ENDOTHELIAL METABOLISM: Pentose Phosphate Pathway drives vascular maturation

STANDFIRST:

Facchinello, Astone et al¹. demonstrate a role for the endothelial oxidative Pentose Phosphate Pathway (oxPPP) in promoting vascular mural cell coverage and maturation during early development by regulating elastin expression, establishing a critical role of oxPPP in the formation of the vascular system.

During embryonic development, vasculogenesis, the process of de novo blood vessel formation from angioblasts, and angiogenesis, the process of vessel formation from the preexisting vessels, require high coordination between endothelial cells (EC) and perivascular/mural cells (pericytes and smooth muscle cells). EC form the nascent vessels, followed by recruitment of perivascular cells resulting in maturation and stabilization of the vasculature. Although previous studies have provided abundant knowledge about molecular mechanisms that contributed to EC – perivascular cell interaction leading to maturation of nascent vessels², little is known about the role of EC metabolism in recruiting perivascular cells and vessel maturation.

While the best characterized metabolic pathways in EC are glycolysis and oxidative phosphorylation, the Pentose Phosphate Pathway (PPP) is increasingly attracting attention. The PPP utilizes the intermediate glucose 6-phosphate (G6P) to generate NADPH and ribose 5-phosphate (R5P) through the oxidative and non-oxidative phases of PPP. The first oxidative phase of PPP (oxPPP) consists of two irreversible reactions catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH or PGD) to produce NADPH, known to play an essential role in the inactivation of intracellular Reactive Oxygen Species (ROS). The second reversible phase supplies cells with ribose-5-phosphate (R5P), a precursor of nucleotides and nucleic acids. These two phases are of importance as a defensive cellular mechanism favoring cell proliferation and/or repair. It has been recently shown that both NADPH and R5P are important for EC proliferation and migration in pathologies such as diabetes^3,4.

The study published in the current issue of Nature Metabolism has defined a new function for the oxPPP in vascular development. Facchinello, Astone et al. have provided several lines of extensive evidence that the oxPPP is necessary to drive perivascular cell coverage and maturation of the dorsal aorta in both zebrafish and mouse models of embryonic development. They demonstrate that inhibition of oxPPP in cultured EC and in vivo models perturbed extracellular matrix (ECM) signaling and, specifically, the ECM protein elastin that is known to be a critical ECM protein regulating vascular smooth muscle cell activity (Figure 1)⁵.

Figure 1. Endothelial oxPPP shunts development towards vascular maturation through elastin:

Normal blood flow induces the side-pathway of glycolysis, the pentose phosphate pathway (PPP) in endothelial cells (EC). The PPP has two different phases: the irreversible oxidative PPP (oxPPP) and the non-oxidative phases. The final product of oxPPP, R5P, regulates elastin expression, leading to perivascular cell recruitment. G6P – glucose-6-phosphate; F6P – fructose-6-phosphate; G3P – glyceraldehyde-3-phosphate; G6PD – glucose 6-phosphate dehydrogenase; 6GPD – 6-phosphogluconate dehydrogenase; NADP+/NADPH – nicotinamide adenine dinucleotide phosphate; GSH/GSSG – glutathione/glutathione disulphide; ROS – reactive oxygen species; R5P – ribose-5-phosphate; TKT – transketolase; LSS – laminar shear stress.

The authors previously demonstrated that hemodynamic forces are ultimately required for vascular myogenesis in zebrafish since the genetic and pharmacological perturbation of blood flow selectively impaired mural cell coverage of the arterial vessels⁶. In this study, the authors have discovered that oxPPP metabolism is activated in response to the blood flow-induced laminar shear stress (LSS), which is critical for normal vascular functioning, thereby connecting hemodynamic forces, EC metabolism, ECM deposition, and perivascular cell coverage into one evolutionally conserved developmental mechanism.

Facchinello, Astone et al. use several independent animal and cell culture models to test their hypotheses. First, they use several EC- and perivascular cell-reporter zebrafish models that allow applying pharmacological inhibitors to study metabolic mechanisms, while simultaneously perform cell lineage tracing studies, as well as g6pd and pgd mutant zebrafish models to study oxPPP in zebrafish vascular development. Second, to validate zebrafish findings in mammalian models, authors induce EC-specific knockout of Pgd during development and in postnatal retinal angiogenesis using mouse models. Third, lost-of-function (shRNA) and gain-of-function (overexpressing lentiviruses) approaches are used to investigate LSS in regulating oxidative and non-oxidative phases of PPP in endothelium and the functional consequences on vascular maturation.

Elastin is a key ECM protein in the arteries that is known to play a critical role in arterial development and vascular disease by providing reversible artery elasticity and strength⁷. However, it was not known whether EC metabolism regulates elastin during development. Facchinello, Astone et al. demonstrate that hemodynamic forces during development control perivascular cell coverage and maintenance by regulating oxPPP-dependent expression of elastin. Notably, the authors found that the final product of oxPPP, R5P, is required for elastin expression in that the addition of R5P sugars to G6PD- and PGD-deficient EC rescued the elastin expression in the absence of functional oxPPP.

It is interesting whether dysregulation of the oxPPP in adult vasculature might cause the loss of mural cells and/or vascular leakage. Such dysregulation might be triggered by the disturbance of laminar flow or by metabolic problems. Undoubtedly, these findings will inspire new studies on vascular dysfunction in diabetes, inflammation, and cancer. Another interesting application of these new findings is for the field of vascular engineering from induced pluripotent stem (iPS) cells, where the most challenging issue is the longevity of vascular implants in vivo. Both LSS and oxPPP appear to be attractive targets for the development of technologies aimed to increase the stability of engineered vasculature by promoting elastin expression⁸.

While the PPP in various cells, including erythrocytes, macrophages, adipocytes, smooth muscle cells, and hepatocytes, was implicated in pathologies^9–11, substantially less is known regarding the oxPPP in the endothelium. Most studies associate the shift from glycolysis to PPP and back with injections and inflammation mainly due to detoxifying effects of NADPH. In the endothelium, while stimulation of glycolysis generally promotes inflammatory responses, upregulation of the PPP suppresses inflammation showing that the PPP is a counter-regulatory anti-inflammatory response¹². Together with the present study, these results reveal an important role for the PPP in vascular protection and stability. Likewise, the studies on the human “Mediterranean” polymorphism of G6PD suggested that the PPP might have a protective function in cardiovascular diseases¹³. Another remarkable functional consequence of the PPP pathway is its role in the relaxation of coronary arteries in an endothelium-dependent manner¹⁴. At the same time, over-activation of PPP was suggested to serve as a mechanism responsible for pro-inflammatory changes and vascular damage caused by hyperglycemia¹⁵. Together, the PPP continues attracting attention as a potentially important therapeutic target in metabolic and inflammatory diseases.

In summary, this study provides a novel mechanism of oxPPP-dependent ECM deposition and subsequent vascular maturation in development that is initiated by the blood flow. Furthermore, these findings reinforce the importance of the various aspects of endothelial metabolism in physiology and vascular pathologies.