Hutchinson-Gilford progeria syndrome is a progeroid disorder of children caused by de novo mutations in LMNA that lead to the synthesis of an internally truncated form of prelamin A (commonly called progerin). The production of progerin causes multiple disease phenotypes, including an unusual vascular phenotype characterized by the loss of smooth muscle cells in the arterial media and fibrosis of the adventitia. In this report, we show that progerin expression, combined with mechanical stress, promotes smooth muscle cell death. Disrupting the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex in smooth muscle cells ameliorates the toxic effects of progerin on smooth muscle cells and limits the accompanying adventitial fibrosis.

Deficiencies in either lamin B1 or lamin B2 cause both defective migration of cortical neurons in the developing brain and reduced neuronal survival. The neuronal migration abnormality is explained by a weakened nuclear lamina that interferes with nucleokinesis, a nuclear translocation process required for neuronal migration. In contrast, the explanation for impaired neuronal survival is poorly understood. We hypothesized that the forces imparted on the nucleus during neuronal migration result in nuclear membrane (NM) ruptures, causing interspersion of nuclear and cytoplasmic contents—and ultimately cell death. To test this hypothesis, we bredLmnb1-deficient mice that express a nuclear-localized fluorescentCrereporter. Migrating neurons within the cortical plate of E18.5Lmnb1-deficient embryos exhibited NM ruptures, evident by the escape of the nuclear-localized reporter into the cytoplasm and NM discontinuities by electron microscopy. The NM ruptures were accompanied by DNA damage and cell death. The NM ruptures were not observed in nonmigrating cells within the ventricular zone. NM ruptures, DNA damage, and cell death were also observed in culturedLmnb1−/−andLmnb2−/−neurons as they migrated away from neurospheres. To test whether mechanical forces on the cell nucleus are relevant to NM ruptures in migrating neurons, we examined culturedLmnb1−/−neurons when exposed to external constrictive forces (migration into a field of tightly spaced silicon pillars). As the cells entered the field of pillars, there were frequent NM ruptures, accompanied by DNA damage and cell death.

The processing of triglyceride-rich lipoproteins (TRLs) in capillaries provides lipids for vital tissues, but our understanding of TRL metabolism is limited, in part because TRL processing and lipid movement have never been visualized. To investigate the movement of TRL-derived lipids in the heart, mice were given an injection of [H]triglyceride-enriched TRLs, and the movement of H-labeled lipids across capillaries and into cardiomyocytes was examined by NanoSIMS. TRL processing and lipid movement in tissues were extremely rapid. Within 30 s, TRL-derived lipids appeared in the subendothelial spaces and in the lipid droplets and mitochondria of cardiomyocytes. Enrichment ofH in capillary endothelial cells was not greater than in cardiomyocytes, implying that endothelial cells may not be a control point for lipid movement into cardiomyocytes. Remarkably, a deficiency of the putative fatty acid transport protein CD36, which is expressed highly in capillary endothelial cells, did not impede entry of TRL-derived lipids into cardiomyocytes.

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a "lawn" of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL.