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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Graduate Group in Molecular and Biochemical Nutrition, Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, California
Submitted 17 April 2006 ; accepted in final form 19 May 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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atherosclerosis; gas chromatography-mass spectrometry; stable isotopes; animal model
Current techniques for measuring cell proliferation in vivo depend on the incorporation of nucleoside analogs, such as [3H]thymidine and bromodeoxyuridine (BrdU), via the nucleoside salvage pathway, into replicating DNA (12, 14) (Fig. 1A) or immunostaining for cell cycle-associated proteins, such as proliferating cell nuclear antigen (PCNA) (22) and Ki67 (4). These methods have several disadvantages in terms of obtaining quantitative and qualitative results. The salvage pathway of labeled deoxyribonucleosides (dN) competes with unlabeled sources for incorporation into newly synthesized DNA, with variable and unpredictable dilution of label (8, 32). Labeling intensity is thereby affected by artifacts when analyzed by autoradiography, specific activity measurements, or antibody staining. Furthermore, BrdU and [3H]thymidine are genotoxic and are known to suppress proliferation of various cell populations when given in long-term studies (1, 8, 14, 17, 31, 32, 34). Cell cycle-associated markers may be present in nonreplicating cells (3, 11) and in cells that never proceed through the S-phase, thus contributing to an overestimation of dividing cells. These methods of analysis are also time-consuming, with high interobserver variability (26, 27, 34). Moreover, these labeling indexes represent short-term measurements that do not reveal integrated proliferation kinetics over time. As a consequence of these technical limitations and the belief that the turnover rate of VSMC is very slow (5, 7, 9), VSMC proliferation and turnover rates have not been definitively established. The quantitative impact of various genetic or nutritional factors on cell proliferation in atherogenic lesions has also not been characterized.
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To test this method for vascular wall applications, experiments were designed to characterize VSMC proliferation and intimal-medial cell (including atheroma) proliferation in different settings. In the first experiment, differences in VSMC turnover/proliferation between young and adult mice were demonstrated. The second experiment consisted of labeling and then delabeling VSMC DNA to establish a die-away curve. The third experiment measured cell proliferation from the intima-media in atherosclerosis-prone apolipoprotein E (ApoE)-knockout (ApoE–/–) mice fed a high-fat diet and labeled continuously from 5 wk of age. In the fourth experiment, atheroma cell proliferation over 3-wk labeling periods was compared in ApoE–/– mice and C57BL/6J controls fed low- or high-fat diets to characterize more precisely the kinetic stages of atherogenesis relative to histological changes. Finally, we conducted a VSMC proliferation experiment in vitro to validate the 2H2O method by comparing label incorporation with cell counting and BrdU proliferative index.
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MATERIALS AND METHODS |
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Cell culture and media. Rat VSMC (catalog no. CRL-2018) were obtained from American Type Culture Collection (Manassas, VA) and cultured in modified DMEM (catalog no. 30-2002, American Type Culture Collection) containing 10% FBS and neomycin (0.2 mg/ml) in a 5% CO2 humidified environment at 37°C. For the method validation experiments, media for rat VSMC were formulated to contain 10% 2H2O by addition of powdered DMEM (catalog no. 50-003-PB, CellGro, Herndon, VA), sodium bicarbonate (1.5 g/l), and 70% 2H2O (Cambridge Isotopes, Andover, MA).
In vivo labeling of VSMC and intimal-medial (atheroma) cell DNA with 2H2O.
Under isoflurane (Abbott Laboratories, Chicago, IL) anesthesia, the mice were injected, intraperitoneally with a 2H2O-NaCl mixture to attain a body water enrichment of 
2% (based on an estimated 60% body weight as water) and then maintained on 4% 2H2O in drinking water ad libitum for 3 wk, unless stated otherwise. In the initial atherogenesis study, the mice were labeled continuously from 5 wk of age until they were killed at monthly time points.
Delabeling VSMC DNA. The mice were injected with 2H2O at 15 wk of age as described above and maintained on 4% 2H2O drinking water for 10 wk. Then the mice were switched to nonlabeled drinking water to eliminate residual 2H2O in body water. Beginning 10 days after discontinuation of 2H2O, the mice were killed at 2-wk intervals to determine VSMC dR enrichment.
Plasma, urine, and tissue sampling. The mice were euthanized by cervical dislocation. Serum was obtained for assessment of body water enrichment. Urine, if present, was drawn from the urinary bladder as an alternative source of body water.
The aorta, from the heart to the iliac bifurcation, and femur bone marrow were excised for dR enrichment. Bone marrow cells were collected by centrifugation in Hanks' balanced salt solution. For histological purposes, aortae were stained with hematoxylin and eosin or immunostained against PCNA (Histo-tec, Hayward, CA).
