Thromb Haemost 2018; 118(01): 210-213
DOI: 10.1160/TH17-07-0494
Letter to the Editor
Schattauer GmbH Stuttgart

The ADAM17 Metalloproteinase Maintains Arterial Elasticity

Alexandros Nicolaou
,
Bernd H. Northoff
,
Zhen Zhao
,
Alexander Kohlmaier
,
Kristina Sass
,
Stefan Rose-John
,
Sabine Steffens
,
Christian Weber
,
Daniel Teupser
,
Lesca M. Holdt
Further Information

Publication History

21 July 2017

24 October 2017

Publication Date:
05 January 2018 (online)

ADAM17 is a transmembrane protease that regulates inflammatory cytokines, receptors, or adhesion molecules by shedding.[1] [2] [3] ADAM17 is also expressed in atherosclerotic plaques,[4] [5] but although inflammatory pathways accelerate atherosclerosis,[6] we have recently identified an unexpected atheroprotective role of ADAM17.[7] [8] In Adam17 mutants, likely due to a deficit in tumour necrosis factor receptor 2 (TNFR2) shedding, the activity of membrane-tethered TNFR2 increased and led to increased adhesive properties and overproliferation.[7] This atheroprotective function could be attributed to a specific role in cells of the myeloid lineage.[9] To date, Adam17 has also been studied in other vascular diseases, such as aneurysms. Its role therein remained controversial, with one study reporting reduced ADAM17 expression in aneurysms,[10] and other studies showing a positive correlation between expression and aneurysm risk or occurrence.[11] [12] [13] In aneurysms, the integrity of the arterial wall is compromised by inflammatory processes and other extracellular matrix (ECM)-degrading factors, impairing elastin lamellae that support and transmit mechanical load to the cellular cytoskeleton.[14] [15] Whether and how ADAM17 links to these processes is unknown.

To investigate whether mouse Adam17 regulated arterial wall integrity, we studied effects of ADAM17 depletion by using Adam17 hypomorphic mutants in an LDL-receptor–deficient background (Adam17 ex/ex.Ldlr−/− ),[16] and myeloid-specific or endothelial-specific conditional knockouts in an apolipoprotein E-deficient background (ApoE−/−.LysMCreADAM17fl/fl; ApoE−/− .BmxCreADAM17fl/fl).[9] Vascular size was determined by quantifying intra-arterial lumen area in the aortic root, or, separately, arterial wall circumference and wall width in the brachiocephalic artery (BCA). Arterial ECM properties were determined by assessing elastic fibres with Verhoeff-van Gieson staining and by quantifying total collagen fibre content by PicroSirius Red imaging using polarized light (AxioVision KS400, Zeiss).[17] Atherosclerotic plaque size was measured as the area of Oil Red-stained tissue in sagittal arterial tissue sections.

We found that aortic root lumen area in hypomorphic Adam17 ex/ex.Ldlr−/− mutants was increased by 30 and 40% in males and females, respectively ([Fig. 1A]–[C]). In contrast, myeloid-specific Adam17 knockouts showed only a trend toward arterial dilation, which did not reach statistical significance, and endothelial-specific depletion did not show any dilation ([Fig. 1D], [E]). To test if ADAM17-dependent cell wall control was a more general function, we analysed the dimensions of the BCA of Ldlr−/− mice. BCAs from ApoE-deficient conditional Adam17 knockouts were not available for comparative analysis. Indeed, BCAs of hypomorphic Adam17 ex/ex.Ldlr−/− mutants were 24 to 39% wider than controls ([Fig. 1F]–[I]). In contrast, atherosclerotic lesion size was not larger in Adam17 hypomorphic mutants than in controls ([Fig. 1J]). This suggested that atherosclerotic plaque development and aneurysm onset were regulated independently by ADAM17, at least in the BCA of Ldlr−/− mice. Based on Picrosirius Red imaging,[17] and not distinguishing between different classes of fibres via birefringence colour discrimination,[18] total collagen fibre content in the intima and in the adventitia was found to be unaffected in the dilated Adam17 mutant BCAs ([Fig. 1K], [L]). Also, we did not observe arterial wall thinning in the mutant BCA ([Fig. 1M]). However, elastin fibres were altered: In wild type, BCAs elastin fibres were arranged in wavy bundles, while those in Adam17 ex/ex.Ldlr−/− mice were stretched-out and showed reduced undulation ([Fig. 1N]). To quantify this phenotypic switch, we determined the length of individual elastic fibres within subsegments of identical length (20 μm) in BCAs of mutants and wild-type mice (n = 3/3) and found a reduced unit fibre length ([Fig. 1O]). Thus, ADAM17 may protect the integrity of the arterial wall by regulating elastin fibre organization. To get first insights into how ADAM17 might control arterial wall integrity, we performed mRNA microarray profiling in aortic tissues of wild-type and Adam17 ex/ex.Ldlr−/− hypomorphic mutants (GSE80000). We did find not only a set of bona-fide atherosclerosis regulators among the top differentially abundant transcripts but also several cytoskeletal contractility regulators and factors required for ECM elastogenesis. The latter included the elastin microfibril-interface Emilin2, the elastin-binding laminin/fibronectin-interacting Fibulin 2, matrix metalloproteinases MMP3/12, the proteoglycan Syndecan 3, and the adhesion molecule and mechanoresponsive Pecam1 ([Fig. 1P]).

