[Outcomes after surgical repair of pulmonary atresia with ventricular septal defect and major aortopulmonary collateral arteries: a series of 104 cases].

Objective: To examine the early and midterm surgical outcome of pulmonary atresia with ventricular septal defect and major aortopulmonary collateral arteries (PA/VSD/MAPCA) using revised surgical strategies. Methods: A retrospective analysis of clinical data, surgical methods, and follow-up results was performed of 104 cases of PA/VSD/MAPCA in Department of Cardiovascular Surgery, Guangzhou Women and Children's Medical Center from January 2017 to September 2022. There were 55 males and 49 females, aged (M(IQR)) 33.9(84.0) months (range: 0.5 to 209.6 months) at the first surgical procedures. The anatomical classification included 89 cases of type B and 15 cases of type C. The number of major aortopulmonary collateral arteries was 4.2 (3.0) (range: 1 to 8). The Kaplan-Meier method was used for survival estimation. Results: In the first stage of surgery, 50 patients underwent a complete primary repair, 12 patients underwent partial repair, 32 patients underwent palliative right ventricular-pulmonary artery connection, and only 10 patients chose the Blalock-Taussig shunt. There were 10 cases of early death. In the second stage, 14 patients underwent complete repair and 4 patients underwent partial repair with no early death. The interval between the two surgeries was 19 (10) months (range: 9 to 48 months). Finally, during the 40 (34) months follow-up period, a total of 64 patients were complete repair and the right/left ventricular pressure ratio after complete repair was 0.63±0.16 (range: 0.36 to 1.00). Survival analysis showed that survival rates at 1 and 5 years after first-stage surgery were both 89.4% (95%CI: 83.5% to 95.3%). At 28 (34) months (range: 1 to 67 months) of follow-up after complete repair, the survival analysis showed that the survival rates at 1 and 5 years were both 95.2% (95%CI: 89.9% to 100%). Conclusions: Using combined approaches tailored to individual patients and optimized unifocalization strategy, the complete repair rate at one stage and the cumulative complete repair rate at 5 years improved significantly with a lower right/left ventricular pressure ratio and satisfactory early and intermediate survival.

[The early results of pulmonary autograft mitral valve replacement (Ross â ¡) in infants].

Objective: To evaluate the early results of pulmonary autograft mitral valve replacement (Ross Ⅱ procedure) in infants with intractable congenital mitral valve lesions. Methods: Between August 2018 and September 2019, 6 infants underwent mitral valve replacement with a pulmonary autograft in Department of Cardiovascular Surgery, Guangzhou Women and Children's Medical Center. There were 2 males and 4 females.The age at surgery ranged from 50 days to 1 year old.Preoperative diagnoses included severe to critic mitral valve insuffiency in all patients, moderate mitral valve stenosis in 3 patients, and mitral valve prolapse in one. When the pulmonary autograft was harvested, a cuff of bovine or autologous pericardium was sewn onto the proximal (infundibular) end of the autograft ( "top-hat" configuration). The distal (pulmonary) end of the autograft was secured to the mitral annulus.At the level of the left atrial free wall, the pericardial cuff was progressively tailored and sewn onto the atrial wall to remain away from the ostia of the pulmonary veins and to maintain normal morphology of the autograft. The bovine jugular valved conduit was used to reconstruct the right ventricular outflow tract. Results: There was one early death due to sudden cardiac arrest at the night of surgery day. The remaining 5 patients were successfully recovered and discharged. Follow-up of survivors ranged from 3 to 13 months. Echocardiographic follow-up demonstrated the flow velocity across the mitral valve position was 1.5 to 2.3 m/s, with a means gradient of 4 to 6 mmHg (1 mmHg=0.133 kPa). Four patients showed mild mitral insuffiency, normal left atrium and ventricle size and left ventricle ejection fraction.One patient had moderate mitral insuffiency, pulmonary valve endocarditis, and reduced left ventricle ejection fraction. The clinical symptoms of all survivals improved significantly and the weight gain were satisfactory. Conclusion: Pulmonary autograft mitral valve replacement may be a feasible and effective remedial surgical strategy for young infants with intractable congenital mitral valve lesions.

[Early- and midterm outcomes of pulmonary artery band as an initial palliation in patients with single ventricle associated with unrestricted pulmonary blood flow].

