Central Role of PKCβ in Neointimal Expansion Triggered by Acute Arterial Injury
We tested the hypothesis that PKCβ contributes to vascular smooth muscle cell (SMC) migration and proliferation; processes central to the pathogenesis of restenosis consequent to vascular injury. Homozygous PKCβ null (−/−) mice or wild-type mice fed the PKCβ inhibitor, ruboxistaurin, displayed significantly decreased neointimal expansion in response to acute femoral artery endothelial denudation injury compared with controls. In vivo and in vitro analyses demonstrated that PKCβII is critically linked to SMC activation, at least in part via regulation of ERK1/2 MAP kinase and early growth response-1. These data highlight novel roles for PKCβ in the SMC response to acute arterial injury and suggest that blockade of PKCβ may represent a therapeutic strategy to limit restenosis.
Key evidence for the inducibility of early growth response-1 (Egr-1) in acute arterial injury first emerged from studies in the rat aorta. Egr-1 and a number of its target genes were induced at the wound margins.1–2 Application of a DNA enzyme that specifically cleaves Egr-1 mRNA in rats and pigs subjected to arterial injury attenuated neointima formation.3–4 However, these studies did not elucidate the pathway by which Egr-1 was upregulated after vascular injury, nor did they delineate the downstream implications of this molecule in vascular repair.
Previous studies provided insights into a mechanism by which Egr-1 was upregulated by activation of PKCβ, particularly the β II isoform, in acute hypoxic stress.5–6 Hypoxia induces rapid activation of PKCβII, leading to activation of Raf, mitogen-activated protein kinase/extracellular signal regulated protein kinase kinase and mitogen-activated protein kinases7: events central to PKCβ-induced transcriptional regulation of Egr-1. In a murine model of single lung ischemia/reperfusion (I/R), PKCβ null (−/−) mice displayed enhanced protection against the adverse effects of I/R compared with Egr-1−/− or wild-type mice.8
Increased activation of the diacylglycerol (DAG)-PKCβ signal transduction pathway has been identified in vascular tissues retrieved from human subjects and animals with experimental diabetes and in vascular cells incubated in elevated concentrations of glucose.9 Administration of the PKCβ inhibitor ruboxistaurin (LY333531) to animals with diabetes resulted in significant improvement in microvascular complications in key target organs of diabetes, the retina, kidney, nerve, and heart.10–12 In this study, we tested the hypothesis that PKCβII centrally modulated the response to chronic vascular stress, even in euglycemia. We performed femoral arterial endothelial denudation in mice and show for the first time that PKCβII is critically linked to smooth muscle cell (SMC) activation and pathological expansion of the neointima triggered by acute arterial injury in euglycemia, at least in part, via activation of ERK1/2 MAP kinases and regulation of Egr-1.
Materials and Methods
Animals and Induction of Vascular Injury
All procedures were approved by the IACUC at Columbia University. Male C57BL/6 mice (age, 8 to 12 weeks; Jackson Laboratories, Bar Harbor, Maine) were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The femoral artery injury was performed as published.13 Egr-1−/− mice,14 a generous gift of Dr Jeffrey Milbrandt (Washington University School of Medicine, St. Louis, Mo), and PKCβ−/− mice,15 were backcrossed 10 generations into C57BL/6. Littermates were used as controls.
C57BL/6 mice were fed PKCβ inhibitor ruboxistaurin-containing chow from 3 days before injury to day 7 or 28 after injury. Ruboxistaurin was generously supplied by Dr Louis Vignati (Eli Lilly & Company; Indianapolis, Ind). For BrdU labeling, mice received two intraperitoneal injections of BrdU (2.5 mg/injection; Sigma-Aldrich), 12 hours and 1 hour before euthanasia. Tissues were fixed and processed as described later.
Harvesting of vessel segments was performed as published.13 The section (5 μm) at the midportion of each femoral artery was treated with Van Giesson staining kit (Sigma-Aldrich), and the degree of intimal thickening was analyzed quantitatively using a Zeiss microscope and image analysis system (Media Cybernetics Inc). Three types of measurements were made, including luminal area, the area encircled by the internal elastic lamina (IEL) and the area encircled by the external elastic lamina (EEL). All measurements were performed by one of the investigators blinded to the experimental protocol. Intimal area was calculated by subtracting the area encircled by IEL from that encircled by the EEL.
