GTP Cyclohydrolase 1 Expression Research Articles

GCH1 reduces LPS-induced alveolar macrophage polarization and inflammation by inhibition of ferroptosis.

GTP cyclohydrolase 1(GCH1) was reported to protect against ferroptosis. However, it is not clear whether GCH1 reduced lipopolysaccharide (LPS)-induced macrophage polarization and inflammation by inhibition of ferroptosis. Bioinformatics analysis was used to screen differential expression genes (DEGs) and obtain the different pathways and biological features. Lasso cox regression analysis with ferroptosis related DEGs was established to screen the most relevant genes for disease risk. LPS induced Raw264.7 macrophage polarization model and GCH1-specific siRNA oligos transfection were performed to confirm the function of GCH1. Immunofluorescence staining, western blot and quantitative real-time PCR were performed to detect the expression of iNOS, CD206, GCH1, IL6, SLC2A6, F4/80, IL1β, TNFα, IL10, GPX4, ACSL4, AMPK and p-AMPK in macrophages. The levels of ROS, SOD, MDA and GSH were detected according to the instructions of the reagent kit, respectively. 542 DEGs were screened from GSE40885 microarray. GO and KEGG pathway enrichmentanalysis showed that the upregulated DEGs induced by LPS in alveolar macrophage were closely associated with inflammatory and immune responses, the downregulated DEGs were related to lipid metabolism, insulin resistance and AMPK signal pathway. Lasso cox regression analysis screened GCH1, IL6, and SLC2A6. Our experimental results showed that the expression of GCH1 and IL6 in the LPS group was higher than that in the control group, but there was no difference in the expression of SLC2A6. Bioinformatics analysis with GSE112720 observed that ferroptosis was enriched in GCHfl/fl + LPS group compared with GCHfl/flTie2cre + LPS group and GCHfl/fl + control group. Silence of GCH1 increased the levels of IL6, TNF-α and IL-1β and decreased IL10 level. Silence of GCH1 increased iNOS level and decreased CD206 level. Moreover, silence of GCH1 raised ferroptosis induced by LPS in macrophages and suppressed the activity of AMPK pathway. GCH1 inhibited ferroptosis in LPS-stimulated macrophages, reduced macrophage toward to M1 polarization and inflammatory response.

Suppression of GCH1 Sensitizes Ovarian Cancer and Breast Cancer to PARP Inhibitor.

Breast and ovarian cancers are common malignancies among women, contributing to a significant disease burden, and are characterized by a high level of genomic instability, owing to the failure of homologous recombination repair (HRR). Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) could elicit the synthetic lethal effect of tumor cells in patients with homologous recombination deficiency, ultimately achieving a favorable clinical benefit. However, primary and acquired resistance remain the greatest hurdle, limiting the efficacy of PARP inhibitors; thus, strategies conferring or augmenting tumor cell sensitivity to PARP inhibitors are urgently required. Our RNA-seq data of niraparib-treated and -untreated tumor cells were analyzed by R language. Gene Set Enrichment Analysis (GSEA) was applied to assess the biological functions of GTP cyclohydrolase 1 (GCH1). Quantitative real-time PCR, Western blotting, and immunofluorescence were applied to confirm the upregulation of GCH1 upon niraparib treatment at transcriptional and translational levels. Immunohistochemistry of patient-derived xenograft (PDX)-derived tissue sections further validated that niraparib increased GCH1 expression. Tumor cell apoptosis was detected by flow cytometry, while the superiority of the combination strategy was confirmed in the PDX model. The expression of GCH1 was aberrantly enriched in breast and ovarian cancers and increased after niraparib treatment via JAK-STAT signaling. GCH1 was also demonstrated to be associated with the HRR pathway. Subsequently, the enhancement of the tumor-killing effect of PARP inhibitors induced by GCH1 suppression using siRNA and GCH1 inhibitor was validated by flow cytometry in vitro. Finally, using the PDX model, we further demonstrated that GCH1 inhibitors markedly potentiated PARP inhibitors' antitumor efficacy in vivo. Our results illustrated that PARP inhibitors promote GCH1 expression via the JAK-STAT pathway. We also elucidated the potential relationship between GCH1 and the homologous recombination repair pathway and proposed a combination regimen of GCH1 suppression with PARP inhibitors in breast and ovarian cancers.

