Author + information
- Received January 17, 2012
- Revision received April 9, 2012
- Accepted April 16, 2012
- Published online August 21, 2012.
- Deepanjana Das, BS⁎,
- Joseph Gawdzik, BS⁎,
- Lisa Dellefave-Castillo, MS⁎,
- Elizabeth M. McNally, MD, PhD⁎,†,
- Aliya Husain, MD‡,
- Jai Raman, MD, PhD§ and
- Marion A. Hofmann Bowman, MD, PhD⁎,⁎ ()
- ↵⁎Reprints requests and correspondence:
Dr. Marion A. Hofmann Bowman, Section of Cardiology, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC 6088, Chicago, Illinois 60637
Objectives The purpose of this study was to determine the relevance of S100A12 expression to human thoracic aortic aneurysms and type A thoracic aortic aneurysm dissection and to study mechanisms of S100A12-mediated dysfunction of aortic smooth muscle cells.
Background Transgenic expression of proinflammatory S100A12 protein in murine aortic smooth muscle causes thoracic aneurysm in genetically modified mice.
Methods Immunohistochemistry of aortic tissue (n = 50) for S100A12, myeloperoxidase, and caspase 3 was examined and S100A12-mediated pathways were studied in cultured primary aortic smooth muscle cells.
Results We found S100A12 protein expressed in all cases of acute thoracic aortic aneurysm dissection and in approximately 25% of clinically stable thoracic aortic aneurysm cases. S100A12 tissue expression was associated with increased length of stay in patients undergoing elective surgical repair for thoracic aortic aneurysm, despite similar preoperative risk as determined by European System for Cardiac Operative Risk Evaluation. Reduction of S100A12 expression in human aortic smooth muscle cells using small hairpin RNA attenuates gene and protein expression of many inflammatory- and apoptosis-regulating factors. Moreover, genetic ablation of the receptor for S100A12, receptor for advanced glycation end products (RAGE), in murine aortic smooth muscle cells abolished cytokine-augmented activation of caspase 3 and smooth muscle cell apoptosis in S100A12-expressing cells.
Conclusions S100A12 is enriched in human thoracic aortic aneurysms and dissections. Reduction of S100A12 or genetic ablation of its cell surface receptor, the receptor for advanced glycation end products (RAGE), in aortic smooth muscle resulted in decreased activation of caspase 3 and in reduced apoptosis. By establishing a link between S100A12 expression and apoptosis of aortic smooth muscle cells, this study identifies novel S100A12 signaling pathways and indicates that S100A12 may be a useful molecular marker and possible target for treatment for human aortic diseases.
- aortic smooth muscle cells
- cell death
- receptor for advanced glycation end products
- S100 proteins
- thoracic aortic aneurysm and dissection
Thoracic aortic aneurysms (TAA) encompass a broad range of degenerative, genetic, structural, and acquired disease states and can be complicated by potentially life-threatening type A thoracic aortic aneurysm dissection (TAAD). There is growing evidence of genetic variation in genes known to be critically important in the development of TAA, including FBN1 encoding fibrillin 1, TGFBR1/2 encoding transforming growth factor-β receptors, and ACTA2 and MHY11, which encode smooth muscle α-actin and myosin heavy chain, respectively, that predispose humans to aortic diseases (1). We recently reported that transgenic mice engineered to express human S100A12 in the aortic smooth muscle develop thoracic aneurysms (2), similar to mouse models of Marfan syndrome (3). S100A12 is a proinflammatory protein that activates the receptor for advanced glycation end products (RAGE) (4) and accelerates atherosclerosis in apolipoprotein A-deficient mice (5). Moreover, S100A12 gene expression in human peripheral blood cells is one of the most predictive genes for obstructive coronary artery disease (6), and S100A12 is expressed in smooth muscle and inflammatory cells in ruptured coronary artery plaque of victims of sudden cardiac death (7), suggesting a critical role of S100A12 in vascular disease and atherothrombosis. Although the expansion of the aortic wall occurs slowly and often is asymptomatic clinically, dissection of the medial layer with bleeding within and along the aortic wall occurs suddenly and leads to major complications, including rupture, ischemia, and arterial thrombosis with embolization, accounting for at least 16,000 deaths annually in the United States (1,8). The precipitating mechanisms underlying dissection are incompletely understood and include hemodynamic factors, endothelial factors, as well as dysfunctional smooth muscle cells (SMC). Moreover, inflammation and the presence of inflammatory cells within the aortic media recently were emphasized as potential mediator for dissections (8,9). Because S100A12 is a potent proinflammatory and chemoattractant protein, and TAA develops in S100A12 transgenic mice, we wished to examine S100A12 expression in human aortic diseases. We found strong expression of S100A12 in inflammatory cells and in SMC in all cases of thoracic aneurysms with type A dissection and in approximately 25% of cases of clinically stable TAA. Furthermore, studies on cultured human aortic smooth muscle cells (HASMC) obtained from patients with TAA or TAAD showed a direct role of S100A12 in mediating apoptosis. Together, these data demonstrate that S100A12 is up-regulated in TAAD and may contribute to the pathogenesis of TAAD by initiating apoptosis of SMC, at least in part via increased oxidative stress.
