Published online before print July 5, 2007, doi: 10.1161/CIRCRESAHA.107.151399
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Attenuated Expression of Profilin-1 Confers Protection From Atherosclerosis in the LDL Receptor–Null Mouse
From the The Schepens Eye Research Institute (G.R.R., A.K.), the Vascular Biology Program, Children’s Hospital (K.S.M.), and the Cardiovascular Division, Brigham and Women’s Hospital (K.S.M.), Harvard Medical School, Boston, Mass.
Correspondence to Giulio R. Romeo, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114. E-mail giulio.romeo{at}schepens.harvard.edu
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Abstract |
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Atherosclerosis-related events are a major cause of morbidity and death worldwide, but the mechanisms underlying atherogenesis are not fully understood. We showed in previous studies that the actin-binding protein profilin-1 (pfn) was upregulated in atherosclerotic plaques and in endothelial cells (ECs) treated with oxidized low-density lipoproteins (oxLDL). The present study addressed the role of pfn in atheroma formation. To this end, mice with heterozygous deficiency of pfn, Pfn+/–, were crossed with Ldlr–/– mice. After 2 months under a 1.25% cholesterol atherogenic diet, Pfn+/–Ldlr–/– (PfnHet) exhibited a significant reduction in lesion burden compared with Ldlr–/– control mice (PfnWT), whereas total cholesterol and triglyceride levels were similar in the 2 groups. Relevant atheroprotective changes were identified in PfnHet. When compared with PfnWT, aortas from PfnHet mice showed preserved endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO)-dependent signaling, and reduced vascular cell adhesion molecule (VCAM)-1 expression and macrophage accumulation at lesion-prone sites. Similarly, knockdown of pfn in cultured aortic ECs was protective against endothelial dysfunction triggered by oxLDL. Finally, bone marrow–derived macrophages from PfnHet showed blunted internalization of oxLDL and oxLDL-induced inflammation. These studies demonstrate that pfn levels modulate processes critical for early atheroma formation and suggest that pfn heterozygosity confers atheroprotection through combined endothelial- and macrophage-dependent mechanisms.
Key Words: profilin • atherosclerosis • eNOS • vascular inflammation
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Introduction |
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The 15-kDa protein Profilin-1 (pfn) is ubiquitously expressed in a broad range of organisms1 and plays a critical role early in development.2 Notably, homozygous deficiency of pfn in the mouse leads to embryonic lethality at the 2-cell stage.3 Initially isolated as a 1:1 complex with G-actin,4 pfn modulates actin dynamics by promoting both actin-filament assembly when barbed ends are free5 and depolymerization when F-actin filaments are capped.6 Pfn also binds phospatidylinositol 4,5-biphosphate (PIP2)7 in a mutually-exclusive fashion to G-actin. Finally, pfn associates with poly-L-proline and a growing list of proline-rich proteins8 that includes vasodilator-stimulated phosphoprotein (VASP).9 It is thought that the interaction with this diverse array of ligands may allow pfn to integrate signaling cues with actin cytoskeleton remodeling. For instance, a proteomic-based approach in mouse brain revealed that pfn complexes with signal transduction proteins as well as members of the endocytic machinery.10
Surprisingly, little is known regarding the role of pfn in disease. We found previously that pfn expression was increased in endothelial cells (ECs) and macrophages of atherosclerotic lesions, and that oxidized LDL (oxLDL) upregulated pfn in cultured ECs.11 Also, forced expression of pfn triggered endothelial dysfunction, which is a promoting factor for atherosclerosis.12 Together, these findings prompted us to test whether pfn played a direct role in atherosclerotic lesion formation. To this end, we bred pfn heterozygous mice, Pfn+/–, which are viable and have a 
50% reduction in pfn protein levels,3 into the atherosclerosis-susceptible Ldlr–/– strain.13 Here, we report that, when fed an atherogenic high-cholesterol diet (HCD) for 2 months, Pfn+/–Ldlr–/– mice showed a marked reduction in atherosclerotic lesion burden, when compared with control Ldlr–/– mice. In addition, we identify endothelial- and macrophage-dependent mechanisms that could account for the atheroprotective phenotype of Pfn+/–Ldlr–/– mice.
