Limits...
Fibulin-4 deficiency increases TGF-β signalling in aortic smooth muscle cells due to elevated TGF-β2 levels.

Ramnath NW, Hawinkels LJ, van Heijningen PM, te Riet L, Paauwe M, Vermeij M, Danser AH, Kanaar R, ten Dijke P, Essers J - Sci Rep (2015)

Bottom Line: Fibulins are extracellular matrix proteins associated with elastic fibres.These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels.TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-β signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-β pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-β neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-β signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-β isoforms. These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels. Significantly increased TGF-β2 levels were also detectable in plasma from homozygous Fibulin-4(R/R) mice, not in wild type mice. TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-β signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-β1, but especially TGF-β2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-β pathway regulation in the pathogenesis of aortic aneurysms.

No MeSH data available.


Related in: MedlinePlus

Strong increase of TGF-β2 levels in Fibulin-4 deficient SMCs.(a) Fibulin-4+/R and Fibulin-4R/R SMCs show equal Tgf-β1 mRNA expression levels compared to Fibulin-4+/+ SMCs. (b) Increased TGF-β1 levels measured in conditioned medium (CM) from Fibulin-4R/R SMCs compared to Fibulin-4+/+ CM on day 1–4 after serum starvation. Fibulin-4+/R SMCs showed significant increased TGF-β1 levels on day 2 and 4 after serum starvation compared to Fibulin-4+/+ SMCs. Furthermore, on day 4 Fibulin-4R/R SMCs show significant increased TGF-β1 levels compared to Fibulin-4+/RSMCs (n = 4 per day for each genotype). Two-way ANOVA analysis for genotype and between days p < 0.05. (c) Gradually increased TGF-β1 is also observed in aortic arch lysates of Fibulin-4+/R (n = 6) and Fibulin-4R/R mice (n = 5) compared to Fibulin-4+/+ aortas (n = 5). This increase is significant in Fibulin-4R/R aortic arch lysates compared to Fibulin-4+/+ aortic arch lysates. (d) Fibulin-4+/R and Fibulin-4R/R SMCs show gradual increased Tgf-β2 mRNA expression levels compared to Fibulin-4+/+ SMCs. (e) Measurement of TGF-β2 revealed markedly increased levels in CM of Fibulin-4+/R and Fibulin-4R/R SMCs, while TGF-β2 was undetectable in CM of Fibulin-4+/+ SMCs (experiments were performed in at least 3 independent experiments). Two-way ANOVA analysis for genotype and between days p < 0.05. (f) Measurements in aortic arch lysates display significantly increased TGF-β2 in Fibulin-4R/R aortas compared to Fibulin-4+/R and Fibulin-4+/+ aortas (*p < 0.05, **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660353&req=5

f4: Strong increase of TGF-β2 levels in Fibulin-4 deficient SMCs.(a) Fibulin-4+/R and Fibulin-4R/R SMCs show equal Tgf-β1 mRNA expression levels compared to Fibulin-4+/+ SMCs. (b) Increased TGF-β1 levels measured in conditioned medium (CM) from Fibulin-4R/R SMCs compared to Fibulin-4+/+ CM on day 1–4 after serum starvation. Fibulin-4+/R SMCs showed significant increased TGF-β1 levels on day 2 and 4 after serum starvation compared to Fibulin-4+/+ SMCs. Furthermore, on day 4 Fibulin-4R/R SMCs show significant increased TGF-β1 levels compared to Fibulin-4+/RSMCs (n = 4 per day for each genotype). Two-way ANOVA analysis for genotype and between days p < 0.05. (c) Gradually increased TGF-β1 is also observed in aortic arch lysates of Fibulin-4+/R (n = 6) and Fibulin-4R/R mice (n = 5) compared to Fibulin-4+/+ aortas (n = 5). This increase is significant in Fibulin-4R/R aortic arch lysates compared to Fibulin-4+/+ aortic arch lysates. (d) Fibulin-4+/R and Fibulin-4R/R SMCs show gradual increased Tgf-β2 mRNA expression levels compared to Fibulin-4+/+ SMCs. (e) Measurement of TGF-β2 revealed markedly increased levels in CM of Fibulin-4+/R and Fibulin-4R/R SMCs, while TGF-β2 was undetectable in CM of Fibulin-4+/+ SMCs (experiments were performed in at least 3 independent experiments). Two-way ANOVA analysis for genotype and between days p < 0.05. (f) Measurements in aortic arch lysates display significantly increased TGF-β2 in Fibulin-4R/R aortas compared to Fibulin-4+/R and Fibulin-4+/+ aortas (*p < 0.05, **p < 0.01).

Mentions: Since we observed increased basal TGF-β signalling in Fibulin-4 deficient SMCs, we analysed whether this was due to increased TGF-β levels. Subconfluent Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs were serum-starved and conditioned medium was collected for 4 consecutive days to determine TGF-β1, − β2 and −β 3 levels. TGF-β3 levels were very low and did not differ between the different genotypes (data not shown). Although Tgf-β1 mRNA levels in SMCs did not differ between the genotypes (Fig. 4a), TGF-β1 levels in Fibulin-4R/R SMCs conditioned medium were higher compared to Fibulin-4+/+ SMCs (Fig. 4b). Conditioned medium from Fibulin-4+/R SMCs showed intermediate TGF-β1 levels. To analyse whether the increased TGF-β1 levels were also observed in vivo, we prepared lysates from aortic arches of Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R mice and measured TGF-β1 levels. These data revealed a similar gradual increase in TGF-β1 levels in aortic arch lysates of Fibulin-4+/R and Fibulin-4R/R mice (Fig. 4c).


