Identification of a Novel Redox-Sensitive Gene, Id3, Which Mediates Angiotensin II–Induced Cell Growth
Background— Reactive oxygen species, such as superoxide (O2−), are involved in the abnormal growth of various cell types. Angiotensin II (Ang II) is one of the most potent inducers of oxidative stress in the vasculature. The molecular events involved in Ang II–induced proliferation of vascular smooth muscle cells (VSMCs) are only partially understood.
Methods and Results— Ang II as well as xanthine/xanthine oxidase (X/XO) led to enhanced DNA synthesis and proliferation of VSMCs. The effect of Ang II was abolished by diphenylene iodonium. Consequently, VSMCs were incubated with X/XO, and modulation of gene expression was monitored by differential display, leading to the identification of a novel redox-sensitive gene, the dominant-negative helix-loop-helix protein Id3, which was upregulated within 30 minutes by X/XO and Ang II. Superoxide dismutase but not catalase inhibited this effect. Overexpression of antisense Id3 via transfection in VSMCs completely abolished Ang II– and X/XO-induced cell proliferation. Ang II, X/XO, and overexpression of sense Id3 downregulated protein expression of p21WAF1/Cip1, p27Kip1, and p53. Overexpression of antisense Id3 abrogated the effect of Ang II on the expression of p21WAF1/Cip1, p27Kip1, and p53. Ang II and overexpression of sense Id3 caused hyperphosphorylation of the retinoblastoma protein. Ang II–induced phosphorylation of the retinoblastoma protein was decreased by overexpression of antisense Id3.
Conclusions— Ang II induces proliferation of VSMCs via production of superoxide, which enhances the expression of Id3. Id3 governs the downstream mitogenic processing via depression of p21WAF1/Cip1, p27Kip1, and p53. These findings reveal a novel redox-sensitive pathway involved in growth control.
Received July 26, 2001; revision received March 1, 2002; accepted March 1, 2002.
Proliferation of vascular smooth muscle cells (VSMCs) is a key event of atherogenesis and coronary restenosis.1,2⇓ Growth of VSMCs is induced by various agonists such as growth factors (eg, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor), cytokines, and hormones.1–4⇓⇓⇓ Angiotensin II (Ang II) causes cell growth via activation of AT1 receptors.3,5⇓ It shares with growth factors numerous mitogenic signaling pathways, including, eg, tyrosine phosphorylation, mitogen-activated protein (MAP) kinase activation, stimulation of the Janus kinase/signal transducers and activators of transcription system, and induction of proto-oncogenes.3,5⇓ Whether Ang II exerts predominantly hypertrophic or also hyperplastic effects on VSMCs, however, is the subject of ongoing controversy.5–11⇓⇓⇓⇓⇓⇓ In this regard, the influence of Ang II on factors involved in cell cycle progression have not been completely understood.
Reactive oxygen species (ROS) are thought to mediate the mitogenic effects of Ang II via activation of, eg, MAP kinases.6 ROS, such as superoxide (O2−), are involved in a multiplicity of pathological settings, such as inflammation, cancer development, and vascular lesions.12–16⇓⇓⇓⇓ ROS exert direct cell damage and mitogenicity, serve as intracellular second messengers, and scavenge vasoprotective nitric oxide.17–20⇓⇓⇓ The induction of cell growth in tumor cells and VSMCs by ROS is of fundamental relevance. O2− induces a wide array of second messengers typical of mitogens.17–20⇓⇓⇓ Proliferation, growth arrest, and apoptosis are key steps in various states of atherogenesis. Therefore, it is crucial to dissect the molecular events that govern ROS-induced regulation of proliferation.
Thus, we explored the effects of Ang II and X/XO on VSMC growth, investigated superoxide-elicited modulations of gene expression in VSMCs with the differential display method, and characterized the role of a newly identified gene in Ang II– and superoxide-caused cell proliferation.
