C-reactive Protein In Vascular Smooth Muscle Cells Research Articles

Radiation-induced C-reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref-1 complex.

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C‐reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation‐induced CRP. NOX inhibition using lentiviral sh‐p22phox prevented apoptosis upon radiation‐induced CRP. CRP overexpression reduced the amount of STAT3/Ref‐1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref‐1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co‐overexpressed with Ref‐1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP‐Ref‐1 complex. Immunofluorescence showed co‐localization of CRP, Ref‐1 and p53 with α‐actin‐positive VSMC in human atherosclerotic plaques. In conclusion, radiation‐induced CRP increased the VSMCs apoptosis through Ref‐1, which dissociated the STAT3/Ref‐1 complex, interfered with JAK/STAT3 activity, and interacted with CRP‐Ref‐1, thus resulting in transcription‐independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.

Downregulation of linc00961 contributes to promote proliferation and inhibit apoptosis of vascular smooth muscle cell by sponging miR-367 in patients with coronary heart disease.

Atherosclerosis is one of the most important risk factors for coronary heart disease (CHD), and growing evidence has shown that long non-coding RNAs (lncRNAs) can serve as prospective markers for atherosclerosis. In this study, we mainly focused on the potential roles of linc00961 in CHD patients. qRT-PCR was used to detect the expressions of linc00961 and miR-367 in CHD patients and ApoE-/-mice, and the correlations were analyzed. Then, HA-VAMC was respectively treated with 5 inflammatory factors and hypoxia conditions to explore the factors that affect linc00961 levels. Furthermore, the linc00961 overexpression lentivirus (LV-linc00961) and linc00961 downregulation lentivirus (LV-sh linc00961) were purchased and transfected into human vascular smooth muscle cells (VSMCs). CCK8 assay was carried out to measure the cell proliferation of VSMC, and the levels of Cyclin D1, Bcl-2, Bax, and cleaved caspase-3 were detected by RT-PCR and Western blot. Moreover, the Luciferase assay was performed to explore the binding site of linc00961 and miR-367. Finally, the miR-367 inhibitor was transfected into LV-sh linc00961 VSMCs to confirm the linc00961 functions via miR-367. We found that linc00961 was significantly decreased in patients with CHD and ApoE-/-mice. Additionally, linc00961 was reduced in VSMCs at the conditions of hypoxia and C-reactive protein (CRP). Most importantly, the overexpression of linc00961 significantly inhibited the VSMCs proliferation, repressed the levels of Cyclin D1 and Bcl-2, and increased the levels of Bax and cleaved caspase-3. However, the downregulation of linc00961 promoted VSMCs proliferation, increased the levels of Cyclin D1 and Bcl-2, and repressed the levels of Bax and cleaved caspase-3. We also found that miR-367 was downregulated following the upregulation of linc00961, while it was upregulated following the downregulation of linc00961. The Luciferase gene reporter assay indicated that linc00961 could directly bind with miR-367 in VSMCs. Finally, we found that linc00961 could inhibit proliferation and promote apoptosis of VSMCs via binding with miR-367. According to the results, our study revealed that linc00961 was significantly decreased in patients with CHD and ApoE-/-mice. Furthermore, our findings firstly uncovered that linc00961 was reduced by hypoxia and CRP in VSMCs. The downregulation of linc00961 contributed to promote proliferation and inhibit apoptosis of VSMCs by sponging miR-367 in CHD patients, which might provide a potential target for treating atherosclerosis.