Objective: To examine the early- and midterm outcomes of pulmonary artery banding as an initial palliation in patients with single ventricle associated with unrestricted pulmonary blood flow. Methods: Between January 2008 and December 2017, 49 patients with single ventricle and unrestricted pulmonary blood flow underwent pulmonary artery banding at Department of Cardiac Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University. There were 29 males and 20 females. The age at the time of surgery was 5.6 (11.5) months (M(Q(R))), and the weight was 5.2 (3.9) kg. The medical records and results after pulmonary artery banding (death/reoperation, transition to the Glenn procedure) and subsequently after the Glenn procedure (death, transition to the Fontan procedure) were reviewed retrospectively. Actuarial survivals were estimated by the Kaplan-Meier curve. Relative factors for affecting outcomes were analyzed using the Cox regression hazard model. Results: There were 8 early deaths, with a mortality of 16.3%, including 4 cases who received simultaneous arch repair. There were 5 late deaths. During the follow-up of 47(62) (M(Q(R))) months, 11 patients (22.4%) underwent pulmonary artery banding adjustment, 29 patients (59.2%) underwent the Glenn procedure, 21 patients (42.8%) underwent the Fontan procedure. The survival of patients after the initial pulmonary artery banding were 77.4% (95%CI: 65.6% to 89.2%) and 72.6% (95%CI: 59.9% to 85.3%) at 1 year and 5 years, respectively. Multivariate Cox regression analysis revealed that systemic ventricular outflow tract obstruction (HR=4.25, 95%CI: 1.50 to 12.03, P=0.006) and total anomalous pulmonary venous connection (HR=6.49, 95%CI: 3.24 to 12.98, P=0.000) were relative factors for death. Conclusions: The early and midterm outcomes of pulmonary artery banding as an initial palliative strategy is not satisfactory. Systemic ventricular outflow tract obstruction and total anomalous pulmonary venous connection are associated with high mortality.

[Effect of multistage surgery in patients with functional single ventricle and risk factors of postoperative death].

Objective: To investigate the efficacy of multistage surgery in patients with functional single ventricle (FSV) and risk factors of postoperative death. Methods: The clinical data of all consecutive patients with FSV undergoing multistage single ventricle palliation surgery in Guangzhou women and children's medical center from January 2008 to December 2017 were retrospectively reviewed. The study included 289 patients. The age was 10.0 (6.0, 35.4) months,and there were 198 male and 91 female patients. The patients were followed up at outpatient clinic. Survival rates were calculated with Kaplan-Meier. Multivariate Cox regression analysis was made to determine the risk factors of postoperative death. Results: Seventy-nine patients required the first stage palliation surgery, 232 patients received the Glenn shunt surgery, and 162 patients completed the Fontan procedure. Overall, postoperative death occurred in 39 patients including 21 after the first stage palliation surgery (early stage 13 cases, late stage 8 cases) , 10 after the Glenn shunt surgery (early stage 5 cases, late stage 5 cases) , and 8 following the Fontan procedure (early stage 6 cases, late stage 2 cases) . Kaplan-Meier analysis showed that survival rate of the entire cohort was 90.2% (95%CI 86.7%-93.7%) , 85.9% (95%CI 81.8%-90.0%) ,and 84.6% (95%CI 79.7%-89.5%) at 1 year, 5 years and 10 years post operation. Survival rate was 74.4% (95%CI 64.8%-84.0%) , 73.0% (95%CI 63.2%-82.8%) , and 73.0% (95%CI 63.2%-82.8%) at 1 year, 5 years, and 10 years post the first stage palliation surgery, 97.8% (95%CI 95.8%-99.7%) , 95.2% (95%CI 92.3%-98.1%) , and 95.2% (95%CI 92.3%-98.1%) at 1 year, 5 years, and 10 years post Glenn shunt surgery, 95.6% (95%CI 92.5%-98.7%) and 93.7% (95%CI 88.8%-98.6%) at 1 year and 5 years post Fontan surgery. Multivariate Cox regression analysis revealed that total anomalous pulmonary venous connection (HR=5.47, 95%CI 2.71-11.04, P<0.001), atrioventricular valve regurgitation more than moderate (HR=2.52, 95%CI 1.32-4.79, P=0.005), systemic ventricular outflow tract obstruction (HR=3.47, 95%CI 1.30-9.29, P=0.013), and required the first palliation surgery (HR=3.12, 95%CI 1.59-6.15, P=0.001) were risk factors of postoperative death. Conclusions: The multistage surgery can effectively improve the survival of patientswith functional single ventricle and is associated with satisfactory long-term prognosis. Total anomalous pulmonary venous connection, atrioventricular valve regurgitation more than moderate, systemic ventricular outflow tract obstruction, and required the first palliation surgery are risk factors of postoperative death in these patients.

