Dr Catherine McClintock

Introduction: Acute respiratory distress syndrome (ARDS) is one of the main causes of Intensive Care Unit morbidity and mortality. Metabolic biomarkers of mitochondrial dysfunction are correlated with disease development and high mortality in many respiratory conditions, however it is not known if they can be used to assess risk of mortality in patients with ARDS.

Objectives: The aim of this systematic review was to examine the link between recorded biomarkers of mitochondrial dysfunction in ARDS and mortality.

Methods: A systematic review of CINAHL, EMBASE, MEDLINE, and Cochrane databases was performed. Studies had to include critically ill ARDS patients with reported biomarkers of mitochondrial dysfunction and mortality. Information on the levels of biomarkers reflective of energy metabolism and mitochondrial respiratory function, mitochondrial metabolites, coenzymes, and mitochondrial deoxyribonucleic acid (mtDNA) copy number was recorded. RevMan5.4 was used for meta-analysis. Biomarkers measured in the samples representative of systemic circulation were analyzed separately from the biomarkers measured in the samples representative of lung compartment. Cochrane risk of bias tool and Newcastle-Ottawa scale were used to evaluate publication bias (Prospero protocol: CRD42022288262).

Results: Twenty-five studies were included in the systematic review and nine had raw data available for follow up meta-analysis. Biomarkers of mitochondrial dysfunction included mtDNA, glutathione coupled mediators, lactate, malondialdehyde, mitochondrial genetic defects, oxidative stress associated markers. Biomarkers that were eligible for meta-analysis inclusion were: xanthine, hypoxanthine, acetone, N-pentane, isoprene and mtDNA. Levels of mitochondrial biomarkers were significantly higher in ARDS than in non-ARDS controls (P = 0.0008) in the blood-based samples, whereas in the BAL the difference did not reach statistical significance (P = 0.14). mtDNA was the most frequently measured biomarker, its levels in the blood-based samples were significantly higher in ARDS compared to non-ARDS controls (P = 0.04). Difference between mtDNA levels in ARDS non-survivors compared to ARDS survivors did not reach statistical significance (P = 0.05).

Conclusion: Increased levels of biomarkers of mitochondrial dysfunction in the blood-based samples are positively associated with ARDS. Circulating mtDNA is the most frequently measured biomarker of mitochondrial dysfunction, with significantly elevated levels in ARDS patients compared to non-ARDS controls. Its potential to predict risk of ARDS mortality requires further investigation.

Background: MSC Extracellular Vesicles (EVs) are tested as novel therapy for ARDS. We previously showed that EVs can modulate host cells via mitochondrial transfer. Mitochondrial dysfunction underpins pathogenesis of many respiratory diseases but its role in ARDS is unclear.

Aim: To investigate the impact of acute lung injury on mitochondrial homeostasis in vivo and the ability of EVs to restore it via mitochondrial transfer.

Methods: Mice were injured by LPS intratracheally and treated intravenously with control EVs or with RhoEVs depleted from mitochondria. 24 hrs after injury lungs were harvested for single cell mRNA sequencing on 10X Illumina platform. In vitro, Human Pulmonary Endothelial Cells (HPMECs) were co-cultured with EVs in the presence of LPS and expression of key regulators of mitochondrial biogenesis was assessed by Western blot.

Results: Transcriptomic analysis of the lung tissue revealed that LPS significantly inhibited key pathways of mitochondrial biogenesis and mitophagy, compared to healthy controls. Vascular endothelial cells was the largest population identified by unsupervised clustering, within this population expression of the master regulator of mitochondrial biogenesis (PGC-1a) as well as downstream mediators (TFAM, NRF-1, SIRT3&4) was decreased by LPS and restored with control EVs but not with RhoEVs. In vitro, LPS reduced protein expression levels of PGC-1a and TFAM in HPMECs, which were restored by control EVs but not by RhoEVs.

Conclusion: Mitochondrial homeostasis is disrupted in acute lung injury in vivo. EV mitochondrial transfer is critical for restoration of mitochondrial biogenesis in pulmonary endothelial cells in vivo and in vitro.

