CuSO4/H2O2induced polydopamine/polysulfobetaine methacrylate co-deposition on poly(amino acid) membranes for improved anti-protein adsorption and antibacterial activity.

Stopping postoperative soft tissue adhesions is one of the most challenging clinical problems that needs to be addressed urgently to avoid secondary injury and pain to patients. Currently, membrane materials with anti-protein adsorption and antibacterial activity are recognized as an effective and promising anti-adhesion barrier to prevent postoperative adhesion and the recurrent adhesion after adhesiolysis. Herein, poly(amino acid) (PAA), which is structurally similar to collagen, is selected as the membrane base material to successfully synthesize PAA-5 membranes with excellent mechanical and degradation properties by in-situ melt polymerization and hot-melt film-forming technology. Subsequently, the co-deposition of polydopamine/polysulfobetaine methacrylate (PDA/PSBMA) coatings induced by CuSO4/H2O2on PAA-5 membranes results in the formation of PDC-5S and PDC-10S, which exhibit excellent hemocompatibility, protein antifouling properties, and cytocompatibility. Additionally, PDC-5S and PDC-10S demonstrated significant antibacterial activity againstEscherichia coliandStaphylococcus aureus, with an inhibition rate of more than 90%. As a result, this study sheds light on newly discovered PAA membranes with anti-protein adsorption and antibacterial activity can sever as one of the promising candidates for the prevention of postoperative peritoneum adhesions.

Adenosine Deaminase as a Potential Diagnostic and Prognostic Biomarker for Severe Fever with Thrombocytopenia Syndrome.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a serious infectious disease caused by the Dabie bandavirus, with a high mortality rate. Currently, there are no effective vaccines or specific treatments for SFTS. Early diagnosis and accurate severity assessment are crucial. METHODS: This study included 171 cases of SFTS, COVID-19, and hepatitis B virus (HBV) patients and healthy controls. We compared the serum adenosine deaminase (ADA) activity across these groups. The diagnostic and prognostic efficiency of serum ADA for SFTS was evaluated by using receiver operating characteristic (ROC) curve analysis. We also examined the correlation between serum ADA in SFTS patients and clinical lab parameters as well as serum cytokines. RESULTS: SFTS patients had significantly higher serum ADA activity than those of COVID-19, HBV patients, and healthy controls. Nonsurvivor SFTS patients had notably higher ADA than survivors. ROC analysis indicated ADA as an effective SFTS diagnostic and prognostic biomarker. ADA correlated with prognosis, viral load, APTT, PT, AST, ferritin, negatively with HDL-c and LDL-c, and positively with cytokines like IL-6, TNF-α, and IL-1ß. Multiorgan failure patients showed significant ADA increase. CONCLUSION: Elevated serum ADA activity in SFTS patients is linked with disease severity and prognosis, showing potential as a diagnostic and prognostic biomarker for SFTS.

GTP Cyclohydrolase Drives Breast Cancer Development and Promotes EMT in an Enzyme-Independent Manner.

GTP cyclohydrolase (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. The catalysis of BH4 biosynthesis is tightly regulated for physiological neurotransmission, inflammation, and vascular tone. Paradoxically, BH4 has emerged as an oncometabolite regulating tumor growth, but the effects on tumor development remain controversial. Here, we found that GCH1 potentiated the growth of triple-negative breast cancer (TNBC) and HER2+ breast cancer and transformed nontumor breast epithelial cells. Independent of BH4 production, GCH1 protein induced epithelial-to-mesenchymal transition by binding to vimentin (Vim), which was mediated by HSP90. Conversely, GCH1 ablation impaired tumor growth, suppressed Vim in TNBC, and inhibited EGFR/ERK signaling while activating the p53 pathway in estrogen receptor-positive tumor cells. GCH1 deficiency increases tumor cell sensitivity to HSP90 inhibition and endocrine treatments. In addition, high GCH1 correlated with poor breast cancer survival. Together, this study reveals an enzyme-independent oncogenic role of GCH1, presenting it as a potential target for therapeutic development. SIGNIFICANCE: GTP cyclohydrolase functions as an oncogene in breast cancer and binds vimentin to induce epithelial-to-mesenchymal transition independently of its enzyme activity, which confers targetable vulnerabilities for developing breast cancer treatment strategies.

