HMGB1/TLR4 signaling pathway enhances abdominal aortic aneurysm progression in mice by upregulating necroptosis

The age-associated increases in aseptic inflammation and necroptosis are closely related to the emergence of various age-associated diseases. In this study, the role of HMGB1/TLR4-induced necroptosis in abdominal aortic aneurysm (AAA) formation was investigated. First, the levels of sterile inflammatory mediators (HMGB1, TLR4) and necroptosis markers were measured in the abdominal aortas of young and old C57BL/6JNifdc mice. We observed that sterile inflammatory mediators and necroptosis markers were greatly increased in the abdominal aortas of old mice. Then, angiotensin II (Ang II)-induced AAA model in APOE−/− mice was used in this study. Mice AAA models were treated with the RIP1 inhibitor necrostatin-1 (Nec-1) or the TLR4 inhibitor TAK-242, respectively. We found that HMGB1, TLR4, and necroptosis markers were elevated in old mice compared with those in young mice. Same elevation was also found in the development of AAA in APOE−/− mice. In addition, the necroptosis inhibitor Nec-1 alleviated Ang II-induced AAA development while downregulating the expression of HMGB1/TLR4. After blocking TLR4 with TAK-242, the expression of necroptosis markers decreased significantly, and the progression of AAA was also alleviated in APOE−/− mice. Our results indicated that HMGB1/TLR4-mediated necroptosis enhances AAA development in the Ang II-induced AAA model in APOE−/− mice and that TLR4 might be a potential therapeutic target for AAA management.


Introduction
Abdominal aortic aneurysm (AAA) is a degenerative process of the abdominal aorta. The morbidity of AAA in individuals over 65 years old is up to 5-10%, and rupture is a common cause of death [1,2]. The mortality of ruptured AAA is 85-90% [3,4]. Currently, open surgery and endovascular repair remain effective methods to prevent aortic rupture [5]. Pharmacological methods remain incapable to limit the further expansion of small-and medium-sized AAAs [6,7].
Necroptosis is a regulated form of necrosis and plays a vital important role in the pathogenesis of various cardiovascular diseases, including myocardial infarction, atherosclerosis, restenosis, and aneurysm. Necroptosis is also the reason for the depletion of aortic smooth muscle cells in aneurysms [8][9][10]. Receptor-interacting protein kinase 1 (RIP1), receptor-interacting protein kinase 3 (RIP3), and mixed lineage kinase domain-like protein (MLKL) are important signaling molecules associated with necroptosis [11]. These common cell membrane receptors can initiate necroptosis include toll-like receptor 4 (TLR4), toll-like receptor 3 (TLR3), and tumor necrosis factor receptor 1 (TNFR1) [12]. Endogenous damage-associated molecular pattern molecules (DAMPs) can activate the cell membrane receptor TLR4 [13]. Studies have shown that necroptosis can be triggered by DAMPs, including the high mobility group protein 1 (HMGB1) and S100 family proteins [14,15]. New evidence suggests that TLR4-mediated necroptosis has a significant role in inflammatory diseases [16,17]. However, it is still unclear whether HMGB1/TLR4-mediated necroptosis is involved in the occurrence of AAA. Therefore, the aim of this study was to examine the mechanism and role of HMGB1/TLR4-mediated necroptosis in an Ang II-induced AAA model in APOE −/− mice.

Mice
Male C57BL/6JNifdc mice were divided into a young group (2 months) and an old group (18 months). Male APOE −/− mice (2 months) with a C57BL/6JNifdc background were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised in the Experimental Animal Center of Shandong Provincial Hospital. All animal experimental procedures were carried out according to the National Institute of Health's guidelines and were approved by Shandong Provincial Hospital's Committee of Animal Protection and Use.