Representative sections were stained with monoclonal anti-smooth muscle actin IgG (1:100, Sigma-Aldrich). Sections were deparaffinized and blocked with hydrogen peroxide (3%) in methanol for 10 minutes. For BrdU staining, slides were immersed in citrate buffer (0.01 mol/L, pH 6) and microwaved twice for 5 minutes. Blocking was performed with goat serum (4%) and bovine serum albumin (1%) in PBS. Primary antibodies were added to slides and incubated overnight at 4°C. They were incubated with secondary affinity-purified peroxidase-conjugated goat anti-mouse IgG (Sigma).
SMC replication in the media and intima of arterial segments was evaluated by staining sections with rat monoclonal anti-BrdU IgG (1:100; Sigma). Numbers of stained and total nuclei were counted, and BrdU labeling index was calculated (BrdU-labeled nuclei/total nuclei×100%).
Analysis of Myeloperoxidase Activity
Two femoral vessel segments per mouse were pooled and homogenized in hexadecyltrimethylammonium bromide followed by three freeze/thaw cycles to release myeloperoxidase from leukocyte granules. Myeloperoxidase activity was measured as described.16
Femoral artery segments were snap frozen in liquid nitrogen, pooled, and stored at −80°C. Cytosolic and membrane proteins were prepared from six pooled arteries as described.17 To isolate total protein extracts, two pooled arteries, or cultured cells, were homogenized and incubated in ice-cold lysis buffer (Cell Signaling Technology). Lysate protein concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories). Equal amounts of protein were subjected to SDS-PAGE (7.5% or 12%) followed by electrophoretic transfer to nitrocellulose membranes. Nonspecific binding was blocked by incubation of membranes with nonfat dry milk or BSA (5%) for 1 hour at RT or overnight at 4°C. Blots were incubated with anti-PKCβI IgG, anti-PKCβII IgG, anti-PKCα IgG, anti-PKCδ IgG, or anti-PKCε IgG; and anti–phospho-ERK1/2 IgG, anti–total-ERK1/2 IgG, anti–phospho-JNK IgG, or anti–total-JNK IgG (Cell Signaling Technology); and anti–phospho-Jak2 IgG (Affinity BioReagents Inc), anti-Jak2 IgG (Santa Cruz Biotechnology Inc), anti–phospho-Stat3 IgG (Cell Signaling Technology), or anti-Stat3 IgG (Santa Cruz Biotechnology Inc); respectively, each at a dilution of 1:1000 for 1 to 3 hours or overnight according to the manufacturer’s instructions. HRP-conjugated donkey anti-rabbit IgG secondary antibody (1:1000, Amersham Biosciences) was used to identify sites of binding of primary antibody.
RNA Extraction and Real-Time PCR
Femoral artery segments were snap frozen in liquid nitrogen. Total RNA was extracted from at least four vessel segments using Trizol reagent (Life Technologies Inc). Total RNA (1 μg) was processed directly to cDNA synthesis using the TaqMan Reverse Transcription Reagents kit (Applied Biosystem) according to the manufacturer’s protocol. All PCR primers and TaqMan probes were designed using software PrimerExpress (Applied Biosystem) and published sequence data from the NCBI database. The sequences of forward and backward primer for mouse Egr-1 or β-actin are 5′-GCCTCGTGAGCATGACCAAT-3′ and 5′-GCAGAGGAAGACGATGAAGCA-3′; 5′CCTGAGCGCAAGTACTCTGTGT-3′ and 5′-GCTGATCCACATCTGCTGGAA-3′; respectively. The sequence of TaqMan probes for mouse Egr-1 or β-actin is 5′-CTCCGACCTCTTCATCCTCGGCG-3′ or 5′-CGGTGGCTCCATCTTGGCCTCAC-3′. Primers were synthesized, and TaqMan probes for mouse Egr-1 or β-actin were labeled with the reporter dye 6FAM or VIC in the 5′ end, and quencher dye TAMRA in both of the 3′ end from Applied Biosystem. Primers and probe for 18s rRNA were purchased from Applied Biosystem. All reactions were performed in triplicate in ABI PRISM 7900HT Sequence Detection System; 18sRNA or β-actin was used as an endogenous control. Data are calculated by 2−ΔΔCT method18 and are presented as the relative proportion induction of mRNA for Egr-1 in injured arteries normalized to 18s rRNA or β-actin, compared with uninjured arteries.