Determination of body water and rat VSMC media enrichment. Body water from plasma or urine and rat VSMC media 2H2O enrichments were determined by a modification of the protocol developed by Neese et al. (27). Approximately 10 µl of the sample were injected into a sealed vial containing calcium carbide. The acetylene gas that was produced was drawn into a syringe and transferred to another sealed vial containing Br2-CCl4. Tetrabromoethane was analyzed on a mass spectrometer (model 5973) with a gas chromatograph (model 6890) and autosampler with use of a 30-m column (model DB-225) at 220°C and methane chemical ionization with selected ion monitoring (Hewlett-Packard, Palo Alto, CA). Ions at mass-to-charge ratio (m/z) 265 (M0), representing the derivatized product without deuterium label, and m/z 266 (M1), representing the derivatized product with a 2H label, were monitored. Calculations of body water 2H2O enrichments were based on standard curves of known concentrations of 2H2O.
Analysis of VSMC or atheroma cell turnover/proliferation. VSMC or atheroma cells were obtained according to a modification of the protocol by Ramos and Cox (30). Briefly, the aorta was cleaned of surrounding fat tissue and treated with 70 U of collagenase (type 1; Sigma Chemical, St. Louis, MO) for 25 min at 37°C to remove the endothelial and adventitial layers. Under a dissecting microscope, the adventitia was stripped using forceps. The remaining sleeve-like preparation represents the subendothelial intimal-medial layer and was used for subsequent analyses.
DNA was extracted from the intimal-medial layer and bone marrow cells (DNeasy tissue kit, Qiagen, Valencia, CA) and enzymatically hydrolyzed into dN. Deoxyadenosine (dA) was isolated by reverse-phase chromatography using LC 18 SPE tubes (Supelco, Bellefonte, PA) and exposed to mild acid hydrolysis to release the base from the dR. The dR group was derivatized to pentane tetraacetate (27) and analyzed by GC-MS containing a 20-m DB-225 column (0.18 mm ID, 0.2 µm film thickness; J & W Scientific, Folsom, CA) and chemical ionization mass spectrometry of m/z 245 (M0) and m/z 246 (M1). Standards of unlabeled dA were run before and after samples to ensure accuracy of isotope ratios for each GC-MS run, as described previously (16, 27).
Rat VSMC growth curve analyses. For the growth curve experiment, rat VSMC were seeded at 1.5 x 105 cells per well in covered six-well plates, with one entire plate representing a time point (i.e., days 1–6). After an overnight incubation, one plate (day 1) was selected to represent average baseline dR natural isotope abundance and starting cell numbers. Unlabeled media in all other plates were replaced with 10% 2H2O-containing media. Thereafter, on each day, the wells were washed twice with PBS, and the cells were incubated with 0.25% trypsin-EDTA to form cell suspensions. One hundred microliters of each suspension were used for cell counting with a hemocytometer. The remaining cell suspension from each well was placed in a separate tube and spun in a microcentrifuge to pellet cells. The supernatant was removed, and DNA was extracted using the Blood and Cell Culture DNA Midi kit (Qiagen) according to the manufacturer's instructions. In addition to the growth curve experiment, rat VSMC were passaged four times in 10% 2H2O-containing media to allow cells to reach maximum 2H dR enrichments (100%-labeled cells). At final passage, the cells were washed and trypsinized, and DNA was extracted. DNA from all samples was subjected to hydrolysis and derivatization for GC-MS analyses (see above).
Determination of rat VSMC proliferation by BrdU and 2H incorporation. Rat VSMC were plated onto six-well plates with or without coverslips at 1 x 105 cells per well. After overnight incubation, the cells were serum starved for 40 h. The medium was then replaced with medium containing 10% FBS. Thereafter, on each day, medium from several wells with coverslips was replaced with medium containing BrdU (1:100 dilution; Zymed BrdU Labeling Reagent, Invitrogen, Carlsbad, CA); medium from plates without coverslips was replaced with medium containing 10% H2O and covered. The cells were labeled each day with 2H2O for 24 h or with BrdU for 6 h. DNA from cells labeled with 2H2O was analyzed for 2H enrichment (see above). The cells on coverslips were stained for BrdU incorporation into nuclei using a streptavidin-biotin system (Zymed BrdU Staining Kit, Invitrogen), according to the manufacturer's protocol, and counterstained with hematoxylin.
Calculations. PCNA and BrdU labeling indices (%) were calculated as 100 x (no. of PCNA- or BrdU-stained nuclei ÷ total no. of nuclei).
The fraction of newly divided cells (f) is calculated on the basis of the precursor-product relationship (16, 21, 44). VSMC or atheroma cell enrichment is compared with the bone marrow enrichment of the same animal as a representation of a nearly 100% turned-over tissue.
