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Molecular mechanisms mediating development of pulmonary cachexia in COPD
Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cigarette smoking (CS) represents the main causative agent underlying development and progress of COPD. Recently, involvement of CS in the pathogenesis of COPDassociated muscle abnormalities is becoming increasingly evident. Nevertheless, involved triggers and underlying mechanisms remain largely unknown. This study was conceived in order to examine effects of cigarette smoke exposure on skeletal muscle morphology, vascular supply and function. For this purpose, we have specifically designed murine COPD/emphysema model and gastrocnemius muscle was examined, while in vitro experiments were conducted using murine C2C12 skeletal muscle myocytes.

In addition to the mild emphysematous changes present in the lungs of CS-exposed mice, our results demonstrated evident signs of muscle atrophy reflected by decreased fiber cross-sectional area, profound fiber size variation and reduced body mass. Furthermore, we have observed impairment in terminal myogenesis and lower number of myonuclei in skeletal muscles of CS-exposed animals despite evident activation of muscle repair process. Additionally, our results demonstrate capillary rarefaction in skeletal muscles of CS-exposed animals which was associated with deregulation of hypoxia-angiogenesis signaling, reduced levels of angiogenic factors such as HIF1-α and VEGF and enhanced expression of VHL and its partner proteins PHD2 and Ube2D1. The results of our in-vitro experiments demonstrated that VHL and its ubiquitination machinery can be synergistically regulated by TNF and hypoxia consequentially impairing angiogenic potential of skeletal muscle myocytes. Finally, we have shown that CS elicits chronic ER stress in murine skeletal muscles which is associated with activation of ERAD and apoptotic pathways as mirrored by elevated expression of Usp19, caspase 12 and caspase 3 in skeletal muscles of CSexposed animals. Moreover, molecular and morphological alterations in CS-exposed mice resulted in impairment of muscle function as reflected by their impaired exercise capacity.

Taken together, from our results it is evident that cigarette smoke exposure elicits set of morphological, vascular and functional changes highly resembling those observed in COPD. Additionally, CS induces wide range of molecular alterations and signaling pathway deregulations suggesting profound effects of cigarette smoke exposure on skeletal muscle cell homeostasis.

Place, publisher, year, edition, pages
Örebro: Örebro university , 2014. , 76 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 107
Keyword [en]
COPD, cachexia, atrophy, cigarette smoke, myogenesis, angiogenesis
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine; Biochemistry
Identifiers
URN: urn:nbn:se:oru:diva-36104ISBN: 978-91-7529-031-7 (print)OAI: oai:DiVA.org:oru-36104DiVA: diva2:740447
Public defence
2014-09-16, Universitetssjukhuset, hörsal C2, Södra Grev Rosengatan, Örebro, 09:15 (English)
Opponent
Available from: 2014-08-25 Created: 2014-08-25 Last updated: 2017-10-17Bibliographically approved
List of papers
1. Exposure to cigarette smoke induces overexpression of von Hippel-Lindau tumor suppressor in mouse skeletal muscle
Open this publication in new window or tab >>Exposure to cigarette smoke induces overexpression of von Hippel-Lindau tumor suppressor in mouse skeletal muscle
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2012 (English)In: American Journal of Physiology - Lung cellular and Molecular Physiology, ISSN 1040-0605, E-ISSN 1522-1504, Vol. 303, no 6, L519-L527 p.Article in journal (Refereed) Published
Abstract [en]

Cigarette smoke (CS) is a well established risk factor in the development of chronic obstructive pulmonary disease (COPD). In contrast, the extent to which CS exposure contributes to the development of the systemic manifestations of COPD, such as skeletal muscle dysfunction and wasting remains largely unknown. Decreased skeletal muscle capillarization has been previously reported in early stages of COPD and might play an important role in the development of COPD-associated skeletal muscle abnormalities. To investigate the effects of chronic CS exposure on skeletal muscle capillarization and exercise tolerance a mouse model of CS exposure was used. The129/SvJ mice were exposed to CS for 6 months, and the expression of putative elements of the hypoxia-angiogenic signaling cascade as well as muscle capillarization were studied. Additionally, functional tests assessing exercise tolerance/endurance were performed in mice. Compared to controls, skeletal muscles from CS-exposed mice exhibited significantly enhanced expression of von Hippel-Lindau tumor suppressor (VHL), ubiquitin-conjugating enzyme E2D1 (UBE2D1) and prolyl hydroxylase-2 (PHD2). In contrast, hypoxia-inducible factor-1 (HIF1-α) and vascular endothelial growth factor (VEGF) expression was reduced. Furthermore, reduced muscle fiber cross-sectional area, decreased skeletal muscle capillarization, and reduced exercise tolerance were also observed in CS-exposed animals. Taken together, the current results provide evidence linking chronic CS exposure and induction of VHL expression in skeletal muscles leading towards impaired hypoxia-angiogenesis signal transduction, reduced muscle fiber cross-sectional area and decreased exercise tolerance.