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Materials and Methods |
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Mice
Profilin-1 heterozygotes mice (Pfn+/–) were originally provided by W. Witke (Mouse Biology Programme, EMBL Monterotondo, Italy)3 on a C57BL/6J background and were crossed with C57BL/6J Ldlr–/– mice from Jackson Laboratory (Bar Harbor, Maine) to obtain Pfn+/– Ldlr+/–. These mice were backcrossed 5 generations into the C57BL/6J background and intercrossed to obtain Pfn+/– Ldlr–/–. Beginning at 6 weeks of age, Pfn+/+ Ldlr–/– (PfnWT) and Pfn+/– Ldlr–/– (PfnHet) were fed a HCD for 8 to 9 weeks (TD96121: 1.25% cholesterol [wt/wt], no sodium cholate; Harlan Teklad). For some experiments, PfnWT and PfnHet mice were fed a regular rodent chow. At the time of euthanasia, blood and tissues were collected for analysis. All experiments were approved by the Schepens Eye Research Institute Animal Care and Use Committee and were performed in accordance with the PHS Guide for the Care and Use of Laboratory Animals and USDA regulations under the Animal Welfare Act.
Tissue Preparation and Measurement of Atherosclerosis
Mice were anesthetized by i.p. injection of ketamine (62.5 mg/kg body weight) and xylazine (12.5 mg/kg body weight), and perfused first with 15 mL saline, then with 15 mL methanol-free 4% formaldehyde (Polysciences). The aorta was collected and freed of periadventitial tissue. Quantitation of atherosclerotic lesions was performed using en face preparations of the descending aorta stained with Sudan IV (Sigma-Aldrich), and sections were obtained from the aortic arch. Lesion area in the descending aorta was determined essentially as described.14 Briefly, after washes in 70% and 80% ethanol, the aorta was stained with 0.5% Sudan IV (35% ethanol, 50% acetone) for 15 minutes, destained with 80% ethanol, and kept in water. After longitudinal dissection, the stained aorta was pinned en face on a polymerized matrix (Sylgard 184, Dow Corning) using 0.1-mm Minutien micropins (Fine Science Tools). Images were collected and analyzed using a Zeiss Axioskop2 Mot Plus. Atherosclerotic burden was calculated as the percent area of Sudan IV+ lesions in the whole descending aorta, including iliac bifurcation. Analysis of the lesion burden was performed in a masked fashion by a single observer (G.R.R). With regard to the aortic arch, specimens were embedded in low-melting point agarose and sectioned through the midline using a Vibratome. This 500-µm-thick slice was embedded in OCT (Tissue-Tek) and further sectioned (8 to 10 µm) for plaque assessment and immunostaining. After staining with Oil red O,11 lesions along the inner curve of the arch and at the right brachiocephalic artery bifurcation were analyzed as described above. At least 3 nonconsecutive sections per specimen were used. Counterstaining with Hematoxylin or Light Green was performed as described previously.11
NO Assay
After perfusion with PBS, the whole descending aorta was isolated from PfnWT and PfnHet fed regular chow or HCD for 8 to 9 weeks. The cleaned specimen was incubated in a tissue culture humidifier for 16 hours in 350 µL DMEM supplemented with 0.5% FBS. The medium was collected, spun, and tested for nitrites using a Griess reagent kit according to manufacturer’s instructions (Molecular Probes).
Statistical Analysis
Data are presented as the mean±SD. Comparisons between 2 groups were performed using unpaired t test. Comparisons for multiple groups were performed using 1-way ANOVA followed by Bonferroni test. A probability value of less than 0.05 was considered significant.
For other methods and procedures, please refer to the supplemental material section (available online at http://circres.ahajournals.org).