Procyanidin B2 ameliorates endothelial dysfunction induced by nicotine via the induction of tetrahydrobiopterin synthesis

• Nicotine decreases the expression of DHFR and GCH1 to inhibit BH 4 and NO production. • PCB2 prevents nicotine-induced DHFR and GCH1 loss. • PCB2 improves endothelium-dependent vasorelaxation impaired by nicotine. • PCB2 induces vasorelaxation through a PPARδ-BH 4 dependent manner. Smoking is an independent risk factor for the cardiovascular diseases. Nicotine, a major component of tobacco, is responsible for the impaired endothelial-dependent vasorelaxation in smokers. Procyanidin B2 (PCB2), a natural flavonoid, has been reported to possess several potential beneficial effects on the cardiovascular system. However, whether PCB2 prevents nicotine-induced endothelial dysfunction remains unknown. In this study, we demonstrated that PCB2 improved nicotine-impaired endothelium-dependent vasorelaxation in mouse thoracic aortas. Mechanistically, PCB2 increased the expression levels of dihydrofolate reductase (DHFR) and GTP cyclohydrolase 1 (GCH1), two important enzymes in the synthesis of tetrahydrobiopterin (BH 4 ), in mouse aortas and cultured ECs. GSK0660, a selective antagonist of peroxisome proliferator-activated receptor δ (PPARδ), abolished the PCB2-induced effects on BH 4 synthesis, nitric oxide (NO) production and vasodilation. Collectively, we demonstrated that PCB2 mitigates nicotine-induced endothelial dysfunction through a PPARδ-BH 4 dependent manner, thereby suggesting a novel role of PCB2 in preventing vascular dysfunction caused by nicotine exposure.

Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer.

Increased tetrahydrobiopterin (BH4) generated in injured sensory neurons contributes to increased pain sensitivity and its persistence. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the de novo BH4 synthetic pathway, and human single-nucleotide polymorphism studies, together with mouse genetic modeling, have demonstrated that decreased GCH1 leads to both reduced BH4 and pain. However, little is known about the regulation of Gch1 expression upon nerve injury and whether this could be modulated as an analgesic therapeutic intervention. We performed a phenotypic screen using about 1000 bioactive compounds, many of which are target-annotated FDA-approved drugs, for their effect on regulating Gch1 expression in rodent injured dorsal root ganglion neurons. From this approach, we uncovered relevant pathways that regulate Gch1 expression in sensory neurons. We report that EGFR/KRAS signaling triggers increased Gch1 expression and contributes to neuropathic pain; conversely, inhibiting EGFR suppressed GCH1 and BH4 and exerted analgesic effects, suggesting a molecular link between EGFR/KRAS and pain perception. We also show that GCH1/BH4 acts downstream of KRAS to drive lung cancer, identifying a potentially druggable pathway. Our screen shows that pharmacologic modulation of GCH1 expression and BH4 could be used to develop pharmacological treatments to alleviate pain and identified a critical role for EGFR-regulated GCH1/BH4 expression in neuropathic pain and cancer in rodents.

M6 A-mediated regulation of PBX1-GCH1 axis promotes gastric cancer proliferation and metastasis by elevating tetrahydrobiopterin levels.

