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(Circulation. 1996;94:2620-2625.)
© 1996 American Heart Association, Inc.

Articles

Homocysteine Increases Cyclin-Dependent Kinase in Aortic Rat Tissue

Barbara Lubec, MD; Olga Labudova, PhD; Harald Hoeger, PhD; Adolf Muehl, PhD; Susanne Fang-Kircher, MD; Manfred Marx, MD; Wilhelm Mosgoeller, MD; J. Gialamas, PhD

the University of Vienna (Austria), Department of Pediatrics, Division of Neonatology (B.L.); the Institute of Animal Breeding (H.H., J.G.); the Institute of Histology (W.M.); the Institute of Medicinal Chemistry (S.F.-K.); and the Department of Pediatrics, Division of Cardiology (O.L., M.M., A.M.), Vienna, Austria.

Correspondence to Dr Barbara Lubec, University of Vienna, Department of Pediatrics, Division of Neonatology, Waehringer Guertel l8, A lo9o Vienna, Austria.

	Abstract

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Background Hyperhomocyst(e)inemia is strongly associated with occlusive arterial disease. A direct effect of homocysteine on the proliferation of smooth muscle cells was proposed recently. This observation led us to examine the effect of homocysteine on cyclin-dependent kinase, the starter of mitosis and reflecting proliferation.

Methods and Results Seventy Him:OFA rats were divided into seven groups. For 12 weeks, 10 rats were fed homocysteine 25 mg/kg body weight per day, 10 were fed 50 mg/kg body wt per day, and 10 were fed 100 mg/kg body weight per day; 10 were given homocysteic acid 100 mg/kg body weight per day, 10 were administered cysteine 100 mg/kg body weight per day, and 10 were given ascorbic acid 270 mg/kg body weight per day. Ten remained untreated and served as controls. Aortic cyclin-dependent kinase was determined at the transcriptional (mRNA) and protein levels. Phosphokinase C and aortic homocyst(e)ine also were evaluated in aortic tissue. Aortic cyclin-dependent kinase protein was significantly (P=.0001) elevated in the three homocysteine-treated groups, and mRNA cyclin-dependent kinase levels were significantly elevated in the rats given the 50 and 100 mg/kg body weight per day protocol. Endothelial damage was shown at higher homocysteine doses as reflected by circulating ACE and von Willebrand factor changes. Proliferation of cells of the aortic wall by bromodeoxyuridine incorporation could be shown in the high-dose homocysteine group only.

Conclusions Our findings indicate that homocysteine specifically stimulates aortic cyclin-dependent kinase at the transcriptional level, with the possible consequence of proliferation of aortic cells as revealed by incorporation of bromodeoxyuridine in the aortic wall.

Key Words: homocysteine • cyclin-dependent kinase • proliferation

	Introduction

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The association between homocyst(e)ine and cerebrovascular or peripheral arterial diseases has been reported by several groups,^{1 2 3 4 5 6} and several mechanisms for the development of vessel wall changes have been described.^{7 8 9 10} Working on homocysteine and its derivatives in radiation protection,¹¹ we found that homocysteines significantly stimulated cellular growth in a clonogenic assay.¹² Tsai and coworkers¹³ recently reported on the promotion of vascular smooth muscle cell growth by homocysteine. They observed an increase in DNA synthesis by as little as 0.l mmol/L homocysteine and found increased mRNA levels of cyclin D_l and A, possibly reflecting the reentry of quiescent aortic smooth muscle cells into the cell cycle.

Because proliferation of arterial tissue presenting with intimal-medial wall thickening seems to be a main feature of vascular changes in hyperhomocyst(e)inemia, we set out to determine whether homocyst(e)ines could be associated with or even activate CDK, the system-starting mitosis and known marker for proliferation,¹⁴ at the protein and transcriptional levels.

