Growth Hormone–Releasing Hormone in Endothelial Inflammation

Abstract

The discovery of hypothalamic hormones propelled exciting advances in pharmacotherapy and improved life quality worldwide. Growth hormone–releasing hormone (GHRH) is a crucial element in homeostasis maintenance, and regulates the release of growth hormone from the anterior pituitary gland. Accumulating evidence suggests that this neuropeptide can also promote malignancies, as well as inflammation. Our review is focused on the role of that 44 - amino acid peptide (GHRH) and its antagonists in inflammation and vascular function, summarizing recent findings in the corresponding field. Preclinical studies demonstrate the protective role of GHRH antagonists against endothelial barrier dysfunction, suggesting that the development of those peptides may lead to new therapies against pathologies related to vascular remodeling (eg, sepsis, acute respiratory distress syndrome). Targeted therapies for those diseases do not exist.

Growth Hormone–Releasing Hormone

Growth hormone–releasing hormone (GHRH) is a 44 - amino acid hormone that binds to specific GHRH receptors on somatotrophs of the anterior pituitary gland to regulate the synthesis and secretion of growth hormone (1). The ectopic production of GHRH by carcinoid and pancreatic tumors facilitated the isolation, characterization, and sequencing of this neuropeptide (2, 3). The full biological activity of GHRH is retained in the first 29 - amino acid sequence (3). The predominant source of GHRH production is the hypothalamus, but it can be also expressed in a diverse variety of tissues (e.g. lung, ovary, testis, kidney, liver) (4).

The pituitary-type GHRH receptor (GHRHR) is a member of the seven-transmembrane class B G-protein coupled receptor family, which includes receptors for vasoactive intestinal peptide, pituitary adenylyl cyclase-activating peptide, secretin, glucagon, glucagon-like peptides, calcitonin, and gastric inhibitory polypeptide (5). GHRHR activates adenylate cyclase-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA), calcium (Ca²⁺)-calmodulin complex, and inositol phosphate-diacylglycerol-protein kinase C. cAMP stimulates PKA to activate the cAMP response element binding protein to regulate GHRHR gene transcription (6).

GHRHR and its splice variants (SVs) have been identified in various human cancers such as lymphoma, glioblastoma, pancreatic cancer, and small cell lung carcinoma, underscoring the role of GHRH and its receptors in cancer pathophysiology (7). Inhibition of intrinsic GHRH expression by small interfering RNA suppressed breast, lung, and prostate cancers. The exogenous GHRH supplementation restored their growth, supporting the involvement of GHRH-related pathways in tumor enlargement (8).

GHRH antagonists (GHRHAnt) (eg, MZ-4-71, MZ-5-156, JV-1-62, and JV-1-63) were developed to oppose the growth-factor activities of GHRH in malignancies (9, 10). Those peptides suppress a diverse variety of malignancies, including pleural mesothelioma (11), ovarian (12), lung, breast, and prostate cancer (13–15). The antitumor activities of GHRHAnt are partially exerted due to insulin-like growth factor I (IGF-I) inhibition, which has been involved in malignancies. A positive correlation between plasma IGF-I levels and a risk to develop colorectal, breast, and prostate cancer was also reported (15).

GHRHAnt can also act by blocking GHRHR/SVs activation (16, 17). SV1, the main SV of GHRHR, was reported to possess ligand-independent activities and exist in hypothalamic tissues (8, 18, 19). GHRH activates the extracellular signal-regulated kinase 1/2 (ERK1/2) (20) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) (21); promoting malignancies. GHRHAnt counteract those effects (21-23). Those peptides can also inhibit the proliferation of non-metastatic cells (eg, benign prostate hyperplasia) (24, 25). Interestingly, recent evidence supports the implication of GHRHAnt in endothelial barrier function (26, 27), introducing a novel field of investigation for diseases related to endothelial inflammation and dysregulation [eg, sepsis, acute respiratory distress syndrome (ARDS)] (28, 29).

