Vascular AFs are much more sensitive to environmental stresses and AFs have been noted to display the earliest and most rapid increase in proliferation compared to vascular SMCs and ECs [24]. Once AFs are activated, they differentiate into MFs, which synthesize and release more proinflammatory cytokines, ROS, and neovascular growth factors, further aggravating vascular remodeling [25]. Emerging evidence suggests that targeting the aggressive phenotypic transformation and proinflammatory properties of AFs is a novel approach for treatment of pulmonary vascular remodeling in PAH [11–13], suggesting that the identification of new molecules involved in the phenotypic transformation of AFs may provide a scientific basis for uncovering potential therapeutic targets for treatment of CHD-PAH.
The ubiquitin-proteasome system (UPS), the most important pathway for intracellular, selective protein degradation, can modulate TGF-β1-induced phenotypic transformation and proliferation of AFs and SMCs, as well as the functions of ECs at multiple levels and with multiple targets, thus participating in the regulation of vascular remodeling [26, 27]. CYLD, a deubiquitinating enzyme, was initially identified as a gene involved in familial cylindromatosis. CYLD directly mediates multiple key signaling pathways, such as NF-κB and MAPK signaling pathways, by its catalytic activity on polyubiquitinated key intermediates [28]. Our previous study indicated that CYLD was overexpressed in CHD-PAH-associated lung tissues and positively correlated with the degree of pulmonary vascular remodeling, while its suppression mitigated increased PAH and its associated pulmonary vascular remodeling in rats with flow-related PAH [18]. However, little is known regarding its role in the phenotypic transformation of HPAAFs. This study demonstrated that treatment with TGF-β1 significantly increased CYLD expression in HPAAFs, while CYLD suppression not only attenuated the expression of proinflammatory cytokines, ECM, α-SMA, SM22a, and ROS in HPAAFs but also decreased cell proliferation and migration, as well as facilitated cell apoptosis, implying that the phenotypic transformation of HPAAFs is cross-regulated by CYLD and TGF-β1, which was consistent with a previous report on aortic AFs [17]. On the contrary, it has been reported that this system is controlled by an autoregulatory feedback loop, in which TNF-α-induced CYLD expression in turn results in suppression of TNF-α-induced inflammation [29, 30]. These discrepancies might be because of the diverse roles of CYLD and the complex effects of CYLD in the phenotypic transformation of different cells, which needs further exploration.
Importantly, our study revealed that TGF-β1 not only enhanced the expression of Nrf2 in HPAAFs but also increased nuclear translocation of Nrf2, which was consistent with a previous study showing that TGF-β1 induced the activation of Nrf2 in human aortic AFs [23]. TGF-β1 enhances the formation of ROS, which can promote the phenotypic transformation of SMCs and AFs, contributing to pulmonary vascular remodeling [17, 31–33]. Huang et al. recently demonstrated that TGF-β1 led to strongly increased ROS production in mice [34]. Briefly, TGF-β1 can elicit Nrf2 expression and ROS formation in HPAAFs.
Nrf2 is the major mediator of ROS-induced fibroblast activation, playing a key role in TGF-β1 signaling, ultimately resulting in the transformation of fibroblasts into an active MF phenotype [21–23]. Nrf2 knockdown exacerbated pulmonary vascular remodeling by increasing the production of ROS to enhance the phenotypic transformation of vascular SMCs and endothelial- to-mesenchymal transition [22, 35]. Our study showed that CYLD knockdown increased TGF-β1-induced activation of Nrf2 in HPAAFs, while additional Nrf2 knockdown in CYLD-knockdown HPAAFs restored TGF-β1-induced ROS formation and production of proinflammatory cytokines, ECM, α-SMA, and SM22α; this strongly authenticated that CYLD exaggerated pressure overload-induced cardiac maladaptive remodeling and dysfunction via downregulation of Nrf2 and ROS [36]. Overall, these data showed that CYLD knockdown enhanced the activation of Nrf2, thus reducing ROS production, which led to the amelioration of TGF-β1-induced transdifferentiation of HPAAFs into MFs. However, it is unlikely that CYLD can directly regulate Nrf2 expression, and the precise mechanisms underlying their interaction remain unclear and require further study.