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Modulating effective lymphatic drainage in aortic adventitia has anti-atherosclerotic effects: a new mechanism of FOXC2 to improve lymphatic endothelial cells inflammatory damage

https://doi.org/10.21203/rs.3.rs-4827875/v1

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Atherosclerosis is a chronic multifactorial cardiovascular disease. The disruption of lymphatic drainage function, which is induced by inflammatory damage to lymphatic endothelial cells (LECs), plays an essential role in atherosclerosis. However, the detailed regulatory mechanisms remain largely unknown. Since FOXC2 has become a major regulator of lymphatic vessel development and maturation, we explored the possibility that FOXC2 contributes to lymphatic drainage and atherosclerosis. Herein, we identified FOXC2 mitigates atherosclerosis by restoring efficiently lymphatic drainage via regulating LECs inflammatory responses. Atherosclerosis leds to aortic lymphatic drainage dysfunction, and an down-regulation of FOXC2 expression in the aorta, especially in aortic adventitia, and an inhibition of the biological activity of FOXC2. FOXC2 overexpression improved the aortic lymphatic drainage function which led to reducing the accumulation of inflammatory factors in aortic adventitia, lymph nodes and lymphatic fluid. However, disruption of lymph flow by left carotid artery ligation abrogated the beneficial effects by FOXC2 overexpression on atherosclerosis. Western blot and co-immunoprecipitation experiment revealed that TRAF2 as a key downstream target in TNF-α-induced LECs inflammatory damage. FOXC2 overexpression down-regulated TRAF2, inhibited interaction of TNF-α with TRAF2, reduced the inflammatory damage to LECs, and restored LECs functions, which promoted the function of lymphatic drainage. FOXC2 knockout aggravated TNF-α-induced LECs inflammatory damage, TRAF2 up-regulation, and increased TNF-α interaction with TRAF2. Collectively, we have demonstrated that FOXC2 serves a central role in the atherogenesis induced by lymphatic drainage dysfunction.

Biological sciences/Physiology

Health sciences/Cardiology

Atherosclerosis

lymphatic drainage function

FOXC2

Lymphatic endothelial cell

Inflammation

Maintaining effective lymphatic drainage in aortic adventitia is a key strategy for inhibiting atherosclerosis. However, the capacities of aortic lymphatic vessels draining the diseased aorta and the significance of lymphatic drainage in atherogenesis remain little understood. Our research clarifies previously unanswered questions about the function of lymphatic vessels draining the aorta during atherosclerosis and its effects on disease development. We conducted measurements on the transport of lymphatic fluid in the aorta and found that it is hindered when atherosclerosis advances. However, when FOXC2 overexpression, it improves the transport of lymphatic fluid during the regression of lesions. In our study, we found that increased FOXC2 expression in inflammatory damaged LECs restored the function of LECs and regulated lymphatic drainage function. Finally, we dissected how FOXC2 engage in the regulation of lymphatic drainage function and the mechanism of action. Our research indicates that the promotion of aortic adventitial lymphatic drainage by FOXC2 was an effective treatment for atherosclerosis.

Atherosclerosis is a vascular disease characterized by the accumulation of lipids and inflammatory cells in arterial walls, which is a common pathological basis of many cardiovascular and cerebrovascular diseases (1). Traditionally, atherosclerotic lesions develop from the luminal side of the artery, which involves several mechanisms triggered by endothelial activation, subendothelial retention of plasma apolipoprotein in the intima, and subsequent inflammation (2). Although atherosclerosis mainly affects the arterial wall intima, it also affects all three layers (3). Increasing evidence suggests that adventitia is a significant contributor to atherosclerosis development and progression. Adventitial lymphatic vessels have a beneficial role in promoting the migratory egress of these immune cells to the lymph nodes to bring about resolution of the plaque and adventitial inflammatory response (4). Despite several decades passing since the proposal of a connection between adventitial lymphangiogenesis and atherosclerosis, there have been very few studies directly examining the relationship between lymphatic transport and atherosclerosis

lymphatic circulation has aroused considerable interest in cardiovascular research for its role in transporting immune cells, clearing peripheral cholesterol-rich high-density lipoproteins via reverse cholesterol transport, and overall modulating inflammatory responses (5). Lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. It is play a crucial role in regulating the drainage of interstitial fluid, which significantly contribute to immune surveillance by removing inflammatory mediators and transporting immune cells to draining lymph nodes, thereby aiding in the resolution of inflammation (6). Functional studies have identified that the adventitia of aorta is rich in lymphatic vessels tissue and the lymphangiogenic response negative correlated with atherosclerotic plaque progression(7). Nevertheless, few of these studies examined the role of lymphatic drainage during atherosclerosis progression. Until recently, animal studies following experimental lymphostasis suggest a protective role for lymphatic vessels in atherosclerosis, and dysfunction of lymphatic vessels could impede the drainage of lipids, growth factors, inflammatory cytokines, and immune cells, further aggravating atherosclerosis lesions (8). Accordingly, atherosclerotic plaque progression may attributed not only to elevated levels of atherogenic factors entering arterial wall, but also to impaired lymphatic clearance caused by aortic lymph stasis. Nonetheless, molecular mechanisms that regulate lymphatic drainage in atherosclerosis are largely unknown. Therefore, regulating lymphatic function is expected to be an important therapeutic method for AS.

Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature, involving LECs proliferate, migrate and closely arrange (9), helping to maintain fluid homeostasis by returning interstitial fluid to blood circulation. The accumulation of inflammatory factors increases LECs damage, leading to LECs molecular characteristics alter, inhibiting LECs functions, destroy the tight connection of lymphatic vascular endothelial cells, and affecting the formation of lymphatic valves and drainage function of lymphatic vessels (10). Therefore, maintenance of normal LECs function, which is important in the process of functional lymphatic remodeling and lymphatic drainage.

Forkhead Box Protein C2 (FOXC2), a member of the transcription factor FOX family, is essential for the maturation of lymphatic vessels by affecting LECs (11). FOXC2 is expressed at high levels in LECs and plays a crucial role in lymphangiogenesis and lymphatic function (12). In contrast, deletion of FOXC2 leads to impaired connections between LECs, impaired lymphatic valves development and focal vascular lumen collapse, causing lymphatic vessel dysfunction and lumen collapse (13). In spite of this information, few studies have investigated how FOXC2 regulates lymphangiogenesis in atherosclerosis, and whether the underlying mechanism is related to the inhibition of inflammatory injury in LECs has not been reported.

Taking into account the research on atherosclerosis mostly focused on the aortic media, we extended our interests to the aortic adventitia lymphatic vessels and to observe whether FOXC2 regulation of aortic lymphatic drainage functions plays a critical role in resistance to atherosclerosis. In the present study, we evaluated the level of FOXC2 in ApoE^−/− mice with HFD-induced atherosclerosis model. We further elucidated that FOXC2 as a key regulator of lymphatic drainage in atherosclerotic mice, which inhibits inflammatory response in vascular walls, and reduces plaque area in atherosclerotic mice. Mechanistically, we found that FOXC2 inhibits the LECs inflammatory injury via inhibiting TRAF2 expression and TNF-α-TRAF2 interaction, leading to recovered the inflammatory damage to LECs, and restored LECs functions, which promoted the function of lymphatic drainage. Our study clarified the role of lymphatic drainage in atherosclerosis disease and its regulatory mechanism, which provided a basis for targeted drug treatment. The results are also expected to stimulate new regulation of lymphangiogenesis in other inflammatory diseases to aid the resolution of inflammation and restoration of tissue homeostasis.

Down-regulation of FOXC2 expression in aorta of patients with atherosclerosis

In order to explore whether FOXC2 is a key factor in regulating atherosclerosis, we performed differential gene expression analysis using the GDS3018 dataset from the Gene Expression Omnibus database (GEO), and found that FOXC2 expression was down-regulated in aortas of atherosclerotic patients, indicting that FOXC2 is expected to become a new therapeutic target for atherosclerosis (Fig. 1A-C).

Low levels of FOXC2 are associated with atherosclerosis in ApoE mice

To determine whether FOXC2 levels are dysregulated in atherosclerosis, we measured its expression in aortas with atherosclerosis-prone ApoE^−/− mice. Firstly, ApoE^−/− mice were fed with HFD to establish an atherosclerosis animal model. Atherosclerosis was diagnosed with thoracic aortic and carotid ultrasonography. As shown in Fig. 2A, ultrasound results demonstrated thickened blood vessel walls and plaque formation in both the carotid artery and aortic arch in the model group compared to the control group. We further investigated the pathological changes in the aortic arch in model group, which showed an distinct accumulation of lipid and atherosclerotic lesions, indicating successful establishment of the model (Fig. 2B-D).

Next, we examined FOXC2 protein expression in HFD-induced ApoE^−/− mice. As shown in Fig. 2E-F, a downregulation of FOXC2 in HFD-induced ApoE^−/− mice compared with control group. The subcellular localization of a protein is closely related to its function and the activated FOXC2 remained mainly in the nucleus fraction (14–15). As shown in Fig. 2G-H, the nucleic/cytoplasm localization of FOXC2 was lower in model group but higher in control group, indicating FOXC2 activation is significantly inhibited during the progression of atherosclerosis. Taken together, these findings showed that reduced levels of FOXC2 and in inhibition of FOXC2 activation are a common feature in atherosclerosis.

FOXC2 overexpression inhibits aortic inflammation and alleviates atherosclerotic lesions in HFD-induced ApoE^−/− mice

Given the aforementioned results, we postulated that overexpression FOXC2 in HFD-induced ApoE-/- mice could minimize atherosclerotic lesions. In order to test whether this hypothesis is true, we delivered FOXC2 cDNA to the abdominal aorta of HFD-induced ApoE^−/− mice using AAV8 vector through the tail vein (Fig. 3A-B). In the AAV-FOXC2 group, the levels of lipid accumulation and atherosclerotic lesions significantly decreased in the aorta (Fig. 3C-E). Next, evaluation of inflammatory cytokines in the aorta after overexpression of FOXC2 in HFD-induced ApoE^−/− mice showed a striking inhibition of the release of TNF-α, ox-LDL, IL-1β and IL-18 (Fig. 3F). Futhermore, the levels of TNF-α and VCAM-1 expression were increases during the development of atherosclerosis but decreases after treating with FOXC2 overexpression (Fig. 3G-I). Collectively, these results implied that FOXC2 could inhibit aorta inflammation and thus alleviate atherosclerotic lesions.

