NO-Releasing Nanoparticles Ameliorate Detrusor Overactivity in Transgenic Sickle Cell Mice via Restored NO/ROCK Signaling.
- 作者列表："Karakus S","Musicki B","Navati MS","Friedman JM","Davies KP","Burnett AL
:Sickle cell disease (SCD) is associated with overactive bladder (OAB). Detrusor overactivity, a component of OAB, is present in an SCD mouse, but the molecular mechanisms for this condition are not well-defined. We hypothesize that nitric oxide (NO)/ ras homolog gene family (Rho) A/Rho-associated kinase (ROCK) dysregulation is a mechanism for detrusor overactivity and that NO-releasing nanoparticles (NO-nps), a novel NO delivery system, may serve to treat this condition. Male adult SCD transgenic, combined endothelial NO synthases (eNOSs) and neuronal NOS (nNOS) gene-deficient (dNOS-/-), and wild-type (WT) mice were used. Empty nanoparticle or NO-np was injected into the bladder, followed by cystometric studies. The expression levels of phosphorylated eNOS (Ser-1177), protein kinase B (Akt) (Ser-473), nNOS (Ser-1412), and myosin phosphatase target subunit 1 (MYPT1) (Thr-696) were assessed in the bladder. SCD and dNOS-/- mice had a greater (P < 0.05) number of voiding and nonvoiding contractions compared with WT mice, and they were normalized by NO-np treatment. eNOS (Ser-1177) and AKT (Ser-473) phosphorylation were decreased (P < 0.05) in the bladder of SCD compared with WT mice and reversed by NO-np. Phosphorylated MYPT1, a marker of the RhoA/ROCK pathway, was increased (P < 0.05) in the bladder of SCD mice compared with WT and reversed by NO-np. nNOS phosphorylation on positive (Ser-1412) regulatory site was decreased (P < 0.05) in the bladder of SCD mice compared with WT and was not affected by NO-np. NO-nps did not affect any of the measured parameters in WT mice. In conclusion, dysregulation of NO and RhoA/ROCK pathways is associated with detrusor overactivity in SCD mice; NO-np reverses these molecular derangements in the bladder and decreases detrusor overactivity. SIGNIFICANCE STATEMENT: Voiding abnormalities commonly affect patients with sickle cell disease (SCD) but are problematic to treat. Clarification of the science for this condition in an animal model of SCD may lead to improved interventions for it. Our findings suggest that novel topical delivery of a vasorelaxant agent nitric oxide into the bladder of these mice corrects overactive bladder by improving deranged bladder physiology regulatory signaling.
: 镰状细胞病 (SCD) 与膀胱过度活动症 (OAB) 相关。逼尿肌过度活动 (OAB的一种成分) 存在于SCD小鼠中，但这种情况的分子机制尚未明确。我们假设一氧化氮 (NO)/ ras同源基因家族 (Rho) A/Rho相关激酶 (ROCK) 失调是逼尿肌过度活动的一种机制，NO释放纳米颗粒 (NO-nps) 是一种新型的NO递送系统，可能有助于治疗这种疾病。使用雄性成年SCD转基因、联合内皮NO合酶 (eNOSs) 和神经元NOS (nNOS) 基因缺陷型 (dNOS-/-) 和野生型 (WT) 小鼠。将空纳米颗粒或NO-np注射到膀胱中，随后进行膀胱测量研究。评估膀胱中磷酸化eNOS (Ser-1177) 、蛋白激酶B (Akt) (Ser-473) 、nNOS (Ser-1412) 和肌球蛋白磷酸酶靶亚基1 (MYPT1) (Thr-696) 的表达水平。与WT小鼠相比，SCD和dNOS-/-小鼠具有更多 (P < 0.05) 数量的排尿和无尿收缩，并且它们通过NO-np处理正常化。与WT小鼠相比，SCD膀胱中eNOS (Ser-1177) 和AKT (Ser-473) 磷酸化降低 (P <0.05)，并被NO-np逆转。与WT相比，SCD小鼠膀胱中RhoA/ROCK通路的标志物磷酸化MYPT1增加 (P <0.05)，并被NO-np逆转。阳性 (Ser-1412) 调节位点上的nNOS磷酸化降低 (P <0.05)与WT相比，SCD小鼠的膀胱中没有NO-np的影响。NO-nps不影响WT小鼠中的任何测量参数。总之，NO和RhoA/ROCK通路的失调与SCD小鼠逼尿肌过度活动有关; NO-np逆转了膀胱中的这些分子紊乱，降低了逼尿肌过度活动。意义声明: 排尿异常通常影响镰状细胞病 (SCD) 患者，但治疗有问题。在SCD动物模型中阐明这种情况的科学可能导致对其的改进的干预。我们的研究结果表明，新的血管舒张剂一氧化氮局部递送到这些小鼠的膀胱中，通过改善紊乱的膀胱生理调节信号来纠正膀胱过度活动症。
METHODS:BACKGROUND:Thalassemia is one of the most common monogenetic diseases in the south of China and Southeast Asia. Hemoglobin Bart's hydrops fetalis syndrome was caused by a homozygous Southeast Asian deletion (-/-) in the HBA gene. Few studies have proved the potential of screen for Bart's hydrops fetalis using fetal cell-free DNA. However, the number of cases is still relatively small. Clinical trials of large samples would be needed. OBJECTIVE:In this study, we aimed to develop a noninvasive method of target-captured sequencing and genotyping by the Bayesian method using cell-free fetal DNA to identify the fetal genotype in pregnant women who are at risk