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Recoupling of eNOS with folic acid prevents abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E mice.

Siu KL, Miao XN, Cai H - PLoS ONE (2014)

Bottom Line: This resulted in a reduction of AAA rate to 19.5%.This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability.Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Molecular Medicine and Cardiology, Departments of Anesthesiology and Medicine, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
We have previously shown that eNOS uncoupling mediates abdominal aortic aneurysm (AAA) formation in hph-1 mice. In the present study we examined whether recoupling of eNOS prevents AAA formation in a well-established model of Angiotensin II-infused apolipoprotein E (apoE) mice by targeting some common pathologies of AAA. Infusion of Ang II resulted in a 92% incidence rate of AAA in the apoE animals. In a separate group, animals were treated orally with folic acid (FA), which is known to recouple eNOS through augmentation of dihydrofolate reductase (DHFR) function. This resulted in a reduction of AAA rate to 19.5%. Imaging with ultrasound showed that FA markedly inhibited expansion of abdominal aorta. FA also abolished elastin breakdown and macrophage infiltration in the AAA animals. The eNOS uncoupling activity, assessed by L-NAME-sensitive superoxide production, was minimal at baseline but greatly exaggerated with Ang II infusion, which was completely attenuated by FA. This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability. Furthermore, the expression and activity of DHFR was decreased in Ang II-infused apoE mice specifically in the endothelial cells, while FA administration resulted in its recovery. Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.

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Folic acid treatment restores endothelial specific DHFR expression and activity in Ang II-infused apoE  mice.DHFR expression (A, n = 8–11) and activity (B, n = 5–7) measured from endothelial cells (ECs) and EC-denuded aortas isolated from apoE  mice after 4 weeks of infusion. Data show that DHFR expression and activity was restored in FA treated animals specifically in the endothelial cells. eNOS was stained to show that isolation/removal of ECs from the vessels was successful. *p<0.05.
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pone-0088899-g007: Folic acid treatment restores endothelial specific DHFR expression and activity in Ang II-infused apoE mice.DHFR expression (A, n = 8–11) and activity (B, n = 5–7) measured from endothelial cells (ECs) and EC-denuded aortas isolated from apoE mice after 4 weeks of infusion. Data show that DHFR expression and activity was restored in FA treated animals specifically in the endothelial cells. eNOS was stained to show that isolation/removal of ECs from the vessels was successful. *p<0.05.

Mentions: The above data show that restoration of eNOS coupling, which is tied to the bioavailability of H4B, may be important in FA's protection against Ang II induced AAA development in the apoE animals. Previous studies have shown that FA treatment can recouple eNOS through the improvement of endothelial DHFR function, which is essential in salvaging H4B [18], [21]. Here, we examined endothelial DHFR activity and expression in Ang II-infused apoE mice to test whether DHFR is also improved during FA prevention of AAA in this model. Endothelial cells were isolated from freshly prepared aortas, and Western blots and HPLC were performed to assess DHFR expression and activity respectively on both the isolated ECs and EC-denuded aortas. The top panels of Figure 7A shows the representative Western blots for eNOS (144 kD), actin control (42 kD), and DHFR (21 kD) (n = 8–11 each). eNOS was stained for quality control to examine the effectiveness of the EC isolation/removal. The increase in eNOS expression in the Ang II-infused groups was expected and well documented in previous works [22]–[24]. The summarized data indicate that DHFR was significantly decreased in aortic ECs of Ang II infused apoE animals, while FA treatment restored this level back to baseline. Similarly, DHFR activity (Figure 7B, n = 5–7) was also markedly reduced in ECs specifically in Ang II infused apoE mice, but then fully recovered to above baseline levels with oral FA treatment. Of note, DHFR expression and activity remained unchanged in EC-denuded aortas.


Recoupling of eNOS with folic acid prevents abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E mice.

Siu KL, Miao XN, Cai H - PLoS ONE (2014)

Folic acid treatment restores endothelial specific DHFR expression and activity in Ang II-infused apoE  mice.DHFR expression (A, n = 8–11) and activity (B, n = 5–7) measured from endothelial cells (ECs) and EC-denuded aortas isolated from apoE  mice after 4 weeks of infusion. Data show that DHFR expression and activity was restored in FA treated animals specifically in the endothelial cells. eNOS was stained to show that isolation/removal of ECs from the vessels was successful. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928303&req=5

pone-0088899-g007: Folic acid treatment restores endothelial specific DHFR expression and activity in Ang II-infused apoE mice.DHFR expression (A, n = 8–11) and activity (B, n = 5–7) measured from endothelial cells (ECs) and EC-denuded aortas isolated from apoE mice after 4 weeks of infusion. Data show that DHFR expression and activity was restored in FA treated animals specifically in the endothelial cells. eNOS was stained to show that isolation/removal of ECs from the vessels was successful. *p<0.05.
Mentions: The above data show that restoration of eNOS coupling, which is tied to the bioavailability of H4B, may be important in FA's protection against Ang II induced AAA development in the apoE animals. Previous studies have shown that FA treatment can recouple eNOS through the improvement of endothelial DHFR function, which is essential in salvaging H4B [18], [21]. Here, we examined endothelial DHFR activity and expression in Ang II-infused apoE mice to test whether DHFR is also improved during FA prevention of AAA in this model. Endothelial cells were isolated from freshly prepared aortas, and Western blots and HPLC were performed to assess DHFR expression and activity respectively on both the isolated ECs and EC-denuded aortas. The top panels of Figure 7A shows the representative Western blots for eNOS (144 kD), actin control (42 kD), and DHFR (21 kD) (n = 8–11 each). eNOS was stained for quality control to examine the effectiveness of the EC isolation/removal. The increase in eNOS expression in the Ang II-infused groups was expected and well documented in previous works [22]–[24]. The summarized data indicate that DHFR was significantly decreased in aortic ECs of Ang II infused apoE animals, while FA treatment restored this level back to baseline. Similarly, DHFR activity (Figure 7B, n = 5–7) was also markedly reduced in ECs specifically in Ang II infused apoE mice, but then fully recovered to above baseline levels with oral FA treatment. Of note, DHFR expression and activity remained unchanged in EC-denuded aortas.

Bottom Line: This resulted in a reduction of AAA rate to 19.5%.This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability.Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Molecular Medicine and Cardiology, Departments of Anesthesiology and Medicine, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
We have previously shown that eNOS uncoupling mediates abdominal aortic aneurysm (AAA) formation in hph-1 mice. In the present study we examined whether recoupling of eNOS prevents AAA formation in a well-established model of Angiotensin II-infused apolipoprotein E (apoE) mice by targeting some common pathologies of AAA. Infusion of Ang II resulted in a 92% incidence rate of AAA in the apoE animals. In a separate group, animals were treated orally with folic acid (FA), which is known to recouple eNOS through augmentation of dihydrofolate reductase (DHFR) function. This resulted in a reduction of AAA rate to 19.5%. Imaging with ultrasound showed that FA markedly inhibited expansion of abdominal aorta. FA also abolished elastin breakdown and macrophage infiltration in the AAA animals. The eNOS uncoupling activity, assessed by L-NAME-sensitive superoxide production, was minimal at baseline but greatly exaggerated with Ang II infusion, which was completely attenuated by FA. This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability. Furthermore, the expression and activity of DHFR was decreased in Ang II-infused apoE mice specifically in the endothelial cells, while FA administration resulted in its recovery. Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.

Show MeSH
Related in: MedlinePlus