Research ArticleVASCULAR BIOLOGY

TFEB inhibits endothelial cell inflammation and reduces atherosclerosis

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Science Signaling  31 Jan 2017:
Vol. 10, Issue 464, eaah4214
DOI: 10.1126/scisignal.aah4214

Protected from atherosclerosis by TFEB

Atherosclerosis, or the buildup of fatty plaques in blood vessels, can lead to high blood pressure and heart attacks. Lu et al. found that, in cultured endothelial cells, the transcription factor TFEB reduced oxidative stress and inflammation, two processes thought to contribute to the development of atherosclerosis. When fed a high-fat diet, mice that overexpressed TFEB in endothelial cells developed smaller atherosclerotic lesions than their control littermates on the same diet. Thus, treatments that enhance the activity of TFEB in endothelial cells could reduce the development of atherosclerosis. Furthermore, because the anti-inflammatory effect of TFEB in endothelial cells was independent of its role in autophagy, a process in which cells digest macromolecules and organelles, these results highlight new roles for this transcription factor.

Abstract

Transcription factor EB (TFEB) is a master regulator of autophagy and lysosome biogenesis. We investigated the function of TFEB in vascular biology and pathophysiology and demonstrated that TFEB in endothelial cells inhibited inflammation and reduced atherosclerosis development. Laminar shear stress, which protects against atherosclerosis, increased TFEB abundance in cultured primary human endothelial cells. Furthermore, TFEB overexpression in these cells was anti-inflammatory, whereas TFEB knockdown aggravated inflammation. The anti-inflammatory effect of TFEB was, at least, partially due to reduced oxidative stress because TFEB overexpression in endothelial cells decreased the concentrations of reactive oxygen species and increased the expression of the antioxidant genes HO1 (which encodes heme oxygenase 1) and SOD2 (which encodes superoxide dismutase 2). In addition, transgenic mice with endothelial cell–specific expression of TFEB exhibited reduced leukocyte recruitment to endothelial cells and decreased atherosclerosis development. Our study suggests that TFEB is a protective transcription factor against endothelial cell inflammation and a potential target for treating atherosclerosis and associated cardiovascular diseases.

INTRODUCTION

Every year, more than 30% of all deaths in the United States are attributable to cardiovascular diseases (CVDs), such as myocardial infarction, stroke, or ischemic heart failure (World Health Organization), and the total direct and indirect cost of CVDs and stroke in the United States for 2010 was estimated to be more than $315 billion (1). Atherosclerosis, a progressive disorder of the vascular wall characterized by abnormal accumulation of lipid and immune cells in the subendothelial region, causes most of the pathogenesis in CVDs (2). Growing evidence suggests that endothelial cell dysfunction occurs in the initial stage of atherogenesis and contributes to the formation, progression, and complication of the atherosclerotic plaque (3). Atherosclerosis is considered to be an inflammatory disease (4, 5), and inflammatory response is an important hallmark of endothelial dysfunction (6). Many proinflammatory factors, such as oxidized low-density lipoprotein, tumor necrosis factor–α (TNFα), and interleukin-1 (IL1), activate endothelial cells and lead to recruitment of circulatory monocytes and leukocytes.

Accumulating evidence reveals a causal relationship between oxidative stress and endothelial inflammation (7, 8). In the cardiovascular system, reactive oxygen species (ROS) exert critical physiological roles in controlling endothelial cell function and vascular tone and pathophysiological roles in inflammation, hypertrophy, proliferation, apoptosis, migration, fibrosis, angiogenesis, and vascular remodeling (912). Excessive ROS lead to inflammation and endothelial dysfunction in vitro and in vivo (9, 13). Endogenous ROS promote atherosclerosis by increasing the abundance of adherent molecules such as E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 (VCAM1) and chemotactic factors such as IL6 and monocyte chemoattractant protein 1 (MCP1) (8), leading to recruitment of inflammatory cells.

Transcription factor EB (TFEB) is a basic helix-loop-helix transcription factor that regulates lysosomal biogenesis and autophagy function in many cell types (14). In macrophages, TFEB induces lysosomal biogenesis and rescues lipid-induced lysosomal dysfunction in atherosclerotic lesion (15). In the heart, oxidative stress induced by monoamine oxidase A impairs the transcriptional activity of TFEB (16), and TFEB deficiency suppresses autophagy (17) and leads to cell death (18).

Here, we report that TFEB abundance in endothelial cells was increased by laminar shear stress. Overexpression of TFEB in endothelial cells potently inhibited inflammation, whereas knockdown of TFEB aggravated inflammation. TFEB reduced intracellular ROS by increasing the abundance of antioxidant genes such as heme oxygenase 1 (HO1) and superoxide dismutase 2 (SOD2). Mice overexpressing TFEB in an endothelial cell–specific manner exhibited decreased endothelial cell–leukocyte adhesion under inflammatory conditions in vivo. In addition, EC-TFEB transgene inhibits atherosclerotic lesion formation in apolipoprotein E–deficient (ApoE−/−) mice.

