Published online before print December 20, 2007, doi: 10.1161/ATVBAHA.107.156810
© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
CaMKII-
Isoform Regulation of Neointima Formation After Vascular Injury
From the Center for Cardiovascular Sciences, Albany Medical College (MC-8), Albany, NY.
Correspondence to Harold A. Singer, PhD, Center for Cardiovascular Sciences (MC8), Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208-3479. E-mail singerh{at}mail.amc.edu
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Abstract |
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Objective— The purpose of this study was to test the function of the calcium/calmodulin-dependent protein kinase II
2 isoform (CaMKII2) in regulating vascular smooth muscle (VSM) cell proliferation and migration in response to vascular injury.
Methods and Results— CaMKII isoform content was assessed in rat carotid arteries after balloon angioplasty–induced injury by Western blotting with isoform specific antibodies. Within 3 days after injury, a significant increase in CaMKII2 and decrease in CaMKII
isoform content was observed in both medial smooth muscle and adventitial fibroblasts. Neointimal VSM cells expressed primarily the 2 isoform. Incubation of the injured vessel with adenovirus encoding siRNA targeting CaMKII isoforms prevented upregulation of the 2 isoform in the media and adventitia; inhibited cell proliferation assessed by PCNA expression in both layers and markedly inhibited neointima formation and adventitial thickening.
Conclusions— CaMKII2 is specifically induced in VSM and adventitial fibroblasts during the response of an artery to injury and is a positive regulator of proliferation and migration in the vessel wall contributing to neointima formation and vascular remodeling. This provides a potential mechanism for Ca2+-dependent regulation of VSM and myofibroblast proliferation and migration in response to vascular injury or disease.
We observed CaMKII isoform modulation in rat carotid artery in response to vascular injury. Attenuating CaMKII2 upregulation using siRNA significantly decreased neointima formation and adventitial thickening. This finding is significant in that it provides a link whereby alterations in Ca2+ signaling directly contribute to vascular injury and disease.
Key Words: CaMKII • VSM proliferation • vascular injury • restenosis • neointima
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Introduction |
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Vascular smooth muscle (VSM) cell phenotype modulation and proliferation is a hallmark of disease or injury-induced remodeling of the vascular wall and is regulated by diverse environmental stimuli including peptide growth factors, extracellular matrix, vasoactive factors, mechanical forces, and oxidative stress.1 Identification of intracellular signaling pathways that integrate this information to regulate VSM cell phenotype and proliferative responses is an active field of research. Ca2+ signals can coordinate multiple functions in VSM including contractile activity, metabolism, and gene transcription.2 There is evidence to suggest that different sources or spatiotemporal patterns of Ca2+ signals may be processed to result in specific patterns of gene expression.3 Similar "excitation-transcription" coupling mechanisms have been proposed in neurons and striated muscle.4 Of particular interest with respect to vascular disease mechanisms is recent evidence indicating that injury-induced changes in VSM Ca2+ dynamics may contribute to development of a migratory/proliferative phenotype associated with neoinitma formation.3,5
We have investigated the multifunctional serine/threonine kinase Ca2+/calmodulin-dependent protein kinase II (CaMKII) as a mediator of Ca2+ signals regulating VSM cell function. A large number of splice variants from 4 differentially expressed CaMKII genes (
, β, , and ) have been identified,6 and there is increasing evidence that isoform-specific structural variations can affect subcellular targeting and function of the kinase.7 Differentiated VSM cells express mRNA transcripts for both CaMKII and variants.8,9 CaMKII isoforms have been implicated in control of differentiated VSM contractile function10–12 whereas the 2 isoform, the main isoform expressed in cultured VSM,8 is an intermediate in the Ca2+-dependent activation of nonreceptor tyrosine kinases and ERK1/2.13,14 CaMKII2 also facilitates adhesion-dependent activation of ERK1/2 in cultured VSM through integrin-dependent and -independent mechanisms15 and is involved in the regulation of VSM cell migration16,17 and proliferation,18 although the mechanisms and relevant substrates are not yet clear.