Animal model
First, C57BL/6JNifdc mice were divided into two groups, Young group (2 month old) and Old group (18 month old). These mice were used to observe degeneration in the abdominal aorta and age-related changes in aseptic inflammatory mediators and necroptosis. Second, APOE −/− mice were used to establish the experimental AAA model. The modeling method was as follows: Alzet osmotic pumps (Model 2004, Durect, CA, USA) were implanted subcutaneously in the interscapular region and released 1.44 mg/kg/day Ang II (GL Biochem). Subcutaneous infusion was continued for 28 days [18]. The mice were grouped into the sham group (Saline), AAA group (Ang II), RIP1 inhibitor group (Ang II/ Necrostatin-1), and TLR4 inhibitor group (Ang II/TAK-242) (n = 8 per group). The RIP1 inhibitor necrostatin-1 (3.2 mg/ kg/day, i.p. [19]) or the TLR4 inhibitor TAK-242 (0.3 mg/ kg, i.p., twice a week [20]) was administered for 28 days. Any animal that died before the end of the experiment was autopsied. After 28 days, the mice were killed for tissue harvesting and image acquisition.

Histology
Paraformaldehyde-fixed aortas were imbedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, USA). Cryostat sections were cut into cross-Sections (6 μm) and then dyed with hematoxylin and eosin (H&E) to analyze the structural integrity or medial aortic wall cell density (in cells/mm 2 ). Verhoeff's Van Gieson (EVG) staining was performed to detect the degradation of elastin. Cell density (cells/mm 2 ) in the aortic medulla was measured by counting the number of nuclei and dividing it by the total observed tissue area. The volume fraction of elastin in the aortic medulla was calculated as the ratio of the EVG stain area to the total observed tissue area [21]. Data quantification was performed using at least 8 sections per artery.

Determination of the aortic diameter
The suprarenal aorta diameter at 0, 7, 14, and 28 days after the implantation of osmotic micropumps was examined using a VEVO2100 Ultrasound system (Visual Sonics Inc., Canada). All suprarenal aorta diameters were independently measured by two researchers who were blinded to the group assignments. Aneurysm is defined as an increase in the maximal diameter of an aorta beyond 50% of the baseline diameter and is characterized by progressive dilatation and rupture of the aortic wall [22].

Isolation of RNA and real-time quantitative polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the aortic tissues of surviving mice with an RNA extraction kit (AG21017, China) and reverse-transcribed into cDNA with an Evo M-MLV RT Mix Kit (AG11728, China). qRT-PCR analysis was performed with the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, China). The primer sequences are shown in Table 1.

Statistical analysis
The data are shown as the mean ± SD. Shapiro-Wilk test was used to determine whether all datasets were normally distributed. All the data were normally distributed in this Table 1  manuscript. Significant differences were determined by Student's t test or one-way ANOVA according to the data type. Time serial diameter data were tested using two-way ANOVA. Statistically significant differences were considered when P < 0.05.

Aging aortic degenerative changes
To determine the cell density, the aortas of young and old mice were dyed with hematoxylin and eosin, and the nuclei were counted. Compared with the young group [(5.36 ± 0.49)×10 3 /mm 2 ], the density of aortic middle layer cells in the old group [(3.84 ± 0.48)×10 3 /mm 2 ] was significantly decreased (Fig. 1A). To evaluate the elastic structure, EVG staining of aortic sections was performed. Compared with the young group, the middle elastin volume fraction was decreased in the old group (Fig. 1B).

Necroptosis or TLR4 inhibition prevents experimental AAA formation in mice
To verify the relationship and mechanism of TLR4 and necroptosis in experimental AAA, mouse AAA models were treated with the necroptosis inhibitor necrostatin-1 and the TLR4 inhibitor TAK-242. Ultrasound examination was performed at a specified time point to observe the change in the luminal diameter and the efficiency of modeling. Overall survival curves of the different groups were shown in the Fig. 4A. The ultrasound results showed that the lumen diameter in the AAA group (1.78 ± 0.21 mm) was significantly larger than that in the sham group (0.98 ± 0.03 mm), and necrostatin-1 (1.30 ± 0.09 mm) or TAK-242 (1.22 ± 0.13 mm) treatment significantly inhibited abdominal aortic dilatation. These results indicated that inhibiting necroptosis or TLR4 could prevent AAA formation induced by Ang II (Fig. 4B-E). Meanwhile, we examined whether necrostatin-1 or TAK-242 could inhibit aortic wall remodeling and elastin degradation in Ang II-induced APOE −/− mice. Histological analysis showed a relatively intact aortic structure and fewer fragmented elastic fibers in necrostatin-1-or TAK-242-treated aortae, while the elastic fibers in the AAA group were fragmented, and the arrangement was disordered (Fig. 4F).