RESULTS

TFEB is an inducible transcription factor under laminar shear stress in endothelial cells

Shear stress affects various processes in endothelial cells, including inflammation, proliferation, and survival (19, 20). In contrast to oscillatory shear stress, which promotes atherosclerosis, laminar shear stress protects against atherosclerosis (21). In human umbilical vein endothelial cells (HUVECs), TFEB abundance was significantly increased by 48-hour laminar shear stress compared with static conditions or oscillatory shear stress at the levels of both mRNA (5.19- ± 0.76-fold compared to static conditions and 5.11- ± 0.76-fold compared to oscillatory shear stress) and protein (2.38- ± 0.17-fold compared to static conditions and 1.78- ± 0.20-fold compared to oscillatory shear stress) (Fig. 1, A and B). We found that laminar shear stress induced TFEB nuclear translocation (Fig. 1, C and D) and decreased mammalian target of rapamycin (mTOR) activity as indicated by impaired phosphorylation of 4E-BP1 at Thr37/46, a downstream effector of mTOR (Fig. 1E). To determine TFEB distribution pattern in vivo, we performed en face staining of TFEB on different sites of the rabbit aorta (22, 23). Our results showed that atherosclerosis-resistant regions (greater curvature of aortic arch and descending aorta) showed higher TFEB protein abundance compared to atherosclerosis-prone regions (lesser curvature of aortic arch) (Fig. 1F). In addition, we determined whether TNFα treatment affected TFEB abundance. In both time-course and dose-response experiments, TNFα did not change TFEB mRNA and protein abundance in endothelial cells (fig. S1, A to D). Together, our data suggest that TFEB is a shear stress–responsive gene with a potentially critical role in endothelial cell biology.

Fig. 1 Laminar shear stress increased TFEB mRNA and protein abundance.

(A and B) HUVECs were exposed to static conditions, oscillatory shear stress, or laminar shear stress for 48 hours. (A) TFEB mRNA was determined by quantitative polymerase chain reaction (qPCR) and normalized against 18S RNA. (B) TFEB protein was determined by Western blot. (C and D) HUVECs were infected with adenovirus encoding Flag-TFEB. After 4-hour shear stress treatment, TFEB protein abundance was determined in the cytoplasmic and nuclear fractions. (E) After 4-hour shear stress treatment, phosphorylated (p) and total 4E-BP1 protein in HUVECs was determined by Western blot. Data in (A) to (E) were from three independent experiments and are presented as means ± SEM. *P < 0.05; **P < 0.01. (F) TFEB protein abundance in different areas of the rabbit aorta was determined by en face immunostaining. Data are representative of three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

Overexpression of TFEB inhibits endothelial cell inflammation

Laminar shear stress suppresses inflammation in endothelial cells both in vitro (24) and in vivo (21). Endothelial cell inflammation is characterized by the induction of various adhesion molecules and cytokines (5). TFEB overexpression suppressed the expression of SELE (which encodes E-selectin), MCP1 (which encodes MCP1), and VCAM1 (which encodes VCAM1) in HUVECs in response to the proinflammatory stimuli TNFα, IL1β, or lipopolysaccharide (LPS) (Fig. 2A). Consistent with these results, TFEB also potently inhibited TNFα-induced transcription of IL1β, IL8 (which encodes IL8), and IL6 (fig. S2A). TFEB also significantly decreased E-selectin and VCAM1 protein abundance by 49 ± 12% and 46 ± 11%, respectively (Fig. 2B). We further demonstrated that TFEB decreased the increase in E-selectin, MCP1, and VCAM1 mRNA and protein abundance induced by TNFα in a dose-dependent manner (fig. S2, B to D). The anti-inflammatory function of TFEB was not limited to HUVECs because TFEB overexpression also exerted a similar anti-inflammatory effect in primary human coronary artery endothelial cells (HCAECs) (Fig. 2, C and D). Thus, TFEB exerted a potent inhibitory effect on endothelial cell inflammation in the presence of various proinflammatory factors.

Fig. 2 TFEB potently inhibits inflammation in endothelial cells.

(A and B) HUVECs were infected with adenovirus encoding green fluorescent protein (GFP) or human TFEB and treated with TNFα, IL1β, or LPS. (A) SELE, VCAM1, and MCP1 mRNAs were determined by qPCR. (B) TFEB, E-selectin, and VCAM1 proteins were determined by Western blot. Band densities were quantitatively analyzed and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (C and D) HCAECs were infected with Ad-GFP or Ad-TFEB and treated with TNFα, IL1β, or LPS. (C) SELE, VCAM1, and MCP1 mRNAs were determined by qPCR. (D) TFEB, E-selectin, and VCAM1 proteins were determined by Western blot. Data in (A) to (D) were from three independent experiments and, where applicable, are presented as means ± SEM. *P < 0.05; **P < 0.01.

TFEB knockdown aggravates inflammation in endothelial cells

To further establish the essential role of TFEB in regulating inflammation, we used a small interfering RNA (siRNA) strategy and achieved >90% knockdown of TFEB mRNA and protein in HUVECs (Fig. 3A). The knockdown of TFEB increased the expression of SELE, VCAM1, MCP1, and IL6 by 2.67- ± 0.18-fold, 8.44- ± 2.2-fold, 1.42- ± 0.08-fold, and 2.26- ± 0.35-fold, respectively (Fig. 3B), and the protein abundance of E-selectin and VCAM1 by 0.65- ± 0.12-fold and 0.3- ± 0.07-fold, respectively, upon TNFα stimulation (Fig. 3C). To exclude off-target effects of siRNA, we used an additional TFEB siRNA (siTFEB #2) that also increased inflammation in endothelial cells (fig. S2, E and F). To determine whether TFEB mediates the anti-inflammatory effect of laminar shear stress in endothelial cells, we conducted TFEB knockdown before shear stress treatment (Fig. 3, D and E). We found that laminar flow suppressed proinflammatory adhesion molecules in the presence of TNFα in the control cells. However, TFEB knockdown significantly attenuated the anti-inflammatory effect of laminar flow on endothelial cells (Fig. 3, D and E). Together, our results suggest that TFEB suppresses inflammation in response to proinflammatory stimuli and mediates the protective effect of laminar shear stress in endothelial cells.

