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Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo.

ABSTRACT: Aseptic loosening is a significant impediment to joint implant longevity. Prosthetic wear particles are postulated to play a central role in the onset and progression of periprosthetic osteolysis, leading to aseptic loosening of the prosthesis.We investigated the inhibitory effects of a lentivirus-mediated short hairpin RNA that targets the TNF-alpha gene on the particle-induced inflammatory and osteolytic changes via macrophages both in vitro and in vivo. An siRNA sequence targeting the mouse TNF-alpha gene from four candidates, transcribed in vitro, was screened and identified. A lentivirus vector expressing short hairpin RNA (shRNA) was then constructed in order to facilitate efficient expression of TNF-alpha-siRNA. Lentivirus-mediated shRNA was transduced into cells of the mouse macrophage line RAW 264.7. Ceramic and titanium particles were introduced 24 h after lentivirus transduction to stimulate cells. TNF-alpha expression, represented by both mRNA and protein levels, was quantified with real-time PCR and ELISA at all time intervals. Lentivirus-mediated shRNA suspension was locally administered into the murine calvarial model, followed by local injection of particles. A multi-slice spiral CT scan was used to evaluate the osteolysis of the calvaria by detecting the width of the cranial sutures.Macrophages developed pseudopods when co-cultured with particles. Lentivirus-mediated shRNA was shown to effectively inhibit the expression of TNF-alpha at both the mRNA and protein levels in RAW 264.7. The multi-slice spiral CT scan showed that the lentivirus-mediated shRNA significantly suppressed osteolysis of mouse calvaria.Our investigation highlighted the results that lentivirus-mediated shRNA targeting the TNF-alpha gene successfully inhibited particle-induced inflammatory and osteolytic changes both in vitro and in vivo. Therefore, lentivirus-mediated gene therapy may provide a novel therapeutic approach to aseptic joint loosening.

SUBMITTER: Qin CQ

PROVIDER: S-EPMC5069821 | biostudies-other | 2016 Oct

REPOSITORIES: biostudies-other

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Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo.

Qin Chu-Qiang CQ Huang Dong-Sheng DS Zhang Chi C Song Bin B Huang Jian-Bin JB Ding Yue Y

BMC musculoskeletal disorders 20161018 1

<h4>Background</h4>Aseptic loosening is a significant impediment to joint implant longevity. Prosthetic wear particles are postulated to play a central role in the onset and progression of periprosthetic osteolysis, leading to aseptic loosening of the prosthesis.<h4>Methods</h4>We investigated the inhibitory effects of a lentivirus-mediated short hairpin RNA that targets the TNF-alpha gene on the particle-induced inflammatory and osteolytic changes via macrophages both in vitro and in vivo. An s ...[more]

PMID: 27756280

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Project description:During hypoxia, upregulation of hypoxia inducible factor-1 alpha transcriptional factor can activate several downstream angiogenic genes. However, hypoxia inducible factor-1 alpha is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging.PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter followed by a separate hypoxia response element-incorporated promoter driving a firefly luciferase reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared with the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of hypoxia inducible factor-1 alpha protein and several downstream angiogenic genes by >30% (P<0.01). Afterward, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in mice. Animals were randomized into shPHD2 experimental group (n=25) versus shScramble control group (n=20). Bioluminescence imaging detected plasmid-mediated transgene expression for 4 to 5 weeks. Echocardiography showed the shPHD2 group had improved fractional shortening compared with the shScramble group at Week 4 (33.7%+/-1.9% versus 28.4%+/-2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of hypoxia inducible factor-1 alpha protein.This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility by in vitro and in vivo experiments. With further validation, the combination of shRNA therapy and molecular imaging can be used to track novel cardiovascular gene therapy applications in the future.

Project description:Small wear particles (0.1-10 ?m) in total joint replacement are generally considered as the major causative agent leading to periprosthetic inflammation and osteolysis. However, little is known about the roles of larger wear particles (10-100 ?m) in periprosthetic inflammation and osteolysis. Additionally, although ample studies demonstrated that increased oxidative stress is critically involved in particle-induced inflammation and osteolysis, detailed changes in antioxidant enzymes expression in the disease development remain largely unclear. Herein, we used a rat knee prosthesis model to assess effects of polyethylene (PE) particles (20-60 ?m) on the levels of oxidative stress markers such as malondialdehyde (MDA) and total antioxidant capacity (TAC) in blood plasma, and on the expression profiles of antioxidant enzymes in knee joint tissues. In combination with a forced-exercise intervention for all surgical rats, we found that the rat groups treated with both artificial joint and PE particles exhibited higher MDA levels and lower TAC levels, together with lower levels of physical activity and higher levels of inflammatory markers, than the sham group and the groups receiving artificial joint or PE particles alone at weeks 20-24 post-operatively. Dose-response relationships between the exposure to PE particles and the induction of oxidative stress and inflammation were also observed in the artificial joint/PE groups. Under such conditions, we unexpectedly found that most of antioxidant enzymes displayed pronounced up-regulation, with concomitant induction of inflammatory and osteoclast-inducing factors (including IL-1?, NF-?B and RANKL), in the artificial joint/PE groups as compared to the sham, artificial joint only, or PE only group. Only a few antioxidant enzymes including SOD2 and GPx2 showed down-regulation. Collectively, our findings demonstrate that implantation of artificial joint along with large PE particles synergistically trigger the induction of oxidative stress; however, down-regulation of many antioxidant enzymes may not necessarily occur during the disease development.

Dataset Information

Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo.

Publications

Lentivirus-mediated short hairpin RNA interference targeting TNF-alpha in macrophages inhibits particle-induced inflammation and osteolysis in vitro and in vivo.

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