Recently, we demonstrated acute modulation of CaMKII isoform expression from to 2 isoforms within hours after enzymatic dispersion of aortic medial VSM cells, coincident with or preceding initial VSM cell proliferation in primary culture.18 Preventing acute upregulation of CaMKII2 using siRNA inhibited cell proliferation in primary culture. In passaged VSM cells, siRNA suppression of CaMKII2 or inhibition of activity using a kinase-negative mutant inhibited proliferation rates.18 In the present studies we used the in vivo balloon-injured rat carotid artery model and demonstrated similar CaMKII isoform modulation in medial wall VSM and adventitial fibroblasts within 3 days after injury. Adenoviral transduction of siRNA targeting CaMKII isoforms attenuated injury-induced CaMKII2 upregulation, inhibited VSM proliferation and migration, and nearly prevented neoinitma formation. Based on these results, we conclude that CaMKII isoform modulation, specifically upregulation of the 2 isoform, is an important component of the response to injury, resulting in Ca2+-dependent regulation of VSM cell or adventitial fibroblast proliferation and subsequent neointima formation.
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Methods |
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For expanded materials and methods, please see http://atvb.ahajournals.org.
Antibodies and Other Materials
Production and specificity of the antibodies used for detection of the 2 isoform of CaMKII,8 pan-CaMKII isoforms,9 and CaMKII18 were described previously. Monoclonal antibody specific for β-actin was purchased from Sigma. Polyclonal antibodies specific for Cdc25C, p-Cdc25C (ser216), and vascular cell adhesion molecule (VCAM)-1 (H-276) were purchased from Santa Cruz Biotechnology. Polyclonal antibodies specific for PCNA and GFP were purchased from Abcam. Monoclonal antibody specific for Histone HI was purchased from Chemicon International. All cell culture media and supplies were obtained from Fisher Scientific unless otherwise specified. All other chemicals were purchased from Sigma. Western blot analysis by standard protocols was previously described.18
Balloon Angioplasty and Adenoviral Infection
Male Sprage-Dawley rats (350 g; Taconic Farms, Germantown, NY) were anesthetized with Xylazine 4.6 mg/kg and Ketamine 70 mg/kg via intraperitoneal injection and balloon angioplasty carried out as previously described.19 Briefly, a 2F Fogarty balloon embolectomy was inserted through a small arteriotomy in the external carotid artery and passed into the common carotid artery. After balloon inflation at 2 atm of pressure (6X), the catheter was partially withdrawn and reinserted 3 times. Animals recovered after receiving a postoperative dose of analgesic (Buprenex 0.20 mg/kg intramuscular). For surgeries involving local gene transfer (adapted from Schulik et al20): after injury, 5 µL of virus (shDELTA2 or shLuciferase) was brought to a final volume of 25 µL in 0.9% normal saline and inserted through the arteriotomy with a 20G catheter and incubated for 30 minutes. A ligation placed around the internal carotid artery proximal to the arteriotomy maintained pressure. The previously described shDELTA2 siRNA18 construct targeted a conserved sequence in CaMKII variants: 5'ATAAACCAATCCACACTAT-3', nt 1543–1562. shDELTA2 was cloned as a short hairpin into an adenoviral construct using the AdTrack vector (a generous gift from Dr Ling-Jun Zhao, St. Louis University School of Medicine, St. Louis, MD21) and AdEasy system (Stratagene). An siRNA sequence targeting the firefly Luciferase gene22 was generated in the same manner. The use of experimental animals for these procedures was reviewed and approved by the Albany Medical College Institutional Care and Use Committee and Institutional Biosafety Committee.
Immunohistochemistry
Animals were euthanized by asphyxiation with CO2 and carotid arteries perfusion fixed with 10% formalin. Sections measuring 6 to 8 µm were cut and recovered on ProbeOn Plus microscope slides and stained using the Vectastain Elite ABC Kit (Vector Laboratories). Sections were mounted with 50% glycerol and 60X phase contrast images were obtained using a Leica DM IRB inverted microscope.
Measurement of Carotid Artery Sections
ImageJ software (NIH) and magnified 10X phase contrast micrographs were used. The neointimal area was measured as the tissue extending from the internal elastic lamina to the lumen. The medial area was measured as the tissue between the external and internal elastic lamina whereas the adventitial area was measured as the tissue outside of the external elastic lamina. Area is expressed either as the neointimal/medial ratio (I/M) or arbitrary units.
In Vivo Migration Assay
Cell migration 3 days after injury was measured by counting intimal cell nuclei stained en face with Histone H1 as previously described.23 Sections were mounted with 50% glycerol and 40X images obtained using a Leica DM IRB inverted microscope. 10 random fields from 1 cm of excised injured vessel were counted and total number of cells was used for analysis.
