C. The tissue sections were treated with 95% formamide+5% sodium citrate for 35 minutes at 70°C and 20 minutes at 90°C to denature the double-stranded DNA, and then sections were blocked with 10% goat serum (Sigma) and incubated with mouse antibody to BrdU (1:8 dilution) (Becton-Dickinson) for 60 minutes at 40°C. Incubation with a biotinylated secondary antibody for anti-BrdU (Biostain kit, Biomeda) for 20 minutes at 40°C was followed by treatment with avidin alkaline phosphatase (Ultraprobe kit, Biomeda) for 20 minutes at 40°C. Fast red/naphthol phosphate solution was used as a chromogen to detect nuclei that had incorporated BrdU. The cell nuclei were counterstained with hematoxylin. Fast red–positive cells and total cells were counted to determine smooth muscle and endothelial cell replication rates.

Statistical Analysis
Data are presented as mean±SEM. To test whether blood pressure or left or right common carotid artery blood flow was affected by left external carotid ligation, distal left common carotid ligation, or sham surgeries, the data for these groups and for unmanipulated controls were subjected to one-way ANOVA. When ANOVA indicated that significant differences existed between groups, these were identified using Scheffé's multiple-comparison test. A one-sample t test was used to test whether the ratio of DNA fragmentation between the left carotid artery (flow-reduced) and the right carotid artery (control) was significantly greater than 1.28 A paired sample t test was used to test for significant differences in the percentage of cells labeled by uptake of propidium iodide between left and right carotid arteries after left carotid flow reduction.28 A paired-sample t test also was used to test for significant differences in cell replication rates in the left carotid arteries compared with the right carotid arteries. All statistical computations were performed using Statview 4.5 (Abacus Concepts) for Macintosh computers.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Hemodynamics
Mean arterial pressure in the rabbits used in the present study was 49±2 mm Hg, and this pressure was not significantly affected by any of the surgical procedures.

The effects of surgical interventions on arterial blood flow rates are presented in Table 1Down. ANOVA indicated that carotid blood flow rates in sham-operated animals were not significantly different from control values and that right carotid blood flows were unaffected 2 days after surgical manipulations of the left carotid artery. In contrast, blood flow rates in left common carotid arteries were reduced below control levels by 71% after left external carotid ligation and by 89% after distal left common carotid ligation.


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  		Table 1. Carotid Blood Flows: 2 Days of Flow Reduction

Morphometry
One week after left external carotid ligation, the diameters of left common carotid arteries were 13% smaller than those of right carotid arteries (P<.05), and medial cross-sectional areas were reduced by 18% (P<.05) (Table 2Down). Although the mean number of smooth muscle cell nuclei per histological cross section was slightly lower in the left versus right carotid artery, this difference was not statistically significant (P=.21). Similarly, no significant difference in the number of endothelial cell nuclei per histological cross section was detected.


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  		Table 2. Vessel Morphometry: 7 Days of Flow Reduction

The Effect of Experimental Reduction of Blood Flow on Cell Proliferation
Large numbers of BrdU-positive cells were consistently observed in the crypts of intestinal villi, where cells replicate at a high rate (positive control, Fig 1Down). No positive cells were detected in crypts of villi or in carotid arteries when the primary antibody was omitted from processing of the sections (negative control). Consequently, we inferred that BrdU uptake was reliably labeling S-phase cells.



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  		Figure 1. Replicating cells (arrowheads) in the left carotid (A) and right carotid (B) arteries. Cells were labeled with 5-bromo-2'-deoxyuridine at 2 days after flow reduction in the left carotid artery. Also shown is a section of small intestine (C) showing cell replication in the crypts of the villi, as a positive control. L indicates lumen.

Replicating cells were detected by BrdU labeling in all carotid arteries of 3-week-old rabbits (Fig 1Up), but BrdU-positive endothelial and smooth muscle cells were much less frequent after flow reduction (Figs 1Up and 2Down). After 2 days of flow reduction, the smooth muscle cell replication rate in the left carotid artery was only 21.5% of that observed in the right carotid artery (P<.05), and the replication rate for these cells remained similarly low at 7 days after the ligation (P<.05). Similarly, endothelial cell proliferation rates were significantly reduced by {approx}85% in the flow-reduced artery at 2 days after surgery (Fig 2Down, P<.05), and replication rates remained low after 7 days of flow reduction (P<.05).



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  		Figure 2. Percentage of smooth muscle cells (left) and endothelial cells (right) replicating per day, as indicated by 5-bromo-2'-deoxyuridine uptake, at 2 and 7 days after left carotid flow reduction. Flow reduction caused a large reduction in cell replication rate at both time points and for both cell types. RCA indicates right carotid artery; LCA, left carotid artery.

No significant differences in either smooth muscle cell or endothelial cell replication rates were observed between the left carotid and right carotid arteries at 2 days after sham surgery (data not shown).

Effect of Blood Flow Reduction on Cell Death
Gel electrophoresis of end-labeled DNA from both left and right carotid arteries of 3-week-old rabbits revealed a "ladder" pattern, with DNA bands appearing at multiples of 180 to 200 bp (Fig 3Down), a hallmark of apoptosis. A DNA ladder was detected in the arteries of experimental, sham-operated, and unmanipulated control animals (not shown). The intensity of low molecular weight bands of the DNA samples varied substantially, even when the DNA from several arteries was pooled. Variability of signal was reduced when radioactivity of the ladder was normalized to radioactivity of the band corresponding to the 30-bp oligonucleotide that was added to control for variability in loading of the gels, but it remained appreciable. Nonetheless, for each paired sample, the carotid arteries carrying reduced blood flow consistently yielded a more intense DNA ladder after gel electrophoresis than did the vessels carrying normal flow. This qualitative impression was confirmed when DNA degradation was quantified by scintillation counting; DNA degradation into oligonucleosomes was significantly elevated after 2 days of flow reduction compared with degradation in the contralateral control arteries (Fig 4Down, P<.05). At this time, the signal from arteries delivering reduced blood flow was {approx}2.5 times that of control arteries. Differences in DNA degradation were less marked after 4 and 7 days of reduced blood flow, but they remained statistically significant (P<.05). The increased cell death in the carotid arteries with reduced blood flow was not an artifact of the surgical manipulations, nor was it due to intrinsic differences between left and right carotid arteries, because no difference in DNA fragmentation in left versus right carotid arteries was observed in sham-operated rabbits (Fig 4Down).



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  		Figure 3. Autoradiogram of DNA gel showing fragmentation into oligonucleotides of DNA isolated from carotid arteries of 3-week-old rabbits 2 days after reducing blood flow rate by 70% in the left carotid artery (LCA). DNA was end-labeled with [32P]dCTP. Lane A shows DNA isolated from the LCA, lane B shows DNA from the right carotid artery (RCA), and lane C shows DNA standards. The bottom band in each lane represents a 30-bp oligonucleotide added to all samples to control for variability in loading of the gels. The other bands in the right lane are multiples of 100 bp.



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  		Figure 4. Degradation of DNA into oligonucleotides of 100 to 800 bp in carotid arteries carrying reduced blood flow (left carotid arteries) is expressed relative to DNA degradation in the opposite (right) carotid arteries. Significantly enhanced DNA degradation (P<.05), indicative of increased apoptosis, was observed at 2, 4, and 7 days after flow reduction in the flow-reduced carotid arteries, with maximal DNA degradation occurring at 2 days. Sham-operated animals showed no differences in the two carotid arteries (bar at extreme right). Sample sizes are n=6 (1 and 4 days), n=7 (2 days), and n=4 (7 days and 2-day sham), and each sample is derived from pooled tissue from four arteries.