BackgroundMethyltransferase 3 (METTL3)‐mediated N6‐methyladenosine (m6A) RNA modification has been demonstrated to be a potential factor in promoting gastric cancer (GC). METTL3 regulates a series of signaling pathways by modifying various mRNAs. This study aimed to identify novel METTL3‐mediated signaling pathways and explored possible targets for use in the clinical setting of gastric cancer.MethodsTo investigate the proliferation and metastatic capacity of GC cell lines with METTL3 knockdown, a xenograft, lung metastasis, and popliteal lymph node metastasis model was used. The m6A‐modified RNA immunoprecipitation (Me‐RIP) sequence was utilized to explore the target mRNAs of METTL3. Cell counting kit 8 and transwell assays were performed to investigate the promoting function of pre‐B cell leukemia homeobox 1 (PBX1) and GTP cyclohydrolase 1 (GCH1). Western blotting and chromatin immunoprecipitation were employed to confirm the involvement of the METTL3‐PBX1‐GCH1 axis. ELISA and liquid chromatography‐mass spectrometry were used to explore the biological function of tetrahydrobiopterin (BH4).ResultsKnockdown of METTL3 suppressed xenograft tumor growth and lung/lymph node metastasis in vivo. Mechanistically, we found that METTL3 combined with and stabilized PBX1 mRNAs. Chromatin immunoprecipitation (ChIP) and further experiments suggested that PBX1 acted as a transcription factor inducing GCH1 expression. Moreover, the METTL3‐PBX1‐GCH1 axis increased BH4 levels in GC cells, thereby promoting tumor progression.ConclusionsThis study suggested that METTL3 enzymes promote tumor growth and lung/lymph node metastasis via METTL3‐PBX1‐GCH1 axis increasing BH4 levels in GC.

GCH1 induces immunosuppression through metabolic reprogramming and IDO1 upregulation in triple-negative breast cancer

PurposeRegulatory T cells (Tregs) heavily infiltrate triple-negative breast cancer (TNBC), and their accumulation is affected by the metabolic reprogramming in cancer cells. In the present study, we sought to identify...

Apolipoprotein A-I mimetic peptide inhibits atherosclerosis by increasing tetrahydrobiopterin via regulation of GTP-cyclohydrolase 1 and reducing uncoupled endothelial nitric oxide synthase activity

Background and aimsThe apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. MethodsLow-density lipoprotein (LDL) receptor null (LDLr−/−) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. ResultsUnexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. ConclusionsHypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

Epigenetic silencing of GCH1promotes hepatocellular carcinoma growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting tetrahydrobiopterin de novo biosynthesis

Metabolic reprogramming is a hallmark of cancer, including hepatocellular carcinoma (HCC). However, its role in HCC remains to be elucidated. Herein, we identified GTP cyclohydrolase 1 (GCH1), the first rate-limiting enzyme in tetrahydrobiopterin (BH4) de novo biosynthesis, as a novel metabolic regulator of HCC. GCH1 was frequently down-regulated in HCC tissues and cell lines by promoter methylation. Low GCH1 expression was associated with larger tumor size, increased tumor number, and worse prognosis in two independent cohorts of HCC patients. Functionally, GCH1 silencing promoted HCC growth in vitro and in vivo, while GCH1 overexpression exerted an opposite effect. The metabolite BH4 inhibited HCC growth in vitro and in vivo. GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis. Mechanistically, GCH1 silencing activated ASK1/p38 signaling; pharmacological or genetic inhibition of ASK1 or p38 abolished GCH1 silencing-induced growth-promoting effect. Further mechanistic studies found that GCH1 silencing-induced BH4 reduction resulted in an increase of intracellular superoxide anion levels in a dose-dependent manner, which mediated the activation of ASK1/p38 signaling. Collectively, our study reveals that epigenetic silencing of GCH1 promotes HCC growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting BH4 de novo biosynthesis, suggesting that targeting GCH1/BH4 pathway may be a promising therapeutic strategy to combat HCC.

GTP Cyclohydrolase 1/Tetrahydrobiopterin Counteract Ferroptosis through Lipid Remodeling.

Ferroptosis is an iron-dependent form of regulated cell death linking iron, lipid, and glutathione levels to degenerative processes and tumor suppression. By performing a genome-wide activation screen, we identified a cohort of genes antagonizing ferroptotic cell death, including GTP cyclohydrolase-1 (GCH1) and its metabolic derivatives tetrahydrobiopterin/dihydrobiopterin (BH4/BH2). Synthesis of BH4/BH2 by GCH1-expressing cells caused lipid remodeling, suppressing ferroptosis by selectively preventing depletion of phospholipids with two polyunsaturated fatty acyl tails. GCH1 expression level in cancer cell lines stratified susceptibility to ferroptosis, in accordance with its expression in human tumor samples. The GCH1-BH4-phospholipid axis acts as a master regulator of ferroptosis resistance, controlling endogenous production of the antioxidant BH4, abundance of CoQ10, and peroxidation of unusual phospholipids with two polyunsaturated fatty acyl tails. This demonstrates a unique mechanism of ferroptosis protection that is independent of the GPX4/glutathione system.