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Results |
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CaMKII Isoform Modulation After Vascular Injury
Western blotting with a pan-CaMKII isoform antibody indicated expression of 2 major CaMKII subunits in uninjured (right side) or sham-injured carotid medial VSM (Figure 1A). The media, adventitia, and 14-day neointima were dissected and analyzed separately. Based on size, comigration with a recombinant standard (not shown), and reactivity with CaMKII isoform-specific antibodies, the predominant 54-kDa band was identified as a
isoform, most likely c, and the less abundant 52 kDa band was identified as CaMKII2. This expression pattern is similar to that found in differentiated medial VSM cells from rat aorta.18 Two weeks after balloon injury, the relative abundance of the 2 isoforms in the injured (left) medial layer reversed and the 2 isoform predominated. A time course analysis using the isoform specific antibodies indicated large increases in CaMKII2 and decreases in CaMKII content in the medial and neointimal layers within 3 days after injury with maximal changes after 7 to 14 days (Figure 1B).
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<0.01).
To quantify the relative CaMKII isoform content in the adventitial layer, we used a volume analysis of Western blot signals using the pan-CaMKII antibody. The relative expression of 2 was higher in uninjured adventitial layer fibroblasts (Figure 1C) compared with medial VSM (supplemental Figure I, see http://atvb.ahajournals.org). Significantly, isoform content modulation was also observed in adventitia after injury with rapid upregulation of 2 and downregulation of isoforms. The CaMKII isoform content in injured vessels gradually returned to control levels between 2 and 10 weeks after injury. Analysis of CaMKII isoform expression in the 14-day neointimal layer, which is largely composed of synthetic phenotype VSM cells,1 indicated a predominance of the CaMKII2 isoform (Figure 1D), an expression profile that closely resembles that in the injured left media and primary cultures of rat carotid (supplemental Figure II) and aortic VSM cells.16,18 We conclude from these observations that CaMKII2 isoform upregulation and isoform downregulation after balloon injury in both medial VSM cells and adventitial fibroblasts is concurrent with, or precedes neointima formation.
Suppression of CaMKII2 Inhibits VSM and Fibroblast Proliferation After Injury
CaMKII2 expression rapidly increases after enzymatic dispersion and culture of rat aortic VSM cells18 and carotid artery medial VSM cells (supplemental Figure II). Suppressing CaMKII2 expression using siRNA or inhibiting activity by overexpressing a kinase-negative mutant in either system indicated a positive role for the kinase in regulating cell proliferation18 (supplemental Figure III). To inhibit CaMKII2 expression in response to injury, an adenovirus expressing a short hairpin siRNA construct, previously shown to target CaMKII isoforms (shDELTA2)18 was infused into the lumen of the common carotid artery immediately after injury. Adenovirus expressing a short hairpin siRNA targeting firefly luciferase was used as a negative control. Both adenoviruses expressed GFP under a separate promoter, allowing verification of infection. Immunostaining carotid artery sections with antibody recognizing GFP demonstrated transduction with the control adenovirus in the medial and adventitial layers 3 days after injury (Figure 2A and 2B). A gradient of expression across the medial layers was observed, suggesting incomplete penetration of the adenovirus under the conditions used. Access of advenovirus to the adventitia using a similar approach has previously been documented and is believed to be facilitated by the vasa vasorum.24 Infusion of the control adenovirus had no effect on CaMKII isoform modulation at 3 (supplemental Figure IV) or 7 days (Figure 2C and 2D) after injury. However, incubation with adenovirus encoding shRNA targeting CaMKII attenuated injury-induced CaMKII isoform modulation by inhibiting CaMKII2 upregulation, an effect which was statistically significant within 7 days (Figure 2C and 2D).
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The consequence of CaMKII2 gene silencing on VSM cell proliferation was assessed by expression of proliferating nuclear cell antigen (PCNA). Medial VSM PCNA expression was markedly increased 3 days after injury an effect that persisted through at least 7 days (Figure 3A and 3C). Adenoviral transduction with shRNA targeting CaMKII2 significantly inhibited PCNA expression at both time points (Figure 3B and 3D), indicating a positive effect of the kinase on VSM cell proliferation in vivo. Consistent with immunohistochemistry demonstrating some adenoviral transduction and GFP expression in the adventitia (Figure 2A), CaMKII isoform modulation assessed as relative expression of 2 and isoforms in the adventitia was inhibited by exposure to adenovirus with shRNA targeting CaMKII2 (Figure 4A and 4B). Adventitial cell proliferation, indicated by PCNA expression, was also inhibited in the left adventitia expressing shRNA targeting CaMKII2 (Figure 4C).
