Trichostatin D as a Novel KLF2 Activator Attenuates TNFα-Induced Endothelial Inflammation

Krüppel-like factor 2 (KLF2) is an atherosclerotic protective transcription factor that maintains endothelial cell homeostasis through its anti-inflammatory, anti-oxidant, and antithrombotic properties. The aim of this study was to discover KLF2 activators from microbial secondary metabolites and explore their potential molecular mechanisms. By using a high-throughput screening model based on a KLF2 promoter luciferase reporter assay, column chromatography, electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) spectra, trichostatin D (TSD) was isolated from the rice fermentation of Streptomyces sp. CPCC203909 and identified as a novel KLF2 activator. Real-time-quantitative polymerase chain reaction (RT-qPCR) results showed that TSD upregulated the mRNA level of KLF2 in endothelial cells. Functional assays showed that TSD attenuated monocyte adhesion to endothelial cells, decreased vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression, and exhibited an anti-inflammatory effect in tumor necrosis factor alpha (TNFα)-induced endothelial cells. We further demonstrated through siRNA and western blot assays that the effects of TSD on monocyte adhesion and inflammation in endothelial cells were partly dependent on upregulating KLF2 expression and then inhibiting the NOD-like receptor protein 3 (NLRP3)/Caspase-1/interleukin-1beta (IL-1β) signaling pathway. Furthermore, histone deacetylase (HDAC) overexpression and molecular docking analysis results showed that TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 activities. Taken together, TSD was isolated from the fermentation of Streptomyces sp. CPCC203909 and first reported as a potential activator of KLF2 in this study. Furthermore, TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 and attenuated endothelial inflammation via regulation of the KLF2/NLRP3/Caspase-1/IL-1β signaling pathway.


Introduction
Atherosclerosis is a chronic inflammatory disease caused by lipid metabolism disorder and is the pathological basis of many cardiovascular diseases, such as coronary heart disease and myocardial infarction [1][2][3]. Endothelial cells play an important role in vascular homeostasis, thrombosis, inflammation, and other processes [4,5]. Endothelial dysfunction caused by endothelial cell damage is an important initiating event in the occurrence and development of atherosclerotic plaques. Damaged endothelial cells secrete a variety of adhesion molecules and chemokines to attract circulating monocytes to adhere to endothelial cells [6,7]. Monocytes migrate to the subcutaneous space through endothelial junctions and differentiate into macrophages, which proceed to take up various forms of modified low-density lipoprotein (LDL), especially oxidized LDL, to form foam cells, an early process in the formation of fat streaks and atherosclerotic plaques [8,9]. 2 of 15 Krüppel-like factor 2 (KLF2), a member of the Krüppel-like factors (KLFs), is a zinc finger transcription factor. The zinc finger domain of KLFs is located at the C-terminal and plays a role in DNA binding and nuclear localization. The N-terminal region of KLFs is a highly diverse non-DNA binding region that mediates protein-protein interactions to activate or inhibit transcription [10][11][12]. KLF2 is an atherosclerotic protective transcription factor that regulates endothelial cell inflammation, thrombosis, and angiogenesis [4,[13][14][15][16]. Therefore, discovery of new small molecule compounds that are KLF2 activators may provide new therapeutic strategies for atherosclerosis.
The NOD-like receptor protein 3 (NLRP3) inflammasome complex is composed of NLRP3, an adaptor protein, apoptosis-associated speck-like protein containing CARD (ASC), and an effector protein, Caspase-1 [17,18]. Through this inflammasome complex, pro-caspase-1 is cleaved into its active isomer, Caspase-1, and pro-interleukin-1beta (pro-IL-1β) is broken down into its active isomer IL-1β [19]. In turn, the resultant release of IL-1β leads to a dysfunctional endothelium and an increase in inflammatory mediators in the form of vascular cell adhesion protein 1 (VCAM-1), which contributes to atherosclerosis [20]. Previous studies showed that simvastatin increased the expression of forkhead box P transcription factor 1 (Foxp1) and KLF2 in endothelial cells and thus inhibited the expression of NLRP3, Caspase-1, and IL-1β and the activation of endothelial cell inflammation [4].
In this study, we obtained Streptomyces sp. CPCC 203909, which upregulated KLF2 luciferase expression, using a previously established high-throughput screening model based on a KLF2 promoter luciferase reporter assay [21,22]. We isolated an active compound trichostatin D (TSD) from its fermentation products that upregulated KLF2 mRNA levels, and we demonstrated that TSD alleviated monocyte adhesion and exhibited anti-inflammatory effects in tumor necrosis factor alpha (TNFα)-induced endothelial cells. Furthermore, we demonstrated that TSD upregulated KLF2 expression by inhibiting histone deacetylase (HDAC) 4, 5, and 7 and attenuated endothelial inflammation through regulation of the KLF2/NLRP3/Caspase-1/IL-1β signaling pathway.