FOXC2 overexpression promotes lymphatic drainage in HFD-induced ApoE mice

To clarify the exact role of FOXC2 in atherosclerosis, we used immunostaining experiment to determine in which layer of the atherosclerotic arterial wall FOXC2 levels are downregulated. As shown in Fig. 4A-B, a decrease in FOXC2 staining in adventitial layer of model group compared with the control group. Previous studies have shown that the adventitia of arteries is rich in lymphatic vessels and insufficient lymphatic drainage could thus exacerbate atherosclerotic lesions. Given the inhibitory effect of effective lymphatic drainage in the aortic adventitial on atherosclerosis and the role of FOXC2 in promoting lymphatic vessel development, we hypothesized that FOXC2 might exert its anti-atherosclerotic effect by restoring lymphatic drainage in HFD-induced ApoE^−/− mice.

To test this hypothesis, periaortic lymph nodes and the lymphatic vessels between periaortic lymph nodes can be clearly visualized by Evans Blue staining. In model mice, transport of Evans Blue from footpad to aortic lymphatic vessels was inhibited and no blue dye was visible and only the lymph nodes were stained with blue dye, indicting lymphatic drainage dysfunction in HFD-induced ApoE^−/− mice. However, after AAV-mediated FOXC2 overexpression, the transport of Evans Blue dye was recoverl and lymphatic vessels was visible in aortic (Fig. 4D). Meanwhile, lymph vessel contraction frequency has now been shown to be another important indicator of lymphatic drainage function. After injecting mice with ICG in the footpad, we quantified lymph vessel contraction frequency by measuring the fold-change in the mean fluorescence intensity of the lymphatic vessels from before to after mechanostimulation. Consistent with Evans blue staining result, NIR fluorescence imaging system also found that the lymphatic clearance by increasing the lymph vessel contraction frequency in aortic in HFD-induced ApoE^−/− mice after AAV-mediated FOXC2 overexpression (Fig. 4E-F). Collectively, we demonstrate that FOXC2 is required for lymphatic drainage function in HFD-induced ApoE^−/− mice.

FOXC2-mediated atherosclerotic plaque regression depends on effective lymphatic drainage in HFD-induced ApoE^−/− mice

To further evaluate the implication of the lymphatic drainage contribution of FOXC2 and whether adequate lymphatic drainage is necessary to mediate regression of atherosclerosis by FOXC2. First, we ligated the left carotid artery (LCA) and removed the left carotid artery (LCA)-draining deep cervical lymph nodes to disrupt lymphatic drainage in atherosclerotic mice after AAV-mediated FOXC2 overexpression (Fig. 4C) (6). As shown in Fig. 4D, after LCA ligation, there were no blue dye was visible around aorta and only the lymph nodes were stained with blue dye. Furthermore, LCA ligation significantly decreased the lymph vessel contraction frequency in aortic (Fig. 4E-F), indicting LCA ligation reversed he promoting effects of FOXC2 overexpression on lymphatic drainage function in HFD-induced ApoE^−/− mice.

Next, we studied the impact of LCA ligation on atherosclerosis. As shown in Fig. 5A-C, LCA ligation abolished the beneficial effects of AAV-mediated FOXC2 overexpression in inhibiting atherosclerosis, and remarkably increased the lipid deposition and plaque area of aortic arch. Lymphatic vessels were stained by podopliain and CD68⁺ were used to label macrophages. LCA ligation reduced the podopliain and increased CD68⁺ in aortic adventitia, which indicated that effective lymphatic drainage inhibits aortic adventitia inflammatory cell accumulation (Fig. 5D-E). Moreover, LCA ligation significantly promoted the level of TNF-α, ox-LDL, IL-1β and IL-18 in lymph fluid (Fig. 5F). In addition, as shown in Fig. 5G, para-aortic draining lymph nodes of LCA ligation had significantly higher percentage of CD11c + and CD45 + compared to AAV-mediated FOXC2 overexpression. Collectively, these findings showed that the efficiency of the regression of atherosclerosis induced by FOXC2 treatment relies on a functional aortic lymphatic drainage.

Expression and functional inhibition of FOXC2 in TNF-α-induced LECs inflammatory injury

The lymphatic vasculature originates from a network of vessels composed of tubular LECs and involves the proliferation, migration and sprouting of LECs. To investigate the effect of FOXC2 on lymphatic drainage function in vitro, TNF-α was used to induce LECs to establish a LECs inflammatory injury model. First, we cultured LECs with different concentrations and times of TNF-α. As shown in Fig. 6A, CCK8 analyses demonstrated that TNF-α-treatments significantly reduced the cell viability and presented a dose-dependent and time-dependent. It is worth noting that cell viability was maintained at 50% at TNF-α 10 ng/mL and 24h, which indicated that LECs inflammatory injury model was established successfully (16). Moreover, TNF-α-treatments significantly upregulated the expression levels of inflammatory factors VCAM-1, ICAM-1, IL-18 and IL-1β in LECs (Fig. 6B-6D). Next, we further examine the effects of TNF-α-induced inflammatory injury on the function of LECs to form lymphatic vessels. Compared to control group, a significantly decreased number of Ki67 positive cells in the TNF-α-treated group, which indicated that the TNF-α-treatments obviously inhibit LECs proliferation in time and concentration-dependent (Fig. 6F and H). Transwell results show that the migration of LECs decreased when TNF-α concentration and time increases (Fig. 6G and I).

