Deletion of MFGE8 Inhibits Neointima Formation upon Arterial Damage

Atherosclerosis, a chronic inflammation of the vessel wall, is a major cause for vascular mortality due to narrowing (stenosis) of the arterial wall. In part, artery stenosis is therapeutically addressed by widening the vessel through angioplasty. Although well-established, angioplasty can damage the arterial endothelium, giving rise to an inflammatory response that leads to a neointimal hyperplasia with consequent recurrence of stenosis.[1] [2] Key players in this process are leukocytes and smooth muscle cells (SMCs), as leukocyte recruitment and SMC proliferation and migration are determinants of neointimal hyperplasia.[3]

MFGE8 (milk fat globule-epidermal growth factor 8) or lactadherin is mostly regarded as a bridging molecule with a critical function in efferocytosis and, hence, during resolution of inflammation.[4] However, MFGE8 also plays a major role in promoting neovascularization,[5] and, more recently, arterial MFGE8 expression emerged as a molecular hallmark of adverse cardiovascular remodelling upon aging.[6] In this context, MFGE8 is directly associated with SMC proliferation and migration,[6] [7] suggesting its participation in neointima formation. We here show that MFGE8 negatively impacts on arterial restenosis and its neutralization may therefore be a potential therapeutic strategy.

To study the role of MFGE8 in arterial restenosis, we subjected two groups of mice, Apoe^−/− and Apoe^−/−Mfge8^−/− , to wire injury of the left carotid artery. To simulate hypercholesterolemia, a condition often present in atherosclerotic patients, the mice were fed a high-fat diet, starting 1 week prior to injury. Two weeks after the injury, the mice were euthanized and the blood and carotids were collected. To assess neointima formation, the carotids were stained for elastic tissue fibres with Verhoeff–Van Gieson stain. Neointima area was significantly smaller throughout the injured carotid in mice lacking MFGE8 as compared with the Apoe^−/− group ([Fig. 1A]–[C]). No differences in blood counts were observed between the groups ([Supplementary Table S1], available in the online version), suggesting that the distinct neointima areas are mediated by local cells. However, cholesterol levels in the blood were higher in Apoe^−/−Mfge8^−/− mice as compared with Apoe^−/− , likely a consequence of decreased fatty acid uptake by the liver as well as small intestine, as MFGE8 has been reported to promote fatty acid uptake.[8] [9] To determine which cells contributed the most for the larger neointima observed in Apoe^−/− mice, we stained the carotids with antibodies against macrophages and SMCs, since MFGE8 has been reported to be expressed in these cells.[10] [11] Lack of MFGE8 did not affect neointimal macrophages, while SMC areas were vastly reduced ([Fig. 1D], [E]). To assess whether this was the result of reduced cell proliferation, the carotid arteries were stained with an antibody against Ki67, a cellular marker of proliferation. The staining revealed significantly less SMC proliferation in mice lacking MFGE8 as compared with their wild-type littermates, suggesting a determinant role of lactadherin in restenosis formation. To confirm this observation, and verify MFGE8 as a therapeutic target, we subjected Apoe^−/− mice to arterial injury combined with the local application of siRNA, either against MFGE8 or scrambled. siRNA against MFGE8 resulted in 50% reduced expression of the protein both in the intima and in the neointima (lesion) area of the carotid artery ([Supplementary Fig. S1], available in the online version). Similar to what was observed in the knockout animal models, analysis of the injured carotids of mice treated with siRNA directed to MFGE8 showed a decreased neointima sizes as compared with mice treated with control siRNA ([Fig. 1G]–[I]). Equally in accordance with the studies in the knockout animal models, no differences in blood counts were observed between the groups ([Supplementary Table S1], available in the online version). Blood cholesterol levels remained unchanged ([Supplementary Fig. S2], available in the online version), supporting the argument presented earlier for the difference observed in the blood of the knockout animal models: since the siRNA effect is strictly local, it does not affect fatty acid uptake. Consistent with the observations in the carotids of Apoe^−/− versus Apoe^−/−Mfge8^−/− mice, the administration of siRNA against MFGE8 affected SMCs' content in the neointima ([Fig. 1K]) and its proliferation ([Fig. 1L]) but the macrophage composition remained unchanged ([Fig. 1J]).