Fig. 3 TFEB knockdown aggravates inflammation in endothelial cells.

HUVECs were transfected with control siRNA (siCt) or siRNA directed against TFEB (siTFEB) before treatment with TNFα. (A) TFEB knockdown efficiency was determined by qPCR and Western blot. (B) SELE, VCAM1, MCP1, and IL6 mRNAs were determined by qPCR. (C) E-selectin and VCAM1 proteins were determined by Western blot. (D and E) HUVECs were transfected with siCt or siTFEB before exposure to static conditions or laminar shear stress for 24 hours and then treated with TNFα. VCAM1 and E-selectin abundance was determined by qPCR (D) and Western blot (E). Data in (A) to (E) were from three independent experiments and are presented as means ± SEM. *P < 0.05; **P < 0.01.

TFEB reduces intracellular ROS in HUVECs

Oxidative stress induced by ROS in endothelial cells triggers the production of proinflammatory molecules and cytokines (8, 10, 2527). We found that overexpression of TFEB reduced ROS production, whereas TFEB knockdown augmented ROS production, both basally and in response to TNFα treatment, using 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) (Fig. 4, A and B). We confirmed these results using luminol chemiluminescence as an additional method to determine ROS concentrations (Fig. 4, C to E). We also measured superoxide by dihydroethidium (DHE) fluorescence in TFEB-overexpressing or TFEB-deficient endothelial cells. Our data show that TFEB significantly inhibited intracellular superoxide in endothelial cells (Fig. 4, F and G).

Fig. 4 TFEB reduces intracellular ROS concentrations in HUVECs.

(A and B) HUVECs were infected with adenoviruses encoding LacZ or TFEB (A) or transfected with siCt or siTFEB (B). Cells were loaded with DCFH-DA to probe for ROS before treatment with TNFα. Fluorescence was determined by fluorescence microscopy (left panel; representative image of three independent experiments) or a microplate reader (right panel). (C and D) HUVECs were infected with adenoviruses encoding GFP or TFEB before treatment with TNFα for the indicated dosages (C) and time (D), followed by luminol loading. Luminescence was detected with a luminometer. (E) HUVECs were transfected with siCt or siTFEB, treated with TNFα, and loaded with luminol. Luminescence was detected with a luminometer. (F) HUVECs were infected with adenoviruses encoding LacZ or TFEB or (G) transfected with siCt or siTFEB before treatment with TNFα and DHE loading. Fluorescence was determined by a microplate reader. Data in (A) to (G) were from three independent experiments and are presented as means ± SEM. *P < 0.05; **P < 0.01. Scale bars, 100 μm.

TFEB increases the transcription of genes encoding antioxidant factors

In endothelial cells, enzymatic and nonenzymatic antioxidant systems prevent cells from oxidative damage. Major enzymatic antioxidants include SOD, catalase, glutathione peroxidase, HO1, thioredoxin 1 (TXN1), and peroxiredoxin (9, 28). HO1 and SOD2 protect against endothelial dysfunction and atherosclerosis. We found that TFEB overexpression increased the transcription of antioxidant genes, including HO1, SOD2, and TXN1 (Fig. 5A and fig. S3, A to G), and the protein abundance of HO1 and SOD2 (Fig. 5, B and C). TFEB binds to a palindromic 10–base pair GTCACGTGAC motif and induces the transcription of its target genes (29). We found a putative TFEB binding site located at −19/−12 in the HO1 promoter (Fig. 5D). A transcriptional activity reporter containing this binding site from the HO1 promoter displayed higher luciferase activity in TFEB-overexpressing cells when compared to cells expressing endogenous TFEB (vector control), an effect that was abolished by mutation of this motif (Fig. 5E). Chromatin immunoprecipitation (ChIP) assays also demonstrated that TFEB bound the HO1 promoter in the area that harbors this motif (Fig. 5F). We also found a putative TFEB binding site in the intron2 region of SOD2 (Fig. 5G), which is consistent with evidence that intron2 harbors several elements that regulate SOD2 expression (3032). Luciferase assays confirmed that a reporter driven by this region harboring the binding site was activated by TFEB overexpression, an effect that was lost by mutation of the binding site (Fig. 5H), and TFEB bound to this site in SOD2 intron2, as determined by ChIP assays (Fig. 5I). To determine whether SOD2 or HO1 were necessary for TFEB-dependent inhibition of inflammation in endothelial cells, we knocked down HO1 and SOD2 in the TFEB-overexpressing endothelial cells. We found that HO1 knockdown largely attenuated the anti-inflammatory effect of TFEB, whereas SOD2 knockdown had a modest effect on TFEB-mediated suppression of inflammation (Fig. 5, J to L). The nuclear factor κB (NF-κB) pathway is a proinflammatory signaling pathway (33). However, our data suggested that TFEB overexpression did not inhibit the TNFα-induced activation of the NF-κB pathway in endothelial cells, as assessed by NF-κB luciferase reporter, inhibitor of NF-κBα (IκBα) degradation, and p65 nuclear translocation (fig. S4, A to C). Together, these results indicate that TFEB directly increases the transcription of the HO1 and SOD2 genes. Although other anti-inflammatory signaling pathways cannot be excluded, enhanced antioxidative capacity through increasing HO1 and SOD2 abundance could partially contribute to the anti-inflammatory effect of TFEB in endothelial cells.

Fig. 5 TFEB increases mRNA and protein abundance of antioxidant genes.

