Tunica Media

In standard liquid-based cell culture systems, the amount of particles associated with cells at any time is a function of the rate of delivery of particles to the cells, how strongly particles adhere to the cell surface, and the rates of cellular uptake and loss by degradation or exocytosis.
ISDD applies well established, long-used principles of diffusional and gravitational transport of particles in viscous media to calculate the movement of particles from the media to the bottom of a vessel where cells reside. The net rate of transport downward toward the bottom of the vessel is calculated within a single partial differential equation, which is solved numerically to calculate the fraction of material transported from media to the bottom of the vessel. Simulations are conducted using commonly available inputs for monodisperse particles: temperature, media density and viscosity, media height, hydrodynamic particle size in the test media, and particle density. Simulations of agglomerates also require two additional parameters describing how the primary particles are packed to form the agglomerate. The model produces a time-course of particle surface area, number and mass transported to the bottom of the vessel, referred to as the delivered dose, which can be compared to measured values in a cell free environment. The delivered dose can also be compared to measured amounts associated with cells (in or adhered to), which is an appropriate, but possibly less certain comparison because the roles of cellular uptake, adherence, and loss of adhered material during washing are not accounted for explicitly in the current formulation of ISDD. ISDD focuses on particle transport because this process can be rate limiting, is very valuable for the experimentalist to understand, can be simulated with a relatively small set of easy to access parameters, and is independent of cell type and other experimental conditions that affect cellular uptake. Moreover, at this time, it is experimentally difficult to separate particle uptake (particles in a cell) from cell associated particles (on a cell or in a cell). If necessary, modifications to the boundary conditions or assumptions regarding fractional uptake can be used to account for cellular uptake. Thus, ISDD calculates the delivered dose, which is equivalent to particles associated with the cell (on a cell or in a cell), the only commonly available experimental measure of target cell dose.

Thickness and stiffness of the eGC were determined using the Atomic Force Microscope (AFM) nanoindentation technique. Preservation of the endothelial cell layer on aorta preparations was approved by immunostaining of PECAM-1/CD31 (Figure 1). Figure 2 A, B illustrates the basic principles of this method. By using a Multimode AFM (Veeco, Mannheim, Germany) with a feedback-controlled heating device (Veeco) measurements were performed at 37°C as described previously [29 (link)].
In brief, the central component of the AFM is a very sensitive mechanical nanosensor – a triangular cantilever with a mounted spherical tip (here: electrically uncharged polystyrene, diameter = 10 µm, Novascan, Ames, IA, USA) that is utilized to periodically indent the cells. A spherical tip was used for this AFM approach instead of a sharp tip because of a larger interaction area between tip and sample that decreases the effective pressure and results in less mechanical noise [30 (link)]. The cantilever functions as a soft spring (spring constant = 11 pN/nm). The xyz-position of the tip is precisely controlled by a piezo-element (Figure 2 A). A laser beam is reflected by the gold-coated backside of the cantilever to a position-sensitive quadrupled photodiode allowing measurements of the cantilever deflection (V). Determination of the spring constant (K_cant) by the thermal tuning method and measurement of the deflection sensitivity (α) of the cantilever on bare glass coverslips facilitate the calculation of the force (F) acting on the cantilever and, in turn, the force exerted by the cantilever to the sample.
Since the piezo displacement (x_piezo) and the deflection sensitivity (α) are known, the indentation depth (deformation) of the sample (x_sample) can be calculated.
For reasons of readability the indentation depth is hereafter called “thickness”. It should be noted that the indentation depth rather represents an apparent thickness, rather than the exact anatomical thickness.
Force indentation curves of a single cell were obtained by plotting the force (F) necessary to indent the cell (indentation depth, x_sample). The sample stiffness can be derived from Hook´s law.
The stiffness (K) is the mechanical resistance of a sample against a defined deformation (e.g. indentation). K depends strongly on the indentation depth and the location, because cells contain a variety of substructures and organelles. The experimental parameters including an indentation velocity of 1 µm/s, a loading force of approximately 400 pN, an indentation frequency in the range of 0.25 - 0.5 Hz, a ramp size of 2 µm, a trig threshold of 35 nm and a tip velocity of 0.5 - 1 µm/s.
Previous experiments, using 1 µm AFM-tips, showed that the glycocalyx thickness is somewhat variable [29 (link),31 (link)]. Since we were interested in the overall condition of the glycocalyx and especially in its changes induced by different stimuli, we here chose larger tips (10 µm), as they indent a larger area. Thus they provided “more averaged” results and enabled us to avoid the data being influenced by the spatial distribution of the eGC thickness. All measurements were performed in HEPES-buffered solution [standard composition in millimolars: 140 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 5 Glucose, 10 HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.4] supplemented with 1% FCS in order to prevent eGC collapse [32 (link)].
Light microscopy was used to ensure that the tip position of the mechanical nanosensor was located neither at the nuclear, nor at the junctional region of cultured endothelial cells. However, this approach was not feasible in (thick) explanted aortas due to the lack of transparency of sub-endothelial layers such as the Tunica media and T. externa.
Figure 3 A, B show typical force indentation curves of an untreated as well as heparinase-treated aortic endothelial cell (“overview mode”). Each force indentation curve was then analyzed separately with a higher magnification (“working mode”) by using a linear approximation for determination of the eGC nanomechanics (Figure 3 C).