The University of Chicago Medical Center Pathology Tissue Bank was searched between 2007 and 2010, and 20 cases of elective TAA repair were chosen randomly. To validate our findings from the initial 20 randomly chosen patients, 30 additional patients with TAA or TAAD were studied with either emergent surgery for type A aortic dissections or elective surgery for large aneurysms (>5.5 cm). All patients had no pre-operative diagnosis or known coronary artery diseases or peripheral artery disease. Retrospective data were collected by reviewing patient medical records, and operative risk before surgery was calculated using the additive and the logistic European System for Cardiac Operative Risk Evaluation (EuroSCORE). Normal control aortic tissue was obtained from heart donors (n = 3, 2 men, age: 44 ± 12 years), aortic valve replacement for stenotic tricuspid aortic valve with normal aorta (n = 2, 2 men, age: 48 ± 8 years), and elective left ventricular assist device (n = 1, woman, age: 32 years). Serial histological sections were prepared from paraffin-embedded aortic tissue blocks and were stained with hematoxylin and eosin, Verhoeff Van Giessen, and Movat, and immunochemistry analyses were carried out with staining with αS100A12 immunoglobulin G (IgG, Abcam 37657, Abcam Cambridge, United Kingdom), α-myeloperoxidase (MPO) IgG (Abcam 9535, Abcam, Cambridge, Massachusetts), and α-smooth muscle actin (Sigma-Aldrich). Staining for S100A12 was semiquantified by 2 blinded investigators (D.D., M.A.H.B.) as absent (0), mild (1+), moderate (2+), or intense (3+) in cells that demonstrated either positive or negative results for MPO staining. Loss of aortic medial elastic fibers was graded as 1 (trace), 2 (mild), 3 (moderate), and 4 (full-thickness loss), as previously reported by Roberts et al. (10,11).
HASMCs were cultured from operatively excised aortic tissue (bicuspid aortic valve: n = 3, TGFBR1 mutation: n = 2, FBN1 mutation: n = 2, unknown cause: n = 3), as previously described (2). Control HASMC were obtained from heart donors (n = 3), aortic valve replacement for stenotic tricuspid aortic valve with normal aorta (n = 2), and elective left ventricular assist device (n = 1). Murine aortic smooth muscle cells (MASMC) were obtained from hemizygous S100A12 transgenic mice of the C57BL6/J strain previously generated (2) and from mice mated with C57BL6/J mice lacking RAGE (RAGE−/−), supplied by Dr. Ann Marie Schmidt (New York University, New York, New York). Only cell cultures demonstrating negative results for contaminating leukocytes (α-CD45.2 antibody from Pharmingen no. 559985 and α-CD68 antibody from Pharmingen no. 556059, BD Biosciences Pharmingen, San Diego, California) were propagated. SMC characterized by staining for smooth muscle actin (Sigma-Aldrich) from passage 4–7 were used for experiments. If indicated, soluble RAGE, the extra cellular ligand binding domain of RAGE (20 μg/ml, R&D Systems), bovine serum albumin (20 μg/ml, Sigma-Aldrich), α lipoic acid (1 μM, 10 μM, and 100 μM, AstraZeneca), or diphenyleneiodonium (DPI) (1 and 10 μM, Sigma-Aldrich) was added before stimulation with lipopolysaccharide (LPS) (100 ng/ml, Sigma-Aldrich), tumor necrosis factor alpha (TNFα) (10 ng/ml, Pierce, Rockford, Illinois), or αFas IgG (CH 11, EMD Millipore). Where indicated, HASMC were transfected with small hairpin RNA (shRNA) encoding for S100A12 and control shRNA (SABiosciences) using the lipofectamine method (Clontech). Transfected HASMC were selected 3 days later by fluorescence-activated cell sorting (FACS) using the GFP-tag coexpressed in the shRNA plasmids before further analysis.
