Resolving the design principles that control postnatal vascular growth and scaling

After birth, tissues grow continuously until reaching adult size, with each organ exhibiting unique cellular dynamics, growth patterns, and (stem or non-stem) cell sources. Using a suite of experimental and computational multiscale approaches, we found that aortic expansion is guided by specific biological principles and scales with the vertebral column rather than animal body weight. Expansion proceeds via two distinct waves of arterial cell proliferation along blood flow that are spatially stochastic, yet temporally coordinated. Each wave exhibits unique cell cycle kinetics and properties, with the first wave exhibiting cell cycle durations as fast as 6 hours. Singlecell RNA sequencing showed changes in fatty acid metabolism concomitant with an increase in cell size. Mathematical modeling and experiments indicated endothelial cell extrusion is essential for homeostatic aortic growth and balancing excess proliferation. In a genetic model of achondroplasia, the aorta achieves proper scaling through enhanced cell extrusion while maintaining normal proliferation dynamics. Collectively, these results provide a blueprint of the principles that orchestrate aortic growth which depends entirely on differentiated cell proliferation rather than resident stem cells. Overall design: Animals were sacrificed through CO2 inhalation before perfusion through the left ventricle with warm Versene solution (0.2g EDTA, 8g sodium chloride, 0.2g potassium chloride, 1.15g anhydrous sodium phosphate, 0.2g anhydrous potassium phosphate, dissolved in distilled water, pH 7.5) The aorta was dissected from the body and adventitia removed before being cut open to expose the endothelium. For the P5 and P10 timepoints, whole aortas were pooled into digestion solution (Liberase TH 70ug/mL, Sigma-Aldrich; DNase I, Qiagen; 10mM HEPES, Corning) and incubated at 37°C for 20 minutes while pipetting up and down every 2 minutes to generate a single-cell suspension. For P20 and P30 timepoints, aortas were cut open and pinned down with the endothelium facing up. After adding warm 1X trypsin (Gibco) for 5 minutes, a microfeather blade was used to gently scrape the endothelium of the aorta, as in previously established methods 17. The samples were incubated in 1X trypsin for an additional 5 minutes while pipetting up and down to generate a single-cell suspension. After neutralizing the digestion solution with DMEM containing 10% FBS, the cell suspension was filtered through a 20 uM cell strainer. Post-digestion cleanup was done prior to cell counting and viability assessment. Cell suspensions were loaded onto a Chromium Next GEM Chip G and run using the Chromium Controller iX (10X Genomics) to generate gel beads in emulsion (GEMs). Singlecell RNA sequencing libraries were constructed using the Chromium Next GEM Single Cell 3' v3.1 (Dual Index). Sequencing was performed on a NextSeq2000 Sequencing System (Illumina). Gene expression matrices were generated by de-multiplexing, aligning, barcode processing, and UMI counting using CellRanger pipeline.