Endothelial Responses in Inflammation

The endothelial cells (EC) form a semipermeable monolayer between blood and the surrounding tissues (30). They cover basement membrane with their basolateral side and are exposed to lumen with their apical side (31). In resting conditions, the endothelium maintains blood fluidity and controls vessel permeability to permit the passage of solutes and small molecules (32). It also restricts extravasation of larger components, regulates blood flow, and quiesces circulating leukocytes (33). In sepsis and ARDS the junctions of the endothelium become dysfunctional, inducing hyperpermeability responses (28, 34). Endothelial barrier dysfunction facilitates neutrophil reverse transendothelial cell migration by promoting the movement of interstitial chemokines into the bloodstream. That effect causes systemic dissemination of reverse transendothelial cell migration neutrophils to induce pathological effects in remote organs, especially the lungs (35).

Two categories of EC activation exist (33, 36). Type 1 includes rapid responses that export Weibel-Palade bodies to release the von Willebrand factor and P-selectin. Those events initiate the interaction of leukocytes and platelets with EC. Type 2 activation is associated with more sustained inflammatory responses, requiring expression of proinflammatory cytokines and adhesion molecules (36). The prototypic mediators of type 2 activation are the tumor necrosis factor α (TNFα) and interleukin (IL)-1, derived primarily from activated leukocytes (33).

Inflammation and GHRH

Since cancer and inflammation frequently coexist (37, 38), it wasexpected that the anti-inflammatory activities of GHRHAnt contribute to their anticancer effects (23). Inflammation engages with endothelial barrier dysfunction in a reciprocal manner, to induce endothelial leak and injury. Below we describe major factors affecting endothelial function in the context of GHRH.

Reactive Oxygen Species), Endothelial Function and GHRH

Reactive oxygen species in the vasculature

Reactive oxygen species (ROS) are generated from partial reduction of oxygen molecules during normal cellular metabolism and exert a pivotal role in redox homeostasis. However, excessive ROS accumulation impairs barrier function, contributing to disease development (39, 40). The endothelium is a source of ROS and reactive nitrogen species; which regulate endothelial cell migration, metabolism, proliferation, angiogenesis, and barrier function (41). The exact molecular mechanisms of ROS-induced endothelial hyperpermeability are not fully understood.

GHRHAnt and ROS

GHRHAnt exert antioxidative effects in the lung and brain microvascular endothelial cells (42, 43). Those peptides can suppress hydrogen peroxide–induced breakdown of brain endothelium by suppressing cofilin and inhibiting the RhoA-mediated formation of F actin. Hence, those observations suggest that GHRHAnt may exert the potential to treat blood-brain barrier dysfunction–related pathologies, including Parkinson's and Alzheimer's disease (43). GHRHAnt reduce ROS (44), and the capacity of those peptides to cross the blood-brain barrier enhances the enthusiasm regarding that possibility (45).

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, increases endothelial oxidative stress (46, 47) and augments bronchoalveolar lavage fluid protein concentration. The GHRHAnt MIA-602 was able to inhibit LPS-induced endothelial leakage, as indicated by the reduction of bronchoalveolar lavage fluid protein concentration in vivo (48). In the brain endothelium, treatment with GHRHAnt significantly reduces LPS-induced pro-oxidative markers including lactate dehydrogenase, nitrite, and cyclooxygenase-2 (COX2) (49). The aged brain encounters toxic levels of ROS, linked to age-associated cognitive decline (50). The GHRHAnt MZ-5-156 reduced oxidative stress in aging mice. That was reflected in glutathione and glutathione peroxidase expression levels and improved cognition (51). Previous studies have reported the suppression of COX2 and cytochrome c oxidase IV due to GHRHAnt, introducing that important concept in the relevant field (4, 52).