(A to C) HUVECs were infected with adenoviruses encoding GFP or TFEB before treatment with TNFα or IL1β. (A) HO1 (which encodes HO1) and SOD2 (which encodes SOD2) mRNAs were determined by qPCR. (B) HO1 and SOD2 proteins were determined by Western blot. (C) Band densities in (B) were quantitatively analyzed and normalized against GAPDH. (D) Wild-type (WT) and mutant HO1 promoter or (G) SOD2 intron2 region was cloned into pGL4.11 luciferase reporter vector. (E to H) Luciferase (luc) activity was determined in AD-293 cells transfected with HO1 pGL4.11 (E) or SOD2 pGL4.11 plasmids (H), together with pcDNA3.1 empty vector or pcDNA3.1 encoding human TFEB. (F and I) HUVECs were infected with adenoviruses encoding LacZ or Flag-TFEB. The binding of TFEB to the HO1 promoter (F) or SOD2 intron2 region (I) was determined by ChIP assay with an antibody recognizing Flag tag (anti-Flag). (J to L) HUVECs were transfected with siCt, siHO1, or siSOD2 before TNFα treatment. (J and K) HO1 and SOD2 knockdown efficiency was determined by qPCR and Western blot. (L) SELE and VCAM1 mRNA was determined by qPCR. Data in (A) to (L) were from three independent experiments and, where applicable, are presented as means ± SEM. *P < 0.05; **P < 0.01.

Inhibition of autophagy does not diminish the inhibitory effect of TFEB on endothelial cell inflammation

Autophagy is an evolutionarily conserved process that degrades protein and damaged organelles. TFEB is a master regulator of lysosomal biogenesis and autophagy in various cell types (14, 29). TFEB-overexpressing HUVECs showed significantly increased mRNA expression of various autophagy genes, such as ATG3 (which encodes autophagy-related 3), ATG9B (which encodes autophagy-related 9B), SQSTM1 (which encodes sequestosome 1 or p62), LAMP1 (which encodes lysosomal-associated membrane protein 1), ATP6V1H [which encodes adenosine triphosphatase (ATPase) H+ transporting V1 subunit H], GNS [which encodes glucosamine (N-acetyl)-6-sulfatase], GLA (which encodes α-galactosidase), and HEXA (which encodes hexosaminidase A) (Fig. 6A). TFEB overexpression also increased the protein abundance of ATG3, microtubule-associated protein 1A and 1B light chain 3–I (LC3-I) and LC3-II (Fig. 6B).

Fig. 6 Inhibition of autophagy does not attenuate the inhibitory effect of TFEB on inflammation in endothelial cells.

(A and B) HUVECs were infected with adenoviruses encoding GFP or TFEB. (A) mRNA for autophagy- and lysosome biogenesis–related genes was determined by qPCR. (B) ATG3, LC3-I, and LC3-II proteins were determined by Western blot (representative of three blots). (C and D) HUVECs were transfected with siCt or siATG5, infected with adenoviruses encoding GFP or TFEB, and treated with TNFα. (C) ATG5 knockdown efficiency was determined by qPCR and Western blot. (D) SELE, MCP1, and VCAM1 mRNAs were determined by qPCR. (E) HUVECs were infected with adenoviruses encoding GFP or TFEB and pretreated with the autophagy inhibitors 3-MA or bafilomycin A1 for 30 min before treatment with TNFα. (F) HUVECs were infected with Ad-GFP or Ad-TFEB and pretreated with the autophagy inhibitor 3-MA, CQ, or bafilomycin A1 for 16 hours before treatment with TNFα. SELE, VCAM1, and MCP1 mRNAs were determined by qPCR. Data in (A) to (F) were from three independent experiments and, where applicable, are presented as means ± SEM. *P < 0.05; **P < 0.01.

We examined whether the anti-inflammatory effect of TFEB in endothelial cells was autophagy-dependent. ATG5 is required for the formation of autophagosomes (34). In HUVECs with ATG5 knockdown (Fig. 6C), TFEB overexpression still potently decreased the mRNA expression of SELE, VCAM1, and MCP1 to a comparable extent as in control endothelial cells (Fig. 6D). In addition, pharmacological inhibition of autophagy by pretreatment with 3-methyladenine (3-MA) (35), chloroquine (CQ) (36), or bafilomycin A1 (37) did not attenuate the inhibitory effect of TFEB on inflammation in endothelial cells (Fig. 6, E and F). Therefore, TFEB inhibition of endothelial cell inflammation may be independent of its activation of the canonical autophagy pathway.

Overexpression of TFEB in endothelial cells inhibits endothelial cell inflammation and reduces atherosclerosis development in mice

