Growth hormone-releasing hormone antagonists protect against hydrochloric acid-induced endothelial injury in vitro.

Growth hormone-releasing hormone (GHRH) regulates the synthesis of growth hormone from the anterior pituitary gland, and it is involved in inflammatory responses. On the other hand, GHRH antagonists (GHRHAnt) exhibit the opposite effects, resulting in endothelial barrier enhancement. Exposure to hydrochloric acid (HCL) is associated with acute and chronic lung injury. In this study, we investigate the effects of GHRHAnt in HCL-induced endothelial barrier dysfunction, utilizing commercially available bovine pulmonary artery endothelial cells (BPAEC). Cell viability was measured by utilizing 3-(4,5-dimethylthiazol2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, fluorescein isothiocyanate (FITC)-dextran was used to assess barrier function. Our observations suggest that GHRHAnt exert protective effects against HCL-induced endothelial breakdown, since those peptides counteract HCL-triggered paracellular hyperpermeability. Based on those findings, we propose that GHRHAnt represent a new therapeutic approach towards HCL-induced endothelial injury.

Protective effects of GHRH antagonists against hydrogen peroxide-induced lung endothelial barrier disruption.

PURPOSE: Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone, which regulates growth hormone release from the anterior pituitary gland. GHRH antagonists (GHRHAnt) are anticancer agents, which also exert robust anti-inflammatory activities in malignancies. GHRHAnt exhibit anti-oxidative and anti-inflammatory effects in vascular endothelial cells, indicating their potential use against disorders related to barrier dysfunction (e.g. sepsis). Herein, we aim to investigate the effects of GHRHAnt against lung endothelial hyperpermeability. METHODS: The in vitro effects of GHRHAnt in H2O2-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Electric cell-substrate impedance sensing (ECIS) was utilized to measure transendothelial resistance, an indicator of barrier function. RESULTS: Our results demonstrate that GHRHAnt protect against H2O2-induced endothelial barrier disruption via P53 and cofilin modulation. Both proteins are crucial modulators of vascular integrity. Moreover, GHRHAnt prevent H2O2 - induced decrease in transendothelial resistance. CONCLUSIONS: GHRHAnt represent a promising therapeutic intervention towards diseases related to lung endothelial hyperpermeability, such as acute respiratory distress syndrome - related or not to COVID-19 - and sepsis. Targeted medicine for those potentially lethal disorders does not exist.

Growth Hormone-Releasing Hormone in Endothelial Inflammation.

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 Antagonist JV-1-36 Suppresses Reactive Oxygen Species Generation in A549 Lung Cancer Cells.

Growth hormone-releasing hormone (GHRH) and its receptors are expressed in a variety of human cancers, and have been involved in malignancies. GHRH antagonists (GHRHAnt) were developed to suppress tumor progression and metastasis. Previous studies demonstrate the involvement of reactive oxygen species (ROS) in cancer progression. Herein, we investigate the effect of a commercially available GHRH antagonist, namely JV-1-36, in the redox status of the A549 human cancer cell line. Our results suggest that this peptide significantly reduces ROS production in those cells in a time-dependent manner and counteracts H2O2-induced ROS. Our study supports the anti-oxidative effects of JV-1-36 and contributes in our knowledge towards the in vitro effects of GHRHAnt in cancers.

Hsp90 inhibition protects brain endothelial cells against LPS-induced injury.

Dysfunction of the blood-brain barrier (BBB) endothelium increases infiltration of lymphocytes and innate immune cells in the brain, leading to the development of neurological disorders. Heat shock protein 90 (Hsp90) inhibitors are anti-inflammatory agents and P53 inducers, which reduce the production of reactive oxygen species (ROS) in a diverse variety of human tissues. In this study, we investigate the effects of those compounds in LPS-induced brain endothelial inflammation, by utilizing human cerebral microvascular endothelial cells (hCMEC/D3). Our results suggest that Hsp90 inhibitors suppress inflammation by inhibiting the LPS-induced signal transducer and activator of transcription 3 (STAT3); and P38 activation. Moreover, those compounds reduce the P53 suppressors murine double minute 2 (MDM2) and murine double minute 4 (MDM4). Immunoglobulin heavy chain binding protein/glucose-regulated protein 78 (BiP/Grp78)-a key element of endothelial barrier integrity-was also increased by Hsp90 inhibition. Hence, we conclude that application of Hsp90 inhibitors in diseases related to BBB dysfunction may deliver a novel therapeutic possibility in the affected population.

