(Hypertension. 1996;28:177-182.)
© 1996 American Heart Association, Inc.
Articles |
Effect of Long-term Treatment With Propionyl-L-Carnitine on Smooth Muscle Cell Polyploidy in Spontaneously Hypertensive Rats
the Cattedra di Anatomia Patologica, Universita' di Roma Tor Vergata, Rome, Italy, and Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minn (G.S.).
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Abstract |
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Experimental studies suggest that DNA content is increased in the smooth muscle cells of the arteries of hypertensive animals. It is unclear whether an increase in DNA content occurring in the smooth muscle cells of hypertensive rats represents a pressure-dependent effect. To evaluate the antihypertensive effect of long-term treatment with propionyl-L-carnitine and the possible morphological changes in thoracic smooth muscle cells correlated with this effect, we studied 4-month-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) randomly divided into five groups. One group of SHR was treated with propionyl-L-carnitine for 12 months; the other four groups of SHR and WKY received no treatment and were controls. We used static and flow cytometry to evaluate the polyploid cell content in thoracic aorta smooth muscle cells. Systolic pressure in untreated SHR progressively increased during the experiment. Treatment did not significantly influence pressure values in SHR. In WKY, blood pressure was significantly lower than that in treated and untreated age-matched SHR (2P<.02). The number of polyploid smooth muscle cells was significantly lower in the propionyl-L-carnitine–treated SHR than in the untreated rats (2P<.04) and similar to values for WKY. The reduction of polyploid cells in treated SHR was paralleled by a significant decrease of the aortic total DNA content, whereas no modifications occurred in smooth muscle cell mass. Long-term treatment with propionyl-L-carnitine may interfere with cellular mechanisms regulating the secondary responses involved in DNA synthesis.
Key Words: polyploidy • antihypertensive agents • propionyl-L-carnitine • rats, inbred SHR
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Introduction |
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Several experimental studies1 2 3 4 have suggested that arteries of hypertensive animals, compared with those of normotensive age-matched animals, show an increase in the volume of tunica media, mainly resulting from an increase in SMC mass. Depending on the type of hypertension and the vascular segment, three different modifications of SMCs have been observed: hypertrophy, hyperplasia, and polyploidy.5 6 7 Although hyperplasia is common in resistance arteries, hypertrophy and polyploidy occur mainly in elastic arteries, such as the aorta.8 9 Recently, similar changes have been shown also in mesenteric resistance vessels.10
The increase in polyploidy seems to be related to the failure of SMC mitotic division after DNA content duplication.11 Moreover, aortic SMCs of SHR present an enhanced incorporation of thymidine in vitro compared with SMCs of normotensive rats.12 13 It is unclear whether the increase in DNA content occurring in the SMCs of hypertensive rats represents a pressure-dependent or pressure-independent effect. Furthermore, several studies14 15 16 with different classes of antihypertensive drugs have shown that the decrease in pressure levels is associated with a decrease in polyploidy. Although there is considerable evidence that polyploidy is a secondary phenomenon related to pressure levels, some observations15 17 18 suggest that aortic SMC polyploidy is not simply a response to increased blood pressure or wall stress.
We have recently demonstrated19 that PLC, a short-chain acyl derivative of carnitine,20 showed an antiproliferative effect on intimal cells in vivo in hypercholesterolemic rabbits. The effect of PLC may be due to stabilization of cell membrane21 22 and prevention of intracellular Ca2+ overload.23 24 The same effect was also found in human endothelial cells.25 Furthermore, in vitro studies26 on rat thoracic aorta indicated that norepinephrine-induced contraction was inhibited by addition of PLC to the bathing solution.
We studied the effect of long-term treatment with PLC in SHR to verify whether this molecule exerts a hypotensive effect and possible morphologically related changes. Surprisingly, although SHR treated with long-term administration of PLC showed no significant decrease in arterial pressure, the number of polyploid cells was dramatically decreased compared with that in the control group.