Cell Culture and In Vitro Assays on Cultured SMCs
Human vascular SMCs (T/G HAVSMC) from American Type Culture Collection (Manassas, Va) were cultured in F12K media with supplements per ATCC protocol. Mouse primary vascular SMCs were cultured from aortas using a modification of the procedure of Tarvo and Barret.19 Experiments were conducted on SMCs after five to eight passages in culture. Cells were >95% SMCs based on SM-actin immunostaining. SMCs were seeded at a density of 2×104 cells/well in 24-well tissue culture-treated plates and incubated in serum-free DMEM for 48 hours. After 60 minutes preincubation with the PKCβ inhibitor LY379196, cells were exposed to serum-free DMEM containing the prototypic stimulus of PKCβ, PMA (100 ng/mL), along with [3H]-thymidine (1 μCi/well, Perkin Elmer). After 12 hours, cells were harvested and cellular proliferation was determined based on the incorporation of [3H]-thymidine. Migration assays were performed as described.20 LY379196 was generously provided by Dr Louis Vignati (Eli Lilly & Company). Northern and Western analysis of transcripts and protein for Egr-1 were performed as described.6
All data are expressed as the mean±SEM. All analyses were performed using the Statview Statistical package (version 5.0.1). Values of P<0.05 were considered statistically significant.
Effects of Genetic Deletion of PKCβ on Neointimal Expansion
To test the premise that PKC, particularly the βII isoform, would be activated by acute arterial injury, we assessed membrane localization of distinct PKC isoforms after endothelial denudation in mice. Activation of PKCβ after denuding injury was rapid; increased PKCβII antigen was observed in the membranous fraction of injured femoral arteries compared with sham (≈7.5 times higher than sham, P<0.001), with a peak at 30 minutes after denuding injury (Figure 1a). In contrast, immunoblotting with an antibody specific to the PKCβI isoform showed no change (not shown). By 30 minutes after denudation, no changes in PKCα, PKCδ, and PKCε isoforms were detected in the membranous fraction from wild-type and PKCβ−/− mice versus sham (Figure 1b through 1d). Similar findings were observed at 60 minutes and 3 days after arterial denudation (data not shown).
To elucidate the proximate triggers that led to rapid activation of PKCβII, we examined the production of reactive oxygen species (ROS).21 Previous studies established that on denuding arterial injury, polymorphonuclear leukocytes (PMNs) rapidly adhere to the injured wall.13 Consistent with these concepts, by 25 minutes after denuding injury, myeloperoxidase (MPO) activity, an enzyme located in PMNs and a source for generation of ROS, was approximately three times higher in injured wild-type PKCβ+/+ femoral artery segments versus sham-treated animals (P=0.006; Figure 1e). In PKCβ−/− mice, MPO activity, although significantly higher than that observed in sham-treated vessels in wild-type mice (P=0.008), was not significantly different than that observed in injured wild-type mice (Figure 1e). These data suggest that PKCβ does not contribute to generation of ROS in arterial injury.
Based on these findings, we tested the impact of acute arterial injury in homozygous PKCβ−/− mice.15 PKCβ−/− animals displayed significantly lower intima/media (I/M) ratio on day 28 after injury compared with wild-type mice (P<0.0001; Figure 2a, 2c through 2e). In Figure 2d, the adjacent section to that shown in Figure 2c was stained with an antibody to smooth muscle actin, thus identifying that the principal cells forming the expanding neointima in wild-type mice were SMCs. A representative sham-treated vessel (wild-type mice) is shown in Figure 2b.