VSMCs were isolated from rat thoracic aorta (male Sprague-Dawley rats, 6 to 10 weeks old, Charles River Wega GmbH, Sulzfeld, Germany) by enzymatic dispersion and cultured over several passages. Cells were grown in a 5% CO2 atmosphere at 37°C in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 1% nonessential amino acids (100×), and 10% FCS. Experiments were performed with cells from passages 5 to 15. For each experiment, cells were serum deprived 24 hours before the indicated treatment.
Assessment of Cell Proliferation
The rate of cell proliferation was determined with a cell proliferation ELISA (Roche Molecular Biochemicals) via incorporation of bromodeoxyuridine (BrdU) into newly synthesized DNA according to the manufacturer’s protocol. Briefly, VSMCs were plated on microtiter plates at a density of 104 cells per well. After 12 hours, cells were serum starved for 24 hours and treated with ROS for 24 hours. BrdU was added at a final concentration of 10 μmol/L, and cells were reincubated for an additional 12 hours at 37°C. Cells were fixed with fixation solution for 30 minutes at room temperature and incubated with 100 μL anti-BrdU peroxidase-labeled antibody for 90 minutes. After 3 washing steps with the washing solution provided, the substrate solution for the colorimetric quantification was added at a final concentration of 100 μL/mL and left at room temperature for 5 to 30 minutes until color development was sufficient for photometric detection.
For cell counting, VSMCs were serum-starved for 24 hours and treated as indicated. Cells were removed from the tissue culture dish by addition of trypsin, pelleted, resuspended in 1 mL of DMEM, and counted in a Neubauer chamber.
Isolation of RNA and Differential Display
Total RNA from ROS-treated and control cells was isolated by use of PEQGold RNAPure (PeqLab) according to the manufacturer’s protocol. To eliminate false-positive results generated from genomic DNA, the RNA was treated with 10 U RNase-free DNase I (Roche Molecular Biochemicals) for 30 minutes at 37°C and extracted with phenol/chloroform. Differential display of mRNA was carried out with the RNAimage mRNA differential display system (GenHunter Corp). Purified total RNA (2 μg) was reverse transcribed by use of 1 of the 3 provided H-T11-M primers (M = A, T, or G), and cDNA representing 200 ng of RNA was submitted to differential display polymerase chain reaction (PCR) by use of the respective H-T11-M primer and 1 of 8 arbitrary primers provided, 1 U of AmpliTaq DNA polymerase (Perkin-Elmer Corp), and 2 μCi [α-33P]dATP (ICN Biomedicals) per reaction. PCR was carried out with 40 cycles of 94°C for 30 seconds, 42°C for 2 minutes, and 72°C for 30 seconds, followed by a final extension step at 72°C for 5 minutes. PCR products were resolved on a 6% denaturing polyacrylamide gel in 1× Tris-borate-EDTA buffer followed by autoradiography. Reproducibly differentially expressed bands were cut out of the gel, and the cDNA was eluted by boiling for 15 minutes. The gel debris was pelleted by centrifugation, and the supernatant was transferred to a fresh microcentrifuge tube. The DNA was then precipitated by addition of 100% ethanol, incubation on dry ice for 30 minutes, and centrifugation at 4°C in an Eppendorf microcentrifuge. The pellet was washed with 85% vol/vol ethanol, dried, and resuspended in deionized distilled H2O. Reamplification of the purified cDNA was performed with the same primer set and PCR conditions as used for the differential display PCR. Reamplified cDNAs were either directly sequenced with the respective arbitrary primer or after cloning into the pCR2.2-vector (Invitrogen BV) via the thymidine-adenosine (TA) cloning method.
