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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

Increased levels of TGF-β2 are detectable in plasma from 100 days old Fibulin-4R/R mice, which reduces on Losartan treatment.(a) TGF-β1 measurements in plasma samples showed no difference between placebo treated Fibulin-4+/+ (n = 9), Fibulin-4+/R (n = 8) and Fibulin-4R/R mice (n = 10). (b) TGF-β2 was detectable in plasma of 12 out of 24 placebo treated Fibulin-4R/R mice compared to only 2 out of 15 in placebo treated Fibulin-4+/+ mice and 2 out of 19 in Fibulin-4+/R mice. TGF-β2 levels are significantly higher in Fibulin-4R/R mice compared to placebo treated Fibulin-4+/+ and Fibulin-4+/R mice. Losartan treatment of Fibulin-4R/R mice seemed to reduce the TGF-β2 levels (0 out of 9) as compared to placebo treated Fibulin-4R/R mice. The red line indicates the detection of the ELISA (Chi-square p < 0.001). (c) Kaplan-Meier survival curve shows an increased survival of Losartan treated Fibulin-4R/Rmice compared to placebo treated Fibulin-4R/Rmice. (d) Aortic diameter of 160 days old placebo and losartan treated Fibulin-4+/+, Fibulin-4+/R mice and Losartan treated Fibulin-4R/R mice. Losartan treated Fibulin-4R/Rmice have significantly enlarged aortic diameters compared to wild type mice, while there are no differences between placebo and Losartan Fibulin-4+/Rmice and wild type mice. (e) HE, elastin and αSMA staining of ascending thoracic aortas. Placebo and losartan treated Fibulin-4+/R mice (160 days old) show an increase in aortic wall thickness and some elastin breaks compared to Fibulin-4+/+mice. Losartan treated Fibulin-4R/Rmice show despite of their survival disrupted aortic wall architecture. In addition, despite losartan treatment there is loss of smooth muscle cell content in the media of Fibulin-4R/R mice. (*p < 0.05, **p < 0.01).
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f5: Increased levels of TGF-β2 are detectable in plasma from 100 days old Fibulin-4R/R mice, which reduces on Losartan treatment.(a) TGF-β1 measurements in plasma samples showed no difference between placebo treated Fibulin-4+/+ (n = 9), Fibulin-4+/R (n = 8) and Fibulin-4R/R mice (n = 10). (b) TGF-β2 was detectable in plasma of 12 out of 24 placebo treated Fibulin-4R/R mice compared to only 2 out of 15 in placebo treated Fibulin-4+/+ mice and 2 out of 19 in Fibulin-4+/R mice. TGF-β2 levels are significantly higher in Fibulin-4R/R mice compared to placebo treated Fibulin-4+/+ and Fibulin-4+/R mice. Losartan treatment of Fibulin-4R/R mice seemed to reduce the TGF-β2 levels (0 out of 9) as compared to placebo treated Fibulin-4R/R mice. The red line indicates the detection of the ELISA (Chi-square p < 0.001). (c) Kaplan-Meier survival curve shows an increased survival of Losartan treated Fibulin-4R/Rmice compared to placebo treated Fibulin-4R/Rmice. (d) Aortic diameter of 160 days old placebo and losartan treated Fibulin-4+/+, Fibulin-4+/R mice and Losartan treated Fibulin-4R/R mice. Losartan treated Fibulin-4R/Rmice have significantly enlarged aortic diameters compared to wild type mice, while there are no differences between placebo and Losartan Fibulin-4+/Rmice and wild type mice. (e) HE, elastin and αSMA staining of ascending thoracic aortas. Placebo and losartan treated Fibulin-4+/R mice (160 days old) show an increase in aortic wall thickness and some elastin breaks compared to Fibulin-4+/+mice. Losartan treated Fibulin-4R/Rmice show despite of their survival disrupted aortic wall architecture. In addition, despite losartan treatment there is loss of smooth muscle cell content in the media of Fibulin-4R/R mice. (*p < 0.05, **p < 0.01).

Mentions: Next, we analysed TGF-β2 expression in the SMCs. Fibulin-4+/R and Fibulin-4R/R SMCs showed >5-fold increased Tgf-β2 mRNA expression levels (Fig. 4d). ELISA analysis on conditioned medium from Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs revealed strongly increased TGF-β2 levels in medium from Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, in which TGF-β2 was undetectable (Fig. 4e). TGF-β2 levels were already significantly higher in conditioned medium from Fibulin-4+/R SMCs. Increased TGF-β2 levels were also detectable in Fibulin-4R/R aortic arch lysates, when compared to aortic arch lysates from Fibulin-4+/+ and Fibulin-4+/R mice (Fig. 4f). Given the increased TGF-β levels in SMCs and aortic tissue derived from Fibulin-4 deficient mice, we determined TGF-β1 and TGF-β2 levels in plasma samples from these mice. TGF-β1 levels were not significantly different among plasma from Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R mice (Fig. 5a). In contrast, plasma TGF-β2 levels were very low in wild type mice and could be detected in 2 out of 15 Fibulin-4+/+ mice and 2 out of 19 Fibulin-4+/R mice (Fig. 5b). TGF-β2 levels could be detected in plasma from 12 out of 24 Fibulin-4R/R mice with significantly higher concentrations compared to Fibulin-4+/+ and Fibulin-4+/R mice. These data show that specifically TGF-β2 levels in aortic tissue and plasma of Fibulin-4 deficient mice are strongly increased.


