Vascular Smooth Muscle Cell Plasticity and Autophagy in Dissecting Aortic Aneurysms

Supplemental Digital Content is available in the text.

T he response of vascular smooth muscle cells (VSMCs) to injury is a major determinant of the development and progression of vascular diseases, including atherosclerosis, restenosis, and aneurysm. [1][2][3] In response to injury and inflammation, VSMCs undergo phenotypic switching from a quiescent contractile phenotype to a proliferative, and migratory synthetic phenotype and can acquire molecular and cellular features of mesenchymal stem cells and macrophages. 4,5 VSMC plasticity is well-documented during atherosclerosis and neointima formation and has been confirmed using lineage-tracing experiments. [6][7][8][9] More recently, using multicolor lineage labeling, we demonstrated that VSMC accumulation in atherosclerotic plaques and injury-induced neointimal lesions results from extensive proliferation of a small subset of differentiated but highly plastic medial VSMCs, a variable proportion of which undergo phenotypic switching to phagocyte-like cells. 10 VSMCs also play important roles in the pathophysiology of aortic aneurysm (AA), and recent studies suggested a role for some aspects of VSMC phenotypic switching in AA. 11,12 However, the plasticity of VSMCs during AA formation has not been assessed.
Here, we used multicolor lineage labeling of VSMCs to characterize the behavior of VSMCs during the development and progression of Ang II (angiotensin II)-induced dissecting AA. We report the occurrence of clonal expansion of a subset of VSMCs in the media, which can outgrow into the adventitia (including the false-channel's borders) of the dissecting AA. The expanded VSMCs undergo phenotypic switching to phagocyte-like cells and can upregulate autophagy and endoplasmic reticulum (ER) stress markers. Importantly, loss of autophagy in VSMCs promotes VSMC death and ER stress-dependent VSMC inflammation and aggravates the aortic disease.

Data Disclosure Statement
The data that support the findings of this study are available from the corresponding authors on reasonable request.

Animals
All experiments were performed according to the Home Office, UK regulations and approved by the local ethics committee. For lineage tracing, Myh11-CreERt2/Rosa26-Confetti males were subjected to 10 intraperitoneal injections of 1 mg tamoxifen over 2 weeks followed by at least 1-week washout. Tagln Cre+ mice (Jax n°004746) and Atg5 flox/flox mice (kindly provided by Noburu Mizushima, University of Tokyo 13 ) were bred in house. Tagln Cre+ /Atg5 flox/flox animals were used to assess the role of autophagy in VSMC. Infusion of 1µg/(min·kg) Ang II, with or without treatment with 10 mg/kg anti-TGF (transforming growth factor) β (clone 1.D.11, BioXCell) was used to induce dissecting AA. Animals were analyzed as described in the online-only Data Supplement.

Statistical Analysis
Values are shown as average±SEM. Differences between groups were evaluated using Mann-Whitney test (2 groups), Kruskal-Wallis test followed by uncorrected Dunn test (> 2 groups), 2-way ANOVA (cell proliferation/survival), or χ 2 test (distribution between 2 groups), as indicated in figure legends. Results were considered statistically significant at P<0.05.

Characterization of VSMCs During Aortic Dissection Induced by Ang II
VSMCs downregulate contractile gene expression during AA formation. 11,12 However, the plasticity of VSMCs during AA formation has not been fully characterized. To this end, we used a prototypical model of dissecting AA induced by Ang II, with or without TGFβ inhibition. 14, 15 We first stained for αSMA (α smooth muscle actin) on cross-sections of mice with aortic dissections. We observed accumulation of αSMA + cells in the false channel in 5 out of 5 animals displaying aortic dissection in this experiment. These αSMA + cells seemed to expand from the media ( Figure 1A) and accumulated in hemorrhagic/thrombotic areas in contact with iron (Perls staining, Figure 1B) and red blood cells, which may explain their acquisition of HMOX (heme oxygenase) 1 expression ( Figure 1C). αSMA + cells detected in the thrombotic/hemorrhagic region also showed increased expression of the phagocytic marker CD68 ( Figure 1D) and the lysosomal marker LAMP2 (lysosomal-associated membrane protein 2; Figure 1E), suggesting that some VSMCs switched towards phagocyte-like cells. We further confirmed our results using flow cytometry analysis of aortic cells isolated from Apoe −/− mice infused with Ang II for 21 days (Figure 2). The proportion of VSMCs (αSMA high CD90 -) with high expression of αSMA markedly decreased in dissected aortas compared with controls, whereas a substantial proportion of αSMA int CD90 high (myofibroblasts) and αSMA low CD90 high (fibroblasts; Figure 2A) was observed in diseased aortas. VSMCs, myofibroblasts, and fibroblasts acquired phagocytic markers LAMP2 ( Figure 2B) and CD68 ( Figure 2C and 2D) proportionally to the severity of aortic disease, and cells from dissecting aneurysms were positive for Ter-119, suggesting an association with red blood cells ( Figure 2F and 2G). These results suggest that a substantial proportion of VSMCs, myofibroblasts, and fibroblasts adopt a phagocyte-like phenotype in dissecting AA.

