Expression and function of an IL-12R type in the human aortic smooth muscle cells Heesu Lee, Sang Won Kang 1 Department of Life Science, Ewha Womans University, Seoul 03760, Republic of Korea; BACKGROUND AIM VSMCs are known to play important roles throughout all stages IL12Rβ2 expression in immune cells is well known, but its of atherosclerosis. Especially, VSMCs proliferation and migration expression in vascular cells is unclear. Because endothelial cells are important for the progression of arteriosclerosis. Also, are almost denuded or detached in the balloon injury model, VSMCs
proliferation contributes to in-stent restenosis. Balloon VSMCs are considered a source of IL12Rβ2, but it is still injury model is a model that mimics this phenomenon. In the uncertain. Therefore, this data shows whether VSMCs express previous study, proteomics was performed in the balloon-injured IL12Rβ2 and how IL12Rβ2 expression level increases rat carotid artery to identify signaling molecules organizing the METHOD VSMC hyperplasia. The differential proteomics analysis revealed the protein that the expressions are changed within 3 days post - Cell culture and Transfection injury. One of
which was Interleukin-12 receptor β2 (IL12Rβ2). - Quantitative real time PCR IL12Rβ2 expression increased during 7 days post injury. IL12Rβ2 - Reverse transcription-PCR is one of Interleukin-12 family receptor, and its ligand is - Immunoprecipitation(IP) and Immunoblotting (IB) Interleukin 12 (IL-12) or Interleukin 35 (IL-35). RESULTS 293T 293T VCAM-1 mRNA expression (Fold chnage) 6 IL12Rb2 mRNA expression (Fold change) IB : Flag IB : Flag pre-treated si-IL12Rb2 Vector Vector HASMC 10 8 8 6 si-CON siRNA : control IL12Rb2 IL12Rb2 IL12Rb2 siRNA : control IL12Rb2 IL12Rb2 IL12Rb2 TNF-α (10ng/mL) :
0 3 5 10 30 (min) DNA : DNA : IκB 4 4 IP : Flag IL12Rb2 IP : IL12Rb2 IL12Rb2 Tubulin 2 2 Flag Flag 0 TNF-α (10ng/mL) : 0 4 8 12 24 (hours) 0 IB IL12Rb2 IB IL12Rb2 IFN-γ : - + + Tubulin HASMC Tubulin HASMC Figure 2. IL12Rβ2 mRNA expression in HASMCs is upregulated by IFN-γ. VCAM-1 mRNA expression (Fold chnage) (50ng/ml) for 48 hours. IB : Flag 6 IL12Rβ2 mRNA expression level was measured by qRT-PCR. (n=3) (**P<0.005). HASMC 5 (A) Time course of IL12Rβ2 mRNA expression following treatment with indicated cytokines. siRNA : control IL12Rb2 siRNA : control IL12Rb2 (B) IL12Rβ2 mRNA expression level
was measured by qRT-PCR. HASMCs were transfected IL-1β (10ng/mL) : 0 5 10 15 30 60 (min) 4 with siIL12Rβ2 using Lipofectamine® RNAiMAX Reagent for 6 hours and then treated with IFN-γ Virus : Vector IL12Rb2 IL12Rb2 Virus : Vector IL12Rb2 IL12Rb2 IκB 3 Full length primer Primer 1 Primer 2 IP : Flag IB : Flag IP : IL12Rb2 Exon : 1 Tubulin 2 1 variant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 IL12Rb2 IL12Rb2 Flag Flag variant 2 0 IL-1β (10ng/mL) : 0 4 8 12 24 (hours) variant 3 IB IL12Rb2 IB IL12Rb2 variant 4 Tubulin Tubulin HASMC 5 * variant 5 Figure 4. Exogenous IL12Rβ2 expression on 293T and
HASMCs. variant 6 293T cells were overexpressed with the empty vector (pQ) or vector encoding human IFN-γ (50ng/mL) : 0 5 10 15 30 60 (min) 4 variant 7 IL12Rβ2 with Flag tag using turbofect reagent for 24 hours and then transfected with pSTAT1 * U937 HASMC siIL12Rβ2 using Lipojet reagent for 48 hours. The immunoprecipitated (A) Flag or (B) (Y701) T-bet mRNA expression (Fold chnage) 6 3 ** IFN-γ : - - + U937 U937 IL12Rβ2 proteins were subjected to the immunoblotting. HASMCs were overexpressed with human IL12Rβ2 with Flag tag encoding retrovirus for 24 STAT1 2 IL12Rb2 colony : 2 primer 1 18 (bp)
colony : 2 primer 2 18 hour and then transfected with siIL12Rβ2 using Lipofectamine®RNAiMAX reagent for 48 13 13 hours. The immunoprecipitated (C) Flag or (D) IL12Rβ2 proteins were subjected to the Tubulin 1 0 β-actin (bp) 500 400 immunoblotting. HASMC HASMC 400 300 200 300 IFN-γ (50ng/mL) : 0 24 48 72 (hours) HASMC+IFN-γ HASMC virus : vec IL12Rb2-Flag Virus : vec IL12Rb2-Flag Figure 1. Effect of pro-inflammatory cytokines on HASMCs primer 1 siRNA : control IL12Rb2 PDGF-BB : 0 5 10 30 0 5 10 30 (min) IL-12: 0 5 10 30 0 5 10 30 (min) HASMCs were treated with (A) TNF-α (10ng/ml) or (B) IL-1β