An antagonist of growth hormone-releasing hormone protects against LPS-induced increase of bronchoalveolar lavage fluid protein concentration.

Growth Hormone-Releasing Hormone (GHRH) is a neuropeptide regulating the release of Growth Hormone (GH) from the anterior pituitary gland, and acts as a growth factor in a diverse variety of tissues. GHRH antagonists (GHRHAnt) have been developed to counteract those events, and the beneficial effects of those peptides toward homeostasis have been associated with anti-inflammatory activities. Our lab is interested in delineating the mechanisms governing endothelial barrier function. Our goal is to establish new grounds on the development of efficient countermeasures against Acute Respiratory Distress Syndrome (ARDS), which has been associated with thousands of deaths worldwide due to COVID-19. Herein we demonstrate in vivo that GHRHAnt suppresses LPS-induced increase in bronchoalveolar lavage fluid (BALF) protein concentration, thus protecting the lungs against edema and inflammation.

Iron Deficiency Anemia as a Factor in Male Infertility: Awareness in Health College Students in the Jazan Region of Saudi Arabia.

Male contribution towards couple infertility is increasing but is less discussed. We aimed to assess the knowledge about iron deficiency anemia (IDA) as a contributor to male infertility in students at health colleges of Jazan University. A multicentric, cross-sectional survey included 910 participants and 768 participants qualified as per our inclusion criteria. The questions were categorized as: Model 1-knowledge about IDA-induced male infertility; Model 2-knowledge about IDA. The average knowledge of IDA causing male infertility is very low among students. The 18-20 years age group had a lesser score for either knowledge of IDA (M2; p-value = 0.047) or total (p-value < 0.0001) compared to the older group. In addition, female students were significantly more likely to be better in achieving higher total scores (p-value = 0.023) as well as M2 scores (p-value < 0.0001) when compared to the respective male category. On the other hand, males were significantly better in scoring for M1 (p-value = 0.004) compared to females. Awareness about iron deficiency anemia as a factor in male infertility may reduce the infertility burden, arising from a preventable factor, in the Jazan region.

P53 mediates the protective effects of metformin in inflamed lung endothelial cells.

The endothelial barrier regulates interstitial fluid homeostasis by transcellular and paracellular means. Dysregulation of this semipermeable barrier may lead to vascular leakage, edema, and accumulation of pro-inflammatory cytokines, inducing microvascular hyperpermeability. Investigating the molecular pathways involved in those events will most probably provide novel therapeutic possibilities in pathologies related to endothelial barrier dysfunction. Metformin (MET) is an anti-diabetic drug, opposes malignancies, inhibits cellular transformation, and promotes cardiovascular protection. In the current study, we assess the protective effects of MET in LPS-induced lung endothelial barrier dysfunction and evaluate the role of P53 in mediating the beneficial effects of MET in the vasculature. We revealed that this biguanide (MET) opposes the LPS-induced dysregulation of the lung microvasculature, since it suppressed the formation of filamentous actin stress fibers, and deactivated cofilin. To investigate whether P53 is involved in those phenomena, we employed the fluorescein isothiocyanate (FITC) - dextran permeability assay, to measure paracellular permeability. Our observations suggest that P53 inhibition increases paracellular permeability, and MET prevents those effects. Our results contribute towards the understanding of the lung endothelium and reveal the significant role of P53 in the MET-induced barrier enhancement.

Suppression of reactive oxygen species in endothelial cells by an antagonist of growth hormone-releasing hormone.

Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone, which regulates the secretion of growth hormone (GH) from the anterior pituitary gland. The effects of GHRH extend beyond the GH-insulin-like growth factor I axis, and that neuropeptide has been involved in the potentiation of several malignancies and other inflammatory disorders. The development of GHRH antagonists (GHRHAnt) delivers an exciting possibility to counteract the pathogenesis of the GHRH-related effects in human pathophysiology, especially when considered that GHRHAnt support endothelial barrier integrity. Those GHRHAnt-mediated effects are exerted at least in part due to the suppression of major inflammatory pathways, and the modulation of major cytoskeletal components. In the present study, we measured the production of reactive oxygen species (ROS) in bovine pulmonary artery endothelial cells, human cerebral microvascular endothelial cells, and human lung microvascular endothelial cells exposed to GHRH or a commercially available GHRHAnt. Our findings reveal the antioxidative effects of GHRHAnt in all three cell lines, which express GHRH receptors. The redox status of NIH/3T3 cells, which do not produce GHRH receptors, was not significantly affected by GHRH or GHRHAnt. Hence, the application of GHRHAnt in pathologies related to increased ROS production should be further investigated.

Induction of the NEK family of kinases in the lungs of mice subjected to cecal ligation and puncture model of sepsis.

Endothelial barrier dysfunction (EBD) is the hallmark of Acute Respiratory Distress Syndrome (ARDS), a potentially lethal respiratory disorder associated with the COVID-19 - related deaths. Herein, we employed a cecal ligation and puncture (CLP) murine model of sepsis, to evaluate the effects of sepsis-induced EBD in the expression of the never in mitosis A (NIMA)-related kinases (NEKs). Members of that family of kinases regulate the activity and expression of the tumor suppressor P53, previously shown to modulate the actin cytoskeleton remodeling. Our results introduce the induction of NEK2, NEK3, NEK4, NEK7, and NEK9 in a CLP model of sepsis. Hence, we suggest that NEKs are involved in inflammatory processes and are holding the potential to serve as novel therapeutic targets for pathologies related to EBD, including ARDS and sepsis. Further studies will delineate the underlying molecular events and their interrelations with P53.

Hsp90 inhibition protects the brain microvascular endothelium against oxidative stress.

The brain endothelium is an integral element of the blood-brain barrier (BBB). Dysfunction of this formation due to increased generation of reactive oxygen species (ROS) progresses the establishment of neurological disorders including stroke and traumatic brain injury. Heat shock protein 90 inhibitors are anti-inflammatory agents, and their activities are mediated, at least in part, by P53. This is a tumor suppressor protein which regulates the opposing activities of Rac1 and RhoA in the cellular cytoskeleton. In the present study we investigated the role of Hsp90 inhibitors in the H2O2-induced brain endothelium breakdown, by employing human cerebral microvascular endothelial cells (hCMEC/D3). Our findings suggest that H2O2 downregulates P53 by enhancing the P53 suppressor mouse double minute 2 homolog (MDM2), as well as by increasing the apyrimidinic endonuclease 1/redox factor 1 (APE1/Ref1). The H2O2 - triggered violation of the brain endothelium barrier was reflected in measurements of transendothelial resistance, and the increased expression of the key cytoskeletal modulators cofilin and myosin light chain 2 (MLC2). Treatment of the hCMEC/D3 cells with Hsp90 inhibitors counteracted those events, and reduced the generation of the hydrogen peroxide - induced reactive oxygen species. Hence, our study suggests that Hsp90 inhibition supports the BBB integrity, and may represent a promising therapeutic approach for disorders associated with brain endothelium breakdown; including COVID-19.

Synthesis, biological activity and POM/DFT/docking analyses of annulated pyrano[2,3-d]pyrimidine derivatives: Identification of antibacterial and antitumor pharmacophore sites.