Northern Blot and Generation of Radiolabeled Probes
Total RNA (15 μg) was electrophoresed on a 1.2% agarose/0.67% formaldehyde gel. After electrophoresis, RNA was transferred onto Hybond N nylon membrane (Amersham Pharmacia Biotech). PCR fragments of 897 bp (Id3) and 1456 bp (GKLF) were radiolabeled with [α-32P]dCTP (ICN Biomedicals) by use of the Prime-It II random-primer labeling kit (Stratagene). Membranes were prehybridized in a solution containing 50% formamide, 6× SSC, 0.5% SDS, 5× Denhardt’s solution, and 100 μg/mL salmon testes DNA (Sigma-Aldrich GmbH) for ≥30 minutes at 42°C. Hybridization was carried out in hybridization solution containing 50% formamide, 6× SSC, 0.5% SDS, 100 μg/mL salmon testes DNA, and the denatured radiolabeled probe in an overnight incubation at 42°C. Membranes were washed twice with 2× SSC and 2 to 4 times with 2× SSC/0.1% SDS at 50°C to 65°C, sealed in a plastic bag, and submitted to autoradiography.
Semiquantitative Reverse Transcription–PCR
Total RNA (2 μg) from ROS-treated and control cells were reverse transcribed by use of 100 pmol p(dN6) oligonucleotide primer (Roche Molecular Biochemicals) and 200 U of Moloney murine leukemia virus reverse transcriptase (Gibco BRL Life Technologies) in the supplied buffer for reverse transcription and 10 U RNasin (Promega). For PCR, 1 μL of each reaction was used with 50 pmol each of the respective primers for amplification of Id3 (sense, 5′-CGACATGAACCACTGCTACTC-3′; antisense, 5′-GGTCAGTGGCAAAAACTCCTC-3′) in Red Taq PCR buffer, dNTP mix, and 1.25 U of Red Taq DNA polymerase (Sigma-Aldrich GmbH). PCR conditions were one 5-minute cycle of 95°C, followed by 25 cycles (GAPDH, 23 cycles) of 94°C for 30 seconds, 57°C (Id3) or 60°C (GAPDH) for 45 seconds, 72°C for 45 seconds, and a final extension for 10 minutes at 72°C. Twenty to 40 μL of each PCR reaction was analyzed on a 1% agarose Tris-acetate-EDTA gel and visualized by ethidium bromide staining, and optical densities of the cDNA bands were quantified. The same cDNA samples were used for the amplification of a 452-nt fragment of GAPDH (sense, 5′-ACCACAGTCCATGCCATCAC-3′; antisense, 5′-TCCACCA-CCCTGTTGCTGTA-3′) to confirm that equal amounts of RNA were reverse transcribed.
ROS-treated and control cells were washed twice with ice-cold PBS, scraped in 1 mL of ice-cold lysis buffer (100 mmol/L Tris, pH 6.8, 4% SDS, 20% glycerol, 0.1 mmol/L PMSF, 1 μg/mL leupeptin, 1 μg/mL aprotinin), heated to 95°C for 5 minutes, and stored at −20°C until use. Aliquots (40 μg) of the cell lysate were electrophoresed through a 0.1% SDS/10% polyacrylamide gel. Proteins were blotted to nitrocellulose membranes in a semidry blotting chamber (Pharmacia Biotech). Blot membranes were stained with Ponceau red to verify appropriate protein transfer and equal loading for each lane. Immunoblotting was performed overnight at 4°C. Antibody dilutions were p53 (Pab 240 mouse monoclonal IgG, sc-99, Santa Cruz Biotechnology Inc) 1:300, p27 (F-8 mouse monoclonal IgG, sc-1641, Santa Cruz) 1:300, p21 (mouse mixed monoclonal IgG, No. 05-345, Upstate Biotechnology, Biomol Hamburg) 1:600, Rb (M-153 rabbit polyclonal, sc-7905, Santa Cruz) 1:100, and Id3 (C-20 rabbit polyclonal, sc-490, Santa Cruz) 1:100. Immunodetection was done with the appropriate secondary antibody for 1 hour at room temperature (1:20 000 dilution, Sigma Chemical) and the enhanced chemiluminescence kit (Amersham). Autoradiography was performed at room temperature.