(10ng/ml) for the indicated colony : 4 8 13 14 16 pSTAT4 time. IκB and tubulin were analyzed by immunoblotting. Tubulin is loading control. (bp) 500 pTyr (Y693) (C) After the serum starvation, HASMCs were treated with IFN-γ (50ng/ml) for the 400 300 indicated time. STAT1, phospho-STAT1, and tubulin were analyzed by immunoblotting. IL12Rb2(G465D) IL12Rb2(G465D) PDGFR STAT4 Tubulin is loading control. The fold change of VCAM-1 mRNA levels in (D) TNF-α (10ng/ml) or (E) IL-β (10ng/ml) 0, 4, HASMC+IFN-γ Vector IL12Rb2 IL12Rb2 pErk 1/2 Flag 8, 12, 24 hours treated HASMCs. Time course of mRNA
expression measured by qRT- primer 2 Virus : PCR. colony : 4 8 13 14 16 Tubulin (F) The fold change of T-bet mRNA levels in IFN-γ (50ng/ml) 0, 24, 48, 72 hours treated (bp) 400 IL12Rb2 Erk 2 HASMCs. Time course of mRNA expression measured by qRT-PCR. (n=3) (*P<0.05, 300 200 **P<0.005). Tubulin Flag HASMC Virus : vec IL12Rb2-Flag IL12Rb2 pSTAT1 0 5 10 30 (min) IL-35 : 0 5 10 30 IL12Rb2 mRNA expression (Fold chnage) 10 8 6 4 TNFα IL12Rb2 mRNA expression (Fold chnage) 10 8 6 4 (A) The transcription variant and isoform of IL12Rβ2. Figure 5. IL12Rb2 affects signaling in HASMC. STAT1 12 12 Tubulin
(Y701) Figure 3. IL12Rβ2 transcription variant 1 is expressed in HASMCs ** IFNγ IL12 IL1β (B) RT-PCR analysis of IL12Rβ2 mRNA expressed in U937, HASMCs, and IFN-γ (50ng/ml) 48hr treated HASMCs. β-actin is an endogenous control gene. Cloning of IL12Rβ2 full length PCR product in (C) U937 and (D) IFN-γ treated HASMCs was Flag performed. Colony PCR to separate the IL12Rβ2 transcriptions variant based on primer from Tubulin (A). (E) Sequencing data of IL12Rβ2 full length in IFN-γ treated HASMCs. 2 2 retrovirus for 24 hour and then transfected with siIL12Rβ2 using Lipofectamine® RNAiMAX hour and
then, after starvation, treated with (A) PDGF-BB (25ng/ml), (B) IL-12 (10ng/ml) or (C) 0 0 Reagent for 48 hours. The protein expression was detected by immunoblotting. IL-35 (10ng/ml) for the times indicated. pTyr, PDGFR, pERK, ERK2, pSTAT4, STAT4, 0 24 48 72 (hours) IFN-γ (50ng/mL) : 0 24 48 72 (hours) (F) HASMCs were overexpressed with IL12Rβ2 wild type or variant G465D encoding HASMCs were overexpressed with human IL12Rb2 with Flag tag encoding retrovirus for 24 pSTAT1, STAT1, Flag, IL12Rβ2 and tubulin were analyzed by immunoblotting. Tubulin is loading control. CONCLUSION REFERENCES
Pro-inflammatory cytokines promote proliferation in VSMCs, [1]. Ross, R. and J.A. Glomset, Atherosclerosis and the arterial among them,, IFN-γ increases the IL12Rβ2 mRNA expression smooth muscle cell: Proliferation of smooth muscle is a key event level in VSMCs. The IL12Rβ2 mRNA increased by IFN-γ is not a in the genesis of the lesions of atherosclerosis. Science, 1973. non-coding mRNA but a transcription variant 1 of IL12Rβ2. 180(4093): p. 1332-9. Overexpressed IL12Rβ2 was not affect PDGF and IL-12 signal [2] Kang, D.H., et al., Vascular Proteomics Reveal Novel Proteins but increase IL-35
signal in VSMCs. Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Because IL-35 has been reported to be involved in the adhesion Regulating Proliferation and Inflammatory Response. PLoS One, molecules expression and proliferation in vascular cells, it is 2015. 10(8): p. e0133845. necessary to confirm that the overexpressed IL12Rβ2 contribute [3] Vignali, D.A. and V.K. Kuchroo, IL-12 family cytokines: to affecting the adhesion molecules expression and proliferation immunological playmakers. Nat Immunol, 2012. 13(8): p. 722-8. of VSMCs via cell assay. Therefore, this study suggest a
