Post-stroke edema is one of the fatal consequences of ischemic stroke where increased intracranial pressure can exacerbate neurological dysfunction leading to mortality. To date, conventional post-stroke therapy has failed to manage cerebral edema and render neuroprotection. Earlier, our lab reported the benefits of intra-arterially administered mesenchymal stem cells (IA MSCs) in alleviating post-stroke vasogenic edema. Recently, post-stroke neuroendocrine regulation involving growth hormone releasing hormone receptor (GHRH-R) analog is gaining attention as one of the potential targets for stroke intervention, adjunctive to the existing conventional therapies. Therefore, the current study aims to explore the combined therapeutic potential of IA MSCs and GHRH-R analogs in resolution of cerebral vasogenic edema following ischemic stroke. Middle cerebral artery occlusion (MCAo) method was used to induce focal ischemic stroke in male Sprague Dawley rats. Further rats were treated with 1*105 IA MSCs in combination with GHRH-R analogs at a dose of 10 µg/rat/day. In the short-term study, animals were subjected to neurobehavioral assessments following euthanasia at 24 h post-stroke. Next, infarct size, neuronal viability and extent of apoptosis were assessed by triphenyl-tetrazolium-chloride staining, Nissl staining and TUNEL assay. Protein expressions were evaluated by western blot and immunofluorescence assay, and finally morphological analysis of cellular organelles were performed by transmission electron microscopy. The study was extended for 3-days and 7-days with sustained release GHRH-R agonist (MR-409) using osmotic pump following IA MSCs administration post-stroke. Animals were subjected to different cognitive and behavioral tests. Further blood specimens and vital organs were subjected to toxicological analysis. Finally, expressions of different water channel genes and proteins were checked using transcriptomic analysis and immunofluorescence assay, respectively. Post-stroke administration of IA MSCs and MR-409 combination reduced mitochondria-endoplasmic reticulum (ER) distance, modulated AQP4 expression, leading to reduction in blood-brain-barrier (BBB) disruption in the short term along with resolving vasogenic edema in the long term. The study provides indirect preliminary evidence of combined administration of IA MSCs with sustained release GHRH-R agonist towards increasing the mitochondria-endoplasmic reticulum association, altering the expression of AQP4, maintaining BBB integrity and alleviating post-stroke vasogenic edema.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β (Aβ) accumulation, tau hyperphosphorylation, neuroinflammation, and synaptic loss. Existing therapies provide only modest symptomatic relief and fail to slow disease progression. Beyond its role in promoting pituitary growth hormone (GH) secretion, growth hormone-releasing hormone (GHRH) has shown neuroprotective effects in experimental ischemic stroke and spinal muscular atrophy. Here, we explored the therapeutic potential of GHRH and its agonist MR-409 in AD models. In vitro, GHRH(1-44)NH₂ promoted survival, proliferation, and neuronal differentiation of rat hippocampal neural stem cells (NSCs) and human SH-SY5Y neuroblastoma cells under growth factor deprivation and amyloid beta (Aβ)1-42 exposure. These effects involved the cAMP/PKA/CREB, ERK1/2, and PI3K/Akt signaling pathways. GHRH also attenuated Aβ-induced neurotoxicity by reducing apoptosis, suppressing GSK-3β activity and tau phosphorylation, restoring nuclear β-catenin, and inhibiting NF-κB-mediated inflammation. In vivo, subcutaneous administration of MR-409 in 5xFAD mice reduced Aβ deposition, tau phosphorylation, gliosis, and proinflammatory cytokine expression. In addition, MR-409 mitigated neuronal and synaptic loss, activated survival and neurogenic pathways, and improved cognitive performance, without altering systemic GH and IGF1 levels. MR-409 also elevated NRF2 mRNA expression while reducing its negative regulator KEAP1. Overall, these findings indicate that GHRH and its analog MR-409 exert neuroprotective effects by modulating key pathological features of AD, including neurodegeneration, impaired neurogenesis, neuroinflammation, and oxidative stress. Given their ability to modulate multiple pathological pathways, GHRH agonists may represent promising therapeutic candidates for AD and other neurodegenerative disorders.