Protein folding may cause ROS generation during electron transfer between protein disulfide isomerase and endoplasmic reticulum (ER) oxidoreductase 1α. Unfolded protein response (UPR) promotes antioxidant responses via modulation of nuclear factor erythroid 2-related factor 2 and nicotinamide adenine dinucleotide phosphate oxidase (53). Recent findings on the activation of UPR by GHRHAnt (26, 54) suggest a novel pathway by which GHRHAnt exert their antioxidative effects in human tissues, which also involves P53 (55). P53 induction suppresses the lipid peroxidation marker malondialdehyde in lung (56) and brain (57) endothelial cells, and enhances barrier function (55, 58).

GHRHAnt induce the expression of P53 in endothelial cells (27), indicating that the antioxidant effects of these peptides may depend on P53 and its phosphorylation (59). Interestingly, UPR regulates endothelial P53 expression. Bovine cells exposed to the UPR inducers brefeldin A, dithiothreitol, and thapsigargin increased P53 abundance, while the UPR inhibitors N-acetyl cysteine, Kifunensine, and adenosine triphosphate-competitive inositol-requiring enzyme-1α (IRE1α) kinase-inhibiting RNase attenuator produced the opposite effects (60). Hence, the delineation of GHRHAnt/UPR/P53 interrelation in the context of redox regulation may provide novel insights towards mechanisms mediating the oxidative stress–induced endothelial hyperpermeability.

Ca²⁺ and GHRHAnt

The binding of GHRH to its receptors results in the activation of adenylyl cyclase, which produces cAMP, leading to activation of PKA. That kinase stimulates an influx of calcium through activation of voltage-sensitive Ca²⁺ channels and plasma membrane depolarization in pituitary somatotrophs (6). GHRH agonists can also affect cytosolic free Ca²⁺ levels in somatotrophs (61). Besides somatotrophs, GHRH and its analogs affect intracellular Ca²⁺ levels in other cell types, such as human embryonic kidney 293 cells (62) and GABAergic neurons (63). Interestingly, those peptides (e.g., JI-34 and JI-36) evoke a biphasic increase of Ca²⁺ concentration in human embryonic kidney 293 cells, which express ghrelin receptor. The GHRHAnt JV-1–42 and JMR-132 suppress those effects in a dose-dependent manner (62). Moreover, intracellular Ca²⁺ regulates the release of insulin from pancreatic beta cells. Those cells can sense changes of blood glucose concentration and respond by secreting insulin. Glucose-stimulated insulin secretion is Ca²⁺-dependent. GHRH agonists increase the level of cAMP in the rat pancreatic β-cell line (64), increasing cytoplasmic Ca²⁺ concentration (65). The GHRH agonists MR-409 and MR-356 upregulate cell proliferation and increase the expression of cellular insulin and insulin-like growth factor 1 (IGF-I) in rat pancreatic β-cell line cells (64). Those analogs can also dramatically reduce the severity of streptozotocin-induced diabetes in vivo (64). Those findings suggest potential therapeutic effectiveness of GHRH agonists in diabetes and its complications.

A rapid increase in cytosolic free Ca²⁺ concentration activate Ca²⁺-dependent nucleases, which degrade chromosomal DNA (66). GHRHAnt can also promote apoptosis in LNCaP human prostate cancer cells through Ca²⁺-dependent pathways. The GHRHAnt JV-1-38 increases intracellular free Ca²⁺ concentration in LNCaP, which in turn elevates phosphatidylserine externalization, a marker of early apoptosis (61, 67). The Ca²⁺ chelator EDTA or voltage-gated Ca²⁺ channel blocker nifedipine significantly reduces Ca²⁺ concentration and early-stage apoptosis, induced by JV-1-38. Those results suggest that the antiproliferative effects of JV-1-38 in prostate cancer cells are primarily mediated by extracellular Ca²⁺ entry through voltage-operated Ca²⁺ channels (61).