When propidium iodide uptake was used to label nonviable cells in vivo, labeled nuclei of nonviable cells showed two distinct nuclear patterns. Most labeled nuclei were ovoid and uniformly stained and appeared nearly normal, although they were slightly smaller than nuclei of viable cells that we observed after counterstaining all nuclei (not shown). In contrast, 10% to 20% of nuclei displayed clumping of fluorescently labeled DNA, a pattern indicative of the chromatin condensation at the periphery of the nucleus that is a structural hallmark of apoptosis (Fig 5Down). Nonviable smooth muscle cells were more frequent after flow reduction, and they more often appeared as clusters. After 2 days of flow reduction, approximately one smooth muscle cell per full thickness field was labeled after propidium iodide injection. We counted 773±19 smooth muscle cells per full thickness field; thus, the propidium iodide labeling index was {approx}0.13%. The number of smooth muscle cells labeled with propidium iodide at 2 days after flow reduction was {approx}5-fold higher than the labeling index in control arteries (Fig 6Down, P<.05). Similarly, the percentage of endothelial cells that were positive for propidium iodide was increased after flow reduction (Fig 6Down, P<.05).



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  		Figure 5. Confocal micrograph of nonviable smooth muscle cells (A and B) and endothelial cells (C) that have taken up propidium after intravenous injection. Most (80% to 90%) of the labeled cells showed nearly normal nuclear morphology (see text) with ovoid, uniformly stained nuclei (A), but the remainder showed clumping of DNA, indicating condensation of chromatin, a marker of later stages of apoptosis (B and C). Bars=10 µm.



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  		Figure 6. Number of cells per full thickness field that were labeled with propidium iodide at 2 days after flow reduction in the left carotid artery (LCA). Flow reduction enhanced the labeling index for smooth muscle cells and endothelial cells. Absolute values for labeling indexes for smooth muscle cells are lower than for endothelial cells, because there are many more smooth muscle cells per full thickness field. RCA indicates right carotid artery. *P<.05.

We measured the clearance of labeled cells from the vessel wall using a pulse-chase strategy. When animals were killed at different times after propidium iodide injection, we found that the number of positive endothelial and smooth muscle cells declined rapidly after injection, with nearly total clearance being achieved in 1 to 2 hours (Fig 7Down). Some labeled smooth muscle cells were observed at 4 hours, but this was due entirely to the detection of a large number of such cells in one animal. Importantly, all cells observed at later time points (2 hours or later) exhibited clumping (condensation) of chromatin.



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  		Figure 7. Number of endothelial and smooth muscle cells labeled with propidium iodide that were detected when animals were killed at different times after propidium iodide injection. All smooth muscle cells that were positive for propidium iodide at 255 minutes were from one animal.

It is possible to make a reasonable estimate from the above data of the relative impact of regulation of cell death versus proliferation on arterial cell populations after blood flow reduction. Two days after flow reduction in the rabbit carotid artery, the propidium iodide labeling index for smooth muscle cells was {approx}0.13%. Since the half-life of these labeled cells in the vessel wall was 1 to 2 hours, daily cell death rates were on the order of 2% per day. After 2 days of flow reduction, cell proliferation rates had fallen from 2% to 5% per day to values that were typically {approx}0.5% per day, one quarter of the cell death rate; therefore, there apparently was a net loss of cells during the early response to flow reduction. Cell death rates subsequently decreased; thus, inhibition of cell proliferation became more important at later times.

Propidium Iodide Labeling of Apoptotic Endothelial Cells In Vitro
Very few cells were positive for propidium iodide or annexin V-FITC in control endothelial cell cultures; however, endothelial cell death was frequent after 2 days of treatment with sphingomyelinase. Isolated cells or (rarely) small islands of cells stained positively with the apoptosis marker, annexin V-FITC, and a subpopulation of these cells stained positively for propidium iodide (Fig 8Down). Some nuclear staining displayed clear evidence of condensation of chromatin, but other nuclei showed normal ovoid morphology (Fig 8Down). On average, 36±9% of fields in triplicate experiments contained annexin-positive cells. Usually one or two positive cells (maximum=6) were detected in these fields (total cells per field=50±2). Of annexin V-FITC–positive cells, 68±11% were positive for propidium iodide. Of propidium iodide–positive cells, 63±19% exhibited nuclear fluorescence that indicated condensation of chromatin; the remainder displayed normal nuclear morphology. Only four cells in 150 fields were positive for propidium iodide but negative for annexin V-FITC.



