Age and Sex in the Development of Hepatic Encephalopathy: Role of Alcohol.

Hepatic encephalopathy (HE) is a neurological condition linked to liver failure. Acute HE (Type A) occurs with acute liver failure, while chronic HE (Type C) is tied to cirrhosis and portal hypertension. HE treatments lag due to gaps in understanding its development by gender and age. We studied how sex and age impact HE and its severity with combined liver toxins. Our findings indicate that drug-induced (thioacetamide, TAA) brain edema was more severe in aged males than in young males or young/aged female rats. However, adding alcohol (ethanol, EtOH) worsens TAA's brain edema in both young and aged females, with females experiencing a more severe effect than males. These patterns also apply to Type A HE induced by azoxymethane (AZO) in mice. Similarly, TAA-induced behavioral deficits in Type C HE were milder in young and aged females than in males. Conversely, EtOH and TAA in young/aged males led to severe brain edema and fatality without noticeable behavioral changes. TAA metabolism was slower in aged males than in young or middle-aged rats. When TAA-treated aged male rats received EtOH, there was a slow and sustained plasma level of thioacetamide sulfoxide (TASO). This suggests that with EtOH, TAA-induced HE is more severe in aged males. TAA metabolism was similar in young, middle-aged, and aged female rats. However, with EtOH, young and aged females experience more severe drug-induced HE as compared to middle-aged adult rats. These findings strongly suggest that gender and age play a role in the severity of HE development and that the presence of one or more liver toxins may aggravate the severity of the disease progression.

Exploring the role of GHRH antagonist MIA-602 in overcoming Doxorubicin-resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the rapid proliferation of mutagenic hematopoietic progenitors in the bone marrow. Conventional therapies include chemotherapy and bone marrow stem cell transplantation; however, they are often associated with poor prognosis. Notably, growth hormone-releasing hormone (GHRH) receptor antagonist MIA-602 has been shown to impede the growth of various human cancer cell lines, including AML. This investigation examined the impact of MIA-602 as monotherapy and in combination with Doxorubicin on three Doxorubicin-resistant AML cell lines, KG-1A, U-937, and K-562. The in vitro results revealed a significant reduction in cell viability for all treated wild-type cells. Doxorubicin-resistant clones were similarly susceptible to MIA-602 as the wild-type counterpart. Our in vivo experiment of xenografted nude mice with Doxorubicin-resistant K-562 revealed a reduction in tumor volume with MIA-602 treatment compared to control. Our study demonstrates that these three AML cell lines, and their Doxorubicin-resistant clones, are susceptible to GHRH antagonist MIA-602.

Effects of GHRH Deficiency and GHRH Antagonism on Emotional Disorders in Mice.

Growth hormone (GH)-releasing hormone (GHRH) has been suggested to play a crucial role in brain function. We aimed to further investigate the effects of a novel GHRH antagonist of the Miami (MIA) series, MIA-602, on emotional disorders and explore the relationships between the endocrine system and mood disorders. In this context, the effects induced by MIA-602 were also analyzed in comparison to vehicle-treated mice with GH deficiency due to generalized ablation of the GHRH gene (GHRH knock out (GHRHKO)). We show that the chronic subcutaneous administration of MIA-602 to wild type (+/+) mice, as well as generalized ablation of the GHRH gene, is associated with anxiolytic and antidepressant behavior. Moreover, immunohistochemical and Western blot analyses suggested an evident activation of Nrf2, HO1, and NQO1 in the prefrontal cortex of both +/+ mice treated with MIA-602 (+/+ MIA-602) and homozygous GHRHKO (-/- control) animals. Finally, we also found significantly decreased COX-2, iNOS, NFkB, and TNF-α gene expressions, as well as increased P-AKT and AKT levels in +/+ MIA-602 and -/- control animals compared to +/+ mice treated with vehicle (+/+ control). We hypothesize that the generalized ablation of the GHRH gene leads to a dysregulation of neural pathways, which is mimicked by GHRH antagonist treatment.

Growth hormone-releasing hormone receptor antagonist MIA-602 attenuates cardiopulmonary injury induced by BSL-2 rVSV-SARS-CoV-2 in hACE2 mice.

