Growth hormone-releasing hormone antagonists counteract interferon-γ - induced barrier dysfunction in bovine and human endothelial cells.

GHRH regulates the secretion of GH from the anterior pituitary gland. An emerging body of evidence suggests that the activities of that neuropeptide are not limited to the GH/IGF-I axis, but they expand towards the mediation of inflammatory processes. GHRHAnt were developed to oppose the activities of GHRH in malignancies, and have been associated with strong anti-inflammatory and anti-oxidative effects in a diverse variety of tissues, including the lungs. In the present study we report that GHRHAnt oppose interferon-γ - induced paracellular hyperpermeability and reactive oxygen species generation in bovine and human pulmonary endothelial cells; and suppress interferon-γ - triggered STAT3, cofilin and ERK1/2 activation. Our observations substantiate previous findings on the protective effects of GHRHAnt in endothelial inflammation and barrier break-down.

Involvement of NEK2 and NEK9 in LPS - induced endothelial barrier dysfunction.

Endothelial hyperpermeability is the hallmark of severe lung injury, including acute respiratory distress syndrome. Despite the fact that Never In Mitosis A (NIMA)-related kinase 2 (NEK2) and NEK9 mediate fundamental cellular processes, our knowledge on their role in barrier function is limited. Herein we show that NEK2 and NEK9 inhibition suppresses LPS-induced paracellular hyperpermeability and myosin light chain 2 activation in endothelial cells. Moreover, the expression levels of both kinases were elevated in inflamed mouse lungs. Based on those findings, we raise the possibility that NEK2 and NEK9 may serve as novel therapeutic targets in lung inflammatory disease.

NEK-mediated barrier regulation.

Endothelial dysfunction has been associated with devastating outcomes which can eventually lead to permanent disability and death. Elucidation of the meticulously devised network orchestrating endothelial responses, provides information to develop new therapies towards endothelial-related disorders. NEK kinases - which have been involved in the development of human disease - promote vascular leak; suggesting the possibility that their inhibition may ameliorate medical conditions related to barrier derangement.

Ceapin-A7 potentiates lipopolysaccharide-induced endothelial injury.

Barrier dysfunction is the hallmark of severe lung injury, including acute respiratory distress syndrome. Efficient medical countermeasures to counteract endothelial hyperpermeability do not exist, hence the mortality rates of disorders related to barrier abnormalities are unacceptable high. The unfolded protein response is a highly conserved mechanism, which aims to support the cells against endoplasmic reticulum stress, and ATF6 is a protein sensor that triggers its activation. In the current study, we investigate the effects of ATF6 suppression in LPS-induced endothelial inflammation. Our observations suggest that Ceapin-A7, which is an ATF6 suppressor, potentiates LPS-induced STAT3 and JAK2 activation. Hence ATF6 activation may serve as a new therapeutic possibility toward diseases related to barrier dysfunction.

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.

P53 in endothelial function and unfolded protein response regulation.

Vascular barrier dysfunction due to endothelial hyperpermeability has been associated with the pathophysiology of sepsis and severe lung injury, which may inflict acute respiratory distress syndrome (ARDS). Our group is focused on the mechanisms operating towards the regulation of endothelial permeability, to contribute in the development of efficient and targeted countermeasures against ARDS. Unfortunately, the number of ARDS-related deaths in the intensive care units has dramatically increased during the COVID-19 era. The findings described herein inform the corresponding scientific and medical community on the relation of P53 and stress responses in barrier function.

Brefeldin A and kifunensine modulate LPS-induced lung endothelial hyperpermeability in human and bovine cells.

Endothelial hyperpermeability is the hallmark of acute respiratory distress syndrome (ARDS). Laborious efforts in the investigation of the molecular pathways involved in the regulation of the vascular barrier shall reveal novel therapeutic targets toward that respiratory disorder. Herein, we investigate in vitro the effects of the α-1,2-mannosidase 1 inhibitor kifunensine (KIF) and brefeldin A (BFA) in the lipopolysaccharides (LPS)-induced endothelial breakdown. Our results suggest that BFA opposes the deteriorating effects of KIF [unfolded protein response (UPR) suppressor] toward the lung microvasculature. Since KIF is a UPR suppressor, and brefeldin A is a UPR inducer, we suggest that a carefully devised UPR manipulation may deliver novel therapeutic avenues in diseases related to endothelial barrier dysfunction (e.g., ARDS and sepsis).