Endothelial cell activation is an early event in atherogenesis, and inhibition of this step can largely attenuate the development of atherosclerosis (38). The adhesion of leukocytes to vascular endothelium is a hallmark of endothelial cell inflammation. We generated transgenic mice that overexpressed TFEB under the mTie2 promoter in endothelial cells (EC-TFEB) (fig. S5, A to C). TFEB protein abundance was not increased in peritoneal or bone marrow–derived macrophages from EC-TFEB mice (fig. S5, D and E). After administration of LPS, EC-TFEB mice demonstrated significantly decreased leukocyte rolling and adhesion on endothelial cells by 68 ± 19% and 59 ± 20%, respectively, in the vessels in the cremaster muscle (Fig. 7, A and B). Administration of CQ did not prevent this decrease in leukocyte rolling or adhesion in EC-TFEB mice (Fig. 7, A and B). Furthermore, compared with the aortas from littermate control mice, the aortas from EC-TFEB mice showed decreased VCAM1 abundance after LPS administration, as assessed by immunostaining (Fig. 7C). Consistent with these findings, Sele and Vcam1 mRNA abundance after LPS administration were significantly decreased in the aortas of EC-TFEB mice when compared with those of control mice (Fig. 7D). To determine whether TFEB overexpression in endothelial cells prevented atherosclerosis development, we crossbred EC-TFEB mice with atherosclerosis-prone ApoE−/− mice, and mice were fed a high-cholesterol diet to induce atherosclerosis. Measurement of the atherosclerotic lesion areas revealed that EC-TFEB/ApoE−/− mice exhibited significantly decreased atherosclerotic lesion formation by 46 ± 16% compared with littermate ApoE−/− mice (Fig. 7, E and F). Plasma samples from the two groups showed no significant differences in total cholesterol, high-density lipoprotein cholesterol (HDL-c), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-c) concentrations (fig. S6). These results indicate that TFEB is a critical suppressor of endothelial cell inflammation and atherosclerosis in vivo.

Fig. 7 EC-TFEB transgene inhibits endothelial cell inflammation and reduces atherosclerosis development.

(A and B) EC-TFEB transgenic and littermate WT mice were treated with saline or CQ for 7 days and then with LPS. (A) Leukocyte recruitment was analyzed with intravital microscopy. Scale bar, 50 μm. (B) The adhesion and rolling of leukocytes on vascular walls were quantitatively analyzed. n = 5 to 6 mice for each group in (A) and (B). (C) Immunostaining for VCAM1 (excitation/emission, 590/617) and CD31 (excitation/emission, 650/665) in thoracic aortas from EC-TFEB and littermate WT mice after LPS administration. Normal rabbit immunoglobulin G (IgG) was used as a negative control. Images are representative of four mice for each group. (D) Sele and Vcam1 mRNA in the aortas from EC-TFEB and littermate control mice after LPS injection was determined by qPCR (normalized against 18S RNA). n = 5 mice for each group. (E and F) EC-TFEB/ApoE−/− and ApoE−/− mice were fed a high-cholesterol diet. (E) En face analysis of atherosclerotic lesions in the aortic tree was performed after Oil Red O staining. (F) The area of atherosclerotic lesions was quantified. n = 10 to 11 mice for each group in (E) and (F). Data are presented as means ± SEM. *P < 0.05; **P < 0.01. Scale bar, 50 μm.

DISCUSSION

CVDs are the leading cause of death in the United States (39). Current evidence supports the central role of endothelial cell inflammation in atherosclerosis (40). Laminar shear stress protects against atherosclerosis by inhibiting endothelial cell inflammation both in vitro and in vivo (41). The induction of TFEB in endothelial cells by laminar shear stress, but not static conditions or oscillatory shear stress, prompted us to investigate the relationship between TFEB and endothelial cell inflammation. Our data suggest that TFEB is indispensable to modulate the inflammatory status and enhance the antioxidative capacity of endothelial cells (Fig. 8).

Fig. 8 The role of TFEB in endothelial inflammation and atherosclerosis.

The diagram summarizes the findings indicating that endothelial TFEB protects against atherosclerosis by inhibiting oxidative stress and inflammation.

We found that laminar shear stress induced TFEB nuclear translocation, which induces TFEB transcription in an autoregulatory loop (42). Furthermore, when TFEB is phosphorylated by mTOR complex 1 (mTORC1), it is retained in the cytoplasm (4345). We found that laminar shear stress decreased mTORC1 activity (Fig. 1E), thus allowing TFEB nuclear translocation in endothelial cells and likely accounting for the increased TFEB abundance induced by laminar flow in endothelial cells both in vitro and in vivo.

Recruitment of leukocytes to endothelial cells is an initial stage of atherogenesis and is mediated by adhesion molecules such as E-selectin and VCAM1 and proinflammatory cytokines such as MCP1 and IL6. We used both gain-of-function and loss-of-function strategies to demonstrate that TFEB was an anti-inflammatory transcription factor that was induced in endothelial cells by various proinflammatory stimuli. Consistent with our in vitro data, endothelial cell–specific expression of a TFEB transgene also inhibited endothelial cell–leukocyte adhesion in vivo, reinforcing the notion that TFEB promoted an anti-inflammatory phenotype in endothelial cells. We also observed decreased atherosclerotic lesion formation in EC-TFEB/ApoE−/− mice compared to littermate ApoE−/− mice, indicating that TFEB activation protected against atherosclerosis in vivo.

TFEB reduced ROS in endothelial cells both basally and upon TNFα treatment. This phenomenon could be attributed to increased transcription of antioxidant genes in endothelial cells, including HO1, SOD2, and TXN1. We demonstrated that HO1 and SOD2 were direct targets of TFEB in endothelial cells. ROS are key signaling molecules in the progression of inflammation (46). Thus, enhanced TFEB-mediated antioxidative capacity could partially explain its anti-inflammatory function. Although the NF-κB pathway plays a critical role in the inflammatory response in many different cell types (47), many proteins regulate endothelial cell inflammation in an NF-κB–independent manner (20, 25, 48). Our data revealed that neither IκBα degradation nor p65 translocation was altered in the TFEB-overexpressing endothelial cells, suggesting that the role of TFEB in endothelial cells was NF-κB–independent. Besides NF-κB, ROS induce a broad spectrum of proinflammatory pathways in endothelial cells, such as atypical protein kinase Cζ (49), apoptosis signal–regulating kinase 1 (20, 50), mitogen-activated protein kinase (51), and c-Jun N-terminal kinases (52). TFEB may modulate these ROS-dependent proinflammatory signaling pathways in endothelial cells. On the basis of our present study, we cannot rule out that TFEB affects other inflammatory pathways in endothelial cells, which will require further examination in follow-up studies.