MiR-124 inhibits spinal neuronal apoptosis through binding to GCH1.

To explore the effect of micro ribonucleic acid (miR)-124 on the spinal neuronal apoptosis and to explore its related mechanism. The rat model of spinal cord injury (SCI) was established, agomir-124 was injected intrathecally and the effect of agomir-124 on motor function recovery of rats was evaluated using the Basso-Beattie-Bresnahan (BBB) score. The gene expression levels of miR-124 and GTP-cyclohydrolase 1 (GCH1) in spinal cord tissues were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and the correlation between them was detected using the Pearson correlation coefficient. Then, the direct interaction between miR-124 and GCH1 mRNA was detected using the TargetScan software and luciferase reporter assay. The changes in apoptosis in each group were examined via flow cytometry and Western blotting. Moreover, the changes in the tetrahydrobiopterin (BH4) content in each group were detected via high-performance liquid chromatography, and the changes in the nitrite level in the supernatant in each group were detected using the Griess reagent. Finally, the changes in the activity of the inducible nitric oxide synthase (iNOS) protein were detected using the iNOS kit. Compared with that in the model group, the BBB score was significantly increased in agomir-124 group at 21, 28, 35 and 42 d. In the agomir-124 group, the relative expression level of miR 124 in spinal cord tissues was significantly increased at 7-28 d and reached the peak at 21 d, while the mRNA level of GCH1 in spinal cord tissues declined and touched the bottom at 21 d. According to the Pearson correlation coefficient, there was a significant negative correlation between the expression of miR-124 and mRNA expression of GCH1 (r =- 0.87, p = 1.5e-6). It was found in the prediction using TargetScan software that GCH1 might be a potential target for miR-124, which was further verified by the luciferase reporter assay. The results of flow cytometry and Western blotting showed that miR-124 significantly reduced the LPS-induced primary spinal neuronal apoptosis, while the miR-124 inhibitor remarkably increased the primary spinal neuronal apoptosis. Moreover, it was also found that the knockout of GCH1 reduced the LPS-induced spinal neuronal apoptosis. In addition, the GCH1 overexpression assay revealed that miR-124 inhibited spinal neuronal apoptosis by suppressing the GCH1 expression. LPS + miR-124 remarkably decreased the BH4 content, nitrite level, and iNOS activity while LPS + miR-124 + GCH1 remarkably increased the BH4 content, nitrite level, and iNOS activity. MiR-124 inhibits neuronal apoptosis in SCI by binding to GCH1. The results in the present study may provide a new mechanism for the therapeutic effect of miR-124, and miR-124 may have a potential therapeutic value in the treatment of SCI in the future.

Exercise training augments neuronal nitric oxide synthase dimerization in the paraventricular nucleus of rats with chronic heart failure

Exercise training (ExT) is an established non-pharmacological therapy that improves the health and quality of life in patients with chronic heart failure (CHF). Exaggerated sympathetic drive characterizes CHF due to an imbalance of the autonomic nervous system. Neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) produce nitric oxide (NO•), which is known to regulate the sympathetic tone. Previously we have shown that during CHF, the catalytically active dimeric form of nNOS is significantly decreased with a concurrent increase in protein inhibitor of nNOS (PIN) expression, a protein that dissociates dimeric nNOS to monomers and facilitates its degradation. Dimerization of nNOS also requires (6R)-5,6,7,8-tetrahydrobiopterin (BH4) for stability and activity. Previously, we have shown that ExT improves NO-mediated sympathetic inhibition in the PVN; however, the molecular mechanism remains elusive. We hypothesized; ExT restores the sympathetic drive by increasing the levels and catalytically active form of nNOS by abrogating changes in the PIN in the PVN of CHF rats. CHF was induced in adult male Sprague-Dawley rats by coronary artery ligation, which reliably mimics CHF in patients with myocardial infarction. After 4 weeks of surgery, Sham and CHF rats were subjected to 3 weeks of progressive treadmill exercise. ExT significantly (p < 0.05) decreased PIN expression and increased dimer/monomer ratio of nNOS in the PVN of rats with CHF. Moreover, we found decreased GTP cyclohydrolase 1(GCH1) expression: a rate-limiting enzyme for BH4 biosynthesis in the PVN of CHF rats suggesting that perhaps reduced BH4 availability may also contribute to decreased nNOS dimers. Interestingly, CHF induced decrease in GCH1 expression was increased with ExT. Our findings revealed that ExT rectified decreased PIN and GCH1 expression and increased dimer/monomer ratio of nNOS in the PVN, which may lead to increase NO• bioavailability resulting in amelioration of activated sympathetic drive during CHF.