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Results |
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Because Pfn–/– mice die during embryonic development,3 we tested the role of pfn in early atheroma formation by crossing adult Pfn+/–, which are viable and fertile, into the Ldlr–/– mouse model of atherosclerosis. Pfn+/–Ldlr–/– (hereafter, PfnHet) were generated at a ratio lower than expected (
1:9 versus 1:6, observed versus expected), in keeping with decreased survival of Pfn+/– through embryonic development.3 Adult PfnHet appeared healthy and did not show differences in mortality either under normal chow or HCD, when compared with Ldlr–/– (hereafter, PfnWT).
PfnHet Are Protected From Atherosclerosis
To test the consequences of reduced pfn expression on atherosclerotic plaque formation, PfnHet and PfnWT were fed a HCD for 2 months beginning at 6 weeks of age. PfnHet and PfnWT showed a similar increase in total cholesterol and triglyceride levels when compared with littermates fed a regular chow diet13 (supplemental Table I). Also, overall lipid absorption was comparable in PfnHet and PfnWT (supplemental material).
En face preparations of the descending aorta were stained with the lipid dye Sudan IV to identify atheromatous lesions. PfnWT aortas showed lipid-laden lesions occurring preferentially at intercostal, kidney, and iliac artery bifurcations (Figure 1). When compared with PfnWT, the total area occupied by Sudan IV+ lesions in PfnHet was dramatically decreased (Figure 1A), by 75% in females (1.1±0.1% versus PfnWT 4.17±0.49%, P<0.0001) and 60% in males (1.34±0.22% versus PfnWT 3.4±0.36%, P<0.001). In the aortic arch, the extent of atheromas was evaluated on 3 nonconsecutive coronal sections cut from the midline. After staining with Oil red O, PfnHet specimens showed minimal lipid deposition and atheroma formation even at sites of high susceptibility such as the inner arch and the brachiocephalic artery bifurcation. Quantification by image analysis demonstrated a 60% decrease of the arch lesion area in PfnHet when compared with PfnWT (0.08±0.03 mm2 versus PfnWT 0.20±0.05 mm2; P<0.001; Figure 2). In addition to a reduced area, aortic arch lesions from PfnHet displayed a significant decrease in the percent area occupied by macrophages, measured as area staining for the macrophage marker MOMA-2 (15.1±3.3% versus PfnWT 23.8±4.9%; P=0.005; Figure 3). Thus, PfnHet showed a substantial reduction of atherosclerotic burden and macrophage accumulation despite lipid levels comparable to those of PfnWT. These studies provided direct evidence for a role of pfn in the pathogenesis of atherosclerosis. Next, we set out to identify the mechanisms contributing to the antiatherogenic phenotype observed in PfnHet.
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The Aorta of PfnHet Shows Attenuated Binding/Uptake of oxLDL and Macrophage Accumulation
The lesser macrophage percent content observed in PfnHet lesions (Figure 3) suggested that reduced pfn could limit the endothelial-mediated recruitment of monocytes or their differentiation into foam cells in the sub-endothelial space, both critical events in lesion initiation and progression.15,16
We first tested this hypothesis by examining macrophage infiltration and expression of VCAM-1, a critical mediator of leukocyte adhesion,17 at branch points of intercostal arteries, which are preferential sites for plaque formation. To address early changes, mice were fed a HCD for only 2 weeks. En face preparations of PfnHet aorta showed a weaker staining for VCAM-1 (Figure 4A), when compared with PfnWT. Confocal microscopy demonstrated that attenuated focal expression of VCAM-1 in PfnHet was associated with reduced macrophage infiltration, evaluated as MOMA-2 staining (Figure 4B). In addition, PfnHet aortas exposed ex vivo to Dil-labeled oxLDL exhibited a blunted uptake/binding of oxLDL at or in proximity of intercostal artery branch points (Figure 4C), which correlated with the defective accumulation of macrophages in the subendothelial space (Figure 4D). Thus, PfnHet displayed attenuated accumulation of monocyte-derived macrophages at atherosclerosis-prone sites that coincided with a weaker expression of endothelial VCAM-1 and a reduced uptake of oxLDL. These changes were detected after only 2 weeks on HCD, suggesting that pfn levels modulated early stages of macrophage recruitment. Because both VCAM-1 expression and engulfment of oxLDL are inciting factors for atherosclerosis,18,19 their attenuation could account in part for the atheroprotective phenotype of PfnHet mice.