Dual-targeted and dual-sensitive self-assembled protein nanocarrier delivering hVEGI-192 for triple-negative breast cancer.

Breast cancer is a highly prevalent malignancy worldwide among women with an increasing incidence in recent years. Triple-negative breast cancer (TNBC), a specific type of breast cancer, occurs primarily in young women and exhibits large tumor size, high clinical stage, and extremely poor prognosis with a high rate of lymph node, liver, and lung metastases. TNBC is insensitive to endocrine therapy and trastuzumab treatment, and there is an urgent need for effective therapeutics and treatment guidelines. However, investigations into antiangiogenic agents for the treatment of TNBC are ongoing. In this study, we successfully engineered a self-assembled protein nanocarrier TfRBP9-hVEGI-192-ELP fusion protein (TVEFP) to deliver the therapeutic protein, human vascular endothelial growth inhibitor (hVEGI-192). This was found to be effective in inhibiting tumor angiogenesis in vivo. The protein nanocarrier effectively inhibited the progression of TNBC in vivo and showed the behavior of self-assembly, thermoresponsiveness, enzyme stimulation-responsiveness, tumor-targeting, biocompatibility, and biodegradability. Near-infrared imaging studies showed that fluorescent dye-stained TVEFP effectively aggregated at the tumor site. The TVEFP nanocarrier significantly expands the application of the therapeutic protein hVEGI-192 and improves the imaging and biotherapeutic effects in TNBC, chiefly based on anti-angiogenesis effects.

Novel therapeutic strategies: targeting epithelial-mesenchymal transition in colorectal cancer.

Epithelial-mesenchymal transition (EMT) is a process during which cells lose their epithelial characteristics, for instance apical-basal cell polarity and cell-cell contact, and gain mesenchymal properties, such as increased motility. In colorectal cancer, EMT has an important role in tumour progression, metastasis, and drug resistance. There has been accumulating evidence from preclinical and early clinical studies that show that EMT markers might serve as outcome predictors and potential therapeutic targets in colorectal cancer. This Review describes the fundamentals of EMT, including biology, newly partial EMT, and associated changes. We also provide a comprehensive summary of therapeutic compounds capable of targeting EMT markers, including drugs in preclinical and clinical trials and those with repurpose potential. Lastly, we explore the obstacles of EMT bench-to-bedside drug development.

Operative ubiquitin-specific protease 22 deubiquitination confers a more invasive phenotype to cholangiocarcinoma.

Oncogenic ubiquitin-specific protease 22 (USP22) is implicated in a variety of tumours; however, evidence of its role and underlying molecular mechanisms in cholangiocarcinoma (CCA) development remains unknown. We collected paired tumour and adjacent non-tumour tissues from 57 intrahepatic CCA (iCCA) patients and evaluated levels of the USP22 gene and protein by qPCR and immunohistochemistry. Both the mRNA and protein were significantly upregulated, correlated with the malignant invasion and worse OS of iCCA. In cell cultures, USP22 overexpression increased CCA cell proliferation and mobility, and induced epithelial-to-mesenchymal transition (EMT). Upon an interaction, USP22 deubiquitinated and stabilized sirtuin-1 (SIRT1), in conjunction with Akt/ERK activation. In implantation xenografts, USP22 overexpression stimulated tumour growth and metastasis to the lungs of mice. Conversely, the knockdown by USP22 shRNA attenuated the tumour growth and invasiveness in vitro and in vivo. Furthermore, SIRT1 overexpression reversed the USP22 functional deficiency, while the knockdown acetylated TGF-ß-activated kinase 1 (TAK1) and Akt. Our present study defines USP22 as a poor prognostic predictor in iCCA that cooperates with SIRT1 and facilitates tumour development.

Paracrine effect of GTP cyclohydrolase and angiopoietin-1 interaction in stromal fibroblasts on tumor Tie2 activation and breast cancer growth.