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For the rat VSMC growth curve experiment, new rat VSMC (%) was calculated as determined by hemocytometry
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Statistical analysis. Values are means ± SD. Statistical analysis was determined by one-way ANOVA followed by Tukey's comparison, with P < 0.05 indicating statistically significant differences.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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0% (0.09 ± 0.01%) after 10 days, 0.07 ± 0.01% after 24 days, and 0.02 ± 0.02% after 38, 52, and 64 days of delabeling (Fig. 3). In contrast, VSMC dR enrichments remained relatively stable at 0.40% for the first 52 days and fell to 0.32 ± 0.09% at 64 days after discontinuation of 2H2O intake (Fig. 3), consistent with the extremely low turnover rates determined by the 2H2O incorporation experiments.
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Cell proliferation in aortic intimal-medial layers in C57BL/6J and ApoE–/– mice studied at 3-wk labeling intervals. To further characterize the proliferation time course in atherosclerotic lesion cells, the above-described study was repeated, but with 3-wk labeling periods, instead of a continuous 2H2O administration protocol. Low-fat diet groups were also included. Thirty ApoE–/– and 19 C57BL/6J mice began the high-fat diet at 5 wk of age, while 19 ApoE–/– and 20 C57BL/6J mice continued the low-fat rodent chow after 5 wk of age. The four groups were given 2H2O at consecutive periods every 3 wk before death (e.g., weeks 0–3, 3–6, 6–9, 9–12, 12–15, 15–18, and 18–21 on the diets, n = 4–5/group).
Significant differences in cell proliferation became apparent after 12 wk on the atherogenic diet in ApoE–/– mice and remained elevated compared with C57BL/6J mice on the low-fat diet: 7.3 ± 1.9% at 12 wk (P < 0.05), 8.8 ± 3.0% at 15 wk (P < 0.01), 14.0 ± 4.9% at 18 wk (P < 0.01), and 17.0 ± 4.3% at 21 wk [P < 0.01 vs. low-fat C57BL/6J (1.6–2.5%); Fig. 4]. In ApoE–/– mice on a low-fat diet, atheroma cell proliferation also began to increase significantly after 21 wk compared with VSMC proliferation of C57BL/6J on a high-fat diet: 7.0 ± 1.3% and 1.30 ± 0.47%, respectively (P < 0.05; Fig. 4).
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6 days. Beginning on day 1, which represents baseline 2H dR enrichment values and initial cell numbers, cells were counted with a hemocytometer and DNA was extracted for 2H enrichments. 2H2O enrichment in media was 10%. Growth curves representing percent new cells determined by cell counting or by 2H enrichment analysis of DNA are shown in Fig. 6. A comparable growth trend approached 100% new cells, with day 6 values at 88 ± 15%, as calculated by hemocytometry, and at 85 ± 6%, as calculated by the 2H2O method. Furthermore, the best-fit curves were similar.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We have adapted the technique to measure cell proliferation/turnover in the aortic intimal-medial layer in mice. The intima (endothelium removed) and media consist essentially exclusively of VSMC in normal mammals but also include cells characteristic of atheromatous lesions as the plaques evolve in the course of atherogenesis (37, 40). We began with testing of the method in young (3- to 9-wk-old) and adult (15- to 21-wk-old) male C57BL/6J mice. VSMC proliferation rates were significantly higher (>4-fold) in younger mice than in adult mice. In a previous report, the adult rat VSMC turnover rate was estimated to be 0.06%/day (9). The calculated replacement rate constant from our results in adult mice was 0.09%/day, representing a turnover time of 3.1 yr. It is possible that VSMC proliferation in mice represents the accumulation of VSMC as the aorta remodels to increases in growth, blood pressure, and force in growing mammals (2, 10, 23, 39, 42). Alternatively, true turnover in adult mice could represent VSMC in anatomic locations subjected to high pressure and turbulence, such as intimal thickenings (42).
To further validate this estimated slow turnover of adult VSMC, we delabeled adult male C57BL/6J mice after prior labeling for 10 wk until body water enrichments decreased to near 0% (
10 days) and then killed the mice at 2-wk intervals. VSMC dR enrichment remained relatively stable for up to 64 days, thus confirming the notion that normal adult VSMC are long-lived. Numerous reports have described the phenotype of adult VSMC as in a contractile state containing little protein-producing machinery compared with a synthetic state (i.e., producing extracellular matrix and prone to proliferation) (5, 23), as commonly observed in fetal and atherosclerotic VSMC. Our labeling results confirm this quiescent status directly in vivo.