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  		Figure 8. A, Confocal micrograph showing ovoid (normal) nuclear morphology displayed by a nonviable (propidium iodide–positive) endothelial cell in a confluent monolayer culture after treatment with sphingomyelinase for 48 hours. B, Same field as in panel A showing double staining with propidium iodide and annexin V-FITC. C, Apoptotic cell stained with propidium iodide and displaying nuclear fluorescence indicative of chromatin condensation after treatment with sphingomyelinase for 48 hours. D, Same field as in panel C showing double staining for propidium iodide and annexin V-FITC. Bars=50 µm.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Previously, we showed that accumulation of cells in the carotid arteries of immature rabbits was suppressed in the weeks following experimental reductions in carotid blood flow rates. The present study has demonstrated that both upregulation of cell death and downregulation of cell proliferation contribute to this suppression of arterial cell growth. Thus, {approx}70% reductions in blood flow rates caused 2- to 3-fold increases in cell death rates and 70% to 90% decreases in proliferation. We infer from these experimental observations that spontaneous developmental changes in blood flow rates also affect vascular cell proliferation and cell death and that both processes modulate arterial growth in accord with changing blood supply demands of developing peripheral tissues. The latter inference is supported by our observation that both cell proliferation and apoptosis, as indicated by degradation of DNA into oligonucleosomes, occurred spontaneously in carotid arteries of control animals. Cell death was further indicated by uptake of propidium iodide7 ; furthermore, cells labeled with propidium iodide frequently displayed clumping of propidium iodide–labeled DNA, which indicates chromatin condensation, a structural feature of apoptotic cells.29 Failure to exclude propidium iodide may be due to loss of membrane integrity in apoptotic cells; however, disrupted cell membranes have not been observed in ultrastructural studies of apoptosis. Alternatively, the extensive and very dynamic membrane blebbing that occurs during apoptosis30 may be associated with endocytotic-like uptake of dye.

An important aspect of the present study was that we were able to estimate daily cell death rates by combining measurements of the percentage of cells labeled with the viability marker, propidium iodide, with estimates of the time over which nonviable cells were capable of being labeled with this marker (discussed below). Accordingly, cell death rates were {approx}0.5% to 1% per day in unmanipulated carotids of 3-week-old rabbits, ie, {approx}20% of cell proliferation rates. Cell death rates rose to {approx}2% per day at 2 days after flow reduction, values that substantially exceeded cell proliferation rates, which had declined to well below 1% per day at this time. Thus, a net loss of cells apparently occurred early after flow reduction. These findings indicate that cell death was not a minor epiphenomenon during flow-related remodeling of developing arteries but that it contributed on a scale that was comparable to that of cell proliferation.

The estimates of cell death rates cited above were based on measurements of the percentage of cells labeled with propidium iodide. In order to convert the resulting labeling indexes into daily cell death rates, we needed to know the time interval over which dying cells could be labeled with the dye. This time interval could not be measured directly in our experiments. However, if apoptotic cells irreversibly lose their capacity to exclude propidium iodide, then the interval over which they can be labeled is equal to the time it takes labeled cells to be cleared from the vessel wall. We assumed irreversible uptake for two reasons. First, uptake of propidium iodide (and also ethidium bromide31 ) reportedly occurs late in apoptosis,24 25 when cells are unlikely to be able to restore dye exclusion. Second, in pulse-chase experiments, the number of propidium iodide–labeled cells in the vessel wall decreased approximately exponentially with time. Such first-order clearance kinetics are inconsistent with reestablishment of dye exclusion in the final stages of apoptosis. For example, if cells could take up dye for only 1 hour, between 3 hours and 2 hours before they are ultimately cleared, then the number of propidium iodide–positive cells would remain constant for 2 hours after labeling, after which the labeled cells would be deleted over the following hour.

