Sex-dependent attenuation of plaque growth after treatment with bone marrow mononuclear cells.

There are clinically relevant differences in symptomatology, risk stratification, and efficacy of therapies between men and women with coronary artery disease. Sex-based differences in plaque attenuation after administration of bone marrow mononuclear cells (BMNCs) are unknown. Forty-five male and 57 female apolipoprotein-E knockout (apoE(-/-)) mice were fed a high-fat diet. At 14 weeks of age, animals received 4 biweekly intravenous sex-matched (males, n=11; females, n=13) or -mismatched (males, n=12; females, n=14) BMNCs obtained from C57BL6/J mice. The rest of the apoE(-/-) mice were vehicle treated (males, n=13; females, n=20) or were age-matched untreated controls (males, n=9; females, n=10). Aortic plaque burden, progenitor cell profiles in bone marrow (BM) and 22 circulating cytokines/chemokines were examined 1 week following the final injection. Only female BMNCs infused into male apoE(-/-) recipients significantly decreased plaque formation (P<0.001). This reparative response univariately correlated with increased CD34(+) (P=0.02), CD45(+) (P=0.0001), and AC133(+)/CD34(+) (P=0.001) cell percentages in the BM of recipients but not with total serum cholesterol or percentage of BM-CD31(+)/CD45(low) cells. In a multivariate analysis, BM-AC133(+)/CD34(+) and BM-CD45(+) percentage counts correlated with a lower plaque burden (P<0.05). Increased granulocyte colony-stimulating factor levels highly correlated with plaque attenuation (r=-0.86, P=0.0004). In untreated apoE(-/-) mice of either sex, BM-AC133(+)/CD34(+) cells rose initially and then fell as plaque accumulated; however, BM-AC133(+)/CD34(+) percentages were higher in females at all times (P

Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin.

We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.