VSMC Clonal Expansion and Phenotypic Switching in Dissecting AA
To test whether the αSMA + cells that have accumulated in the adventitia have originated from preexisting VSMCs, we used multicolor lineage tracing in Myh11-CreERt2/Rosa26-Confetti mice to track VSMCs and their progeny. 10 VSMCs were labeled by tamoxifen injections before the induction of AA by Ang II infusion and TGFβ inhibition ( Figure 3). Stochastic labeling of VSMCs using this method results in a mosaic pattern in the noninjured aortic media. 10 We found that αSMA + cells that accumulated in the adventitia and the false channel in mice with aortic dissection were also positive for Confetti fluorescent reporters (5 out of 6 mice) indicating that they were VSMC-derived cells coming from the media ( Figure 3A). Interestingly, in contrast to the stochastic mosaic labeling of the normal aortic media, VSMC-derived cells in the adventitia displayed a nonrandom color distribution ( Figure 3A). We observed large regions containing lineage-labeled cells of a single color or intermixed single colors in all (5/5) animals with VSMC-derived Confetti + cells outside the medial layer, suggesting that these cell outgrowths are derived from clonal expansion of a small number of cells ( Figure 3A). We also found monochromatic patches of VSMCs in the medial layer of 5 out of the 6 animals analyzed (>5 cells per patch, Figure 3B and Figure Figure 3D) confirmed that VSMCs were proliferating both in the media ( Figure 3E) and in the adventitial outgrowth areas of dissected aortas ( Figure 3F and Figure IV in the online-only Data Supplement). Confetti + cells in the media expressed almost no phagocytic markers, but the Confetti + cells that have expanded into the adventitia started to express HMOX1, CD68, and LAMP2 ( Figure 3G). CD90 expression was undetectable in Confetti + cells, except for a few cells with very low expression (data not shown). Our data indicate that clonal proliferation and phenotypic switching of medial VSMCs are important features of Ang II-induced aortic dissection.

Atg5 Deficiency in VSMCs Promotes the Development of Severe Aortic Dissection
The lysosomal pathway, and particularly LAMP2, controls autophagosome maturation. 16 Moreover, autophagy plays critical roles in VSMC biology 17 and has recently been linked with VSMC phenotypic switching. 18 Defective autophagy in VSMC is associated with accelerated VSMC senescence, neointima formation, and atherogenesis, 17,19 but its role in the pathophysiology of dissecting AA is still uncertain. 20 Studying aortic cross-sections, we found increased expression of ATG16L1 (autophagy-related protein 16 like 1) in medial and adventitial αSMA + cells of dissecting AA (5 out of 5) compared with very limited staining in VSMCs of healthy aortas ( Figure 4A), suggesting a potential role of autophagy in this disease condition. We confirmed that ATG16L1 is expressed in VSMC-derived cells, using the Confetti lineage tracing animals ( Figure VA in the onlineonly Data Supplement). ATG5 (autophagy protein 5) is essential for all types of autophagy, and we found that ATG5 was also expressed in VSMC-derived Confetti + cells ( Figure VB and VC in the online-only Data Supplement). Furthermore, Atg5 gene expression was upregulated in primary VSMCs at passage 4 in culture compared with ex vivo primary VSMCs  Thus, defective autophagy in VSMCs increases the incidence and severity of aortic dissection.

Atg5 Deficiency in VSMCs Impedes Autophagosome Formation and Enhances Cell Death
To confirm that Atg5 deficiency inhibits autophagy in VSMCs, we analyzed the expression of LC3 (microtubule-associated protein 1 light chain 3) in αSMA + cells after the induction of AA. Punctate LC3 staining, associated with autophagosome formation, was significantly reduced in Tagln Cre+ /Atg5 flox/flox mice compared with Tagln Cre-/Atg5 flox/flox control animals ( Figure 5A). Conversely, Atg5 deficiency in VSMCs led to a significant accumulation of the autophagosome cargo protein SQSTM1 (sequestosome 1)/p62 ( Figure 5B) as well as LAMP2 ( Figure 5C). Loss of Atg5 was associated with an increase of apoptotic VSMCs in the media, as shown by active CASPASE-3 staining ( Figure 5D), suggesting that autophagy promotes cell survival. This was confirmed using in vitro experiments, which revealed a substantial reduction of VSMC survival ( Figure 5E) and proliferation ( Figure 5F) in response to serum, and an increased susceptibility to ER stress-induced cell death ( Figure 5G) in the absence of Atg5.