Immunoblotting was performed on whole cell lysate (Pierce) for S100A12 (Abcam 37657), b-actin, caspase 3, Fas (Cell Signaling Technology, Danvers, Massachusetts), Fas ligand (Cell Signaling Technology, Beverly, Massachusetts), and deoxyribonucleic acid (DNA) fragmentation factor A (DFFA, Millipore), and semiquantitative analysis was performed using ImageQuant TL software (Amersham/GE Healthcare, Little Chalfont, United Kingdom). Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed from high-quality total RNA (Qiagen) after transcription to complementary DNA (cDNA, Qiagen whole transcriptome assay) using SYBR GreenER (Invitrogen, Carlsbad, California) amplification with an IQ5 cycler (Bio-Rad, Hercules, California). Pathway focused microarray RT-PCR was purchased from SABiosciences. Primers were designed on the primer bank MGH library. Relative levels of gene expression were calculated: relative mRNA expression = 2exp (ΔCT target gene − ΔCT housekeeping gene). Immunofluorescent assays for caspase 3 (BioVision, Milpitas, California) were analyzed on a Fluostar Optima plate reader (BMG Labtech, Ortenberg, Germany). For immunofluorescence microscopy, SMC were grown on glass cover slips, fixed with 20% ice cold methanol, or analyzed for apoptosis using a assay for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) from Roche, Applied Science, Cells were imaged by confocal laser microscopy (Zeiss 500 Meta), and fluorescent intensity was analyzed in 5 high-power fields, at least in 200 cells.
The study and all procedures were carried out with the approval of the Chicago Institutional Review Board and the Institutional Animal Care and Use Committee.
Descriptive statistics (percentage, mean, and SD), crude comparisons based on chi-square statistics, and plots were used to describe the patient cohort. All continuous data are reported as mean ± SD, and discrete variables are summarized by percentage. All experiments were performed in at least 3 replicates per group, and all cell culture experiments were repeated 3 times. The independent-sample t test and 1-way analysis of variance were used for mean comparisons between 2 or multiple groups, respectively. The Spearman rank correlation, 2-sided chi-square test for 2 independent proportions, and Fisher exact test also were performed to describe association between different outcome variables. We applied the Bonferroni correction to the 0.05 significance level to adjust for multiple comparisons.
S100A12 protein is significantly up-regulated in human thoracic aortic type A dissection
We first studied S100A12 expression in aortic tissue collected from 20 patients undergoing elective repair for TAA (Table 1). All patients had a computed tomography image of the chest or an angiogram showing a TAA of at least 4.5 cm and were referred for nonurgent surgery. Of those 20 patients, 7 had a bicuspid aortic valve, 2 had Marfan syndrome, 1 had familial TAA, and 10 patients had no specific diagnosis before surgery. We found S100A12-positive cells within the medial layer in 7 cases, and representative images are shown in Figure 1. S100A12 expression was observed in necrotizing aortitis (Fig. 1E), in atherosclerosis (Fig. 1H), and in cases with histological evidence of subacute dissection (Fig. 1K), whereas we did not observe S100A12 expression in the medial layer in the other 13 cases of TAA (Fig. 1B) or in nonaneurysmal control aortic tissue (n = 5, not shown). Importantly, all patients in this group underwent elective and nonurgent repair of TAA. Notably, the 7 patients with S100A12 expression in the aorta had a significantly increased length of hospitalization after surgery, with a mean duration of 24 days (range, 9 to 49 days) compared with a mean duration of 8 days (range, 5 to 13 days) in the 13 S100A12-negative TAA patients (p = 0.04). The post-operative complications we encountered included prolonged ventilation resulting from respiratory failure (n = 2) and multiorgan failure with need for dialysis (n = 1). One patient died on post-operative day 15 of multiorgan failure occurring after revision for aortic graft dehiscence in the S100A12-positive group; this is compared with no deaths in the S100A12-negative group. To account for pre-operative comorbidities, we calculated the EuroSCORE, a widely used score to predict mortality resulting from cardiac surgery (12). We found no difference in the EuroSCORE between the S100A12-positive and the S100A12-negative cases (10.0 and 8.7, respectively, p = 0.62) (Fig. 1N). Moreover, there was also no difference in age between the 2 groups. This suggests that expression of S100A12 in aortic tissue in TAA is a marker of post-operative complications with increased length of hospital stay.