[Surgical management of atrioventricular valve regurgitation in functional single ventricle].

Objective: To evaluate the early- and mid-term outcomes of surgical atrioventricular valve (AVV) intervention in patients with functional single ventricle (FSV). Methods: The clinical data of 40 consecutive FSV patients who underwent surgical AVV intervention between January 2008 and December 2017 at Department of Cardiac Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University were reviewed retrospectively. There were 25 male and 15 female patients. The median age at AVV intervention was 4.5 to 204.0 months (M(Q(R)): 42.5 (59.7) months), and the median body weight was 6.0 to 55.4 kg (M(Q(R)): 13.8 (8.1) kg). The systemic AVV morphology included common AVV in 21 patients, mitral valve in 7 patients, tricuspid valve in 3 patients, and both mitral and tricuspid valve in 9 patients. At first surgical intervention, 15 patients had severe, 25 patients had moderate AVV regurgitation. The timings of the initial surgical intervention were at the first palliative, Glenn, between Glenn and Fontan, and Fontan stage in 5, 23, 4, and 8 patients, respectively. The methods of the initial surgical intervention were AVV repair in 31 patients, AVV replacement in 9 patients. Actuarial survivals were estimated by the Kaplan-Meier method. Prognosis factors for atrioventricular valve reoperation were analyzed using the Cox regression hazard model. Results: There were 6 early deaths, with a mortality of 15.0%. Thirty-four survival patients received a follow-up of 1 to 117 months (M(Q(R)): 44 (34) months). Survival of patients after the initial surgical intervention were 85.0% (95% CI: 74.0% to 95.9%), 79.3% (95% CI: 66.6% to 92.0%), and 79.3% (95% CI: 66.6% to 92.0%) at 1 year, 5 years and 10 years, respectively. In all, only 13 patients completed the Fontan procedure. Six patients underwent 8 reoperations, including AVV replacement in 5 patients, redo AVV repair in 3 patients. Freedom from AVV reoperation at 1 and 5 years was 89.8% (95% CI: 78.8% to 100%) and 79.4% (95% CI: 64.7% to 94.1%), respectively. Multivariate Cox regression analysis revealed that common AVV (HR=3.53, 95% CI: 1.63-7.67, P=0.020) was the prognosis factors for AVV reoperation. Conclusions: The early- and mid-term outcomes of surgical AVV intervention in FSV patients are not satisfactory. The mortality and reoperation rate are still high, and common AVV predicts the AVV reoperation after the initial surgical intervention.

[Improving outcomes of patients with heterotaxy and functional single ventricle: a 10-year follow-up of 70 cases in a single institution].

Objective: To review current-era palliation outcomes of patients with heterotaxy and functional single ventricle in a single institution. Methods: The clinical data of 70 consecutive patients with heterotaxy undergoing multistage single ventricle palliation in Guangzhou Women and Children's Medical Center from January 2008 to December 2017 were retrospectively reviewed, and the prognosis factors for mortality were analyzed. There were 53 male and 17 female patients.The median age was 13.3 months (range: 6 days to 150 months). Single ventricle multistage palliation included 1(st) stage palliation surgery, 2(nd) bidirectional Glenn shunt, and 3(rd) stage modified Fontan.The Kaplan-Meier method was used to estimate the probability of survival. Multivariate analysis was performed by Cox regression model. Results: Sixty-two patients had right atrial isomerism while eight had left atrial isomerism. Eighteen patients (25.7%) required the first stage palliation. Fifty-five patients received the Glenn shunt, and the Fontan procedure was completed in 27 patients. Overall, mortality occurred in 17 patients (24.3%) including 9 after the first stage palliation, 6 after the Glenn shunt, and 2 following the Fontan procedure. Survival estimates for the entire cohort following surgery were 81% (95%CI: 72% to 90%), 74%(95%CI: 64% to 85%), and 74% (95%CI: 64% to 85%) at 1 year, 5 years and 10 years, respectively. Survival estimates following the first stage palliation were 56% (95%CI: 33% to 79%) and 49% (95%CI: 26% to 73%) at 1 year and 5 years, respectively. Multivariate Cox regression analysis revealed that total anomalous pulmonary venous connection (TAPVC) (HR=6.16, 95%CI: 1.65 to 22.95, P=0.007), atrioventricular valve regurgitation more than moderate (HR=3.81, 95%CI: 1.32 to 10.94, P=0.013) and required the first palliation surgery (HR=4.58, 95%CI: 1.34 to 15.72, P=0.016) were prognosis factors for overall mortality. Conclusions: The management of heterotaxy patients with functional single ventricle remains challenging, and the outcomes are continously improving in china. TAPVC, atrioventricular valve regurgitation more than moderate and required the first palliation surgery still are prognosis factors for overall mortality in heterotaxy syndrome.