Because of the known changes in humoral immune function in PKCβ−/− mice,15 it was important to use a distinct strategy to test the impact of this pathway in acute arterial injury. We used pharmacological inhibition of PKCβ to suppress its effects in vivo. Selective inhibitors of PKCβ (ruboxistaurin and LY379196) were tested for their ability to modulate neointimal expansion and SMC properties. In wild-type mice subjected to femoral artery injury, daily administration of ruboxistaurin resulted in significantly less neointimal expansion on day 28 compared with vehicle-treated controls (P=0.02; Figure 2f, 2h, and 2i). Figure 2g depicts a representative sham-treated vessel retrieved from wild-type mice.
PKCβ Modulates SMC Proliferation
To address the mechanisms by which blockade/deletion of PKCβ resulted in reduced neointimal expansion, we assessed the expanding neointima at an early time after injury, at which point SMC proliferation was previously found to be accelerated.13 Incorporation of Bromodeoxyuridine (BrdU) was significantly decreased in SMCs of the expanding neointima in PKCβ−/− versus wild-type mice on day 7 (Figure 3a, 3c, and 3d) (32% versus 60%; P<0.0001). In wild-type mice subjected to femoral artery injury, administration of ruboxistaurin decreased incorporation of BrdU on day 7 compared with vehicle (Figure 3e, 3g, and 3h) (30% versus 60%; P<0.0001). Figure 3b and 3f indicate that BrdU incorporation in sham-treated vessels was essentially not detected.
Downstream Targets of PKCβ and the Role of Egr-1 in SMCs
Egr-1 was previously found to be regulated, at least in part, by PKCβII in acute hypoxic stress. On acute femoral artery endothelial denudation in C57BL/6 mice, a time-dependent increase in mRNA transcripts encoding Egr-1 was found, with a peak 1 to 2 hours after injury (Figure 4a). By 3 and 6 hours, transcripts for Egr-1 had significantly declined (Figure 4a).
To definitively assess the role of Egr-1 in the response to vessel injury, we used mice deficient in Egr-1 (Egr-1−/−). Compared with wild-type mice, Egr-1−/− mice displayed significantly less neointimal expansion on day 28 after denuding injury (Figure 4b, 4d, and 4e). Figure 4c represents sham-treated wild-type vessels.
Consistent with an essential role for PKCβ in regulation of Egr-1 in arterial injury, arteries retrieved from PKCβ−/− mice displayed significantly lower transcript levels for Egr-1 compared with wild-type mice (Figure 4f). No differences in Egr-1 transcripts were observed at baseline between the two groups of mice (Figure 4f). Furthermore, immunohistochemistry revealed that the principal Egr-1–expressing cells after acute arterial injury were SMCs (Figure 4h), as demonstrated by colocalization with anti-smooth muscle actin IgG (Figure 4i). In contrast, sham control arteries from wild-type (Figure 4g), PKCβ−/− (Figure 4j) mice, and injured arteries from PKCβ−/− mice (Figure 4k) did not express detectable levels of Egr-1 antigen. Immunohistochemistry at multiple time points for detection of mononuclear phagocytes, using rat anti-mouse anti-F4/80 IgG, failed to identify significant numbers of these cells (not shown), consistent with published studies.13
PKCβII-Mediated SMC Activation: In Vitro Analyses
To delineate the mechanisms underlying the impact of PKCβII on regulation of Egr-1 after arterial injury, we studied primary cultures of SMCs and used a prototypic stimulus for PKCβ, phorbol myristate acetate (PMA). The PKCβ inhibitor LY379196 was used to inhibit the effect of this PKC isoform. At concentrations less than 600 nmol/L, this inhibitor is selective for the PKCβI and II isoforms.22
Both transcripts and protein for Egr-1 were lower in primary cultures of human aortic SMCs exposed to PMA, 100 ng/mL in the presence of the PKC inhibitor LY379196 (Figure 5a and 5b, respectively). Similarly, in primary cultures of murine SMCs, PMA-triggered upregulation of transcripts for Egr-1 was suppressed by pretreatment with LY379196 (Figure 5c).