Fig. 1 MFGE8 promotes postinjury neointima formation by fostering smooth muscle cell proliferation. Mice were fed a high-fat diet starting 1 week prior to carotid injury induction. Two weeks after injury, mice were euthanized and the blood and carotids were collected. Carotids were fixed in paraformaldehyde and embedded in paraffin. For each antibody staining, three sections per mouse were analysed. (A–F) Assessment of neointima size and composition in Apoe^−/− versus Apoe^−/−Mfge8^−/− mice. (A) Neointima area was analysed by elastic tissue fibres staining, with Verhoeff–Van Gieson stain (EVG), and quantified throughout the carotids. (B) To better quantify the difference in neointima area between the two groups, the area under the curve (AUC), corresponding to the data points presented in (A), was calculated. (C) Representative images of EVG-stained injured carotids. To identify possible differences in neointima composition between the two mice groups, carotids were stained with antibodies against (D) macrophages (anti-Galectin-3 or anti-Mac2) or (E) smooth muscle cells (SMCs) (anti-α-SMA). Each data point indicates the average of positive area per section in one mouse. To determine whether the increased numbers of SMCs in the neointima of Apoe^−/−Mfge8^−/− mice originated from cell proliferation or migration, carotids were stained with an antibody against a cell proliferation marker, Ki67 (F). n = 8 (Apoe^−/− mice) or n = 11 (Apoe^−/−Mfge8^−/− mice) throughout panels A–F. Statistical comparisons were made with t-test following D'Agostino–Pearson omnibus normality test. (G–L) Assessment of neointima size and composition in Apoe^−/− mice treated with control siRNA or directed to MFGE8. (G) Neointima area was quantified throughout the carotids, and (H) the AUC, corresponding to the data points presented in (G), was calculated. (I) Representative images of EVG-stained injured carotids. Carotids were stained with antibodies against (J) macrophages (anti-Galectin-3 or anti-Mac2) or (K) SMCs (anti-α-SMA). Each data point indicates the average of positive area per section in one mouse. (L) Carotids were stained with an antibody against a cell proliferation marker, Ki67. n = 7 mice per group throughout panels G–L. Statistical comparisons were made with t-test following Kolmogorov–Smirnov normality test. All data are represented as mean ± SEM. Scale bar represents 200 µm.

Overall, these results strongly point toward a relevant role of MFGE8 in postinjury arterial wall remodelling, with the potential to be exploited for therapeutic purposes. Our studies suggest these effects to be SMC-mediated, more specifically: by stimulating SMC proliferation, MFGE8 promotes the formation of neointima possibly leading to hyperplasia and consequent stenosis.

Note: The review process for this paper was fully handled by Gregory Y. H. Lip, Editor-in-Chief.

• References

• 1 Dangas G, Kuepper F. Cardiology patient page. Restenosis: repeat narrowing of a coronary artery: prevention and treatment. Circulation 2002; 105 (22) 2586-2587
• 2 Nicolais C, Lakhter V, Virk HUH. , et al. Therapeutic options for in-stent restenosis. Curr Cardiol Rep 2018; 20 (02) 7
• 3 Soehnlein O, Wantha S, Simsekyilmaz S. , et al. Neutrophil-derived cathelicidin protects from neointimal hyperplasia. Sci Transl Med 2011; 3 (103) 103ra98
• 4 Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature 2002; 417 (6885): 182-187
• 5 Silvestre JS, Théry C, Hamard G. , et al. Lactadherin promotes VEGF-dependent neovascularization. Nat Med 2005; 11 (05) 499-506
• 6 Fu Z, Wang M, Gucek M. , et al. Milk fat globule protein epidermal growth factor-8: a pivotal relay element within the angiotensin II and monocyte chemoattractant protein-1 signaling cascade mediating vascular smooth muscle cells invasion. Circ Res 2009; 104 (12) 1337-1346
• 7 Wang M, Fu Z, Wu J. , et al. MFG-E8 activates proliferation of vascular smooth muscle cells via integrin signaling. Aging Cell 2012; 11 (03) 500-508
• 8 Khalifeh-Soltani A, Gupta D, Ha A. , et al. Mfge8 regulates enterocyte lipid storage by promoting enterocyte triglyceride hydrolase activity. JCI Insight 2016; 1 (18) e87418
• 9 Khalifeh-Soltani A, McKleroy W, Sakuma S. , et al. Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids. Nat Med 2014; 20 (02) 175-183
• 10 Bagnato C, Thumar J, Mayya V. , et al. Proteomics analysis of human coronary atherosclerotic plaque: a feasibility study of direct tissue proteomics by liquid chromatography and tandem mass spectrometry. Mol Cell Proteomics 2007; 6 (06) 1088-1102
• 11 Ait-Oufella H, Kinugawa K, Zoll J. , et al. Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosis in mice. Circulation 2007; 115 (16) 2168-2177