[End-to-side anastomosis for interrupted aortic arch in neonates and infants].

Objective: To review the early and mid-term results of end-to-side anastomosis technique for interrupted aortic arch in neonates and infants. Methods: Clinic data of 46 patients were diagnosed as interrupted aortic arch in Department of Cardiac Surgery, Guangzhou Women and Children's Medical Center between January 2010 and December 2016 were analyzed retrospectively. Twenty-six cases were neonates. The median age underwent surgery was 23 days (range: 2 days to 8 years). Anatomical subtypes included 36 cases of type A and 10 cases of type B. There was no type C case. The reconstruction of the aortic arch was completed by an extended end-to-side anastomosis technique between the descending aorta and the undersurface of the proximal aortic arch. In 42 patients, all with intracardiac anomalies, had concomitant complete repair of intracardiac anomalies through a median sternotomy. The remaining 4 patients, all without intracardiac anomalies, an end-to-side anastomosis was constructed through a left thoracotomy. During follow-up, aortic arch recurrent obstruction, left ventricular outflow tract obstruction (LVOTO) and tracheal stenosis were focused. Results: There were 3 surgical deaths, with amortality rate of 6.5%. The remaining 43 patients survived after surgery. In 39 of these patients, deep hypothermic cardiac arrest (DHCA) strategy was used for brain protection, and the mean time of DHCA was (16±3) minutes. Eight patients underwent delayed sternal closure. The mean mechanical ventilation time and ICU stay time were (3.4±1.6) days (range: 2 to 12 days) and (6.4±2.7) days (range: 3 to 16 days) respectively. In 16 patients, all with tracheal or bronchial stenosis before surgery, mechanical ventilation was successfully evacuated, and no new airway stenosis occurred. There was no residual pressure difference between upper and lower extremity arterial blood pressure at discharge. Echocardiography showed normal arterial blood flow velocity in aortic arch. At a mean follow-up of (36.2±18.9) months (range: 6 months to 7 years), there were two patients lost and one late date. Four patients developed a recurrent stenosis at the aortic arch, of which two were severe, and the other two were mild. In 2 patients, both with mild LVOTO before surgery, no significant increase in the degree of obstruction was found during the follow-up. Two patients developed new mild to moderate LVOTO without clinical symptoms, and continued to follow up. In all patients, the tracheal or bronchial stenosis were extenuated, and there was no new progressive airway stenosis by regular bronchoscopy. Conclusions: The end-to-side anastomosis technique for the reconstruction of the aortic arch achieved excellent early and mid-term results in neonates and infants suffered from interrupted aortic arch. Reducing the anastomotic tension by extensive mobilization is the key to prevent postoperative early complications and late recurrent arch obstruction.

Deletion of endothelial cell-specific liver kinase B1 increases angiogenesis and tumor growth via vascular endothelial growth factor.

Liver kinase B1 (LKB1) is a serine/threonine protein kinase ubiquitously expressed in mammalian cells. It was first identified in Peutz-Jeghers syndrome as a tumor suppressor gene. Whether endothelial LKB1 regulates angiogenesis and tumor growth is unknown. In this study, we generated endothelial cell-specific LKB1-knockout (LKB1endo-/-) mice by crossbreeding vascular endothelial-cadherin-Cre mice with LKB1flox/flox mice. Vascular endothelial growth factor (VEGF) level was highly co-stained in endothelial cells but not in macrophages in LKB1endo-/- mice. Consistently, LKB1endo-/- mouse tissues including the lung, skin, kidney and liver showed increased vascular permeability. Tumors implanted in LKB1endo-/- mice but not macrophage-specific LKB1-knockout mice grew faster and showed enhanced vascular permeability and increased angiogenesis as compared with those implanted in wild-type mice. Injection of VEGF-neutralizing antibody but not the isotype-matched control antibody decreased endothelial-cell angiogenesis and tumor growth in vivo. Furthermore, LKB1 deletion enhanced mouse retinal and cell angiogenesis, and knockdown of VEGF by small-interfering RNA decreased endothelial cell proliferation and migration. Re-expression of LKB1 or knockdown of VEGF receptor 2 decreased the overproliferation and -migration observed in LKB1endo-/- cells. Mechanistically, LKB1 could bind to the VEGF transcription factor, specificity protein 1 (Sp1), which then inhibited the binding of Sp1 to the VEGF promoter to reduce VEGF expression. Endothelial LKB1 may regulate endothelial angiogenesis and tumor growth by modulating Sp1-mediated VEGF expression.