Isolation and Identification of TSD from Streptomyces sp. CPCC 203909
In the preliminary screening assay, we found that the fermentation of Streptomyces sp. CPCC 203909 increased the luciferase activity of KLF2 in COS-7 cells. Following bioassayguided isolation using silica column chromatography, octadecyl-silica RP flash column chromatography, and semi-preparative HPLC, one positive compound was obtained as a KLF2 activator. A comparison of the ultra violet (UV), electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) data with previous references showed that the structure of the positive compound was TSD ( Figure 1B) [23].

TSD Increases KLF2 Activity and mRNA Levels
To confirm whether TSD increased KLF2 activity, a dose-response curve was generated using KLF2 promoter luciferase assays in COS-7 cells, as described in the Methods section. It was observed that TSD significantly increased the luciferase activity of KLF2 by more than 40-fold at 2.5 µM, with an EC 50 value of 0.15 µM ( Figure 1C). We then evaluated the mRNA level of KLF2 in human umbilical vein endothelial cell (HUVECs) after treatment with TSD. The real-time-quantitative polymerase chain reaction (RT-qPCR) data indicated that TSD significantly increased KLF2 mRNA levels at 5 and 10 µM and by more than 4-fold at 10 µM ( Figure 1D). In addition, cell viability experiments showed that TSD caused no obvious cytotoxicity at the experimental concentration from 1 µM to 40 µM in HUVECs ( Figure 1E).

TSD Attenuated Monocyte Adhesion to HUVECs and Exhibited Anti-Inflammatory Effects
VCAM1 and intercellular adhesion molecule 1 (ICAM-1) are two adhesion molecules secreted after endothelial cell injury that reflect the degree of endothelial cell injury. KLF2 is a potential anti-inflammatory transcription factor that plays an important role in maintaining endothelial cell function by inhibiting the expression of VCAM-1 and ICAM-1 [4,[24][25][26].
To evaluate the anti-inflammatory effect of TSD, an endothelial cell adhesion model induced by TNFα was constructed. When compared with the vehicle control (TSD at 0 µM), TNFα significantly induced THP-1 monocyte adhesion to HUVECs, while TSD (5 and 10 µM) treatment significantly attenuated TNFα-induced monocyte adhesion (Figure 2A,B), which indicated that TSD has an anti-inflammatory effect. potential anti-inflammatory transcription factor that plays an important role in maintaining endothelial cell function by inhibiting the expression of VCAM-1 and ICAM-1 [4,[24][25][26].
To evaluate the anti-inflammatory effect of TSD, an endothelial cell adhesion model induced by TNFα was constructed. When compared with the vehicle control (TSD at 0 μM), TNFα significantly induced THP-1 monocyte adhesion to HUVECs, while TSD (5 and 10 μM) treatment significantly attenuated TNFα-induced monocyte adhesion ( Figure  2A,B), which indicated that TSD has an anti-inflammatory effect.  quantification results (B) of monocyte adhesion to HUVECs after treatment with TSD. The cells were plated in 6-well plates and treated with TSD (5, 10 µM) or DMSO for 18 h; then, tumor necrosis factor alpha (TNFα) (10 ng/mL) was added to the cells for 6 h. THP-1 cells were co-incubated for another 30 min. Values are represented as the mean ± SEM; n = 3; one-way ANOVA was used for analysis. *** p < 0.0001. (C-E) mRNA expression (C), representative western blot (D), and quantification of protein expression of vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) (E) after treatment with TSD. The cells were plated in 6-well plates before treatment with TSD (0, 0.1, 1, 5, and 10 µM) for 18 h. TNFα (10 ng/mL) was added to the cells for 6 h, and then, RT-qPCR was performed to assess the mRNA level of KLF2, VCAM1, and ICAM1. Western blot was performed to assess VCAM-1 and ICAM-1 protein levels. The mRNA and protein levels were normalized to ACTB (β-actin). Values are represented as the mean ± SEM; n = 3; one-way ANOVA was used for analysis. * p < 0.05, ** p < 0.001, *** p < 0.0001. The potential molecular mechanism by which TSD reduced monocyte adhesion was investigated. As shown in Figure 2C, TNFα significantly decreased KLF2 mRNA levels when compared with the vehicle control, while TSD upregulated KLF2 mRNA levels in TNFα-induced HUVECs when compared with the TNFα-only group. In addition, TNFα significantly increased VCAM1 and ICAM1 mRNA levels when compared with the vehicle control, while TSD significantly decreased the mRNA ( Figure 2C) and protein levels of VCAM-1 and ICAM-1 ( Figure 2E) in the TNFα-treated cells when compared with the TNFαonly group. Taken together, these results indicated that TSD exerted an anti-inflammatory effect on HUVECs.