Subsequently, we examined the changes in the level of FOXC2 in TNF-α induced LECs. Western blot analysis showed that the FOXC2 levels decreased in time and concentration-dependent manners (Fig. 6J). Meanwhile, TNF-α decreased FOXC2 expression in nucleus and increased in cytoplasm (Fig. 6K). Immunofluorescence results were consistent with the results mentioned above. As shown in Fig. 6L-6M, TNF-α treatment led to significant inhibition of FOXC2 nuclear translocation in a concentration- and time-dependent manners. Collectively, these results showed that the expression and function of FOXC2 are significantly reduced in TNF-α induced LECs, suggesting that FOXC2 may be involved in inflammatory injury of LECs.

FOXC2 improves TNF-α-induced inflammatory injury in LECs

To further verify whether FOXC2 regulating LECs inflammation, we explored the influence of FOXC2 on TNF-α-induced LECs. First, we overexpressed or knock down FOXC2 expression in LECs and verified changes in protein abundance by Western blot (Fig. 7A-D). As shown in Fig. 7E, FOXC2 overexpression significantly enhanced the cell vitality stimulated with TNF-α. Furthermore, FOXC2 overexpression inhibited the expression and release of TNF-α-induced inflammatory cytokines VCAM-1, ICAM-1, IL-18 and IL-1β in LECs (Fig. 7F-H). At the same time, FOXC2 overexpression expression increased the number of ki-67 positive cells in TNF-α-induced LECs and enhanced cells proliferative ability (Fig. 7I-J). Transwell results also showed that FOXC2 overexpression enhanced TNF-α-induced cell migration (Fig. 7K-L). However, when FOXC2 was knocked down in LECs, which further aggravated TNF-induced LECs proliferation and migration reduced, and increased expression and release of inflammatory cytokines, indicating that FOXC2 could inhibit the inflammatory injury of LECs and promote the recovery of LEC function.

TRAF2 targetes at binding with TNF-α and induces inflammation in LECs

TNF-α is an important mediator of inflammatory processes, which mediates its inflammatory signals by binding with high affinity to two distinct cell surface receptors, known as TRAF1 and TRAF2 (17). We found that the TRAF2 expression were markedly increased in concentration-dependent manners in LECs induced by TNF-α, while no statistical difference was detected in the expression levels of TRAF1 (Fig. 8A-B). Then, we quantified TRAF2 expression in aortic adventitia, as expected, atherosclerotic mice exhibited elevated TRAF2 expression levels, compared with normal mice (Fig. 8C-D). To further examine whether TRAF2 affected inflammatory response of TNF-α-induced LECs, we silenced TRAF2 by specific siRNA (Fig. 8E), and then detected the molecular interaction between TNF-α and TRAF2 in LECs. As shown in Fig. 8F, decreasing TRAF2 expression significantly restrained the interaction between TNF-α and TRAF2 in TNF-α-stimulated LECs. At the same time, LECs-specific knockout of TRAF2 expression notably decreased the inflammatory cytokines VCAM-1, ICAM-1, IL-18 and IL-1β expression and release in TNF-α-induced LECs (Fig. 8G-H). Moreover, knockout of TRAF2 significantly promoted the number of ki-67 positive cells in TNF-α-induced LECs (Fig. 8I-J). Consistently, TRAF2 knockdown increased the migration capability of LECs stimulated by TNF-α (Fig. 8K-L). It is worth noting that decreasing TRAF2 significantly promoted FOXC2 nuclear translocation in TNF-α-induced LECs (Fig. 8L-M). These results suggest that TRAF2 is critical in the TNF-induced inflammatory response of LECs and FOXC2 might suppress the development of cellular inflammatory responses by regulating TRAF2.

FOXC2 attenuates inflammatory damage in LECs and atherogenesis by inhibiting TNF-α-TRAF2 interaction

Based on the above results, we further examined the effect of FOXC2 on TRAF2 expression in aortic adventitia of HFD-induced ApoE^−/− mice. As expected, we found that FOXC2 overexpression significantly inhibits TRAF2 protein levels in atherosclerotic aortic adventitia (Fig. 9A-B). Similarly, FOXC2 overexpression inhibited TRAF2 expression in TNF-α-mediated inflammation in LECs, but this effect was reversed by TRAF2 overexpression (Fig. 9C).

The formation of lymphatic valves is necessary for promoting lymphatic drainage (18). Next, we measured the lymphatic valves formation-related proteins Laminin α5 and Integrin α9 levels. Western blot assays showed the Laminin α5 and Integrin α9 expression was significantly upregulated in TNF-α-induced LECs after overexpression of FOXC2 treatment, while this effect was inhibited after TRAF2 overexpression (Fig. 9D-E).

To further clarify whether the effect of FOXC2 in regulating TNF-α-mediated inflammation in LECs and promoting lymphatic valve formation is related to inhibition of TNF-α and TRAF2 interactions. As shown in Fig. 9F, FOXC2 overexpression displayed inhibitory effects on the interaction between TNF-α and TRAF2 in TNF-α-mediated inflammation in LECs, indicating that FOXC2 overexpression suppresses inflammatory damage in LECs and atherogenesis by preventing TNF-α-TRAF2 interaction.