Aptamer-modified PLGA nanoparticle delivery of triplex forming oligonucleotide for targeted prostate cancer therapy.

Presented study aimed to prepare A10 aptamer-modified poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles loaded with triplex forming oligonucleotides(TFO) for targeted prostate cancer therapy. We first synthesized a PLGA-PEG-Apt copolymer. The PLGA-PEG-Apt nanoparticles (NP-Apt) were loaded with TFO using double emulsion solvent evaporation method. Carboxy-fluorescein labeled TFO-NP-Apt, TFO-NP and TFO were prepared for cellular uptake experiments. Cell counting kit-8 (CCK-8) test was used to determine the ability of TFO-NP-Apt to inhibit LNCaP cell proliferation. RT-PCR and Western blot was conducted to analyze AR gene expressing. Then, a mouse model of prostate cancer was used to evaluate the anti-cancer effect of TFO-NP-Apt in vivo. We confirmed that the PLGA-PEG-Apt conjugation was successful. The TFO encapsulation efficiency and drug loading percentage were 46.1± 3.6% and 40.8±5.3%, respectively. TFO-NP-Apt showed a more efficient cellular uptake than TFO-NP or TFO in LNCaP cells. TFO-NP-Apt was significantly more cytotoxic than TFO-NP and TFO in the CCK-8 test (p<0.001). TFO-NP-Apt silenced the AR gene better than unconjugated Apt, naked TFO, NP or saline. TFO-NP-Apt were more effective than TFO-NP, naked TFO, NP and saline at inhibiting prostate cancer growth in vivo (p<0.05). Aptamer-modified TFO-loaded PLGA nanoparticles may prove useful in targeted therapy for advanced prostate cancer.

Ebselen as a peroxynitrite scavenger in vitro and ex vivo.

We have previously shown that peroxynitrite (PN) selectively impaired prostacyclin (PGI2)-dependent vasorelaxation by tyrosine nitration of PGI2 synthase in an in situ model (Zou MH, Jendral M and Ullrich V, Br J Pharmacol 126: 1283-1292, 1999). By using this established model, we tested whether or not ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), which reacts rapidly with the anionic form of PN, affected PN inhibition of PGI2 synthase. Administration of ebselen (1 to 50 microM) to bovine coronary strips 5 min prior to PN (1 microM) treatment neither prevented PN-triggered vasoconstriction nor the inhibition of PGI2 release. In line with these results, ebselen affected neither PN inhibition of the conversion of [14C]-PGH2 into 6-keto-PGF1 alpha nor the nitration of PGI2 synthase in bovine aortic microsomes. Following the hypothesis that a reaction of ebselen with cellular thiols could have caused the inefficiency of ebselen, we observed that free ebselen quickly reacted with thiols in both coronary strips and in aortic microsomes to form two metabolites, one of which was identified as the ebselen-glutathione adduct, whereas the other had a similar retention time to that of the ebselen-cysteine adduct. The nitration of phenol by PN in a metal-free solution could be blocked more efficiently in the presence of ebselen or glutathione alone than in the presence of both, indicating that like selenomethionine and other selenocompounds, ebselen-thiol adducts were less reactive towards PN than ebselen itself. Further evidence came from the results that ebselen became effective in preventing the inhibition and nitration of PGI2 synthase after thiol groups of microsomal proteins were previously oxidized with Ellman's reagent. We conclude that in cellular systems ebselen is present as thiol adducts and thus loses its high reactivity towards PN, which is required to compete with the nitration of PGI2 synthase.

Distribution and cellular localization of prostacyclin synthase in human brain.