Fibulin-4 deficiency increases TGF-β signalling in aortic smooth muscle cells due to elevated TGF-β2 levels.

Ramnath NW, Hawinkels LJ, van Heijningen PM, te Riet L, Paauwe M, Vermeij M, Danser AH, Kanaar R, ten Dijke P, Essers J - Sci Rep (2015)

Strong increase of TGF-β2 levels in Fibulin-4 deficient SMCs.(a) Fibulin-4+/R and Fibulin-4R/R SMCs show equal Tgf-β1 mRNA expression levels compared to Fibulin-4+/+ SMCs. (b) Increased TGF-β1 levels measured in conditioned medium (CM) from Fibulin-4R/R SMCs compared to Fibulin-4+/+ CM on day 1–4 after serum starvation. Fibulin-4+/R SMCs showed significant increased TGF-β1 levels on day 2 and 4 after serum starvation compared to Fibulin-4+/+ SMCs. Furthermore, on day 4 Fibulin-4R/R SMCs show significant increased TGF-β1 levels compared to Fibulin-4+/RSMCs (n = 4 per day for each genotype). Two-way ANOVA analysis for genotype and between days p < 0.05. (c) Gradually increased TGF-β1 is also observed in aortic arch lysates of Fibulin-4+/R (n = 6) and Fibulin-4R/R mice (n = 5) compared to Fibulin-4+/+ aortas (n = 5). This increase is significant in Fibulin-4R/R aortic arch lysates compared to Fibulin-4+/+ aortic arch lysates. (d) Fibulin-4+/R and Fibulin-4R/R SMCs show gradual increased Tgf-β2 mRNA expression levels compared to Fibulin-4+/+ SMCs. (e) Measurement of TGF-β2 revealed markedly increased levels in CM of Fibulin-4+/R and Fibulin-4R/R SMCs, while TGF-β2 was undetectable in CM of Fibulin-4+/+ SMCs (experiments were performed in at least 3 independent experiments). Two-way ANOVA analysis for genotype and between days p < 0.05. (f) Measurements in aortic arch lysates display significantly increased TGF-β2 in Fibulin-4R/R aortas compared to Fibulin-4+/R and Fibulin-4+/+ aortas (*p < 0.05, **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660353&req=5

f4: Strong increase of TGF-β2 levels in Fibulin-4 deficient SMCs.(a) Fibulin-4+/R and Fibulin-4R/R SMCs show equal Tgf-β1 mRNA expression levels compared to Fibulin-4+/+ SMCs. (b) Increased TGF-β1 levels measured in conditioned medium (CM) from Fibulin-4R/R SMCs compared to Fibulin-4+/+ CM on day 1–4 after serum starvation. Fibulin-4+/R SMCs showed significant increased TGF-β1 levels on day 2 and 4 after serum starvation compared to Fibulin-4+/+ SMCs. Furthermore, on day 4 Fibulin-4R/R SMCs show significant increased TGF-β1 levels compared to Fibulin-4+/RSMCs (n = 4 per day for each genotype). Two-way ANOVA analysis for genotype and between days p < 0.05. (c) Gradually increased TGF-β1 is also observed in aortic arch lysates of Fibulin-4+/R (n = 6) and Fibulin-4R/R mice (n = 5) compared to Fibulin-4+/+ aortas (n = 5). This increase is significant in Fibulin-4R/R aortic arch lysates compared to Fibulin-4+/+ aortic arch lysates. (d) Fibulin-4+/R and Fibulin-4R/R SMCs show gradual increased Tgf-β2 mRNA expression levels compared to Fibulin-4+/+ SMCs. (e) Measurement of TGF-β2 revealed markedly increased levels in CM of Fibulin-4+/R and Fibulin-4R/R SMCs, while TGF-β2 was undetectable in CM of Fibulin-4+/+ SMCs (experiments were performed in at least 3 independent experiments). Two-way ANOVA analysis for genotype and between days p < 0.05. (f) Measurements in aortic arch lysates display significantly increased TGF-β2 in Fibulin-4R/R aortas compared to Fibulin-4+/R and Fibulin-4+/+ aortas (*p < 0.05, **p < 0.01).
Mentions: Since we observed increased basal TGF-β signalling in Fibulin-4 deficient SMCs, we analysed whether this was due to increased TGF-β levels. Subconfluent Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs were serum-starved and conditioned medium was collected for 4 consecutive days to determine TGF-β1, − β2 and −β 3 levels. TGF-β3 levels were very low and did not differ between the different genotypes (data not shown). Although Tgf-β1 mRNA levels in SMCs did not differ between the genotypes (Fig. 4a), TGF-β1 levels in Fibulin-4R/R SMCs conditioned medium were higher compared to Fibulin-4+/+ SMCs (Fig. 4b). Conditioned medium from Fibulin-4+/R SMCs showed intermediate TGF-β1 levels. To analyse whether the increased TGF-β1 levels were also observed in vivo, we prepared lysates from aortic arches of Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R mice and measured TGF-β1 levels. These data revealed a similar gradual increase in TGF-β1 levels in aortic arch lysates of Fibulin-4+/R and Fibulin-4R/R mice (Fig. 4c).

Bottom Line: Fibulins are extracellular matrix proteins associated with elastic fibres.These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels.TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-β signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-β pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-β neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-β signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-β isoforms. These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels. Significantly increased TGF-β2 levels were also detectable in plasma from homozygous Fibulin-4(R/R) mice, not in wild type mice. TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-β signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-β1, but especially TGF-β2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-β pathway regulation in the pathogenesis of aortic aneurysms.

No MeSH data available.


Related in: MedlinePlus