GHRHAnt and Inflammatory Mediators

Nuclear Factor Kappa B and GHRHAnt

GHRHR activation and nuclear factor kappa B (NF-κB) are interconnected. In human ciliary epithelial cells, LPS activates GHRHR through the NF-κB subunit p65. Similarly, ectopic expression of p65 dramatically increases the mRNA levels of GHRHR, which suggests that p65 is a positive regulator of GHRHR expression (68). In a rat model of endotoxin-induced uveitis, as well as in human ciliary epithelial cells, GHRHAnt can partially alleviate inflammatory responses. Those findings indicate a potential therapeutic approach to alleviate acute ocular inflammation (68). Moreover, LPS triggers NF-κB and increases the expression of inflammatory and pro-oxidative markers in mouse prefrontal cortex. GHRHAnt exert anti-inflammatory and antioxidative effects, and demonstrate anxiolytic and antidepressant-like effects in the brain (49).

GHRHAnt were initially developed to suppress cancers. The anti-cancer effects of those peptides are mediated by (1) the inhibition of pituitary growth hormone release, which results in a decrease of hepatic IGF-I secretion; (2) suppression of autocrine or paracrine GHRH binding to the GHRH receptors; and (3) blockage of autocrine and/or paracrine production of tumoral IGF-I and IGF-II (15). Moreover, the Ikappa B kinase/NF-κB signaling is often altered in human cancers. Aberrant NF-κB regulation promotes cancers through transcriptional activation of genes associated with cell proliferation, angiogenesis, metastasis, inflammation, and suppression of apoptosis (69).

GHRHAnt significantly inhibit the activation of NF-κB in different cancers. MIA-690, a GHRHAnt, downregulates this pathway to attenuate colon cancer progression in vivo (70). The GHRHAnt JMR-132 inhibits NF-κB activation by suppressing the phosphorylated form of NF-κB p50 in prostate cancer cells, while GHRH exerts opposite results (52). NF-κB suppression is also associated with the antineoplastic effects of GHRHAnt in gastric cancer (71) as well as esophageal squamous cell carcinoma (72). P53 is the potential mediator of GHRHAnt-induced NF-κB suppression. The transcriptional activation of NF-κB is downregulated by P53 (73), and GHRHAnt induce P53 expression (74). Thus, the inhibitory function of GHRHAnt on NF-κB may be mediated by P53 (23, 75, 76); however, more intense investigations are needed to elucidate those events.

Mitogen-Activated Protein Kinase and GHRHAnt

GHRHAnt suppress P38 mitogen-activated protein kinase (MAPK) and ERK1/2. Both pathways increase endothelial permeability (77, 78). ERK1/2 activation mediates vascular endothelial growth factor and histamine-triggered injury in porcine coronary venules (79). Activation of c-Jun N-terminal kinase and P38 MAPK results in monocyte chemoattractant protein-1 (MCP-1), TNF-α, IL-6, and IL-1β increased protein expression both in vitro and in vivo (80). It can also trigger the activation of NF-κB (81). GHRHAnt suppress the phosphorylation and activation of ERK1/2 in lung endothelial cells, while GHRH and MR-409—a GHRH agonist—exert the opposite effects (27). Similar effects were observed in prostate cancer cells (82). Of note, the inhibition of heat shock protein 90 results in suppression of MAPK (83).

JAK2/STAT3 and GHRHAnt

Treatment of endothelial cells with cholinergic agonist (84) or laminar shear stress (85) exert protective effects via JAK2/STAT3 suppression. GHRHAnt significantly inhibit the phosphorylation of JAK2/STAT3 in bovine pulmonary artery endothelial cells, whereas GHRH and GHRH agonists activate that pathway (27). These antagonists can also counteract LPS-induced endothelial damage, suggesting that inhibition of JAK2/STAT3 is involved in GHRHAnt-mediated endothelial protection (27).

The role of GHRHR in acute ocular inflammation has also been investigated. In a rat model of endotoxin-induced uveitis, LPS induces the expression of GHRHR, which physically interacts with JAK2 and activates the JAK2/STAT3 pathway. That effect augments the production of IL-6, IL-17A, COX2, and inducible nitric oxide synthase (iNOS) (68). MIA-602—a GHRHAnt—counteracted JAK2/STAT3 activation (68). JAK2 is associated with the signal transduction of growth hormone receptor (86). It has been reported that JAK/STAT pathway can be activated by GHRH, and GHRHAnt oppose those effects in cancer cells (21). Moreover, MIA-602 reduces the risk of gastric cancer by downregulating PAK1 and STAT3 (71).