Prostacyclin (PGI(2)) is a labile, lipid-derived metabolite of arachidonic acid synthesized through the sequential action of cyclo-oxygenase (COX) and prostacyclin synthase (PGIS). In addition to its well-characterized vasodilatory and thrombolytic effects, an increasing number of studies report an important role of PGI(2) in nociception in various animal species. In this study we investigated the regional distribution of PGIS in human brain by immunohistochemistry and in situ hybridization. PGIS-immunoreactive (ir) protein was localized to blood vessels throughout the brain. Neuronal cells and glial cells, such as microglia and oligodendrocytes, also showed intense labeling. The strongest expression of PGIS was seen in large principal neurons, such as pyramidal cells of the cortex, pyramidal cells of the hippocampus, and Purkinje cells of the cerebellum. Abundance of PGIS mRNA was observed in blood vessels and large neurons and correlated well with the immunohistochemical findings. The expression of PGIS in human brain was further demonstrated by immunoblotting and detection of 6-keto-PGF (1alpha), the stable degradation product of prostacyclin in human brain homogenate. These results demonstrate a widespread expression of PGIS in the central nervous system and suggest a potentially important role of prostacylin in modulating neuronal activity in human brain.

Rapid reactions of peroxynitrite with heme-thiolate proteins as the basis for protection of prostacyclin synthase from inactivation by nitration.

Prostacyclin (PGI(2)) synthase is a heme-thiolate (P450) protein which reacts with low levels of peroxynitrite (PN) under tyrosine nitration and inactivation. Studying heme proteins as models, we have found the heme-thiolate protein NADH-NO reductase (P450(NOR)) to be highly efficient in decomposing PN under concomitant nitration of phenol. The present study investigates two other P450 proteins, P450(BM-3) and chloroperoxidase, in order to test for the specific role of the thiolate ligand in the reaction with PN. A comparison with horseradish peroxidase and microperoxidase gives evidence of kinetic differences that classify heme-thiolate proteins, but not other heme proteins, as effective inhibitors of PGI(2) synthase nitration and inactivation. P450(BM-3) with PN catalyzes phenol nitration and nitration of its own tyrosine below 10 microM PN, whereas chloroperoxidase and P450(NOR) at such concentrations also nitrate phenol but not enzyme-bound tyrosine residues. We conclude that heme-thiolate proteins in general exhibit high reactivity with PN and turnover, probably due to the special electronic structure of the presumed thiolate-ferryl intermediate.

Hypoxia-reoxygenation triggers coronary vasospasm in isolated bovine coronary arteries via tyrosine nitration of prostacyclin synthase.

The role of peroxynitrite in hypoxia-reoxygenation-induced coronary vasospasm was investigated in isolated bovine coronary arteries. Hypoxia-reoxygenation selectively blunted prostacyclin (PGI2)-dependent vasorelaxation and elicited a sustained vasoconstriction that was blocked by a cyclooxygenase inhibitor, indomethacin, and SQ29548, a thromboxane (Tx)A2/prostaglandin H2 receptor antagonist, but not by CGS13080, a TxA2 synthase blocker. The inactivation of PGI2 synthase, as evidenced by suppressed 6-keto-PGF1 alpha release and a decreased conversion of 14C-prostaglandin H2 into 6-keto-PGF1 alpha, was paralleled by an increased nitration in both vascular endothelium and smooth muscle of hypoxia-reoxygenation-exposed vessels. The administration of the nitric oxide (NO) synthase inhibitors as well as polyethylene-glycolated superoxide dismutase abolished the vasospasm by preventing the inactivation and nitration of PGI2 synthase, suggesting that peroxynitrite was implicated. Moreover, concomitant administration to the organ baths of the two precursors of peroxynitrite, superoxide, and NO mimicked the effects of hypoxia-reoxygenation, although none of them were effective when given separately. We conclude that hypoxia-reoxygenation elicits the formation of superoxide, which causes loss of the vasodilatory action of NO and at the same time yields peroxynitrite. Subsequently, peroxynitrite nitrates and inactivates PGI2 synthase, leaving unmetabolized prostaglandin H2, which causes vasospasm, platelet aggregation, and thrombus formation via the TxA2/prostaglandin H2 receptor.

Selective nitration of prostacyclin synthase and defective vasorelaxation in atherosclerotic bovine coronary arteries.

