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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Cell Signal. 2018 Jun 18;50:1–8. doi: 10.1016/j.cellsig.2018.05.009

Pharmacological inhibition of MALT1 protease activity suppresses endothelial activation via enhancing MCPIP1 expression

Yong Li 1,2,#, Shengping Huang 1,#, Xuan Huang 1,2, Xiuzhen Li 1, Adrian Falcon 1, Adele Soutar 1, Frederic Bornancin 3, Zhisheng Jiang 4, Hong-Bo Xin 2, Mingui Fu 1,*
PMCID: PMC6086570  NIHMSID: NIHMS977898  PMID: 29913212

Abstract

Mucosa associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is not only an intracellular signaling scaffold protein but also a paracaspase that plays a key role in the signal transduction and cellular activation of lymphocytes and macrophages. However, its role in endothelial cells remains unknown. Here we report that pharmacological inhibition of MALT1 protease activity strongly suppresses endothelial activation via enhancing MCPIP1 expression. Treatment with MALT1 protease inhibitors selectively inhibited TNFα-induced VCAM-1 expression in HUVECs and LPS-induced VCAM-1 expression in mice. In addition, Inhibition of MALT1 protease activity also significantly inhibited TNFα-induced adhesion of THP-1 monocytic cells to HUVECs. To explore the mechanisms, MALT1 inhibitors does not affect the activation of NF-κΒ signaling pathway in HUVEC. However, they can stabilize MCPIP1 protein and significantly enhance MCPIP1 protein level in endothelial cells. These results suggest that MALT1 paracaspase also targets MCPIP1 and degrade MCPIP1 protein in endothelial cells similar as it does in immune cells. Taken together, the study suggest inhibition of MALT1 protease activity may represent a new strategy for prevention/therapy of vascular inflammatory diseases such as atherosclerosis.

Keywords: endothelial activation, inhibitors of MALT1, VCAM-1, MCPIP1, vascular inflammation

1. Introduction

Vascular endothelial activation, featured by the expression of adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), is an early and important event in atherosclerosis [1]. Although the use of preventive pharmacological approaches, such as lipid lowering and antihypertensive drugs, have been beneficial, there is no treatment available to reduce these early steps of atherogenesis. VCAM-1 is a member of the immunoglobulin gene superfamily that mediates leukocyte binding to endothelial cells through interaction with its integrin counterreceptor, very late activation antigen-4 (VLA-4). Because selective expression of VLA-4 on monocytes and lymphocytes, VCAM-1 plays an important role in mediating mononuclear leukocyte adhesion, and recruit them to the activated endothelium, leading to a chronic inflammatory state [2]. Thus, targeting the expression of VCAM-1 gene may provide novel therapeutic strategy for vascular inflammatory diseases.

MALT1 (mucosa associated lymphoid tissue lymphoma translocation protein 1) is a component of a signalosome CARMA1-BCL10-MALT1, which is critical for the signal transduction in immune cells [3–5]. However, the role of MALT1 in endothelial cells remains unknown. Recent studies demonstrated that MALT1 also acts as an arginine-specific protease, by which it contributes to the activation of immune cells via cleaving the negative regulators of inflammatory signaling such as CYLB and A20 [6, 7]. MCPIP1 is a newly identified RNase that acts as a master controller of macrophage inflammation, immune cell activation as well as endothelial activation [8–10]. Recent studies suggest that MCPIP1 is also a target of MALT1 protease in lymphocytes and macrophages [11, 12]. Pharmacological inhibition of MALT1 protease activity with MI-2 (a reported covalent inhibitor of MALT1) can selectively enhance the expression of MCPIP1 in macrophages and reverse the LPS-induced systemic inflammation and acute lung injury [12].

In this study, we have further examined the effect of pharmacological inhibition of MALT1 protease activity on endothelial activation. We found that inhibition of MALT1 protease activity significantly suppressed the expression of VCAM-1 both in vitro and in vivo. As a result, MALT1 inhibitors also significantly repressed monocyte adherence to the activated endothelial cells. The effect of MALT1 inhibitors on endothelial activation is dependent on the increased expression of MCPIP1 but not NF-kB signal pathway. These results suggest that pharmacological inhibition of MALT1 protease activity may provide a new therapeutic strategy for vascular inflammatory diseases such as atherosclerosis.

