Di Nolfo 2017 MiPschool Obergurgl
Event: MiPschool Obergurgl 2017
Marfan syndrome is a connective tissue disorder caused by mutations in FBN1 encoding fibrillin-1 and characterized by cardiovascular alterations, predominantly involving thoracic aorta aneurysms. Although with a lower prevalence, an association of Marfan syndrome with neurovascular complications has been reported[1-6]. However, cerebral artery properties in Marfan syndrome have never been explored. Marfan syndrome is linked to enhanced TGFβ signaling which is known to induce Nox4 isoform of NADPH oxidase expression, a major reactive oxygen species-producing enzyme in the brain[7-10].
Previous studies of our laboratory show that the Middle Cerebral Arteries from Marfan mice develop modest structural changes along the progression of disease.
Characterize for the first time properties of the middle cerebral arteries composition from a mouse displaying the most common class of mutation observed in 6-month-old Marfan syndrome Fbn1 (C1039G/+). The work also aims to evaluate the protective effects of the TGFβ-NOX4 pathway in the same four conditions. For these two aims we worked with mice from four conditions: mice control NOX4 (+/+), MARFAN Fbn1 (C1039G/+) NOX4 (+/+), CONTROL NOX4 (-/-) , MARFAN mice Fbn1(C1039G/+) NOX4 (-/-).
Analysis with MetaMorph Image Analysis software of two stacks of confocal images of several regions from each arterial segment demonstrated that the only variation of nuclei number is detectable in MARFAN mice Fbn1 (C1039G/+) NOX4 (-/-) with respect to MARFAN mice Fbn1(C1039G/+), in particular the significant difference is present only in the number of adventitia cells.
The amount of elastin was assessed by the analysis of the fluorescence that it emits naturally. Total elastin fluorescence in MCA cross section showed no variation among groups. In the same manner, analysis of internal elastic lamina thickness showed no different among groups. The only variation was observed in the average fluorescence intensity per pixel between MARFAN NOX4(-/-) and MARFAN NOX4(+/+).
We measured the expression and content of collagen and MMP implicated in extracellular matrix (ECM) modification. The Marfan condition compared with control mice showed no difference in mRNA levels of collagen 1A1 and in collagen deposition. However, a Marfan associated increase in matrix metalloproteinase MMP-9 mRNA levels was observed. NOX4(-/-) mice showed an intrinsic decreased of Collagen 1A1 mRNA levels, with unchanged collagen deposition. Importantly, MARFAN NOX4(-/-) mice led to augmented collagen deposition, despite diminished collagen 1A1 and augmented MMP-9 mRNA levels.
• Bioblast editor: Kandolf G
Labels: MiParea: nDNA;cell genetics, Genetic knockout;overexpression Pathology: Other
We next measured expression levels of the most relevant NADPH oxidases of brain vasculature. We evaluated mRNA levels of NADPH oxidase catalytic (Nox1, Nox2, Nox4) and regulatory (p22phox) subunits. Marfan cerebral arteries did not show differences in Nox1 and p22phox mRNA expression levels compared with wild-type mice. In contrast, mRNA levels of Nox4 were significantly higher in Marfan mice. Deletion of Nox4 per se did not alter either Nox1 or p22phox mRNA levels. However, Nox4 deficiency in Marfan mice led to higher Nox1 mRNA expression levels than in wild-type mice.
ROS levels were evaluated by DHE-derived fluorescence, and they were higher along the MCA wall of Marfan mice than in wild-type mice. The only variation was observed between MARFAN NOX4(+/+) and control. This suggests that the superoxide anion is not increased via Nox4, but probably through other Nox such as Nox1.
We analyzed TGF-β expression in cerebral arteries from the four conditions. Marfan showed higher mRNA level than in control mice in quantitative analysis of TGF-β mRNA level. However, the absence of Nox4 reduce TGF-β mRNA expression levels.
Our study contributes to understanding of the role of the TGF-β/Nox4 signaling pathway in cerebral vasculature in healthy, and particularly in Marfan patients, since we show that genetic depletion of Nox4 in Marfan mice induces collagen deposition which contributes to arterial wall hypertrophy, thereby causing overt MCA structural alterations. Overall, the results of the present study suggest that Nox4 has a role in regulating cerebrovascular resistance in Marfan animals. Thus, our findings lead to the hypothesis that an overall reduction in TGF-β signaling, an ongoing therapeutic approach against aortic aneurysm formation in MFS, might have a negative impact on brain circulation.
- (1,2)Di Nolfo, (2)Jimenez Altajo F, (1)Belluardo N
- Univ degli studi Palermo, Dept Biotecnol mediche Medicina Molec, Italy
- Univ Autonoma Barcelona, Dept Darmacologia Toxicologia. - [email protected]
Figure 1. Schematic representation of ECMdisintegration inMFS. Fibrillin-1 aggregates into functionalmicrofibrils and sequesters TGF-β in the ECM, regulating its signaling. Loss of fibrillin-1 releases TGF-β and its constitutive stimulation activates genes like MMPs which induce elastolysis, decreasing ECM stability.
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