Increased ROS have been observed virtually in every aspect of atherosclerotic plaque formation (53). As a result, there are numerous methods to measure ROS, although each method has its own pitfalls. DCFH-DA is the most commonly used probe for intracellular ROS. It is cleaved and trapped in the cell. Upon oxidation, it becomes the highly fluorescent product dichlorofluorescein (DCF). A major concern is that photoreduction of DCF will generate superoxide radicals, amplifying oxidative stress (54). Luminol is a cell-permeable chemiluminescent probe used to detect various kinds of ROS. However, it is oxidized by not only ROS but also ONOO. The use of luminol has also been criticized because it can undergo redox cycling and may overestimate ROS (55). To overcome these limitations, we used multiple assays, including DCFH-DA, luminol, and DHE, to assess intracellular ROS and obtained consistent results that TFEB inhibits intracellular ROS independently of the method used.

TFEB is a master transcription factor that regulates lysosomal biogenesis and autophagy. The role of autophagy in inflammation is not fully understood, especially in the cardiovascular system (56). Autophagy not only protects cells from inflammation-related cell death (57) but also serves an indispensable role in inflammation and immunity against infection (58). We used three autophagy inhibitors that block autophagy at different steps (3-MA inhibits the initial step of autophagy, and bafilomycin and CQ inhibit autophagosome acidification) and found that autophagy was not involved in the anti-inflammatory effects of TFEB. Although most studies of TFEB focus on autophagy-related processes, TFEB also regulates other types of genes, such as lipid degradation and β-oxidation genes in the liver (42, 59). Our data demonstrated that TFEB inhibited endothelial inflammation independent of the canonical autophagy pathway as well, which indicates that TFEB has critical functions beyond acting as an autophagy master gene. There are certain obvious limitations when using the autophagy inhibitors that we used in our study. 3-MA blocks autophagy by inhibiting class I and class III phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) (35). However, 3-MA may modulate autophagy in a nutritional condition and treatment time-dependent manner (60, 61). In addition, inhibiting PI3K affects diverse signaling pathways and trafficking process, besides autophagy (62). CQ is a weak base and inhibits autophagy by impairing lysosomal acidification. In addition to blocking autophagy, CQ also affects other cellular processes such as mitosis and endocytosis (62). Bafilomycin A1 inhibits the V-ATPase (vacuolar H+-ATPase) proton pump on the lysosome membrane, thus reducing vesicle acidification (6365). However, this proton pump is also involved in intracellular ion transport (66), vesicle trafficking (67), proliferation, and migration (68). These limitations confound the a priori interpretation of our data. We found that bafilomycin A1 treatment enhanced the anti-inflammatory effect of TFEB in endothelial cells. Perturbation of lysosomal function by bafilomycin A1 promotes TFEB nuclear translocation (45). In addition, adenosine triphosphate hydrolysis by the V-ATPase is critical to regulate the V-ATPase–Ragulator interaction and promote mTORC1 lysosomal translocation (64). Inhibition of lysosome function decreases mTORC1 activity in a feedback loop that induces autophagy (69). Bafilomycin A1 may decrease mTORC1 activity, possibly induce TFEB nuclear translocation, and therefore enhance TFEB anti-inflammatory activity. Thus, we used ATG5 knockdown in vitro to demonstrate that impaired autophagy did not attenuate the anti-inflammatory effect of TFEB, reminiscent of how TFEB-mediated inhibition of IL1β secretion induced by LPS and cholesterol crystals is independent of Atg5 in macrophages (15).

In summary, we demonstrated that TFEB is an anti-inflammatory factor that inhibits leukocyte recruitment and atherosclerosis development. This finding extends our understanding of TFEB in inflammation, oxidative stress, and vascular disease and reveals TFEB as a potential molecular target for treatment of atherosclerosis and associated CVDs.

MATERIALS AND METHODS

Animal procedures

Endothelial cell–specific TFEB transgenic mice (EC-TFEB) were generated with an mTie2 promoter–driven human TFEB coding region on C57BL/6 mice background. Mice had free access to water and rodent chow diet. EC-TFEB/ApoE−/− mice were generated by breeding EC-TFEB mice with ApoE−/− mice (The Jackson Laboratory). Eight- to 10-week-old EC-TFEB/ApoE−/− mice and littermate ApoE−/− mice were fed a high-cholesterol diet (17.3% protein, 21.2% fat, 48.5% carbohydrate, 0.2% cholesterol by mass, and 42% calories from fat; TD.88137, Envigo) for 8 weeks. Blood samples were sent to the Chemistry Laboratory of the Michigan Diabetes Research and Training Center to determine total cholesterol, TGs, LDL-c, and HDL-c. All animal work was performed in accordance with guidelines set by the University of Michigan Animal Care and Use Committee.

Materials and reagents

Antibodies against E-selectin, VCAM1, GAPDH, p65, and actin were from Santa Cruz Biotechnology. Antibodies against phosphorylated 4E-BP1, 4E-BP1, TFEB, and histone H3 were from Cell Signaling Technology (CST). Flag antibody was from Sigma-Aldrich. Recombinant human TNFα and IL1β were from R&D Systems. CQ, 3-MA, and LPS were from Sigma-Aldrich. Bafilomycin A1 was from Cayman Chemical.

