# Restoration of NOX4 signalling reverses endothelial colony-forming cell angiogenic dysfunction associated with experimental and clinical diabetes

## TL;DR

Restoring NOX4 signaling can reverse blood vessel repair issues in cells from diabetic patients, offering a new therapeutic approach for cardiovascular disease.

## Contribution

This study is the first to show that reduced NOX4 expression causes angiogenic dysfunction in ECFCs from diabetic donors and that restoring NOX4 can rescue this function.

## Key findings

- NOX4 overexpression rescues impaired migration and tube formation in high-glucose-exposed ECFCs.
- NOX4OE in diabetic ECFCs enhances pro-angiogenic signaling via endoglin/SERPINE1/E2F1 and reduced p53 phosphorylation.
- Clinical relevance is supported by impaired ECFC function in gestational diabetic donors, which is rescued by NOX4 overexpression.

## Abstract

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.

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.

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.

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.