Growth Hormone Releasing Hormone in Endothelial Barrier Function

Abstract

Growth hormone releasing hormone (GHRH) is the integral regulator of the growth hormone (GH)–insulin-like growth factor 1 (IGF-1) axis. It exerts mitogenic effects in a plethora of progressive cancers. Recent evidence suggests the emerging role of that 44-amino acid (aa) neuropeptide in lung endothelial barrier function (EBF), which will be discussed herein.

GHRH is a 44-aa peptide released by the hypothalamus to regulate the secretion of GH from the anterior pituitary gland via binding to the pituitary-type GHRH receptor (GHRH-R). GHRH-R is a 423-aa G protein-coupled receptor, predominantly expressed in the somatotrophs, essential for human growth. GH regulates the secretion of IGF-1 in the liver and it is involved in the paracrine production of IGF-1 in other tissues. Hence, GHRH possesses the capacity to affect human homeostasis in multifarious ways [1].

GHRH was first isolated and sequenced from pancreatic tumors, suggesting its possible involvement in the establishment and progression of malignancies. GHRH antagonists were developed to inhibit the tumor-promoting activities of IGF-1. Active splice variants (SVs) of the GHRH-R as well as the ligand itself (GHRH) were detected in a diverse variety of cancers. SV1 exerts ligand-independent activities, and the silencing of the extra-hypothalamic GHRH in breast, prostate, and lung cancers strongly suppressed them. GHRH antagonists counteract the tumor-promoting activities of GHRH and GHRH-specific receptors in malignancies and have been associated with antioxidative activities, at least in part, by inducing P53. P53 is a tumor suppressor protein involved in the protection of the lung microvasculature against inflammatory insults and the maintenance of the lung endothelial barrier [2].

The lung endothelial barrier is a dynamic semi-permeable structure, which regulates the bidirectional transport of biological components (e.g., fluids, proteins, gases) through the vascular wall and the interstitium. The normal function of this barrier is compromised during the development and establishment of severe inflammatory events, including but not limited to direct or indirect lung injury, such as extrapulmonary sepsis and trauma, shock, burn injury, and mass transfusion. A severe form of acute lung injury (ALI) is acute respiratory distress syndrome (ARDS), which represents a lethal form of lung inflammatory disease. Lung endothelial hyperpermeability is the hallmark of ARDS and a prominent effect in phase II of coronavirus disease 2019 (COVID-19). The anti-inflammatory effects of corticosteroids are beneficial in cases of ARDS, but their side effects, which include fluid retention, high blood pressure, mood swings, weight gain, and osteoporosis, burden their long-term clinical applications [3].

The expression of GHRH-specific receptors is not limited to lung cancers. Human and bovine pulmonary microvascular endothelial cells express GHRH-Rs and respond to the corresponding ligand. Hence, we investigated the possibility that the GHRH antagonist MIA-602 enhances the EBF, since it induces the endothelial defender P53, previously found to balance the opposing activities of Rac1/Ras homolog family member A (RhoA) in the lung microvasculature. The Rac1/pCofilin pathway enhances the formation of cortical actin (barrier enhancement) and RhoA/myosin light chain 2 (MLC2) triggers the formation of filamentous actin (F actin) inducing hyperpermeability responses [4].

GHRH antagonists enhanced the transendothelial resistance of endothelial monolayers, while GHRH and the GHRH agonist MR-409 exerted the opposite effects. The quantification of pCofilin and MLC2 protein levels in cells exposed to MIA-602 prior to lipopolysaccharide (LPS), demonstrated the ability of the GHRH-related analogs to affect microvascular integrity through actin cytoskeleton remodeling. The endotoxin lipopolysaccharides (LPS) is found in the outer membrane of the Gram-negative bacteria and triggers the Toll-like receptor 4 (TLR4) signal transduction pathway to activate the extracellular signal-regulated kinases (ERK) 1/2, the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3), the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), as well as the corresponding cytokine and chemokine storm. GHRH-R activation resulted in similar inflammatory events, opposed by the GHRH antagonists. In conclusion, this study associated GHRH and GHRH antagonists with lung EBF, introducing the strong possibility that those antagonists may be of major therapeutic value in diseases related to endothelial barrier dysfunction (EBD) [5].