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Endothelial Function Is Preserved in PfnHet Fed a HCD
Reduced VCAM-1 expression in the PfnHet aorta suggested that the endothelium of PfnHet could mount a protective response after exposure to HCD. A growing body of evidence indicates that LDL-mediated vascular injury is in part mediated by eNOS inhibition20 and decreased NO bioavailability, which leads to expression of endothelial adhesion molecules, eg, VCAM-1.21
Thus, we studied eNOS phosphorylation at residues indicative of the enzyme activation, ie, Ser1177, or inhibition, ie, Thr495. Western blot analysis of aortic arch specimens showed a significant decrease of phospho-S1177 in PfnWT fed HCD, but not in PfnHet, when compared with littermates on a regular chow (1.8±0.26 versus PfnWT 0.51±0.12 relative D.U., when normalized for total eNOS; P<0.0001; Figure 5A). Conversely, phosphorylation at the inhibitory site T495 was increased in PfnWT on HCD further indicating eNOS inhibition in these mice. Akt activation, evaluated as phosphorylation at S473, was comparable in all conditions, suggesting that kinases other than Akt mediated changes in eNOS activation (data not shown). Next, we investigated effects of NO action in vivo by studying the phosphorylation at Ser239 of the 46/50kDa vasodilator-stimulated phosphoprotein (VASP). Phosphorylation at Ser239 has been validated as a useful indicator of NO-dependent signaling both in cultured cells22 and in tissues.11,23 In agreement with the pattern of eNOS activation, phospho-S239 VASP was markedly reduced in the aorta of PfnWT fed HCD, when compared with littermates on a regular chow, whereas it was preserved in PfnHet. Also, an antibody that detects both phosphorylated (50kDa) and unphosphorylated VASP (46kDa) showed HCD-induced decrease of the phosphorylated/unphosphorylated ratio in PfnWT, but not in PfnHet (1.94±0.17 versus PfnWT 0.49±0.13 relative D.U. of the 50kDa/46kDa ratio; P<0.0001; Figure 5B). VASP phosphorylation was comparable in PfnWT and PfnHet fed regular diet, indicating that pfn deficiency per se was not sufficient to induce these changes. Also, the aorta of PfnWT, but not PfnHet, fed HCD showed a significant decrease in NO secretion when compared with littermates on regular diet (0.22±0.04 versus PfnWT 0.11±0.05 µmol/L nitrites; Figure 5C). In keeping with the profile of eNOS activation and NO production, HCD resulted in increased blood pressure in PfnWT, but not in PfnHet, when compared with littermates on regular chow (supplemental Figure I). Finally, aortic SMCs isolated from PfnWT and PfnHet mice showed similar levels of phosphorylated VASP on treatment with the NO-donor SNAP, suggesting that the response to NO was not directly affected by pfn levels (supplemental Figure II). Taken together, these studies demonstrate that PfnHet mice are protected from the decline in endothelial function occurring in PfnWT on the atherogenic diet.
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Decreased CD36 Levels in the PfnHet Aorta
As presented in Figure 4, the aorta of PfnHet exhibited a blunted binding/uptake of oxLDL. We hypothesized that pfn deficiency, in addition to preserving endothelial function, limited the process leading to oxLDL uptake by macrophages.
We studied protein levels for CD36 and other scavenger receptors (SR), which mediate unregulated cholesterol accumulation and play a role in atherogenesis.19,24 CD36 was significantly decreased in the adipose tissue and aortic arch from PfnHet (A.Arch: 1.08±0.21 versus PfnWT 1.96±0.13 relative D.U., when corrected for RasGAP loading control; P<0.001) whereas levels for SR-B1 were comparable in the two groups within all tissues examined (Figure 6A). As shown previously,3 pfn levels were halved in heterozygote tissues, with almost undetectable levels in the muscle. Moreover, CD36 levels were comparable in mice fed a regular chow (Figure 6B).
