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Interleukin-1β-Induced Senescence Promotes Osteoblastic Transition of Vascular Smooth Muscle Cells

interleukin-1β senescence osteoblastic transition vascular calcification

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#1 Engadin

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Posted 14 July 2020 - 06:40 PM


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O P E N    A C C E S S   S O U R C E (.PDF) :    Kidney and Blood Pressure Research

 
 
 
 
 
 
Highlights
 
  •  Phosphate inducing senescence-associated calcification is concerned with up-regulating p53 expression.
 
  •  IL-6 induces senescence-associated calcification through activating IL-6/sIL-6/STAT3/p53/p21 signaling pathway.
 
  •  The synergistic action of phosphate and IL-6 enhances senescence-associated calcification in a p53-dependent manner.
 
  •  The synergistic action of phosphate and IL-6 is inhibited by anti-aging agents in a dose-dependent manner.
 
 
 
Abstract
 
Introduction: Interleukin (IL)-1β, as a key biomarker and mediator of vascular calcification in patients with end-stage renal disease (ESRD), may be involved in the process of premature senescence of vascular smooth muscle cells (VSMCs). This work sought to investigate whether IL-1β-induced premature senescence contributes to the process of osteoblastic transition and vascular calcification in VSMCs.
 
Methods: Eighty-eight patients with ESRD (aged 25-81 years), 11 healthy individuals, and 15 cases of lesion-free distal radial arteries from dialysis ESRD patients with angiostomy were collected in this study. Immunohistochemical analysis was performed to detect expression of IL-1β, p21, and bone morphogenetic protein-2 (BMP2) in the distal radial arteries. Primary human VSMCs from healthy neonatal umbilical cords were incubated with test agents for 1-3 days. Intracellular levels of reactive oxygen species (ROS) and senescence-associated-β-galactosidase (SA-β-gal) staining were used to detect senescent cells. Alizarin red staining and the calcium content of the cell layer were used to detect mineral deposition in VSMCs.
 
Results: Coincident with positive staining of IL-1β, p21, and BMP2 in the lesion-free distal radial arteries, 66.67% patients showed mineral deposition. Serum IL-1β was 0.24 ± 0.57, 1.20 ± 2.95, and 9.41 ± 40.52 pg/mL in 11 healthy individuals, 20 patients without calcification, and 53 patients with calcification, respectively. Analysis of the cross-table chi-square test showed cardiovascular calcification is not correlated with levels of serum IL-1β in patients with ESRD (p = 0.533). In response to IL-1β, VSMCs showed a senescence-like phenotype, such as flat and enlarged morphology, increased expression of p21, an increased activity of SA-β-gal, and increased levels of ROS. IL-1β-induced senescence of VSMCs was required for the activation of IL-1β/NF-κB/p53/p21 signaling pathway. IL-1β-induced senescent VSMCs underwent calcification due to osteoblastic transition mainly depending upon the upregulation of BMP2. Resveratrol, an activator of sirtuin-1, postponed the IL-1β-induced senescence through blocking the NF-κB/p53/p21 pathway and attenuated the osteoblastic transition and calcification in VSMCs.
 
Conclusions: High levels of IL-1β in medial smooth muscles of arteries may play roles in inducing senescence-associated calcification. IL-1β-induced senescence depending on the activation of the NF-κB/p53/p21 signaling pathway and contributing to osteoblastic transition of VSMCs.
 
 
 
Abbreviations
 
ESRD: end-stage renal diseases
CKD: chronic kidney disease
NDD-CKD: non-dialysis-dependent chronic kidney disease
VSMCs: vascular smooth muscle cells
IL-6: interleukin-6
STAT3: signal transducer and activator of transcription 3
p-STAT3: phosphorlated-STAT3
SA-β-Gal: senescence-associated-β-galactosidase
L-Re: slow concentrations of resveratrol
H-Res: high concentrations of resveratrol
α-SMA: α-smooth muscle actin
VEGFR2: vascular endothelial growth factor receptor 2
SIRT1: sirtuin 1
Ac-p53: acetylated-p53
ROS: reactive oxygen species.
 
 
 
Resveratrol-represses-osteoblastic-trans
 
Resveratrol represses osteoblastic transition in IL-1β-induced senescent VSMCs. A Immunoblotting analysis for expression of p21, ɑ-SMA, RUNX2, and BMP2 in VSMCs by exposure to phosphate or phosphate/resveratrol over 3 days. B Immunoblotting analysis for the expression of p21, ɑ-SMA, RUNX2, and BMP2 in VSMCs cultured in medium with phosphate/IL-1β or phosphate/ IL-1β/resveratrol over 3 days. Pi, phosphate; Ab, anti-IL-1β neutralization antibody; Res, resveratrol; 3-day vehicle denotes 3-passage VSMCs cultured in complete DMEM/F-12 medium for 3 days; ɑ-SMA, α-smooth muscle actin; RUNX2, runt-related transcription factor-2; BMP2, bone morphogenetic protein-2; IL, interleukin.
 