To further immerse into the unexplored depths of the GHRH-related regulation of the endothelial barrier, we tested the effects of those compounds on the activation of the unfolded protein response (UPR). UPR is triggered upon the accumulation of misfolded proteins above a critical threshold. Three endoplasmic reticulum (ER) transmembrane protein stress sensors comprise this highly conserved molecular machinery (UPR), namely inositol-requiring enzyme 1α (IRE1α), pancreatic endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). They can restore the protein-folding demand and capacity by increasing the ER via the generation of more ER components, by enhancing the efficacy of molecular chaperones, and by degradation of the improperly folded ER polypeptides. When UPR activation fails to alleviate ER stress, then the affected cells will undergo apoptosis [6].

UPR has been associated with the regulation of the EBF and mild UPR activation supports endothelial barrier integrity. Kifunensine (a UPR suppressor) induces hyperpermeability responses and UPR induction due to heat shock protein 90 inhibition counteracted the kifunensine-induced endothelial barrier disruption [7]. Silencing of binding immunoglobulin protein (BiP) (an ER stress marker) in human pulmonary artery endothelial cells promoted F-actin formation and increased EBD. The low-density lipoprotein (LDL)-induced inflammatory responses in human mesangial cells were reduced after IRE1α silencing. Pretreatment of those cells with tunicamycin (a UPR inducer) attenuated the LDL-induced proinflammatory cytokine storm [8]. Since GHRH antagonists were previously reported to induce CHOP (a UPR activation marker) [9], we investigated the hypothesis that UPR participates in the effects of the GHRH antagonists.

UPR was involved in the protective effects of GHRH antagonists in bovine pulmonary endothelial cells. More specifically, GHRH antagonists activated IRE1a, PERK, and ATF6. That effect was reflected in the expression levels of BiP, protein disulfide isomerase (PDI), and endoplasmic oxidoreductin-1 (ERO1-La). However, the GHRH agonist MR-409 delivered the opposite effects. To examine the effects of the GHRH antagonists in kifunensine-inflicted EBD, we tested the effects of those peptides in cells subjected to kifunensine treatment. GHRH antagonists suppressed the remodeling of the actin cytoskeleton due to this mannosidases inhibitor and counteracted the kifunensine-induced decrease in the transendothelial resistance of those lung cells [10]. Hence, GHRH antagonists employ UPR to enhance the function of the microvasculature. The exact interrelations leading to those events are to be elucidated.

Cancers employ molecular chaperones (e.g., heat shock protein 90) or adaptive mechanisms (e.g., UPR) to overcome environments deprived of oxygen or nutrients. Hence, malignancies depict increased UPR activities. Since GHRH antagonists induce UPR, and ER stress increase beyond a threshold triggers apoptosis, GHRH antagonists may selectively eliminate cancers expressing GHRH-Rs via robust and irreparable increases of ER stress [11]. Furthermore, GHRH has been shown to increase the secretion of several chemokines and cytokines (e.g., interferon-gamma, interleukin 6) from human peripheral blood mononuclear cells [12], which express GHRH-Rs [13]. GHRH antagonists opposed those effects [12]. Thus, GHRH antagonists may suppress the pathogenesis of ARDS through the obstruction of the corresponding inflammatory pathways.

Dr Schally’s group suggested that GHRH agonists suppress the growth of lung cancers in vivo, when they are applied for prolonged periods. That unexpected finding was explained by providing evidence that the GHRH agonist MR409 downregulates GHRH-Rs. A similar effect appears in the context of pituitary receptors chronically exposed to luteinizing hormone-releasing hormone (LHRH) agonists and has been applied towards the management of LHRH-dependent cancers [14]. Since GHRH-Rs modulate lung inflammation and fibrosis [15], the possibility that chronic administration of GHRH agonists may deliver protective effects against EBD cannot be excluded.

The development of medical countermeasures to oppose ARDS is of the utmost need. Pharmacological interventions to stochastically accelerate the recovery of the impaired lung microvasculature will provide exciting opportunities in therapeutics. GHRH-related analogs may serve that purpose, since they are involved in the regulation of EBF and the secretion of several inflammatory mediators (Figure 1).

Figure 1. Growth Hormone Releasing Hormone Antagonists (GHRHAnt) Support Endothelial Barrier Function.

GHRHAnt activate the unfolded protein response (UPR), which in turn induces P53. That tumor suppressor possesses anti-inflammatory and antioxidative activities in the lungs, suppresses generation of reactive oxygen species (ROS), and enhances endothelial barrier integrity via Rac1-mediated cofilin deactivation and the suppression of filamentous actin (F-actin). The latter event is mediated through the blocking of the Ras homolog family member A (RhoA)/myosin light chain 2 (MLC2) pathway. Thus, GHRHAnt oppose the endothelial barrier dysfunction (EBD) due to lung hyperpermeability. This feature is the hallmark of acute respiratory distress syndrome (ARDS).