Place, publisher, year, edition, pages
Bethesda, USA: American Physiological Society, 2012
Keyword
Capillaries, chronic obstructive pulmonary disease, hypoxia inducible factor-1 alpha, pulmonary cachexia syndrome, vascular endothelial growth factor
National Category
Medical and Health Sciences Physiotherapy
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-24177 (URN)10.1152/ajplung.00007.2012 (DOI)000309109300005 ()22842216 (PubMedID)2-s2.0-84866418091 (Scopus ID)
Funder
NIH (National Institute of Health)
Note

Funding Agencies:

Olle Engkvist Byggmastare Fund, Sweden

NIEHS Environmental Health Science Center grant 

Available from: 2012-07-31 Created: 2012-07-31 Last updated: 2017-12-07Bibliographically approved
2. Chronic cigarette smoke exposureimpairs skeletal muscle regenerative capacity in murineCOPD/emphysema model.
Open this publication in new window or tab >>Chronic cigarette smoke exposureimpairs skeletal muscle regenerative capacity in murineCOPD/emphysema model.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Cigarette smoke (CS) is a well established risk factor in the development of COPD and irreversible airflow limitation. In contrast, the extent to which CS exposure contributes to development of peripheral skeletal muscle dysfunction and wasting remains largely unknown. Decline in skeletal muscle regenerative capacity has been previously reported in COPD patients.

Methods: To investigate effects of chronic CS exposure on skeletal muscle regenerative capacity, 129/SvJ mice were exposed to CS for 6 months. The expression levels of myogenin, Jarid2, Znf496, Notch1, Pax7, Fgf1 and Myh3, which are known to regulate skeletal muscle myogenesis, were studied. Additionally, number of fibers with central nuclei, myonuclei number and mean fiber cross-sectional area were assessed.

Results: Compared to controls, skeletal muscles from CS-exposed mice exhibited significantly decreased expression of Jarid2, coupled with enhanced expression of Znf496, Notch1, Pax7, Fgf1 and Myh3. Expression of myogenin, a marker of terminally differentiated myofibers, was reduced. Furthermore, reduced muscle fiber crosssectional area, increased number of fibers with central nuclei and reduced myonuclei number were also observed in CS-exposed animals.

Conclusions: Taken together, current results provide evidence linking chronic CS exposure and an ongoing damage/repair process as well as impaired regenerative capacity in skeletal muscles of CS-exposed mice.

Keyword
cigarette smoke, chronic obstructive pulmonary disease, skeletal muscle dysfunction, skeletal muscle regeneration
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:oru:diva-38189 (URN)
Note

Funding and support:

Olle Engkvist Byggmästare Fund,

Åke WibergFoundation, Sweden

and the research funds of the Department of Medicine,Danderyd Hospital, Stockholm(to S.M.A-H),

Örebro university grant to doctoralsstudents (We thank the Developmental Studies Hybridoma Bank (DSHB, Universityof Iowa, IA, USA)) for antibody against Pax7

Available from: 2014-10-27 Created: 2014-10-27 Last updated: 2017-10-17Bibliographically approved
3. TNF stimulation induces VHL overexpression and impairs angiogenic potential in skeletal muscle myocytes
Open this publication in new window or tab >>TNF stimulation induces VHL overexpression and impairs angiogenic potential in skeletal muscle myocytes
2014 (English)In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 34, no 1, 228-236 p.Article in journal (Refereed) Published
Abstract [en]

Decreased skeletal muscle capillarization is considered to significantly contribute to the development of pulmonary cachexia syndrome (PCS) and progressive muscle wasting in several chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). It is unclear to which extent the concurrent presence of systemic inflammation contributes to decreased skeletal muscle capillarization under these conditions. The present study was designed to examine in vitro the effects of the pro-inflammatory cytokine, tumor necrosis factor (TNF), on the regulation of hypoxia-angiogenesis signal transduction and capillarization in skeletal muscles. For this purpose, fully differentiated C2C12 skeletal muscle myocytes were stimulated with TNF and maintained under normoxic or hypoxic conditions. The expression levels of the putative elements of the hypoxia-angiogenesis signaling cascade were examined using qPCR, western blot analysis and immunofluorescence. Under normoxic conditinos, TNF stimulation increased the protein expression of anti-angiogenic von-Hippel Lindau (VHL), prolyl hydroxylase (PHD)2 and ubiquitin conjugating enzyme 2D1 (Ube2D1), as well as the total ubiquitin content in the skeletal muscle myocytes. By contrast, the expression levels of hypoxia-inducible factor 1‑α (HIF1-α) and those of its transcriptional targets, vascular endothelial growth factor (VEGF)A and glucose transporter 1 (Glut1), were markedly reduced. In addition, hypoxia increased the expression of the VHL transcript and further elevated the VHL protein expression levels in C2C12 myocytes following TNF stimulation. Consequently, an impaired angiogenic potential was observed in the TNF-stimulated myocytes during hypoxia. In conclusion, TNF increases VHL expression and disturbs hypoxia-angiogenesis signal transduction in skeletal muscle myocytes. The current findings provide a mechanism linking systemic inflammation and impaired angiogenesis in skeletal muscle. This is particularly relevant to further understanding the mechanisms mediating muscle wasting and cachexia in patients with chronic inflammatory diseases, such as COPD.