Reduced β2GPI Inhibiting Glomerular Mesangial Cells VEGF-NO Axis Uncoupling Induced by High Glucose.

VEGF-NO axis uncoupling is an important pathogenesis for DN. Reduced β2GPI could play a part in VEGF signaling pathway and has a protective effect on diabetic vascular disease. This study investigates the effect of reduced β2GPI on glomerular mesangial cells VEGF-NO axis uncoupling induced by high glucose. Compared to control group, glomerular mesangial cell line HBZY-1 cells treated with high glucose expressed higher levels of VEGF mRNA and protein and produced more ROS but less NO. The related proteins related to VEGF-NO axis were assayed. High glucose could significantly increase the expression of the level of VEGFR2 and obviously increase phosphorylation of Akt and eNOS but significantly decrease the expression of GTP cyclohydrolase 1 (GCH-1), reducing the production of eNOS dimer. Both β2GPI and reduced β2GPI partly reverse these effects caused by high glucose. Reduced β2GPI had stronger effect than β2GPI. GCH-1 is the speed limit of tetrahydrobiopterin (BH4) synthesis enzyme. As the key part of eNOS cofactors, BH4 could partly restore eNOS dimer induced by high glucose. Our results indicated that high glucose could interfere with eNOS dimer formation. β2GPI and reduced β2GPI can partly reverse the VEGF-NO axis uncoupling by restoring the GCH-1 expression level and then promote eNOS dimer formation.

Abstract 163: Glioblastoma, cancer stem cells, and reactive species balances: A case for GTP cyclohydrolase 1

Abstract Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. Previous studies have shown the importance of reactive species balances, partially controlled by the coupling of nitric oxide synthases (NOS) with their cofactor, in maintenance of glioma stem cell (GSC) phenotype as well as survival of cancer cells in general. In this study, we investigated the roles of GTP cyclohydrolase 1 (GCH1), which is the first and rate-limiting enzyme of the pathway producing of NOS cofactor producing pathway, in GBM stem cell phenotypes via redox balances. We found that GCH1 RNA and protein expression were increased in GSCs in comparison to non-GSCs, but that GCH1 was not exclusive to the GSC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and neurosphere-forming capability and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. GCH1 increased CD44 expression and was upregulated in the detrimental mesenchymal GBM subtype in which CD44 served as a marker. Mechanistically, we found that the expression of GCH1 increased BH4 production, as well as augmented multiple antioxidant pathways, including the expression of PARK7, was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in GSCs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM, and GCH1 pathway is a potential target for therapy. Citation Format: Anh N. Tran, Kiera Walker, David G. Harrison, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee Sedaka, Jennifer Pollock, Matthew S. Goldberg, Dolores Hambardzumyan, Sara J. Cooper, G Yancey Gillespie, Anita B. Hjelmeland. Glioblastoma, cancer stem cells, and reactive species balances: A case for GTP cyclohydrolase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 163.

Stachydrine protects eNOS uncoupling and ameliorates endothelial dysfunction induced by homocysteine

BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases (CVDs). Stachydrine (STA) is an active component in Chinese motherwort Leonurus heterophyllus sweet, which has been widely used for gynecological and cardiovascular disorders. This study is aimed to examine the effects of STA on homocysteine (Hcy)-induced endothelial dysfunction.MethodsThe effects of STA on vascular relaxation in rat thoracic aortas (TA), mesenteric arteries (MA) and renal arteries (RA) were measured by using Multi Myograph System. The levels of nitric oxide (NO), tetrahydrobiopterin (BH4) and guanosine 3′, 5′ cyclic monophosphate (cGMP) were determined. Endothelial nitric oxide synthase (eNOS) dimers and monomers were assayed by using Western blotting. GTP cyclohydrolase 1 (GTPCH1) and dihydrofolate reductase (DHFR) expressions were measured by using quantitative reverse transcriptase-PCR (qRT-PCR) and Western blotting.ResultsSTA effectively blocked Hcy-induced impairment of endothelium-dependent vasorelaxation in rat TA, MA and RA. STA-elicited arterial relaxations were reduced by NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or the NO-sensitive guanylyl cyclase inhibitor 1H- [1, 2, 4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), but not by inducible iNOS inhibitor 1400 W nor the nonselective COX inhibitor indomethacin. Hcy caused eNOS uncoupling and decreases in NO, cGMP and BH4, which were attenuated by STA. Moreover, STA prevented decreases of GTPCH1 and DHFR levels in Hcy-treated BAECs.ConclusionWe demonstrated that STA effectively reversed the Hcy-induced endothelial dysfunction and prevented eNOS uncoupling by increasing the expression of GTPCH1 and DHFR. These results revealed a novel mechanism by which STA exerts its beneficial vascular effects.

Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo.

GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation. Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization. We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin. Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

Abstract 17215: Exercise Training Restores Dimeric nNOS by Regulating PIN Expression in the Paraventricular Nucleus of Chronic Heart Failure Rats

Introduction and Hypothesis: Exercise training (ExT) is an established non-pharmacological therapy that improves the health and quality of life in patients with chronic heart failure (CHF). CHF is characterized by exaggerated sympathetic drive due to an imbalance of the autonomic nervous system. Neuronal nitric oxide synthases (nNOS) in the paraventricular nucleus (PVN) produce nitric oxide (NO), which is known to regulate the sympathetic tone. Previously we have shown that during CHF, catalytically active dimeric form of nNOS is significantly decreased (~40% decrease in dimer/monomer ratio) with a concurrent increase in PIN expression (~50%), a protein that uncouples dimeric nNOS to monomers and facilitate its degradation. Dimerization of nNOS also requires tetrahydrobiopterin (BH4) for stability and activity. Previously, we have shown that ExT improve NO-mediated sympathetic inhibition in the PVN, however, the molecular mechanism remains elusive. We hypothesized; ExT restores the sympathetic drive by reinstating the levels and catalytically active form of nNOS by abrogating changes in PIN in the PVN of CHF rats. Methods and Results: CHF was induced in adult male Sprague-Dawley rats by coronary artery ligation, which reliably mimics CHF in patients with myocardial infarction. After 4-weeks, Sham and CHF rats were subjected to 3-4 weeks of progressive treadmill exercise. ExT significantly (p &lt; 0.05) restored PIN expression (0.68±0.05 CHF vs 0.40±0.06* ExT-CHF), and reinstated dimer/monomer ratio (0.67±0.04 CHF vs 0.90±0.07* ExT-CHF), in the PVN of rats with CHF. Moreover, we found decreased GTP cyclohydrolase 1(GCH1) expression: a rate-limiting enzyme for BH4 biosynthesis in the PVN of CHF rats (0.74±0.03 Sham vs 0.48±0.02* CHF), suggesting that perhaps reduced BH4 availability may also contribute to decreased nNOS dimers. Interestingly, CHF induced decrease in GCH1 expression was ameliorated with ExT (0.48±0.02 CHF vs 0.70±0.07* ExT-CHF). Conclusions: Our findings revealed that ExT- rectified, decreased PIN and GCH1 expression and improved dimer/monomer ratio of nNOS in the PVN, which may lead to increase NO resulting in amelioration of activated sympathetic drive during CHF. Acknowledgements: AHA- 14SDG19980007 and NIH-HL62222 grants.

Inhibition of proteasome-mediated glucocorticoid receptor degradation restores nitric oxide bioavailability in myocardial endothelial cells in vitro.