Necroptosis or TLR4 inhibition decreases the expression of HMGB1 and TLR4 and attenuates necroptosis in Ang II-induced AAA
As shown in Fig. 5, protein or RNA was extracted from the aortic tissue and analyzed by Western blotting or RT-PCR, respectively. In the necrostatin-1-treated group, much lower  Fig. 5A, B). RT-PCR analysis also showed reduced HMGB1, TLR4, and necroptosis markers mRNA in Nec-1 group compared with AAA group (Fig. 5C). IF staining also obtained similar results (Fig. 5D). The TAK-242-treated group also showed the same trend. Overall, these results confirm that TLR4 inhibition reduced the inflammatory reaction and necroptosis in mice with Ang II-induced AAA.

Discussion
AAA, which is a degenerative process of the aorta, is a frequent cardiovascular disease in senior citizens [23,24]. Clinically, AAA is repairable by endovascular techniques and open surgery [25]. However, these two treatments are only applicable to patients with an AAA diameter greater than 5.5 cm [26]. If an AAA with a diameter between 3 and 5.5 cm is left uncontrolled, it can lead to extensive dilatation of the abdominal aorta and ultimately rupture [27,28]. Therefore, it is very important to understand the pathogenesis of AAA and identify new targets for the treatment of small-and medium-diameter AAA.
Aortic structural changes occur throughout life, but most degenerative changes occur after middle age [29,30]. Vascular remodeling and medial layer degeneration are typical age-related aortic structural changes [31,32]. The incidence of these pathological changes is significantly increased in the elderly population and shows a growing trend with aging [33]. The aortic medial layer, which is composed of VSMCs, Fig. 4 Inhibition of TLR4 (or necroptosis) by TAK-242 (or Necrostatin-1) attenuates arterial wall damage and inhibits AAA formation in mice. Following Ang II subcutaneous infusion, different treatment groups mice were monitored for AAA formation and progression via serial ultrasound measurements of maximal suprarenal aorta diameters. Observation of tissue morphology in different groups by HE and EVG staining. A Overall survival curve of each group. B Incidence of AAA in each group of mice 28 days after pump implantation. C Ultrasound images of the abdominal aorta diameter at 0 d, 7 d, 14 d, and 28 days after pump implantation. D The maximum diameter of the abdominal aorta was measured at 0 d, 7 d, 14 d, and 28 d after pump implantation. E Representative images of the aorta ectasia in each group. F Representative HE staining and EVG staining images of the abdominal aorta sections. Data are shown as mean ± SD (n = 6/ group). Two-way ANOVA, **p < 0.01, ****P < 0.0001, vs. the Sham group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. the AAA group elastin and concentric collagen bands, is the most important degenerative reconstruction site for aneurysm formation [34,35]. Medial VSMC loss is the main pathological feature of AAA and has been observed in clinical pathology and mouse AAA models [9].
In this study, aortic degenerative changes occurred in old mice, and the density of mesothelial cells decreased, which was consistent with Wheeler et al. [21]. In addition, there was an increase in the expression of aseptic inflammatory mediators and key necroptosis proteins in the aortas of old mice. This phenomenon was consistent with Aleah and Sathyaseelan et al. [36,37]. The same trend was also observed in the AAAs of APOE −/− mice induced by Ang II. Chronic aseptic inflammation that occurs with aging is involved in Fig. 5 Effects of Necrostatin-1 and TAK-242 on the expression of HMGB1, TLR4, and necroptosis marker proteins in the Ang IIinduced AAA in mice. Aortae were collected 28 days after Ang II subcutaneous infusion. Western blot and RT-PCR were performed to evaluate the HMGB1, TLR4, and necroptosis markers of different treatment groups mice. Localization of HMGB1, TLR4 and RIP3 by immunofluorescence staining. A, B Western blot of extracts from the aortas of each mouse group for analysis of HMGB1, TLR4, RIP1, RIP3, and MLKL levels (n = 6/group). C Relative mRNA expressions of HMGB1 (n = 6/group), TLR4, RIP1, RIP3, and MLKL (n = 6/ group) in the aortas of each mouse group. D Representative double immunofluorescence staining of HMGB1, TLR4, RIP3 (green), and α-SMA (red) in APOE −/− mouse aortic tissue. Scale = 50 μm. Data are shown as mean ± SD. One-way ANOVA, ****P < 0.0001, vs. the Sham group; ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. the AAA group the pathogenesis of various age-related diseases, including cardiovascular aging and related diseases, such as atherosclerosis and aneurysms [38]. Age-related aortic remodeling is the result of aseptic inflammation [39]. The accumulation of age-related DAMPs, which can bind to cell surface receptors on innate immune cells, is an important trigger for aseptic inflammation [40,41].
