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Cerebrovascular abnormalities are a common feature of sickle cell disease that may be associated with risk of vaso-occlusive pain crises, microinfarcts, and cognitive impairment. An activated endothelium and adhesion factors, VCAM-1 and P-selectin, are implicated in sickle cell vasculopathy, including abnormal hemodynamics and leukocyte adherence. This study examined the association between cerebral expression of these adhesion factors and cortical microvascular blood flow dynamics by using in-vivo two-photon microscopy. We also examined the impact of blood transfusion treatment on thesemarkers of vasculopathy. Results showed that sickle cell mice had significantly higher maximum red blood cell (RBC) velocity (6.80 ± 0.25 mm/sec, p 0.01 vs. 5.35 ± 0.35 mm/sec) and more frequent blood flow reversals (18.04 ± 0.95/min, p = 0.01 vs. 13.59 ± 1.40/min) in the cortical microvasculature compared to controls. In addition, sickle cell mice had a 2.6-fold (RFU/mm2) increase in expression of VCAM-1 and 17-fold (RFU/mm2) increase in expression of P-selectin compared to controls. This was accompanied by an increased frequency in leukocyte adherence (4.83 ± 0.57/100 µm/min vs. 2.26 ± 0.37/100 µm/min, p = 0.001). We also found that microinfarcts identified in sickle cell mice were 50% larger than in controls. After blood transfusion, many of these parameters improved, as results demonstrated that sickle cell mice had a lower post-transfusionmaximumRBC velocity (8.30 ± 0.98mm/sec vs. 11.29 ± 0.95 mm/sec), lower frequency of blood flow reversals (12.80 ± 2.76/min vs. 27.75 ± 2.09/min), and fewer instances of leukocyte adherence compared to their pre-transfusion imaging time point (1.35 ± 0.32/100 µm/min vs. 3.46 ± 0.58/100 µm/min). Additionally, we found that blood transfusion was associated with lower expression of adhesion factors. Our results suggest that blood transfusion and adhesion factors, VCAM-1 and P-selectin, are potential therapeutic targets for addressing cerebrovascular pathology, such as vaso-occlusion, in sickle cell disease. [ABSTRACT FROM AUTHOR]

Genome-wide association studies have identified the single nucleotide polymorphism (SNP) rs3278 in the human SLC4A7 gene as one of the marker loci for addiction vulnerability. This marker is located in an intron of the gene, and its genomic role has been unknown. In this study, we examined rs3278 and three adjacent SNPs prevalent in alcoholics for their effects on an alternative promoter that would lead to the production of the NH2-terminally truncated protein NBCn1ΔN450, missing the first 450 amino acids. Analysis of the transcription start site database and a promoter prediction algorithm identified a cluster of three promoters in intron 7 and two short CpG-rich sites in intron 6. The promoter closest to rs3278 showed strong transcription activity in luciferase reporter gene assays. Major-to-minor allele substitution at rs3278 resulted in increased transcription activity. Equivalent substitutions at adjacent rs3772723 (intron 7) and rs13077400 (exon 8) had negligible effect; however, the substitution at nonsynonymous rs3755652 (exon 8) increased the activity by more than twofold. The concomitant substitution at rs3278/rs3755652 produced an additive effect. The rs3755652 had more profound effects on the promoter than the upstream regulatory CpG sites. The amino acid change E326K caused by rs3755652 had negligible effect on transporter function. In HEK 293 cells, NBCn1ΔN450 was expressed in plasma membranes, but at significantly lower levels than the nontruncated NBCn1-E. The pH change mediated by NBCn1ΔN450 was also low. We conclude that rs3278 and rs3755652 stimulate an alternative transcription of the SLC4A7 gene, increasing the production of a defective transporter. [ABSTRACT FROM AUTHOR]