Place, publisher, year, edition, pages
Spandidos Publications, 2014
National Category
Medical Biotechnology
Research subject
Medical Disability Research
Identifiers
urn:nbn:se:oru:diva-35141 (URN)10.3892/ijmm.2014.1776 (DOI)000338178000027 ()24820910 (PubMedID)2-s2.0-84902649801 (Scopus ID)
Available from: 2014-05-25 Created: 2014-05-25 Last updated: 2017-12-05Bibliographically approved
4. Cigarette smoke exposure up-regulates Ubiquitin specific protease 19 in murine skeletal muscles as an adaptive response to prolonged ER stress
Open this publication in new window or tab >>Cigarette smoke exposure up-regulates Ubiquitin specific protease 19 in murine skeletal muscles as an adaptive response to prolonged ER stress
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Enhanced protein degradation via ubiquitin proteolytic system (UPS) was demonstrated to play an important role in the pathogenesis of cachexia syndrome and muscle wasting in patients with COPD and animal models of the disease. The role of cigarette smoke (CS) exposure in eliciting these abnormalities remains largely unknown. Usp19 is a member of UPS suggested to be involved in progressive muscle wasting in different catabolic conditions. However, factors regulating Usp19 expression, activity and correlation/s with CS-induced muscle atrophy remainunclear.

Methods: To address these questions, 129 SvJ mice were exposed to cigarette smoke for 6 months and the gastrocnemius muscles were collected. Expression levels of Usp19 as well as pivotal mediators of ER stress response have been studied using PCR, qPCR, western blot and immunofluorescence. Factors regulating muscle Usp19 expression were studied using in-silico analysis of Usp19 promoter as well as by stimulating C2C12 myocytes with different inducers of ER stress including hypoxia, TNF and tunicamycin. Finally, Usp19 expression was depleted in C2C12 myocytes using specific Usp19 siRNA quadriplex and the expression of pivotal myogenic regulators were analyzed.

Results: Usp19 mRNA expression was enhanced in skeletal muscles of CS-exposed mice. Concurrently, ER stress-associated Caspase 12 and Caspase 3 were activated in the CS-exposed group. Analysis of Usp19 promoter sequence revealed binding sites for ER stress response transcription factors such as HSF, STRE1 and AML1-α. Exposure of C2C12 myocytes to tunicamycin but not hypoxia elevated expression levels of Usp19. TNFstimulation elevated Usp19 protein expression but inhibited its RNA transcription in a dose- and time-dependent manner. Finally, Usp19 overexpression in tunicamycin-treated myocytes was accompanied by reduced expression of myosin heavy chain and tropomyosin and their levels were increased after knocking down Usp19 in C2C12 myocytes.

Conclusions: In summary, our data demonstrated elevated expression of Usp19 in skeletal muscles of CS-exposed 129 SvJ mice. Moreover, Usp19 overexpression was associated with muscle adaptations to ER stress and suppression of myogenesis. Taken together; our results might provide further insight into molecular mechanisms underlying development and progression of skeletal muscle abnormalities in response to chronic cigarette smoke exposure.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-38194 (URN)
Available from: 2014-10-27 Created: 2014-10-27 Last updated: 2017-10-17Bibliographically approved
5. The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation
Open this publication in new window or tab >>The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation
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2013 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 14, 770- p.Article in journal (Refereed) Published
Abstract [en]

Background: Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function.

Results: AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change >|1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways.

Conclusions: This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.

Keyword
Porphyromonas gingivalis, Aortic smooth muscle cells, Proliferation, Gene expression profiling
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-33287 (URN)10.1186/1471-2164-14-770 (DOI)000328639800002 ()24209892 (PubMedID)2-s2.0-84887327838 (Scopus ID)
Funder
Swedish Research Council, 2008-2459Swedish Heart Lung Foundation, 2011-0632
Note

Funding Agency: Foundation of Olle Engkvist; Foundation of Mats Kleberg (se även Forskningsfinansiär)

Available from: 2014-01-24 Created: 2014-01-24 Last updated: 2017-12-06Bibliographically approved

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