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The relative expression of CD36 in the aortic arch was tested by immunohistochemistry, which revealed a preferential staining of foam cells within atheromatous lesions and, at a much lesser extent, of the adventitia and outer portion of the media (Figure 6C). As reported previously, aortic endothelium was virtually negative for CD36. Thus, decreased CD36 levels in the aortic arch of PfnHet mice correlated with the anti-atherogenic phenotype and the attenuated lipid uptake in macrophages.
Decreased CD36 Levels and Binding/Uptake of oxLDL in Isolated Macrophages
We verified the effects of pfn on CD36 in bone marrow (BM)–derived macrophages. Under unstimulated conditions, macrophages derived from PfnHet showed a 50% decrease in total CD36 levels (0 hours: 0.24±0.04 versus PfnWT 0.46±0.08 relative D.U., when corrected for RasGAP; P<0.001; Figure 7A). The difference was enhanced by oxidized oxLDL that were able to upregulate CD36 in PfnWT cells, as reported previously,25 but only marginally in PfnHet (3 hours: 0.37±0.06 versus PfnWT 1.02±0.13 relative D.U., when corrected for RasGAP; P<0.0001). SR-B1 levels were not affected by oxLDL in either genotype.
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Also, flow cytometry analysis showed that macrophages from PfnHet had a defective oxLDL binding/uptake, when compared with PfnWT (Figure 7B). Together, these studies showed that the reduction in oxLDL binding and CD36 levels occurring in the aorta of PfnHet in situ were recapitulated in BM-derived macrophages.
Finally, CD36 internalization was significantly decreased in PfnHet macrophages (supplemental Figure IV).
PfnHet Macrophages Show Attenuated oxLDL-Mediated Inflammation
Inflammatory mechanisms contribute to initiation and progression of atherosclerotic disease.12 We reasoned that attenuated oxLDL uptake in PfnHet aorta and macrophages would correlate with decreased signaling through inflammatory pathways activated by oxLDL such as p3826 and JNK.27 Indeed, activation of p38 and of its downstream target ATF-2 was reduced in PfnHet macrophages when compared with PfnWT (Figure 8A). Similarly, expression of the proatherogenic enzyme iNOS,28 which is another p38-regulated gene,29 was significantly attenuated in PfnHet macrophages exposed to oxLDL. The activation of JNK1/2 was comparable in PfnHet and PfnWT cells on oxLDL.
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Changes in the activation of p38 were observed also in vivo. Under HCD, phospho-p38 levels in the aortic arch of PfnHet were significantly lower than in PfnWT (Figure 8B; 0.21±0.04 versus PfnWT 0.35±0.09 relative D.U., when normalized for total p38; P<0.005). These studies demonstrated that the activation of the stress-induced p38 pathway on modified lipids was partially abrogated in both PfnHet macrophages and PfnHet aorta.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study addressed the role of pfn in atherosclerotic lesion onset. Pfn heterozygosity resulted in a dramatic decrease in lesion burden, thus indicating that pfn levels modulate processes critical for atherogenesis. We identified 2 mechanisms that could account for the protection in PfnHet: (1) a preserved endothelial function, and (2) a defective uptake of oxLDL in macrophages.
Pfn and Endothelial Function
When exposed to HCD, the PfnHet aorta showed preserved endothelial-dependent NO signaling and tamed VCAM-1 expression at lesion-prone sites, compared with PfnWT. Similarly, inhibition of de novo pfn synthesis in cultured ECs resulted in reduced injury on oxLDL. How does attenuated pfn expression protect from endothelial dysfunction? The comparison of PfnHet fed normal diet versus HCD indicated that pfn levels modified the response of the endothelium to atherogenic injury rather than its basal function, suggesting that pfn is a relevant component of the stress response triggered by modified lipids.