In the S100A12-positive cases, we found S100A12 mostly expressed in the medial aortic layer associated with inflammatory changes or histological evidence of subacute dissection (Figs. 1D to 1L). By contrast, in TAA cases showing medial degeneration, but without histological evidence of dissection, S100A12 was not expressed or only minimally expressed; except in 1 of the 13 S100A12-negative TAA cases, we found histological evidence of chronic dissection. Therefore, to test directly the hypothesis that S100A12 is a marker of aortic dissection, we selected 14 patients with from our histology archive in whom TAAD was diagnosed prior undergoing urgent surgical repair. As expected, these TAAD patients had higher pre-operative EuroSCOREs (mean: 17.3, range: 9 to 21) (Table 1), and on histological examination, all 14 cases had S100A12-positive cells in the medial layer of the ascending thoracic aorta. Costaining for MPO, a peroxidase enzyme expressed in all myeloid cells and abundantly enriched in neutrophil granulocytes, revealed S100A12 expression in MPO-positive inflammatory cells and also in MPO-negative SMC (Fig. 2). To account for factors possibly related to disease severity, we selected 16 more cases of advanced TAA with aneurysms exceeding 5.5 cm undergoing elective and nonurgent repair of TAA. We found S100A12 expression in 4 cases (25%) of TAA of more than 5.5 cm, compared with 100% in TAAD (p < 0.0001). The S100A12 expression in those cases of large TAA was localized to a tear with dissecting split of the medial layer (n = 2), and in 2 cases, it was localized to medial degeneration without dissection. The intensity of S100A12 stain overall was less in the clinically stable TAA cases, compared with the cases with acute dissection. The specificity of the staining for S100A12 was verified by immunoblot analysis of protein isolated from the aortic medial layer showing the expected 12 kDa size (Fig. 2D). To determine further a possible association of S100A12 expression in aneurysmal aortic tissue with other key processes of aortic remodeling, we graded the integrity of aortic medial fibers. The morphological features of elastic fibers serve as a good marker of chronic aortic remodeling, such as replacement with fibrous tissue and deposits of mucoid material, and are easily discernible, and the grading of elastic fiber loss is highly reproducible. This is in contrast to acute type A dissection, where abnormal ascending aortic elastic fibers uncommonly precede acute dissection (10,11). Online Figure 1 shows the frequency of elastic fiber degradation in elective TAA with regard to S100A12 protein expression. For neurysms smaller than 5.5 cm, intact or only minimal fiber loss (grade 0 to 1) is more prevalent among S100A12-negative aneurysms (61% vs. 0%, p = 0.007), whereas advanced elastolysis grade 3 or 4 is more prevalent among S100A12-positive TAA (57% vs. 8%, p = 0.015). This relationship was not observed among larger TAAs, and there was no difference in high-grade elastolysis in cases of large TAA of more than 5.5 cm with and without S100A12 expression (50% and 33%, respectively, p = 0.55). These data suggest that S100A12 expression in aneurysmal aorta is associated with proteolysis of elastic fibers, because all S100A12-positive TAAs had evidence of grade 2 or higher elastic fiber degradation.
Moreover, expression of activated caspase 3, an enzyme known to play a central role in the execution phase of cell apoptosis, also was present in all TAAD tissue and colocalized with S100A12 to the area near the dissection tear in the tunica media. Caspase 3 was not detected in S100A12-negative cases (Fig. 2).
Furthermore, we examined cultured aortic SMC and found increased mRNA and protein expression of cell death receptor Fas and Fas ligand in the cell lines that also express S100A12 (3.5-fold increase in Fas protein and 2.6-fold increase in Fas ligand protein, p < 0.01) (Online Fig. 2). As expected, we found enhanced mRNA for several members of the transforming growth factor β signaling pathways in HASMC harvested from TAA compared with control HASMC; however, there was no difference with regard to S100A12 expression in the different TAA-HASMC lines (Online Fig. 2).