2. Materials and methods

2.1. Cell culture

The primary human vascular endothelial cells including human aortic ECs (HAEC), human coronary artery ECs (HCAEC), human dermal microvascular ECs (HDMEC), human lung microvascular ECs (HLMEC) and human umbilical vein ECs (HUVEC) were purchased from Lonza Walkersville Inc. (Walkersville, MD) and cultured in EGM or EGM2 medium according to the manufacturer’s instruction. These cells were used for no more than five passages. The human acute monocytic leukemia cell line THP-1 was obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium (Corning) containing 10% FBS (Sigma-Aldrich).

2.2. Reagents

Human recombinant TNFα and LPS were purchased from Sigma (Saint Louis, MO). VCAM-1 (sc-13160), ICAM-1 (sc-1511-R) and β-actin (sc-1616) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). MCPIP1 rabbit polyclonal antibody was purchased from GeneTex (GTX110807). MALT1, BLC10, A20, CYLD, RelB, p-P65, P65, pIΚΚα/β, IKKα, IKK β, pΙΚΒα, and ΙΚΒα antibodies were purchased from Cell Signaling Technology. Small interfering RNAs (siRNAs) targeting MALT1 and control siRNAs were purchased from Santa Cruz Biotechnology. QPCR primers were from IDT. MI-2 was purchase from R&D systems. MLT-827 was synthesized at Novartis (13). Mepazine was from Calbiochem.

2.3. Western blot

Cells or lung tissues from mice were solubilized in the extraction buffer containing 0.5% Brij® L23 solution (Sigma-Aldrich), 50 mM KCl, 2 mM CaCl2, 20% glycerol, 50 mM Tris-HCl, and proteases inhibitors and phosphatases, pH 7.4. Cell lysates were cleaned by centrifugation at 12,000 rpm for 10 min and assayed for protein concentration using a standard BCA protein assay kit (Pierce, Rockford, IL). 20 μg protein samples were separated using SDS-PAGE and then transferred onto a NC membrane (Corning). The membrane was blocked for 20 min at 4 °C with 5% skim milk dissolved in PBST (PBS, pH 7.4, containing 0.1% Tween 20). The membrane was incubated overnight with primary antibodies at 4 °C with gentle rocking. Anti-rabbit IgG or anti-mouse IgG conjugated with horseradish peroxidase (Thermo) was incubated for 1 hours. The membranes were detected with HRP chemiluminescence kit (Pierce, Rockford, IL) and exposed to X-ray film.

2.4. Real-time PCR analysis

Total RNA from HUVEC cells was harvested using TRIzol® Reagent (Life Technologies™) for Real-Time PCR analysis. Briefly, 2 μg of RNA was reverse-transcribed (RT) to synthesize cDNA using High-capacity cDNA Reverse Transcription Kit (Life Technologies). 1/400th of RT reaction was used as a template for real-time quantification using SYBR Green master mix (ABI). Data was acquired using ABI StepOne Plus real-time PCR system instrument. Data was normalized to β-actin and compared to control sample. Data represents mean ± SEM from at least three independent biological experiments.

2.5. Monocyte adhesion assay

Monocyte adhesion assay was performed as previously described [13]. HUVECs were grown in 6-well plate and treated with 10 ng/mL TNFα at 37 °C for 6 h after pretreatment with MI-2 for 30 min, and then washed twice with PBS. THP-1 cells were labeled with fluorescein isothiocyanate using a PKH67 fluorescent staining kit (Zynaxis, Inc., Malvern, PA) according to the instruction of the manufacturer. Add 5×105 fluorescence dye-labeled THP-1 cells into each well and allowed to interact with HUVECs for 1 hour at 37°C. Non-adherent cells were removed by gently washing with cold PBS. The number of adherent THP-1 cells was determined by measuring the fluorescence intensity under Cytation 3 Cell Imaging Multi-mode Reader (Biotek Instruments).