of having hemoglobin Bart hydrops fetalis in a large-scale study. STUDY DESIGN:In total, 192,173 couples from 30 hospitals were enrolled in our study and 878 couples were recruited, among whom both the pregnant women and their husbands were detected to be carriers of Southeast Asian type (-/αα) of α-thalassemia. Prenatal diagnosis was performed by chorionic villus sampling, amniocentesis, or cordocentesis using gap-polymerase chain reaction considered as the golden standard. RESULTS:As a result, we found that the sensitivity and specificity of our noninvasive method were 98.81% and 94.72%, respectively, in the training set as well as 100% and 99.31%, respectively, in the testing set. Moreover, our method could identify all of 885 maternal samples with the Southeast Asian carrier and 36 trisomy samples with 100% of sensitivity in T13, T18, and T21 and 99.89% (1 of 917) and 99.88% (1 of 888) of specificity in T18 and T21, respectively. CONCLUSION:Our method opens the possibility of early screening for maternal genotyping of α-thalassemia, fetal aneuploidies in chromosomes 13/18/21, and hemoglobin Bart hydrops fetalis detection in 1 tube of maternal plasma.
METHODS:OBJECTIVES:Sickle cell disease (SCD) is a serious illness with disabling acute and chronic pain that needs better therapies, but insufficient patient participation in research is a major impediment to advancing SCD pain management. The purpose of this article is to discuss the challenges of conducting an SCD study and approaches to successfully overcoming those challenges. DESIGN:In a repeated-measures, longitudinal study designed to characterize SCD pain phenotypes, we recruited 311 adults of African ancestry. Adults with SCD completed 4 study visits 6 months apart, and age- and gender-matched healthy controls completed 1 visit. RESULTS:We recruited and completed measures on 186 patients with SCD and 125 healthy controls. We retained 151 patients with SCD with data at 4 time points over 18 months and 125 healthy controls (1 time point) but encountered many challenges in recruitment and study visit completion. Enrollment delays often arose from patients' difficulty in taking time from their complicated lives and frequent pain episodes. Once scheduled, participants with SCD cancelled 49% of visits often because of pain; controls canceled 30% of their scheduled visits. To facilitate recruitment and retention, we implemented a number of strategies that were invaluable in our success. CONCLUSION:Patients' struggles with illness, chronic pain, and their life situations resulted in many challenges to recruitment and completion of study visits. Important to overcoming challenges was gaining the trust of patients with SCD and a participant-centered approach. Early identification of potential problems allowed strategies to be instituted proactively, leading to success.
METHODS:OBJECTIVES:Sickle cell anemia is the commonest genetic disorder in India, and the frequency of the sickle cell gene is very high in the remote tribal areas where facilities are generally limited. Therefore, a rapid and affordable point-of-care test for sickle cell disease is needed. METHODS:The diagnostic accuracy of HemoTypeSC was evaluated against automated high-performance liquid chromatography (HPLC) as the gold standard for its efficacy in a newborn screening program. RESULTS:A total of 1,559 individuals (980 newborns and 579 adults) from four participating centers were analyzed by both methods. HemoTypeSC correctly identified 209 of 211 total hemoglobin (Hb) SS cases, for a 99.1%/99.9% total HbSS sensitivity/specificity. Overall, HemoTypeSC exhibited sensitivity and specificity of 98.1% and 99.1% for all possible phenotypes (HbAA, HbAS, and HbSS) detected. HPLC is relatively expensive and not available in most laboratories in remote tribal areas. CONCLUSIONS:We conclude that the rapid, point-of-care testing device HemoTypeSC test is suitable for population and newborn screening for the HbS phenotype.