Cancer-associated fibroblasts (CAFs) play a key role in promoting tumor growth, acting through complex paracrine regulation. GTP cyclohydrolase (GTPCH) expression for tetrahydrobiopterin synthesis in tumor stroma is implicated in angiogenesis and tumor development. However, the clinical significance of GTPCH expression in breast cancer is still elusive and how GTPCH regulates stromal fibroblast and tumor cell communication remains unknown. We found that GTPCH was upregulated in breast CAFs and epithelia, and high GTPCH RNA was significantly correlated with larger high grade tumors and worse prognosis. In cocultures, GTPCH expressing fibroblasts stimulated breast cancer cell proliferation and motility, cancer cell Tie2 phosphorylation and consequent downstream pathway activation. GTPCH interacted with Ang-1 in stromal fibroblasts and enhanced Ang-1 expression and function, which in turn phosphorylated tumor Tie2 and induced cell proliferation. In coimplantation xenografts, GTPCH in fibroblasts enhanced tumor growth, upregulating Ang-1 and alpha-smooth muscle actin mainly in fibroblast-like cells. GTPCH inhibition resulted in the attenuation of tumor growth and angiogenesis. GTPCH/Ang-1 interaction in stromal fibroblasts and activation of Tie2 on breast tumor cells could play an important role in supporting breast cancer growth. GTPCH may be an important mechanism of paracrine tumor growth and hence a target for therapy in breast cancer.

A case-cohort study of postoperative myocardial infarction: impact of anemia and cardioprotective medications.

OBJECTIVE: Postoperative myocardial infarction (poMI) is a serious and costly complication. Multiple risk factors for poMI are known, but the effect of anemia and cardioprotective medications have not been defined in real-world surgical practice. METHODS: Patients undergoing inpatient elective surgery were assessed at 17 hospitals from 2008 to 2011 for the occurrence of poMI (American Heart Association definition). Non-MI control patients were chosen randomly on the basis of case type. Descriptive, univariable, and multivariable statistical analysis were performed for primary outcomes of poMI and death at 30 days. RESULTS: Compared with controls (N = 304), patients with poMI (N = 222) were older (72 ± 11 vs 60 ± 17 years, P < .0001), had a lesser preoperative hematocrit (37 ± 6 vs 39 ± 5, P < .0001), more often were smokers, had a preoperative T-wave abnormality (21% vs 9%, P < .0001), and had a preoperative stress test with a fixed deficit (26% vs 3%; P < .001). Preoperative factors associated with poMI included peripheral vascular disease (odds ratio 2.6; 95% confidence interval 1.3-5.3), tobacco use (1.7; 1.01-2.9), history of percutaneous coronary angioplasty (2.8; 1.6-5.0), and age (1.05; 1.03-1.07), whereas hematocrit >35 (0.51; 0.32-0.82) and preoperative acetylsalicylic acid, ie, aspirin (0.59; 0.4-0.97) were protective. Preoperative ß-blockade, statin, and use of angiotensin-converting enzyme inhibitors were not associated with lesser rates of poMI. Non-MI complication rates were 23-fold greater in the poMI group compared with the control group (P < .0001). Mortality with poMI within 30 days was 11% compared with 0.3% in non-MI control patients (P < .0001). In patients with poMI, factors independently associated with death included use of epidurals (3.5; 1.07-11.4) and bleeding (4.2; 1.1-16), whereas preoperative use of aspirin (0.29; 0.1-0.88), and postoperative ß-blockade (0.18; 0.05-0.63) were protective. Cardiac catheterization, percutaneous coronary intervention, or coronary artery bypass grafting after poMI was performed in 34% of those alive and 20% of those who died (P = .16). CONCLUSION: In the current era, poMI patients have a markedly increased risk of death. This risk is decreased with preoperative use of acetylsalicylic acid and post MI ß-blockade. Further study is warranted to explore the role of anemia and cardiac interventions after poMI.

A statewide colectomy experience: the role of full bowel preparation in preventing surgical site infection.