Plasmid Construction and Electroporation of VSMCs
Full-length cDNAs of Id3 were generated by PCR amplification of reverse-transcribed RNA derived from VSMCs (see above). Primers for Id3 were sense, 5′-CTCCAACCTCCAACATGAAGG-3′; antisense, 5′-GTTCAAAAATGGTTTATTATGCAAAATGTT-3′. PCR products were checked on a 1% Tris-acetate-EDTA agarose gel and cloned into the pCR2.2 vector (Invitrogen BV) via TA cloning. The orientation and validity of the insert were determined by automated sequencing, and Id3 sense and antisense constructs for electroporation were generated by cloning 912-bp BamHI/EcoRV fragments (NM_013058) into the pcDNA3 vector. For electroporation, VSMCs grown at a confluent monolayer were removed from the culture dish by addition of trypsin and pelleted. The pellet was resuspended in 200 μL of Optimem I (Gibco BRL Life Technologies), and cells were counted. For each sample, 106 cells were incubated with 20 μg of the respective DNA in precooled cuvettes (Promega) for 30 minutes on ice. After the cuvette had been warmed to 37°C for 30 seconds in a water bath, electroporation was performed for 16 ms at 0.3 kV and 500 μF. After an additional incubation for 30 minutes at room temperature, cells were plated on the appropriate culture dishes or microtiter plates.
The rate of apoptosis in Id3-transfected cells was assessed with the cell death detection ELISAPLUS System (Molecular Biochemicals). The test principle is based on the determination of the amount of nucleosomes generated during the apoptotic fragmentation of cellular DNA. Cells were scraped and collected by centrifugation for 5 minutes at 1500 rpm (Heraeus Megafuge 1.0) and washed in 1 mL DMEM. The pellet was then resuspended in 0.5 mL incubation buffer and left at 4°C for 30 minutes; after centrifugation for 10 minutes at 15 000 rpm and 4°C in a microcentrifuge, 200 μL of supernatant was diluted in 1.8 mL of incubation buffer. From each sample, 100 μL was incubated in anti-histone–coated microtiter-plate wells for 90 minutes, the wells were washed 3 times with incubation buffer, and 100 μL of anti–DNA-peroxidase–linked antibody was added, followed by further incubation for 90 minutes. After 3 washing steps with incubation buffer, 100 μL of ABTS substrate solution for the peroxidase was added, and after 10 to 20 minutes, the rate of apoptosis was determined by photometric measurement at 492 nm.
Cultured rat VSMCs were incubated with 1 μmol/L Ang II with or without 30 minutes of pretreatment with 100 μmol/L diphenylene iodonium (DPI) and with xanthine 100 μmol/L, xanthine oxidase 1.6×10−3 U/mL (X/XO) to induce production of O2−. After a 24-hour incubation, DNA synthesis and cell proliferation were assessed. Figure 1, A and B, shows that Ang II caused a significant increase in DNA synthesis and cell proliferation that was inhibited by pretreatment with DPI. Consequently, X/XO also caused a significant increase in DNA synthesis and cell proliferation. Both Ang II–induced (maximal at 1 μmol/L) and X/XO-induced (maximal at 100 μmol/L) mitogenicities were concentration dependent (data not shown). These data suggest that Ang II exerts potent mitogenic effects via release of ROS.