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Methods |
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Animals and Blood Pressure Measurements
We used 4-month-old male SHR (n=15) and male normotensive WKY (n=10) (Charles River, Calco, Italy). Rats were housed in single cages with a 12-hour light/dark cycle and room temperature of 25°C, were fed a standard diet and water ad libitum, and were periodically weighed. Five SHR (4-mo SHR) and five WKY (4-mo WKY) were killed at the beginning of the experiment. The remaining 10 SHR were divided into two groups—one composed of 4-month-old treated rats and the other of 4-month-old untreated rats—and scheduled in pairs to begin the experiment at 15-day intervals to avoid overlap in the death of rats at the end of the experiment. One SHR of each subgroup was treated with 100 mg/kg per day PLC (Sigma-Tau) in the drinking water for 12 months (final age, 16 months; SHR+PLC). The remaining SHR of each pair received no treatment for 12 months (final age, 16 months; 16-mo SHR), and they were used as controls. The group composed of five WKY received no treatment for 12 months and was used as a control group (final age, 16 months; 16-mo WKY).
Systolic pressure was measured at monthly intervals with a piezoelectric tail-cuff pulse transducer (Basile) in conscious rats maintained at room temperature. Rats were identified by numbers; the blood pressure technician was not aware of the experimental grouping.
Experimental Procedures
Rats were killed by injection of 30 mg/kg body wt thiopental sodium IP (Abbott). KCl (1 mL; 1 mmol/mL) was injected intravenously to arrest the heart in diastole.3 The length of the aorta was measured in situ. Then, the aorta and heart were removed in a single block and fixed in 0.1 mol/L phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 12 hours at room temperature. The aorta was separated from the heart and weighed after removal of the adventitia. The thoracic aorta was selected; serial segments were used for static cytometry, flow cytometry, and electron microscopy. For electron microscopy, blocks of tissue were postfixed in 1% phosphate-buffered osmium and embedded in epoxy resin (Epon 812). Ultrathin sections were examined with an electron microscope (Philips 301).
This study was conducted in accordance with the Human and Animal Research Committee guidelines of our institution.
DNA Polyploidy
DNA content was evaluated by static and flow cytometry techniques. Densitometric analysis by static cytometry was performed on histological sections stained by the Feulgen method27 with the Quantimet 920 image analyzer (Cambridge Instruments Ltd). We used 8-µm sections.27 All measurements were performed with a monochromatic light of 546-nm wavelength. For each section, 200 nuclei were randomly measured. Chicken erythrocytes were stained the same way and used as a standard because each erythrocyte is known to contain 2.5 pg DNA.9 Under our measurement conditions and according to the method of Lee et al,28 the integrated optical density was divided into various intensity units, each equivalent to 0.46 pg DNA. Cells with 20 intensity units or less (ie, <9.20 pg DNA per cell) were considered diploid cells (2C); cells with 21 to 40 intensity units (9.66 to 18.40 pg DNA per cell), tetraploid cells (4C); and those with more than 41 intensity units (>18.86 pg DNA per cell), octaploid cells (8C). These values are within the range previously reported2 14 for WKY and SHR SMCs.
Flow cytometry was performed with an Epics Profile cytometer (Coulter Electronics, Inc). Segments of thoracic aorta were sectioned, broken into small pieces, and dispersed separately into single cells by the enzymatic action of elastase (Sigma Chemical Co, 1 mg in 1.5 mL PBS [pH 8.4]) and protease (Sigma, 1 mg in 1.5 mL PBS, pH 7.2) for 90 minutes at 37°C with continuous mixing (vortex). After aspiration (21-gauge needle) and filtration through a 50-mesh nylon gauze, the material was centrifuged at 1500 rpm for 10 minutes. The pellet was then washed, resuspended in PBS buffer, and centrifuged at 1500 rpm for 10 minutes. The nuclei were counted through a Toma-Zeiss chamber (Carl Zeiss Inc) to obtain a final concentration of 1x106 nuclei/mL and then were stained with propidium iodide (Sigma) solution containing 50 µg/mL RNase (Sigma) at room temperature for 10 minutes. Approximately 20 000 nuclei were counted for each case. The variability coefficient for each case ranged between 2.5 and 8.