Anti-Inflammatory Effects of TSD in TNFα-Induced HUVECs Depend on KLF2
TSD upregulated KLF2 expression and exhibited anti-inflammatory effects in endothelial cells. To investigate whether the anti-inflammatory effects of TSD on HUVECs were dependent on KLF2, HUVECs were transfected with KLF2 siRNA (siKLF2) for 24 h, and then, TNFα-induced monocyte adhesion assays were performed. As shown in Figure 3A, siKLF2 downregulated the mRNA level of KLF2 to about 30% compared with the siControl group. The same as above, siControl and TNFα-treated group significantly increased monocyte adhesion compared with the siControl group ( Figure 3B), and TSD remarkably decreased the TNFα-induced enhancement of monocyte adhesion ( Figure 3B). siRNA knockdown of KLF2 resulted in a higher level of monocyte adhesion compared with the TSD treatment group ( Figure 3A-C). In addition, the decreased protein levels of VCAM-1 and ICAM-1 induced by TSD were partially increased when KLF2 was knocked down ( Figure 3D,E). Thus, these data indicate that TSD exerted anti-inflammatory effects on endothelial cells in part by upregulating KLF2 expression. HUVECs were cultured in 6-well plates and were transfected with siControl or siKLF2 for 6 h. Then, the medium was replaced with endothelial cell medium and incubated for another 18 h. The cells were then treated with or without TSD (10 μM) for 18 h, followed by 10 ng/mL TNFα for 6 h. Monocyte adhesion assays and western blot analysis were performed, as described in the Methods section. A representative image of each group is shown. The protein levels were normalized to β-tubulin.

TSD Exerts Anti-Inflammatory Effects on Endothelial Cells through the KLF2/NLRP3/Caspase-1/IL-1β Signaling Pathway
KLF2 inhibited activation of the NLRP3 inflammasome [17,18,27], and inhibition of NLRP3 expression reversed the inflammation of endothelial cells [28][29][30]. To investigate whether TSD affected NLRP3, Casapase-1, and IL-1β protein levels, western blot experiments were performed. TNFα stimulation significantly increased NLRP3, Casapase-1, and IL-1β protein levels, while TSD reversed this increase in a dose- , and quantification of protein expression of VCAM-1 and ICAM-1 (E) after treatment with siControl, KLF2 siRNA (siKLF2), or TSD. HUVECs were cultured in 6-well plates and were transfected with siControl or siKLF2 for 6 h. Then, the medium was replaced with endothelial cell medium and incubated for another 18 h. The cells were then treated with or without TSD (10 µM) for 18 h, followed by 10 ng/mL TNFα for 6 h. Monocyte adhesion assays and western blot analysis were performed, as described in the Methods section. A representative image of each group is shown. The protein levels were normalized to β-tubulin. Values are represented as the mean ± SEM; n = 3; Student's t test (A) or one-way ANOVA (C,E) was used for analysis. * p < 0.05, ** p < 0.001, *** p < 0.0001.