We then applied the method to measurement of atheroma cell proliferation in the intimal-medial area of the vessel wall in atherosclerosis-prone ApoE–/– mice compared with VSMC proliferation of their background strain C57BL/6J. Two labeling strategies were used. In the first study, mice were fed a high-fat diet, containing 42% of kilocalories from fat, and labeled continuously from 5 wk of age. The labeling continued until the animals were killed at monthly time points. Results indicated a surge in cell proliferation (f from 15 to 40%) in ApoE–/– mice fed the high-fat diet at weeks 15–21 that remained elevated through week 24, whereas in the control animals, VSMC fractional proliferation showed a slow increase, reaching maximal values of 10% new cells. The increase in fractional proliferation values observed in ApoE–/– mice may represent the initiation of atheroma proliferation with subsequent accumulation of cells. These results did not indicate the exact time at which proliferation occurred, however, and whether there was constant turnover or no further proliferation at weeks 21-24. Lutgens and colleagues (20) observed decreases in DNA synthesis markers in human atherosclerotic lesions with severity of disease, suggesting that atheroma proliferation may be a characteristic of early atherosclerotic lesions and may represent the lesions formed in ApoE–/– mice at weeks 15–21, whereas more advanced lesions at weeks 21–24 may signify a transition to apoptosis. Alternatively, the plateau observed during continuous labeling might have represented a period of low cellular activity, in which VSMC may have reached senescence or, if cell proliferation is episodic, a hiatus in the development of atheroma (20).
Accordingly, we labeled mice at 3-wk intervals to create "windows" of cell proliferation measurements. These studies were also extended to include measurements of intimal-medial cell turnover/proliferation in ApoE–/– and C57BL/6J mice fed a low-fat diet, in addition to the high-fat diet, to determine whether this method detected interactions or degrees of hyperproliferation. Significantly augmented levels of aortic intimal-medial cell proliferation were apparent in ApoE–/– mice after 12 wk on the high-fat diet and, less strikingly, on the low-fat diet, compared with proliferation in C57BL/6J on low- and high-fat diets. The timing of cell proliferation corresponded to the histological time line of atherosclerosis, as previously characterized by Nakashima et al. (25). Furthermore, the morphological observations in this study correlated with the increase in atheroma cell proliferation in ApoE–/– mice in the first high-fat diet study (Fig. 2). The initial rise in DNA proliferation may correlate with events in type II lesions [American Heart Association classification], because immunohistochemical findings from Lutgens et al. (20) showed that this lesion state has the highest proliferation (2.7 ± 0.5% vs. 0.02 ± 0.02% nondiseased, P < 0.05) in a biphasic pattern of proliferation in atherosclerosis in human specimens.
The proliferative signals, therefore, remain present through the evolution of complex atherosclerotic plaques observed by week 21 in these animals. However, the cells responsible for the elevated proliferation rates that we observed in atheromatous lesions may include cell types other than VSMC. In particular, monocyte migration into lesions from the bloodstream (29) can contribute to high proliferation rates, because circulating monocytes have been shown to exhibit rapid turnover (24, 26, 27).
As demonstrated by our in vivo experiments, the 2H2O method for measuring VSMC and atheroma cell proliferation can be successfully applied to mouse models. To further support the validity of the technique, we designed a cell culture experiment using rat VSMC grown in medium containing 10% 2H2O to compare cell proliferation using the 2H2O method with direct cell counting and with BrdU incorporation. The percentage of new cells corresponding to a specific time point was essentially identical by the 2H2O method and hemocytometry (Fig. 6). Thus data generated by 2H2O labeling are as dependable as those deduced from a direct cell-counting method. Moreover, it is evident that the percentage of 2H used in vitro (10%) does not alter cell proliferation, which occurred at expected rates. In addition, trends in proliferation were similar and stable over time in VSMC labeled with 2H or BrdU. We expected the proliferation indexes to be the same within an experiment, because the cells were labeled at the same time on each day.
In cardiovascular diseases that involve high rates of VSMC endoreplication (i.e., DNA replication during the S-phase of the cell cycle without subsequent completion of mitosis and/or cytokinesis), such as hypertension, this method may overestimate the percentage of new cells. In this case, the fractional proliferation value can be interpreted to be percentage of new DNA, which is still useful in determining effects on vascular wall DNA replication.
In conclusion, we have presented a relatively simple method for measurement of aortic intimal-medial cell (VSMC and atheroma) turnover/proliferation in vivo. The trends in proliferation with use of the 2H2O method correlated with those shown by PCNA immunostaining in vivo and by BrdU immunostaining in vitro and were quantitatively identical to results from direct cell counting in vitro. The combination of this stable isotope-mass spectrometric approach and morphological observations provides a more quantitative portrayal of atheroma cell kinetics than is currently available to investigators. This technique may prove useful, in combination with other methodologies, in a variety of vascular conditions, because VSMC and lesion cell hyperplasia are characteristics of many vasculopathies.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Present address for A. Chu: Cognia Corporation, 117 East 55th St., New York, NY 10022.
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FOOTNOTES |
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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.
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REFERENCES |
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