Reduction of endogenous S100A12 in cultured HASMC attenuates apoptosis
The relationship of S100A12 to markers of apoptosis was studied in cultured HASMC harvested from patients undergoing surgical repair for TAA or TAAD and in control HASMC from patients undergoing cardiac surgery without TAA or TAAD. We found variable expression of S100A12 in HASMC from TAA or TAAD by immunoblotting and no expression of S100A12 in control HASMC (data not shown). Three independent HASMC lines with endogenous S100A12 expression were studied; one carried a known mutation in the FBN1 gene (R529X) harvested from a patient with Marfan syndrome, and one cell line carried a mutation in the TGFBR1 gene (977A→C, K326T) from a patient with familial TAA, and a third TAA-HASMC line was without specific genetic diagnosis. HASMC were transfected transiently with interfering short hairpin RNA (shRNA) to knock down endogenous S100A12 or with control shRNA. After isolation of the cells positive for green-fluorescent protein (GFP) by flow cytometry, we confirmed complete absence of S100A12 in the HASMC transfected with shRNA-S100A12 and endogenous S100A12 expression in HASMC transfected with control shRNA (Fig. 3). We next examined gene expression in the FBN1R529X HASMC endogenously expressing S100A12 (shRNA-control transfected cells) and compared this with FBN1R529X HASMC with reduction of S100A12 (shRNA-S100A12 transfected cells) using pathway-focused RT-PCR microarrays for apoptosis and inflammation (SABiosciences). We identified broad down-regulation of apoptosis- and inflammation-regulating genes. Of the 84 genes present on the microarray plate, 23 genes were more than 2-fold and 49 genes were <2-fold down-regulated on the apoptosis array, and 19 genes were more than 2-fold and 56 genes were <2-fold down-regulated on the inflammation microarray (Online Table 1). Because we found strong reduction in Fas, caspase 3, and DFFA, we next examined these genes using quantitative RT-PCR in independent complementary DNA samples of the 3 S100A12-expressing HASMC lines. As shown in Figure 3D, reduction of S100A12 abolished mRNA for Fas, caspase 3, and DNA-fragmentation factor (p < 0.0001). Moreover, reduction of S100A12 suppressed protein expression for Fas and cleaved caspase 3 and DNA- fragmentation factor by 40% to 60% in S100A12-expressing HASMC (p = 0.02), but had no effect on S10012-negative HASMC (Figs. 3E and 3F). To confirm a critical role of S100A12 on caspase 3 activation, we stimulated S100A12-expressing HASMC with apoptosis-initiating monoclonal Fas IgG and observed a 2.8-fold increase in active caspase 3 using an immunofluorescent active caspase 3 assay. shRNA reduction of S100A12 reduced Fas-IgG–stimulated active caspase 3 by 80% (p < 0.001) (Fig. 3G). These data suggest that S100A12 is a critical nodal point in many apoptosis- and inflammation-regulating genes.
S100A12 is induced in HASMC by cytokines, and knockdown of S100A12 attenuates cytokine-induced activation of caspase 3 in a partial RAGE-dependent manner
We previously showed that LPS induces S100A12 in HASMC in agreement with other studies that showed induction of S100A12 in response to cytokine stimulation (13,14). Moreover, studies of the S100A12 gene indicated that CCAAT/enhancer-binding protein beta and protein kinase C are critical regulators of transcription in response to LPS (15). As shown in Figure 4A, stimulation with low-dose LPS or with the distinct cytokine TNFα induced S100A12 in normal HASMC. PCR amplification detected complementary DNA for Fas, caspase 3, and DFFA in both control HASMC (bovine serum albumin) and in cytokine-treated HASMC (LPS or TNFα), with significantly higher gene expression for Fas, caspase 3, and TNFα in cytokine-treated HASMC compared with control HASMC (p < 0.05 for each gene). Importantly, cotreatment with shRNA-S100A12 attenuated cytokine-induced gene expression for Fas, caspase 3, and DFFA by 70% to 90% (p < 0.01 for each gene), whereas control shRNA had no effect on cytokine-induced up-regulation of apoptosis-controlling genes (Fig. 4B). Quantification of active caspase 3 showed a 1.6-fold increase in LPS-treated cells and a 1.9-fold increase in TNFα-treated cells compared with baseline; this was attenuated in HASMC cotreated with shRNA-S100A12 by 74% and 65%, respectively (p < 0.05) (Fig. 4C).
