Unfolded protein response in endothelial injury

ABSTRACT

Endothelial barrier dysfunction is associated with sepsis and lung injury, both direct and indirect. We discuss the involvement of unfolded protein response in the protective effects of heat shock protein 90 inhibitors and growth hormone releasing hormone antagonists in the vascular barrier, to reveal new possibilities in acute respiratory distress syndrome treatment.

KEYWORDS:

• Growth hormone
• heat shock proteins
• lung injury
• sepsis

Endothelial barrier dysfunction is associated with sepsis and lung inflammatory disease, including acute respiratory distress syndrome (ARDS) [1]. Targeted therapies for those disorders do not exist, as evident by the mortality rates ofARDS related to COVID-19. Delineation of the intracellular pathways regulating endothelial permeability will contribute toward identification of novel therapeutic targets, to support – and accelerate – the recovery of the impaired endothelium of those hospitalized individuals in need [2]. The current medical countermeasures do not sufficiently reduce the number of ARDS-related deaths, and their corresponding side effects limit long-term use [3].

P53 is a tumor suppressor protein involved in lung health and disease [4,5], which protects the endothelium against LPS-induced breakdown via Rac1 and RhoA modulation [6,7]. Transgenic mice which do not express P53 were more susceptible to lung injury and inflammation, as compared to the wild-type littermates, in bold contrast to rodents expressing more P53 [8]. The antioxidative effects of P53 contribute in those protective events [9], which involve the apurinic/apyrimidinic endonuclease 1/redox effector factor-1 [10]. Interestingly, this endothelial defender [11] is induced due to the application of heat shock protein 90 (Hsp90) inhibitors and growth hormone releasing hormone (GHRH) antagonists (GHRHAnt) in bovine and human lung endothelial cells, as well as in mouse lungs [6,12–14]. The importance of P53 in vascular defense is underscored by recent findings on its role toward the enhancing effects of Metformin – a drug used in diabetes 2 patients – in barrier function [15,16].

Hsp90 is a molecular chaperone, which aids toward the maturation of a plethora of proteins in the intracellular niche, and participates in inflammatory processes [17]. The inhibition of the corresponding cascades suppresses the progression of inflammation [18]. Hence, the application of those compounds is not limited only to cancers, but possess the potential to be helpful in counteracting ARDS, partially due to P53 induction [19]. GHRHAnt are being developed to inhibit malignancies, and cardiovascular complications [20,21]. Those antagonists act through GHRH-specific receptors widely expressed in human tissues, including the lungs [22,23]. GHRH regulates the secretion of growth hormone (GH) from the anterior pituitary gland [24], but its actions are not limited to the GHRH-GH-IGF1 axis [25,26]. Hsp90 inhibitors and GHRHAnt oppose cancers, exert anti-inflammatory activities, protect against LPS-induced hyperpermeability, and reduce bronchoalveolar lavage fluid protein concentration in models of LPS-induced acute lung injury (ALI) [18,27–31].

To identify a common – and potent – mechanism by which those compounds act, we decided to investigate the possibility that can affect the unfolded protein response (UPR). This is a highly conservative mechanism, consisted of three sensors, namely the inositol-requiring enzyme-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) [32]. Upon increased endoplasmic reticulum (ER) stress, cells activate UPR so to resolve unmatured protein aggregation. If that attempt fails, then alternative UPR-mediated pathways are engaged to proceed with cell death via apoptosis [33]. UPR also serves as a highly efficient adaptive mechanism [34], which increases rough ER-mediated activities and cell repair [35,36]. The positive outcomes of ER activation in the endothelial context were largely unknown [37], and it was suggested that global ATF6-mediated protection against disease [38].

We first examined whether Hsp90 inhibitors activate UPR in the lungs, both in vitro and in vivo. We utilized three different inhibitors, each representing a different generation of those compounds [39]. Our observations revealed that UPR branches were activated – as well as their downstream targets – and that those events were not associated with toxic effects [12,14]. GHRHAnt also induced UPR in vitro [13]. However, the effects of targeted UPR manipulation in the barrier function were not interrogated.

To proceed with this task, endothelial cell monolayers were exposed to Kifunensine, a potent inhibitor of the mannosidase I enzyme and UPR reducer. The transendothelial resistance of BPAEC was reduced, and that effect was substantiated by the formation of filamentous actin and enhancement of the severing activity of cofilin [40]. That was the first indication that UPR inhibition negatively affects the stability of the endothelial barrier, an event counteracted by the Hsp90 inhibitor Luminespib and GHRHAnt [13,41]. We also tested the effects of Brefeldin A – a UPR inducer – and Kifunensine in LPS-induced endothelial hyperpermeability. LPS reduces IRE1α in mouse lungs [42]. It was revealed that UPR is a crucial modulator of LPS-mediated breakdown in both human and bovine cells [43]. In a mouse model of LPS-inflicted injury, Tunicamycin – a UPR inducer – suppressed inflammation. Moreover, this compound reduced the paracellular and transendothelial permeability of endothelial monolayers, supporting the protective effects of UPR activation in the compromised endothelium [44]. Interestingly, the previously mentioned effects apply to commercially available brain endothelial cells [45–47], which reside in the blood–brain barrier (BBB). Its breakdown associates with severe neurodegenerative disease [42,48].

Many aspects of the UPR-related barrier function are unknown, as well as the exact role of P53 in those events. P53 interrelates with UPR [49], and the involvement of never-in-mitosis A (NIMA)-related kinases – which affect the stability of P53 [50] – in those events cannot be excluded. In an experimental model of sepsis, it was revealed that the abundance of those kinases – which are involved in cell motility [51,52] – was increased in septic lungs [53]. Efforts to investigate the great depths of those phenomena will most probably lead to new and promising therapeutic avenues toward pathologies related to endothelial barrier function, including ARDS related – or not – to sepsis (e.g. hydrochloric acid-induced ARDS).

Data availability statement

The manuscript does not include data.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Our research is supported by the R&D, Research Competitiveness Subprogram (RCS) of the Louisiana Board of Regents through the Board of Regents Support Fund (LEQSF(2019-22)-RD-A-26) to NB.