Prognostic Value of HIF-1α-Induced Genes in Sepsis/Septic Shock.

Hypoxia is characterized as one of the main consequences of sepsis, which is recognized as the leading cause of death in intensive care unit (ICU) patients. In this study, we aimed to examine whether the expression levels of genes regulated under hypoxia could be utilized as novel biomarkers for sepsis prognosis in ICU patients. Whole blood expression levels of hypoxia-inducible factor-1α (HIF1A), interferon-stimulated gene 15 (ISG15), hexokinase 2 (HK2), lactate dehydrogenase (LDHA), heme oxygenase-1 (HMOX1), erythropoietin (EPO), and the vascular endothelial growth factor A (VEGFA) were measured on ICU admission in 46 critically ill, initially non-septic patients. The patients were subsequently divided into two groups, based on the development of sepsis and septic shock (n = 25) or lack thereof (n = 21). HMOX1 mRNA expression was increased in patients who developed sepsis/septic shock compared to the non-septic group (p < 0.0001). The ROC curve, multivariate logistic regression, and Kaplan-Meier analysis demonstrated that HMOX1 expression could be utilized for sepsis and septic shock development probability. Overall, our results indicate that HMOX1 mRNA levels have the potential to be a valuable predictive factor for the prognosis of sepsis and septic shock in ICU patients.

The Interplay between Aquaporin-1 and the Hypoxia-Inducible Factor 1α in a Lipopolysaccharide-Induced Lung Injury Model in Human Pulmonary Microvascular Endothelial Cells.

Aquaporin-1 (AQP1), a water channel, and the hypoxia-inducible factor 1α (HIF1A) are implicated in acute lung injury responses, modulating among others pulmonary vascular leakage. We hypothesized that the AQP1 and HIF1A systems interact, affecting mRNA, protein levels and function of AQP1 in human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS). Moreover, the role of AQP1 in apoptosis and wound healing progression was examined. Both AQP1 mRNA and protein expression levels were higher in HPMECs exposed to LPS compared to untreated HPMECs. However, in the LPS-exposed HIF1A-silenced cells, the mRNA and protein expression levels of AQP1 remained unaltered. In the permeability experiments, a statistically significant volume increase was observed at the 360 s time-point in the LPS-exposed HPMECs, while LPS-exposed HIF1A-silenced HPMECs did not exhibit cell swelling, implying a dysfunctional AQP1. AQP1 did not seem to affect cell apoptosis yet could interfere with endothelial migration and/or proliferation. Based on our results, it seems that HIF1A silencing negatively affects AQP1 mRNA and protein expression, as well as AQP1 function, in the setting of lung injury.

Increased Autotaxin Levels in Severe COVID-19, Correlating with IL-6 Levels, Endothelial Dysfunction Biomarkers, and Impaired Functions of Dendritic Cells.

Autotaxin (ATX; ENPP2) is a secreted lysophospholipase D catalyzing the extracellular production of lysophosphatidic acid (LPA), a pleiotropic signaling phospholipid. Genetic and pharmacologic studies have previously established a pathologic role for ATX and LPA signaling in pulmonary injury, inflammation, and fibrosis. Here, increased ENPP2 mRNA levels were detected in immune cells from nasopharyngeal swab samples of COVID-19 patients, and increased ATX serum levels were found in severe COVID-19 patients. ATX serum levels correlated with the corresponding increased serum levels of IL-6 and endothelial damage biomarkers, suggesting an interplay of the ATX/LPA axis with hyperinflammation and the associated vascular dysfunction in COVID-19. Accordingly, dexamethasone (Dex) treatment of mechanically ventilated patients reduced ATX levels, as shown in two independent cohorts, indicating that the therapeutic benefits of Dex include the suppression of ATX. Moreover, large scale analysis of multiple single cell RNA sequencing datasets revealed the expression landscape of ENPP2 in COVID-19 and further suggested a role for ATX in the homeostasis of dendritic cells, which exhibit both numerical and functional deficits in COVID-19. Therefore, ATX has likely a multifunctional role in COVID-19 pathogenesis, suggesting that its pharmacological targeting might represent an additional therapeutic option, both during and after hospitalization.

Alteration of L-Dopa decarboxylase expression in SARS-CoV-2 infection and its association with the interferon-inducible ACE2 isoform.

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.

Selection of the Appropriate Control Group Is Essential in Evaluating the Cytokine Storm in COVID-19.

BACKGROUND/AIM: Lately, studies have reported contradicting results on the cytokine storm seen in critically-ill COVID-19 patients. Depending on the control group used, cytokines have been found to be higher, similar or even lower in COVID-19 compared to critical illnesses associated with elevated cytokine concentrations. However, most of these studies do not take into account critical illness severity. Hence, we decided to compare cytokine levels in critically-ill COVID-19 patients and critically-ill patients of a general intensive care unit (ICU), who did not have sepsis or septic shock, but had an equal disease severity. PATIENTS AND METHODS: Interleukin (IL)-6, IL-8, IL-10 and tumour necrosis factor-α (TNF-α) were measured on ICU admission in mechanically ventilated, COVID-19 (N=36) and non-COVID-19 (N=30) patients, who had not received dexamethasone, and had equal critical illness severity. Non-COVID-19 patients did not have sepsis or septic shock. RESULTS: In our case control study, circulating IL-6 and IL-10 were lower, while TNF-α and IL-8 levels were higher in critically-ill COVID-19 patients, compared to critically-ill non-COVID-19 patients. CONCLUSION: It is difficult to infer whether the cytokine storm seen in COVID-19 differs from other critical conditions. It is important to recognize that the conclusions of related studies may depend on control group selection.