To study potential mechanisms involved in S100A12-mediated apoptosis, we used MASMC. Briefly, transgenic mice with expression of human S100A12 in aortic smooth muscle were generated previously (2) and then crossed with mice lacking RAGE (16). We previously reported that transgenic S100A12-MASMC had higher levels of oxidative stress and interleukin-6 secretion, among other changes, characteristic of a phenotypic switch from a contractile to a synthetic phenotype, and these changes were attenuated by cotreatment with soluble RAGE (2), the S100A12 ligand-binding domain of RAGE, thus preventing activation of cell surface RAGE. We then expanded these findings by measuring activated caspase 3 in S100A12-expressing MASMC and found a slight increase compared with wild-type MASMC (1.3-fold, p = 0.04). Importantly, on stimulation with low-dose LPS (100 ng/ml) or low-dose TNFα (20 ng/ml), the S100A12-MASMC showed strong activation of caspase 3 (4- and 2.9-fold, respectively) compared with a 1.3- and 1.4-fold increase in wild-type MASMC (Fig. 5A). Moreover, cytokine stimulation caused enhanced DNA fragmentation as visualized by TUNEL staining in S100A12-MASMC, but only minimally in wild type-MASMC at the studied concentrations for LPS and TNFα (15% vs. 7% and 12% vs. 7%, respectively, p < 0.01) (Fig. 5B). Most importantly, MASMC lacking RAGE were protected from the synergistic effects of S100A12 and LPS or TNFα stimulation on caspase 3 activation and cell apoptosis. Activated caspase 3 was reduced in cytokine-stimulated S100A12- MASMC by 58% and 52% in cells lacking RAGE compared with S100A12- MASMC with intact RAGE signaling (p < 0.01) (Fig. 5A). This was paralleled with a significant reduction of TUNEL-positive MASMC (Fig. 5B). Together, we found that smooth muscle targeted forced expression of human S100A12 in MASMC markedly enhanced cytokine-induced apoptosis, and this was dependent on RAGE, the cell surface receptor for S100/calgranulins endogenously expressed on smooth muscle cells.
Given the importance of reactive oxygen species to inflammation, cell death, and apoptosis, we probed oxidative stress-related changes. We found an increase in oxidized DNA using immunofluorescence microscopy with anti–8-hydroxy-2-deoxyguanosine IgG with approximately 60% of all nuclei staining positive for this marker of oxidative damage to the DNA in S100A12 transgenic MASMC, compared with a less than 2% baseline in wild-type MASMC (p < 0.001) (Fig. 5C). S100A12-MASMC lacking RAGE had reduced significantly the number of oxidized nuclei (25%, p = 0.01) compared with S100A12-MASMC with intact RAGE signaling, confirming that the pro-oxidative effect of S100A12 is mediated, at least in part, by RAGE. Treatment with DPI, an inhibitor of the nicotinamide adenine dinucleotide phosphate-oxidase NADPH oxidase also attenuated DNA oxidation, even in cells lacking RAGE (12% and 25%, p = 0.04). These data suggest that oxidative stress in cultured smooth muscle cells is a critical downstream effect of S100A12 mediated in large part by RAGE and by NADPH-oxidase. Therefore, we tested the effects of antioxidant compounds on apoptosis markers in HASMC with endogenous S100A12 expression (harvested from TAA or TAAD cases) and in control HASMC without S100A12 expression. As shown in Figures 5D to 5F, pre-treatment with α-lipoic acid or DPI reduced active caspase 3, the apoptosis index determined by TUNEL assay, and reduced the index for cells with oxidized DNA in a dose-dependent manner.
An inflammatory component was identified previously in TAA and TAAD, composed primarily of T cells and macrophages (9). Our study demonstrates that S100A12, a cytokine-like protein with proinflammatory signaling, is highly enriched in myeloid and in SMC in type A aortic dissection. On the basis of our cell culture data and the known pathological effects of S100A12 on smooth muscle cells, we speculate that S100A12 is highly pathogenic in TAA and may predispose patients to aortic dissection. S100A12 is also known as calgranulin C or EN-RAGE (extracellular newly identified RAGE binding protein) because it activates pattern recognition receptor RAGE (4) and is likely also to activate Toll-like receptor 4, because this was shown recently for the homolog S100A8/9 (17). Moreover, S100 proteins are expressed endogenously in myeloid cells and at high levels in neutrophil granulocytes and are implicated in the antibacterial and antifungal defense (18) by activating the p67phox/NADPH-oxidase system to initiate a respiratory burst in phagocytes (19). Similar to its endogenous role in phagocytes, our laboratory recently showed binding of S100A12 to NADPH-oxidase subunit Nox-1 in vascular smooth muscle cells (5), and importantly, Nox-1 inhibition attenuated S100A12-mediated dysfunction of aortic SMC (20).