Glucocorticoids (GCs), including the synthetic GC derivate dexamethasone, are widely used as immunomodulators. One of the numerous side effects of dexamethasone therapy is hypertension arising from reduced release of the endothelium-derived vasodilator nitric oxide (NO). Herein, we described the role of dexamethasone and its glucocorticoid receptor (GR) in the regulation of NO synthesis in vitro using the mouse myocardial microvascular endothelial cell line, MyEND. GC treatment caused a firm decrease of extracellular NO levels, whereas the expression of endothelial NO synthase (eNOS) was not affected. However, GC application induced an impairment of tetrahydrobiopterin (BH4 ) concentrations as well as GTP cyclohydrolase-1 (GTPCH-1) expression, both essential factors for NO production upstream of eNOS. Moreover, dexamethasone stimulation resulted in a substantially decreased GR gene and protein expression in MyEND cells. Importantly, inhibition of proteasome-mediated proteolysis of the GR or overexpression of an ubiquitination-defective GR construct improved the bioavailability of BH4 and strengthened GTPCH-1 expression and eNOS activity. Summarising our results, we propose a new mechanism involved in the regulation of NO signalling by GCs in myocardial endothelial cells. We suggest that a sufficient GR protein expression plays a crucial role for the management of GC-induced harmful adverse effects, including deregulations of vasorelaxation arising from disturbed NO biosynthesis.

Continuous DOPA synthesis from a single AAV: dosing and efficacy in models of Parkinson's disease

We used a single adeno-associated viral (AAV) vector co-expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) to investigate the relationship between vector dose, and the magnitude and rate of recovery in hemi-parkinsonian rats. Intrastriatal injections of >1E10 genomic copies (gc) of TH-GCH1 vector resulted in complete recovery in drug-naïve behavior tests. Lower vector dose gave partial to no functional improvement. Stereological quantification revealed no striatal NeuN+ cell loss in any of the groups, whereas a TH-GCH1 dose of >1E11 gc resulted in cell loss in globus pallidus. Thus, a TH-GCH1 dose of 1E10 gc gave complete recovery without causing neuronal loss. Safety and efficacy was also studied in non-human primates where the control vector resulted in co-expression of the transgenes in caudate-putamen. In the TH-GCH1 group, GCH1 expression was robust but TH was not detectable. Moreover, TH-GCH1 treatment did not result in functional improvement in non-human primates.

Role of tetrahydrobiopterin in pulmonary vascular remodelling associated with pulmonary fibrosis

BackgroundPulmonary hypertension in idiopathic pulmonary fibrosis (IPF) is indicative of a poor prognosis. Recent evidence suggests that tetrahydrobiopterin (BH4), the cofactor of nitric oxide synthase (NOS), is involved in pulmonary...

Regulation of β-adrenergic control of heart rate by GTP-cyclohydrolase 1 (GCH1) and tetrahydrobiopterin.

Clinical markers of cardiac autonomic function, such as heart rate and response to exercise, are important predictors of cardiovascular risk. Tetrahydrobiopterin (BH4) is a required cofactor for enzymes with roles in cardiac autonomic function, including tyrosine hydroxylase and nitric oxide synthase. Synthesis of BH4 is regulated by GTP cyclohydrolase I (GTPCH), encoded by GCH1. Recent clinical studies report associations between GCH1 variants and increased heart rate, but the mechanistic importance of GCH1 and BH4 in autonomic function remains unclear. We investigate the effect of BH4 deficiency on the autonomic regulation of heart rate in the hph-1 mouse model of BH4 deficiency. In the hph-1 mouse, reduced cardiac GCH1 expression, GTPCH enzymatic activity, and BH4 were associated with increased resting heart rate; blood pressure was not different. Exercise training decreased resting heart rate, but hph-1 mice retained a relative tachycardia. Vagal nerve stimulation in vitro induced bradycardia equally in hph-1 and wild-type mice both before and after exercise training. Direct atrial responses to carbamylcholine were equal. In contrast, propranolol treatment normalized the resting tachycardia in vivo. Stellate ganglion stimulation and isoproterenol but not forskolin application in vitro induced a greater tachycardic response in hph-1 mice. β1-adrenoceptor protein was increased as was the cAMP response to isoproterenol stimulation. Reduced GCH1 expression and BH4 deficiency cause tachycardia through enhanced β-adrenergic sensitivity, with no effect on vagal function. GCH1 expression and BH4 are novel determinants of cardiac autonomic regulation that may have important roles in cardiovascular pathophysiology.