Phosphoinositide 3-kinase/Akt and permeability

Phosphoinositide 3-kinase (PI3K)/Akt regulates cell growth, proliferation, metabolism, and migration. The Akt kinase family includes Akt1, Akt2, and Akt3 (87). PI3K/Akt activation is involved in multiple sclerosis, rheumatoid arthritis, psoriasis, autoimmune diabetes, and chronic obstructive pulmonary disease (88). The major Akt isoform—namely Akt1—is expressed in endothelial cells and promotes microvascular leakage in response to histamine. Akt1 knockout mice are resilient against leukocyte infiltration (89). GHRHAnt prevented androgen-independent prostate cancer progression via Akt and ERK inhibition (82). Moreover, JMR-132 inhibits the epidermal growth factor receptor and Akt pathways, suppressing the growth of ovarian cancer cells (90). GHRHAnt MIA-602 blocked bleomycin-induced activation of the PI3K-Akt pathway, resulting in amelioration of lung fibrosis (91). GHRHAnt can also enhance the tumor suppressor AMP-activated protein kinase in A549 lung cancer cells (92).

UPR and GHRHAnt

UPR

ER is a tubular-reticular network separated from the surrounding cytosol by a single lipid bilayer ER membrane. Its major function is to synthesize and fold secreted and transmembrane proteins, which constitute approximately one-third of all protein content. ER is involved in calcium homeostasis, lipid and steroid synthesis, as well as carbohydrate metabolism (93). Disruption of ER homeostasis due to increased secretory load, hypoxia, nutrient deprivation, viral infection, and oxidative stress results in the accumulation of misfolded or unfolded proteins, a condition referred to as ER stress (94). To restore ER homeostasis, cells developed an evolutionarily conserved signal transduction mechanism, namely UPR (95).

UPR functions via protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and IRE1α (94). Upon activation, UPR increases ER volume, augments the expression of ER chaperons to enhance protein folding capacity, and reduces protein synthesis. It also triggers the ER-associated degradation pathway to remove misfolded proteins from the ER lumen (96).

UPR and barrier function

UPR exerts a prominent role in the maintenance of pulmonary (97) and cardiovascular system (98, 99). Vascular dysfunction is a pivotal cause for the pathogenesis of ARDS (100). The role of UPR in endothelial barrier function is the focus of recent investigations, since it is involved in endothelial permeability (101). The UPR suppressor Kifunensine potentiates cytoskeletal remodeling of lung endothelial cells by activating MLC2 and cofilin (102, 103). Moreover, it appears that the heat shock protein 90 inhibitors (104, 105) and GHRHAnt (54) utilize UPR to reduce ROS generation and to suppress inflammation (23, 68) in the endothelium. Binding immunoglobulin protein (BiP) is a downstream UPR target (106). Knockdown or inactivation of BiP stimulates actin cytoskeletal reorganization, indicating that BiP is involved in vascular function and inflammation (107).

In mouse mammary gland tumors, PERK is downregulated due to Akt activation (108). Cardiac-specific PERK knockout mice exhibit increased left ventricular fibrosis and cardiomyocyte apoptosis, and exacerbated lung remodeling in response to chronic traverse aortic constriction (109). In addition, ATF6 exhibits protection against lung endothelial hyperpermeability due to LPS. Targeted pharmacological activation of this pathway by AA147 prevents LPS-induced cofilin and MLC2 activation, as well as VE-cadherin phosphorylation. This ATF6 activator can also counteract the LPS-triggered decrease in transendothelial resistance, and increased paracellular permeability (110). Inhibition of ATF6 facilitates endothelial barrier disruption, suggesting that it may serve as a possible therapeutic target for diseases related to barrier dysfunction (110).

