Link, Patrick A.

AbstractPulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell‐specific approach. Our previous work demonstrated that targeting non‐canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non‐canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH‐associated PASMC remodeling, with longer‐lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non‐canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.

Aberrant vascular remodeling contributes to the progression of many aging-associated diseases, including idiopathic pulmonary fibrosis (IPF), where heterogeneous capillary density, endothelial transcriptional alterations, and increased vascular permeability correlate with poor disease outcomes. Thus, identifying disease-driving mechanisms in the pulmonary vasculature may be a promising strategy to limit IPF progression. Here, we identified Ccn3 as an endothelial-derived factor that is upregulated in resolving but not in persistent lung fibrosis in mice, and whose function is critical for vascular homeostasis and repair. Loss and gain of function experiments were carried out to test the role of CCN3 in lung microvascular endothelial function in vitro through RNAi and the addition of recombinant human CCN3 protein, respectively. Endothelial migration, permeability, proliferation, and in vitro angiogenesis were tested in cultured human lung microvascular endothelial cells (ECs). Loss of CCN3 in lung ECs resulted in transcriptional alterations along with impaired wound-healing responses, in vitro angiogenesis, barrier integrity as well as an increased profibrotic activity through paracrine signals, whereas the addition of recombinant CCN3 augmented endothelial function. Altogether, our results demonstrate that the matricellular protein CCN3 plays an important role in lung endothelial function and could serve as a promising therapeutic target to facilitate vascular repair and promote lung fibrosis resolution.

AbstractPulmonary arterial hypertension (PAH) is a progressive, devastating disease, and its main histological manifestation is an occlusive pulmonary arteriopathy. One important functional component of PAH is aberrant endothelial cell (EC) function including apoptosis‐resistance, unchecked proliferation, and impaired migration. The mechanisms leading to and maintaining physiologic and aberrant EC function are not fully understood. Here, we tested the hypothesis that in PAH, ECs have increased expression of the transmembrane protein integrin‐β5, which contributes to migration and survival under physiologic and pathological conditions, but also to endothelial‐to‐mesenchymal transition (EnMT). We found that elevated integrin‐β5 expression in pulmonary artery lesions and lung tissue from PAH patients and rats with PH induced by chronic hypoxia and injection of CD117+ rat lung EC clones. These EC clones exhibited elevated expression of integrin‐β5 and its heterodimerization partner integrin‐αν and showed accelerated barrier formation. Inhibition of integrin‐ανβ5 in vitro partially blocked transforming growth factor (TGF)‐β1‐induced EnMT gene expression in rat lung control ECs and less in rat lung EC clones and human lung microvascular ECs. Inhibition of integrin‐ανβ5 promoted endothelial dysfunction as shown by reduced migration in a scratch assay and increased apoptosis in synergism with TGF‐β1. In vivo, blocking of integrin‐ανβ5 exaggerated PH induced by chronic hypoxia and CD117+ EC clones in rats. In summary, we found a role for integrin‐ανβ5 in lung endothelial survival and migration, but also a partial contribution to TGF‐β1‐induced EnMT gene expression. Our results suggest that integrin‐ανβ5 is required for physiologic function of ECs and lung vascular homeostasis.

Retinal pigmented epithelial (RPE) cells play an important role in retinal fibrotic diseases such as proliferative vitreoretinopathy (PVR). The purpose of this study was to elucidate the involvement of dopamine receptor signaling in regulating the fibrotic activation of RPE cells. Dopamine receptor expression, the effect of dopamine on fibrotic activity, and dopamine production were measured in the human RPE cell line ARPE-19. The fibrotic activation of RPE cells was evaluated in response to treatments with selective dopamine receptor agonists and antagonists by measuring gene expression, migration, proliferation, and fibronectin deposition. DRD2 and DRD5 are the dominant dopaminergic receptors expressed in ARPE-19 cells and TGF-β stimulation enhances the autocrine release of dopamine, which we show further exasperates fibrotic activation. Finally, treatment with D2 dopamine receptor antagonists or D5 dopamine receptor agonists inhibits profibrotic gene expression, migration, proliferation, and fibronectin deposition and thus may serve as effective mechanisms for treating retinal fibrosis including PVR.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcription cofactors implicated in the contractile and profibrotic activation of fibroblasts. Fibroblast contractile function is important in alveologenesis and in lung wound healing and fibrosis. As paralogs, YAP and TAZ may have independent or redundant roles in regulating transcriptional programs and contractile function. Using IMR-90 lung fibroblasts, microarray analysis, and traction microscopy, we tested whether independent YAP or TAZ knockdown alone was sufficient to limit transcriptional activation and contraction in vitro. Our results demonstrate limited effects of knockdown of either YAP or TAZ alone, with more robust transcriptional and functional effects observed with combined knockdown, consistent with cooperation or redundancy of YAP and TAZ in transforming growth factor β1 (TGFβ1)-induced fibroblast activation and contractile force generation. The transcriptional responses to combined YAP/TAZ knockdown were focused on a relatively small subset of genes with prominent overrepresentation of genes implicated in contraction and migration. To explore potential disease relevance of our findings, we tested primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and confirmed that YAP and TAZ combined knockdown reduced the expression of three cytoskeletal genes, ACTA2, CNN1, and TAGLN. We then compared the contribution of these genes, along with YAP and TAZ, to contractile function. Combined knockdown targeting YAP/TAZ was more effective than targeting any of the individual cytoskeletal genes in reducing contractile function. Together, our results demonstrate that YAP and TAZ combine to regulate a multigene program that is essential to fibroblast contractile function.

AbstractYes‐associated protein (YAP) and PDZ‐binding motif (TAZ) have emerged as important regulators of pathologic fibroblast activation in fibrotic diseases. Agonism of Gαs‐coupled G protein coupled receptors (GPCRs) provides an attractive approach to inhibit the nuclear localization and function of YAP and TAZ in fibroblasts that inhibits or reverses their pathological activation. Agonism of the dopamine D1 GPCR has proven effective in preclinical models of lung and liver fibrosis. However, the molecular mechanisms coupling GPCR agonism to YAP and TAZ inactivation in fibroblasts remain incompletely understood. Here, using human lung fibroblasts, we identify critical roles for the cAMP effectors EPAC1/2, the small GTPase RAP2c, and the serine/threonine kinase MAP4K7 as the essential elements in the downstream signaling cascade linking GPCR agonism to LATS1/2‐mediated YAP and TAZ phosphorylation and nuclear exclusion in fibroblasts. We further show that this EPAC/RAP2c/MAP4K7 signaling cascade is essential to the effects of dopamine D1 receptor agonism on reducing fibroblast proliferation, contraction, and extracellular matrix production. Targeted modulation of this cascade in fibroblasts may prove a useful strategy to regulate YAP and TAZ signaling and fibroblast activities central to tissue repair and fibrosis.