Prostacyclin synthase (PCS) is an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature, and inactivation of PCS by tyrosine nitration may favor atherosclerotic processes. Here, we show that PCS is nitrated and inactivated in early stage atherosclerotic lesions (focal intimal thickenings). Immunoprecipitation with antibodies raised against nitrotyrosine yielded PCS as the main nitrated protein in blood vessels. Moreover, we identified two nitrated degradation products of PCS with molecular mass of 30 and 46 kd, which were selective for atherosclerotic tissue. Agonist (acetylcholine, angiotensin II)-induced prostacyclin formation was decreased in atherosclerotic vessels compared with normal tissue, whereas PGE2 formation was increased and cyclooxygenase activity remained unchanged. A selective loss of PCS activity was confirmed by direct measurement of enzymatic activity. In line with this, we observed defective relaxation of early atherosclerotic vessels following vasoconstrictive stimulation. This functional impairment was completely reversed by coincubation with an antagonist of the thromboxane/PGH2 receptor but not by a thromboxane synthase inhibitor. These data suggest that reduced PCS activity in atherosclerotic arteries prevents the rapid use of PGH2, which accumulates and acts as an agonist on the vasoconstrictive thromboxane receptor.

Interleukin 1beta decreases prostacyclin synthase activity in rat mesangial cells via endogenous peroxynitrite formation.

We have reported that peroxynitrite (PON) selectively inactivated prostacyclin synthase (PGIS) by a mechanism of tyrosine nitration at the active site [Zou, Martin and Ullrich (1997) Biol. Chem. Hoppe-Seyler 378, 707-713]. We have now extended our studies on rat mesangial cells (RMC) and show that nitration can occur under the influence of cytokines. Pretreatment of RMC with interleukin 1beta (IL-1beta), which up-regulated cyclo-oxygenase 2 and inducible nitric oxide synthase (NOS-2), significantly attenuated the conversion of [14C]prostaglandin H2 (PGH2) into the stable prostacyclin (PGI2) metabolite 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha). The presence of superoxide dismutase (SOD, 100 units/ml) or the NOS synthase inhibitor Nomega-monomethyl-l-arginine (100 microM) as well as cycloheximide (10 microM) plus actinomycin (10 microM) abolished IL-1beta-mediated down-regulation of 6-oxo-PGF1alpha from PGH2. At the same time, 6-oxo-PGF1alpha production from arachidonate (AA) increased at the expense of prostaglandin E2 (PGE2). Neither NO alone generated from different NO donors nor superoxide from xanthine/xanthine oxidase (1-100 m-units/ml) inhibited PGI2 synthesis, either from PGH2 or from AA. Bolus additions of chemically synthesized PON or the PON generator 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) exhibited a potent inhibition of 6-oxo-PGF1alpha release from both PGH2 and AA. In addition, immunoprecipitation of nitrotyrosine-containing proteins from PON- and SIN-1-treated RMC yielded distinct nitrated PGIS bands but also from IL-1beta-pretreated cells alone, compared with a lack of nitrated PGIS in control cells. Taken together, our results strongly suggest that IL-1beta pretreatment of RMC via NOS-2 leads to the production of PON with the consequence of a partial nitration and inhibition of PGIS.

The comparative trial of TCu 380A IUD and progesterone-releasing vaginal ring used by lactating women.

The objective of this paper was to compare the efficacy, acceptability, safety, and bleeding pattern of TCu 380A intrauterine device (IUD) and progesterone-releasing vaginal ring used by breastfeeding women. The study population included 97 breastfeeding women using IUD and 100 women using vaginal ring. Of the IUD users, no insertion failure, perforation, or accidental pregnancy occurred in 12 months. There was one IUD expulsion. There were no discontinuations of IUD due to medical reasons other than expulsion. The total discontinuation rate was 2.3%. In the ring group, no accidental pregnancy occurred. The major reasons for discontinuation were ring use-related problems and vaginal problems. The total discontinuation rate was 65.4% within 1 year. The frequency of any one complaint among the ring users was higher than that among the IUD users. There were no differences in the proportion of women having no sexual activity and in the weight of their babies between the two groups. Compared with the IUD users, the median number of bleeding/spotting (B/S) episodes and B/S days of the vaginal ring users were fewer; consequently, the mean length of B/S-free interval was longer in all four reference periods; the mean length of B/S episode and segment were the same; the occurrence of amenorrhea was more frequent; in contrast, the proportions of normal bleeding patterns were fewer. The frequencies of prolonged bleeding, frequent bleeding, and infrequent bleeding patterns did not differ between the two groups. The percentage of irregular bleeding was fewer only in the first two reference periods. It is concluded that the TCu 380A IUD and progesterone-releasing vaginal ring used by breastfeeding women are safe and effective. The higher discontinuation rate of the ring users was mainly because of use-related problems. Breastfeeding women with TCu 380A IUD had better tolerance and acceptability. The TCu 380A IUD does not, but the progesterone-releasing vaginal ring does, suppress the recovery of ovarian function. However, once return of menstruation occurred, there were no differences in bleeding patterns between the two contraceptive methods.