	Methods

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Animals
Seventy 79- to 82-day-old white, female Him:OFA rats (Institute of Animal Breeding, Himberg, Austria) were used in the experiments. They were kept at room temperature (23°C) under a day/night rhythm and had free access to rat chow (Altromin). Ten rats in the control group had free access to tap water. The experimental groups were divided into groups of 10 rats each and for 12 weeks were given orally 25, 50, or 100 mg/kg body weight per day homocysteine (Sigma Chemical Co), 100 mg·kg^-1·d^-1 HCA (Sigma), 100 mg·kg^-1·d^-1 cysteine (Sigma), or 270 mg·kg^-1·d^-1 ascorbic acid in tap water supplied in light-protected bottles. The rats were killed at the end of the experiment by CO₂ treatment, and the thoracic and abdominal aortic vessels were taken. We fixed 5 mm of the thoracic part in 4% paraformaldehyde in isotonic PBS; the rest of the aortic tissue was put immediately into liquid nitrogen and kept frozen at -70°C until chemical analyses. To rule out toxic unspecific effects, body weight and fluid and food intake were evaluated and did not differ between groups (data not shown).

Sample Preparation for Phosphokinase Determinations
Aliquots of aortic tissue were weighed and homogenized by a Potter in an ice bath in the presence of 1% SDS in a 0.01-mol/L Tris buffer, pH 7.4. The samples were spun down in a refrigerated centrifuge at 8000g, and the clear supernatant was used for phosphokinase determinations.

Determination of CDK Protein
The detection of p34 cdc2 kinase (CDK) was achieved by an ELISA with the commercially available In Vivo CDK ELISA (Paracelsian Biotechnology for Toxicological Testing and Research).

The principle of the assay is that proliferative stimulation is determined through the detection of CDK. In the G₀ stage of the cell cycle, expression of CDK is minimal, but as cells are stimulated into G₁, the expression of CDK increases.

The standards cd RLHCl–positive control and the cd RLLCl–negative CDK control were used for calibration. The supplier's instructions were followed.

Determination of mRNA CDK
Aortic samples were taken from liquid nitrogen and ground. mRNA extraction was performed by use of the Quick Prep Micro mRNA Purification kit (Pharmacia). Subsequently, mRNA was applied onto 1.4% agarose gel after denaturation with glyoxal and dimethyl sulfoxide according to the method by McMaster and Carmichael¹⁵ and electrophoresed at 3 to 4 V/cm for 2.5 hours in circulating 0.01-mol/L phosphate buffer, pH 7.0. RNA was then transferred to a positively charged nylon membrane (Hybond N+, Dupont, NEF 986) by capillary blotting,¹⁶ fixed with 0.05 N sodium hydroxide for 5 minutes at room temperature, and finally equilibrated at pH 7.0 with three washes in 2xSSC.

cDNA probes for human ß-actin (ATCC 9800) and CDK cdc p34 (ATCC 63161) were used for Northern and dot blots.

For transfection, an aliquot of frozen competent cells (Escherichia coli HB 101) was thawed to 0°C, and 5 µL of the plasmid (5 µg/100 µL) was incubated with the competent cells on ice, followed by incubation for 90 seconds at 42°C. The tubes were returned to the ice bath for 2 minutes. Subsequently, 1 mL of prewarmed (37°C) sodium citrate medium was added and incubated for 60 minutes on a shaker. Then, tubes were centrifuged for 10 minutes at 4500g, and the pellet was resuspended in LB medium at three different dilutions, plated in LB Agar medium plus ampicillin (100 µg/mL), and allowed to grow overnight at 37°C.

A single bacterial colony was transferred to 4 mL LB medium containing ampicillin, and the culture was incubated overnight at 37°C with vigorous shaking. Then, 1 mL of the tube was inoculated in 500 mL of LB medium containing ampicillin prewarmed to 37°C in a 2-L flask; this culture was incubated under vigorous shaking until the OD at 600 nm was 0.4.

For the isolation of plasmid, the Plasmid Maxi kit (Omega 12145), which is based on a modified alkaline lysis procedure, was used, followed by the binding of plasmid DNA to an anion exchange resin under appropriate low-salt and low-pH conditions. Plasmid DNA is eluted by this principle by a high-salt buffer and concentrated by isopropanol precipitation.