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.

Signaling mechanism of growth hormone-releasing hormone receptor.

The hypothalamic peptide growth hormone-releasing hormone (GHRH) stimulates the secretion of growth hormone (GH) from the pituitary through binding and activation of the pituitary type of GHRH receptor (GHRH-R), which belongs to the family of G protein-coupled receptors with seven potential membrane-spanning domains. Splice variants of GHRH-Rs (SV) in human tumors and other extra pituitary tissues were identified and their cDNA was sequenced. Among the SVs, splice variant 1 (SV1) possesses the greatest similarity to the full-length GHRH-R and remains functional by eliciting cAMP signaling and mitogenic activity upon GHRH stimulation. A large body of work have evaluated potential clinical applications of agonists and antagonists of GHRH in diverse fields, including endocrinology, oncology, cardiology, diabetes, obesity, metabolic dysfunctions, Alzheimer's disease, ophthalmology, wound healing and other applications. In this chapter, we briefly review the expression and potential function of GHRH-Rs and their SVs in various tissues and also elucidate and summarize the activation, molecular mechanism and signalization pathways of these receptors. Therapeutic applications of GHRH analogs are also discussed.

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells.

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1ß, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1ß secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities.

Alveolar epithelial cell growth hormone releasing hormone receptor in alveolar epithelial inflammation.

Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells' gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-ß and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-ß, TNF-α, TGFß-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.

Growth hormone releasing hormone signaling promotes Th17 cell differentiation and autoimmune inflammation.

Dysregulation of Th17 cell differentiation and pathogenicity contributes to multiple autoimmune and inflammatory diseases. Previously growth hormone releasing hormone receptor (GHRH-R) deficient mice have been reported to be less susceptible to the induction of experimental autoimmune encephalomyelitis. Here, we show GHRH-R is an important regulator of Th17 cell differentiation in Th17 cell-mediated ocular and neural inflammation. We find that GHRH-R is not expressed in naïve CD4+ T cells, while its expression is induced throughout Th17 cell differentiation in vitro. Mechanistically, GHRH-R activates the JAK-STAT3 pathway, increases the phosphorylation of STAT3, enhances both non-pathogenic and pathogenic Th17 cell differentiation and promotes the gene expression signatures of pathogenic Th17 cells. Enhancing this signaling by GHRH agonist promotes, while inhibiting this signaling by GHRH antagonist or GHRH-R deficiency reduces, Th17 cell differentiation in vitro and Th17 cell-mediated ocular and neural inflammation in vivo. Thus, GHRH-R signaling functions as a critical factor that regulates Th17 cell differentiation and Th17 cell-mediated autoimmune ocular and neural inflammation.

The Application of GHRH Antagonist as a Treatment for Resistant APL.

GHRH is a hypothalamic peptide shown to stimulate the proliferation of malignant cells in humans. We have previously shown that the use of GHRH antagonist MIA-602 successfully suppressed the growth of many human cancer cell lines, spanning more than 20 types of cancers. In this study, we demonstrate the presence of GHRH-R in the NB4, NB4-RAA, and K-562 model cell lines. Furthermore, we demonstrate the inhibited proliferation of all three cell lines in vitro after incubation with MIA-602. The treatment of xenografts of human APL cell lines with MIA-602 led to a significant reduction in tumor growth. Additionally, combination therapy with both doxorubicin (DOX) and MIA-602 showed a marked synergistic effect in reducing the proliferation of the K-562 AML cell line. These findings suggest that MIA-602 could be utilized to address resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) therapies, as well as in augmenting anthracycline-based regimens.

GHRH agonist MR-409 protects ß-cells from streptozotocin-induced diabetes.

