Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture

Aaron James, Jia Shen, Kevork Khadarian, Shen Pang, Greg Chung, Raghav Goyal, Greg Asatrian, Omar Velasco, Jung Kim, Xinli Zhang, Kang Ting, Chia Soo

Research output: Contribution to journalArticle

Abstract

Introduction: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPARγ) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARγ shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.

Methods: PPARγ shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPARγ shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPARγ, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).

Results: In vitro, PPARγ shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPARγ shRNA treatment.

Discussion: These findings suggest that acute loss of PPARγ in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARγ shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARγ shRNA on OB and OC cell types, and it may help determine whether PPARγ antagonists are potential therapeutic agents for osteoporotic bone loss.

Original languageEnglish (US)
Pages (from-to)2699-2710
Number of pages12
JournalTissue Engineering - Part A
Volume20
Issue number19-20
DOIs
StatePublished - 2014
Externally publishedYes

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Adipogenesis
PPAR gamma
Osteogenesis
Small Interfering RNA
Bone
Bone and Bones
Osteoblasts
Osteoclasts
Phosphatases
Bone Marrow
Adipocytes
X-Ray Microtomography
Lentivirus
Osteocalcin
Acid Phosphatase
Mesenchymal Stromal Cells
Tomography
Peroxisome Proliferator-Activated Receptors
Cathepsin K
Fatty Acid-Binding Proteins

ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • Biomaterials
  • Biomedical Engineering

Cite this

Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture. / James, Aaron; Shen, Jia; Khadarian, Kevork; Pang, Shen; Chung, Greg; Goyal, Raghav; Asatrian, Greg; Velasco, Omar; Kim, Jung; Zhang, Xinli; Ting, Kang; Soo, Chia.

In: Tissue Engineering - Part A, Vol. 20, No. 19-20, 2014, p. 2699-2710.

Research output: Contribution to journalArticle

James, A, Shen, J, Khadarian, K, Pang, S, Chung, G, Goyal, R, Asatrian, G, Velasco, O, Kim, J, Zhang, X, Ting, K & Soo, C 2014, 'Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture', Tissue Engineering - Part A, vol. 20, no. 19-20, pp. 2699-2710. https://doi.org/10.1089/ten.tea.2013.0736
James, Aaron ; Shen, Jia ; Khadarian, Kevork ; Pang, Shen ; Chung, Greg ; Goyal, Raghav ; Asatrian, Greg ; Velasco, Omar ; Kim, Jung ; Zhang, Xinli ; Ting, Kang ; Soo, Chia. / Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture. In: Tissue Engineering - Part A. 2014 ; Vol. 20, No. 19-20. pp. 2699-2710.
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T1 - Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture

AU - James, Aaron

AU - Shen, Jia

AU - Khadarian, Kevork

AU - Pang, Shen

AU - Chung, Greg

AU - Goyal, Raghav

AU - Asatrian, Greg

AU - Velasco, Omar

AU - Kim, Jung

AU - Zhang, Xinli

AU - Ting, Kang

AU - Soo, Chia

PY - 2014

Y1 - 2014

N2 - Introduction: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPARγ) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARγ shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.Methods: PPARγ shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPARγ shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPARγ, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).Results: In vitro, PPARγ shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPARγ shRNA treatment.Discussion: These findings suggest that acute loss of PPARγ in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARγ shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARγ shRNA on OB and OC cell types, and it may help determine whether PPARγ antagonists are potential therapeutic agents for osteoporotic bone loss.

AB - Introduction: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPARγ) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARγ shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.Methods: PPARγ shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPARγ shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPARγ, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).Results: In vitro, PPARγ shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPARγ shRNA treatment.Discussion: These findings suggest that acute loss of PPARγ in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARγ shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARγ shRNA on OB and OC cell types, and it may help determine whether PPARγ antagonists are potential therapeutic agents for osteoporotic bone loss.

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