 
 
Introduction
 
Chronic kidney disease (CKD) is highly correlated with cardiovascular mortality and vascular calcification. In anatomy, vascular calcification includes medial and intimal calcification. Intimal calcification, which is associated with atheromatous lipid-rich flow-limiting lesions, generally occurs in medium-sized conduit arteries with atherosclerotic plaques [1]. Medial calcification, associated with a diffuse granular pattern of mineral deposition, usually occurs in older popu-lation and patients with diabetic nephropathy [2, 3]. Approximately 50% of patients with CKD show vascular calcification before hemodialysis [4]. Multiple recent studies have shown that disturbed phosphate/calcium metabolism, vascular smooth muscle cells (VSMCs) apoptosis, and osteogenic transition of VSMCs are associated with vascular calcification [4, 5]. In particular, inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1α, IL-1β, IL-4, and interferon-γ,  play  substantial  roles  in  vascular  calcification  [6].  Among  them,  IL-1β,  a  key biomarker and mediator of vascular calcification due to increased serum IL-1β levels in patients with chronic stable coronary heart diseases [7], has been considered to induce osteogenic tran-sition of VSMCs in the presence of hyperphosphatemia [5, 8].IL-1β is one of the most potent activators of the transcription factor NF-κB. In mammalian cells, classical NF-κB exhibits transactivation activities in the form of a heterodimer with p50 and p65/RelA proteins involved in inflammation and immune responses [9]. Activated NF-κB is associated with the pathogenesis of CKD. Moreover, NF-κB activation induced by a tumor necrosis factor-like weak inducer of apoptosis directly favors phosphorous-induced osteo-genic  transition  and tissue  nonspecific alkaline  phosphatase (ALP)  activity and  promotes phosphorous-induced VSMC calcification [10].Normal human cells undergo senescence in response to persistent DNA damage. There are no specific markers for senescent cells. Many cells express a pH-labile enzyme called senescence-associated-β-galactosidase (SA-β-Gal) when they undergo senescence because of multiple stimuli. Cellular senescence is divided into 3 different types: replicative senescence, oncogene-induced senescence, and stress-induced senescence. Chronic stimulation of cells with cytokines causes enough stress to induce stress-induced senescence. Interestingly, there are some pieces of evidence that support correlation of stress-induced premature senescence with IL-1β stimulation [11, 12]. In comparison with vehicle-treated controls, human osteoar-thritic chondrocytes exposed to IL-1β exhibits an increased number of SA-β-Gal-positive cells [13]. Dai et al. [14] reported that the p53/p21 pathway mediates the features of chondrocyte senescence induced by IL-1β and hydrogen peroxide.
 
As CKD is considered as an age-related disease, senescence-associated vascular calcifi-cation is becoming an interesting topic of study. Calcification of VSMCs is inhibited in the presence of macrophages and lipids [15], indicating the possible role of senescent VSMCs in the formation of vascular calcification. Compared with control young cells, VSMC calcification was reported to be dramatically enhanced in senescent cells. It is suggested that senescent VSMCs  are  apt to  osteoblastic  transition. Runt-related  transcription  factor-2 (RUNX2),  an important transcription factor that regulates osteoblastic differentiation, is involved in the senescence-associated osteoblastic transition in VSMCs [16–18]. bone  morphogenetic protein-2 (BMP2), as a molecular marker of osteoblasts, is highly expressed in VSMCs in the process of osteoblastic transition.
 
However, little is known about senescence in VSMCs induced by IL-1β. The involvement of IL-1β in the senescence-associated osteoblastic transition causing vascular calcification in VSMCs remains unclear. In addition, it remains to be determined whether pharmacological intervention can prevent senescence-associated osteoblastic transition that occurs in senescent VSMCs. In the present study, we examined whether plasma IL-1β coincided with vascular calcification caused by senescence-associated osteoblastic transdifferentiation of VSMCs in patients with end-stage renal disease (ESRD). We found that high levels of IL-1β in medial smooth muscles of arteries from patients with ESRD may play roles in inducing senes-cence-associated calcification. IL-1β-induced senescence of VSMCs was required for the acti-vation of the IL-1β/NF-κB/p53/p21 pathway, and senescent VSMCs enhanced the suscepti-bility to calcification  by  increasing osteoblastic  transition.  Furthermore,  we demonstrated that resveratrol effectively inhibited vascular calcification associated with osteoblastic tran-sition in IL-1β-induced senescent VSMCs.

 


Edited by Engadin, 14 July 2020 - 06:41 PM.






Also tagged with one or more of these keywords: interleukin-1β, senescence, osteoblastic transition, vascular calcification

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