Patients with type 1 diabetes (T1D) suffer from insufficient functional ß-cell mass, which results from infiltration of inflammatory cells and cytokine-mediated ß-cell death. Previous studies demonstrated the beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R), such as MR-409 on preconditioning of islets in a transplantation model. However, the therapeutic potential and protective mechanisms of GHRH-R agonists on models of T1D diabetes have not been explored. Using in vitro and in vivo models of T1D, we assessed the protective propertie of the GHRH agonist, MR409 on ß-cells. The treatment of insulinoma cell lines and rodent and human islets with MR-409 induces Akt signaling by induction of insulin receptor substrate 2 (IRS2), a master regulator of survival and growth in ß-cells, in a PKA-dependent manner. The increase in cAMP/PKA/CREB/IRS2 axis by MR409 was associated with decrease in ß-cell death and improved insulin secretory function in mouse and human islets exposed to proinflammatory cytokines. The assessment of the effects of GHRH agonist MR-409 in a model of T1D induced by low-dose streptozotocin showed that mice treated with MR-409 exhibited better glucose homeostasis, higher insulin levels, and preservation of ß-cell mass. Increased IRS2 expression in ß-cells in the group treated with MR-409 corroborated the in vitro data and provided evidence for the underlying mechanism responsible for beneficial effects of MR-409 in vivo. Collectively, our data show that MR-409 is a novel therapeutic agent for the prevention and treatment of ß-cells death in T1D.

vPIF-1 is an insulin-like antiferroptotic viral peptide.

Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin-like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone-releasing hormone antagonist-induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms.

Expression Levels of GHRH-Receptor, pAkt and Hsp90 Predict 10-Year Overall Survival in Patients with Locally Advanced Rectal Cancer.

BACKGROUND: Rectal cancer constitutes nearly one-third of all colorectal cancer diagnoses, and certain clinical and molecular markers have been studied as potential prognosticators of patient survival. The main objective of our study was to investigate the relationship between the expression intensities of certain proteins, including growth-hormone-releasing hormone receptor (GHRH-R), Hsp90, Hsp16.2, p-Akt and SOUL, in specimens of locally advanced rectal cancer patients, as well as the time to metastasis and 10-year overall survival (OS) rates. We also investigated whether these outcome measures were associated with the presence of other clinical parameters. METHODS: In total, 109 patients were investigated retrospectively. Samples of pretreatment tumors were stained for the proteins GHRH-R, Hsp90, Hsp16.2, p-Akt and SOUL using immunhistochemistry methods. Kaplan-Meier curves were used to show the relationships between the intensity of expression of biomarkers, clinical parameters, the time to metastasis and the 10-year OS rate. RESULTS: High levels of p-Akt, GHRH-R and Hsp90 were associated with a significantly decreased 10-year OS rate (p = 0.001, p = 0.000, p = 0.004, respectively) and high expression levels of p-Akt and GHRH-R were correlated with a significantly shorter time to metastasis. Tumors localized in the lower third of the rectum were linked to both a significantly longer time to metastasis and an improved 10-year OS rate. CONCLUSIONS: Hsp 90, pAkt and GHRH-R as well as the lower-third localization of the tumor were predictive of the 10-year OS rate in locally advanced rectal cancer patients. The GHRH-R and Hsp90 expression levels were independent prognosticators of OS. Our results imply that GHRH-R could play a particularly important role both as a molecular biomarker and as a target for the anticancer treatment of advanced rectal cancer.

Efficacy of a growth hormone-releasing hormone agonist in a murine model of cardiometabolic heart failure with preserved ejection fraction.

Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major unmet medical need owing to its diverse pathophysiology and lack of effective therapies. Potent synthetic, agonists (MR-356 and MR-409) of growth hormone-releasing hormone (GHRH) improve the phenotype of models of HF with reduced ejection fraction (HFrEF) and in cardiorenal models of HFpEF. Endogenous GHRH exhibits a broad range of regulatory influences in the cardiovascular (CV) system and aging and plays a role in several cardiometabolic conditions including obesity and diabetes. Whether agonists of GHRH can improve the phenotype of cardiometabolic HFpEF remains untested and unknown. Here we tested the hypothesis that MR-356 can mitigate/reverse the cardiometabolic HFpEF phenotype. C57BL6N mice received a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (l-NAME) for 9 wk. After 5 wk of HFD + l-NAME regimen, animals were randomized to receive daily injections of MR-356 or placebo during a 4-wk period. Control animals received no HFD + l-NAME or agonist treatment. Our results showed the unique potential of MR-356 to treat several HFpEF-like features including cardiac hypertrophy, fibrosis, capillary rarefaction, and pulmonary congestion. MR-356 improved cardiac performance by improving diastolic function, global longitudinal strain (GLS), and exercise capacity. Importantly, the increased expression of cardiac pro-brain natriuretic peptide (pro-BNP), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor-A (VEGF-A) was restored to normal levels suggesting that MR-356 reduced myocardial stress associated with metabolic inflammation in HFpEF. Thus, agonists of GHRH may be an effective therapeutic strategy for the treatment of cardiometabolic HFpEF phenotype.NEW & NOTEWORTHY This randomized study used rigorous hemodynamic tools to test the efficacy of a synthetic GHRH agonist to improve cardiac performance in a cardiometabolic HFpEF. Daily injection of the GHRH agonist, MR-356, reduced the HFpEF-like effects as evidenced by improved diastolic dysfunction, reduced cardiac hypertrophy, fibrosis, and pulmonary congestion. Notably, end-diastolic pressure and end-diastolic pressure-volume relationship were reset to control levels. Moreover, treatment with MR-356 increased exercise capacity and reduced myocardial stress associated with metabolic inflammation in HFpEF.

Growth hormone-releasing hormone agonist attenuates vascular calcification in diabetic db/db mice.

Introduction: Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. VC increases mortality of all-causes. VC is one of most common cardiovascular complications in type II diabetes. So far, no therapy has been proven to be effective in treatment of clinical VC. The present study investigated the therapeutic effects of MR409, an agonistic analog of growth hormone-releasing hormone (GHRH-A), on VC in diabetic db/db mice. Method and result: Diabetic mice were injected with MR409 subcutaneously every day for 8 weeks. Long-term treatment with MR409 improved serum lipid profile and endothelium-dependent relaxation to acetylcholine, and reduced vascular structural injury in diabetic mice without affecting serum growth hormone level. Echocardiography showed that calcium plaques present in heart valve of diabetic mice disappeared in diabetic mice after treatment with MR409. MR409 inhibited vascular calcium deposition associated with a marked reduction in the expressions of osteogenic-regulated alkaline phosphatase (ALP) and transcription osteogenic marker gene Runx2 in diabetic mice. MR409 also inhibited vascular reactive oxygen species (ROS) generation and upregulated the expressions of anti-calcifying protein Klotho in diabetic mice. Discussion: Our results demonstrate that GHRH-A MR409 can effectively attenuate VC and heart valve calcification, and protect against endothelial dysfunction and vascular injury in diabetic mice without significantly affecting pituitary-growth hormone axis. The mechanisms may involve upregulation of anti-calcifying protein Klotho and reduction in vascular ROS and the expression of redox sensitive osteogenic genes Runx2 and ALP. GHRH-A may represent a new pharmacological strategy for treatment of VC and diabetics associated cardiovascular complications.

Agonist of growth hormone-releasing hormone improves the disease features of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is a severe autosomal recessive neuromuscular disease affecting children and young adults, caused by mutations of the survival motor neuron 1 gene (SMN1). SMA is characterized by the degeneration of spinal alpha motor neurons (αMNs), associated with muscle paralysis and atrophy, as well as other peripheral alterations. Both growth hormone-releasing hormone (GHRH) and its potent agonistic analog, MR-409, exert protective effects on muscle atrophy, cardiomyopathies, ischemic stroke, and inflammation. In this study, we aimed to assess the protective role of MR-409 in SMNΔ7 mice, a widely used model of SMA. Daily subcutaneous treatment with MR-409 (1 or 2 mg/kg), from postnatal day 2 (P2) to euthanization (P12), increased body weight and improved motor behavior in SMA mice, particularly at the highest dose tested. In addition, MR-409 reduced atrophy and ameliorated trophism in quadriceps and gastrocnemius muscles, as determined by an increase in fiber size, as well as upregulation of myogenic genes and inhibition of proteolytic pathways. MR-409 also promoted the maturation of neuromuscular junctions, by reducing multi-innervated endplates and increasing those mono-innervated. Finally, treatment with MR-409 delayed αMN death and blunted neuroinflammation in the spinal cord of SMA mice. In conclusion, the present study demonstrates that MR-409 has protective effects in SMNΔ7 mice, suggesting that GHRH agonists are promising agents for the treatment of SMA, possibly in combination with SMN-dependent strategies.