It has been documented that in both people and mice, lymphangiogenesis and atherosclerosis are related (22). Immunity, lipid clearance, and tissue fluid homeostasis all depend on a functioning lymphatic vasculature (23). While adventitial lymphangiogenesis and atherosclerosis have been associated, little is known about the function of the aortic lymphatic arteries that drain the diseased aorta and the significance that lymphatic drainage plays in atherogenesis. Recent reports have demonstrated that the lymphatic system plays a crucial role in eliminating cholesterol from atherosclerotic arteries by reversing cholesterol transport. They also showed that immune cells can leave atherosclerotic lesions through lymphatic vessels (24). Moreover, when transgenic mouse models with lymphatic insufficiency are bred with atherogenic mice, it results in increased development of atherosclerosis (25). However, existing research has focused less on investigating the role of the aortic lymphatics compared to the peritoneal and cutaneous lymphatics. Here, our results observed that the reduction of lymphangiogenesis in aortic adventitia in atherosclerotic mice resulted in aortic lymph stasis, which led to the accumulation and infiltration of various inflammatory factors in the aortic adventitia, emphasizing the significance of assessing lymphatic functioning in the therapy of atherosclerosis.

Lymphangiogenesis is a complex process that is related to the proliferation, migration, and network formation of LECs (26). Although LECs are static in most cases, when involved in inflammation, tumours and trauma, LECs are significantly inhibited in their ability to migrate, proliferate and inhibit lymphangiogenesis under the influence of growth chemokines (27). Thus, LECs inflammatory damage led to inhibition of lymphangiogenesis is crucial to atherosclerosis development. TNF-α is known to stimulate LECs inflammatory damage (28). Our study reveals that the LECs proliferation and migration abilities significantly inhibits in TNF-α induced LECs, and FOXC2 expression and nuclear translocation levels markedly decrease in TNF-α-damaged LECs. The overexpression of FOXC2 led to inhibiting the release and expression of inflammatory factors and promoting proliferation and migration abilities in TNF-α induced LECs. The present study provides evidence that FOXC2 inhibited cell inflammation damage and restore cellular function in LECs stimulated with TNF-α.

LECs inflammatory damage and the subsequent disruption of lymphatic drainage result in adventitial inflammation and atherosclerotic plaque rupture. Hence, strategies aimed to relieve inflammation in LECs and efficiently restore lymphatic drainage delay the atherosclerosis lesion progression, potentially serving as a therapeutic intervention. In this study, we reveals that aortic lymphatic transport is restricted during atherosclerosis, and this sluggish lymphatic drainage result in adventitial inflammation and atherosclerosis exacerbation. FOXC2 significantly reversed inflammatory response of aortic arteries via restoring lymphatic drainage in HFD-induced ApoE^−/− mice. Moreover, we observed that TNF-α led to inflammation and functional inhibition in LECs by binding to TRAF2. We also shed light on the mechanism of how FOXC2 benefits atherosclerosis by inhibiting inflammation in LECs and thereby promoting lymphatic drainage. These findings implicate FOXC2 as a potential therapeutic target for atherosclerosis.

FOXC2, a forkhead transcription factor, is present in diverse embryonic tissues and plays a crucial role in the development of mesodermal tissues, such as bone tissue, vascular tissue, and lymphatic tissue (29). Established data shows the link of FOXC2 with many malignancies, including prostate cancer, colon cancer, colorectal cancer, and human schwann cell tumors (30). In spite of these findings, the function of FOXC2 in the onset of atherosclerosis has not been investigated in any prior research. Through GEO database, we found FOXC2 is downregulated in atherosclerosis patients. Extremely, little is known about the role of FOXC2 in atherosclerosis. This motivates us to further reveal the role of FOXC2 in the atherosclerosis progression. Here, our results show that an decreased expression of FOXC2 in HFD-induced ApoE^−/− mice artery with atherosclerotic plaques. Under normal circumstances, FOXC2 is located in the cytoplasm and is activated after entering the nucleus, which is implicated in lipid metabolism via sustaining survival of preadipocytes and increasing oxidation of fatty acid in mouse adipose (31). Similar to previously reported research, our results presented suggest that following the development of atherosclerosis, the expression of FOXC2 in the nucleus was also sharply reduced. When FOXC2 was overexpressed in aortic, the aortic inflammatory responses were remarkably alleviated and the disease progression was shown to be delayed and the survival rate to be increased. Based on these results, we postulate high expression and functional activation of FOXC2 plays a pivotal role in the development of atherosclerosis.

To investigate FOXC2's role in atherosclerosis, we examined the distribution of FOXC2 in atherosclerotic and non-atherosclerotic arteries in mice. Interestingly, in this study, we observed that decreased FOXC2 expression in adventitial of mice atherosclerotic arteries, suggesting that the anti-atherosclerosis effect of FOXC2 may related to the promotion of adventitial lymphangiogenesis. Furthermore, overexpressing FOXC2 enhanced aortic lymphatic drainage and got rid of aortic tissue inflammatory response and atherosclerotic plaques. This indicates that FOXC2 has the potential to address lymphatic dysfunction. However, the precise mechanism by which FOXC2 achieves this effect is yet unknown and requires further investigation. To better understand how FOXC2 promotes lymphangiogenesis in atherosclerosis, we ligated the left carotid artery (LCA) to disrupt aortic lymph flow. We observed that when aorta lymph flow is disrupted, it leads to adventitial inflammation and the formation of atherosclerotic plaque in mice. This disruption inhibits the regression of atherosclerosis that is produced by FOXC2. Lymph nodes are situated along the lymphatic vessels and serve as filters to eliminate infections and antigens found in the lymphatic fluid, which is of great significance to immune monitoring (32). The dysfunction of lymphatic drainage will be accompanied by the damage of lymph nodes, resulting in the inability to clear excess inflammatory factors and trigger the body's inflammatory response (33). This finding is consistent with the our study that disruption of aortic lymph flow weakened the beneficial effects of promoting lymphatic drainage by FOXC2, increased the infiltration of macrophages in aortic adventitia, inhibited inflammatory factors elimination in lymph, and enhenced the T cells accumulation in lymph nodes. These results indicated that functional lymphatic vessels play a crucial role in preventing adventitial inflammation and atherosclerosis progression by promoting the clearance of immune cells from the aortic wall and efficient lymphatic drainage of aorta is necessary for therapeutic effect of FOXC2 on atherosclerotic plaque. Herein, we present a novel atherosclerosis inhibitory strategy of FOXC2 that suppresses atherosclerosis by alleviating lymphatic dysfunction.