New annulated pyrano[2,3-d]pyrimidine derivatives were synthesized with hydroxyl, methoxy, bromine, nitrile and nitro substituents on its skeleton. The correlated electronic effect of substituents on the magnitude of antibacterial activity was noted. The electron donating substituents (namely; 4-OH, 4-OCH3, 4-Br) and electron withdrawing substituents (4-NO2) on phenyl ring in the pyrano[2,3-d]pyrimidine skeleton exerted different influence on its antimicrobial activity against some Gram-positive and Gram-negative bacteria such as Pseudomonas aureus, E. coli, Staphylococcus aureus, Klebsiella pneumonia and Bacillus cereus. All the pyrano[2,3-d]pyrimidines were characterized by spectroscopic analyses. Antibacterial screening revealed that the presence of heteroaryl, cyano and amino groups on pyrano[2,3-d]pyrimidine skeleton increases its penetrating power on the bacterial cell wall so that the product becomes more biologically active. So the the nature of electron withdrawing or electro-donnor Impact of substituents should be taken in consideration in drug design. Hydrolysis of -CRN to amide restored vital Intramolecular interaction like ortho-nitrophenyl and ONOδ-…NHδ+/amide link, offering a crucial template for antibacterial NH, HO-pharmacophore sites, which ultimately elevated innate antimicrobial profiles. POM combinatorial analysis of tangible electronic contributions due to armed annulated pyrano[2,3-d]pyrimidines concluded their broad antimicrobial activity and viable/prominent drug score index through perspective parameters particularly: inter atomic distance/linkers, steric, electronic, polar parameters, and with a different polarising effect of electron donating/withdrawing environments of substituents. Furthermore, an anti-Kinase pharmacophore site (OCNHCO) was evaluated in continuation of the POM investigations. All synthesized products verified fewer side effects than standard streptomycin, but facile implication in selective cancer media (viz. breast or leucemia still needs to be screened).

Autophagy, Unfolded Protein Response and Lung Disease.

Acute Respiratory Distress Syndrome is a severe disorder affecting thousands of individuals worldwide. The available medical countermeasures do not sufficiently suppress the unacceptable high mortality rates associated with those in need. Thus, intense efforts aim to delineate the function of the lung endothelium, so to deliver new therapeutic approaches against this disease. The present manuscript attempts to shed light on the interrelations between the unfolded protein response and autophagy towards lung disease, to deliver a new line of possible therapeutic approaches against the ferocious Acute Respiratory Distress Syndrome.

GHRH Antagonists Protect Against Hydrogen Peroxide-Induced Breakdown of Brain Microvascular Endothelium Integrity.

Growth hormone releasing hormone is a hypothalamic neuropeptide, which regulates the release of growth hormone from the anterior pituitary gland. Growth hormone releasing hormone antagonists are anticancer agents, associated with strong anti-inflammatory activities. In the present study, we investigated the effects of the GHRH antagonist MIA-602 in the integrity of the brain microvascular endothelium in vitro. Our observations suggest that MIA-602 protects against the H2O2-induced breakdown of the brain endothelium and enhances its integrity by inducing P53, deactivating cofilin, and suppressing the RhoA inflammatory pathway. Thus, GHRH antagonists may offer an exciting possibility for the treatment of pathologies related to the blood brain barrier dysfunction, including the Parkinson's and Alzheimer's diseases.

P53 versus inflammation: an update.

Etiologies of human pathophysiology have been associated with states of severe inflammation. The endothelium defender P53 supports cellular functions, by orchestrating anti-inflammatory responses. The purpose of the present article is to provide an update on the mechanisms enforcing the protective actions of P53 in human homeostasis, and to discuss current efforts on the development of new therapies against inflammatory abnormalities.

P53 Regulates the Redox Status of Lung Endothelial Cells.

The anti-inflammatory activities of P53 in the vasculature have been associated with the enhancement of the endothelial barrier function. In the present study, we employed human and bovine lung endothelial cells, to investigate whether P53 expression levels affect the redox status of pulmonary cells. Moreover, we tested the possibility that those events affect the endothelial integrity of the lung microvascular monolayers. Our observations suggest that P53 suppression by LPS, pifithrin, or small interfering RNA increased the expression of the redox marker malondialdehyde. In contrast, P53 induction by Nutlin or the Hsp90 inhibitor AUY922 exerted the opposite effects, namely, suppressed that lipid oxidation marker. The direct measurement of the reactive oxygen species by 2,7-Dichlorodihydrofluorescein diacetate confirmed the antioxidant activity of P53 in the vasculature. Furthermore, the increased reactive oxygen species production due to P53 suppression was associated with lung hyperpermeability responses. In conclusion, P53 supports endothelial barrier function, at least in part, via the modulation of the reactive oxygen species.