TSD Exerts Anti-Inflammatory Effects on Endothelial Cells through the KLF2/NLRP3/Caspase-1/IL-1β Signaling Pathway
KLF2 inhibited activation of the NLRP3 inflammasome [17,18,27], and inhibition of NLRP3 expression reversed the inflammation of endothelial cells [28][29][30]. To investigate whether TSD affected NLRP3, Casapase-1, and IL-1β protein levels, western blot experiments were performed. TNFα stimulation significantly increased NLRP3, Casapase-1, and IL-1β protein levels, while TSD reversed this increase in a dose-dependent manner ( Figure 4A,B). Furthermore, by knocking down KLF2 with siRNA, the inhibitory effect of TSD on NLRP3, Casapase-1, and IL-1β proteins was reversed ( Figure 4C,D), which indicated that the TSD-mediated inhibition of the NLRP3/Caspase-1/IL-1β signaling pathway was partially dependent on KLF2.  after treatment with TSD. Cells in 6-well plates were treated with or without TSD (0, 0.1, 1, 5, and 10 μM) for 18 h, and TNFα (10 ng/mL) was added for 6 h. Western blot was performed to assess NLRP3, Casapase-1, and IL-1β protein levels. (C, D) Representative western blots (C) and quantification (D) of the protein expression of NLRP3, Casapase-1, and IL-1β after treatment with siControl and siKLF2. HUVECs were cultured in 6-well plates and transfected with siControl or siKLF2 with Lipofectamine RNAi MAX for 6 h. Then, the medium was replaced with endothelial cell medium and incubated for another 18 h. Then, the cells were treated with or without TSD (10 μM) for 18 h, followed by 10 ng/mL of TNFα for 6 h. Western blot was performed to assess NLRP3, Casapase-1, and IL-1β protein levels. Each group was represented by a representative image. Values are represented as the mean ± SEM; n = 3; one-way ANOVA was used for analysis. * p < 0.05, ** p < 0.001, *** p < 0.0001.

TSD Upregulates the Expression of KLF2 by Inhibiting HDAC4, HDAC5, and HDAC7
Histone deacetylases catalyze DNA deacetylation and play a crucial role in the modulation of atherosclerosis [31]. TSD is an analog of trichostatin A (TSA), which is a pan-HDAC inhibitor [32][33][34]. Studies have found that inhibition of HDAC activity induced KLF2 expression, which then contributed to reducing inflammation of endothelial cells [35,36]. We wondered whether the upregulation of KLF2 induced by TSD was related to the inhibition of a subtype of HDACs.
To investigate whether TSD upregulated KLF2 and exerted anti-inflammatory effects through a subtype of HDACs, we first transfected eight HDAC plasmids into COS-7 cells. for 18 h, and TNFα (10 ng/mL) was added for 6 h. Western blot was performed to assess NLRP3, Casapase-1, and IL-1β protein levels. (C,D) Representative western blots (C) and quantification (D) of the protein expression of NLRP3, Casapase-1, and IL-1β after treatment with siControl and siKLF2. HUVECs were cultured in 6-well plates and transfected with siControl or siKLF2 with Lipofectamine RNAi MAX for 6 h. Then, the medium was replaced with endothelial cell medium and incubated for another 18 h. Then, the cells were treated with or without TSD (10 µM) for 18 h, followed by 10 ng/mL of TNFα for 6 h. Western blot was performed to assess NLRP3, Casapase-1, and IL-1β protein levels. Each group was represented by a representative image. Values are represented as the mean ± SEM; n = 3; one-way ANOVA was used for analysis. * p < 0.05, ** p < 0.001, *** p < 0.0001.