IVUS was performed using the IVUS imaging system (Volcano Corporation) and a 20 MHz 3.5 F Visions PV 0.035 Digital IVUS Catheter (Volcano Corporation). For the current study 20 mm of IVUS pullback segment distal to the ostia of the RCA and the LAD was selected. The area circumscribed by the outer border of the echolucent tunica media and the luminal border was manually traced on each 1 mm IVUS frame within selected fragment. The following indices of vessel morphology were assessed: 1) lumen volume (mm³) = lumen area (the area bounded by the luminal border) × length of fragment; 2) vessel volume (mm³) = EEM area × length of fragment; 3) plaque + media volume (mm³) = vessel volume – lumen volume; 4) relative atheroma volume (%) = plaque plus media volume divided by the vessel volume × 100%.

Five gram-positive bacteria [Bacillus subtilis^X, Enterococcus faecalis (ATCC 25922), Staphylococcus aureus (ATCC 27659), Bacillus cereus^x and Mycobacterium smegmatis^x) and five gram-negative [(Proteus mirabilis^x, Salmonella typhimurium (ATCC 14028), Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 13883)] were examined in this experiment (^x denotes that ATCC number is not available). All the bacteria, except E. faecalis were sub-cultured on Tryptic soy agar (TSA) media at 37°C for 24hours. E. faecalis was grown on 5% sheep blood agar media. These subcultures were kept at 4°C to guarantee bacterial viability and purity.

All statistical analyses were performed using Origin 9.01 or OriginPro, GraphPad Prism v7.03 or v9.4, Cytoscape 3.5.1, and R 4.0.3 with the ggpubr 0.4.0 and tidyverse 1.3.0 packages. The significance of network proximity was evaluated by creating 1,000 random modules of the same size and comparing the observed proximity value with the null model (random control) through fitting normal distributions and P values were obtained by z test. Data are presented as the mean ± SEM unless otherwise indicated. Comparison between 2 groups was performed using the Student’s unpaired 2-tailed t test. The paired 2-tailed Student’s t test was used for analyses comparing metabolic tracer signal differences in the pulmonary endothelium and vascular media within the same rat. Hypergeometric testing was applied using the fgsea R package to identify key MSigDB Hallmark pathways distinguishing control versus inflammatory PAH (as described further in the legend of Supplemental Figure 3B). The Mann-Whitney and Kruskal-Wallis nonparametric tests were used to compare 2 or more non-normally distributed groups. Cell type–specific differences in ¹⁵N-proline or ²H-glucose labeling between MCT and control rats were assessed by nested ANOVA. The Pearson or Spearman (for experiments with small sample size) correlation coefficient is reported for linear regression. Graphical representation of comparisons including N ≥ 3 uses the box or violin plot format inclusive of mean, median, IQR, and maximum and minimum. A P value < 0.05 and FDR < 0.05 were considered statistically significant.
All data were included for analysis with the following exception: the right ventricular systolic pressure could not be measured in 1 rat due to periprocedural mortality from hemorrhage. MIMS and accompanying histologic experiments used 3 biological replicates per condition. No nonlinear adjustments were made to representative images. Primary data were reviewed in a blinded manner when possible.