Zoom Image
Fig. 1 (A) Representative Oil-Red O staining of the aortic root of homozygous hypomorphic ADAM17 mutant mice (Adam17 ex/ex.Ldlr−/−) and ADAM17 wild-type Adam17wt/wt.Ldlr−/− . (BC) Quantification of arterial lumen area in the aortic root of (B) male and (C) female homozygous Adam17ex/ex.Ldlr−/− (ex/ex; n = 18/16), heterozygous Adam17wt/ex.Ldlr−/− (wt/ex; n = 19/21) and wild-type Adam17wt/wt.Ldlr−/− (wt/wt; n = 17/21) mice. (DE) Quantification of the lumen area of the aortic root of (D) myeloid-specific ApoE−/−LysMCreADAMfl/fl (n = 8) and control ApoE−/−-ADAMfl/fl (n = 5) and (E) inducible endothelial-specific ApoE−/−BmxCreADAMfl/fl (n = 5) and control ApoE−/−-ADAMfl/fl (n = 4) mice. (F) Representative Oil-Red O staining of the brachiocephalic artery (BCA) of homozygous mutant Adam17ex/ex.Ldlr−/− and wild-type Adam17wt/wt.Ldlr−/− mice. (G) For the quantification of the circumference of the BCA, transverse cryo-sections of 10 μm were prepared at three positions (200, 400 and 600 μm proximal to the bifurcation of the BCA with the right common carotid artery and right subclavian artery). (HI) Quantification of the circumference of the BCA at the three positions in (H) male and (I) female homozygous Adam17ex/ex.Ldlr−/− (ex/ex; n = 15/16/16; n = 14/14/14), heterozygous Adam17wt/ex.Ldlr−/− (wt/ex; n = 17/18/18; n = 21/21/21) and wild-type Adam17wt/wt.Ldlr−/− (wt/wt; n = 18/18/18; n = 19/19/19 mice. (J) Quantification of Oil Red-positive atherosclerotic plaque size in the BCAs of wild-type, heterozygous or homozygous Adam17 mutants (Adam17 wt/wt.Ldlr−/−; Adam17 wt/ex.Ldlr−/−; Adam17 ex/ex.Ldlr−/−). (K) Representative PicroSirius Red staining for visualizing collagen density of the BCA. (L) Quantification of PicroSirius Red intensity by polarization imaging in the intima and adventitia of homozygous Adam17ex/ex.Ldlr−/− (n = 6) and wild-type Adam17wt/wt.Ldlr−/− (n = 6) mice. (M) Quantification of the thickness of the arterial wall in BCA sections from Adam17 wt/wt.Ldlr−/− and Adam17et/ex.Ldlr−/− mice. (N) Representative Verhoeff-van Gieson staining visualizing elastin fibres in the BCA. (O) Quantification of elastin fibre length of homozygous Adam17ex/ex.Ldlr−/− (n = 3) and wild-type Adam17wt/wt.Ldlr−/− (n = 3) mice. Five fibres were quantified of each animal. (P) Volcano plot of mRNAs differentially abundant in aortas of wild-type Adam17wt/wt.Ldlr−/− (n = 4) and homozygous Adam17ex/ex.Ldlr−/− (n = 4) mice (transcriptome-wide expression profiling by microarrays). Highlighted are genes with a fold change >0.8 which are associated with atherosclerosis (purple; exemplary: uncoupling protein Ucp1, regulator of G protein signaling Rgs1, C-X-C motif chemokine ligand Cxcl16, metalloproteinase Mmp3, C-C motif chemokine ligand 5 Ccl5, adipose differentiation-related protein Adfp) or with ECM/cytoskeletal regulation (yellow; exemplary: metalloproteinases Mmp3 and Mmp12, Myosin Myo1f, Coronin Coro1a, Emilin2, Syndecan Sdc3, Pecam1, Fibulin Fbln2, Enah/Vasp-like Evl, leupaxin Lpxn, melanoma inhibitory activity Mia1, Unc5c, Fbxl22, Pip5k1b, Sarcolipin Sln). Graphs in B–E, H, I, L, M, and O show mean and standard error of the mean, boxplots in J show median and interquartile range. Normality of distribution was assessed by Kolmogorov-Smirnov testing in PRISM6 statistical software (GraphPad). Comparison of multiple groups was done using ANOVA, and Tukey as a post-test. Comparison of two groups was done using the t-test for normally distributed data (n.s.—statistically not significant, p < 0.05). The Mann–Whitney test was used for non-normally distributed data (marked with asterisks [****] p < 0.0001; [*]p < 0.05).