PIP: The efficacy, acceptability, and bleeding patterns associated with use of the Copper T 380A IUD and the progesterone-releasing vaginal ring during lactation were compared in a 12-month study conducted in Beijing City, China. Enrolled were 97 breast-feeding IUD users and 100 lactating vaginal ring acceptors. The devices were inserted 29-64 days postpartum. In the IUD group, there was one expulsion during the study period, but no cases of perforation, pelvic inflammatory disease, or pregnancy. The two IUD discontinuations (2.3/100 woman-years) were attributable to user rather than medical-related reasons. There were no accidental pregnancies in the ring group either. However, there were 50 medical-related and four user-related discontinuations among ring users, for a 12-month discontinuation rate of 65.4/100 woman-years. Three women discontinued ring use due to menstrual problems, 10 requested removal for vaginal problems such as increased discharge and vaginitis, seven experienced frequent ring expulsion, 12 left the device out for more than 48 hours, and 13 found the ring unpleasant to use. Significantly more ring than IUD users had at least one complaint at the 1-, 3-, and 6-month post-insertion visits. Menstruation diaries revealed bleeding/spotting immediately post-insertion in both groups, followed by a variable period of amenorrhea until resumption of menses. The median number of bleeding/spotting episodes and the occurrence of amenorrhea were more frequent in the vaginal ring group. Once menstruation resumed, there were no differences in bleeding patterns between the two contraceptive methods. The results indicated progesterone suppresses the recovery of ovulation in breast-feeding women but does not interrupt the regulation of menstruation.

Inhibition of cyclooxygenase-1 and -2 by R(-)- and S(+)-ibuprofen.

Since the discovery of a cytokine-inducible isozyme of cyclooxygenase (COX-2), its pharmacologic inhibition has been the subject of recent investigations. These include tests for the selectivity of known nonsteroidal antiinflammatory drugs (NSAIDs) on the constitutive enzyme of cyclooxygenase (COX-1) compared with the inducible enzyme COX-2. The interesting question arose whether the R(-)- and S(+)-isomers exhibited different inhibitory potencies for ibuprofen. Results with isolated COX-1 and COX-2 isozymes confirmed the known higher efficacy of S(+)-compared with R(-)-ibuprofen. The R(-)-isomer is almost inactive in inhibiting COX-2. In addition, the S(+) form has a several times lower potency with COX-2 than with COX-1. These data were evaluated in platelets containing mainly the constitutive COX-1, with interleukin-1, pretreated, rat mesangial cells which almost exclusively express COX-2.

Peroxynitrite formed by simultaneous generation of nitric oxide and superoxide selectively inhibits bovine aortic prostacyclin synthase.

The effect of various oxidants on bovine aortic prostacyclin synthase was tested with 14C-labelled prostaglandin endoperoxide as substrate. No sensitivity against hydrogen peroxide, superoxide or hydroxyl radicals was observed but hypochlorite inhibited with an IC50 value of 7 microM. Among the reactive nitrogen species nitric oxide and nitrogen dioxide radicals were ineffective, but peroxynitrite irreversibly blocked prostacyclin biosynthesis with an IC50 value of 50 nM. Peroxynitrite acted within seconds whereas hypochlorite required up to 30 min for completion. Simultaneous generation of nitric oxide and superoxide also caused inhibition which suggested that under pathological conditions like ischemia-reperfusion not only the vasodilatory effects of nitric oxide but also those of prostacyclin could be eliminated.

Peroxynitrite inhibition of nitric oxide synthases.

Peroxynitrite (PN) can be formed under mainly pathophysiological conditions from nitric oxide (NO) and superoxide anion and may be responsible for oxidative modifications of biomolecules. Preparations of nitric oxide synthases from porcine cerebellum (nNOS), bovine aortic endothelium (eNOS) and cytokine-treated murine macrophages (iNOS) were inhibited by PN in their ability to transform arginine to citrulline and nitric oxide with IC50 values of 15, 28, and 10 microM, respectively. Glutathione, bovine serum albumin and tyrosine provided varying degrees of protection in the three preparations. Intact endothelial cells, upon exposure to PN, rapidly lost their glutathione content but protein-SH groups and eNOS activity remained largely unaffected. Destruction of the heme-thiolate catalytic site was observed when nNOS was exposed to PN suggesting that the irreversible oxidation of this bond may be the common mechanism of NOS inhibition.