Degradation of DNA into oligonucleosomes and frequent condensation of chromatin demonstrate that apoptosis contributes to cell death after flow reduction.29 Furthermore, our finding that all propidium iodide–labeled cells are rapidly cleared from the vessel wall argues that apoptosis is the primary mode of cell death. Propidium iodide stains cells late in apoptosis, which is inherently a very rapid process that is completed in a few hours.32 33 In contrast, this dye stains cells very early after the induction of necrosis,34 which is commonly a prolonged process. We cannot exclude, however, any contribution due to nonapoptotic cell death. Evidence for the latter includes the occasional appearance of a smear of DNA at higher molecular weights in DNA gels, a possible indicator of necrosis. The more frequent absence of these higher molecular weight fragments is evidence that arterial cell necrosis is not a primary response to flow reduction. Also, it does not appear that nonapoptotic cell death (necrosis) can be inferred from the frequent appearance of cells positive for propidium iodide that displayed no chromatin condensation. Such cells were frequently seen in cell cultures after treatment with the apoptosis-inducing agent, sphingomyelinase, and in which apoptosis was independently confirmed by staining with annexin V-FITC. Thus, the staining with propidium iodide of nonviable cells in vivo was qualitatively similar to the staining of apoptotic cells in vitro. We hypothesize that most propidium iodide–positive cells that displayed nuclei with uncondensed chromatin are apoptotic but not yet at the point of chromatin condensation. This hypothesis, while unproved, would account for why virtually all cells that were positive for propidium iodide 2 hours after labeling showed chromatin condensation.

There were quantitative differences between propidium iodide staining patterns in vivo and after sphingomyelinase treatment in vitro. Approximately two thirds of apoptotic cells in vitro displayed nuclear fluorescence suggestive of chromatin condensation, whereas this fluorescence pattern was seen in <=20% of the cells that were propidium iodide positive in vivo. These differences may indicate different kinetics of cell death in the in vivo versus in vitro models, or they may reflect differences in the timing of final clearance (sloughing or phagocytosis) of late apoptotic cells in the two systems, since final clearance would affect how long cells with condensed chromatin were available for labeling.

The finding that apoptosis contributes to postnatal development of arteries is noteworthy, since previous investigations of postnatal arterial growth have focused on cell proliferation. However, justification for this focus was questionable, since there is extensive evidence for deletion of vascular cells, including complete vessels or vascular beds,35 during earlier development. Furthermore, endothelial cell apoptosis has been reported in remodeling of the adult microvasculature during regression of the corpus luteum.36 Failure to recognize the importance of apoptosis in the late development of other organ systems has been attributed to the rapid time course of apoptotic cell death.32 33 Rapid cell death renders detection of dying cells problematic in late development because cell proliferation rates generally have slowed; therefore, relatively few cell deaths per day could have a large impact on structure over the long term. If cell deaths occur very rapidly and are infrequent, then very few dying cells will be detectable at a specific time.

Available data indicate that vascular cell death during development is indeed rapid. In the present study, pulse-chase experiments indicated that nonviable cells labeled in vivo with propidium iodide were cleared rapidly after uptake of the dye. Our previous studies of newborn lambs also provided indirect evidence of rapid cell death. We found that DNA contents in the abdominal aortas of these animals did not change in the first 3 weeks after birth; ie, cell death rates apparently matched cell proliferation rates of 2% to 3% per day. However, when nonviable cells were detected by in situ nick end labeling of DNA strand breaks using terminal deoxytransferase (TUNEL labeling), only {approx}0.06% of the cells were labeled at the time of fixation. These data indicate that the cells could be labeled by TUNEL for <1 hour.

At 3 weeks of age, arteries are developing and remodeling at a relatively slow rate, as indicated by cell proliferation rates that are <5% per day. Consequently, we did not anticipate that changes in cell proliferation or cell death rates would result in large changes in cell number over the 1-week duration of these experiments. Indeed, the modest difference in the number of smooth muscle cells per vessel cross section was not statistically significant at 1 week after flow reduction. However, smooth muscle cells also synthesize and remodel medial extracellular matrix,4 5 and we did observe that the total medial tissue mass, a product of both cell proliferation and elaboration of extracellular matrix by medial cells, was significantly lower in arteries carrying reduced flow versus control vessels at this time. Thus, even over this short time, the consequences of flow reduction on wall structure are discernible.