Synthetic growth hormone-releasing hormone agonist ameliorates the myocardial pathophysiology characteristic of heart failure with preserved ejection fraction.

AIMS: To test the hypothesis that the activation of the growth hormone-releasing hormone (GHRH) receptor signalling pathway within the myocardium both prevents and reverses diastolic dysfunction and pathophysiologic features consistent with heart failure with preserved ejection fraction (HFpEF). Impaired myocardial relaxation, fibrosis, and ventricular stiffness, among other multi-organ morbidities, characterize the phenotype underlying the HFpEF syndrome. Despite the rapidly increasing prevalence of HFpEF, few effective therapies have emerged. Synthetic agonists of the GHRH receptors reduce myocardial fibrosis, cardiomyocyte hypertrophy, and improve performance in animal models of ischaemic cardiomyopathy, independently of the growth hormone axis. METHODS AND RESULTS: CD1 mice received 4- or 8-week continuous infusion of angiotensin-II (Ang-II) to generate a phenotype with several features consistent with HFpEF. Mice were administered either vehicle or a potent synthetic agonist of GHRH, MR-356 for 4-weeks beginning concurrently or 4-weeks following the initiation of Ang-II infusion. Ang-II-treated animals exhibited diastolic dysfunction, ventricular hypertrophy, interstitial fibrosis, and normal ejection fraction. Cardiomyocytes isolated from these animals exhibited incomplete relaxation, depressed contractile responses, altered myofibrillar protein phosphorylation, and disturbed calcium handling mechanisms (ex vivo). MR-356 both prevented and reversed the development of the pathological phenotype in vivo and ex vivo. Activation of the GHRH receptors increased cAMP and cGMP in cardiomyocytes isolated from control animals but only cAMP in cardiac fibroblasts, suggesting that GHRH-A exert differential effects on cardiomyocytes and fibroblasts. CONCLUSION: These findings indicate that the GHRH receptor signalling pathway(s) represents a new molecular target to counteract dysfunctional cardiomyocyte relaxation by targeting myofilament phosphorylation and fibrosis. Accordingly, activation of GHRH receptors with potent, synthetic GHRH agonists may provide a novel therapeutic approach to management of the myocardial alterations associated with the HFpEF syndrome.

Antagonist of Growth Hormone-Releasing Hormone Potentiates the Antitumor Effect of Pemetrexed and Cisplatin in Pleural Mesothelioma.

Pleural mesothelioma (PM) is an aggressive cancer with poor prognosis and no effective therapies, mainly caused by exposure to asbestos. Antagonists of growth hormone-releasing hormone (GHRH) display strong antitumor effects in many experimental cancers, including lung cancer and mesothelioma. Here, we aimed to determine whether GHRH antagonist MIA-690 potentiates the antitumor effect of cisplatin and pemetrexed in PM. In vitro, MIA-690, in combination with cisplatin and pemetrexed, synergistically reduced cell viability, restrained cell proliferation and enhanced apoptosis, compared with drugs alone. In vivo, the same combination resulted in a strong growth inhibition of MSTO-211H xenografts, decreased tumor cell proliferation and increased apoptosis. Mechanistically, MIA-690, particularly with chemotherapeutic drugs, inhibited proliferative and oncogenic pathways, such as MAPK ERK1/2 and cMyc, and downregulated cyclin D1 and B1 mRNAs. Inflammatory pathways such as NF-kB and STAT3 were also reduced, as well as oxidative, angiogenic and tumorigenic markers (iNOS, COX-2, MMP2, MMP9 and HMGB1) and growth factors (VEGF and IGF-1). Overall, these findings strongly suggest that GHRH antagonists of MIA class, such as MIA-690, could increase the efficacy of standard therapy in PM.