Background
Progenitor endothelial colony forming cells (ECFCs) are critical for vascular homeostasis and hold therapeutic potential for ischaemic cardiovascular disease (CVD). As angiogenic capacity and efficacy within diseased tissues is particularly impacted in diabetic patients, who show high incidence of ischaemic CVD, targeting of critical ECFC pathways in this setting represents an innovative focus towards enhancing intrinsic vasoreparative function. We previously reported that NADPH oxidase 4 (NOX4)-derived reactive oxygen species promote cord blood-derived ECFC (CB-ECFC) pro-angiogenic response, whilst NOX4 overexpression (OE) enhances revascularisation capacity. Here, we aimed to investigate specific influence of NOX4-dependent signalling on CB-ECFC angiogenic dysfunction observed upon exposure to both experimental and clinical diabetes to define whether NOX4 may represent a viable therapeutic target in this context.

Methods
CB-ECFCs were cultured in high glucose (D-glucose, 25 mmol/L) or control media (5 mmol/L) ± phorbol 12-myristate 13- acetate (PMA, 500 nmol/L) for 72 h with assessment of migratory/tubulogenic capacity and NOX4 mRNA expression (qRT-PCR). Detailed analysis of angiogenic function and signalling (Western blot, RNA sequencing) was performed in CB-ECFCs isolated from donors with gestational diabetes prior to NOX4 plasmid OE to define rescue potential and key mechanistic pathways (network analysis, proteome profiling). Statistical significance was determined using one-way ANOVA with Bonferroni post-host testing or paired/unpaired Student’s t-test, as appropriate.

Results
PMA-stimulated CB-ECFC migration and tube-forming capacity observed in control cells was suppressed in experimental diabetes in parallel with reduced NOX4 expression and rescued by plasmid NOX4OE. As direct evidence of clinical relevance, CB-ECFCs from gestational diabetic donors showed reduced angiogenic potential associated with attenuated NOX4, eNOS activity and downregulation of key vasoreparative signalling. Furthermore, NOX4OE rescued angiogenic function in chronically diabetic CB-ECFCs via modulation of downstream signalling involving both direct and indirect enhancement of pro-angiogenic protein expression (endoglin/SERPINE1/E2F1) linked to reduced p53 phosphorylation.

Conclusions
Taken together, these data indicate for the first time that reduced NOX4 expression plays a pivotal role in CB-ECFC angiogenic dysfunction linked with diabetes whilst highlighting NOX4-dependent signalling as a potential target to protect and augment their intrinsic vasoreparative capacity towards addressing current translational barriers.
 

J Intensive Care Soc. 2023 Aug; 24(1 Suppl): 1–117. Published online 2023 Apr 21. doi: 10.1177/17511437231156066

Background: MSC Extracellular Vesicles (EVs) offer a novel cell-free therapy for ARDS. We previously showed that EVs could ameliorate host cells acute injury via mitochondrial transfer, yet role of mitochondrial dysfunction in ARDS remains nebulous. 

Aim: To investigate the influence of acute lung injury on mitochondrial homeostasis in preclinical models and the ability of EVs to restore it via mitochondrial transfer. 

Methods:  Mice were injured by intratracheal LPS and treated intravenously with EVs or with mitochondrial depleted RhoEVs. 24 hrs lungs were harvested for single cell mRNA sequencing on 10X Illumina platform. In vitro Human Pulmonary Endothelial Cells (HPMECs) were co-cultured LPS with and without the presence of EVs. Western blots assessed gene expression of mitochondrial biogenesis regulators. HPMEC Barrier integrity was assessed using xCElligence technology. The role of Biogenesis in modulation of HPMEC was studied using specific pharmacological inhibitor (SR18292). Knockdown of protein GPS2 expression in EVs was performed by siRNA treatment of MSCs. 

Results: Transcriptomic analysis of the lung tissue revealed that LPS significantly inhibited key pathways of mitochondrial biogenesis, compared to healthy controls. Vascular endothelial cells was the largest population identified by unsupervised clustering, within this population expression of the master regulator of mitochondrial biogenesis (PGC-1a) as well as downstream mediators  (TFAM and PPARG) was decreased by LPS and restored with control EVs but not with RhoEVs. In vitro, LPS reduced protein expression levels of PGC-1a and downstream mediators in HPMECs, which were restored by control EVs but not by RhoEVs. SR18292 impeded the ability of HPMECs to maintain barrier integrity. GPS2 silenced EVs are unable to restore LPS induced barrier integrity. 

Conclusion: Mitochondrial homeostasis is disrupted in acute lung injury in vivo. EV mitochondrial transfer is critical for restoration of mitochondrial biogenesis in pulmonary endothelial cells in vivo and in vitro. PGC1a is important regulator of endothelial lung barrier integrity and mediator of EV effect.

Late breaking abstractt

Cytotherapy

2022