TNF-α is a focus of treatment for inflammatory diseases, and there are over 20 drugs that target the TNF-α-signaling pathway being used in clinical practice (33). Research indicates that TNF-α inhibitors can lower inflammation in atherosclerotic individuals (34). It is well known that the pleiotropic cytokine TNF-α activates TNF receptors, resulting in pro-inflammatory mediators, necroptosis, and apoptosis activation, which are important in atherosclerotic pathogenesis (35). Multiple clinical and animal investigations have demonstrated that inflammation is linked to a decrease in lymphatic outflow from adjacent tissues, which significantly aggravated atherosclerotic lesions (36). Inflammation-induced lymphatic dysfunction can be the result of many mechanisms. For instance, TNF-α promotes LECs to produce nitric oxide, which directly restricts lymphatic muscle cells functional gene expression (35). Furthermore, compelling evidence has demonstrated that the TNF-α-TRAF2 interaction in macrophages elicits signaling responses that are both inflammatory and promote the development of atherosclerosis (37). Likewise, our experiments in LECs showed that the silence of TRAF2, rather than TRAF1, inhibited TNF- α -induced inflammatory damage, promoted LECs proliferation and migration abilities and induced FOXC2 nuclear translocation, which induced by TNF- α-TRAF2 interaction in LECs. Additionally, we have also discovered that TRAF2 expression was increased in aortic adventitia in atherosclerotic lesions. Consistent with our results, the significant role of TRAF2 in the development of atherosclerosis has been validated in additional mouse models. Deletion of TRAF2 in LDLR^−/− mice fed high cholesterol diet preserved endothelial function and integrity, reduced intima thickness and inflammation and increased expression of protective factors (38). Based on these results, we conducted additional research to explore the molecular mechanisms through which FOXC2 enhanced lymphangiogenesis and facilitated lymphatic drainage. Our data showed that the expression of TRAF2 in aortic adventitia in the atherosclerotic lesions was reduced and TNF-α-TRAF2 interaction was inhibited in LECs by FOXC2 overexpression. In this manner, FOXC2 overexpression attenuated cellular inflammatory factors uptake in LECs, reduced inflammatory response occurs, restored cell function and promoted the expression of lymph valve formation related proteins Laminin α5 and Integrin α9. Thus, these findings add FOXC2 to the TNF- α-TRAF2 interaction axis in the regulation of lymphatic vessel function and atherosclerosis development.

To summary, the present study first revealed the transcription factors FOXC2 inhibited LECs inflammatory injury and promoted lymphatic drainage function via inhibiting TNF-α-TRAF2 interaction dependent mechanisms, and consequently enhances lymphatic drainage of inflammatory response from the arterial wall, relieving atherosclerosis development. These findings indicate that FOXC2-mediated lymphangiogesis may be a potential therapeutic strategy to improve the lymphatic drainage of inflammatory factors, promote resolution of inflammation via removal of inflammatory cytokines and immune cells from the inflamed area, and restore tissue homeostasis (Fig. 10).

Reagents and antibodies

TNF-α and Collagenase type II were bought from Sigma-Aldrich (USA). Rabbit anti-TNF-α, anti-TRAF1, anti-TRAF2, anti-VCAM-1, anti-ICAM-1, anti-IL-18, and anti-IL-1β were bought from Abcam (ab255275, ab300075, ab126758, ab134047, ab282575, ab243091, ab283818, USA). Rabbit anti-CD68 anti-Integrin α9 and FITC-Phalloidin were obtained from Chengdu Must Biotechnology (R381310, R383223, R16003, Chengdu, China). Rabbit anti-Laminin α5 and DAPI solution were bought from Beyotime Institute of Biotechnology. TNF-α ELISA Kit, ox-LDL ELISA Kit, IL-18 ELISA Kit, IL-1β ELISA Kit, ICAM-1 ELISA Kit and VCAM-1 ELISA Kit were purchased from Jianglai Biotechnology. Evans blue stain and Protein A/G beads were purchased from the Shanghai Yuanye Biotechnology. Indocyanine green was purchased from Anhui Zesheng Technology. Crystal violet dye solution were purchased from Solebao Technology. Culture medium (endothelial cell medium, ECM) was obtained from SciencCell (USA). AAV8-GP-FOXC2 mus was designed and synthesized by GenePharma. 24-well Tran-swell chamber were obtained from Merck Millipore (USA).