Heart failure (HF) with preserved ejection fraction (HFpEF) carries a high mortality and remains a major therapeutic challenge. Effective, targeted therapies capable of reversing HFpEF pathophysiology are urgently needed. We previously demonstrated that activation of the cardiac growth hormone-releasing hormone (GHRH) pathway using high potency synthetic agonists of GHRH (GHRH-agonists) improves the HFpEF phenotype in both large and small animal models, including in a murine model of cardiometabolic HFpEF (High fat diet + L-NAME). Here we sought to define the downstream signaling pathways responsible for this effect. A transcriptomic screen in human iPSC-derived cardiomyocytes (hiPSC-CMs) identified the HIF-1α pathway as being activated by GHRH receptor (GHRHR) signaling, revealing an oxygen-independent mechanism of HIF-1α activation. Based on this finding, we investigated the interaction between the cardioprotective effects of the GHRH-agonist MR-356 and HIF-1α pathway activation in the murine HFD+L-NAME model of cardiometabolic HFpEF. We generated a cardiomyocyte-specific HIF-1α knockout (HIF-1αCMKO) mouse line and demonstrated that our previously reported beneficial effects of GHRH-agonist administration were completely abolished in HIF-1αCMKO mice. Together, these findings establish the GHRHR-HIF-1α axis as a putative integrative pathway coupling metabolic and contractile remodeling, suggesting that therapeutic targeting of this axis represents a novel disease-modifying approach to treating cardiometabolic HFpEF.

Recent research underscores the crucial role of hormone regulation in benign prostatic hyperplasia (BPH) and the therapeutic promise of growth hormone-releasing hormone (GH-RH) antagonists. BPH incidence in aging men doubled over three decades, driven by prostatic enlargement and lower urinary tract symptoms (LUTS). Aging-related changes in GH-RH and luteinizing hormone-releasing hormone (LH-RH) biology promote BPH through hormonal and inflammatory processes. Traditional therapies provide symptomatic relief but often fail to prevent progression. This review explores the 50-year extensive development of LH-RH and GH-RH peptide analogs from discovery to delivery and their potential in BPH treatment. In preclinical studies, GH-RH antagonists reduced prostate volume, improved LUTS, and modulated inflammation mediated by NF-κB and IGF-I. Clinical trials are needed to validate antagonist efficacy and safety. Given BPH's public health impact among the aged, and especially among aging Veterans, integrating GH-RH antagonists into management strategies may offer precision-based therapeutic advancements.

Background. Antagonists of GHRH have experimental therapeutic value, but as single agents are not likely to improve clinical outcomes, especially in advanced prostate cancer resistant to androgen deprivation therapy. Our objective is to identify anti-cancer drugs that, in combination with MIA-602 or -690 GHRH antagonists, increase cell death in all types of prostate cancer. Methods/Results. We identified inhibitors of PI3Kα or PI3Kβ that consistently increased cell death when combined with MIA-602/690. The PI3K family is critical in mediating upstream signals from receptors to downstream AKT/mTOR signaling pathways and has an important role in cancer progression. The results revealed that MIA-602/690 alone decreased androgen receptors and likely enhanced PI3K (negative feedback), which was then countered by the addition of PI3K inhibitors. Furthermore, the MIA-602/690 + PI3K inhibitor combination affected multiple signaling pathways, including apoptosis (anti-apoptotic Mcl-1L switching to pro-apoptotic Mcl-1S), proliferation (E2F1, cyclin A), PI3Kα/β, AKT, and ERK. Similar results were obtained with a more clinically relevant acetate salt form of MIA-602/690. The identification of PI3K as a drug target for prostate cancer is significant because PTEN (negative regulator of PI3K) loss of function occurs in 40–50% and PIK3CA mutation/amplification occurs in 60% of prostate cancer patients, leading to a poor prognosis. Conclusion. The ability of the MIA-602/690 + PI3K inhibitor combination to alter multiple signaling pathways may weaken the activation of adaptive mechanisms resulting from each drug and improve efficacy.

The hypothalamic polypeptide 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. Various splice variants of GHRH-R (SV) in human neoplasms and other extrapituitary tissues were demonstrated 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 stimulation by GHRH. In this review, we briefly discuss the activation, regulation, molecular mechanisms and signaling pathways of GHRH-Rs and their SVs in various tissues and also summarize the expression, biological activities and potential function of GHRH, its analogs and their receptors. A large body of work have extensively studied and evaluated potential clinical applications of agonists and antagonists of GHRH in diverse fields, including oncology, endocrinology, obesity, diabetes, other metabolic dysfunctions, cardiology, immune functions, mood disorders, Alzheimer's and lung disease, ophthalmology, inflammation, wound healing and other applications. These results strongly support the potential therapeutic use of GHRH analogs in human medicine in the near future.

