This study demonstrates that BH4 bioavailability is reduced during the early post-TBI phase and that exogenous supplementation of BH4 reduces post-TBI vascular oxidative stress by a mechanism that relates to its eNOS cofactor function. Our study also suggests that at least some of GT3’s radioprophylactic properties may be related to increased generation of BH4 through the de novo synthesis pathway by a mechanism that involves suppression of the regulatory protein, GFRP.
Radiation-induced endothelial dysfunction is believed to play an important role in the pathogenesis of both early and delayed radiation injury (22
). Radiation exposure increases vascular oxidative stress and induces various functional and morphological changes in endothelial cells, such as loss of thromboresistance, increased permeability, and apoptosis. Endothelial nitric oxide synthase (eNOS) is considered to be one of the key regulatory enzymes of endothelial function. Changes in eNOS function are believed to be involved in the development of radiation-induced endothelial dysfunction. eNOS dependent endothelial dysfunction may not only result in inadequate production of the regulatory molecule NO, but also to significant production of the highly reactive oxygen radical O2−
by eNOS in certain situations, referred to as eNOS “uncoupling”. In the uncoupled state, eNOS produces O2−
at the expense of NO.
One of the main causes of eNOS uncoupling is inadequate availability of the eNOS cofactor BH4. BH4 is highly redox sensitive and its availability may be reduced under conditions of oxidative stress due to rapid oxidation of BH4 to BH2. Insufficient BH4 availability and consequential eNOS uncoupling has been shown to play important roles in the pathogenesis of endothelial dysfunction during various conditions characterized by increased oxidative stress, such as hypercholesterolemia, diabetes, and hypertension (16
). Until now, little was known about the effects of radiation exposure on the availability of BH4, the possible importance of BH4 dependent eNOS uncoupling in radiation-induced endothelial dysfunction, and the extent to which radioprotective agents regulate BH4.
Our current data show that radiation exposure induces a reduction in BH4 availability during the early post-irradiation phase. The occurrence and relevance of radiation-induced, BH4 dependent eNOS uncoupling, is supported by the observation that supplementation with GT3 as well as BH4 reduces vascular peroxynitrite production during the early post-TBI phase. In contrast, supplementation with NH4, a compound with similar anti-oxidant properties as BH4 but no NOS cofactor function, does not reduce peroxynitrite production, indicating that BH4 exerts its effect by acting as a NOS cofactor and not by acting as a free radical scavenger.
The beneficial effects of GT3 on post-irradiation vascular peroxynitrite production depend on inhibition of HMG-CoA reductase by GT3. Statins, well known inhibitors of HMG-CoA reductase, have been reported to regulate eNOS function and BH4 availability, thereby reducing eNOS-uncoupling and oxidative stress. The main underlying mechanism for this effect of statins is upregulation of the expression of GTPCH, a key enzyme in BH4 synthesis (28
). Whether GT3 may attenuate post-irradiation vascular peroxynitrite production by modulating BH4 metabolism has, to our knowledge, not been investigated previously. Moreover, because the mechanism by which GT3 inhibits HMG-CoA reductase differs from that of statins (29
), it is interesting to speculate that there may be synergy between statins and GT3 in terms of radioprotective efficacy, just as there is for cholesterol lowering (30
BH4 is synthesized in endothelial cells by de novo
synthesis from GTP or by a salvage pathway that converts BH2 to BH4. The rate limiting enzyme in de novo
BH4 production is GTPCH. GTPCH activity is regulated on multiple levels. Transcriptional and posttranslational changes, like phosphorylation at serine 81, are known to regulate GTPCH activity (31
). Protein-protein interaction is another important regulatory mechanism. In this context, GFRP provides an important negative feedback mechanism for BH4 production (33
). The binding of GFRP to GTPCH enables end-product feedback inhibition by BH4. Conversely, phenylalanine can stimulate GTPCH enzymatic activity via GFRP.
Unlike statins, GT3 does not affect the expression of GTPCH1. On the other hand, GT3 induces a reduction in GFRP protein levels by reducing GFRP gene transcription. Moreover, GT3 not only reduces general cellular GFRP levels, but also reduces GFRP-GTPCH1 protein binding. These effects appear to depend on inhibition of HMG-CoA reductase by GT3. Further research is needed to determine whether GT3 can prevent the post-irradiation decline in BH4 availability by suppressing GFRP production in vivo.
Restoration of BH4 supplies or preventing BH4 shortage appears to be a novel, promising, and interesting approach to ameliorate radiation injury. Since the effects of BH4 supplementation are very unpredictable due the high extracellular concentrations compared to the desired intracellular levels, it seems appropriate to instead focus on strategies that could increase intracellular BH4 without high concentration biopterin supplementation.
In conclusion, exposure to TBI reduces the availability of the eNOS cofactor BH4 during the early post-irradiation phase. Post-irradiation free radical production can be attenuated by increasing BH4 availability. The radioprotective vitamin E analogue, GT3, regulates the expression of GFRP and may thus exert its radioprotective effects partly through regulation of BH4 availability. Clearly, further research is warranted, for example, to explore the effects of GT3 on BH4 metabolism and BH4 related radioprotection in vivo
; to investigate whether the findings in the present study also apply to localized and/or fractionated irradiation as used clinically; whether synergy exists between the effects of GT3 and statins in terms of protection, mitigation, or treatment of radiation-induced normal tissue injury; and to what extent similar mechanisms apply to other antioxidants and vitamin E analogues with HMG-Co reductase inhibitory activities, such as δ-tocotrienol (29