Poster E7 - Collagen and Glycosaminoglycans Accumulation in the Ascending Thoracic Aorta of a mouse model for Marfan Syndrome

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Colin W. Means

Undergraduate Student & Researcher
Yale University
Colleyville, Texas, United States

Co-Author(s)

DW

Dar Weiss, PhD

Assistant Professor
University of Denver, United States

Primary Investigator(s)

JH

Jay D. Humphrey, PhD

Professor
Yale University
New Haven, Connecticut, United States

Introduction: Marfan syndrome (MFS) affects the connective tissue via a mutation in the FBN1 gene that encodes the proteins of the elastic fibers in the extracellular matrix by binding to each other to form microfibrils – fibrillin-1. Mortality from MFS frequently stems from a rupture of the ascending thoracic aorta (ATA). In mice and humans, Marfan syndrome is associated with increased transcripts for fibrillar collagens and increased collagen in the aortic wall, thereby increasing stiffness and strength of the wall as a compensatory effect in the absence of non-mutant elastic ability. While there is much information in the literature comparing wild-type and Marfan mice, there is not a detailed assessment of the histology, transcriptomics, and proteomics as these mouse models age, especially in relation to how the compensatory collagen and glycosaminoglycans change over time. Therefore, this study worked to examine the effect of age on the progression of the Fbn1C1041G/+ mouse model of Marfan syndrome in comparison to wild-type (WT) mice.

Materials and

Methods: Animal Model. Littermate WT and Fbn1C1041G/+ (MFS) mice at 12 weeks, 1 year, and 2 years of age were used to characterize the aging process of the MFS mice in comparison to the WT because 2 years of age mice typically aligns with around 70 years of age in humans.

Histology. The ascending thoracic aorta of the mice was prepared for histological examination. Cross-sections were stained with picrosirius red (PSR) to categorize collagen fibers as thick or thin or Movat pentachrome to measure elastin, cytoplasm, collagen, glycosaminoglycans, and fibrin in the wall. Movat slides were imaged in bright-field. PSR slides were imaged in dark-field with polarized light to separately capture and quantify medial and adventitial collagens.

Transcriptomics. RNA from the ATA was isolated using an RNeasy Mini Kit (Qiagen) and whole-transcriptome sequencing was performed using a NovaSeq 6000 System (Illumina) at the Yale Center for Genome Analysis. Sequenced reads were imported into CLC Genomics Workbench V23 (Qiagen) and processed using trimmed mean of M values normalization and log counts per million (CPM) transformation.

Proteomics. Proteomics were gathered using mass spectrometry by the Yale Keck MS & Proteomics Resource. LC MS/MS data were analyzed with Proteome Discoverer 2.4 (Thermo Fisher Scientific) and the Mascot search engine (v. 2.7 Matrix Science LLC). Reads were normalized using Pierce Peptide Retention Time Calibration Mixture and further analyzed in CLC Genomics Workbench V23 (Qiagen).

Results, Conclusions, and Discussions: Histological examination of transverse cross-sections of the ATA stained with Movat indicated an age-dependent increase in glycosaminoglycans (GAGs) wall area in male MFS (MMFS) (Figure 1C). Additionally, proteomic analysis showed age-dependent increases of proteoglycans aggrecan (Acan) and versican (Vcan) in MMFS mice (Figure 1B). While transcriptomic analysis revealed comparable expression of Vcan and decreased expression of Acan with age (Figure 1A), it also showed decreased expression of Adamts9 and Adamts15 (Figure 1A), enzymes involved in the degradation of extracellular matrix components, including proteoglycans.

Despite unchanging Acan and Vcan gene expression with age, the histological analysis indicated an age-related increase in GAGs wall area and aggrecan and versican protein levels. The decrease in Adamts9 and Adamts15 gene expression likely contributes to the observed increase in GAGs and proteoglycans by reducing their degradation in the extracellular matrix.

PSR staining revealed a significant age-dependent increase in collagen. This dependence was particularly pronounced in MMFS mice between 12 weeks and 1 year of age in which rate of collagen wall area growth was greater than the male WT (MWT) before leveling off and having mild wall area increase from 1 year to 2 years (Figure 1D). On the other hand, MWT maintained a linear increase of collagen wall area before reaching approximately the same collagen wall area as the MMFS at 2 years.

This rapid increase in collagen wall area mimicked accelerated aging in MMFS in comparison to Male WT (MWT) with the MMFS reaching comparable collagen wall area at 1 year to the MWT collagen wall area at 2 years.

This study used multiomics and microstructural analyses to characterize MFS in mice during aging. The results suggest a difference in the aortic aging of MFS and WT mice with the aorta aging rapidly until around 1 year before staying approximately the same from 1 year to 2 years in MFS and aging gradually in WT and an increase in GAGs wall area during aging because of a decrease in GAGs degradation.

Acknowledgements (Optional):