Cell culture and stimulation

HUVECs and HCAECs were purchased from Lonza. HUVECs were cultured in M199 medium supplemented with 16% fetal bovine serum (FBS), recombinant human fibroblast growth factor (1 ng/ml) (Sigma-Aldrich), heparin (90 μg/ml) and 20 mM Hepes, and penicillin/streptomycin mix (50 mg/ml) at 37°C/5% CO2-humidified incubator. HCAEC was cultured in EGM-2MV medium (Lonza). Endothelial cells of passage less than five were used for experiments. Bovine aortic endothelial cells (BAECs) and AD-293 cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS. Thioglycollate-elicited peritoneal macrophages and bone marrow monocytes were isolated from both wild-type and EC-TFEB mice, as described previously (70, 71). Purity of isolated cells was measured by sorting with F4/80 antibody. Endothelial cells were stimulated with TNFα (2 ng/ml), IL1β (5 ng/ml), or LPS (100 ng/ml) for 4 hours, unless otherwise indicated. To block autophagy, Endothelial cells were pretreated with 3-MA (5 mM), bafilomycin A1 (200 nM), or CQ (5 μM) for 30 min or 16 hours and then treated with TNFα (2 ng/ml) for 4 hours.

siRNA-mediated gene knockdown

Endothelial cells were transfected with siRNA or nontargeting siRNA (20 nM; Ambion In Vivo Negative Control #1 siRNA, Thermo Scientific) using Lipofectamine RNAiMAX Reagent (Invitrogen) in accordance with the manufacturer’s protocol. siTFEB (5′-AGACGAAGGUUCAACAUCA-3′), siTFEB #2 (5′-CUACAUCAAUCCUGAAAUG-3′), and siHO1 (pool of 5′-GGCAGAGGGUGAUAGAAGA-3′, 5′-ACACUCAGCUUUCUGGUGG-3′, 5′-AGAGAAUGCUGAGUUCAUG-3′, and 5′-GAGGAGAUUGAGCGCAACA-3′) were from Dharmacon. siAtg5 (SignalSilence Atg5 siRNA I, #6345) was from CST. siSOD2 (5′-CGCUUACUACCUUCAGUAGtt-3′) was from Ambion.

Shear stress model

A detailed protocol has been described previously (72). Briefly, HUVEC monolayers at 80 to 90% confluence in 100-mm tissue culture dishes were exposed to arterial amounts of unidirectional laminar shear stress (15 dynes/cm2), bidirectional oscillatory shear stress at 1-Hz cycle (±5 dynes/cm2) by rotating a Teflon cone (0.5° cone angle) with a stepping motor (Servo Motors) and computer program control (DC Motor Company), and static cultured condition for the indicated time. Endothelial cells were transfected with siRNA-control or siRNA-TFEB (20 nM) for 48 hours before shear stress treatment for 24 hours and then stimulated with TNFα (2 ng/ml) for 4 hours.

DCFH-DA assay

DCFH-DA assay was performed with Cellular ROS/Superoxide Detection Assay Kit (Abcam) in accordance with the manufacturer’s protocol. Briefly, HUVECs were washed with phosphate-buffered saline (PBS) and treated with 20 μM DCFH-DA and TNFα at indicated doses and time. The plate was read with a 488/520 fluorescence filter by a fluorometer (Promega) or visualized with fluorescence microscopy. The fluorescence was normalized to protein content in each well.

DHE superoxide assay

Superoxide production was assessed by DHE (Cayman Chemical) fluorometric assays. Briefly, HUVECs were washed with PBS and treated with 5 μM DHE and TNFα (10 ng/ml) for 1 hour. The plate was read with a 510/595 fluorescence filter by a fluorometer (Promega). The fluorescence was normalized to protein content in each well.

Luminol chemiluminescence assay

Luminol chemiluminescence assay was performed as described previously (25). Briefly, HUVECs were treated with TNFα at the indicated dosage and time. After washing with PBS twice, cells were loaded with 1 mM luminol (Cayman Chemical). Luminescence was detected with a luminometer (Promega) and normalized to protein content as determined by Bradford assay.

Intravital microscopy

Eight- to 10-week-old EC-TFEB and wild-type mice were administered saline or CQ (50 mg/kg, intraperitoneally) for 1 week. Intravital microscopy analysis was performed as described previously (73). Briefly, mice were injected LPS (30 μg/kg) by tail vein. Four hours later, mice were injected rhodamine 6G chloride (Thermo Scientific) to stain leukocytes for 20 min. The cremaster muscle was dissected from surrounding tissues, cut longitudinally, and kept flat by silk suture. The muscle was kept moist by saline at 37°C. Intravital microscopy was used to monitor microcirculation. Leukocyte rolling was quantified by counting the number of cells rolling past a fixed point in a minute. Leukocytes that were stationary for more than 30 s were counted as adherent to endothelial cells. All animal work was performed in accordance with guidelines set by the University of Michigan Animal Care and Use Committee.

