Transcriptional Regulation of Postnatal Aortic Development

Introduction: The aorta undergoes tremendous changes in geometry, composition, and mechanical properties during postnatal development. These changes are necessarily driven by transcriptional changes, either genetically programmed or mechano-responsive, but there has not been a careful comparison of time-course changes in the transcriptional profile and biomechanical phenotype. It is during this period, for example, that most current surgical procedures are directed for treating children born with congenital heart and vascular defects, and therefore there is a pressing need for a better understanding of aortic development from molecular to mechanical perspectives to drive improved treatments for children.
Here, we report general findings from bulk RNA-sequencing (RNA-seq) for ~8000 transcripts plus detailed time courses of expression for ~100 transcripts during postnatal aortic development in wild-type mice, and also relate a subset of these changes to associated evolving biomechanical function.

Materials and

Methods: Male C57BL/6J mice were euthanized either by decapitation (age < 10 days) or CO2 asphyxiation (age > 10 days), and the aorta was rapidly excised en bloc from just distal to the left subclavian artery to the iliac bifurcation and placed in RNA stabilization solution. Using this segment of the aorta provided sufficient RNA for study with n = 9 mice at P2, n = 5 at P10, n = 5 at P21, n = 6 at P42, and n = 6 at P84 (well into maturity). These ages were selected to coincide largely with those reported previously for evolving mural composition, geometry, and mechanical properties of the descending thoracic aorta in developing male C57BL/6J mice. Total RNA was isolated using an RNeasyMini Kit (Qiagen) according to the manufacturer’s protocol and whole-transcriptome sequencing was performed using a NovaSeq 6000 System (Illumina, Inc.) in the Genomics Core at Yale University. RNA sequencing reads were analyzed using CLC Genomics Workbench 23 (Qiagen) and gene ontology using Ingenuity Pathway Analysis (Qiagen).

Results, Conclusions, and Discussions: Principal component analysis revealed that transcripts separated well for postnatal ages P2, P10, and P21 but less well for postnatal ages P42 and P84 suggesting that the period up to weaning (at ~P21) generally includes the most rapid and marked changes in gene expression.
Gene ontology studies highlighted associated biological processes, including many expected in arterial development: proliferation, migration, organization of cytoplasm and cytoskeleton and cell-cell contact. Again, analyses revealed that changes in biological processes were greatest between P2 and P10 and similarly between P10 and P21 (Figure 1).

Consistent with the interpretation of the seminal work of Mecham and colleagues (Kelleher et al., 2004), we confirmed multiple general classes of time-course changes in expression – those that were highest at P2 and decreased to low levels thereafter included transcripts related to proliferation and migration of cells, those that peaked early in the postnatal period and decreased thereafter related to structural protein production, those that increased from early postnatal ages to maturity included contractile proteins and ECM modifiers.
we considered possible correlations between the transcriptional profile and biomechanical phenotype, the latter determined primarily by elastic energy storage, distensibility, and contractile strength. We found strong correlations between the energy storage and the expression of Eln, Fbln5, Lox, Efemp2 and Fbn1 which encode the primary constituents of the elastic fiber unit. Contractility was highly correlated with the expression of Myh11 and Mylk6.

Acknowledgements (Optional): This research was supported, in part, by grants from Additional Ventures (SVRF and AVCC).