Although net changes in tissue accumulation occur slowly after flow reduction, decreases in diameter are rapid, with statistically significant reductions of 13% being observed at 7 days. The rapid nature of these diameter reductions probably means that they are initiated, as in adults,4 by vasoconstriction that is subsequently followed by remodeling. However, 13% diameter reductions are inadequate to renormalize mean wall shear stress after 70% flow reductions. According to estimates based on Poiseuille's law (shear stress proportional to flow and to the inverse cube of diameter), shear stress remains suppressed by {approx}50% at these time points. Poiseuille-based estimates of shear stress are probably realistic in the straight unbranched rabbit carotid artery, where relatively low Reynolds number flow prevails.37 Thus, an ongoing stimulus for remodeling persists beyond the duration of the present study, which probably explains why both proliferation and apoptosis remained different from control at 1 week and why chronic flow reductions lead to substantial effects on accumulation of wall mass and accumulation of specific wall constituents over the long term.4

For endothelial cells, increased apoptosis and decreased proliferation adjust the cell population in accord with effects of decreased flow on vessel diameter1 and therefore on the surface area the monolayer must cover. This negative-feedback control over cell density appears to persist into adulthood. We previously reported that flow reduction in the adult rabbit carotid caused reduction of vessel diameter and therefore reduction of luminal surface area, but normal endothelial cell density was rapidly restored through a decrease in cell number of {approx}20%.4 38 Endothelial cell replication rates for adult arteries are too low for reduced proliferation to account for this decrease in cell number over the duration of those experiments (5 days), so the rate of cell loss from the monolayer must have increased, possibly through apoptosis.

Although both apoptosis and cell proliferation contribute to flow-induced remodeling in immature postnatal arteries, extrapolations to other periods of development and to other situations should be made cautiously. For example, in perinatal sheep preparations, apoptosis of arterial smooth muscle cells correlated well with the large, vessel-specific changes in blood flow that occur at birth; however, proliferation rates did not.7 Instead, smooth muscle cell proliferation rates were relatively invariant among different arteries during this period. Cell replication rates are high shortly after birth, and it may be that potent systemic growth stimuli mask the effects of flow on cell proliferation.

Our findings of suppressed blood flow rates leading to reduced cell accumulation in developing arteries are in marked contrast to the effects of flow rate on the intimal proliferation that occurs in both balloon-injured arteries39 and in vascular grafts.40 In these systems, increased flow inhibits and/or decreased flow enhances intimal proliferation. Why flow stimulates growth in development but inhibits growth in these more pathological settings is not known; however, the cells near the vessel lumen have been activated to proliferate in grafts and after injury. This activation includes autocrine/paracrine stimulation via local production of growth factors41 and possibly factors that stimulate growth factor expression or chemotaxis from media to intima. High flows may contribute to convective loss of these agonists into the vessel lumen, thus suppressing the proliferative response. In developing arteries, remodeling is largely a medial phenomenon; therefore, loss of autocrine regulators to the lumen may be less important. As yet, the effects of convective transport on local mitogen concentrations have not been determined.

The present study does not establish the local mechanisms by which vascular cell apoptosis and proliferation are regulated after blood flow reduction. Shear-sensitive expression of local regulators of proliferation and cell death may be involved. Endothelial expression of platelet-derived growth factor, a mitogen and survival factor42 for vascular smooth muscle, is upregulated by shear stress in vitro,43 44 as are many other candidate genes.45 Alternatively, apoptosis may be secondary to other responses to shear stress. Thus, vasoconstriction caused by flow reduction, or early manifestations of the remodeling response that follows vasoconstriction,4 may inhibit proliferation and trigger apoptosis.


*    Acknowledgments
 
This study was supported by grant GR 13299 of the Medical Research Council of Canada. Dr Langille is a Career Investigator of the Heart and Stroke Foundation of Ontario, and Dr Cho was a trainee of the Heart and Stroke Foundation of Canada.

Received April 21, 1997; accepted June 9, 1997.


*    References
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 *References
 

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