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in Primary Human Endometrial Carcinomas: Novel Therapeutic Approaches.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various tumors, including endometrial carcinomas (EC). However, tumoral receptors that mediate the antiproliferative effects of GHRH antagonists in human ECs have not been fully characterized. In this study, we investigated the expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors (GHRH-R) in 39 human ECs and in 7 normal endometrial tissue samples using RT-PCR. Primers designed for the PCR amplification of mRNA for the full length GHRH-R and SVs were utilized. The PCR products were sequenced, and their specificity was confirmed. Nine ECs cancers (23%) expressed mRNA for SV1, three (7.7%) showed SV2 and eight (20.5%) revealed mRNA for SV4. The presence of SVs for GHRH-Rs could not be detected in any of the normal endometrial tissue specimens. The presence of specific, high affinity GHRH-Rs was also demonstrated in EC specimens using radioligand binding studies. Twenty-four of the investigated thirty-nine tumor samples (61.5%) and three of the seven corresponding normal endometrial tissues (42.9%) expressed mRNA for GHRH ligand. Our findings suggest the possible existence of an autocrine loop in EC based on GHRH and its tumoral SV receptors. The antiproliferative effects of GHRH antagonists on EC are likely to be exerted in part by the local SVs and GHRH system.

Tumorigenic transformation of human prostatic epithelial cell line RWPE-1 by growth hormone-releasing hormone (GHRH).

BACKGROUND: Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In this study, we analyzed the carcinogenetic potential of exposure to GHRH of a nontumor human prostate epithelial cell line (RWPE-1) as well as its transforming effect in a xenograft model. METHODS: We performed cell viability, cell proliferation, adhesion and migration assays. In addition, metalloprotease (MMP)-2 activity by means gelatin zymography, GHRH-R subcellular location using confocal immunofluorescence microscopy and vascular endothelial growth factor (VEGF) levels by enzyme-linked immunoassay were assessed. Besides, we developed an in vivo model in order vivo model to determine the role of GHRH on tumorigenic transformation of RWPE-1 cells. RESULTS: In cell cultures, we observed development of a migratory phenotype consistent with the gelatinolytic activity of MMP-2, expression of VEGF, as well as E-cadherin-mediated cell-cell adhesion and increased cell motility. Treatment with 0.1 µM GHRH for 24 h significantly increased cell viability and cell proliferation. Similar effects of GHRH were seen in RWPE-1 tumors developed by subcutaneous injection of GHRH-treated cells in athymic nude mice, 49 days after inoculation. CONCLUSIONS: Thus, GHRH appears to act as a cytokine in the transformation of RWPE-1 cells by mechanisms that likely involve epithelial-mesenchymal transition, thus reinforcing the role of GHRH in tumorigenesis of prostate.

Antagonist of growth hormone-releasing hormone MIA-690 attenuates the progression and inhibits growth of colorectal cancer in mice.

Colorectal cancer (CRC) is an aggressive tumor in which new treatment options deliver negative results on cure rates and long-term survival. The anticancer effects of growth hormone-releasing hormone (GHRH) antagonists have been reported in various experimental tumors, but their activity in CRC is unknown. In the present study, we demonstrated that chronic treatment with GHRH antagonist of MIAMI class, MIA-690, promoted survival and gradually blunted tumor progression in experimentally induced colitis-associated cancer in mice, paralleled by reduced inflammation in colon tissue. In particular, MIA-690 improved disease activity index score, and reduced loss of weight and mortality, by improving the survival rates, compared with vehicle-treated group. MIA-690 was also found to reduce various inflammatory and oxidative markers, such as serotonin, prostaglandin (PG)E2 and 8-iso-PGF2α levels, as well as COX-2, iNOS, TNF-α, IL-6 and NF-kB gene expression. Moreover, MIA-690 inhibited the protein expression of c-Myc, P-AKT and Bcl-2 and upregulated p53 protein expression. In conclusion, we showed that MIA-690 suppresses CRC progression and growth by reducing inflammatory and oxidative markers and modulating apoptotic and oncogenic pathways. Further investigations are required for translating these findings into the clinics.