Eplerenone inhibits aldosterone-induced CRP generation in rat vascular smooth muscle cells by regulating the MR-ROS-ERK1/2 signal pathway

Atherosclerosis is a chronic inflammatory disease in the vessel wall. As a representative inflammatory cytokine, C-reactive protein (CRP) participates in the formation and development of atherosclerosis. It is demonstrated that aldosterone induces CRP generation in vascular smooth muscle cells (VSMCs). This study explored the inhibitory effect of eplerenone on aldosterone-induced CRP expression in VSMCs and mechanism. In the in vitro experiments, rat VSMCs were cultured and aldosterone (10 nM) was used as a stimulant for CRP generation. VSMCs were pretreated with eplerenone for 1 h prior to the stimulation. Messenger RNA (mRNA) and protein expression were identified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot, respectively. The production of reactive oxygen species (ROS) was observed by a fluorescence microscope. In the in vivo experiment, a model of hyperaldosteronism was established by the subcutaneous administration of aldosterone to rats with the osmotic minipumps for 4 weeks. Serum aldosterone and CRP levels were determined with a radioimmunoassay and ELISA (enzyme-linked immunosorbent assay), respectively. The results showed that eplerenone inhibited aldosterone-induced mRNA and protein expression of CRP in VSMCs in vitro and in vivo, and decreased the circulating CRP level of hyperaldosteronism rats. Meanwhile, eplerenone reduced aldosterone-stimulated ROS generation and aldosterone-activated ERK1/2 phosphorylation in VSMCs. In summary, eplerenone inhibits aldosterone-induced CRP generation in VSMCs by regulating the MR-ROS-ERK1/2 signal pathway. These results provide new evidence for the potential anti-inflammatory effect of eplerenone.

Homocysteine induces the expression of C-reactive protein via NMDAr-ROS-MAPK-NF-κB signal pathway in rat vascular smooth muscle cells

ObjectiveHomocysteine (Hcy) is known as an independent risk factor for atherosclerosis. C-reactive protein (CRP) directly participates in initiation and progression of atherosclerosis. However, there is no direct evidence to demonstrate pro-inflammatory effect of Hcy on vascular smooth muscle cells (VSMCs) through CRP. In the present study, we examined the effect of Hcy on CRP expression and investigated the related mechanism in VSMCs. Methods and resultsProtein expression and secretion were detected by Western blot and ELISA, respectively. mRNA expression was detected by RT-PCR. Superoxide anion was detected by lucigenin chemiluminometry and the immunofluorescence staining was observed by a fluorescence microscope. The results revealed that Hcy significantly induced mRNA and protein expressions of CRP in VSMCs both in vitro and in vivo, and anti-IL-1β or anti-IL-6 neutralizing antibody alone or in combination partially reduced Hcy-induced CRP expression. Hcy increased the expression of NR1 subunit of N-methyl-d-aspartate receptor (NMDAr), and MK-801 alleviated Hcy-induced CRP expression in VSMCs. Further studies showed that Hcy-stimulated superoxide anion generation in VSMCs. Nevertheless, pretreatment of the cells with MK-801, TTFA and DPI significantly reduced Hcy-stimulated superoxide anion generation, and antioxidant NAC decreased Hcy-induced CRP expression in VSMCs. Additionally, PD98059, SB205380 or PDTC antagonized Hcy-induced CRP expression, and MK-801, NAC, PD98059 or SB205380 inhibited Hcy-activated phosphorylations of ERK1/2 and p38. ConclusionThe present study demonstrates that Hcy is able to initiate an inflammatory response in VSMCs by stimulating CRP production, which is mediated through NMDAr-ROS-ERK1/2/p38-NF-κB signal pathway. These findings provide new evidence for a role of Hcy in pathogenesis of atherosclerosis.