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CaMKII2 Silencing Attenuates Neointima Formation
To assess the effects of siRNA-mediated CaMKII2 suppression on neointima formation, the vessels were analyzed 3 and 14 days after injury. Previous reports documented accumulation of neointimal VSM cells between 1 and 4 days after injury, an effect attributed mainly to VSM migration because the response preceded peak medial wall proliferation.23 Luminal cells identified by nuclear histone H1 staining and also staining positively for an endothelial marker for VCAM-1, were nearly completely removed by the balloon injury protocol (Figure 5A). Cells populating the luminal surface after 3 days stained positively for CaMKII2 consistent with a medial VSM origin. Preventing CaMKII2 upregulation with siRNA resulted in attenuation of this early accumulation of intimal cells (Figure 5B). Analysis of the vessel at 14 days, during peak neointima formation, demonstrated that siRNA mediated inhibition of CaMKII isoform modulation persisted through this period (Figure 6A and 6B) and development of neointima was inhibited by 80% (Figure 6C). Analysis of the adventitial layer, also indicated a significant increase in crosssectional area 14 days after injury with a significant inhibition of this response after infection with the siRNA adenovirus targeting CaMKII2 (Figure 6D). Taken together, the data indicate that upregulation of CaMKII2 is an important regulator of vascular wall remodeling in response to injury.
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Discussion |
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Based on our previous results implicating CaMKII
2 as regulator of cell migration and proliferation in cultured rat aortic VSM cells,16,18 we investigated the function of CaMKII2 in the vascular response to injury. Consistent with results using an in vitro system,18 we observed a rapid and marked modulation in vascular wall CaMKII isoform expression after balloon injury in vivo, coinciding with acquisition of a migratory/proliferative phenotype in both carotid artery medial VSM and adventitial fibroblasts. Inhibiting acute upregulation of the CaMKII2 isoform attenuated migration and proliferation, and functionally suppressed neointima formation and adventitial thickening. We conclude from these studies that CaMKII isoforms specifically couple Ca2+ signals to regulate cell migration and proliferation, and that increased expression of these isoforms in VSM or adventitial myofibroblasts is an important component of injury induced vascular wall remodeling.
Potential sources of neointimal VSM or VSM-like cells after balloon injury have been reported to include dedifferentiated medial VSM, adventitial myofibroblasts, and circulating progenitor cells.1 Although in the present study we did not identify the source of neointimal cells, early studies using this model support migratory and proliferating medial VSM as the primary source,19 and more recent studies using mouse models have been interpreted as favoring vascular wall as opposed to circulating sources of cells in atherosclerotic lesions.25 From the standpoint of CaMKII isoform expression, the phenotype of neoinitmal cells was found here to closely resemble that of cells in either the injured medial VSM or adventitial layers, with the CaMKII2 isoform predominating.
Although our adenoviral transduction approaches were limited in that they resulted in incomplete siRNA targeting of CaMKII across the medial layers and partial inhibition of CaMKII2 expression in medial VSM, the intervention was effective in inhibiting neointimal cell accumulation as early as 3 days after injury. High efficiency transduction and strong expression of the siRNA construct in the innermost medial layer may account for this effect, as intimal accumulation of cells at this early stage after injury has been attributed mainly to migration of medial VSM cells across the internal elastic lamina.19 Assuming that this is true, these results are the first linking a specific CaMKII isoform to regulation of VSM cell migration in vivo. However, the relative contribution of VSM cell migration compared with proliferation at this time point is not absolute, and inhibition of VSM cell proliferation after CaMKII2 silencing may contribute to the observed effects on intimal VSM cell accumulation at this early time point. Although we cannot rule out adventitial myofibroblasts as contributing to the response at this stage, earlier reports indicated that migrating adventitial cells were first observed in the neointima 7 days after balloon injury.26
At later stages after injury, there is significant evidence that adventitial fibroblasts can contribute to neointima formation.26–30 Based on the fact that the adventitial layer was targeted after luminal infusion of adenoviral siRNA constructs (Figure 2A), presumably via the vasa vasorum,24 some of the efficacy of the intervention with respect to blocking neointima formation after 14 days that we found here may therefore be attributed to inhibition of myofibroblast migration and proliferation. Inhibition of adventitial cell PCNA expression and adventitial layer thickening after adenoviral infusion CaMKII2 siRNA supports a function for the kinase in regulating myofibroblast proliferation and adventitial layer growth. However, at this point we have only demonstrated a change in relative CaMKII2/ isoform content averaged across the adventitial layer, and our Western blotting analysis of PCNA does not provide information on the cellular source or extent of adventitial cell proliferation. It remains possible that a subset of adventitial fibroblasts, perhaps restricted to the area surrounding vaso vasorum, or a nonfibroblast cell (VSM or endothelium) were activated by injury and inhibited by the siRNA targeting CaMKII2. A greater efficiency of siRNA construct delivery, specifically to the adventitia, perhaps by application of adenovirus suspended in pluronic gel to the perivascular surface of the injured artery26,31 may be a better approach for assessing myofibroblast contribution to neointima formation.
