Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May 28;8(5):e63825.
doi: 10.1371/journal.pone.0063825. Print 2013.

Global gene expression profiling in PAI-1 knockout murine heart and kidney: molecular basis of cardiac-selective fibrosis

Asish K Ghosh  1 , Sheila B Murphy, Raj Kishore, Douglas E Vaughan
Affiliations

Global gene expression profiling in PAI-1 knockout murine heart and kidney: molecular basis of cardiac-selective fibrosis

Asish K Ghosh et al. PLoS One. .

Abstract

Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1 (PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and several other biological processes. The significance of these observations in the light of heart-specific profibrotic signaling and fibrogenesis are discussed.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Raj Kishore is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Relationship among and variability of samples.
Hierarchical clustering and multi-dimensional scaling analyses were performed. The MvA plots showing the small variation in A. WT/HT vs. KO/HT and B. WT/KID vs. KO/KID, and large variation in C. KO/HT vs. KO/KID and D. WT/HT vs. WT/KID.
Figure 2
Figure 2. PAI-1 deficiency is associated with fibrosis in hearts but not in kidneys.
The levels of collagen accumulation in cardiac and renal tissues derived from wildtype and PAI-1 knockout mice were determined by Masson’s trichrome staining. Photographs were taken by an Olympus DP71 camera. A. 12-month old WT/HT; B. 12-month old PAI-1 KO/HT; C. 24-month old WT/HT; D. 24-month old PAI-1 KO/HT; E. 12-month old WT/KID, F. 12-month old PAI-1 KO/KID, G. 24-month old WT/KID, H. 24-month old PAI-1 KO/KID.
Figure 3
Figure 3. Effect of PAI-1 deficiency on differential global gene expression profiling in hearts and kidneys.
Differentially expressed genes are presented by volcano plots with fold induction or repression and statistical significance in four different groups. A. Expression levels of genes in wildtype and PAI-1 knockout hearts. B. Expression levels of genes in wildtype and PAI-1 knockout kidneys. C. Expression levels of genes in PAI-1 knockout hearts and kidneys. D. Expression levels of genes in wildtype hearts and kidneys. E. Venn diagram showing the differentially expressed genes which are common in PAI-1 KO/HT vs. PAI-1 KO/KID group and WT/HT vs. WT/KID group (Blue); and unique in PAI-1 KO/HT vs. KO/KID group (Yellow) and WT/HT vs. WT/KID group (Red).
Figure 4
Figure 4. Biological processes representing genes that were differentially expressed in wildtype and PAI-1 knockout heart and kidney groups.
Percentages of differentially expressed genes (upregulated or downregulated) under different biological processes are shown by Pie charts. A. WT/HT vs. PAI-1 KO/HT; B. WT/KID vs. PAI-1 KO/KID; C. PAI-1 KO/HT vs. PAI-1 KO/KID and D. PAI-1 KO/HT vs. PAI-1 KO/KID (unique); E. WT/HT vs. WT/KID; F. WT/HT vs. WT/KID (unique). Numbers of deregulated genes are shown in parentheses.
Figure 5
Figure 5. Validation of expression levels of mRNAs in aged PAI-1 knockout and wildtype hearts and kidneys by qPCR analysis.
Total RNA from wildtype and PAI-1 knockout heart and kidney tissues were used for quantitation of A. Ankrd1; B. Pi16; C. Egr1: D. Dbp and E. Timp1 by qPCR analysis using gene specific primers. Data represents mean of triplicates ±SEM. P value of each sample was indicated in the Figure A–E.
Figure 6
Figure 6. Levels of Ankrd1, Pi16, Timp4 and Timp1 in TGF-β-treated endothelial cells.
Mouse cardiac endothelial cells were treated with TGF-ß2 (10 ng/ml) for 7 days. At the end of incubation, control and EndMT-derived fibroblast-like cells were harvested and whole lysates were prepared. Equal amounts of proteins were subjected to Western blot using specific antibodies as indicated.
Figure 7
Figure 7. Schematic diagram showing possible involvement of Ankrd1-Egr1 axis and a subset of profibrotic factors as igniters of cardiac fibrosis.
Along with other key profibrogenic factors, Ankrd1-Egr1 axis may play a pivotal role in initiation of cardiac-selective fibrosis; and kidney is protected from PAI-1 deficiency-induced fibrogenesis due to lack of these profibrotic regulators. Antifibrotic Klotho may also protect kidney from fibrogenesis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wynn TA (2007) Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 117: 524–529. - PMC - PubMed
    1. Wynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214: 199–210. - PMC - PubMed
    1. Czubryt MP (2012) Common threads in cardiac fibrosis, infarct scar formation, and wound healing. Fibrogenesis Tissue Repair 5: 19. - PMC - PubMed
    1. Hu B, Phan SH (2013) Myofibroblasts. Curr Opin Rheumatol 25: 71–77. - PMC - PubMed
    1. Ghosh AK, Vaughan DE (2012) PAI-1 in Tissue Fibrosis. J Cell Physiol 227: 493–507. - PMC - PubMed

Publication types

MeSH terms

Substances