Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2′, 7′-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks.

Background: Stargardt disease 1 (STGD1; MIM 248200) is a monogenic form of autosomal recessive genetic disease caused by mutation in ABCA4. This gene has a major role in hydrolyzing N-retinylidene-phosphatidylethanolamine to all-trans-retinal and phosphatidylethanolamine. The purpose of this study is to identify the frequency of putative disease-causing mutations associated with Stargardt disease in a South Indian population. Methods: A total of 28 clinically diagnosed Stargardt-like phenotype patients were recruited from south India. Ophthalmic examination of all patients was carefully carried out by a retina specialist based on the stages of fundus imaging and ERG grouping. Genetic analysis of ABCA4 was performed for all patients using Sanger sequencing and clinical exome sequencing. Results: This study identified disease-causing mutations in ABCA4 in 75% (21/28) of patients, 7% (2/28) exhibited benign variants and 18% (5/28) were negative for the disease-causing mutation. Conclusion: This is the first study describing the genetic association of ABCA4 disease-causing mutation in South Indian Stargardt 1 patients (STGD1). Our findings highlighted the presence of two novel missense mutations and an (in/del, single base pair deletion & splice variant) in ABCA4. However, genetic heterogeneity in ABCA4 mutants requires a larger sample size to establish a true correlation with clinical phenotype.

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