2.6. MTT (3-[4,5-Dimethyl-Triazolyl-2]2,5-Diphenyl Tetrazolium Bromide) Assay

THP-1 monocyte cells were plated in a 96-well plate format and treated with different concentrations of MI-2 for 2 hours. The cells were incubated in serum-free RPMI 1640 containing MTT (0.5 mg/mL) for 4 hours. Then the medium was removed and the cells were incubated for 15 min with equal volume of DMSO to dissolve the formazan crystals. The absorbance of MTT formazan was determined at 570 nm in Cytation 3 Cell Imaging Multi-mode Reader (Biotek Instruments). Viability was expressed as a percentage of absorbance of treated cells to untreated cells.

2.7. LPS challenge in mice

Adult C57BL/6 mice from Jackson Laboratory were challenged by the injection (i.p.) of 25 mg/kg LPS (O127:B8; Sigma-Aldrich) in 200 μl sterile saline. They were divided into three groups. The first group of mice was treated with MI-2 (25 mg/body weight, i.p.) for 2 hours, then the mice were treated with LPS (25 mg/kg body weight, i.p.); the second group of mice were treated with PBS for 2 hours, then the mice were treated with LPS; the third group of mice were treated with PBS only. After LPS injection, the mice were closely monitored for general condition and survival. All mice were sacrificed after 8 hours of LPS injection and the lungs were collected for immunoblotting analysis. All experiments were approved by the Institutional Animal Care and Use Committee of University of Missouri Kansas City.

2.8. Statistical analysis

Data were expressed as the mean ±SD from three independent experiments. Statistical analysis was performed using SPSS18.0 for Windows. Comparisons between two groups were performed by paired t-test. Significance was considered to be p<0.05. Experiments were repeated at least three times.

3. Results

3.1. MALT1 inhibitors suppresses endothelial activation

Endothelial activation is characterized by the expression of multiple chemokines and adhesion molecules such as VCAM-1 and ICAM-1. To explore the role of MALT1 in endothelial activation, first we detected the expression of MALT1 in human vascular endothelial cells. As shown in Figure 1A, MALT1 protein is easy to detect in human endothelial cells from different tissues including aorta, coronary artery, dermal, lung and umbrella vein. Next, we examine the effect of MI-2 treatment (a reported inhibitor of MALT1) on the expression of VCAM-1 and ICAM-1 in activated endothelial cells. As shown in Figure 1B, TNFα, LPS and IL-1β greatly induced both expression of VCAM-1 and ICAM-1. Pretreatment with MI-2 significantly suppressed VCAM-1 expression, but not ICAM-1 expression in activated endothelial cells. Furthermore, MI-2 treatment significantly inhibited TNFα-induced VCAM-1 mRNA expression, but not ICAM-1 mRNA expression. The mRNA levels of TNFα, IL-1β, MCP-1 and CXCL1 were also reduced by MI-2 in activated endothelial cells (Figure 1C). To further confirm the role of MALT1 in the endothelial activation, siRNAs targeting MALT1 were transfected into HUMVECs to knockdown the expression of MALT1. The transfected cells were then activated by TNFα. As shown in Figure 1D, MALT1 expression was efficiently decreased by more than 90% with si-MALT1. The expression of VCAM-1 was also significantly decreased by si-MALT1 treatment. MI-2 is an active site irreversible inhibitor of MALT1 protease activity but it lack specificity (14). Mepazine is the first allosteric MALT1 inhibitor reported (15). MLT-827 is a recent reported potent and selective MALT1 inhibitor (13). To test if MLT-827 and Mepazine also have similar effect on endothelial activation, we performed similar experiments using MLT-827 and mepazine. As shown in Figure 1E and 1F, Pretreatment with MLT-827 or mepazine significantly suppressed TNFα-induced VCAM-1 expression, but not ICAM-1 expression in human endothelial cells. Taken together, these results suggest that MALT1 protease activity is essential for the expression of VCAM-1 in activated endothelial cells. Pharmacological inhibition of MALT1 protease activity represents a new strategy to suppress endothelial activation.