Atg5 Deficiency in VSMCs Promotes an ER Stress Response and Inositol-Requiring Enzyme 1α-Dependent Inflammation
There is a close interplay between autophagy and the ER stress response, and recent studies indicate that autophagy may resolve ER stress responses through direct removal of IRE (inositol-requiring enzyme)1α. 21 Consistent with the latter finding, we observed a substantial accumulation of the ER stress sensor IRE1α in Atg5-deficient VSMCs in vivo ( Figure 6A). In vitro cultured Atg5-deficient VSMCs also showed substantial accumulation of IRE1α in the absence of any external stimulus ( Figure 6B). VSMCs respond to IL (interleukin) 1 stimulation by abundant secretion of inflammatory cytokines 22 and chemokines. 23 Interestingly, IL1β-induced expression of Il6 ( Figure 6C), Cxcl1 (C-X-C motif chemokine ligand 1), and Ccl2 (C-C motif chemokine ligand 2; Figure VII in the online-only Data Supplement) was significantly higher in Atg5-deficient VSMCs compared with wild-type control cells and was abrogated by inhibition of IRE1α kinase activity. Consistent with the increased inflammatory response, aortic sections of Tagln Cre+ /Atg5 flox/flox mice treated with Ang II+anti-TGFβ showed increased neutrophil accumulation compared with Tagln Cre-/Atg5 flox/flox control mice ( Figure 6D). We also found a tendency (P=0.06) towards increased circulating levels of IL-6 in Tagln Cre+ /Atg5 flox/flox compared with Tagln Cre-/Atg5 flox/ flox mice; however, other tested circulating cytokines and chemokines were not different between the 2 groups ( Figure VII in the online-only Data Supplement). These results indicate that autophagy-dependent regulation of ER stress modulates VSMC and local aortic inflammation.

Autophagy and ER Stress Are Features of Human Aortic Dissection
To examine the relevance of our findings to human pathology, we analyzed sections of human aortas with or without dissection, collected from separate patients. We found that 4 out of 5 samples with aortic dissection contained αSMA + and TAGLN + (transgelin) cells ( Figure 7A) in areas devoid of elastic lamellae outside the media, whereas such features could not be detected in nondissected normal aortas (n=5; Figure VIII in the online-only Data Supplement). The vast majority of adventitial αSMA + cells did not express CD90 ( Figure IX in the online-only Data Supplement) indicating that they were not of fibroblast origin and suggesting that they have most likely expanded from the aortic media. Although LC3 expression in VSMCs was similar between nondissected and dissected aortas ( Figure 7B), the latter showed increased accumulation of the autophagosome cargo protein SQSTM1/p62 ( Figure 7C) and increased expression of the ER stress marker GRP78/BiP (glucose-regulated protein 78/binding immunoglobulin protein; Figure 7D). Our data suggest that VSMCs of dissected AAs in humans may have deregulated autophagy resulting in ER stress activation.

