Interleukin-29 Accelerates Vascular Calcification via JAK2/STAT3/BMP2 Signaling

Abstract

Background Vascular calcification (VC), associated with enhanced cardiovascular morbidity and mortality, is characterized by the osteogenic transdifferentiation of vascular smooth muscle cells. Inflammation promotes VC initiation and progression. Interleukin (IL)-29, a newly discovered member of type III interferon, has recently been implicated in the pathogenesis of autoimmune diseases. Here we evaluated the role of IL-29 in the VC process and underlying inflammatory mechanisms. Methods and Results The mRNA expression of IL-29 was significantly increased and positively associated with an increase in BMP2 (bone morphogenetic protein 2) mRNA level in calcified carotid arteries from patients with coronary artery disease or chronic kidney disease. IL-29 and BMP2 proteins are colocalized in human calcified arteries. IL-29 binding to its specific receptor IL-28Rα (IL-28 receptor α) (IL-29/IL-28Rα) inhibited the proliferation of rat vascular smooth muscle cells without altering cell apoptosis or migration. IL-29 promoted the calcification of rat vascular smooth muscle cells and their osteogenic transdifferentiation in vitro as well as the rat aortic ring calcification ex vivo, induced by the calcification medium or osteogenic medium. The procalcification effect of IL-29 was reduced by pharmacological inhibition of IL-29/IL-28Rα binding as well as suppression of janus kinase 2/signal transducer and activator of transcription pathway activation, accompanied by decreased BMP2 expression in the cultured rat vascular smooth muscle cells. Conclusions These results suggest an important role of IL-29 in VC development, at least partly, via activating the janus kinase 2/signal transducer and activator of transcription 3 signaling. Inhibition of IL-29 or its specific receptor, IL-28Rα, may provide a novel strategy to reduce VC in patients with vascular diseases.

Keywords: JAK/STAT pathway; interleukin‐29; vascular calcification; vascular smooth muscle cell.

Figure 1. IL‐29 expression is elevated in calcified arteries from patients with CAD and CKD.

A, Quantification of IL‐29 and BMP2 mRNA expression in the calcified carotid arteries from 4 patients with CAD, 4 patients with CKD, 6 noncalcified arteries, and a Pearson correlation between calcified arteries. The 2^−ΔΔCt method was used, with GAPDH as an internal reference, to calculate the relative gene expression level. Data are mean±SD. Statistical significance was tested using a 2‐tailed unpaired t test. B, Representative images showing ARS staining for the lesions of calcification, as well as IL‐29 and BMP2 protein expression on the lesions of the carotid arteries from 3 patients with CAD, 3 patients with CKD, and 3 noncalcified arteries as control. The magnification is ×200. Arrows showed the calcification deposition. Data are mean±SD. Statistical significance was calculated from an ordinary 1‐way ANOVA with Tukey multiple comparisons test. ARS indicates alizarin red S; BMP2, bone morphogenetic protein 2; CAD, coronary artery disease; CKD, chronic kidney disease; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; and IL‐29, interleukin 29.

Figure 2. IL‐28Rα is highly expressed in calcified mice and specifically expressed in VSMCs.

A, Representative images of ARS staining for the calcification lesions (×200), IL‐28a (red), as well as IL‐28Rα (green) expression in the aortic tissues of vitamin D₃–induced calcified mice (×400) (n=5). Nuclei were stained with DAPI (blue). B and C, Representative images showing the protein expression of IL‐28Rα (green), IL‐10R2 (green), and α‐SMA (red) in the mice aortic tissues, as well as IL‐28Rα (red) and IL‐10R2 (red) in the primary rat and human VSMCs. Nuclei were stained with DAPI (blue). The magnification is ×400. D, The mRNA levels of IL‐28Rα and IL‐10R2 in the primary rat and human VSMCs. Data from a representative experiment were performed in 5 replicates and presented as mean±SEM. Statistical significance was tested using a 2‐tailed unpaired t test. α‐SMA indicates α‐smooth muscle actin; ARS, alizarin red S; DAPI, 4',6‐diamidino‐2‐phenylindole; IL‐10R2, interleukin 10 receptor 2; IL‐28a, interleukin 28a; IL‐28Rα, interleukin 28 receptor α; and VSMC, vascular smooth muscle cell.