For the digestion with restriction enzymes, 3 µg plasmid, 2 µL SuRECut buffers for restriction endonucleases (Boehringer Mannheim cat 1995), 2 µL BamHI, 2 µL EcoRI (Boehringer Mannheim), and 9 µL water were incubated for 3 hours in a thermoblock at 37°C. After the reaction was stopped, the solution was electrophoresed on 1% agarose gel in a 0.15-mol/L Tris borate buffer, pH 8.0, to show the length of fragments. A single band for CDK and ß-actin was shown (Fig 1).

View larger version (126K): [in this window] [in a new window]   Figure 1. Top, Single individual band after hybridization with cDNA CDK p34 cdc, indicating the specificity of the hybridization (lines 1 through 7, in the given order of groups from left to right). The Northern blot of the housekeeping gene, ß-actin, used as a control and for normalization of values calculated on densitometry also showed a single band (not shown). Bottom, Dot blots were used to quantify mRNA CDK. The order of groups of 10 rats (left to right) is (top to bottom) group 1 through 7. Note that group 3, the high-dose homocysteine treatment group, showed the highest density of mRNA.

With QUIAquick gel extraction (QUIAGEN 28704), a method with selective DNA binding properties of a silica gel membrane, the DNA probe was isolated and quantified at 260 nm.

The probe was denatured before labeling by boiling it for 5 minutes, cooling it on ice, and labeling it with fluorescein-12-dUTP with the Renaissance Random Primer Fluorescein-12-dUTP labeling kit (Dupont, NEL 203).

After fixation of bound RNA, the nylon membrane was incubated in prehybridization solution (0.25 mol/L phosphate buffer, pH 7.2, containing 5% SDS [wt/vol], 1 mmol/L EDTA, and 0.5% blocking reagent [from Dupont NEL 203]) for 12 hours at 65°C in a hybridization oven. The blots were hybridized overnight at 65°C with the labeled probes (50 ng/mL prehybridization buffer).

After hybridization, nonspecifically bound material was removed by posthybridization washes with 0.5x and 0.1x prehybridization buffer twice for 10 minutes at 65°C. The 0.5x and 0.1x prehybridization buffer was raised to 65°C before use, and the second wash was performed at room temperature.

Hybridized blots were blocked with 0.5% blocking reagent in 0.1 mol/L Tris HCl, pH 7.2, and 0.15 mol/L NaCl for 1 hour at room temperature. Membranes were then incubated with antifluorescein HRP antibody (Dupont NEL 203) at a 1:1000 dilution in the solution given above for 1 hour under constant shaking.

Membranes were washed four times for 5 minutes in the solution given above.

The Nucleic Acid Chemiluminescence Reagent (Dupont NEL 201) was added to the membranes and incubated for 1 minute. Excess detection reagent was removed by the use of filter paper, and the membrane was placed in Sarawrap paper and exposed to autoradiography Reflection films (Dupont NEF 496) for l5 minutes at room temperature.

Dot blots were performed according to the method of White and Bancroft.¹⁷ This procedure consisted of placing 2 µg total RNA dissolved in 10 µL double-distilled water mixed with 500 µL of 100% formamide, 162 µL of 37% formaldehyde, 100 µL of 10xMOPS buffer. This mixture was incubated for 10 minutes at 65°C and then cooled on ice. Samples were placed onto the membrane by the Manifolds filtration equipment (dot blot apparatus, BIORAD), and the hybridization was performed as described above.

Densitometry of films was performed with the Hirschmann elscript 400 densitometer (Germany).

mRNA CDK values are normalized versus ß-actin (housekeeping gene) levels.

Determination of PKC
For the determination of aortic PKC, the protein kinase C enzyme assay system (Amersham, RPN 77) was used. The system is based on the PKC catalyzed transfer of the -phosphate group of adenosine-5''- triphosphate to a peptide specific for PKC.

Determination of Homocyst(e)ine
Aliquots of aortic tissue were weighed, hydrolyzed, evaporated to dryness, and analyzed by a high-performance liquid chromatography method.^{10 18}

Blood was taken from the retro-orbital plexus, and EDTA plasma was obtained after centrifugation. We used 100 µL for the homocyst(e)ine determination by the principle given above.