OBJECTIVE: To assess the utility of full bowel preparation with oral nonabsorbable antibiotics in preventing infectious complications after elective colectomy. BACKGROUND: Bowel preparation before elective colectomy remains controversial. We hypothesize that mechanical bowel preparation with nonabsorbable oral antibiotics is associated with a decreased rate of postoperative infectious complications when compared with no bowel preparation. METHODS: Patient and clinical data were obtained from the Michigan Surgical Quality Collaborative-Colectomy Best Practices Project. Propensity score analysis was used to match elective colectomy cases based on primary exposure variable-full bowel preparation (mechanical bowel preparation with nonabsorbable oral antibiotics) or no bowel preparation (neither mechanical bowel preparation given nor nonabsorbable oral antibiotic given). The primary outcomes for this study were occurrence of surgical site infection and Clostridium difficile colitis. RESULTS: In total, 2475 cases met the study criteria. Propensity analysis created 957 paired cases (n = 1914) differing only by the type of bowel preparation. Patients receiving full preparation were less likely to have any surgical site infection (5.0% vs 9.7%; P = 0.0001), organ space infection (1.6% vs 3.1%; P = 0.024), and superficial surgical site infection (3.0% vs 6.0%; P = 0.001). Patients receiving full preparation were also less likely to develop postoperative C difficile colitis (0.5% vs 1.8%, P = 0.01). CONCLUSIONS: In the state of Michigan, full bowel preparation is associated with decreased infectious complications after elective colectomy. Within this context, the Michigan Surgical Quality Collaborative recommends full bowel preparation before elective colectomy.

A statewide, community-based assessment of alvimopan's effect on surgical outcomes.

OBJECTIVE: Alvimopan was approved by the Food and Drug Administration in May 2008 and has been shown to accelerate gastrointestinal recovery after colectomy. Our independent study evaluated alvimopan as it is used in actual hospital practice in the state of Michigan. We hypothesized that alvimopan significantly decreases incidence of prolonged ileus and reduces length of stay (LOS) in patients who have undergone colectomy. METHODS: We identified 4749 patients from the Michigan Surgical Quality Collaborative (N = 28 hospitals) database between August 2007 and December 2010 who underwent elective colectomy operations. A total of 528 patients received alvimopan both pre- and postoperatively. We first selected a control group of patients from hospitals that had never administered alvimopan (n = 1833) and used propensity matching to manage differences in patient demographics and clinical characteristics. To control for hospital and surgeon characteristics, we then performed a sensitivity analysis, using a separate group of historical control patients treated before May 2008 in hospitals that would later administer alvimopan (n = 270). The Fisher exact test was used to compare complication rates, and the Student t test was used to compare LOS. RESULTS: Patients who received alvimopan had significantly lower incidence of prolonged ileus (2.3% vs 7.9%; P < 0.001) and a significantly shorter LOS (4.84 ± 4.54 vs 6.40 ± 4.45 days; P < 0.001) than control patients in hospitals that had never administered alvimopan. No differences were noted in these outcomes using sensitivity analysis. CONCLUSION: This study suggests that the actual utilization of alvimopan leads to a reduction in prolonged ileus and LOS in patients who underwent colectomy. By accelerating postoperative recovery, alvimopan has the potential to benefit patients and health care systems by improving outcomes, ensuring patient comfort, and reducing cost.

Preoperative risk factors for postoperative Clostridium difficile infection in colectomy patients.

BACKGROUND: Wide variation among hospitals in the rate of Clostridium difficile infection (CDI) after surgery was hypothesized to be related to different prophylactic antibiotic practices. METHODS: Between March 2008 and March 2010, 30-day confirmed postoperative CDI rates were prospectively collected for patients undergoing colectomy surgery at 23 hospitals participating in a collaborative quality improvement program. Preoperative variables significantly associated with CDI (P ≤ .10) in a bivariate analysis were incorporated into a logistic regression model to test for independent associations. RESULTS: Among 4,936 patients, the overall rate of CDI was 1.6% (range by hospital, 0%-9%). After adjusting for patient comorbidities and hospital site, type of preoperative antibiotics used for prophylaxis was not significantly associated with CDI. Emergency surgery, low albumin, and neurologic and renal comorbidities emerged as independent preoperative predictors of CDI. CONCLUSIONS: Perioperative antibiotic practices did not prove to be independently associated with CDI after colectomy surgery.