To clarify the underlying molecular mechanisms, VSMCs were incubated with 100 μmol/L X/XO for 0, 0.5, 2, and 4 hours before total RNA was extracted and digested with DNase I. Gene expression modulation was monitored by the differential display technique with various supplied primer combinations. Several cDNA amplification products were differentially regulated by X/XO. Isolation, purification, subcloning, and automated sequencing led to the identification of the dominant-negative helix-loop-helix protein Id3. The full-length rat Id3 cDNA was cloned by PCR and subcloned in pcDNA3, and nucleotide composition was checked by automated sequencing. For confirmation of the differential display results, cells were incubated for the indicated time points with either 1 μmol/L Ang II or 100 μmol/L X/XO, RNA was isolated, and semiquantitative PCR and Northern blotting were performed. Id3 is profoundly upregulated within 0.5 hours by both Ang II and X/XO (Figure 2, A and B). This effect is concentration dependent, with a maximum at 1 μmol/L Ang II and 100 μmol/L X/XO (data not shown). Id3 protein expression is comparably upregulated by Ang II and X/XO, as assessed by Western blots (Figure 2, C and D). In addition, it was shown that a 30-minute pretreatment of VSMCs with DPI inhibits the Ang II–caused upregulation of Id3. In addition, cells were incubated with 1 μmol/L Ang II and 100 μmol/L X/XO in the presence of 200 U/L catalase or superoxide dismutase. Thirty minutes later, Id3 mRNA expression was assessed by Northern blots. Figure 2E reveals that superoxide dismutase rather than catalase inhibits the effects of Ang II and X/XO on Id3 expression. These findings demonstrate the identification of a novel redox-sensitive gene, Id3, which is induced in VSMCs on stimulation with Ang II via production of superoxide radicals.
Ang II and O2− both cause proliferation of VSMCs. Id3 is a dominant-negative helix-loop-helix protein expressed in various cell types. To characterize the role of Id3 in Ang II–induced proliferation of VSMC growth, full-length sense and antisense Id3 cDNAs inserted into the expression vector pcDNA3 were transfected into VSMCs via electroporation. By this technology, transfection efficiencies of ≈30% were ensured. Control experiments showed reduced and enhanced expression of Id3 after the respective transfection (Figure 3A). Transfected cells were stimulated with vehicle, 1 μmol/L Ang II with or without 30 minutes of pretreatment with 20 μmol/L DPI, and 100 μmol/L X/XO. Figure 3B demonstrates that Ang II–induced DNA synthesis was abrogated by overexpression of antisense Id3. Overexpression of sense Id3 slightly increased the basal DNA synthesis. Consistently, superoxide-induced VSMC mitogenicity was diminished by antisense Id3 overexpression, as depicted in Figure 3C. Control experiments show that other genes, such as 18S rRNA or the AT1 receptor mRNA, were not influenced by either sense or antisense Id3, suggesting a specific effect of Id3 on the cell growth machinery (data not shown). Therefore, Ang II–driven superoxide release may cause VSMC proliferation via induction of Id3. To test whether the Id3 antisense effects were mediated via an increased rate of cell death, after transfection with the insertless vector, antisense Id3, and sense Id3, apoptosis was quantified in VSMCs. Figure 3D demonstrates that sense Id3 but not antisense Id3 induces increased apoptosis events in VSMCs.
Cyclin-dependent kinase (Cdk) inhibitors (CKIs) and the tumor suppressor protein p53 are essential regulators of growth. To determine whether Ang II– and superoxide-induced VSMC proliferation is associated with the modulation of CKIs, VSMCs were stimulated with either vehicle, 1 μmol/L Ang II, or 100 μmol/L X/XO before cellular proteins were isolated and Western blots were performed to quantify protein expression of p21WAF1/Cip1, p27Kip1, and p53. Figure 4, A and B, shows that Ang II and X/XO depress the expression of p21WAF1/Cip1, p27Kip1, and p53, suggesting that the decreased expression of p53 and Cdks is a prerequisite to the observed VSMC proliferation.
Next, the role of Id3 was defined in the Ang II– and superoxide-induced regulation of Cdks and p53. Cells were transfected with sense and antisense Id3, followed by stimulation with 1 μmol/L Ang II. Figure 5 shows that overexpression of antisense Id3 inhibited Ang II–caused downregulation of p21WAF1/Cip1, p27Kip1, and p53, indicating that induction of Id3 by Ang II or superoxide leads causally to the depression of p53 and the Cdks p21WAF1/Cip1 and p27Kip1.
The retinoblastoma gene product (Rb) is hyperphosphorylated in VSMCs stimulated with 1 μmol/L Ang II (Figure 6). Ang II–induced Rb hyperphosphorylation was diminished by overexpression of antisense Id3, suggesting that Ang II induces Rb through induction of Id3.