Several reports indicate that pfn levels may play a role in endothelial function. Pfn upregulation was required for oxLDL-induced adhesion molecule expression in ECs.11 Pfn overexpression in ECs resulted in increased adhesion and migration.30 Moreover, Dardik et al showed that together homocysteine and turbulent flow conditions, both promoting factors for atherosclerosis, enhanced pfn gene expression in ECs.31 Thus, these studies suggest that biomechanical and metabolic abnormalities relevant to vascular disease elicit the upregulation of pfn, which in turn could integrate these cues into remodeling of EC cytoskeleton. In this view, cytoskeletal changes, other than pfn, were shown to regulate eNOS activation (reviewed in ref.32). Lack of direct interaction between pfn and eNOS either in vitro or in cultured ECs (our unpublished observations) suggests that eNOS activation may be modulated via pfn association to one or more of its binding partners.
Profilin-1, CD36, and oxLDL Uptake
In addition to protecting from endothelial dysfunction, pfn heterozygosity limited oxLDL uptake and alleviated oxLDL-mediated inflammation in macrophages.
PfnHet expressed lower aortic levels of CD36, which plays a major role in modified LDL uptake in macrophages33 and may promote19 or antagonize34 lesion formation. Decreased CD36 in the aorta of PfnHet could have merely reflected lesser vascular accumulation of macrophages, which express high levels of CD36. However, we found that attenuated macrophage recruitment may not be the only explanation for reduced CD36 levels in PfnHet. Indeed, PfnHet macrophages exhibited a defective internalization for CD36, but not for SR-B1, indicating that pfn controls endocytosis of membrane CD36. A proteomic approach in mouse brain demonstrated that pfn could complex with critical components of the endocytotic machinery, including clathrin.10 However, the definitive role of profilins in endocytosis is far from being understood. Recently, Gareus et al showed that reducing pfn-2 levels led to increased endocytosis in neurons.35 The apparent discrepancy between our findings and Gareus’ report could be explained by the distinct sets of endocytosis-related partners engaged by the 2 profilin isoforms10 or the prevalent expression of pfn versus pfn-2 in macrophages (our unpublished observations). Also, the difference between CD36 and SR-B1 is potentially relevant because, albeit structurally related, they rely on distinct mechanisms for lipid transport across the membrane.36
Both aorta and derived macrophages of PfnHet displayed inhibition of inflammatory pathways elicited by lipids, when compared with respective wild-type controls. Specifically, the activation of p38 and 2 of its downstream targets, ie, ATF-2 and iNOS,29 was largely prevented. Decreased activation of p38 pathway in PfnHet may result from attenuated oxLDL uptake, thus limiting upstream events in the proinflammatory cascade that promotes plaque onset and progression (reviewed in ref.12).
Taken together, our results provide evidence that pfn levels modulate early atheroma formation in a mouse model of disease. At least 2 aspects of these studies may be relevant to human cardiovascular disease. First, increased pfn in the aorta of diabetic patients11 could contribute to the heightened risk of atherosclerosis-related events observed in diabetes.37 Second, differences in pfn levels may contribute to atherosclerosis susceptibility in the general population. Ongoing studies in humans should help clarify whether pfn levels can predict atherosclerosis initiation and validate pfn as a marker for cardiovascular disease.
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Acknowledgments |
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We thank Walter Witke (EMBL-Monterotondo, Italy) for the profilin-1 mice and the profilin antibody; Monty Krieger (MIT, Boston) for the SR-B1 antibody; Rita Barcia (SERI, Boston) for help with confocal microscopy; Carla M. Harris (MMPC, VUMC) for lipoprotein analysis; Ronald Jandacek (MMPC, University of Cincinnati) for absorption tests; Tony Walshe (SERI, Boston) for help with blood pressure recording; A. Doria, C. Gerhardinger and M. Lorenzi for critical reading of the manuscript.
Sources of Funding
This work was supported by a CDA-JDRF (#2-2004-609; to G.R.R.) and the "U. Di Mario" Fellowship-FO.Ri.SID (to G.R.R.).
Disclosures
None.
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Footnotes |
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Original received March 1, 2007; revision received May 9, 2007; accepted June 25, 2007.
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References |
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