Figure 1. MALT1 inhibitors suppresses endothelial activation.

Figure 1.

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(A) MALT1 protein levels in HUVEC, HAEC, HCAEC, HDMEC and HLMEC were measured by Western blot. (B) HUVECs were pretreated with DMSO or MI-2 (1 μM) for 30 minutes and then treated with or without inflammatory stimuli (10 ng/mL TNF α, 1 μ g/mL LPS or 10 ng/mL IL1 β ) for 8 hours. The protein levels of VCAM-1, ICAM-1 were detected by Western blot. The bands were quantified by Gel-Pro Analyzer software and presented as fold changes; n=3, *p<0.05 or **p<0.001 vs DMSO treatment. (C) HUVECs were pretreated with MI-2 (1 μM) for 30 minutes and then incubated with or without TNFα (10 ng/mL) for 4 hours. The relative mRNA levels of VCAM-1, ICAM-1, IL1β, TNFα MCP1 and CXCL1 were detected by QPCR using 2△△CT method. Data represent mean±SD, n=4. (D) HUVECs were transiently transfected with short interfering RNA targeting on MALT1 (si-MALT1) or nonspecific short interfering RNA (si-Control) by electroporation (Amaxa). Transfected cells were quiescent for 48 hours and then treated with or without TNFα (10 ng/mL) for 8 hours. Expression of MALT1 and VCAM-1 were detected using Western blot. Band intensity of VCAM-1 was quantified by Gel-Pro Analyzer software; n=3, **p<0.001 vs treated si-Control cells. (E&F) HUVECs were pretreated with DMSO or 1 μM of MLT-827 (E) or mepazine (F) for 30 minutes and then treated with or without inflammatory stimuli (10 ng/mL TNF) for 8 hours. The protein levels of VCAM-1, ICAM-1 were detected by Western blot. The bands were quantified by Gel-Pro Analyzer software and presented as fold changes; n=3, *p<0.05 or **p<0.001 vs DMSO treatment.

3.2. Administration of MI-2 inhibits the expression of VCAM-1 in vivo

To examine whether MI-2 treatment could inhibit VCAM-1 expression in vivo, mice were treated with or without MI-2 (25 mg/body weight, i.p.). After 2 hours of injection, the mice were challenged with LPS (25 mg/kg body weight, i.p.) for 8 hours. Lungs were harvested for Western blotting analysis. As shown in Figure 2, LPS dramatically induced VCAM-1 expression in the lungs. Pretreatment with MI-2 significantly inhibited LPS-induced VCAM-1 expression, but not affected ICAM-1 expression. These results further confirmed the role of MALT1 in the regulation of endothelial activation in vivo.

Figure 2. Administration of MI-2 inhibits the expression of VCAM-1 in vivo.

Figure 2.

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Adult C57BL/6 mice were intraperitoneally injected (i. p.) with PBS or LPS (25 mg/kg body weight) for 8 hours. One group of mice was pretreated with MI-2 (25 mg/kg body weight) for 2 hours and then injected with LPS for 8 hours. Mice were euthanized 8 hours after LPS injection. The lungs were harvested for Western Blot with indicated antibodies, β-actin serves as a loading control. Fold-changes of protein levels were determined by densitometry and normalized to β-actin. Data quantifications from Western blot were expressed as the mean ± SD from three independent experiments. *p<0.05, **p<0.01 by Student’s t-test.