Summarizing, our combined data reveal a function of ADAM17 in arterial elastin network maintenance. This protective role of ADAM17 is corroborated by independent genetic work showing that increased ADAM17 activity reduced coronary arteriole dilation[19] and that conditional deletion of Adam17 in cardiomyocytes increased left ventricular dilation in pressure-overload models.[20] Since neither endothelial nor myeloid Adam17 depletion showed significant arterial dilation, it is possible that ADAM17 executes its role in maintaining the elasticity of the arterial wall through vascular smooth muscle cells (VSMCs) or fibroblasts. Indeed, the elastin network in arteries is known to be deposited largely by VSMCs.[14] Since the full-body Adam17 hypomorphic mutant and the conditional Adam17 knockouts were, however, in different genetic backgrounds that are known to differently contribute to atherosclerosis (Ldlr−/− and ApoE−/−, respectively[21] or could theoretically have differential genetic interactions with Adam17 [22] [23]), we caution that one cannot formally rule out that ADAM17 protected from aneurysms also through other cells than VSMCs.

How ADAM17 regulates arterial elasticity remains open. Our analysis suggests that loss of Adam17 reduces elastin fibre elasticity, which could lead to radial dilation of the vessel —and the latter would be expected to trigger peripheral stretching of the wall.[15] [24] This may trigger fibre straightening without affecting integrity, number, or thickness (see [Fig. 1N]).

For controlling arterial wall elasticity, TNF-α/TNFR signaling, which is an established ADAM17 substrate and de-repressed in the Adam17 mutants,[7] could be one of the many primary effectors in this pathway. Indeed, TNF-α is known to be anti-elastogenic because it increases the expression of metalloproteinases, which are elastolytic and more abundant and active in human aneurysms.[25] [26] Metalloproteases can also impair the expression of tropoelastin.[27] However, other ADAM17 substrates may be effectors and impact the activity of elastin-regulating factors such as emilins or fibulins, both of which are also known genetic determinants in human aneurysmal syndromes.[28] [29] Finally, our study indicates that non-topically administering ADAM17 inhibitors in the clinics may bear side effects in the form of artery wall destabilization and aneurysms.

Sources of Funding

This work was funded by the University Hospital, LMU Munich, by the University of Leipzig, and by the German Research Foundation (DFG) as part of the Collaborative Research Center CRC1123 ‘Atherosclerosis - Mechanisms and Networks of Novel Therapeutic Targets’ (project B1: L.M.H., D.T.; project A1: C.W.; project C1: S.S.). The work of S.R-J. was funded by the DFG as part of the Cluster of Excellence ‘Inflammation at Interfaces’ and as part of the CRC877 (project A1).


 
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