GHRH antagonists support lung endothelial barrier function.

Growth Hormone-Releasing Hormone (GHRH) regulates the release of growth hormone from the anterior pituitary gland. GHRH also acts as a growth and inflammatory factor in a variety of experimental models in oncology. In the current study, we used bovine pulmonary arterial cells in order to investigate the effects of GHRH and its antagonistic and agonistic analogs in key intracellular pathways that regulate endothelial permeability. GHRH antagonists suppressed the activation of MLC2, ERK1/2, JAK2/STAT3 pathway and increased the intracellular P53 and pAMPK levels. In contrast, both GHRH and GHRH agonist MR409 exerted the opposite effects. Furthermore, GHRH antagonists supported the integrity of endothelial barrier, while GHRH and GHRH agonists had the contrary effects, as reflected in measurements of transendothelial resistance. Our observations support the evidence for the anti - inflammatory role of GHRH antagonists in the vasculature. Moreover, our results suggest that GHRH antagonists should be considered as promising therapeutic agents for treating severe respiratory abnormalities, such as the lethal Acute Respiratory Distress Syndrome (ARDS).

P53-induced reduction of lipid peroxidation supports brain microvascular endothelium integrity.

Dysregulation of the blood brain barrier due to oxidative stress causes neurological disorders, such as Alzheimer's and Parkinson's disease. We employed brain microvascular endothelial cells; to investigate the effects of P53 towards the lipid oxidation of the BBB. P53 reduction by LPS, siRNA for P53 and Pifithrin increased the expression levels of malondialdehyde, a marker of oxidative stress and lipid peroxidation. Furthermore, P53 suppression impaired the permeability of the BBB monolayers. In contrast, P53 induction by Nutlin and Hsp90 inhibitor AUY922 enhanced the BBB function. In conclusion, P53 supports BBB integrity, at least in part, by reducing lipid peroxidation.

Unfolded protein response regulates P53 expression in the pulmonary endothelium.

Lung endothelial barrier dysfunction leads to severe pathologies, including the lethal Acute Respiratory Distress Syndrome. P53 has been associated with anti-inflammatory activities. The current study employs a variety of unfolded protein response (UPR) activators and inhibitors to investigate the regulation of P53 by UPR in lung cells. The bovine cells that were exposed to the UPR inductors brefeldin A, dithiothreitol, and thapsigargin; demonstrated elevated expression levels of P53 compared to the vehicle-treated cells. On the contrary, the UPR inhibitors N-acetyl cysteine, kifunensine, and ATP-competitive IRE1α kinase-inhibiting RNase attenuator; produced the opposite effects. The outcomes of the present study reveal a positive regulation between UPR and P53. Since it has been shown that a mild induction of the unfolded protein response opposes inflammation, we suggest that P53 is involved in those protective activities in the lung.

P53 supports endothelial barrier function via APE1/Ref1 suppression.

The tumor suppressor protein P53 is strongly involved in orchestrating cellular defenses in the diverse variety of human tissues. Anomalies to lung endothelium permeability are streaming severe consequences towards human health, often associated with fatal outcomes. Ongoing investigations suggest that P53 exerts a prominent strategic role in crucial signaling cascades, in charge of both the maintenance and defense of pulmonary endothelium against toxic intruders. The current study employs human and bovine lung microvascular cells, as well as pharmacologic and genetic P53 modulators to demonstrate the negative regulation of APE1/Ref1 by P53. Moreover, it includes real time measurements of endothelial permeability, to reveal the disruptive role of APE1/Ref1 towards endothelial integrity. Those findings supports our efforts to elucidate the highly sophisticated regulatory network that enact endothelial adaptations under the plethora of challenging environmental factors.