Growth hormone-releasing hormone (GHRH) antagonists exert antitumor functions in different experimental cancers. However, their role in combination with radiotherapy in non-small cell lung cancer (NSCLC) remains unknown. Therefore, we investigated the radiosensitizing effect of GHRH antagonists in NSCLC. A549 and H522 NSCLC cell lines were exposed to ionizing radiation (IR) and GHRH antagonists MIA-602 and MIA-690, either individually or in combination. Cell viability and proliferation were evaluated by MTT, BrdU, flow cytofluorimetry, and clonogenic assays; gene and protein expression, signaling pathways, and apoptosis were analyzed by real-time PCR, Western blot, annexin staining, and caspase-3 assay. GHRH antagonists showed antitumor effects alone and potentiated IR-induced inhibition of cell viability and proliferation. The combination of MIA-690 and IR decreased the expression of GHRH receptor, its oncogenic splice variant 1, and IGF1 mRNA levels. Additionally, cell cycle inhibitors and proapoptotic markers were upregulated, whereas cyclins, oncogenic MYC, and the antiapoptotic protein Bcl-2 were downregulated. Radioresistance was prevented by MIA-690, which also blunted epithelial-mesenchymal transition by enhancing E-cadherin and reducing mesenchymal, oxidative, and proangiogenic effectors. Finally, both MIA-602 and MIA-690 enhanced radiosensitivity in primary human NSCLC cells. These findings highlight the potential of GHRH antagonists as radiosensitizers in NSCLC treatment.

Growth hormone-releasing hormone (GHRH) and its ability to stimulate the production and release of growth hormone from the pituitary were discovered more than four decades ago. Since then, this hormone has been studied extensively and research into its functions is still ongoing. GHRH has multifaceted roles beyond the originally identified functions that encompass a variety of direct extrapituitary effects. In this Review, we illustrate the different biological activities of GHRH, covering the effects of GHRH agonists and antagonists in physiological and pathological contexts, along with the underlying mechanisms. GHRH and GHRH analogues have been implicated in cell growth, wound healing, cell death, inflammation, immune functions, mood disorders, feeding behaviour, neuroprotection, diabetes mellitus and obesity, as well as cardiovascular, lung and neurodegenerative diseases and some cancers. The positive effects observed in preclinical models in vitro and in vivo strongly support the potential use of GHRH agonists and antagonists as clinical therapeutics.

Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.

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.

Abstract Disclosure: M. Gonzalez Medina: None. R. Louzada: None. V. Pita-Grisanti: None. M. Blandino: None. T. Cui: None. W. Sha: Owner/Co-Owner; Self; WS. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs. R. Cai: Owner/Co-Owner; Self; R.C. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs. A.V. Schally: Owner/Co-Owner; Self; A.V.S. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs.. E. Bernal-Mizrachi: None. Patients with type 1 diabetes (T1D) have decreased functional β-cell mass due to inflammatory cell infiltration and cytokine-induced β-cell death. Clinical trials using a combination of immunomodulatory agents and β-cell protecting medications such as GLP1-R agonists (GLP1-Ra) are being considered. A potential limitation for the clinical efficacy of GLP1-R agonists is the loss of GLP-1-R at the cell surface in states of hyperglycemia. Hence, novel agents that enhance β-cell survival and proliferation through parallel pathways could improve responses to GLP1-R agonists and/or synergistically potentiate β-cell responses. Previous studies have demonstrated beneficial effects of growth hormone-releasing hormone receptor agonists (GHRH-Ra), such as MR-409 on islet preconditioning in transplantation models. Our previous studies showed that GHRH-Ra protects β-cells from autoimmune injury in vitro and improves glucose levels in a model of high-dose streptozotocin-induced diabetes via stimulation of the cAMP/PKA/CREB/IRS2 axis. However, the effects of combination of incretin-based therapy with GHRHRa on β-cell survival in T1D models have not yet been explored. Using the low-dose STZ model in C57/B6 mice, we investigated how MR-409 alone or in combination with GLP1-R agonists (Exendin-4 or Ex-4) promotes β-cell survival and prevents T1D. Low-dose STZ treated mice received daily MR-409 and Ex-4 injections, either alone or in combination for six weeks. We found that, STZ-treated mice that received either MR-409, Ex-4 or combination exhibited better glucose homeostasis while the combination failed to induce any additional effect. Interestingly, glucose tolerance and insulin levels were improved only in the MR-409-treated mice. β-cell mass was similarly increased in mice that received MR-409, Ex-4 and combination groups compared to the vehicle-treated. Further, treatment of human islets with MR-409 and/or Ex-4 induced insulin receptor substrate 2 (IRS2) expression, a master regulator of survival and growth in β-cells, while the combination of both therapies did not have any additional effect. Experiments in human islets exposed to proinflammatory cytokines treatment demonstrated that MR-409 and/or Ex-4 was associated with a decrease in β-cell death and improved insulin secretory function by activation of the cAMP/PKA/CREB/IRS2 axis. These studies demonstrate that although there was no evidence for a synergistic effect between MR-409 and Ex-4, MR-409 appears to have more potent effects on improving glucose tolerance and insulin levels when compared to the long-term effect of Ex-4 in a low-dose STZ model, suggesting that MR-409 could be an alternative therapeutic agent in the treatment of β-cell death in T1D. Presentation: 6/2/2024

