Tracer-Based Metabolomics Reveals Nitric Oxide Metabolic Dynamics in a 3D Microvessels-on-Chip Model of Endothelial Dysfunction

Nitric oxide plays a pivotal role in maintaining vascular homeostasis. It is synthesized primarily by endothelial nitric oxide synthase (eNOS), which converts L-arginine to L-citrulline, producing NO as a byproduct. NO acts as a vasodilator, helping blood vessels relax, and also regulates several other important processes like inflammation and oxidative stress. Disruptions in NO production or bioavailability are commonly observed in endothelial dysfunction, which is associated with various cardiovascular diseases (CVDs) such as atherosclerosis, hypertension, and diabetes. Endothelial dysfunction is often characterized by diminished NO levels, leading to impaired vasodilation and a pro-inflammatory state, making the accurate measurement of NO synthesis and its metabolic pathways vital for both diagnostic and therapeutic developments.  The traditional 2D endothelial cell culture models have limitations when it comes to studying NO metabolism and the associated endothelial responses. These models fail to mimic the physiological fluid dynamics that influence endothelial cell behavior, particularly the influence of laminar shear stress. Shear stress, caused by the flow of blood, is a critical factor in modulating eNOS activity and, consequently, NO production. Without the incorporation of these environmental cues, the static 2D models typically present an inaccurate portrayal of endothelial function, often leading to an overemphasis on a pro-inflammatory state and the generation of oxidative stress. Given these limitations, recent paper published in FASEB Journal and conducted by Dr. Kanchana Pandian, Dr. Luojiao Huang, Dr. Abidemi Junaid, Dr. Amy Harms, Dr. Anton Jan van Zonneveld, and led by Professor Thomas Hankemeier from the Leiden University in Netherlands, the researchers developed 3D microvessels-on-chip system with significant improvement in fluid flow, thereby mimicking the mechanical environment endothelial cells experience in vivo. The innovative aspect of this research lies in the application of tracer-based metabolomics to study NO metabolism. By employing isotope-labeled L-arginine, the researchers were able to trace the metabolic conversion of arginine to citrulline and ornithine key metabolites in NO production. This tracer-based method offers several advantages over traditional techniques, which often suffer from sensitivity issues or difficulty in measuring NO due to its rapid reactivity and short half-life.

In their study, the team emphasized the importance of using a 3D model over a static 2D system. In the 2D model, endothelial cells exhibited characteristics of endothelial dysfunction, such as diminished NO production and a pro-inflammatory state. However, in the 3D microvessels-on-chip model, particularly under unidirectional fluid flow, the endothelial cells showed improved NO production, reflecting a more physiologically accurate state of vascular health. The authors observed the differential impact of flow on NO metabolism. In the 3D model, the ratio of labeled-citrulline to labelled-ornithine was significantly higher under flow conditions, particularly when eNOS was stimulated by VEGF. This suggests that flow, particularly unidirectional flow, enhances eNOS activity and NO production, which is a critical factor in maintaining endothelial function. Conversely, the 2D model showed no significant changes in response to VEGF stimulation, underscoring its limitations in accurately representing endothelial biology. They also highlighted the crucial role of arginase in NO metabolism where inhibition of arginase with BEC led to increased citrulline production, which indicates that arginase may compete with eNOS for the common substrate L-arginine. By inhibiting arginase, more L-arginine is available for NO production, thus enhancing eNOS activity and restoring endothelial function in conditions of endothelial dysfunction.

This research is significant and has important implications for our understanding and treating cardiovascular diseases, where endothelial dysfunction and reduced NO bioavailability are central pathophysiological features. The ability to accurately measure NO production in a physiologically relevant model can provide deeper insights into the metabolic shifts that occur during disease progression and offer potential therapeutic targets to restore NO levels. For example, the application of arginase inhibitors, as demonstrated in this study, could be a promising strategy to increase NO production in patients with CVDs characterized by endothelial dysfunction. Moreover, the use of 3D microvessels-on-chip systems can be extended to other vascular diseases, allowing for a more nuanced study of the effects of various pharmacological interventions under conditions that closely mimic human physiology.

In conclusion, Professor Thomas Hankemeier and his team provided a more accurate in vitro representation of endothelial function by integrating fluid flow, which mimics the physiological conditions experienced by endothelial cells in vivo.  It also addressed a critical gap in cardiovascular disease modeling. Endothelial dysfunction, characterized by impaired NO production, is a key contributor to cardiovascular diseases such as atherosclerosis, hypertension, and diabetes. With this innovative device of a more physiologically relevant model, we have a better tool for investigating endothelial cell responses to metabolic and inflammatory stimuli, particularly in relation to NO synthesis. The ability to trace NO metabolites with precision through the application of stable isotope-labeled arginine provides detailed insights into the metabolic pathways that underlie NO production, revealing the direct impacts of shear stress, enzyme inhibition, and stimulation on endothelial cell function. Moreover, the authors’ findings  that targeting arginase activity could be a potential strategy for restoring NO production in conditions of endothelial dysfunction. Since arginase competes with eNOS for L-arginine, its inhibition leads to higher NO production, presenting a promising avenue for cardiovascular disease treatment. Furthermore, the tracer-based metabolomics approach could be extended to study other diseases characterized by metabolic dysregulation, providing a broader platform for drug discovery and therapeutic interventions. Indeed, we anticipate the use of the 3D microvessels-on-chip model can revolutionize preclinical testing of drugs aimed at modulating endothelial function. It allows for more accurate predictions of how endothelial cells will respond in vivo, making it a valuable tool for pharmaceutical research. Additionally, the integration of this model with various stimulatory and inhibitory compounds, as demonstrated in the study, provides a comprehensive method for understanding how different pathways contribute to endothelial dysfunction and offers new targets for therapeutic intervention.