Pravastatin inhibits C-reactive protein generation induced by fibrinogen, fibrin and FDP in isolated rat vascular smooth muscle cells

The available evidence indicates that C-reactive protein (CRP) participates directly in atherosclerosis formation as an inflammatory molecule. Our previous investigation suggested that fibrinogen, fibrin and fibrinogen degradation products (FDP) produce a pro-inflammatory effect on vascular smooth muscle cells (VSMCs) through inducing CRP generation. In the present study, we observed the effect of pravastatin on CRP generation induced by fibrinogen, fibrin and FDP in rat VSMCs. VSMCs from Sprague-Dawley rats were cultured. Fibrinogen, fibrin and FDP were used as stimulants for CRP generation. VSMCs were preincubated with pravastatin at 10, 30, 100μM for 30min prior to stimulation. CRP mRNA expression was studied by reverse transcription polymerase chain reaction (RT-PCR). CRP levels in the supernatant of VSMCs were measured by enzyme-linked immunosorbent assay (ELISA). CRP expression in VSMCs was examined with immunocytochemical staining. ELISA analysis showed that the pravastatin concentration-dependently reduced fibrinogen-, fibrin- and FDP-stimulated generation of CRP in VSMCs, with maximal inhibition of 56.6, 55.7 and 62.3%, respectively. Immunocytochemical staining and RT-PCR revealed that pravastatin inhibited protein and mRNA expression of CRP in VSMCs significantly. Pravastatin at the concentrations used in the present experiment has ability to relieve vascular inflammation and to restrain atherosclerotic processes via inhibiting the CRP production induced by fibrinogen, fibrin and FDP in VSMCs, which helps explain the beneficial effects of pravastatin on atherosclerosis.

C-Reactive Protein Induces TNF-α Secretion by p38 MAPK–TLR4 Signal Pathway in Rat Vascular Smooth Muscle Cells

Atherosclerosis is a chronic inflammatory disease. C-reactive protein (CRP) not only is an inflammatory marker but also regulates the expressions of other inflammatory cytokines associated with the pathogenesis of atherosclerosis. Toll-like receptor 4 (TLR4) also contributes to atherogenesis via transducting inflammatory signals. Herein, our studies focused on characterizing the effect of CRP on tumor necrosis factor α (TNF-α) production and TLR4-related molecular mechanisms in rat vascular smooth muscle cells (VSMCs). The results showed that CRP stimulated VSMCs to secrete TNF-α and enhanced TLR4 expression in a time-concentration-dependent manner. TLR4 knockdown significantly inhibited CRP-induced TNF-α generation, and p38 mitogen-activated protein kinase (MAPK) blocker SB203580 depressed TLR4 expression and TNF-α production initiated by CRP in VSMCs. The data demonstrate that CRP triggers an inflammatory response in rat VSMCs by inducing TNF-α secretion, which is mediated by p38 MAPK-TLR4 signaling pathway.

(-)-Epigallocatechin Gallate Inhibits Endothelin-1-Induced C-Reactive Protein Production in Vascular Smooth Muscle Cells

Numerous studies have shown that C-reactive protein (CRP), a pro-inflammation cytokine, makes a direct contribution to atherosclerosis, and that (-)-epigallocatechin gallate (EGCG) is able to exert an anti-atherosclerotic effect by anti-oxidative and anti-inflammatory activities. Based on our previous study, the effect of EGCG on endothelin-1 (ET-1)-induced CRP production in rat vascular smooth muscle cells (VSMCs) and the possible mechanism were observed. The in vitro experiments showed that EGCG concentration-dependently inhibited ET-1-stimulated expression of CRP both in protein and mRNA levels in VSMCs as determined by the immunocytochemical staining, the enzyme-linked immunosorbent assay and the real-time quantitative polymerase chain reaction (RT-qPCR). The in vivo investigation with the double-labelled immunofluorescence staining and RT-qPCR displayed that EGCG also prevented ET-1-induced CRP expression in protein and mRNA levels in the aortic VSMCs of rats receiving the subchronic infusion of ET-1. In addition, EGCG suppressed reactive oxygen species (ROS) generation evoked by ET-1 in VSMCs as observed by the fluorescence probe. These demonstrate that EGCG may inhibit ET-1-stimulated generation of CRP in VSMCs so to relieve the inflammatory response and oxidative stress via blocking ROS signal, which provides new evidence for an anti-atherosclerotic effect of EGCG.