Sarcopenia as a prognostic factor among patients with stage III melanoma.

BACKGROUND: Several hypotheses proposed to explain the worse prognosis for older melanoma patients include different tumor biology and diminished host response. If the latter were true, then biologic frailty, and not age, should be an independent prognostic factor in melanoma. METHODS: Our prospective institutional review board (IRB)-approved database was queried for stage III patients with computed tomography (CT) scans at time of lymph node dissection (LND). Psoas area (PA) and density (PD) were determined in semi-automated fashion. Kaplan-Meier (K-M) survival estimates and Cox proportional-hazard models were used to determine PA and PD impact on survival and surgical complications. RESULTS: Among 101 stage III patients, PD was significantly associated with both disease-free survival (DFS) (P = 0.04) and distant disease-free survival (DDFS) (P = 0.0002). Cox multivariate modeling incorporating thickness, age, ulceration, and N stage showed highly significant association with PD and both DFS and DDFS. DDFS was significantly associated with Breslow thickness (P = 0.04), number of positive nodes (P = 0.001), ulceration (P = 0.04), and decreasing muscle density (P = 0.01), with hazard ratio of 0.55 [95% confidence interval (CI) 0.35-0.87]. PD also correlated with surgical complications, with odds ratio (OR) of 1.081 [95% CI 1.016-1.150, P = 0.01]. CONCLUSIONS: Decreased psoas muscle density on CT, an objective measure of frailty, was as important a predictor of outcome as tumor factors in a cohort of stage III melanoma patients. On multivariate analysis, frailty, not age, was associated with decreased disease-free survival and distant disease-free survival, and higher rate of surgical complications.

Progression of noncirrhotic portal hypertension in a pediatric population.

BACKGROUND/OBJECTIVES: The optimal management of children with noncirrhotic portal hypertension is controversial. Some groups suggest early and aggressive surgical intervention, while others report long-term success with conservative management. METHODS: We conducted a retrospective study of 26 patients with noncirrhotic portal hypertension treated at our institution. We compared platelet counts, white blood cell (WBC) counts, spleen size, hospital admissions, gastrointestinal bleeds, and longitudinal trends of specific clinical parameters using standard univariate and time-trend analytic techniques. RESULTS: Mean age at the time of diagnosis was 5.2 years. Portal vein thrombosis was found in 84.6% of patients (n=22). There was one mortality related to malignancy. There was not a progression of hypersplenism in patients that did not receive a shunt and conversely, we did not notice a significant decrease in spleen size following shunt surgery (P=0.2). Platelet and WBC counts trended downward among patients managed medically, while platelets increased and WBC counts remained stable in surgical patients. There was a significant decrease in hospital admissions for gastrointestinal bleeding following surgical intervention in the shunt group compared with nonshunt (P=0.0009). CONCLUSION: While our analysis was limited given small sample sizes and selection bias, it suggests that the majority of pediatric patients with noncirrhotic portal hypertension will do well long-term without surgical intervention.

Roles of tetrahydrobiopterin in promoting tumor angiogenesis.

Nitric oxide (NO), which is derived from endothelial NO synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment. Tetrahydrobiopterin (BH4) is an absolute requirement for eNOS activity. In this study, we investigated whether this activation is both maintained by a wild-type Ras/phosphatidylinositol 3-kinase (PI3K)/Akt-positive feedback loop in endothelial cells and affects tumor angiogenesis. We found that supplementation of BH4 (via the pterin salvage pathway with Sep) increased Akt/eNOS phosphorylation in both human eNOS-transfected COS-7 cells and endothelial cells concomitant with increases in NO production, cell proliferation, migration, and tube formation. This augmentation was abrogated by a PI3K inhibitor. Sepiapterin (Sep) also increased GTP-bound wild-type Ras and PI3K/Akt/eNOS activation, which was prevented by the eNOS inhibitor, Nω-Nitro-L-arginine methyl ester (L-NAME). Furthermore, expression of GTP cyclohydrolase I (the rate-limiting enzyme in de novo BH4 synthesis) under doxycycline control potentiated in vivo tumorigenesis, tumor cell proliferation, as well as angiogenesis. Conversely, both switching off GTP cyclohydrolase I expression as well as inhibiting its enzymatic activity significantly decreased eNOS expression and tumor vascularization. This study demonstrates an important role for BH4 synthesis in angiogenesis by the activation of eNOS for NO production, which is maintained by a PI3K/Akt-positive feedback loop through effects on wild-type Ras in endothelial cells. Our findings suggest that BH4 synthesis may be a rational target for antiangiogenesis therapy for tumors.