3.3. MI-2 treatment attenuates monocyte adherence to activated endothelial cells

Next, we further tested the functional significance of pharmacological inhibition of MALT1 protease activity on endothelial activation by performing monocyte adherence assay. HUVECs were pretreated with or without MI-2 and then activated with TNFα. Fluorescent-labeled THP-1 cells were incubated with activated endothelial cells and the washed twice with cold PBS. The adherent THP-1 cells were imaged and counted by Cytation 3 Cell Imaging Multi-mode Reader. As shown in Figure 3A, TNFα dramatically increased the number of THP-1 cells attached to activated endothelial cells. MI-2 treatment significantly inhibited the adhesion of THP-1 cells to TNFα-stimulated HUVEC cells. Cell viability was measured to demonstrate that the inhibition of THP-1 attachment by MI-2 was not due to its cytotoxicity, as the doses of MI-2 used in the study did not show any cytotoxic effect on THP-1 cells (Figure 3B). To further confirm that VCAM-1 mediates the MI-2-induced monocyte adherence, we transfected VCAM-1 or empty vector into HUVECs and the transfected cells were treated as described above. As shown in Figure 3C, MI-2 treatment significantly inhibited TNFα-induced monocyte adherence, whereas overexpression of VCAM-1 can rescued this inhibition. The expression of MCPIP1, VCAM-1 and ICAM-1 was verified by Western blot (Figure 3D).

Figure 3. MI-2 treatment attenuates monocyte adherence to activated endothelial cells.

Figure 3.

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(A) HUVECs were treated with or without 1μM/L MI-2 for 30 minutes, and then incubated with or without 10 ng/mL TNFα for 8 hours and co-cultured with PHK67-labeled THP-1 cells for another 1 hour. Following three washing with PBS, attached cells were visualized by Cytation 3 Cell Imaging Multi-mode Reader (Biotek Instruments). Adhesive cells were counted and analyzed. (B) Cell viability percentage of THP-1 cells treated with different concentration of MI-2 (0, 0.5μM, 1 μM, 2 μM) for 2 hours was estimated by MTT. Data is shown as mean±SEM of three deparate experiments. Treatments significantly different from the untreated control at p<0.05 are presented as *. (C) HUVECS were transfected with VCAM-1 or empty vector for 24 hours, then the transfected cells were treated and analyzed as described in (A). The cell lysates were also harvested for Western blot analysis (D).

3.4. MI-2 treatment does not affect NF-κΒ signaling in TNFa-induced endothelial cells

Since MALT1 acts as a scaffold protein that plays a key role in NF-κΒ signaling in response to antigen receptor stimulation in lymphocytes [3] and the expression of VCAM-1 is controlled by NF-κB signaling [17], we tested if MI-2 treatment affect TNFα-induced NF-κΒ signaling in endothelial cells. As expected, the phosphorylation of IKK, ΙκΒα and NF-κΒ subunit P65 were increased in TNF-α-induced HUVECs. However, these protein phosphorylation were not affected by MI-2 treatment (Figure 4). These results indicate that MI-2 treatment suppresses endothelial cell activation not through a NF-κΒ-dependent mechanism.

Figure 4. MI-2 treatment does not affect NF-κΒ signaling in TNFα-induced endothelial cells.

Figure 4.

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HUVECs were pretreated with 1 μM MI-2 for 30 minutes then exposed with 10 ng/mL of TNFα for 0, 5, 15 and 30 minutes as indicated. Cell lysates were extracted and Western blot were performed to detect phosphorylation of IκΒα, P65 and IΚΚα/β with specific antibodies as indicated. Total protein levels of ΙκΒα, P65, ΙΚΚα and ΙΚΚβ were also detected. Experiments were repeated three times and showed consistent results.

3.5. MALT1 inhibitors enhances MCPIP1 expression in human endothelial cells

It is well-known that MALT1 can cleave CYLD, A20, BCL10 and RelB through its protease activity, by which it fine-tune lymphocyte activation signaling [6, 7] We recently reported that pharmacological inhibition of MALT1 protease activity enhanced MCPIP1 protein levels in macrophages [12]. To investigate if MALT1 protease also targets MCPIP1 in endothelial cells, HUVECs were incubated with MI-2 and MG132. As shown in Figure 5A, MI-2 treatment can prevent MCPIP1 protein cleavage and enhance its protein levels in a dose-dependent manner. Moreover, MI-2 treatment significantly induced MCPIP1 expression in time-dependent manners in HUVECs (Figure 5B). MI-2 treatment did not affect MCPIP1 mRNA levels (Figure 5C). Interestingly, MI-2 treatment did not affect the protein levels of CYLD, A20, BCL10 and RelB in HUVECs (Figure 5D). Similarly, MLT-827 and mepazine also selective increased MCPIP1 protein levels in human endothelial cells (Figure 5E, 5F and 5G). Taken together, these results suggest that MALT1 protease selectively targets MCPIP1 to degradation in human endothelial cells.