Our laboratory recently showed a proapoptotic function of S100A12 with increased expression of FAS, caspase 10, and caspase 3 in human airway SMC (21) and thereby extended previous studies showing that excess amounts of S100A8/9 induce apoptosis and cell death in a variety of cell types (22,23). Moreover, Boyd et al. (24) found a 40-fold increase in S100A8/9 mRNA in cardiomyocytes in a mouse model of sepsis, and S100B was up-regulated in ischemic cardiomyocytes (25). Importantly, in both studies, up-regulation of S100 proteins was associated with increased cell death and tissue damage that was attenuated by interruption of RAGE signaling. Our studies presented here demonstrate that down-regulation of the ligand S100A12 attenuates cell death of SMC with pathological endogenous expression of S100A12 and in SMC with cytokine-induced expression of S100A12. These findings support the hypothesis that S100A12 is an important regulator of cell survival and cell death.
S100/calgranulins are involved in the regulation of numerous intracellular activities, such as protein phosphorylation, enzyme activities, cell proliferation and differentiation, cytoskeletal rearrangement and membrane organization, protection from oxidative stress, and regulation of intracellular Ca+2 homeostasis, and have been implicated in intercellular regulation of chemotaxis, phagocytosis, and immunoregulation. Therefore, the specificity for S100/calgranulin-mediated effects likely depends on the tissue distribution and cell type. This view is supported by recent findings in mice lacking S100A9 in which constitutive deletion of murine S100A9 in apolipoprotein E-deficient mice attenuated atherosclerosis (26), whereas the lack of S100A9 in bone marrow–derived cells did not attenuate atherosclerosis in low-density lipoprotein receptor–deficient mice (27). Therefore, pathological expression of S100/calgranulins within the vasculature may be an important accelerator of vascular disease, particularly because S100A12 is not present in normal vascular tissue, but is highly up-regulated in smooth muscle cells of diseased blood vessels, such as in human ruptured coronary artery plaques (7), in human atherosclerosis (5) and, as shown in this study, also in type A aortic dissections and, to a lesser degree, in clinically stable TAA. It is intriguing that TAA and TAAD are such focal diseases, and because there is no access to distal vascular tissue other than the surgically removed aneurysmal tissue in a given patient with TAA or TAAD, we cannot exclude the possibility that S100A12 could be expressed in other vascular beds beyond the aneurysmal thoracic aorta in those patients with TAA or TAAD. It is interesting to note that S100A12 expression in explanted SMC persists over several cell culture passages, and this finding deserves further study. We speculate that epigenetic changes occurring within the S100 gene cluster may control S100 protein expression. This view is supported by the presence of several CpG islands within the S100 gene cluster and within the first intron of the S100A12 gene (28). Therefore, methylation and demethylation of regulatory DNA may explain the cell-specific expression of S100 proteins, which frequently is up-regulated or down-regulated in various pathological states, including cancer and vascular diseases.
S100A12 is more than an inflammatory biomarker. It causes dysfunction of vascular smooth muscle with an increase in matrix metalloproteinase-2 protein, interleukin-6, transforming growth factor-β signaling, and reduction of contractile fibers leading to the development of thoracic aortic aneurysms in transgenic mice expressing human S100A12 driven by the smooth muscle 22-α promoter (2), demonstrating a clear pathological effect of S100A12 in vascular smooth muscle. S100 proteins and other ligands to RAGE and Toll-like receptors, such as HMGB1 protein, previously were implicated in the process of sterile inflammation associated with dying cells (29). Our report for the first time shows that knockdown of S100A12 attenuates many apoptosis- and inflammation-regulating genes, including Fas-mediated apoptosis pathways. One interesting finding in our study was the strong attenuation of inflammasome-associated pathways, including PYCARD and caspase 1, after reducing S100A12. Caspase 1 is activated in response to multiple stimuli, but after being activated, results in a conserved program of cell death referred to as pyroptosis (30). S100 proteins and other damage-associated molecular pattern molecules are good candidate molecules to activate caspase 1, and further exploration of these pathways may shed light on the intracellular function of S100/calgranulins.