Immunostaining

EC-TFEB and littermate wild-type mice were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg). The thoracic aorta was harvested and then fixed in 4% paraformaldehyde. The sample was embedded in optimal cutting temperature compound (Thermo Scientific) and cut to an 8-μm section in a Leica cryostat. The section was blocked in 5% goat serum for 1 hour at room temperature and then incubated with primary TFEB antibody (Bethyl Laboratories, A303-673A), VCAM1 (Abcam), CD31 (1:100; HistoBioTec LLC) at 4°C overnight. After washing with PBS, the sample was incubated with Alexa Fluor–labeled secondary antibody (1:1000; Jackson ImmunoResearch Laboratories) at room temperature for 1 hour. Images were obtained with an Olympus IX73 microscope. Background correction was performed using the appropriate IgG negative controls. For en face immunostaining of TFEB on the rabbit aorta, the different regions of aorta were harvested from wild-type New Zealand White rabbits and fixed in 4% paraformaldehyde. The samples were incubated with primary TFEB antibody (1:1000; Biorbyt, orb332323) for 2 days. After washing with PBS, the sample was incubated with Alexa Fluor–labeled secondary antibody (1:1000; Jackson ImmunoResearch Laboratories) at room temperature for 1 hour. Images were obtained with a Nikon A1 confocal microscope. Background correction was performed using the appropriate IgG negative controls.

RNA preparation and reverse transcription qPCR analysis

Total RNA was extracted from cells using RNeasy Kit (Qiagen), followed by reverse transcription with a SuperScript III kit (Invitrogen) and random primers. mRNA was determined by qPCR (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad). The mRNA abundance was normalized to internal control, GAPDH, unless otherwise indicated. The primers used are shown in table S1.

Protein extraction and Western blot

Cells were lysed in radioimmunoprecipitation assay lysis buffer (Thermo Scientific) with a protease inhibitor cocktail (Roche Applied Science). Proteins were resolved in 10% SDS–polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1 hour at room temperature in tris-buffered saline–Tween 20 (TBST) containing 5% fat-free milk and incubated with primary antibody (1:1000) at 4°C overnight. After TBST washing, membranes were incubated with secondary antibody (1:8000; LI-COR Biosciences) for 1 hour at room temperature. After TBST washing, bands were analyzed using an image-processing program (LI-COR Odyssey).

ChIP assay

ChIP assay was performed with EZ-ChIP Kit (Millipore) according to the manufacturer’s protocol. Purified precipitated DNA was used as template for qPCR, and primers used were listed in table S1.

Nuclear and cytoplasmic protein extraction

Nuclear and cytoplasmic protein extraction was performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific) in accordance with manufacturer’s protocol. Histone H3 (CST) served as internal control for nuclear protein. Actin (Santa Cruz Biotechnology) served as internal control for cytoplasmic protein.

Plasmid construction and transfection

Desired DNA fragments of HO1 promoter (−483 to +14) and SOD2 intron2 (787–1769) from human genome were PCR-amplified and cloned into pGL4.11 luciferase reporter vector (Promega). Mutation of the putative binding site was performed using Q5 Site-Directed Mutagenesis Kit (New England Biolabs). TFEB overexpression plasmid was generated by cloning human TFEB coding region to pcDNA3.1 mammalian expression vector (Thermo Scientific). All PCR products were verified by DNA sequencing. BAECs were cotransfected with plasmid at 70 to 80% confluence using Lipofectamine 2000 (Invitrogen) in accordance with the suggested protocol. Promoter activity was detected by firefly luciferase and normalized against Renilla luciferase activity.

Construction of adenoviruses

Adenoviruses encoding GFP and human TFEB were generated by cloning the coding region of human TFEB and control GFP into the AdTrack-CMV (cytomegalovirus) vector (Agilent Technologies). Next, the coding region was cloned from the Ad-track vector to the AdEasy vector by homologous recombination in Escherichia coli. The adenovirus encoding LacZ and human TFEB were generated by cloning the coding region of TFEB and control LacZ into the PCR8/GW/TOPO TA vector (Invitrogen). The adenovirus encoding Flag-TFEB was generated by inserting the Flag tag at the N terminus of human TFEB coding region. The sequence was then cloned from the Entry Vector to the pAd/CMV/V5-DEST Vector (Invitrogen) by LR recombination. The adenoviruses were packaged in human embryonic kidney 293 cells and purified by CsCl2 density gradient ultracentrifugation. Adenovirus titration was determined by the Adeno-XTM qPCR titration kit (Clontech).

Statistics

Data are presented as means ± SEM. Student’s t test or one-way analysis of variance (ANOVA) followed by Holm-Sidak test was used to analyze data. A P value of <0.05 was considered as statistically significant. All results are representative from at least three independent experiments.

SUPPLEMENTARY MATERIALS

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Fig. S1. TNFα did not change TFEB mRNA or protein abundance.

Fig. S2. TFEB inhibits endothelial inflammation.

Fig. S3. The effect of TFEB overexpression on oxidative stress–related genes.

Fig. S4. TFEB did not inhibit the NF-κB pathway in endothelial cells.

Fig. S5. Characterization of EC-TFEB transgenic mice.

Fig. S6. The EC-TFEB transgene did not alter plasma lipid profiles in ApoE−/− mice.

Table S1. Primers used for qPCR and genotyping.

REFERENCES AND NOTES

Acknowledgments: We thank M. T. Garcia-Barrio (Cardiovascular Research Institute, Morehouse School of Medicine) for helpful discussions and critical comments. Funding: This work was supported, in whole or in part, by NIH grants HL068878, HL105114, and HL088391 (to Y.E.C.) and American Heart Association grant 14SDG19880014 (to Y.F.). Author contributions: All authors have been involved in critical review of the manuscript. H.L. and Y.F. obtained, contributed, and analyzed the data. C.Q., W.L., W.H., and T.Z. provided technical and material support. The manuscript was drafted by H.L. and Y.F. and then critically reviewed, including comments and feedback from Y.E.C. and J.Z. The study was conceived and designed by Y.E.C. and Y.F. Competing interests: The authors declare that they have no competing interests.
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