TSD Upregulates the Expression of KLF2 by Inhibiting HDAC4, HDAC5, and HDAC7
Histone deacetylases catalyze DNA deacetylation and play a crucial role in the modulation of atherosclerosis [31]. TSD is an analog of trichostatin A (TSA), which is a pan-HDAC inhibitor [32][33][34]. Studies have found that inhibition of HDAC activity induced KLF2 expression, which then contributed to reducing inflammation of endothelial cells [35,36]. We wondered whether the upregulation of KLF2 induced by TSD was related to the inhibition of a subtype of HDACs.
To investigate whether TSD upregulated KLF2 and exerted anti-inflammatory effects through a subtype of HDACs, we first transfected eight HDAC plasmids into COS-7 cells. Western blot experiments showed that the HDAC1-8 plasmids were successfully transfected and expressed in COS-7 cells. To examine which HDAC type was related to KLF2 expression, we co-transfected HDAC1-8 expression plasmids and KLF2 luciferase reporter plasmids in COS-7 cells, to detect which HDAC subtype acted on KLF2 according to the luciferase activity. As shown in Figure 5A, HDAC4, HDAC5, and HDAC7 inhibited KLF2 transcriptional activity. We then selected HDAC4, HDAC5, and HDAC7 plasmids to examine the effects of TSD on these three subtype HDACs using a KLF2 upregulating screening model. The results showed that TSD reversed KLF2 transcriptional activity inhibited by HDAC4, HDAC5, and HDAC7 ( Figure 5B), which indicated that TSD had an inhibitory effect on HDAC4, HDAC5, and HDAC7. Western blot experiments showed that the HDAC1-8 plasmids were successfully transfected and expressed in COS-7 cells. To examine which HDAC type was related to KLF2 expression, we co-transfected HDAC1-8 expression plasmids and KLF2 luciferase reporter plasmids in COS-7 cells, to detect which HDAC subtype acted on KLF2 according to the luciferase activity. As shown in Figure 5A, HDAC4, HDAC5, and HDAC7 inhibited KLF2 transcriptional activity. We then selected HDAC4, HDAC5, and HDAC7 plasmids to examine the effects of TSD on these three subtype HDACs using a KLF2 upregulating screening model. The results showed that TSD reversed KLF2 transcriptional activity inhibited by HDAC4, HDAC5, and HDAC7 ( Figure 5B), which indicated that TSD had an inhibitory effect on HDAC4, HDAC5, and HDAC7.

Discussion
Endothelial cells regulate vascular tone, endothelium integrity, thrombosis, endothelial injury, and repair [4][5][6][7]. Pro-inflammatory cytokines such as TNFα and IL-1β can trigger endothelial cell inflammation. In response to injury, endothelial cells are activated and produce VCAM-1, ICAM-1, E-selectin, and other molecules that lead to the adhesion of monocytes and neutrophils to endothelial cells [37,38]. It was found that the activation of KLF2 inhibited the expression of VCAM-1 and E-selectin, thereby exerting anti-inflammatory effects [39][40][41]. Therefore, the discovery of KLF2 activators in endothelial cells might prevent and treat cardiovascular diseases associated with endothelial dysfunction, such as atherosclerosis.
In this study, using a high-throughput drug screening model based on a KLF2 luciferase reporter gene, the fermentation products of Streptomyces sp. CPCC 203909 were isolated and purified, and the active compound TSD was obtained. According to a literature search, the regulatory effect of TSD on KLF2 transcription factor expression was identified for the first time. Our previous study reported that TSD had a positive role in anti-atherosclerosis by upregulating the activity of CD36 and lysosomal integral membrane protein II analogous-1 (CLA-1), which is important in cholesterol efflux. The results in this study showed that TSD reversed TNFα-induced monocyte adhesion, upregulated mRNA levels of KLF2 in endothelial cells, and downregulated mRNA and protein levels of the inflammatory adhesion molecules VCAM-1 and ICAM-1 in TNFαinduced endothelial cells. We also demonstrated that the anti-inflammatory effects of TSD on endothelial cells were partially dependent on KLF2. In addition, it was demonstrated that TSD inhibited the expression of the NLRP3/Caspase-1/IL-1β signaling pathway by upregulating KLF2 expression, thereby exerting an anti-inflammatory effect.
In addition, as statins are studied well and proven to attenuate endothelial dysfunction via KLF2 activation [4,27], we then performed KLF2 promoter luciferase