Figure 5. MALT1 inhibitros enhance MCPIP1 expression in human endothelial cells.

Figure 5.

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(A) Western blots for MCPIP1 after 30 minutes pretreatment with increasing concentration of MI-2 (0.25 μM, 0.5 μM, 1 μM and 2 μM) or DMSO, followed by proteasome inhibitor MG-132 (5 μM) treatment for 2 hours in HUVEC. The full-length and cleavage products of MCPIP1 were marked. (B) HUVECs were treated with MI-2 for different durations as indicated. Cell lysates were collected to analysis by Western blots with the MCPIP1 antibody. β-actin served as a loading control. Fold changes of the MCPIP1 protein levels were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. (C)The mRNAs of MCPIP1 in (A) cells were quantified by QPCR. Actin levels were used to normalize. Data represent mean±SD. (D) HUVECs were pretreated with or without MI-2 (1 μM) for 30 minutes and then stimulated with or without TNFα (10 ng/mL) for 8 hours. The protein levels of MALT1 and its targets (MCPIP1, A20, CYLD, BCL10 and RelB) were determined by Western blots with individual antibodies as indicated. β-actin served as a loading control. (E) HUVECs were treated with MLT-827 or mepazine for different durations or doses as indicated. Cell lysates were collected to analysis by Western blots with the MCPIP1 antibody. β-actin served as a loading control. Data are representative of three independent experiments. (F&G)) HUVECs were pretreated with or without MLT-827 (F) or mepazine (G) for 30 minutes and then stimulated with or without TNFα (10 ng/mL) for 8 hours. The protein levels of MALT1 and its targets (MCPIP1, A20, CYLD, BCL10 and RelB) were determined by Western blots with individual antibodies as indicated. β-actin served as a loading control.

3.6. MCPIP1 mediates MI-2-induced inhibition of VCAM-1 expression in human endothelial cells

We previously reported that MCPIP1 can protect ECs against TNFα-induced endothelial activation by attenuating the expression of VCAM-1 other than ICAM-1 [10]. Overexpression of MCPIP1 significantly attenuated TNFα-induced VCAM-1, but not ICAM-1 (Figure 6A), suggesting that MCPIP1 may mediated MI-2-induced inhibition of VC AM-1 expression in human endothelial cells. To test this idea, HUVECs were transfected with si-MCPIP1 or si-Control. The transfected cells were then treated with MI-2 and/or TNFα. As shown in Figure 6B, knocking down of MCPIP1 at least partially reversed MI-2-mediated inhibition on VCAM-1 expression. To further verify that MCPIP1 mediated inhibition of VCAM-1 expression is dependent on its RNase activity, we transfected HUVECs with HA-MCPIP1 and HA-MCPIP1-mutant (which lost its RNase activity). The cells were treated as described above. As shown in Figure 6C, overexpression of wild-type MCPIP1, but not its mutant decreased VCAM-1 expression.

Figure 6. MCPIP1 mediates MI-2-induced inhibition of VCAM-1 expression in human endothelial cells.

Figure 6.

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(A) HUVECs were transiently transfected with Flag-MCPIP1 or empty vector for 48 hours. The cell lysates were harvested, and the protein levels of MCPIP1, VCAM-1 and ICAM-1 were measured by Western blots. The bands of VCAM-1 were quantified by Gel-Pro Analyzer software and presented as fold changes, n=3, *p<0.05 vs DMSO treatment. (B) HUVECs were tansfected with si-Control or si-MCPIP1 for 48 hours. The transfected cells were pretreated with or without MI-2 (1 μM) for 30 minutes and then stimulated with or without TNFα (10 ng/mL) for 8 hours. The protein levels of MCPIP1, VCAM-1 and ICAM-1 were determined by Western blots. β-actin served as a loading control. Fold changes of the MCPIP1 and VCAM-1 protein levels were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments as the mean± SD, **p<0.01 by Student’s t-test. (C) HUVECs were transiently transfected with HA-MCPIP1 or HA-MCPIP1M (mutant) for 48 hours. The cell lysates were harvested, and the protein levels of MCPIP1, VCAM-1 and ICAM-1 were measured by Western blots.