The mechanisms by which CaMKII2 inhibits VSM or myofibroblast migration are under investigation. Previous in vitro studies have pointed to a potential function for the kinase in regulating integrin engagement and signaling or extracellular signal regulated kinase (ERK) signaling.13–15 The effects of CaMKII2 on cell proliferation are potentially mediated through direct effects on the cell cycle because cultured carotid VSM cells as well as adventitial fibroblasts were found to have lower levels of Cdc25 phosphorylation when exposed to siRNA targeting CaMKII2 or when overexpressing an inhibitory kinase-negative mutant KN2 (Supplemental Figure III). These results are consistent with previous reports indicating that this cell cycle regulatory phosphatase is a substrate for CaMKII in HeLa cells,32 and with flow cytometric analysis of CaMKII2 depleted cells which indicated delayed progression through the G2/M transition of the cell cycle.18
Although this is the first study in the literature to identify CaMKII function in response to vascular injury, it is not the first in vivo study indicating the importance Ca2+ and its downstream effectors in this response.5,33 Importantly, work by Lipskaia et al provided evidence that adenoviral overexpression of sarco/endoplasmic reticulum calcium ATPase (SERCA2a) inhibits VSM cell proliferation and subsequent neointima formation after balloon catheter-induced injury in the rat carotid artery.5 The authors concluded that the delivery of SERCA2a to the injured media reversed pathological alterations in Ca2+ handling in proliferating VSM cells.5 Overexpression of SERCA2a in serum-stimulated VSM cells was reported to inhibit proliferation through inactivation of calcineurin and its target NFAT resulting in decreased cyclin D1 levels and cell cycle arrest at the G1 phase.5 A potential link between this study and the present findings is that pharmacological inhibition of CaMKII in NIH3T3 cells reduced cyclin D1 levels and enhanced the association of p27(Kip1) with Cdk2 causing G1 arrest.34
Upregulation of CaMKII isoforms has been described in models of cardiac hypertrophy and heart failure, and mechanisms involving CaMKII-dependent regulation of histone deacetylase have been proposed to account for alterations in gene expression associated with these pathologies.35 Similarly, the CaMKII2 isoform is upregulated in regenerating skeletal muscle after injury.36 Our recent in vitro studies18 and the current in vivo studies have for the first time clearly demonstrated a rapid shift in CaMKII isoform expression, including upregulation of the CaMKII2 isoform on acute transition of vascular smooth muscle cells from a differentiated to synthetic phenotype. By using molecular approaches to inhibit expression of CaMKII2 or inhibit its activity, we have demonstrated that this isoform promotes VSM migration and proliferation and contributes to vascular response to balloon injury. Because atherosclerosis, vein graft failure, and transplant vasculopathy are dependent on pathologic VSM cell proliferation, we predict a role for CaMKII isoform modulation in each of these processes. In addition, increases in VSM cell ploidy, similar to those observed after suppression of endogenous CaMKII2 in cultured aortic VSM cells, have been linked to vascular remodeling associated with hypertension,37 and in this context, it may be interesting to consider a role of CaMKII isoform modulation as a factor contributing to vascular remodeling in hypertensive disease.
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Acknowledgments |
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The authors acknowledge the technical assistance of Edward Lewis and the administrative support of Wendy M. Hobb.
Sources of Funding
This work was supported by NIH Training Grant T32-HL-07194 and R01-HL-49426.
Disclosures
None.
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Footnotes |
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Original received September 26, 2007; final version accepted December 10, 2007.
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