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Fibroblast mechanosignaling and extracellular matrix remodeling in pulmonary arterial hypertension


Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by sustained elevated pulmonary arterial pressures in which the pulmonary vasculature and right ventricle undergo significant structural and functional remodeling. To understand the mechanisms of this disease, we use a multiscale approach to quantify the relationship between cellular signaling and tissue biomechanics, specifically fibrotic cell signals arising due to stretch and changes in substrate stiffness. Since sex differences are well-established in PAH patients, where the prevalence of PAH in women is much higher but their survival rate is better, we also want to examine how biological sex plays a role in how fibroblasts respond to stretch and stiffness. Given the upregulation of profibrotic gene expression of pulmonary arterial adventitial fibroblasts (PAAFs) and right ventricular cardiac fibroblasts in PAH, in-silico models were made of cell signaling, in-vitro experiments with cultured fibroblasts were conducted, and pulmonary artery tissue from in-vivo rat models of pulmonary arterial hypertension were analyzed.

We studied crosstalk of profibrotic pathways in PAAFs as well as teased apart the effects of stretch and substrate stiffness in mechanical activation of these cells. By conducting in-vitro inhibition studies that the model was able to recapitulate, we demonstrated the usefulness of a computational model that was 80% accurate in simulating experimental designs and robust to parameter and epistemic uncertainty. We used this model to propose crucial experiments on the effects of AngII and TGFβ blockade based on the sensitivity analysis determining that those two cytokines along with stiffness and stretch were the most crucial to PAH pathology. The model was able to qualitatively predict 70% of the in-vitro AngII and TGFβ inhibition experiments, and areas of quantitative mismatch were used to propose differential regulation, missing pathways, and determine the relative importance of specific pathways. Pathways determined to be important in more than one gene’s regulation of expression were stiffness and stretch upregulating TGFβ, AngII and the Hippo pathway, stretch activation of TRP, AngII activating MAPK and AP1 to latent TGFβ, and TGFβ activation of smad2/3 phosphorylation and TAK1 activation of p38. This has led to a model that can be used for predictive high-throughput screening of which target species in the PAAF signaling network can be inhibited to reverse adverse remodeling in PAH.

By combining in-vitro and in-silico data, we were able to tease apart profibrotic responses to individual mechanical stimuli and individual inhibitors. Reducing variables have allowed us to come to important conclusions about the importance of genes at varying time points of the disease due to stretch or stiffness, which is important since marked differences are seen at different weeks of PAH treatment. We learned that expression of Col1a1, Col3a1, and Eln might rise early in-vivo as the increased mPAP in PAH increases vascular wall strain and stays elevated, but Loxl1 and Acta2 expression may rise at first but eventually returns to baseline as wall stiffening becomes severe, while Fn1 may be induced after the ECM stiffens. By varying stretch time from 0, 4, 8, and 24 hours, we were able to find that Col1a1 expression increases progressively while other genes increased at 4 or 8 hours but went back down to baseline, indicating profibrotic responses to stretch may be more transient in some genes.

Isolating cells and tissue from both the pulmonary artery and right ventricle have allowed us to tackle the vascular remodeling in PAH in two different organ systems, which is important to understanding the interplay which leads to RV dysfunction. Ultimate failure of the RV shows survival rate sexual dimorphism in PAH, a sex paradox which has not yet been explained. Our studying of male and female-derived fibroblasts in both the pulmonary artery and right ventricle as well as male and female pulmonary artery and right ventricular tissue has led us to conclude that profibrotic genes are more likely to be activated in male-derived cells and tissue. In PAAFs, female-derived fibroblasts require a higher threshold of stiffness before activation and the profibrotic genes primarily only respond to changes in stiffness not stretch while ovariectomized-derived cells had a higher baseline expression. In RV CFBs, male-derived cells primarily respond to stretch, which given there is demonstrated greater end diastolic pressure in male RVs, means this induction by stretch may lead to adverse remodeling via increased ECM remodeling. There is significant upregulation of all six genes in male-derived PA tissue in sugen-hypoxia animal model of PAH, with only four genes upregulated in female-derived PA tissue and one in ovariectomized-derived PA tissue. The observed variability in response of fibroblasts from male, female, and ovariectomized rats and tissue to PAH conditions exhibits why it is so important to consider sexual dimorphism in disease to explain differences in outcome and is crucial headway into why male patients are less likely to survive PAH due to pressure overload in the remodeled pulmonary arteries leading to pressure overload in the right ventricle.

We used our in-silico model to determine that mechanical stimuli were a main driver of PAH, then iterated upon it to separately probe what pathways affected stretch and stiffness separately. In-vivo data in PAs from male rats demonstrated profibrotic activation in all six genes, while in-vitro data in male-derived PAAFs analyzing those same six genes demonstrated their response to both stiffness and stretch. Adding the variable of sex led to conclusions that mechanoregulation in the cells themselves are responsible for changes in response to stretch and stiffness as PAAFs from female rats were less likely to be activated by stretch and PAs from female rats were overall less induced, while PAAFs and PAs from ovariectomized rats had a high baseline expression and were much less likely to be induced. The variable of time in male PAAF response to stretch demonstrates that not all profibrotic genes respond equally, such as Col1a1 increasing while the other genes have a more transient response. By isolating variables of stretch, stiffness, and sex, we can break down PAH into its important pathways in different sexes.

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