Figure 3. IL‐29 inhibits the proliferation of VSMCs without significant change in the apoptosis and migration.

A, Proliferation of primary rat VSMCs (n=3) after treatment with IL‐29 (1, 10, or 100 ng/mL) was monitored by real‐time cellular analysis over 120 hours. Data from 1 representative experiment were performed in triplicate and presented as mean±SEM. B and C, Primary rat VSMCs was treated with different concentrations of IL‐29 (1, 10, 100 ng/mL). The cell apoptosis was examined with flow cytometry at 48 hours, and the migration was determined with transwell analysis at 24 hours. Representative scattergrams show flow cytometry analysis for cell apoptosis, and micrographs show transwell assay for cell migration (×100). Data from 1 representative experiment performed in triplicate. Values are ±SEM. Statistical significance was calculated from ordinary 1‐way ANOVA with Tukey multiple comparisons test. Annexin V‐FITC indicates Annexin V‐fluorescein isothiocyanate; Control, only medium treatment; IL‐29, interleukin 29; ns, not significant; and VSMCs, vascular smooth muscle cells.

Figure 4. IL‐29 promotes the osteogenic differentiation and calcification in vitro and ex vivo.

A and B, Primary rat VSMCs were cultured with IL‐29 (1, 10, or 100 ng/mL) in CaP medium for 3 days (upper) or IL‐29 (100 ng/mL) administration in OGM medium for 17 days (lower), as well as IL‐29 (100 ng/mL) treatment in primary human VSMCs in CaP medium for 3 days B, Representative photos of the ARS staining and quantified OD value were shown. In the other experimental set, cell supernatants and cell lysis were used for the quantification of calcium contents. Data from 1 representative experiment were performed in 4 to 6 replicates and shown as mean±SEM. C, Rat aortic rings were exposed to CaP medium and IL‐29 (100 ng/mL) for 6 days. Representative images and quantification of ARS staining for calcified areas by ImageJ software from 3 sections of each rat (n=3) were displayed. The magnification is ×40. Arrows indicated the calcified area. Values are mean±SEM. D, Primary rat VSMCs were incubated with IL‐29 (100 ng/mL) in the CaP condition for 48 hours. Representative Western blot images for BMP2 and RUNX2 protein expression were shown in duplicate each group (left), and relative expression of them to GAPDH were semiquantified (right). The cumulative data were from 3 independent experiments and expressed as mean±SEM. E, Primary rat VSMCs were incubated with IL‐29 (100 ng/mL) in the CaP condition for 48 hours. mRNA levels of VSMC osteogenic markers BMP2 and OPN, and VSMC phenotype markers α‐SMA and SM22α were analyzed with real time polymerase chain reaction. Pooled data from 3 independent experiments were performed in duplicate and expressed as mean±SEM. All of the statistical significance in this figure was calculated using ordinary 1‐way ANOVA with Tukey multiple comparisons test. α‐SMA indicates α‐smooth muscle actin; ARS, alizarin red S; BMP2, bone morphogenetic protein 2; control, only medium treatment; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; IL‐29, interleukin 29; OD, optical density; OGM, osteogenic calcification medium; OPN, osteoprotegerin; RUNX2, Runt‐related transcription factor 2; SM22α, smooth muscle 22α; and VSMC, vascular smooth muscle cell.

Figure 5. Blocking IL‐29 or IL‐28Rα inhibits the procalcification effect of IL‐29.

