Boosting Cellular NADPH in Blood Vessel Cells Slows Vascular Aging
Scientists discover that raising cytosolic NADPH in endothelial cells—via G6PD or folic acid—reverses hallmarks of vascular aging in mice.
Summary
Researchers used a fluorescent NADPH sensor to reveal that cytosolic NADPH rises in senescent endothelial cells, while mitochondrial NADPH stays unchanged. The rate-limiting enzyme G6PD drives this increase, and its activity is boosted when nitric oxide-mediated inhibition (S-nitrosylation at C385) is reduced. Overexpressing G6PD protected against vascular aging by increasing reduced glutathione and suppressing HDAC3 activity. Screening 1,419 FDA-approved drugs, folic acid emerged as a top candidate; it raises NADPH via MTHFD1 and effectively reversed vascular aging markers in both angiotensin II-infused and naturally aged mice. These findings reframe NADPH metabolism as a causal, targetable driver of vascular aging.
Detailed Summary
Vascular aging underlies much of the cardiovascular disease burden in older adults, yet the metabolic mechanisms governing endothelial cell senescence remain incompletely understood. This study addresses a key gap by mapping how the essential redox cofactor NADPH changes across cellular compartments during endothelial senescence—and whether correcting those changes can slow vascular aging.
Using genetically encoded fluorescent sensors (iNap1 for cytosol, iNap3 for mitochondria) expressed in primary human aortic endothelial cells (HAECs), the team showed that cytosolic NADPH rises substantially during senescence induced by angiotensin II, high glucose, endothelin-1, homocysteine, or simple replicative exhaustion. Strikingly, mitochondrial NADPH was unchanged across all these models. The same cytosolic NADPH elevation was confirmed in endothelial cells freshly isolated from the aortas of old (18-month) versus young (4-month) mice, and in thoracic aortic tissue from 24-month-old naturally aged mice.
Mechanism work identified glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative pentose phosphate pathway, as the driver. During senescence, declining nitric oxide levels reduce S-nitrosylation of G6PD at cysteine-385, which disinhibits the enzyme and raises its activity—producing more cytosolic NADPH. Functionally, G6PD overexpression in HAECs reduced senescence markers (p16, p21, SASP cytokines, β-galactosidase staining), increased reduced glutathione (GSH), and suppressed HDAC3, an epigenetic regulator linked to inflammatory gene expression. Conversely, G6PD knockdown worsened senescence phenotypes. Endothelial-specific G6PD overexpression in mice protected against angiotensin II-induced vascular dysfunction and reduced arterial stiffness.
To translate these findings pharmacologically, the researchers conducted a high-throughput screen of 1,419 FDA-approved drugs using iNap1 fluorescence as a readout. Folic acid (FA) was the top hit. FA elevates cytosolic NADPH through MTHFD1-catalyzed reactions in the folate one-carbon cycle. In both angiotensin II-infused and naturally aged mice, FA supplementation improved endothelial function, reduced vascular stiffness, and suppressed senescence biomarkers—effects abolished when MTHFD1 was knocked down, confirming on-target activity.
Overall, the study establishes that cytosolic NADPH metabolism is an active adaptive response to endothelial senescence, and that amplifying it genetically or with folic acid causally delays vascular aging. The dual NADPH-GSH-HDAC3 axis offers a mechanistic explanation for how NADPH protects against the epigenetic and oxidative changes driving endothelial senescence.
Key Findings
- Cytosolic NADPH rises in senescent endothelial cells across five models; mitochondrial NADPH is unchanged.
- G6PD de-S-nitrosylation at C385 (due to reduced NO) boosts its activity and elevates cytosolic NADPH during senescence.
- G6PD overexpression reduces senescence markers and vascular stiffness; knockdown worsens them.
- NADPH protects against vascular aging by increasing reduced glutathione and inhibiting HDAC3 activity.
- Folic acid, acting via MTHFD1, raised NADPH and reversed vascular aging in both angiotensin II-infused and naturally aged mice.
Methodology
Study used genetically encoded iNap1/iNap3 fluorescent sensors to measure compartment-specific NADPH in live primary human aortic endothelial cells and mouse aortic endothelial cells. Multiple senescence models were employed (angiotensin II, high glucose, endothelin-1, homocysteine, replicative), complemented by in vivo G6PD overexpression/knockdown mouse models, microarray analysis, and an iNap1-based high-throughput screen of 1,419 FDA-approved drugs.
Study Limitations
The in vivo mouse models (angiotensin II infusion and natural aging) may not fully recapitulate human vascular aging complexity. Human plasma NADPH data showed only a trend with age, and causal direction in humans remains unconfirmed. Endothelial-specific G6PD overexpression effects on other cell types and long-term safety of NADPH elevation were not thoroughly characterized.
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