Transgenic mice overexpressing ATF6 in their cardiomyocytes exhibit better functional recovery from ex vivo ischemia/reperfusion and are protected against necrosis and apoptosis (111). Activation of ATF6 exerts protection against acute myocardial infarction (112). Indeed, ATF6 and IRE1 are associated with prosurvival pathways against ischemic injury (111, 113), and similar observations were reported in ischemic stroke brain. Reduced infarct volume and improved functional outcomes were observed in ATF6 knock-in mice 24 hours after stroke. Autophagy at early reperfusion after stroke may contribute to ATF6-mediated neuroprotection (114).

Preconditioning of human retinal microvascular endothelial cells with the UPR inducer tunicamycin suppressed TNFα-induced NF-κB activation and adhesion molecules (i.e., intercellular cell adhesion molecule-1, VCAM1). Hence, that UPR inducer ameliorates retinal vascular leakage. In contrast, silencing of X-box binding protein 1—a downstream target of IRE1α—attenuated the effects of preconditioning in inflammation (115).

GHRHAnt and UPR

The effects of GHRHAnt on the modulation of UPR are not completely understood. Recent in vitro studies on lung endothelial cells demonstrate the association of UPR in GHRH-related endothelial barrier modulation. The GHRHAnt MIA-602 significantly induces the expression of all three UPR sensors (IRE1α, PERK, and ATF6). That effect was also reflected by the upregulation of BiP, protein disulfide isomerase, and endoplasmic oxidoreductin-1. In contrast, GHRH agonist MR-409 exerts the opposite effects (54). Moreover, GHRHAnt suppress the remodeling of actin cytoskeleton due to UPR suppressor Kifunensine and counteract Kifunensine-induced decrease in transendothelial resistance value (54). The IGF-I axis (116) has also been shown to induce RhoA (117), supporting the aforementioned events. In addition, UPR activation prevents TNF-α-induced iNOS expression in human retinal endothelial cells (115), while GHRHAnt suppress iNOS (118). Interestingly, a positive correlation between UPR activation and P53 expression in lung endothelium was observed (60).

P53 supports the endothelium against inflammation by suppressing the apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (119), NF-κB (55), RhoA (120), and lipid peroxidation (56, 57). Hence, the UPR-induced barrier protection may be orchestrated by P53. The exact interrelations of P53 and UPR-related components in the context of the microvasculature are not completely understood. Future studies on identifying the specific pathways involved in endothelial barrier protection will probably assist in discovering novel therapeutic interventions against diseases related to endothelial hyperpermeability. Utilizing GHRHAnt in events of mass casualties (e.g., use of hydrochloric acid as a chemical weapon) to restore barrier function cannot be excluded (121).

Sites of chronic inflammation can promote malignancies (122). During prolonged ER stress, UPR shifts to a proapoptotic mechanism to eliminate impaired cells, and, therefore, GHRHAnt may selectively eradicate cancers via robust ER stress induction (123). In the placental choriocarcinoma cell line (JEG-3), GHRHAnt JMR-132 induces BiP and eIF2α phosphorylation. This peptide inhibits cell viability by inducing apoptosis through activation of caspase-3, as well as by suppressing Akt phosphorylation (124). Pretreatment with eIF2α inhibitor abolishes JMR-132–induced cleaved caspase-3 expression and promotes cell growth. Those events demonstrate that GHRH analogs are involved in JEG-3 cell viability and apoptosis through Akt and eIF2α (124).

ADP-ribosylation factor (Arf) is a small GTP-binding (G) protein highly expressed in a diverse variety of cancers (i.e., breast, lung, colon, ovarian, prostate) (125). Since brefeldin A inhibits Arf activation (126) and GHRHAnt induce UPR (54), it is possible that Arf may participate in GHRHAnt-triggered inhibition of cancer cells. Others have reported the involvement of ER stress in the development and progression of different cancers including prostate, breast, and colon cancer (127–129). In fact, tumor microenvironment such as hypoxic, acidic, and nutrient-deprived milieu can induce ER stress and activate UPR (123). The small molecule IRE1 inhibitor (MKC8866) suppresses triple negative breast cancer growth (130, 131).