novel [4] de Paus, R.A., et al., Differential expression and function of role of IL12Rβ2 in VSMCs and proposes IL12Rβ2 may be human IL-12Rbeta2 polymorphic variants. Mol Immunol, 2013. associated with VSMC hyperplasia. 56(4): p. 380-9. [Y. Vascular biology-2] Expression and function of an IL-12R type in the human aortic smooth muscle cells Heesu Lee¹, Sang Won Kang¹ ¹Life Science, Ewha Womans University, Seoul 03760, Korea Interleukin-12 receptor β2 (IL12Rbeta2) is a type of the Interleukin-12 (IL-12) receptors that forms heterodimer with Interleukin-12 receptor β1 (IL12Rb1). IL-12 binding to
the receptor induces the Th1 differentiation and interferon- gamma (IFN-γ) production through STAT4. IL12Rbeta2 is known to be expressed in the immune cells, like T cell or NK cell, but its expression in vascular cells is largely unknown. The proliferation and migration of Vascular Smooth Muscle Cells (VSMCs) play an important role in the progression of atherosclerosis. We found that IL12Rbeta2 expression is induced by a balloon injury on carotid vessels. Moreover, the knock-down of IL-12Rbeta2 expression resulted in a negative effect on vascular hyperplasia. In this study, we show that the
expression of IL12Rbeta2 is induced in the vascular smooth muscle cells treated with interferon-gamma (IFN-γ). Then, we identify the transcript variant of IL12Rbeta2 in the VSMCs with single nucleotide alteration. Thus, the study suggests a novel role of interleukin receptor in the non-immune cells. The vasodilatory effect of gemigliptin via activation of voltage-dependent K channels and SERCA pumps in aortic smooth muscle + Hee Seok Jung, Won Sun Park Department of Physiology, Kangwon National University School of Medicine, Chuncheon, South Korea Introduction With the increasing prevalence of
diabetes, there is growing pressure on public health resources for diabetes treatment. Cardiovascular disease is a significant complication of type 2 diabetes mellitus (DM) and is the primary cause of mortality in patients with type 2 DM. Most diabetic patients take oral medication to treat type 2 DM. A variety of antidiabetic treatment options are available, including sulfonylureas, dipeptidyl peptidase-4 (DPP-4) inhibitors, thiazolidinediones, meglitinides, glucagon-like peptide-1 (GLP-1) agonists, and sodium glucose co-transporter 2 (SGLT2) inhibitors. DPP-4 inhibitors are an effective
antidiabetic drug that reduce blood glucose by increasing GLP-1 levels, thereby stimulating insulin secretion. Gemigliptin is a potent selective and long-acting DPP-4 inhibitor that is used in clinical treatment in many countries. Gemigliptin lowers albuminuria, which benefits patients with renal insufficiency, particularly older adults. Given the significance of cardiovascular complications in patients with diabetes, many studies have investigated the cardiovascular safety of antidiabetics. Previous studies have shown that gemigliptin plays a protective role in cardiovascular diseases through
the inhibition of ER-stress-induced apoptosis and inflammation in cardiomyocytes. Furthermore, gemigliptin has a protective effect on the proliferation and migration of vascular smooth muscle cells via Nrf2 activation. One area yet to be studied is the effects of gemigliptin on vascular contractility and the associated regulatory factors. Maintenance of vascular tone is critical for vascular function, and improving vascular contractility is often necessary when treating vascular diseases. Potassium (K ) channels are + expressed in vascular smooth muscle cells and play a key role in vascular
tone regulation. There are four type of K channels in vascular smooth muscle cells: 1) inwardly rectifying K (Kir), 2) + + voltage-dependent K (Kv), 3) large-conductance Ca -activated K (BK ), and 4) ATP-sensitive K (K ATP ) channels. Kv channels, which are most prevalent in vascular cells, are known as + + 2+ + Ca important channels for controlling vascular tone. An effective Kv channel inhibitor, 4-aminopyridine (4-AP), induced vasoconstriction in some arteries, suggesting that Kv channels in vascular smooth muscle may be an important therapeutic target for treating cardiovascular diseases
such as hypertension, hypoxia, and diabetes. In t