Next, we examined the impact of PMA/PKCβ on two central functional properties of SMCs, proliferation and migration. In cultured human aortic SMCs, incubation with PMA resulted in increased incorporation of tritiated thymidine (P<0.0001), a process suppressed by LY379196 (Figure 5d). Incubation of primary murine aortic SMCs with PMA triggered increased incorporation of tritiated thymidine (P<0.0001), in a manner reduced by LY379196 (Figure 5e). Further, we studied the role of PKCβ in mediating cellular migration, a key property of SMCs in the expanding neointima after injury. In modified Boyden chambers, addition of PMA to the lower compartment significantly increased the number of migrating primary murine aortic SMCs (P<0.0001), a process significantly suppressed by LY379196 (Figure 5f).
These findings established that Egr-1 was a downstream target of PKCβ in acute arterial injury and that proliferation and migration of SMCs were modulated, at least in part, via PKCβ.
Signal Transduction Pathways Mediating the Impact of PKCβ in Acute Arterial Injury: In Vivo Analyses
Previous studies linked a range of signaling mechanisms, including the mitogen-activated (MAP) kinase pathway, especially extracellular signal-regulated protein kinase (ERK1/2), Janus kinase (Jak) 2, and signal transducer and activator of transcription (Stat) 3, to the response to arterial injury in SMCs.23–24 Homogenates of injured arteries harvested 15 minutes after denudation revealed markedly increased phosphorylated ERK1/2 and phospho-JNK compared with sham-treated arterial segments in wild-type mice (P<0.0001; Figure 6a and 6b). In contrast, PKCβ−/− mice displayed only a small increase in phosphorylation of ERK1/2 and JNK above sham (P<0.0001; Figure 6a and 6b).
In addition, we tested the potential role of PKCβ on the Jak2/Stat3 pathway on day 7 after injury. Although phospho-Jak2 and phospho-Stat3 were significantly increased in injured versus sham mice on day 7 (Figure 6c and 6d), there were no significant differences between phospho-Jak2 and phospho-Stat3 between wild-type versus PKCβ−/− mice after injury. No differences in phospho-Jak2 or phospho-Stat3 were observed between injured versus sham-treated arteries at 24 hours, 3 days, or 5 days after denudation (not shown).
Signal Transduction Pathways Mediating the Impact of PKCβ in SMCs: In Vitro Analyses
As it is not feasible to chronically administer inhibitors of ERK 1/2 and JNK MAP kinases in vivo, we dissected these pathways in vitro using primary cultures of murine aortic SMCs. Incubation of primary murine SMCs with PMA (100 ng/mL) for 15 minutes resulted in a striking increase in phosphorylation of ERK1/2 and JNK (Figure 6e and 6f). Real-time quantitative PCR analysis of RNA from mouse aortic SMCs incubated with PMA demonstrated an ≈7.7 times increase in expression of Egr-1 transcripts compared with untreated SMCs, in a manner suppressed by PD98059, but not by the JNK inhibitor SP600125 (Figure 6g). These findings suggested that PKCβ-mediated regulation of Egr-1 was due, at least in part, to phosphorylation of ERK1/2, but not via phosphorylation of JNK.
Lastly, we examined the impact of PKCβ on proliferation of murine aortic SMCs. Although PMA caused a significant increase in tritiated thymidine incorporation in wild-type SMCs, pretreatment with PD98059 strikingly suppressed this effect, whereas SP600125 caused a statistically significant attenuation in proliferation, albeit to a degree less than that observed by blockade of ERK1/2 MAP kinase (Figure 6h).