Methods for lymphatic vessel culture and gene transfection.

OBJECTIVE: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself. METHODS: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3-12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells. RESULTS: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3-12 days without significant impairment of the parameters of their pumping and effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels. CONCLUSIONS: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.

Copper chelator ATN-224 inhibits endothelial function by multiple mechanisms.

Copper is required for the proliferation of endothelial cells and copper-lowering therapy reduces tumour growth in animal models. It has been reported that ATN-224, a novel copper chelator, potently inhibits the activity of the copper-dependent enzyme superoxide dismutase 1 (SOD1) in endothelial cells. We performed microarray analysis of gene expression in endothelial cells exposed to ATN-224 which revealed upregulation of stress response genes including heme-oxygenase 1 (HO-1) and differential regulation of several genes previously implicated in angiogenesis including CXCR4, ANGP2, PGES2, RHAMM, ITB4 and AQP1 (p<0.01). These changes were confirmed on qPCR. Treatment of HUVEC with ATN-224 caused increased superoxide levels, phospho-ERK signalling, nuclear NRF1 expression, HO-1 expression and induction of the anti-apoptotic proteins P21, BCL2 and BCLXL. There was also nuclear translocation of SOD1. SOD1 RNA interference replicated the effects of ATN-224 on endothelial cell function but did not cause upregulation of HO-1 or PGES2, suggesting additional mechanisms of action of ATN-224. Downregulation of AQP1, which has been shown to have a role in angiogenesis, was seen with both ATN-224 and SOD1 siRNA. AQP1 expression could be rescued after ATN-224 by added copper. RNA interference to AQP1 inhibited endothelial proliferation and migration, confirming the role of AQP1 in endothelial cell function. Therefore regulation of AQP1 may represent an important action of copper chelation therapy.

5-methyltetrahydrofolate rapidly improves endothelial function and decreases superoxide production in human vessels: effects on vascular tetrahydrobiopterin availability and endothelial nitric oxide synthase coupling.

BACKGROUND: The circulating form of folic acid, 5-methyltetrahydrofolate (5-MTHF), may have beneficial effects on endothelial function; however, its mechanisms of action remain uncertain. Decreased nitric oxide (NO) bioavailability and increased vascular superoxide production in vascular disease states are due in part to endothelial NO synthase (eNOS) uncoupling related to deficiency of the eNOS cofactor tetrahydrobiopterin (BH4), but whether this mechanism is important in human atherosclerosis and represents a rational therapeutic target remains unclear. We hypothesized that 5-MTHF would improve endothelial function by decreasing superoxide and peroxynitrite production and by improving eNOS coupling, mediated by BH4 availability. METHODS AND RESULTS: Vascular superoxide/peroxynitrite production and vasomotor responses to acetylcholine and bradykinin were determined in saphenous veins and internal mammary arteries from 117 patients undergoing CABG. The effects of 5-MTHF were examined ex vivo (n = 61) by incubating vessels with 5-MTHF (1 to 100 micromol/L) and in vivo by intravenous infusion of 5-MTHF or placebo before vessel harvest (n = 56). 5-MTHF improved NO-mediated endothelium-dependent vasomotor responses and reduced vascular superoxide, both ex vivo and in vivo. These changes were not explained by direct superoxide scavenging by 5-MTHF in vitro or by changes in plasma total homocysteine in vivo. Rather, 5-MTHF was a strong peroxynitrite scavenger and increased vascular BH4 and the BH4/total biopterin ratio. Furthermore, 5-MTHF reversed eNOS uncoupling, as assessed by NG-nitro-l-arginine methyl ester-inhibitable superoxide production, increased the eNOS dimer:monomer ratio, and enhanced eNOS activity. CONCLUSIONS: 5-MTHF has beneficial effects on endothelial function and vascular superoxide production in human atherosclerosis, by preventing peroxynitrite-mediated BH4 oxidation and improving eNOS coupling.