Animals and study design

One week of adaption feeding was given to male WT C57BL/6 J mice and ApoE knock-out mice (ApoE^−/−, 18–20 g, 6 weeks) on C57BL/6J background from Changzhou Card Vince Laboratory Animal Co., Ltd. The Anhui University of Traditional Chinese Medicine Ethics Committee approved all animal experiments. (Approval number: AHUCM-mouse-2023011). Mice were housed in an SPF environment with a 12-h light and 12-h dark cycle. Male ApoE^−/− mice were fed with a high-fat diet (HFD; 42% fat, 0.2% total cholesterol) for 12 weeks to induce atherosclerosis.

To overexpress FOXC2 in the aortic, AAV8-GFP-FOXC2 vector (1.54×10¹² v.g/mL) and AAV8-GFP-NC vector was prepared by diluting the vector to a viral titre of 1.54×10⁹ v.g/mL using sterile phosphate-buffered saline. (The original titre of the AAV8-GFP-NC vector was 2.28×10¹² v.g/mL) The prepared vector was stored in an ice box to prevent the virus titre from being affected by repeated freeze-thaw cycles. After Apoe^−/− mice were fed HFD for 5 weeks, mice were injected with AAV8 expressing FOXC2 (AAV-FOXC2) via tail vein. Mice injected with AAV8-GFP-NC were used as controls (AAV-ctrl). This injection was performed every 5 weeks for a total of two injections, during which time HFD feeding continued. Two weeks after the last injection, the mice were sacrificed and aortic tissue were analyzed with westren blot to detect the expression level of FOXC2.

Left carotid artery (LCA) ligation-induced mouse lymphatic obstruction mode

11-week-old male ApoE^−/− mice were subjected to partial ligated of left carotid artery after HFD feeding for 5 weeks. In general, mice received anesthesia via isoflurane inhalation and exposed the left carotid artery. The left carotid artery, which includes the external carotid artery and internal carotid artery, was tied off with a 6 − 0 silk suture, during which time HFD feeding continued. After 5 weeks of the left carotid artery ligation procedure, the lymphatic drainage function test was performed to measure whether the lymphatic obstruction model was successful.

Evans Blue Staining

The permeability of lymphatic vessels was evaluated by using Evans Blue dye for tracing the mice hind leg and thoracic aorta lymphatic drainage. Mice were anesthetized with isoflurane and 10 µL of Evans blue dye was intradermally injected into mouse foot pads. After ten minutes of administering the injection, the lymphatic vessels were observed using a stereo-microscope imaging equipment. The outflow of Evans Blue surrounding the vessel indicates the extent of the leakage.

NIR-II fluorescence imaging

Lymphatic vessel contraction capacity was evaluated by injecting 10 µL of indocyanine green (ICG) and using NIR-II fluorescence imaging (Series II 808/900–1700, Suzhou NIR-Optics Technologies Co., Ltd.) to observe the contractility of collecting lymphatic vessels that drain the lower limb and thoracic aorta. To demonstrate the ability of lymphatic to react to a sudden fluid load, mechanostimulation was applied to the injection site using a cotton swab, pressing gently for one second, ten times. An examination of fluorescence intensity was conducted to determine the frequency of contractions in the collecting lymphatic vessels, using a region of interest (ROI) approach.

Lymphatic fluid extraction

For clearer localisation of the lymphatic vessels, mice were gavaged using 1 mL of corn oil. After 1 h of gavage, mice were anaesthetized and dissected with scissors along the midline of the abdomen to expose the milky-white lymphatic vessels. A notch was carefully cut in the lymphatic vessel with microscopic scissors, and the lymphatic fluid was stimulated to flow out by pressing on the mouse intestine. Finally, the lymphatic fluid was sucked out with a 10 µL pipette gun and placed in a 1.5 mL EP tube, which was stored in the refrigerator at -80°C for spare use.

Flow cytometry

Mice were anesthetized with isoflurane, and 10 µL of Evans blue staining solution was injected into the paw pads. Then, mice were dissected to expose the aorta, and Evans blue-stained lymph nodes around the aorta were harvested and used for flow cytometry analysis. The lymph nodes were cut and digested in RPMI medium containing 1 mg/mL type II collagenase and 0.05 mg/mL deoxyribonuclease I solution; the cells were homogenized for 10 minutes to obtain a single cell suspension; the single cell suspension was centrifuged for 10 minutes at 4°C and 1500 rpm. The cells were then analyzed by flow cytometry using a FITC-anti-CD45, APC-anti-CD11c antibody for 10 minutes; CD45⁺ and CD11c⁺ were measured by flow cytometry, and the data were analyzed using FlowJo software.

Immunohistochemistry staining

Aortic paraffin sections were deparaffinized using xylene and then removed from the slices, and sodium citrate buffer was added for antigen repair. After 20 min, goat serum was added for sealing for 1 h and washed with PBS. Then, added with antibodies against FOXC2, VCAM-1, and TNF-α, respectively for 14 h and incubated in fluorescent secondary antibody for 1 h. After stained with DAPI for 15 min, the sections were dried with a droplet of neutral resin sealing solution and photographed for observation under the microscope.

Aortic imaging

Mice were sacrificed by enucleation. Aortas were dissected using blunt forceps and removing surrounding fat and connective tissue. A stereomicroscope was then used to capture images.

HE staining

To observe lesions of atherosclerosis, the tissue slices were fixed in paraffin and then sliced into 7-µm sections. The sections were stained with haematoxylin for a duration of 1 minute and subsequently submerged in an acidic solution of alcohol for a period of 30 seconds. After staining with eosin for 50 s and dehydration via 100%, 95% and 85% ethanol, the slices were mounted in xylene and viewed under a microscope.