Determination of Plasma Markers for Endothelial Injury
The determination of plasma ACE, an in vivo and in vitro marker for endothelial injury, was performed by use of a standard method.¹⁹

The determination of a second marker of endothelial injury, vWF in plasma,^{19 20 21} was performed by use of a commercially available test kit (Boehringer Mannheim).

Incorporation of Bromodeoxyuridine
One hour before the rats were killed, 50 mg/kg body weight 5-bromo-2'-deoxyuridine (Sigma B 5002) dissolved in 5 mL/kg isotonic sodium chloride was injected intravenously (tail vein). The rats were killed by CO₂ treatment, and 5 mm of the thoracic aortic vessel was removed and fixed in 4% paraformaldehyde in isotonic PBS.

Samples were then dehydrated in alcohol (20%-30%-50%-70%-80% at room temperature) and stored in 80% alcohol until further processing. The samples were embedded in paraffin after 96%/100% alcohol, 4xmethylbenzoate (5 minutes each), 3xbenzol (2 minutes each), benzolparaplast saturated at 40°C for 5 minutes, and 4xparaplast (three times at 60 minutes), and 5-µm sections were prepared.

The incorporation of bromodeoxyuridine was demonstrated by a standard immunohistochemical technique on deparaffinized sections²² using the antibody anti-bromodeoxyuridine, monoclonal antibody to bromodeoxyuridine from mouse-mouse hybrid cells, clone BMC 9318, and guidelines from Boehringer Mannheim (1170 376).

Statistical Analysis
Comparison of groups was analyzed by the Kruskal-Wallis test, followed by the Wilcoxon signed rank test. Linear regression analysis was calculated for the pairs. Values of P<.05 were considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Table 1 lists the means and SDs of all parameters of all groups 1 through 7, and Table 2 gives the statistical differences.

View this table: [in this window] [in a new window]   Table 1. Mean±SD of the Parameters Included in the Study

View this table: [in this window] [in a new window]   Table 2. Statistical Differences of the Parameters Between the Groups

In the homocysteine-treated groups, a dose-dependent increase of CDK protein took place; none of the other compounds increased CDK at the protein level.

mRNA CDK normalized versus the housekeeping gene ß-actin (values for ß-actin not shown) was significantly elevated from the homocysteine dose of 50 and 100 mg/kg body weight per day, with a remarkable increase after the high-dose homocysteine administration (Fig 1). mRNA CDK was not elevated in any other study group.

PKC, used as a control to rule out a general effect of homocysteine on phosphokinases, did not differ between all the groups studied.

The parameters for endothelial damage were plasma ACE and vWF; ACE in the low-dose homocysteine group did not differ from that in the untreated control group and was reduced significantly in the groups treated with homocysteine 50 and 100 mg/kg body weight. None of the other groups showed a significant decrease in ACE.

vWF in turn was not statistically different after low-dose homocysteine administration, but the increase was significant from the mean homocysteine dose onward. A moderate but statistically significant increase in vWF was detected in the HCA group.

Plasma and aortic homocyst(e)ine were statistically higher in the homocysteine-treated groups.

ACE and vWF correlated strongly and significantly (P<.0001) with aortic homocyst(e)ine (Fig 2A and 2B). CDK protein correlated strongly with CDK mRNA (P<.0001; Fig 2C), as did mRNA CDK with ACE (P<.0001; Fig 2D) and CDK protein with ACE (P<.0001; Fig 2E). mRNA CDK and CDK protein also showed a strong association with vWF (P<.0001; Fig 2F and 2G). CDK protein and mRNA CDK correlated strongly with aortic homocyst(e)ine (P<.0001; Fig 2H and 2I). The incorporation of bromodeoxyuridine was found in the high-dose homocysteine treatment group only (Fig 3). No nuclear staining for the bromodeoxyuridine incorporation reflecting proliferation was found in all other histological sections.

View larger version (131K): [in this window] [in a new window]   Figure 2. Linear regression analysis of parameters evaluated showing the strong association between the variables CDK, mRNA CDK, aortic homocyst(e)ine, and endothelial damage parameters ACE and vWF.