These findings reinforce the concept that pathways leading to restenosis are programmed by key alterations in the vessel wall that occur within minutes to hours after the initial insult. Activation of PKCβ is underway within the first 15 minutes of arterial injury. A key question that arises is by what biochemical/molecular mechanism does acute arterial injury cause rapid activation of PKCβ? Although previous work suggested that monocyte recruitment to the vessel wall is not a prominent feature of arterial injury in C57BL/6 mice, it has been shown that polymorphonuclear leukocytes are rapidly recruited to the injured vessel.13 One consequence of neutrophil activation is generation of oxidant stress, in part by myeloperoxidase (MPO) activity.25 Our studies suggest that increased MPO activity is measurable rapidly after arterial injury compared with sham controls in C57BL/6 mice and provides a mechanism by which PKC, and especially the β isoform, may be activated by acute endothelial denudation. Specifically, one consequence of MPO activity, generation of advanced glycation endproducts (AGEs), has been linked to PKCβ activation.26–27 These considerations highlight the concept that arterial injury acutely perturbs critical biochemical pathways leading to rapid recruitment of signaling pathways that significantly modify the vascular milieu.
Previous studies have linked the PKCδ isoform to stimulation of SMCs. In those studies, however, the specific stimuli to activation of PKCδ were distinct from those studied here. In vitro, Li and colleagues28 showed that mechanical stress induced SMC migration in cell culture, in a pathways dependent on PKCδ. In vivo, Leitges and colleagues29 showed that vein graft arteriosclerosis was significantly attenuated in homozygous PKCδ−/− mice versus littermates. We conclude that these stimuli are distinct from those studied in the present work, as our data showed clearly that there was no evidence of activation of PKCδ triggered by endothelial denudation in the murine femoral artery. These considerations highlight novel and intricate means of regulation of SMC properties that are dependent, at least in part, on an array of PKC isoforms and single subtypes.
We previously reported that hypoxia/reoxygenation (H/R) treatment of alveolar mononuclear phagocytes triggered rapid activation of PKCβ; downstream consequences of PKCβ activation in these cells included upregulation of Egr-1 transcripts.8 Pretreatment of the alveolar mononuclear phagocytes with inhibitors of PKCβ, ERK1/2 MAP kinase, and JNK MAP kinase before H/R suppressed upregulation of Egr-1.8 In the present studies, however, pretreatment of SMCs with inhibitors of PKCβ and ERK1/2 MAP kinase before exposure to PMA attenuated upregulation of Egr-1. Inhibitors of JNK MAP kinase did not affect Egr-1 regulation. These findings underscore the concept that the impact of PKCβ may vary in distinct cell types and in response to different stimuli. Although the in vitro stimulus to activation of PKCβ used here, PMA, is not specific for activation of PKCβ isoform, the same is true of other stimuli used to trigger activation of this enzyme, such as high concentrations of glucose in cell culture medium. Thus, experiments using LY379196 and PKCβ null mice in the present work confirm the central importance of the PKCβ pathway in triggering neointimal expansion stimulated by endothelial denudation.
In terms of clinical translation, our data highlight the potentially critical difference between strategies such as sirolimus-coated stents versus blockade of PKCβ in restenosis. Certainly, sirolimus-coated stents demonstrated striking efficacy in reduction of restenosis,30–31 yet the beneficial effect may not be complete at least in certain subsets of subjects.32 The recent observations that parenterally administered sirolimus provided only marginal benefit, even at high doses, strongly suggested that more effective targets for untoward neointimal expansion in restenosis and atherosclerosis will be needed in the future.33–34
Taken together, these findings highlight pivotal roles for PKCβ in the SMC response to acute arterial injury and the development of pathological neointimal expansion.
The authors gratefully acknowledge support from the LeDucq Foundation, Surgical Research Fund of Columbia University, Burroughs Wellcome Fund, and grants from the USPHS, National Institutes of Health (RO1 HL075529–01). We thank Dr Jeffrey Milbrandt for the generous gift of Egr-1−/− mice.
Original received June 24, 2004; resubmission received October 14, 2004; revised resubmission received January 11, 2005; accepted January 12, 2005.
Khachigian L, Lindner V, Williams A, Collins T. Egr-1 induced endothelial gene expression: a common theme in vascular injury. Science. 1996; 271: 1427–1431.