Aortic ultrasonography monitoring

The mice were rendered unconscious by administering 2% isoflurane by breathing. The hair on the chest and neck was meticulously removed through shaving, followed by the application of ultrasonic gel to the chest. The images were taken with a micro-imaging equipment (Vevo 2100 system, Canada) utilizing a 30-MHz suprasternal probe on the chest.

Western blot analysis

Lysing samples in RIPA buffer with PMDF on icfor 30 min. The proteins were put on polyvinylidene fluoride (PVDF) membranes and treated with the following antibodies: FOXC2 (1:1000), TNF-α (1:1000), TRAF1 (1:1000), TRAF2 (1:1000), VCAM-1 (1:1000), ICAM-1 (1:1000), IL-18 (1:1000), and IL-1β (1:1000). Finally, a high-sensitivity multi-function imaging device (GE, USA) was used to capture images.

Separation and extraction of aortic adventitia

Mice were sacrificed by enucleation and the aorta was carefully removed using microscopic scissors and microtome forceps. Next, aorta was placed in collagenase type II at a concentration of 1 mg/mL for digestion, which was stopped by placing the aorta in physiological saline after 30 seconds. Finally, the aorta was dissected with microtome scissors and removed from the aorta with microtome forceps.

Immunofluorescence staining

Immunofluorescence staining of aortic adventitia was performed using a whole patch approach. Briefly, aortic adventitia wree placed in slides were washed with PBS and immersed in 0.3% Triton X-100 and 0.5% bovine serum albumin. The appropriate primary antibodies were prepared and incubated overnight at 4°C and the secondary antibodies were incubated for 2 h. Next, the adventitia were incubated in RapiClear 1.52 to increase the optical transparency of the outer membrane; Finally, laser confocal microscope were used to capture images.

Cell culture and transfection

The LECs line were purchased from the Cell Resource Center of China (Beijing, China). The cells were gathered and grown in ECM with 20% FBS at 37°C and 5% CO2 in a humidified environment. The LECs were then divided into ten groups as follows: control, TNF-α group (LECs stimulated with 0.01, 0.1, 1, 10, 100 ng/mL TNF-α), TNF-α + si-FOXC2 group (FOXC2 knockdown LECs cultured with 10 ng/mL TNF-α), TNF-α + pcDNA-FOXC2 group (FOXC2 overexpress LECs cultured with 10 ng/mL TNF-α), TNF-α + si-TRAF2 group (TRAF2 knockdown LECs cultured with 10 ng/mL TNF-α) and TNF-α + pcDNA-FOXC2 + si-TRAF2 group (FOXC2 overexpress LECs cultured with TRAF2 knockdown LECs containing 10 ng/mL TNF-α).

To knock down the FOXC2 or TRAF2, a transfection reagent was used to transfect LECs with FOXC2 or TRAF2 siRNA and non-targeting control siRNA. To overexpress the FOXC2, the FOXC2 cDNA fragments were cloned into the pcDNA plasmid, generating pcDNA-FOXC2. Then, FOXC2 or TRAF2 levels were detected by Western blot.

immunofluorescence staining

Cover slips were put into a 24-well plate, and LECs were then plated on them. Then, the cells permeabilized, blocked, and treated with Ki67 primary antibodies for one night at 4°C. Next, cells were treated with fluorescently labelled secondary antibodies and Alexa Fluor 488-phalloidin. Lastly, pictures were taken with an upside-down confocal camera, and the Image J software was used to count the number of Ki67-positive cells.

Transwell migration assay

LEC migration in response to FOXC2 treatment was investigated using transwell inserts with 8 µm pore polycarbonate membranes in 24-well plate. Briefly, cells were pretreated with/without FOXC2 for 6 h. After 6 hours, 20000 cells were planted in the top chambers using 100 mL of basal medium ECM (0.5% FBS), and the lower chambers were filled with full endothelial cell growth media ECM. After being at 37°C for 12 hours, the membranes were fixed and marked with 0.5% crystal violet. The cells on top were then wiped off. An inverted phase-contrast microscope was used to take pictures of 7–9 randomly chosen fields. Image J was then used to count the cells on the bottom side of the membranes.

Co-immunoprecipitation experiment

Cell extracts were treated in extraction buffer at 4°C with the corresponding antibodies overnight. Protein A-sepharose beads were used to precipitate the immunocomplexes for two hours. After that, they were five times cleaned with extraction buffer and boiled in sodium dodecyl sulfate (SDS) sample buffer. Western blot analysis was carried out as stated after the immunoprecipitated proteins or aliquots containing 40 µg protein were separated on SDS-polyacrylamide gels.

Statistical analysis

Statistical analysis was performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Differences in means between multiple groups were analysed by one-way analysis of variance (ANOVA). Statistical significance was set at P values < 0.05.

Acknowledgments

Youli Bao: Writing original-draft, Investigation. Zhenglong Li: Visualization. Zijian Liu: Formal analysis. Jiahui Liu: Methodology, Validation. Li Zhu: Formal analysis. Xin Sun: Formal analysis. Wencheng Zhao: Writing-review & editing. An Zhou: Methodology and funding acquisition. Hongfei Wu: Writing origina-draft and funding acquisition.

This research was supported by the National Natural Science Foundation of China (82274137), the Natural Science Foundation of Anhui Province (2208085MH275) and the Provincial University Natural Science Research Project of Anhui (2022AH020041).

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