Discussion
Endothelial cells regulate vascular tone, endothelium integrity, thrombosis, endothelial injury, and repair [4][5][6][7]. Pro-inflammatory cytokines such as TNFα and IL-1β can trigger endothelial cell inflammation. In response to injury, endothelial cells are activated and produce VCAM-1, ICAM-1, E-selectin, and other molecules that lead to the adhesion of monocytes and neutrophils to endothelial cells [37,38]. It was found that the activation of KLF2 inhibited the expression of VCAM-1 and E-selectin, thereby exerting anti-inflammatory effects [39][40][41]. Therefore, the discovery of KLF2 activators in endothelial cells might prevent and treat cardiovascular diseases associated with endothelial dysfunction, such as atherosclerosis.
In this study, using a high-throughput drug screening model based on a KLF2 luciferase reporter gene, the fermentation products of Streptomyces sp. CPCC 203909 were isolated and purified, and the active compound TSD was obtained. According to a literature search, the regulatory effect of TSD on KLF2 transcription factor expression was identified for the first time. Our previous study reported that TSD had a positive role in anti-atherosclerosis by upregulating the activity of CD36 and lysosomal integral membrane protein II analogous-1 (CLA-1), which is important in cholesterol efflux. The results in this study showed that TSD reversed TNFα-induced monocyte adhesion, upregulated mRNA levels of KLF2 in endothelial cells, and downregulated mRNA and protein levels of the inflammatory adhesion molecules VCAM-1 and ICAM-1 in TNFα-induced endothelial cells. We also demonstrated that the anti-inflammatory effects of TSD on endothelial cells were partially dependent on KLF2. In addition, it was demonstrated that TSD inhibited the expression of the NLRP3/Caspase-1/IL-1β signaling pathway by upregulating KLF2 expression, thereby exerting an anti-inflammatory effect.
In addition, as statins are studied well and proven to attenuate endothelial dysfunction via KLF2 activation [4,27], we then performed KLF2 promoter luciferase assays in COS-7 cells and TNFα-induced monocyte adhesion assays to compare the effects of statins (Simvastatin and Atorvastatin) with TSD. As shown in Supplementary Figure S1A Figure S1A), and Atorvastatin maximally increased the luciferase activity of KLF2 by about 12-fold, with an EC 50 value of 5.14 µM (Supplementary Figure S1B). Monocyte adhesion assays results showed that Simvastatin (1 and 10 µM) and Atorvastatin (1 and 10 µM) treatment attenuated TNFα-induced monocyte adhesion (Supplementary Figure S2) and exhibited anti-inflammatory effects in endothelial cells (Supplementary Figure S2). Furthermore, our results indicated that TSD is more effective in upregulating KLF2 expression and anti-inflammatory effects than Simvastatin and Atorvastatin according to the results of KLF2 promoter luciferase assay and inhibiting monocyte adhesion (Supplementary Figures S1 and S2).
In conclusion, our work found that TSD is a novel KLF2 activator and that it has a strong anti-inflammatory effect on endothelial cells. In the future, the anti-atherosclerotic effect of TSD will be studied in western diet-induced apolipoprotein E knockout mice. CPCC203909 was cultured on slants of BD Difco ISP Medium 2 (0.4% yeast extract, 1.0% malt extract, 0.4% dextrose, and 2.0% agar; Becton, Dickinson and Company, New Jersey, USA) at 28 • C. Colonies were picked with a plastic inoculating loop and inoculated into 3 mL of A2 liquid medium (1.0% glucose, 3.0% amylogen, 2.0% cottonseed meal, 0.3% yeast extract, 0.3% ammonia sulfate, 0.1% magnesium sulfate, 0.1% dipotassium hydrogen phosphate, 0.1% sodium chloride, and 0.5% calcium carbonate), and the final pH was adjusted to 7.2. Flasks with the inoculated media were incubated at 28 • C on a rotary shaker at 180 rpm for 2 days after sterilization. For the seed culture, the culture was added to 100 mL of A2 liquid medium (at a ratio of 1:100) on a rotary shaker at 180 rpm at 28 • C and incubated for 2 days. Next, two hundred and fifty fermentation bags containing 50 g of rice and 50 mL of distilled water were used for fermentation. The contents were soaked and autoclaved at 121 • C for 30 min. After cooling to room temperature, 5.0 mL of the seed medium was added to each bag at 28 • C for 30 days.

Cell Culture
The COS-7 cell line was obtained from Cell Resource Center (Institute of Basic Medical Sciences, CAMS, Beijing, China) and was cultured in Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). HUVECs was obtained from PromoCell (Heidelberg, Germany) and was grown in endothelial cell medium (ECM) (ScienCell). The THP-1 cells line was provided by Cell Resource Center (Institute of Basic Medical Sciences, CAMS, Beijing, China) and was cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS. All cells were cultured at 37 • C with 5% CO 2 in a cell incubator.