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)

Increased levels of TGF-β2 are detectable in plasma from 100 days old Fibulin-4R/R mice, which reduces on Losartan treatment.(a) TGF-β1 measurements in plasma samples showed no difference between placebo treated Fibulin-4+/+ (n = 9), Fibulin-4+/R (n = 8) and Fibulin-4R/R mice (n = 10). (b) TGF-β2 was detectable in plasma of 12 out of 24 placebo treated Fibulin-4R/R mice compared to only 2 out of 15 in placebo treated Fibulin-4+/+ mice and 2 out of 19 in Fibulin-4+/R mice. TGF-β2 levels are significantly higher in Fibulin-4R/R mice compared to placebo treated Fibulin-4+/+ and Fibulin-4+/R mice. Losartan treatment of Fibulin-4R/R mice seemed to reduce the TGF-β2 levels (0 out of 9) as compared to placebo treated Fibulin-4R/R mice. The red line indicates the detection of the ELISA (Chi-square p < 0.001). (c) Kaplan-Meier survival curve shows an increased survival of Losartan treated Fibulin-4R/Rmice compared to placebo treated Fibulin-4R/Rmice. (d) Aortic diameter of 160 days old placebo and losartan treated Fibulin-4+/+, Fibulin-4+/R mice and Losartan treated Fibulin-4R/R mice. Losartan treated Fibulin-4R/Rmice have significantly enlarged aortic diameters compared to wild type mice, while there are no differences between placebo and Losartan Fibulin-4+/Rmice and wild type mice. (e) HE, elastin and αSMA staining of ascending thoracic aortas. Placebo and losartan treated Fibulin-4+/R mice (160 days old) show an increase in aortic wall thickness and some elastin breaks compared to Fibulin-4+/+mice. Losartan treated Fibulin-4R/Rmice show despite of their survival disrupted aortic wall architecture. In addition, despite losartan treatment there is loss of smooth muscle cell content in the media of Fibulin-4R/R mice. (*p < 0.05, **p < 0.01).
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f5: Increased levels of TGF-β2 are detectable in plasma from 100 days old Fibulin-4R/R mice, which reduces on Losartan treatment.(a) TGF-β1 measurements in plasma samples showed no difference between placebo treated Fibulin-4+/+ (n = 9), Fibulin-4+/R (n = 8) and Fibulin-4R/R mice (n = 10). (b) TGF-β2 was detectable in plasma of 12 out of 24 placebo treated Fibulin-4R/R mice compared to only 2 out of 15 in placebo treated Fibulin-4+/+ mice and 2 out of 19 in Fibulin-4+/R mice. TGF-β2 levels are significantly higher in Fibulin-4R/R mice compared to placebo treated Fibulin-4+/+ and Fibulin-4+/R mice. Losartan treatment of Fibulin-4R/R mice seemed to reduce the TGF-β2 levels (0 out of 9) as compared to placebo treated Fibulin-4R/R mice. The red line indicates the detection of the ELISA (Chi-square p < 0.001). (c) Kaplan-Meier survival curve shows an increased survival of Losartan treated Fibulin-4R/Rmice compared to placebo treated Fibulin-4R/Rmice. (d) Aortic diameter of 160 days old placebo and losartan treated Fibulin-4+/+, Fibulin-4+/R mice and Losartan treated Fibulin-4R/R mice. Losartan treated Fibulin-4R/Rmice have significantly enlarged aortic diameters compared to wild type mice, while there are no differences between placebo and Losartan Fibulin-4+/Rmice and wild type mice. (e) HE, elastin and αSMA staining of ascending thoracic aortas. Placebo and losartan treated Fibulin-4+/R mice (160 days old) show an increase in aortic wall thickness and some elastin breaks compared to Fibulin-4+/+mice. Losartan treated Fibulin-4R/Rmice show despite of their survival disrupted aortic wall architecture. In addition, despite losartan treatment there is loss of smooth muscle cell content in the media of Fibulin-4R/R mice. (*p < 0.05, **p < 0.01).
Mentions: Next, we analysed TGF-β2 expression in the SMCs. Fibulin-4+/R and Fibulin-4R/R SMCs showed >5-fold increased Tgf-β2 mRNA expression levels (Fig. 4d). ELISA analysis on conditioned medium from Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs revealed strongly increased TGF-β2 levels in medium from Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, in which TGF-β2 was undetectable (Fig. 4e). TGF-β2 levels were already significantly higher in conditioned medium from Fibulin-4+/R SMCs. Increased TGF-β2 levels were also detectable in Fibulin-4R/R aortic arch lysates, when compared to aortic arch lysates from Fibulin-4+/+ and Fibulin-4+/R mice (Fig. 4f). Given the increased TGF-β levels in SMCs and aortic tissue derived from Fibulin-4 deficient mice, we determined TGF-β1 and TGF-β2 levels in plasma samples from these mice. TGF-β1 levels were not significantly different among plasma from Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R mice (Fig. 5a). In contrast, plasma TGF-β2 levels were very low in wild type mice and could be detected in 2 out of 15 Fibulin-4+/+ mice and 2 out of 19 Fibulin-4+/R mice (Fig. 5b). TGF-β2 levels could be detected in plasma from 12 out of 24 Fibulin-4R/R mice with significantly higher concentrations compared to Fibulin-4+/+ and Fibulin-4+/R mice. These data show that specifically TGF-β2 levels in aortic tissue and plasma of Fibulin-4 deficient mice are strongly increased.

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