Evaluation of Aortic Medial Hypertrophy
To evaluate the PLC effect on aortic hypertrophy, we determined the volume density of SMCs in aortic tunica media [VV(SMC/MED)] and the total aortic SMC mass. SMC volume density was determined from electron micrographs at a magnification of x7160 with use of a standard point-counting technique.29 Total aortic SMC mass was calculated as Aortic Medial WeightxVV(SMC/MED).15
Evaluation of Total DNA Content
For evaluation of aortic DNA content, three samples of aorta were collected from the ascending, thoracic, and abdominal tracts; embedded in paraffin; and treated as follows.30 The samples were deparaffinized with two 20-minute changes of a clearing agent (TopExilo, Pabish) and rinsed in absolute alcohol for 10 minutes. The samples were then weighed and suspended in 1 mL lysis solution (0.05 mol/L Tris-HCl [pH 9], 0.15 mol/L NaCl, 5% mmol/L EDTA) containing 1% sodium dodecyl sulfate, 500 µg/mL proteinase K (Sigma), and 100 U/mL elastase. The suspended samples were mixed (vortex) at high speed for 3 to 5 minutes and incubated at 37°C for 48 hours. After the first day of incubation, 250 µg/mL proteinase K was added. Nucleic acid was extracted three times with a volume of phenol, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1), respectively. The NaCl concentration of the extracted solution was adjusted to 0.3 mol/L, and DNA was precipitated with the addition of 2 vol cold absolute alcohol. The DNA was incubated at -20°C overnight and centrifuged at 10 000 rpm for 30 minutes in an RC5B Plus Ultracentrifuge equipped with an SS34 rotor (Sorvall DuPont Co). The pellet was washed in 70% ethanol, air-dried, and resuspended. DNA concentration was measured at 260 nm with a spectrometer (DMS-300S, Variant Techtron Pty Ltd) by a standard method. A calibration curve was calculated with DNA concentrations ranging from 0.2 to 10 ng/µL and measured with a 1:100 dilution of the original sample (Fig 1
). For all samples, the ratio between the readings at 260 and 280 nm (OD260/OD280) was more than 1.8, thus certifying the quality of the DNA purification.31 The results were related to the total weight of the aorta. The total DNA content was evaluated in five of the 16-mo SHR and in four of five of the SHR+PLC because the residual embedded tissue was insufficient. For each sample, DNA content was measured twice, and the mean value was used for statistical analysis.
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Statistical Analysis
Statistical analysis was performed with the SPSS program (Statistical Package for the Social Sciences, 4th ed, MJ Norusis/SPSS Inc). For each parameter, the mean, SD, and SE were calculated. Differences among coupled experimental groups were evaluated by paired Student's t test. Values of P<.05 were considered statistically significant.
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Results |
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Systolic Pressure
Systolic pressure in 16-mo SHR progressively increased from 207.8±2.5 mm Hg at the beginning of the experiment to 247.6±8.6 at month 12 and 260.8±5.7 at month 16 (Fig 2
). In SHR+PLC, systolic pressure progressively increased from 205.6±3.3 to 259.6±6.6 mm Hg. Blood pressure did not differ significantly in the treated and untreated SHR groups.
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Systolic pressure was 132.6±2.4 mm Hg in 16-mo WKY at 4 months, and it increased slightly until month 16. These values were significantly lower than those measured in 16-mo SHR and SHR+PLC (2P<.02). Before death, systolic pressure was 195.2±9.8 mm Hg in 4-mo SHR and 135.6±3.2 in 4-mo WKY.
Heart and Body Weights
Long-term treatment with PLC did not prevent the increase in cardiac mass associated with hypertension. Mean heart weight was 2.18±0.21 g in SHR+PLC and 2.24±0.14 in 16-mo SHR (Table 1). Furthermore, heart weight as a percentage of body weight did not differ significantly between these two groups.
