Effects of Empagliflozin on Vascular and Skeletal Mineralization in Hyperlipidemic Mice.

Cardiovascular disease and osteoporosis, major causes of morbidity and mortality, are associated with hyperlipidemia. Recent studies show that empagliflozin (EMPA), an inhibitor of sodium-glucose cotransporter-2 (SGLT2), improves cardiovascular health. In preclinical animal studies, EMPA mitigates vascular calcification in the males but its effects in the females are not known. Thus, we used female mice to test the effects of EMPA on calcification in the artery wall, cardiac function, and skeletal bone. By serial in vivo microCT imaging, we followed the progression of aortic calcification and bone mineral density in young and older female Apoe-/- mice fed a high-fat diet with or without EMPA. The two different age groups were used to compare early vs. advanced stages of aortic calcification. Results show that EMPA treatment increased urine glucose levels. Aortic calcium content increased in both the controls and the EMPA-treated mice, and EMPA did not affect progression of aortic calcium content in both young and older mice. However, 3-D segmentation analysis of aortic calcium deposits on microCT images revealed that EMPA-treated mice had significantly less surface area and volume of calcified deposits as well as fewer numbers of deposits than the control mice. To test for direct effects on vascular cell calcification, we treated murine aortic smooth muscle cells with EMPA, and results showed a slight inhibition of alkaline phosphatase activity and inflammatory matrix calcification. As for skeletal bone, EMPA-treated mice had significantly lower BMD than the controls in both the lumbar vertebrae and femoral bones in both young and older mice. The findings suggest that, in hyperlipidemic female mice, unlike males, SGLT2 inhibition with empagliflozin does not mitigate progression of aortic calcification and may even lower skeletal bone density.

Effects of LP533401 on vascular and bone calcification in hyperlipidemic mice.

Peripheral serotonin levels are associated with cardiovascular disease risk. We previously found that serum serotonin levels are higher in hyperlipidemic mice than wild-type mice. Evidence also suggests that serotonin regulates biomineralization, in that serotonin treatment augments TNF-a-induced matrix calcification of aortic valve interstitial cells and that a selective inhibitor of peripheral serotonin, LP533401, rescues bone loss induced by ovariectomy in mice. Thus, in the present study, we examined the effects of LP533401 on both skeletal bone mineral density (BMD) and aortic calcification in both young and older hyperlipidemic mice susceptible to calcific atherosclerosis and bone loss. By serial in vivo microCT imaging, we assessed BMD and aortic calcification of Apoe-/- mice fed an atherogenic (high cholesterol) diet alone or mixed with LP533401. Results show that in the young mice, LP533401 blunted skeletal bone loss in lumbar vertebrae but not in femurs. LP533401 also blunted the initial development of aortic calcification but not its progression. Echocardiographic analysis showed that LP533401 blunted both hyperlipidemia-induced cardiac hypertrophy and left ventricular dysfunction. In the older mice, LP533401 increased the BMD of lumbar vertebrae but not of femurs. The aortic calcification progressed in both controls and LP533401-treated mice, but, at post-treatment, LP533401-treated mice had significantly less aortic calcification than the controls. These findings suggest that LP533401 mitigates adverse effects of hyperlipidemia on skeletal and vascular tissues in site- and stage-dependent manners.

Effects of activity levels on aortic calcification in hyperlipidemic mice as measured by microPETmicroCT.

BACKGROUND AND AIMS: Cardiovascular disease risk is associated with coronary artery calcification and is mitigated by regular exercise. Paradoxically, elite endurance athletes, who have low risk, are likely to have more coronary calcification, raising questions about the optimal level of activity. METHODS: Female hyperlipidemic (Apoe-/-) mice with baseline aortic calcification were subjected to high-speed (18.5 m/min), low-speed (12.5 m/min), or no treadmill exercise for 9 weeks. 18F-NaF microPET/CT images were acquired at weeks 0 and 9, and echocardiography was performed at week 9. RESULTS: In controls, aortic calcium content and density increased significantly. Exercise regimens did not alter the time-dependent increase in content, but the increase in mean density was blunted. Interestingly, the low-speed regimen significantly reduced 18F-NaF uptake, a marker of surface area. Left ventricular (LV) systolic function was lower while LV diameter was greater in the low-speed group compared with controls or the high-speed group. In the low-speed group, vertebral bone density by CT decreased significantly, contrary to expectations. Male hyperlipidemic (Apoe-/-) mice were fed a Western diet and also subjected to low-speed or no exercise followed by imaging at weeks 0 and 9. In males, exercise also did not alter the time-dependent increase in aortic calcification. Exercise did not affect 18F-NaF uptake or bone mineral density, but it blunted the diet-induced LV hypertrophy seen in controls. CONCLUSIONS: These results suggest that, in mice, exercise has differential effects on aortic calcification, cardiac function, and skeletal bone mineral density.

Cell dynamics and developmental bias in the ontogeny of a complex sexually dimorphic trait in Drosophila melanogaster.

The Drosophila sex comb (SC) has been hailed as a powerful tool for integrative studies in development, evolution, and behavior, but its ontogeny is poorly understood, even in the model organism Drosophila melanogaster. Using 4D live imaging and other techniques, we carried out a detailed analysis of the cellular events that take place during the development of the SC. We showed that the comb and other contiguous bristle formations assemble from noncontiguous precursor cells, which join together through intercalation. Most of the rotation of the SC (which has a longitudinal orientation in D. melanogaster but is initially transverse) occurs after this stage, when the structure is a single unit. We have provided evidence that male-specific convergent extension through cell rearrangement is responsible for both this rotation and another sexually dimorphic bristle trait. Contiguous bristle formations act as barriers to cell movement within the epithelium, and we demonstrated that a particularly rapid rotation of the proximal region of the comb is associated with the presence of a constricted area between a portion of the SC and a transverse row of contiguous bristle precursors. Our results suggest that the cell dynamics in the neighborhood of the SC may have biased its evolution.

Developmental constraints and convergent evolution in Drosophila sex comb formation.

The most complex and diverse secondary sexual character in Drosophila is the sex comb (SC), an arrangement of modified bristles on the forelegs of a subclade of male fruit flies. We examined SC formation in six representative nonmodel fruit fly species, in an effort to understand how the variation in comb patterning arises. We first compared SC development in two species with relatively small combs, Drosophila takahashii, where the SCs remain approximately transverse, and Drosophila biarmipes, where two rows of SC teeth rotate and move in an anterior direction relative to other bristle landmarks. We then analyzed comb ontogeny in species with prominent extended SCs parallel to the proximodistal axis, including Drosophila ficusphila and species of the montium subgroup. Our study allowed us to identify two general methods of generating longitudinal combs on the tarsus, and we showed that a montium subgroup species (Drosophila nikananu) with a comb convergently similar in size, orientation and position to the model organism Drosophila melanogaster, forms its SC through a different developmental mechanism. We also found that the protein product of the leg patterning gene, dachshund (dac), is strongly reduced in the SC in all species, but not in other bristles. Our results suggest that an apparent constraint on SC position in the adult may be attributable to at least two different lineage-specific developmental processes, although external forces could also play a role.