Research Article: Extracellular matrix in ascending aortic aneurysms and dissections – What we learn from decellularization and scanning electron microscopy

Date Published: March 18, 2019

Publisher: Public Library of Science

Author(s): Teresa Mimler, Clemens Nebert, Eva Eichmair, Birgitta Winter, Thomas Aschacher, Marie-Elisabeth Stelzmueller, Martin Andreas, Marek Ehrlich, Guenther Laufer, Barbara Messner, Feng Zhao.

http://doi.org/10.1371/journal.pone.0213794

Abstract

Pathological impairment of elastic fiber and other extracellular matrix (ECM) components are described for the aortic media of ascending thoracic aortic aneurysms (aTAA) but the exact pathological impairment of the structure and its degree still needs further investigations. To evaluate the quantity and quality of elastic fiber sheets and other ECM structures (e.g. collagen), cells were removed from different types of aneurysmal tissues (tricuspid aortic valve [TAV] associated-, bicuspid aortic valve [BAV] associated-aneurysmal tissue and acute aortic dissections [AAD]) using 2.5% sodium hydroxide (NaOH) and compared to decellularized control aortic tissue. Likewise, native tissue has been analysed. To evaluate the 2D- (histological evaluation, fluorescence- and auto-fluorescence based staining methods) and the 3D structure (scanning electron microscopic [SEM] examination) of the medial layer we first analysed for a successful decellularization. After proving for successful decellularization, we quantified the amount of elastic fiber sheets, elastin and other ECM components including collagen. Aside from clearly visible focal elastic fiber loss in TAV-aTAA tissue, decellularization resulted in reduction of elastic fiber auto-fluorescence properties, which is perhaps an indication from a disease-related qualitative impairment of elastic fibers, visible only after contact with the alkaline solution. Likewise, the loss of collagen amount in BAV-aTAA and TAV-aTAA tissue (compared to non-decellularized tissue) after contact with NaOH indicates a prior disease-associated impairment of collagen. Although the amount of ECM was not changed in type A dissection tissue, detailed electron microscopic evaluation revealed changes in ECM quality, which worsened after contact with alkaline solution but were not visible after histological analyses. Apart from the improved observation of the samples using electron microscopy, contact of aneurysmal and dissected tissue with the alkaline decellularization solution revealed potential disease related changes in ECM quality which can partly be connected to already published data, but have to be proven by further studies.

Partial Text

According to World Health Organization (WHO), thoracic aortic aneurysms (TAAs) belong to the group of cardiovascular diseases, which are the number one cause of death worldwide.[1] In 2014 in the United States, 9,863 deaths were due to an aortic aneurysm.[2] According to their location, TAAs can be divided into ascending and descending aortic aneurysms. They also show differences in their pathology affecting the proper wall function, which are suspected to be due to the different origins of the vascular smooth muscle cells (SMCs). While in the ascending part of the aorta, SMCs come from the neural crest, the SMCs from the descending aorta origin from the paraxial mesoderm.[3] Aside from these hypotheses, the underlying cause of non-syndromic ascending thoracic aortic aneurysm (aTAA) formation is completely unknown.

The organization of the aortic ECM is very crucial since it is needed for maintaining tensile strength as well as stiffness of the aorta.[15,16,24] Any alterations in the composition of the ECM including collagen and elastin have tremendous effects on the normal function of the ECM which might be involved in the formation of aneurysms or dissections.[25–28] Therefore the present study aimed at analysing the ECM structure/amount/quality of control and aneurysmal/dissection tissue in its native form as well as after decellularization. Using decellularization of aneurysmal and dissection tissue by an alkaline solution (NaOH), subsequent histological and detailed SEM examinations, we describe so far unreported and potential disease-related changes in elastic fiber and collagen quantity/quality apparent in native tissue or which become only evident after contact with alkaline solution. Of note, comparison of native with decellularized and non-aneurysmal with aneurysmal/dissected tissue is necessary to prevent misinterpretation of the data.

By decellularization of aneurysmal and non-aneurysmal tissue using an alkaline solution, differences in the quality of ECM components became obvious, which can partly be connected with already existing data. In terms of TAV-aTAAs, an impaired quality of elastic fibers became obvious, although the amount is not significantly reduced. Since this only occurs with TAV-aTAAs, this could indicate, apart from an already known focal loss of fibers, a previously unknown disease-related impairment of the elastic fiber quality, which of note is not due to a loss in elastin expression. Regarding collagen, a contact with alkaline solutions leads to a loss in the amount of this ECM protein, however only in aneurysmal tissue associated with a BAV or a TAV. As collagen dissolves after contact with alkaline solution and keeping in mind that we used a very low concentration, it is reasonable to assume that a previous disease-related damage to collagen causes the accelerated degradation in BAV- and TAV- associated aneurysmal tissue. The authors are completely aware that alkaline solutions are able to damage collagen and remove it from tissues.[38] However, the unchanged collagen content in the control tissue after decellularization (compared to native tissue) suggests that the differences in aneurysmal tissue after decellularization may indicate a pathological change in the quality of this protein and is no general effect of the alkaline solution treatment. Changes in the quality of ECM components within the ascending aorta of dissection patients were also detectable, however only in electron microscopic analyses.

 

Source:

http://doi.org/10.1371/journal.pone.0213794

 

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