Lowe HC, Fahmy RG, Kavurma MM, Baker A, Chesterman CN, Khachigian LM. Catalytic oligodeoxynucleotides define a key regulatory role for early growth response-factor 1 in the porcine model of coronary in stent restenosis. Circ Res. 2001; 89: 670–677.
Yan SF, Lu J, Zou YS, Soh-Won J, Cohen DM, Buttrick PM, Cooper DR, Steinberg SF, Mackman N, Pinsky DJ, Stern DM. Hypoxia-associated induction of early growth response-1 gene expression. J Biol Chem. 1999; 274: 15030–15040.
Yan SF, Lu J, Zou YS, Kisiel W, Mackman N, Leitges M, Steinberg S, Pinsky D, Stern D. Protein kinase Cβ and oxygen deprivation. J Biol Chem. 2000; 275: 11921–11928.
Danis RP, Bingaman DP, Jirousek M, Yang Y. Inhibition of intraocular neovascularization caused by retinal ischemia in pigs by PKCβ inhibition with LY333531. Invest Ophthalmol Vis Sci. 1998; 39: 171–179.
Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Kashiwagi A, Ways DK, King GL, Kikkawa R. Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J. 2000; 14: 439–447.
Nakamura J, Kato K, Hamada Y, Nakayama M, Chaya S, Nakashima E, Naruse K, Kasuya Y, Mizubayashi R, Miwa K, Yasuda Y, Kamiya H, Ienaga K, Sakakibara F, Koh N, Hotta N. A protein kinase C beta selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats. Diabetes. 1999; 48: 2090–2095.
Roque M, Fallon JT, Badimon JJ, Zhang WX, Taubman MB, Reis ED. Mouse model of femoral artery denudation injury associated with the rapid accumulation of adhesion molecules on the luminal surface and recruitment of neutrophils. Arterioscler Thromb Vasc Biol. 2000; 20: 335–342.
Lee SL, Wang Y, Milbrandt J. Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1). Mol Cell Biol. 1996; 16: 4566–4572.
Leitges M, Schmedt C, Guinamard R, Davoust J, Schaal S, Stabel S, Tarakhovsky A. Immunodeficiency in protein kinase Cβ deficient mice. Science. 1996; 273: 788–791.
Goldblum S, Wu K, Jay M. Lung myeloperoxidase as a measure of pulmonary leukostasis in rabbits. J App Physiol. 1985; 59: 1978–1985.
Scivittaro V, Ganz MB, Weiss MF. AGEs induce oxidative stress and activate protein kinase Cβ II in neonatal mesangial cells. Am J Physiol Renal Physiol. 2000; 278: F676–F683.
Koyama H, Olson N, Dastva F, Reidy M. Cell replication in the arterial wall. Circ Res. 1998; 82: 713–721.
Seki Y, Shibata R, Nagata T, Yasukawa H, Yoshimura A, Imaizumi T. Role of JAK/STAT pathway in rat carotid artery remodeling after vascular injury. Circ Res. 2000; 87: 12–18.
Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban Hayashi E, Perin M, Colombo A, Schuler G, Barragan P, Guagliumi G, Molnar F, Falotico R; RAVEL Study Group. Randomized study with the sirolimus-coated Bx velocity balloon-expandable stent in the treatment of patients with de novo native coronary artery lesions. N Engl J Med. 2002; 346: 1773–1780.
Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, Jaeger JL, Kuntz RE; SIRIUS Investigators. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med. 2003; 349: 1315–1323.
Lemos PA, Saia F, Ligthart JM, Arampatzis CA, Sianos G, Tanabe K, Hoye A, Degertekin M, Daemen J, McFadden E, Hofma S, Smits PC, de Feyter P, van der Giessen WJ, van Domburg RT, Serruys PW. Coronary restenosis after sirolimus-eluting stent implantation: morphological description and mechanistic analysis from a consecutive series of cases. Circulation. 2003; 108: 257–260.
Brara PS, Moussavian M, Grise MA, Reilly JP, Fernandez M, Schatz RA, Teirstein PS. Pilot trial of oral rapamycin for recalcitrant restenosis. Circulation. 2003; 107: 1722–1724.