Augmented BH4 by gene transfer restores nitric oxide synthase function in hyperglycemic human endothelial cells.

OBJECTIVE: Endothelial dysfunction in diabetes is characterized by decreased nitric oxide (NO) bioactivity and increased superoxide (SO) production. Reduced levels of tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase (eNOS), appear to be associated with eNOS enzymatic uncoupling. We sought to investigate whether augmented BH4 biosynthesis in hyperglycemic human aortic endothelial cells (HAEC) by adenovirus-mediated gene transfer of GTP cyclohydrolase I (GTPCH, the rate-limiting enzyme for the de novo BH4 synthesis), would be sufficient to rescue eNOS activity and dimerization. HAEC were cultured in media with low glucose (5 mM) or high glucose (30 mM). METHODS: After 5 days, the cells with/without GTPCH gene transfer (AdeGFP as a control) were prepared for assays of (1) NO with electron paramagnetic resonance (EPR); (2) SO with cytochrome c reduction and dihydroethidine (DHE) fluorescence; (3) BH4 with high-performance liquid chromatography (HPLC); (4) eNOS expression and dimerization with immunoblotting. RESULTS: We found that high glucose decreased HAEC NO and increased SO production, in association with reductions in both total biopterin and BH4 levels. High glucose increased total eNOS protein levels in HAEC 1.5-fold, but this was present principally in the monomeric form. GTPCH gene transfer increased cellular biopterin levels and NO production but decreased SO production. Furthermore, augmenting BH4 increased the eNOS dimer:monomer ratio 2.6-fold. CONCLUSION: This study demonstrates a critical role for BH4 in regulating eNOS function, suggesting that GTPCH is a rational target to augment endothelial BH4 and recover eNOS activity in hyperglycemic endothelial dysfunction states.

GTP cyclohydrolase I gene transfer reverses tetrahydrobiopterin deficiency and increases nitric oxide synthesis in endothelial cells and isolated vessels from diabetic rats.

Nitric oxide (NO) synthesis in endothelial cells is impaired in diabetes. We previously showed that impaired NO synthesis in the spontaneously diabetic BB (BBd) rat is due to decreased levels of tetrahydrobiopterin (BH4), secondary to decreased expression of GTP cyclohydrolase I (GTPCH). The aim of this study was to utilize adenoviral GTPCH gene transfer to reverse BH4 deficiency and repair the ability of endothelial cells to produce NO. GTPCH gene transfer increased BH4 levels in BBd endothelial cells from 0.17 +/- 0.02 (mean +/-SE) to 73.37 +/- 14.42 pmol/million cells and NO production from 0.77 +/- 0.07 to 18.74 +/- 5.52 nmol/24 h/million cells. To demonstrate a functional effect of increasing BH4 concentrations in tissues, we transferred GTPCH into aortic rings from BBd and Zucker diabetic fatty (ZDF) rats, models of human type I and type II diabetes, respectively. GTPCH gene transfer led to a dose-dependent increase in acetylcholine-induced vasorelaxation, preventable by inhibiting NO synthase. Maximal relaxation of virus-treated rings (10(10) virus particles/ml) to acetylcholine was significantly higher than sham-treated rings (BBd 64% vs. 37%, P<0.005; ZDF 80% vs. 44%, P<0.05). This study demonstrates that GTPCH gene transfer can reverse BH4 deficiency in both type I and type II diabetes and provides an experimental basis for using gene therapy to treat cardiovascular complications in diabetic patients.