A and B, Primary rat VSMCs were cultured with IL‐29 (100 ng/mL) in the presence of IL‐29 neutralization antibody (1 and 10 μg/mL) or IL‐28Rα neutralization antibody (0.1, 1, or 10 μg/mL) in the CaP condition for 3 days. Representative images of ARS staining are displayed. The cumulative data for OD value were from 1 representative experiment performed in triplicates or quadruplicates and expressed as mean±SEM. Statistical significance was tested using ordinary 1‐way ANOVA with Tukey multiple comparisons test (A) and Brown‐Forsythe ANOVA followed by Dunnett T3 multiple comparisons test (B). ARS indicates alizarin red S; IL‐28Rα, interleukin 28 receptor α; IL‐29, interleukin 29; CaP, calcification medium; OD, optical density; and VSMC, vascular smooth muscle cell.

Figure 6. IL‐29 selectively activates JAK2/STAT3 signaling pathway in VSMC calcification.

A and B, Representative Western blots and semiquantitative analysis for the change of JAK2/STAT pathways in primary rat VSMCs treated with IL‐29 (100 ng/mL) in the CaP condition for 30 and 60 minutes, respectively. p‐STAT1/STAT1 and p‐STAT4/STAT4 were not detected in this experiment. The cumulative data were from 3 independent experiments and expressed as mean±SEM. Statistical significance was calculated from ordinary 1‐way ANOVA with Tukey multiple comparisons test. C, Representative Western blots for the change of MAPK pathway. No obvious changes were observed after IL‐29 treatment in CaP condition compared with only CaP medium‐treated group. CaP indicates calcification medium; ERK, extracellular regulated protein kinase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; IL‐29, interleukin 29; JAK2, janus kinase 2; JNK, c‐Jun N‐terminal kinase; MAPK, mitogen‐activated protein kinase; P38, p38 mitogen activated protein kinase; p‐ERK, phospho‐ERK; p‐JAK2, phospho‐JAK2; p‐JNK, phospho‐JNK; p‐P38, phospho‐P38; p‐STAT1, phospho‐STAT1;p‐STAT2, phospho‐STAT2; p‐STAT3, phospho‐STAT3; p‐STAT4, phospho‐STAT4; p‐STAT5, phospho‐STAT5; p‐STAT6, phospho‐STAT6; STAT1, signal transducer and activator of transcription 1; STAT2, signal transducer and activator of transcription 2; STAT3, signal transducer and activator of transcription 3; STAT4, signal transducer and activator of transcription 4; STAT5, signal transducer and activator of transcription 5; STAT6, signal transducer and activator of transcription 6; and VSMC, vascular smooth muscle cell.

Figure 7. JAK2 or STAT3 antagonist reduces the procalcification effect of IL‐29 and BMP2 expression.

A and B, Primary rat VSMCs were treated with IL‐29 (100 ng/mL) with or without the presence of JAK2 antagonist (fedratinib, 1 μM) or STAT3 antagonist (niclosamide, 10 μg/mL) in the CaP condition for 3 days. Representative images of ARS staining for calcium deposition were shown. C and D, Representative Western blots and semiquantitative analysis showed the effect of JAK2 or STAT3 antagonist on BMP2 protein expression. The cumulative data were from 4 independent experiments and expressed as mean±SEM. Statistical significance was calculated from ordinary 1‐way ANOVA with Tukey multiple comparisons test. ARS indicates alizarin red S; BMP2, bone morphogenetic protein 2; CaP, calcification medium; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; IL‐29, interleukin 29; JAK2, janus kinase 2; STAT3, signal transducer and activator of transcription 3; and VSMC, vascular smooth muscle cell.

Figure 8. Schematic illustration of the effect of IL‐29 on VSMC calcification process and possible blocking strategies.

IL‐29 accelerates the osteogenic differentiation and calcification of VSMCs under the CaP condition. This process can be interrupted by blocking the binding of IL‐29/IL‐28Rα or activation of JAK2/STAT3 pathway. BMP2 indicates bone morphogenetic protein 2; Ca, calcium; CaP, calicificaiton medium; IL‐28Rα, interleukin 28 receptor α; IL‐29, interleukin 29; JAK2, janus kinase 2; Pi, inorganic phosphate; p‐JAK2, phospho‐JAK2; p‐STAT3, phospho‐STAT3; STAT3, signal transducer and activator of transcription 3; and VSMC, vascular smooth muscle cell.