Discussion
Previous work on the role of VSMCs in AA has focused on the detrimental effects of VSMC death in promoting adverse arterial wall remodeling because of reported medial thinning, degeneration, and extensive apoptosis of VSMCs in very late stages of AA development. 24,25 Notably, however, despite medial thinning and VSMC death, ascending thoracic AAs show an increase in overall medial area and have preserved VSMC density, suggesting a hyperplastic response. 26,27 Animal models of AA also suggest that VSMCs display some aspects of phenotypic switching early during the development of both thoracic and abdominal AA. 11,12 Here, we have tested the hypothesis that a hyperplastic VSMC response could compensate for increased VSMC death during AA development. Using lineage tracing of preexisting VSMCs in mice, we found that in response to Ang II infusion, a subset of VSMCs clonally expand in the media of the thoracic and abdominal aorta, and in the context of aortic dissection, expand through the aortic wall into the adventitia and the newly formed false channel. We propose that the resulting VSMC-derived cells might play a reparative role at several disease stages, from aneurysm development to aneurysm dissection. Interestingly, large foci of VSMCs also accumulate in areas of extensive elastin degradation corresponding to the external medial layers and adjacent adventitia of human thoracic AAs, suggesting similar pathophysiological mechanisms in human AAs. Our lineage-tracing experiments demonstrate that a subset of preexisting VSMCs proliferate, downregulate contractile protein expression, and upregulate proteins associated with a phagocytic-like phenotype in AA. This resembles the VSMC behavior observed in other vascular disease models, 7-10 suggesting that the extensive plasticity is an inherent physiologically relevant feature of VSMCs. Importantly, we found many examples of clonal proliferation resulting in monochromatic patches within the medial layer in animals showing no signs of aortic dissection. This observation suggests that activation of proliferation occurs in a larger proportion of VSMCs than what was estimated from the clonal VSMC contribution to neointima formation after vascular injury. 10 The finding that VSMCs downregulate the contractile phenotype in AA is consistent with and further validates previous work. 11,12,28 Two recent studies reported that interference with molecular pathways involved in VSMC phenotypic switching may have detrimental effects in AA. VSMC-restricted deletion of KLF4 (Kruppel-like factor 4), which has previously been identified as a regulator of several aspects of VSMC phenotypic switching in atherosclerosis, 8 reduced aortic disease severity in mouse models of AA, 28 although it did not abrogate the disease. A more recent study identified a role for HDAC9-MALAT1-BRG1 (histone deacetylase 9-metastasisassociated lung adenocarcinoma transcript 1-brahma-related gene 1) complex in the downregulation of the contractile VSMC phenotype in AAs driven by mutations of the TGFβ pathway; VSMC-restricted deletion of MALAT1 significantly preserved the contractile phenotype of VSMCs and reduced AA development in a mouse model of Marfan with Fbn1 mutation. 12 These studies are consistent with a detrimental role of the downregulation of the contractile phenotype of VSMCs in AAs. However, KLF4 and MALAT1 may impact other VSMC functions beyond, and may be independently, of their role in regulating the contractile phenotype of VSMCs.
Beyond the downregulation of differentiation and contractile markers of VSMCs, VSMC phenotypic switching induces a wide range of functions, which might have opposing functions on AA formation and progression. Here, we assessed the particular role of VSMC autophagy in AA and found that loss of Atg5 in VSMC reduced autophagosome generation and resulted in increased disease progression and mortality in Ang II-treated animals with TGFβ inhibition. Previously, the role of VSMC autophagy in the development of AA was examined in Atg7 flox/flox /Tagln Cre/+ mice. 20 The authors concluded that mice with smooth muscle cell-specific Atg7 deficiency do not develop dissecting abdominal AA in response to Ang II. 20 Importantly, that study was conducted using Ang II infusion under normocholesterolemic conditions where mice are resistant to AA. 14,15 Additional cues, such as the presence of hypercholesterolemia 14 or the concomitant blockade of TGFβ signaling pathway, 15,29 are required to promote the susceptibility of the aorta to aneurysm formation and dissection in response to Ang II infusion. In our present study, the use of a previously validated model of dissecting AA 15,29 revealed a clear detrimental effect of defective autophagy in VSMCs on AA development. The incidence and severity of dissected AAs were significantly higher in mice with Atg5 deletion in VSMCs. Of note, 18% of the mice (25% of the mice that died suddenly) showed evidence of extraaortic hemorrhage in the peritoneum, spleen, and intestine, suggesting that defective autophagy in VSMCs may be associated with widespread impairment of the vascular response to injury. Taken together, the data show that autophagy in VSMCs is critically required for the maintenance of vascular integrity during the development and progression of AAs. This vasculoprotective effect may be explained at least in part, by the role of autophagy in preserving VSMC survival in response to injury. Our data also identify a role of autophagy in the regulation of VSMC inflammation, potentially through the degradation of IRE1α. 21 IRE1α has previously been involved in mediating inflammatory responses downstream of toll-like receptors 30,31 and C-type lectin receptors, 32 but its role in IL1R1 (interleukin 1 receptor 1) signaling pathways has not been addressed. We speculate that this interconnection between autophagy, ER stress responses, and inflammatory pathways is of major importance to the outcome of the reparative process after injury and merits further consideration. Finally, the direct impact of autophagy on the regulation of VSMC clonal proliferation and phenotypic switching will need to be addressed. It will also be interesting to address the direct impact of autophagy deletion on the response of VSMC to Ang II stimulation.

Conclusions
We provide genetic evidence for the activation of VSMC proliferation, selective clonal expansion, and phenotypic switching towards phagocytic-like phenotypes in VSMCs during the development of dissecting AA. We identify a critical role for autophagy in the preservation of vessel integrity, possibly through limitation of VSMC death and ER stress-dependent inflammation. The results advance our understanding of the reparative mechanisms that operate during aneurysm development and progression, which could be exploited clinically. Future studies to identify the precise stimuli responsible for VSMC proliferation and accumulation in this context are important to reveal potential new therapeutic targets.