Cardiovascular complications of atherosclerosis, such as myocardial infarction and stroke, are the primary causes of death in the Western world.21,22⇓ Atherosclerosis represents a vascular process characterized by, among other things, a pathogenic growth of VSMCs.1,2⇓ Proliferation of VSMCs has been extensively investigated within the last few years, and the following events have been identified as essential. Agonists such as growth factors or hormones stimulate their receptors and activate a multiplicity of intracellular pathways, such as various protein kinases, MAP kinases, and proto-oncogenes, which ultimately accelerate the proliferation of VSMCs.3,5⇓ Downstream of these early events, Cdks, CKIs, cofactors, and target genes and proteins are of great relevance for the cell-cycle progress that precedes enhanced cell growth.23–25⇓⇓ CKIs such as p21WAF1/Cip1 and p27Kip1, the tumor suppressor protein p53, and Rb are engaged in the tight control of cell growth.25,26⇓ It has been shown in various cell types that depression of CKIs and p53 expression and the hyperphosphorylation of Rb are sufficient to induce proliferation.25,26⇓
Ang II mediates a wide array of biological effects, including the enhanced production of ROS and cell proliferation.3,5⇓ Several interventional studies, above all the Heart Outcomes Prevention Evaluation (HOPE) Study, have clearly demonstrated that Ang II plays a decisive role in atherogenesis.27 There is a great body of evidence that these growth-stimulating features of Ang II are responsible for the impact of Ang II on the pathogenesis of atherosclerosis,1–3,20⇓⇓⇓ yet it has not been clearly shown whether Ang II leads to hyperplasia rather than to hypertrophy of VSMCs.5,7–11⇓⇓⇓⇓⇓ Moreover, the intracellular pathways governing these mitogenic processes are poorly defined. It is well established that Ang II causes early cellular events, such as stimulation of phospholipase C, protein kinase C, MAP kinases, and proto-oncogenes, which are typical for growth-promoting agonists.26,29⇓ The subsequent cellular steps are less clear, especially with regard to cell cycle progression.
The data presented show that Ang II induces the production of superoxide, which mediates proliferation of VSMCs. This is in good agreement with previous reports demonstrating that free radicals are essential for the mitogenic effects of Ang II.28,30⇓ It was recently reported that Ang II could not depress p27Kip1 but was capable of stimulating Cdk2 activity in VSMCs.8 In renal proximal tubular cells, Ang II induced the expression of p27Kip1.29 A different study was not able to show an effect of Ang II on Rb, Cdks, or p27Kip1,9 whereas another research group detected an increase of Cdk activity and Rb phosphorylation after Ang II stimulation.7 Neither the cellular or molecular events that could explain these conflicting results nor the detailed mechanisms involved in Ang II–evoked growth are known. Here, we report not only that Ang II induces redox-mediated cell proliferation but also that Ang II and superoxide profoundly depress growth-inhibiting factors, such as p53, p21WAF1/Cip1, and p27Kip1 and cause hyperphosphorylation of Rb, which allows binding of the growth-inhibiting factor E2F.
This is governed by the induction of Id3, as elucidated with the differential display gene-hunting method. Id3 antagonizes the function of DNA-binding basic helix-loop-helix transcription factors, such as E2A, leading to decreased cell differentiation and increased proliferation.31 In addition, Id3 has been implicated in the apoptosis of fibroblasts.32 The helix-loop-helix transcription factor E2A, which is scavenged by Id3, stimulates the transcription of p21WAF1/Cip1, suggesting that induction of Id3 could lead to decreased expression of p21WAF1/Cip1 accompanied by proliferation. This is consistent with our data, which demonstrate that induction of Id3 is a prerequisite to Ang II– and superoxide-evoked depression of p21WAF1/Cip1. Moreover, Id3 influences p27Kip1, p53, and Rb in a similar manner, suggesting that Id3 resides in a central position upstream of Cdks, CKIs, p53, and Rb, which ascribes to Id3 a crucial significance for growth control in VSMCs.