Cell Viability Assay
HUVECs were inoculated in a 96-well plate, and TSD at different concentrations (0, 1, 2.5, 5, 10, 20, and 40 µM) was added. The cells were incubated for 24 h at 37 • C with 5% CO 2 . A sensitive colorimetric assay was performed to quantify the viable cells in cytotoxicity assay with a Cell Counting Kit-8 (CCK-8, Applygen, Beijing, China) using a microplate reader. For the assay, 10 µL of the CCK-8 working solution was added to each well, and then cells were incubated at 37 • C for 2 h. The absorbance was measured at 450 nm.

Western Blot Analysis
The total protein from cells was extracted using RIPA buffer (Solarbio, Beijing, China) supplemented with 1 mmol/L phenylmethanesulfonyl fluoride (PMSF) (Solarbio, Beijing, China) for 30 min at 4 • C. Then, the cell lysate was centrifuged (12,000 rpm) at 4 • C for 30 min to collect the protein extracted in the supernatant. The protein concentrations were determined with bicinchoninic acid (BCA) Protein Assay kits (Thermo Fisher Scientific, Waltham, MA, USA). The total cell proteins were separated by SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked with 5% skim milk (Becton, Dickinson and Company, New Jersey, USA) for 1 h at room temperature. Then, the membranes were incubated with appropriate primary antibodies overnight at 4 • C, followed by incubation with appropriate secondary anti-rabbit and antimouse antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. All relative protein levels of target genes were normalized to β-actin or β-tubulin. Densitometric analysis of the blots was carried out with NIH ImageJ software. The primary antibodies included human VCAM-1 (Abcam, Cambridge, UK), human ICAM-1 (Cell Signaling Technology, Danvers, MA, USA), human NLRP3 (Cell Signaling Technology, Danvers, MA, USA), human Caspase-1 (Abcam, Cambridge, UK), human IL-1β (Abcam, Cambridge, UK), human Flag (Applygen, Beijing, China), human β-actin (Proteintech, Beijing, China), and human β-Tubulin (Proteintech, Beijing, China).

Monocyte Adhesion Assay
The monocyte adhesion assay was performed as previously described [21,22]. HU-VECs were plated in 6-well plates and incubated with the vehicle (0.1% DMSO), TSD at different concentrations (0.1 µM, 1 µM, 5 µM, and 10 µM), Simvastatin (1 µM and 10 µM) or Atorvastatin (1 µM and 10 µM) in a CO 2 incubator for 18 h. Recombinant TNFα (R&D Systems, Minneapolis, MN, USA) at a final concentration of 10 ng/mL was then added to the culture for another 6 h. Next, 0.5 mL of THP-1 cells at a density of 4 × 10 6 /mL was added to the culture and co-incubated for 30 min. Then, the cells were gently washed three times with FBS-free medium to remove non-adherent THP-1 cells. Photographs of cells were taken under a microscope (Leica CM1950, Wetzlar and Mannheim, Germany).

Small Interfering RNA (SiRNA) Assay
HUVECs were cultured in 6-well cell culture plates and transfected with siControl (50 nM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or siKLF2 (50 nM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) with Lipofectamine RNAi MAX (Thermo Fisher Scientific, Waltham, MA, USA) in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA). After 6 h, the medium was replaced with ECM and incubated for another 18 h. Then, the cells were treated with or without TSD (10 µM) or TNFα, the same as in Section 4.8. Next, western blot and monocyte adhesion assays were performed.

Molecular Modeling Analysis
Molecular docking studies between TSD and each HDAC, HDAC4 (PDB code: 4A69), HDAC5 (PDB code: 5UWI), and HDAC7 (PDB code: 3C10) were performed using Discovery Studio 4.5 software (NeoTrident Technology Ltd., Beijing, China) with the default settings. ChemDraw 14.0 was applied to generate the 3D structures, and LibDock software was also used. The affinity of the receptor-ligand interaction was evaluated based on the LibDock score.

Statistical Analysis
Data analysis was performed using GraphPad Prism 8 software. Data were presented as the mean with SEM. Student's t test or one-way ANOVA was conducted, and p < 0.05 was regarded as statistically significant.

Conclusions
In this study, TSD was isolated from the fermentation of Streptomyces sp. CPCC203909 and first reported as a potential activator of KLF2. Furthermore, TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 and attenuated endothelial inflammation via regulation of the KLF2/NLRP3/Caspase-1/IL-1β signaling pathway.