Cytokines, Barrier Function and GHRHAnt

Cytokines and vascular function

Cytokines are small, secreted proteins (<40 kDa) (e.g., interleukins, chemokines, interferons, and TNFα) involved in inflammation (132). The release of proinflammatory cytokines activates immune cells to facilitate their accumulation into the inflamed tissues (133). The IL-1 family is divided into 11 subgroups (134), and IL-1β has been shown to alter endothelial permeability through Wnt5A (135). Treatment of EC with IL-1β destabilizes tight junctions (136), increases adhesion molecule expression, and induces a procoagulant phenotype of endothelial tissue via upregulation of tissue factor (137). It also increases lung endothelial permeability by activating RhoA and α_vβ₆ integrin-dependent pathways (138). In addition, IL-1 induces proinflammatory cytokines through MAPK and NF-κB activation, which induce barrier disruption (139). Hence, suppression of IL-1 may promote vascular integrity.

In acute inflammation, IL-6 is released by several types of immune (eg, macrophages and monocytes) and nonimmune cells (eg, EC, mesenchymal cells, and fibroblasts) (140). Those effects are mediated by JAK2-induced STAT3 phosphorylation at Y705 residue, as well as by de novo synthesis of the corresponding RNA and proteins. Therefore, IL-6 is involved in diseases associated with endothelial dysfunction, such as ARDS (141), rheumatoid arthritis, (142, 143) and COVID-19–related respiratory dysfunction (144).

TNFα can promote the interactions between leukocytes and EC by inducing P-selectin, E-selectin, VCAM1, and intercellular cell adhesion molecule-1. Those events result in rolling, firm adhesion, and transmigration of leukocytes from the blood to interstitial tissues (145). TNFα can also facilitate an excessive deposition of fibronectin. Administration of anti-TNFα before reperfusion prevents the generation of oxidative stress in coronary endothelium following ischemia/reperfusion. Hence, TNFα may also cause endothelial dysfunction by escalating oxidative stress (146). In addition, the interaction of TNFα with TNFR1 and TNFR2 activates the NF-κB pathway (147). This pathway is essential for the production of different proinflammatory cytokines and adhesion molecules, and inflicts endothelial dysfunction in many pathological conditions (148).

GHRHAnt and cytokines

Proinflammatory cytokines are released by activated macrophages to upregulate inflammatory reactions. GHRH and its analogs are involved in the regulation of cytokine release during inflammation. Bleomycin can cause lung oxidative stress, inducing inflammatory responses, and promote lung fibrosis (149). This chemotherapeutic agent can also increase IL-1β and IL-18 in mouse lungs. IL-18 knockout mice exert attenuated lung injury due to bleomycin (150). The GHRHAnt MIA-602 suppresses cytokine production and inhibits bleomycin-induced lung inflammation and fibrosis (91), suggesting a potential role of GHRHAnt in those disorders. In addition, severe inflammation may also contribute to the development of prostate cancer (151). The GHRHAnt JMR-132, MIA-313, and MIA-459 suppress IL-1β and NF-κB, to reduce prostate size in testosterone-induced BPH in vivo (152).

GHRHAnt exert anti-inflammatory activities and inhibit cytokine release in a variety of in vitro and in vivo experimental models. In endotoxin-induced uveitis, LPS induces the mRNA levels of GHRHR, SV1, and GHRH in the epithelium of iris and ciliary body, facilitating the infiltration of macrophages and leukocytes in aqueous humor (153). The induction of GHRHR by LPS is mediated by p65 (68). GHRHAnt can also reduce the expression of IL-1β, TNFα, and MCP-1 in ocular inflammation (153). In a dextran sodium sulfate-induced colitis model, the GHRHAnt MIA-690 inhibits IL-6 and TNFα gene expression in colon specimens (154). Similar results were observed in human peripheral blood mononuclear cells, in which GHRH stimulates the secretion of IL-2, IL-6, IL-8, IL-17, and interferon γ. GHRHAnt counteracted those effects (155–158).