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DNA Polyploidy
Static Cytometry
The 16-mo SHR showed a considerable increase in tetraploid (polyploid) cells compared with 4-mo SHR (16.05±2.73% and 4.89±0.41%, respectively; 2P<.02) (Fig 3). In normotensive WKY, a slight yet significant increase of polyploid cell percentage was observed with aging (2P<.02). SHR+PLC showed a clear decrease in polyploid cell content compared with 16-mo SHR (2P<.04) (Table 2). The percentage of polyploid cells in SHR+PLC was about half that in 16-mo SHR and was similar to that in 16-mo WKY.
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Flow Cytometry
In SHR and WKY, a significant increase of thoracic aortic polyploid cell content (tetraploid cell [4C] DNA content) was observed with age (2P<.02), which was similar to the static cytometry results (Fig 4). In SHR+PLC, the percentage of polyploid cells was significantly lower than that in 16-mo SHR (12.91±1.88% and 25.52±5.07%, respectively; 2P<.03) (Table 2
) and similar to that of normotensive 16-mo WKY. Two examples of flow cytometric data obtained from 16-mo SHR and SHR+PLC are shown in Fig 5.
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Evaluation of SMC Mass and Total DNA Content
SMC volume density and total mass did not differ significantly between 16-mo SHR and SHR+PLC (Table 3). Conversely, as reported in Tables 2 and 3, a significant reduction in total aortic DNA content was observed in SHR+PLC compared with 16-mo SHR (2P<.03).
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Discussion |
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We investigated the relationships between blood pressure levels and the development of polyploidy in aortic SMCs in SHR using PLC. Although PLC has no effect on blood pressure levels, its long-term administration inhibits the development of hypertension-induced polyploidy in aortic SMCs. After 12 months of treatment, the polyploid value of SHR+PLC was similar to that of the normotensive controls (16-mo WKY) and approximately half that of the untreated hypertensive rats (16-mo SHR). Although the polyploid cell percentage calculated by flow cytometry was higher than the same percentage calculated by static cytometry, the methods provided comparable results (Figs 3 and 4
). The higher frequency of polyploid cells by flow cytometry than by static cytometry should be ascribed first to the fact that flow cytometry analyzes some 20 000 whole cells, whereas the image analyzer evaluates 200 cells on an 8-µm histological section that, although being the thickness generally considered most adequate for this method,27 may include nonwhole nuclei. Furthermore, the type of stain used to show DNA with flow cytometry (propidium iodide) is different from that used with static cytometry (Feulgen staining). The pathophysiological significance of polyploidy in SHR aortic SMCs is unknown. In agreement with previous studies,9 14 32 our results show that the percentage of polyploid SMCs in the aorta increased along with the duration of the hypertensive state, being significantly higher in 16-mo SHR than in 4-mo SHR (2P<.02). SMCs increased DNA content without progressing to the M phase of the cell cycle. This may represent a mechanism whereby an SMC population maintains a highly differentiated state (ability to synthesize proteins), expanding its gene expression or translational capacity.33 34 Alternatively, these cells may lack a factor necessary to their progression throughout the remaining cell cycle.33 Theoretically, SMC polyploidy could be prevented by inhibiting SMCs from entering the G1 or S phase or by removing the block of mitosis, thus allowing the cells to divide in a normal diploid fashion.