Toll-like receptors (TLRs) are the key transmembrane proteins that recognize extracellular antigens and transmit information into cells to initiate the inflammatory response [42,43]. TLR4 can be activated by endogenous DAMPs, and it is one of the earliest and most studied members of the Toll-like family [13]. TLR4 and its ligand HMGB1 play important roles in promoting aseptic inflammation. Their expression levels are positively correlated with age [44]. HMGB1 is mainly present in the nucleus under physiological conditions. When cells are stimulated or in a pathological state, HMGB1 can be released from the nucleus [45]. It has been reported that TLR4 deficiency can reduce necroptosis in microglia and inhibit retinal inflammation [17]. Necroptosis is a highly regulated cell death pathway that can be triggered by many different cell membrane receptors, including TLR4 [12]. These receptors lead to the phosphorylation and activation of the necrotizing kinase RIPK3. RIPK3 phosphorylates MLKL, causing changes in membrane permeability [46]. After plasma membrane disruption, HMGB1 in the nucleus is released into the cell. Necroptosis can release DAMPs, suggesting that there might be some crosstalk between HMGB1/TLR4 and necroptosis, forming a vicious cycle.
To confirm the relationship and mechanism between the HMGB1/TLR4 pathway and necroptosis in APOE −/− mice with AAA induced by Ang II, the selective TLR4 inhibitor TAK-242 and the necroptosis inhibitor necrostatin-1 were used in this study. TAK-242 can bind to the intracellular domain of TLR4 and inhibit its signal transduction [47]. Necrostatin-1 is a RIP1-targeted inhibitor that can inhibit the necroptosis signaling pathway and inhibit the death of necrotizing cells during the development of diseases [10]. RIP1 blockade by necrostatin-1 prevents AAA formation in mice induced with Ang II [48]. This finding was also verified by our research. Moreover, necrostatin-1 also reduced HMGB1 and TLR4 expression by inhibiting RIP1 signaling. Similarly, we noted that the inhibition of TLR4 signaling by TAK-242 downregulated HMGB1 expression. Notably, after blocking TLR4 with TAK-242, the expression of necroptosis markers decreased significantly, and necrostatin-1 effectively blocked necroptosis. In summary, during degenerative changes in the aging aorta, the expression of TLR4 on the surface of VSMCs and its extracellular ligand HMGB1 were increased, thus initiating necroptosis in VSMCs. After destruction of the plasma membrane, HMGB1 in the nucleus was released into the extracellular space to further perpetuate necroptosis in VSMCs and form a vicious positive feedback loop, which eventually led to the loss of VSMCs and a decrease in elastin and collagen secreted by VSMCs, thus resulting in AAA formation (Fig. 6). In addition, inhibiting TLR4 prevented aortic degeneration and inflammation in APOE −/− mice with AAA induced by Ang II, thus preventing experimental AAA formation.
There are several limitations in the present studies. First, TLR4 signaling was blocked by specific inhibitors. Further studies using conditional TLR4 knockout mice are needed to more accurately assess the relationship between TLR4 necroptosis. Second, in this study, the AAA was AngIIinduced in mouse models. However, considering the pathomorphological differences between animal models and human AAA, it is too early to draw a conclusion about the formation of human AAA. Further research is needed to address these issues.
Taken together, our results suggest that chronic low-grade sterile inflammation, which occurs with aging, exacerbates necroptosis in VSMCs during aortic degenerative changes. Consistent with other findings, the HMGB1/TLR4 pathway and necroptosis play important roles during AAA development in mice induced by Ang II, providing more evidence for the underlying mechanism of AAA formation. TAK-242 alleviates the inflammatory reaction and necroptosis by inhibiting TLR4 signaling and has a protective effect against AAA in mice induced by Ang II, providing further evidence for TLR4 as a potential therapeutic target of AAA.