GHRHAnt in the COVID-19 Context

COVID-19 emerged as a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Thisis a single-stranded RNA-envelope that belongs to the β coronavirus family (159). The Centers for Disease Control and Prevention reported that COVID-19 caused over 1 million deaths in the United States. SARS-CoV-2 is phylogenetically distinct from other coronaviruses, encoding four major structural proteins—spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein (159). The S protein is composed of S1 and S2 subunits, which promote receptor recognition and cell membrane fusion (160).

Inflammatory stimuli activate alveolar macrophages via Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors. In patients with severe COVID-19, hyperactivation of macrophages has been linked to epithelial and endothelial damage in ARDS (161). SARS-CoV-2 binds to the alveolar epithelial receptor angiotensin-converting enzyme 2 receptor through its S protein, which in turn facilitates membrane infusion and endocytosis (162). The S1 subunit of S protein stimulates the production of proinflammatory cytokines and chemokines, including IL-2, IL-6, IL-8, TNFα, interferon γ, MCP-1, and MIP1α. This potent viral pathogen-associated molecular pattern is recognized by TLR2, triggering NF-κB activation (163). Therefore, diminishing those inflammatory responses would be a therapeutic approach to reduce lung damage.

LPS induces lung inflammation by triggering TLR4, which is responsible for recognizing pathogen-associated molecular pattern. The LPS-binding protein is involved in an innate immune response that induces the uptake of LPS to endothelial cells (164). Interestingly, the LPS-binding site was observed in SARS-CoV-2 S protein that exacerbated the inflammatory response in monocytes and peripheral blood mononuclear cells. Recent studies suggest that S protein potentiates LPS-induced upregulation of TNF-α, IL-1β, and IL-6; and MIA-602 counteracted those events. Furthermore, MIA-602 reduced ROS and MMP-9 expression levels, in support of its anti-inflammatory effects (165).

Based on the previous information, we speculate that GHRHAnt may represent a possible therapeutic strategy in COVID-19 patients. Those peptides can enhance barrier function, suppress lung inflammatory responses, and block crucial components of the cytokine storm. However, direct evidence on the protective effects of GHRHAnt against COVID-19 does not exist.

Conclusions

The development of GHRHAnt is a promising strategy to oppose malignancies, inflammation, and endothelial barrier dysregulation in several preclinical models of experimental disease. Ongoing efforts strive to discover the exact molecular pathways streaming those beneficial effects, aiming to support the premise that GHRHAnt exert a strong potential to be useful in clinical settings of inflammatory lung disease, including sepsis-induced ARDS. Targeted medicine for those diseases does not exist.

Abbreviations

ARDS

acute respiratory distress syndrome

Arf

ADP-ribosylation factor

ATF6

activating transcription factor 6

BiP

binding immunoglobulin protein

cAMP

cyclic adenosine monophosphate

Ca²⁺

calcium

COX2

cyclooxygenase-2

EC

endothelial cell

ER

endoplasmic reticulum

ERK1/2

extracellular signal-regulated kinase 1/2

GHRH

growth hormone–releasing hormone

GHRHR

growth hormone–releasing receptor

GHRHAnt

GHRH antagonist

IGF-I

insulin-like growth factor I

IL

interleukin

iNOS

inducible nitric oxide synthase

IRE1α

inositol-requiring enzyme-1α

JAK2

Janus kinase 2

LPS

lipopolysaccharide

MAPK

mitogen activated protein kinase

MCP-1

monocyte chemoattractant protein-1

NF-κB

nuclear factor kappa B

PERK

protein kinase RNA like ER kinase

PI3K

phosphoinositide 3-kinase

PKA

protein kinase A

ROS

reactive oxygen species

SARS

severe acute respiratory syndrome coronavirus 2

STAT3

signal transducer and activator of transcription 3

SV

splice variant

TLR

Toll-like receptor

TNFα

tumor necrosis factor α

UPR

unfolded protein response

Conflict of Interest

None.

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.