The results described have several implications. Ang II causes VSMC growth through superoxide production. This results in depression of the CKIs p21WAF1/Cip1 and p27Kip1, as well as the tumor suppressor gene product p53. Consequently, Rb is hyperphosphorylated. These events are specifically mediated through Id3. The data not only significantly advance our understanding of growth processes in general, but the gene-hunting approach also provided us with a newly described redox-sensitive gene that could be used for the forced induction of proliferation arrest of VSMCs. This ultimately could be implemented in novel treatment strategies for atherosclerosis and restenosis.
This work was supported by the Deutsche Forschungsgemeinschaft and by the Deutsche Herzstiftung.
↵*The first 2 authors contributed equally to this work.
- ↵Badimon JJ, Fuster V, Chesebro JH, et al. Coronary atherosclerosis: a multifactorial disease. Circulation. 1993; 87: 113–116.
- ↵Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. N Engl J Med. 1993; 362: 801–809.
- ↵Berk BC. Angiotensin II signal transduction in vascular smooth muscle: pathways activated by specific tyrosine kinases. J Am Soc Nephrol. 1999; 10: 62–68.
- ↵Pratt RE. Angiotensin II and the control of cardiovascular structures. J Am Soc Nephrol. 1999; 10: 120–128.
- ↵Griendling KK, Ushio-Fukai M, Lassegue B, et al. Angiotensin II signaling in vascular smooth muscle. Hypertension. 1997; 29: 366–373.
- ↵Berk BC. Angiotensin II signal transduction in vascular smooth muscle: pathways activated by specific tyrosine kinases. J Am Soc Nephrol. 1999; 10 (suppl 11): S62–S68.
- ↵Sadoshima J, Aoki H, Izumo S. Angiotensin II and serum differentially regulate expression of cyclins, activity of cyclin-dependent kinases, and phosphorylation of retinoblastoma gene product in neonatal cardiac myocytes. Circ Res. 1997; 80: 228–241.
- ↵Servant MG, Coulombe P, Turgeon B, et al. Differential regulation of p27 (Kip1) expression by mitogenic and hypertrophic factors: involvement of transcriptional and posttranscriptional mechanisms. J Cell Biol. 2000; 148: 543–556.
- ↵Yamakawa T, Tanaka S, Numaguchi K, et al. Involvement of Rho-kinase in angiotensin II–induced hypertrophy of rat vascular smooth muscle cells. Hypertension. 2000; 35: 313–318.
- ↵Grisham MB, Jourd’heuil D, Wink DA. Review article: chronic inflammation and reactive oxygen and nitrogen metabolism: implications in DNA damage and mutagenesis. Aliment Pharmacol Ther. 2000; 14 (suppl 1): 3–9.
- ↵Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis. 2000; 21: 361–370.
- ↵Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
- ↵Irani K. Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res. 2000; 87: 179–183.
- ↵WHO-MONICA Project. Myocardial infarction and coronary deaths in the World Health Organization Project: registration procedures, event rates, and case-fatality rates in 38 populations from 21 countries in four continents. Circulation. 1994; 90: 583–612.
- ↵Tanner FC, Boehm M, Akyurek LM, et al. Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation. Circulation. 2000; 101: 2022–2025.
- ↵Weiss RH, Joo A, Randour C. p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J Biol Chem. 2000; 275: 10285–10290.
- ↵Rao GN, Berk BC. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ Res. 1992; 70: 593–599.
- ↵Hannken TR, Schroeder G, Zahner RA, et al. Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells. J Am Soc Nephrol. 2000; 11: 1387–1397.
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- ↵Prabhu S, Ignatova A, Park ST, et al. Regulation of the expression of cyclin dependent kinase inhibitor p21 by E2A and Id proteins. Mol Cell Biol. 1997; 17: 5888–5896.
- ↵Norton JD, Atherton GT. Coupling of cell growth control and apoptosis functions of Id proteins. Mol Cell Biol. 1998; 18: 2371–2381.