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.

P53 in acute respiratory distress syndrome.

P53 is a tumor suppressor protein, associated with strong anti-inflammatory activities. Recent evidence suggest that this transcription factor counteracts lung inflammatory diseases, including the lethal acute respiratory distress syndrome. Herein we provide a brief discussion on the relevant topic.

Protective Mechanism of the Selective Vasopressin V1A Receptor Agonist Selepressin against Endothelial Barrier Dysfunction.

Sepsis and septic shock are among the most common causes of death in the intensive care unit; advanced therapeutic approaches are thus urgently needed. Vascular hyperpermeability represents a major manifestation of severe sepsis and is responsible for the ensuing organ dysfunction and failure. Vasopressin V1A receptor (V1AR) agonists have shown promise in the treatment of sepsis, increasing blood pressure, and reducing vascular hyperpermeability. The effects of the selective V1AR-selective agonist selepressin have been investigated in an in vitro model of thrombin-, vascular endothelial growth factor-, angiopoietin 2-, and lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial hyperpermeability. Results suggest that selepressin counteracts the effects of all four endothelial barrier disruptors in a concentration-dependent manner, as reflected in real-time measurements of vascular permeability by means of transendothelial electrical resistance. Further, selepressin protected the barrier integrity against the LPS-mediated corruption of the endothelial monolayer integrity, as captured by VE-cadherin and actin staining. The protective effects of selepressin were abolished by silencing of the vasopressin V1AR, as well as by atosiban, an antagonist of the human V1AR. p53 appears to be involved in mediating these palliative effects, since selepressin strongly induced its expression levels, suppressed the inflammatory RhoA/myosin light chain2 pathway, and triggered the barrier-protective effects of the GTPase Rac1. We conclude that V1AR-selective agonists, such as selepressin, may prove useful in the improvement of endothelial barrier function in the management of severe sepsis. SIGNIFICANCE STATEMENT: A cardinal sign of sepsis, a serious disease with significant mortality and no specific treatment, is pulmonary endothelial barrier dysfunction that leads to pulmonary edema. Here, we present evidence that in cultured human lung microvascular endothelial cells, the synthetic, selective vasopressin V1A receptor agonist selepressin protects against endothelial barrier dysfunction caused by four different edemogenic agents, suggesting a potential role of selepressin in the clinical management of sepsis.

Regulation of lung endothelial permeability by NEK kinases.

Dysregulation of lung endothelial barrier function may lead to lethal outcomes, as demonstrated in the case of the acute respiratory distress syndrome (ARDS). p53 participates in the regulation of the lung endothelial barrier, and it has been associated both in vivo and in vitro with protective effects against the LPS-induced hyperpermeability. Family members of the never in mitosis A-related kinases (NEKs) are crucial mediators of fundamental cellular processes, including mitosis, and have been shown to posttranslationally modify p53. Since such modifications affect p53 stability and activity, it is highly probable that NEK kinases are also regulators of lung endothelial permeability. Thus, they may serve as possible therapeutic targets for treatment of pathologies associated with endothelial barrier dysfunction.

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.

The highly interrelated GHRH, p53, and Hsp90 universe.

p53 universe is composed of a complex regulatory network, destined to counteract multifarious challenges threatening cell survival. Imbalance in those responses may result in human disease associated with inevitable consequences. The present work delivers our view of the corresponding phenomena, by involving the endothelium defender in meticulously orchestrated events against inflammatory stimuli. Immersing into the great depths of p53 cosmos may lead to promising therapies against devastating disorders, including acute respiratory distress syndrome.

Endothelial-to-Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction.