The results of our study indicated that the decrease in the number of polyploid cells in SHR+PLC was associated with a significant decrease in total aortic DNA content compared with untreated age-matched SHR; SMC mass was not modified (Table 2). The effect of PLC on SMC polyploidy is likely because of interference in the progression through the cell cycle from G0 to G1 and G1 to S phases. Two pieces of circumstantial evidence seem to support this hypothesis. Recent in vitro studies in our laboratory have demonstrated that thymidine incorporation in SMCs isolated from SHR and treated with PLC added to the culture medium was significantly lower than in SMCs isolated from age-matched untreated SHR (unpublished data, 1995). Moreover, in in vivo aged rabbits fed a hypercholesterolemic diet, long-term administration of PLC significantly decreased cell proliferation in the atherosclerotic plaques compared with that of untreated hypercholesterolemic controls.19
To date, the mechanisms by which PLC affects DNA synthesis or other phases of the cell cycle are not clearly understood. PLC is a short acyl derivative of carnitine20 endowed with a stabilizing action on biomembranes that may result from an activity on fatty acid metabolism21 or be due to a possible interaction with the membrane lipid bilayer.22 In a study by Ferrari et al23 with an animal model of New Zealand White rabbits, PLC protected mitochondrial function by preventing the mitochondrial calcium overload occurring in the myocardium during ischemia and reperfusion. Moreover, Bevilacqua et al24 demonstrated an effect of PLC on L-type calcium channels in human heart sarcolemma in patients with idiopathic dilated cardiomyopathy who underwent myocardial transplantation. PLC added to plasma membrane labeled with the L-type calcium channel blocker isradipine decreased the affinity of this molecule for the calcium channels. This result was consistent with a possible competition between PLC and Ca2+ and showed that L-type calcium channels of human sarcolemma are modulated by PLC, apparently in competition with Ca2+. Van Hinsbergh and Scheffer25 evaluated the effect of PLC treatment on the cytoplasmic calcium level in endothelial cells incubated with oxidative substances. PLC administration lowered the cytoplasmic calcium level, as indicated by the reduction in fluorescence intensity of the intracellular calcium indicator fura 2.
The calcium channel blocking–like activity may be responsible for the PLC effect on the initiation of the cell cycle or DNA synthesis. Other calcium channel blocking agents, such as nifedipine, have been shown to inhibit proliferation of cultured aortic rat SMCs.35
The Ca2+-calmodulin system is one of the crucial components of the pathway through which growth factors lead to DNA synthesis after binding to their specific receptors. In fact, platelet-derived growth factor and epidermal growth factor markedly stimulate Ca2+ release from intracellular stores via inositol triphosphate generated with diacylglycerol by activated phospholipase C.36 37 Elevation of calmodulin levels is required for the entry of quiescent cells into the cell cycle and their progression through G1 and S phases.38 Interestingly, an increased permeability to calcium ion39 and higher calmodulin activity40 have been described in SHR and other models of hypertension. The hypothesis should not be excluded that the inhibition of SMC polyploidy induced by PLC treatment may result from an interference of PLC with the oncogene expression (c-fos and c-myc)41 regulating the cell cycle.
Other drugs have an effect on hypertension-induced polyploidy. Owens14 demonstrated that antihypertensive drug treatment of SHR with a combination of reserpine, hydralazine, and chlorothiazide decreased arterial pressure and prevented the development of polyploidy. Further studies15 16 demonstrated the effectiveness of other antihypertensive agents, such as angiotensin-converting enzyme inhibitors (enalapril, captopril), in preventing the development of polyploidy in SHR. However, in contrast to PLC, the effect of all the above-mentioned drugs was always associated with a lowering of blood pressure levels. Leitschuh and Chobanian17 obtained an effect similar to that obtained with PLC using propranolol in a deoxycorticosterone-salt model of hypertension. In that study, propranolol prevented the development of SMC polyploidy even though it had little or no effect on blood pressure. Those findings as well as our findings suggest that although blood pressure is important for the development of polyploidy, other factors may also play a crucial role in this change.17
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grant 94.00656.PF41 from the National Council of Research (CNR), FATMA Project. We express our gratitude to Dr Aram V. Chobanian for suggestions in a preliminary review of the manuscript. The authors thank Alfredo Colantoni, Renzo Bernabei, Angela Ortenzi, Antonio Volpe, and Sabrina Cappelli for excellent technical assistance.
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Footnotes |
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Reprint requests to Prof Luigi Giusto Spagnoli, Cattedra di Anatomia ed Istologia Patologica, Dipartimento di Chirurgia, Universita' di Roma Tor Vergata, Via della Ricerca Scientifica, 00153 Roma, Italy.
Received July 25, 1995; first decision August 16, 1995; accepted April 1, 1996.
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References |
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