Human L-Dopa decarboxylase interaction with annexin V and expression during apoptosis.

l-Dopa Decarboxylase (DDC) is a pyridoxal requiring enzyme that catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (l-Dopa) to Dopamine (DA). The function of DDC in physiological and pathological biochemical pathways remains poorly understood, while the function and regulation of human DDC isoforms is almost completely elusive. We have shown that Annexin V, a fundamental apoptosis marker, is an inhibitor of l-Dopa decarboxylase activity. Here we show the interaction of both the full-length DDC and the truncated isoform alternative DDC (Alt-DDC) with Annexin V in human tissue and cell lines. Interestingly, DDC isoform expression is enhanced or remains unaffected following staurosporine (STS) treatment, despite increased levels of cytotoxicity and apoptosis. The findings presented here provide novel insights concerning the involvement of DDC in programmed cell death.

Study of inflammatory biomarkers in COPD and asthma exacerbations.

INTRODUCTION: Exacerbations are critical events in the course of asthma and chronic obstructive pulmonary disease (COPD). These events are potentially life-threatening, and the studies have shown that they have tremendous implications on long-term disease control and the overall prognosis of the patients. The aim of this study was to examine adipokines, cytokines and C-reactive protein (CRP) as potential biomarkers in asthma and COPD. MATERIAL AND METHODS: Prospective cohort study of COPD and asthma patients treated for acute exacerbations. Thirty-nine COPD patients and 15 asthmatic patients were included in the study. Leptin, adiponectin, resistin, interleukin (Il)-6, 8, 18, tumor necrosis factor-a (TNF-a), and CRP were measured at three time points: on admission, at resolution and at the stable phase. Pre- and post-bronchodilation spirometry was additionally performed at resolution and at the stable phase. RESULTS: In COPD patients, leptin, leptin/adiponectin (L/A) ratio and resistin were elevated on admission compared to the stable phase. In asthmatic patients, leptin levels were raised on admission compared to the stable phase, and adiponectin was elevated at resolution compared to admission. In both diseases, CRP was significantly increased on admission compared to both resolution and stable disease. Finally, TNF-a could distinguish between asthma and COPD stable phase. CONCLUSIONS: Leptin and CRP levels may be useful biomarkers in monitoring COPD and asthma response to treatment during an exacerbation episode. Hypoadiponectinemia was detected in asthma and COPD during all stages of the diseases. TNF-a could distinguish between asthma and COPD stable phase.

L-Dopa decarboxylase interaction with the major signaling regulator ΡΙ3Κ in tissues and cells of neural and peripheral origin.

L-Dopa decarboxylase (DDC) catalyzes the decarboxylation of L-Dopa to dopamine and 5-hydroxytryptophan (5-HTP) to serotonin. Although DDC has been purified from a variety of peripheral organs, including the liver, kidney and pancreas, the physiological significance of the peripherally expressed enzyme is not yet fully understood. DDC has been considered as a potential novel biomarker for various types of cancer, however, the role of DDC in the development of hepatocellular carcinoma (HCC) remains to be evaluated. Phosphatidylinositol 3-kinase (PI3K), on the other hand, has been shown to play a key role in the tumorigenesis, proliferation, metastasis, apoptosis, and angiogenesis of HCC by regulating gene expression. We initially identified the interaction of DDC with PI3K by means of the phage display methodology. This association was further confirmed in human hepatocellular carcinoma cell lines, human embryonic kidney cells, human neuroblastoma cells, as well as mouse brain, by the use of specific antibodies raised against DDC and PI3K. Functional aspects of the above interaction were studied upon treatment with the DDC inhibitor carbidopa and the PI3K inhibitor LY294002. Interestingly, our data demonstrate the expression of the neuronal type DDC mRNA in HCC cells. The present investigation provides new evidence on the possible link of DDC with the PI3K pathway, underlining the biological significance of this complex enzyme.

Acid-induced acute lung injury in mice is associated with P44/42 and c-Jun N-terminal kinase activation and requires the function of tumor necrosis factor α receptor I.

Aspiration of hydrochloric acid (HCl)-containing gastric juice leads to acute lung injury (ALI) and hypoxemic respiratory failure due to an exuberant inflammatory response associated with pulmonary edema from increased vascular and epithelial permeability. The aim of this study was to determine the role and signaling mechanisms of tumor necrosis factor α (TNF-α) in experimental ALI from HCl aspiration using a combination of genetic animal models and pharmacologic inhibition strategies. To this end, HCl was instilled intratracheally to mice, followed by respiratory system elastance measurement, bronchoalveolar lavage, and lung tissue harvesting 24 h after injection. Hydrochloric acid instillation induced an inflammatory response in the lungs of wild-type mice, evidenced as increased bronchoalveolar lavage total cells, neutrophils, and total protein; histologic lung injury score; and respiratory system elastance, whereas TNF-α receptor I mRNA levels were maintained. These alterations could be prevented by pretreatment with etanercept or genetic deletion of the 55-kd TNF-α receptor I, but not by deletion of the TNF-α gene. Hydrochloric acid induced a 6-fold increase in apoptotic, caspase 3-positive cells in lung sections from wild-type mice, which was abrogated in mice lacking TNF-α receptor I. In immunoblotting and immunohistochemistry studies, HCl stimulated signaling via p44/42 and c-Jun N-terminal kinase, which was blocked in TNF-α receptor I knockout mice. In conclusion, ALI induced by HCl requires TNF-α receptor I function and associates with activation of downstream proinflammatory signaling pathways p44/42 and c-Jun N-terminal kinase.