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The number of people worldwide aged 60 years and older is expected to increase to 2.1 billion by 2050. Wound healing in the elderly is delayed by age-related factors, including decreased collagen synthesis by fibroblasts. Our previous study found that growth hormone releasing hormone (GHRH) agonism accelerated fibroblast-mediated wound healing. The purpose of this study is to evaluate the influence of aging on GHRH and analog, MR-409, stimulation of human fibroblast function and collagen synthesis, and its implications on chronic wound care in the elderly. Senescent human fibroblasts were treated with 0-5μM GHRH(1-29) or MR-409 in serum-free growth medium for 8 days. As control, equal volume of peptide dissolving buffer was used. At concentrations of 5μM, GHRH(1-29) and MR-409 increased the proliferation of fibroblasts by 228±7% (p<0.01), 250±15% (p<0.01), and 166±10% (p<0.01), 174±13% (p<0.01), respectively. Human senescent fibroblasts were also maintained in serum-free medium containing 2μM of GHRH(1-29) or MR-409 for 6 days. Concentration of pro-collagen I α1, a precursor of mature collagen I, was measured. Both GHRH(1-29) and MR-409 showed stimulatory effects on collagen I synthesis for at least 6 days. There was a 23.9%-44.4% increase in the pro-collagen I α1 levels compared to control. MR-409, synthetic GHRH analogue, accelerates wound healing on aged skin that otherwise exhibits delayed healing by promoting collagen synthesis, and proliferation of senescent fibroblasts.

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.

Type C hepatic encephalopathy (Type C HE) is a major and complex neurological condition that occurs following chronic liver failure. The molecular basis of Type C HE remains elusive. Type C HE is characterized by mental confusion, cognitive and motor disturbances. The presence of Alzheimer type II astrocytes (AT2A) is the key histopathological finding observed in Type C HE. However, nothing is currently known regarding AT2A development and its involvement in cognitive, and motor deficits in Type C HE. We, therefore, examined in rats the mechanisms by which liver failure contributes to the progression of AT2A, and its role in the development of cognitive and motor deficits in thioacetamide (TAA) model of Type C HE. We and others earlier reported increased oxidative/nitrosative stress (ONS), JNK1/2, and cMyc activation in ammonia-treated astrocyte cultures, as well as in brains from chronic liver failure. We now found increased levels of astrocytic glia maturation factor (GMF, a factor strongly implicated in neuroinflammation), as well as various inflammatory factors (IL-1β, TNF-α, IL-6, MMP-3, COX2, CXCL1, and PGE2), and reduced levels of GFAP and increased levels of aggregated nuclear protein Lamin A/C in rat brain cortex post-chronic liver failure. We also found increased levels of GMF and inflammatory factors (MMP-3, COX2, CXCL1, and PGE2) in astrocytes post-ammonia treatment in vitro. Additionally, pharmacological inhibition of upstream signaling of GMF (ONS, JNK1/2, and cMyc) or GMF inhibitors W-7 and trifluoperazine significantly reduced the levels of inflammatory factors, the number of AT2A cells, as well as the cognitive and motor deficits in TAA-treated rats. Increased levels of GMF were also identified in human post-mortem brain sections. These findings strongly suggest that increased levels of astrocytic GMF due to elevated levels of ONS, JNK1/2, and cMyc and the subsequent inflammation contribute to the development of AT2A and the consequent cognitive, and motor deficits in chronic liver failure.

The development of resistance remains the primary challenge in treating castration-resistant prostate cancer (CRPC). GHRH receptors (GHRH-R), which are coupled to G-proteins (GPCRs), can mediate EGFR transactivation, offering an alternative pathway for tumour survival. This study aimed to evaluate the effects of the GHRH-R antagonist MIA-690, in combination with the EGFR inhibitor Gefitinib, on cell viability, adhesion, gelatinolytic activity, and the cell cycle in advanced prostate cancer PC-3 cells. The findings demonstrate a synergistic effect between MIA-690 and Gefitinib, leading to the inhibition of cell viability, adhesion, and metalloprotease activity. Cell cycle analysis suggests that both compounds induce cell cycle arrest, both individually and in combination. Furthermore, similar effects of the GHRH-R antagonist MIA-690 combined with Gefitinib were observed in PC-3 tumours developed by subcutaneous injection in athymic nude mice 36 days post-inoculation. These results indicate that combined therapy with a GHRH-R antagonist and an EGFR inhibitor exerts a stronger antitumor effect compared to monotherapy by preventing transactivation between EGFR and GHRH-R in CRPC.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)