View larger version (210K): [in this window] [in a new window]   Figure 3. Incorporation of bromodeoxyuridine into cells of the aortic media after high-dose homocysteine (bottom) compared with the aortic vessel wall of an untreated rat (top).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Oral administration of homocysteine led to significantly increased aortic CDK protein levels. This increase paralleled aortic homocyst(e)ine levels in the corresponding experimental rats. At the transcriptional level, increased mRNA CDK was not found in the very-low-dose homocysteine group. This finding would be compatible with the role of homocyst(e)ine in the induction of growth, proliferation, and vessel wall thickening described in hyperhomocyst(e)inemic states and homocystinuria. In 1988, Draetta and coworkers²³ reported on synthesis and distribution of a 34-kD protein in rat tissues and cell lines. They found that increased synthesis of p34 was due to an increase in abundance of p34 mRNA. Their data were consistent with the possibility that p34 played a role in cell division in higher vertebrates. That same year, Labbe and coworkers²⁴ published that in starfish and amphibian oocytes, the activity of a major protein kinase (CDK) increased dramatically at meiotic and mitotic nuclear divisions. Riabowol and coworkers²⁵ showed 1 year later that cdc2 CDK was a component of M phase–promoting factor in xenopus oocytes and that the homologous kinase in human hela cells was maximally active during mitosis. McGowan and coworkers²⁶ showed that the activity of that kinase was regulated in a cell cycle–dependent manner by reversible phosphorylation and through association with other proteins. A further characterization of CDKs in the human system was shown by Lucas and coworkers.²⁷ Further elucidation of the system was provided by Dutta and Stillman,²⁸ who showed that cdc2 family kinases phosphorylate a human cell DNA replicating factor, RPA, and activate DNA replication.

Although the signal for proliferation as reflected by increased CDK protein was found in all homocysteine-treated groups, endothelial damage followed the transcriptional pattern: the very low dose did not alter ACE and vWF, and transcription of CDK also was not found to be increased at low doses.

In this model, the question of whether moderately elevated homocyst(e)ine levels seen in patients with mild hyperhomocyst(e)inemia would increase CDK in the human system cannot be answered. Furthermore, proliferation as evaluated by bromodeoxyuridine incorporation revealed positive results in the aortic vessel wall only in the group treated with high-dose homocysteine. To answer this question, studies on human vascular tissues are needed, and we provide here the basis for such a protocol.

Feeding of HCA, a metabolite of homocysteine, failed to increase CDK levels, but because vWF was moderately but significantly increased, this compound may show some potential for inducing endothelial injury per se, but not by a CDK-mediated mechanism.

Increased CDK by thiols as homocyst(e)ine could have been expected. Reports from the late 1950s showed that thiols do play a role in growth promotion. Stern²⁹ published that thiol groups are important for cell division and referred to the observations that the nitroprusside reaction was high in proliferating tissues and that thiol blocking agents inhibit cell division. Stern also clearly described the coincidental increase in thiols with mitosis in the lily longiflorum.

Mazia³⁰ published data related to cell division and thiols supporting Stern's observations.

Our data point to a more specific mechanism because cysteine did not influence CDK in our present study and because clonogenic assays performed in our laboratory neither increased CDK nor promoted growth.¹²

In the present study, we have measured CDK protein and mRNA, which means that the increase of CDK was mediated by homocyst(e)ine at the transcriptional level, a phenomenon shown to occur for increased transcription of collagen in hyperhomocyst(e)inemia.³¹

The homocyst(e)ine effect seems to be specific because another kinase, PKC, was not stimulated. The possibility that the reducing effect of homocysteine was responsible for the increase in CDK was ruled out by the observation that ascorbic acid failed to increase CDK at the protein or transcriptional level. The strong association between CDK and aortic homocyst(e)ine fit well into the current concept that homocyst(e)ine directly stimulates growth in vivo.

	Selected Abbreviations and Acronyms

 

CDK = cyclin-dependent kinase HCA = homocysteic acid PKC = phosphokinase C vWF = von Willebrand factor

	Acknowledgments

 
We are highly indebted to the Red Bull Co, Salzburg, Austria, for generous financial assistance and to Prof Dr Gert Lubec, CChem, FRSC (UK), for scientific support and advice.

Received June 27, 1996; revision received August 15, 1996; accepted August 16, 1996.

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