The proapoptotic function of S100A12 is mediated, at least in part, by its cell surface receptor RAGE, which is expressed in many cells, including SMC, endothelial cells, and inflammatory cells (31). There is mounting evidence of RAGE being a disease amplifier for initiation and progression of vascular diseases (reviewed in Yan et al. ), including aortic aneurysms (33). RAGE-mediated cell perturbation is at least in part dependent on redox-sensitive signals such as the translocation of redox-sensitive nuclear factor κ B, which, for example, is attenuated in stimulated endothelial cells by pre-treatment with antioxidant α lipoic acid (34). Using α lipoic acid and DPI, we showed marked inhibition of S100A12-mediated cell death. This is in agreement with a recent study by Kim et al. (35) demonstrating the ability of α lipoic acid to reduce vascular smooth muscle cell apoptosis by restoring intracellular redox status. Indeed, oxidative stress plays a key role in the pathogenesis of thoracic aortic dissections, as recently demonstrated by several studies. For example, Liao et al. (36) used comparative proteomics and identified 126 proteins differentially expressed in the aortic media of patients with dissections and with normal aortas. Interestingly, increased lipid peroxidation and a more than 50% reduction in superoxide dismutase in aortic type A dissections was one of the most important changes noted in this study.
The coexistence of inflammatory cells with markers of apoptotic vascular cell death in the media of ascending aortas with aneurysms and type A dissection was noted previously by several investigators (8,9). However, it remains unknown whether apoptosis of medial smooth muscle cells occurs before dissection as a primary event leading to weakening and rupture of the aortic wall, or alternatively, as suggested by Roberts et al. (11), the intraluminal arterial pressure in the false luminal channel compresses the adjacent media and induces cell death after, not before, acute dissection.
Critical pathways involved in TAA and progression to TAAD include immunological processes, such as T-cell and natural killer cell pathways, oxidative stress, depletion of vascular smooth muscle through the process of apoptosis, and the destruction of the extracellular matrix by matrix metalloproteinases (1). Here we showed evidence that S100A12 is highly expressed in the medial layer in acute type A aortic dissection. Because all samples from TAAD cases expressed S100A12 regardless of the heterogeneity present in this sample group, we speculate that S100A12 expression in aortic smooth muscle is a common response to multifactorial injury. This is supported by our findings in clinically stable TAA, in which we found S100A12 expression in approximately 25%, most commonly associated with histological evidence of dissection. Alternatively, it is possible that S100A12 is a marker of the inflammatory changes that occur in the aortic wall after acute or chronic dissection, rather than being a predisposing factor for dissection. This underscores the complex biology of aortic disease, and histological examination of aneurysmal or dissected aortic tissue is inevitably unlikely to determine the temporal relationship between vascular inflammation and aortic dissection. The mechanisms by which S100A12 becomes up-regulated in human aortic diseases is not understood completely, but it likely is in response to various inflammatory signals, because we demonstrated a robust induction of S100A12 in normal human aortic smooth muscle cells on treatment with LPS and TNFα. Further studies using aortic tissue, rather than in cultured aortic smooth muscle cells from patients with thoracic aneurysms, are needed to examine the impact of S100A12 on key vascular proteins, such as fibrillin, collagen, and others. One limitation of our study is the small number of patients and the heterogeneity within the control group. Importantly, reduction of S100A12 attenuates many apoptosis- and inflammation-regulating genes in HASMC with endogenous S100A12 expression and in cytokine-primed MASMC with forced expression of human S100A12. S100A12-mediated cell death is, at least in part, dependent on RAGE-generated and NADPH-oxidase–generated oxidative stress, because apoptosis is attenuated in smooth muscle cells lacking RAGE and in HASMC treated with the antioxidants α lipoic acid and DPI.
The authors thank Dr. Ann Marie Schmidt for the RAGE−/− mice, Dr. Sydeaka Watson for statistical advice, and the patients and their physicians involved in this study.
For supplemental figures and tables, please see the online version of this article.
This work was supported by funding from the National Institutes of Health (grant K08 HL090917–02 to Dr. Hofmann Bowman and grant 5R01HL078926–05 to Dr. McNally), the American Heart Association (grant 11UFEL 762056 to Ms. Das), the National Marfan Foundation, and the Doris Duke Charitable Foundation (to Drs. Hofmann Bowman and McNally). Dr. Hofmann Bowman is a recipient of the Doris Duke Clinical Scientist Development Award. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. The first two authors contributed equally to this work.
- Abbreviations and Acronyms
- deoxyribonucleic acid
- European System for Cardiac Operative Risk Evaluation
- human aortic smooth muscle cells
- immunoglobulin G
- murine aortic smooth muscle cells
- receptor for advanced glycation end products
- smooth muscle cells
- thoracic aortic aneurysms
- thoracic aortic aneurysm dissection
- tumor necrosis factor
- Received January 17, 2012.
- Revision received April 9, 2012.
- Accepted April 16, 2012.
- American College of Cardiology Foundation
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