4. Discussion

Endothelial activation is a critical process involved in many vascular diseases including atherosclerosis. Upon inflammatory stimulation, such as TNFα, IL-1β and LPS, the endothelial cells express adhesion molecules such as VCAM-1 and ICAM-1, which mediate monocytes and neutrophils recruitment into vascular wall or tissues [18]. Targeting the expression of VCAM-1 and/or ICAM-1 is a potential therapeutic strategy for vascular inflammatory diseases [19]. In this paper, we have reported that pharmacological inhibition of MALT1 protease activity with MI-2 potently suppresses VCAM-1 expression both in vitro and in vivo, which is dependent on MALT1 protease-mediated MCPIP1 protein degradation in vascular endothelial cells. Findings were corroborated with alternative MALT1 inhibitors, in particular MLT-827, which is a recently described potent and selective inhibitor (13). In addition, as long term-treatment with MI-2 is not toxic in mice (20), our study suggest that MI-2 may provide an opportunity for drug development targeting to vascular inflammatory diseases.

It is reported that GPCR ligands such as angiotensin II can trigger NF-κΒ activation via CARMA3-Bcl10-MALT1 signalosome in endothelial cells, which further results in the expression of VCAM-1 and ICAM-1 and recruitment of immune cells to vascular wall or tissues [21–23]. Since the transcription of VCAM-1 gene is controlled by NF-κΒ activation, we have examined the effect of MI-2 treatment on NF-κΒ signaling activation. Our results suggest that MI-2 treatment does not affect NF-κΒ signal activation in endothelial cells.

As several groups have demonstrated that MALT1 targeted MCPIP1 for degradation in lymphocytes and macrophages [11, 12], we hypothesize that pharmacological inhibition of MALT1 may increase MCPIP1 protein expression via stabilizing its protein. We observed that MI-2 treatment, or treatment with MLT-827 or mepazine, selectively enhanced MCPIP1 expression in endothelial cells. Furthermore, siRNA-mediated knocking down of MCPIP1 at least partially reversed the effect of MI-2 on VCAM-1 expression. The other mechanisms by which that MLAT1 inhibitors suppresses endothelial activation may also exist and need to be further explored.

Recently, it is reported that MALT1 could cleave CYLD into two fragments. The fragments can alter endothelial barrier integrity which is unrelated to NF-κΒ signaling [24]. In this study, we observed that MI-2 treatment did not affect CYLD protein level in human endothelial cells. The discrimination may be caused by different cells and cultured conditions.

In conclusion, we here demonstrate that pharmacological inhibition of MALT1 protease activity selectively enhances MCPIP1 expression and by which it suppresses VCAM-1 expression and endothelial inflammation. These results suggest MALT1 as a promising therapeutic target for vascular inflammatory diseases such as atherosclerosis.

Highlights.

  • We studied the role of MALT1 protease in endothelial activation. > Pharmacological inhibition of MALT1 protease activity significantly suppresses VCAM-1 expression and endothelial activation. > Pharmacological inhibition of MALT1 protease activity with MI-2 may be a novel therapeutic strategy for vascular inflammatory diseases such as atherosclerosis.

Acknowledgements

This work was supported by National Institutes of Health grants (AI103618 to MF) and American Heart Association grant (17AIREA33660073 to MF), and the National Natural Science Foundation of China grant (81670429 to ZJ).

Footnotes

Disclosure of Conflicts of Interest

No conflicts of interests were disclosed by all authors.

Authors’ contributions

YL, SH, XH, XL, AF and AS carried out the experiment; FG, SL, ZJ and HX provided consults; YL and MF designed the study and wrote the manuscript.

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