OBJECTIVE: Endothelial cells (EC) in obese adipose tissue (AT) are exposed to a chronic proinflammatory environment that may induce a mesenchymal-like phenotype and altered function. The objective of this study was to establish whether endothelial-to-mesenchymal transition (EndoMT) is present in human AT in obesity and to investigate the effect of such transition on endothelial function and the endothelial particulate secretome represented by extracellular vesicles (EV). Approach and Results: We identified EndoMT in obese human AT depots by immunohistochemical co-localization of CD31 or vWF and α-SMA (alpha-smooth muscle actin). We showed that AT EC exposed in vitro to TGF-ß (tumor growth factor-ß), TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-γ) undergo EndoMT with progressive loss of endothelial markers. The phenotypic change results in failure to maintain a tight barrier in culture, increased migration, and reduced angiogenesis. EndoMT also reduced mitochondrial oxidative phosphorylation and glycolytic capacity of EC. EVs produced by EC that underwent EndoMT dramatically reduced angiogenic capacity of the recipient naïve ECs without affecting their migration or proliferation. Proteomic analysis of EV produced by EC in the proinflammatory conditions showed presence of several pro-inflammatory and immune proteins along with an enrichment in angiogenic receptors. CONCLUSIONS: We demonstrated the presence of EndoMT in human AT in obesity. EndoMT in vitro resulted in production of EV that transferred some of the functional and metabolic features to recipient naïve EC. This result suggests that functional and molecular features of EC that underwent EndoMT in vivo can be disseminated in a paracrine or endocrine fashion and may induce endothelial dysfunction in distant vascular beds.

A glimpse at growth hormone-releasing hormone cosmos.

Growth hormone-releasing hormone is a hypothalamic neuropeptide, which regulates the secretion of growth hormone by the anterior pituitary gland. Recent evidence suggest that it exerts growth factor activities in a diverse variety of in vivo and in vitro experimental malignancies, which are counteracted by growth hormone-releasing hormone antagonists. Those peptides support lung endothelial barrier integrity by suppressing major inflammatory pathways and by inducing the endothelial defender P53. The present effort provides information regarding the effects of growth hormone-releasing hormone in the regulation of P53 and the unfolded protein response. Furthermore, it suggests the possible application of growth hormone-releasing hormone antagonists towards the management of acute lung injury, including the lethal acute respiratory distress syndrome.

P53: The endothelium defender.

P53 represents the paradigm of a multitalented transcription factor, responsible for the cellular defense against a plethora of potentially harmful stimuli. It exercises the ability to strongly oppose both cancer and inflammation, partially due to the fact that both conditions are highly interrelated. Endothelium hyperpermeability is considered the hallmark of severe lung inflammation, and the cardinal feature of the lethal acute respiratory distress syndrome. An emerging body of evidence suggests a strategic role of P53 towards vascular barrier integrity. The "endothelium defender" orchestrates meticulously devised responses; to counteract toxin-induced destructions of endothelium monolayers. The present effort seeks to further our understanding on the expanding P53 universe, discussing the most recent information regarding the involvement of that molecule in the pulmonary function.

Hsp90 inhibitors suppress P53 phosphorylation in LPS - induced endothelial inflammation.

P53 has been recently involved in the defense against inflammation. The "guardian of the genome" appears to orchestrate cellular responses against bacterial toxins, by regulating crucial pathways that orchestrate the vascular barrier functions. Indeed, an emerging body of evidence suggests that this tumor suppressor is involved in the mediation of the beneficial effects of Hsp90 inhibition in the inflamed endothelium. Interestingly, those compounds augment the abundance of P53 in the intracellular niche, while LPS dramatically reduces it. The current study focuses on the outcome of LPS and Hsp90 inhibition on P53 phosphorylation, since this modification negatively affects P53 stability. In an in "vitro" model of LPS - induced vascular leak in bovine pulmonary arterial endothelial cells, LPS induced P53 phosphorylation in four distinct residues, namely Ser. 6, Ser. 15, Ser. 33 and Ser. 392. Furthermore, LPS triggered the activation of the myosin light chain 2, which produces endothelial barrier dysfunction by cellular retraction and intercellular gap formation. Indeed, mice exposed to the toxin demonstrated elevated levels of the pro - inflammatory cytokines IL-2 and IL-10 in the bronchoalveolar lavage fluid. In bold contrast, the HSP90 inhibitor 17-DMAG, counteracted the LPS - induced effects both in vivo and in vitro. Specifically, this hsp90 inhibitor reduced phosphorylated P53 levels and lessened the activation of myosin light chain 2 (phosphorylation) in the bovine endothelium. Moreover, 17 - DMAG suppressed inflammation in mouse lungs, as reflected in reduced IL-2 and IL-10 BALF levels. In summary, the present results support previous observations on the protective role of P53 against inflammation and clarify mechanisms that govern vascular barrier function.

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.