Age-related changes in mitochondria are associated with decline in mitochondrial function. With advanced age, mitochondrial DNA volume, integrity and functionality decrease due to accumulation of mutations and oxidative damage induced by reactive oxygen species (ROS). In aged subjects, mitochondria are characterized by impaired function such as lowered oxidative capacity, reduced oxidative phosphorylation, decreased ATP production, significant increase in ROS generation, and diminished antioxidant defense. Mitochondrial biogenesis declines with age due to alterations in mitochondrial dynamics and inhibition of mitophagy, an autophagy process that removes dysfunctional mitochondria. Age-dependent abnormalities in mitochondrial quality control further weaken and impair mitochondrial function. In aged tissues, enhanced mitochondria-mediated apoptosis contributes to an increase in the percentage of apoptotic cells. However, implementation of strategies such as caloric restriction and regular physical training may delay mitochondrial aging and attenuate the age-related phenotype in humans.

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A cluster of miR-221/222 is a key player in vascular biology through exhibiting its effects on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). These miRNAs contribute to vascular remodeling, an adaptive process involving phenotypic and behavioral changes in vascular cells in response to vascular injury. In proliferative vascular diseases such as atherosclerosis, pathological vascular remodeling plays a prominent role. The miR-221/222 cluster controls development and differentiation of ECs but inhibits their proangiogenic activation, proliferation, and migration. miR-221/222 are primarily implicated in maintaining endothelial integrity and supporting quiescent EC phenotype. Vascular expression of miR-221/222 is upregulated in initial atherogenic stages causing inhibition of angiogenic recruitment of ECs and increasing endothelial dysfunction and EC apoptosis. In contrast, these miRNAs stimulate VSMCs and switching from the VSMC “contractile” phenotype to the “synthetic” phenotype associated with induction of proliferation and motility. In atherosclerotic vessels, miR-221/222 drive neointima formation. Both miRNAs contribute to atherogenic calcification of VSMCs. In advanced plaques, chronic inflammation downregulates miR-221/222 expression in ECs that in turn could activate intralesion neoangiogenesis. In addition, both miRNAs could contribute to cardiovascular pathology through their effects on fat and glucose metabolism in nonvascular tissues such as adipose tissue, liver, and skeletal muscles.

At present, several types of modified LDL have been shown to occur in human blood. Curtiss and Witztum15 have demonstrated the presence of nonenzymatically glycosylated LDL in the blood of hyperglycemic diabetic patients. We have recently shown that LDL isolated from the blood of atherosclerotic patients was able to induce intracellular lipid accumulation in cultured aortic cells1617 and differed from native LDL by a lower content of sialic acid; i.e., it appeared to be a desialylated lipoprotein."11,2 Lipoprotein (a), which differs from LDL by the presence of an additional apoprotein is also considered to play an important role in the deposition of intracellular lipids.18"19 In the present study, we demonstrated that in vivo modified LDL caused lipid accumulation in cultured human intimal smooth muscle cells and monocytes. We tested the hypothesis that modified lipoprotein aggregates but not single particles caused intracellular lipid accumulation. To this end we showed that 1) in vivo modified LDLs were able to form aggregates, 2) these aggregates caused the accumulation of cholesteryl esters in cultured cells, and 3) the removal of aggregates from LDL preparation prevented the intracellular cholesteryl ester accumulation. We also attempted to examine the mechanism underlying the interaction between LDL aggregates and vascular cells. Preparation of Lipoproteins LDL (1.019-1.050 g/cm') was isolated by sequential ultracentrifugation in a preparative ultracentrifuge after appropriate adjustment of density with solid NaBr20 from the pooled blood of 12 healthy subjects, from 12 patients with coronary heart disease (CHD) with angiographically documented stenosis of coronary arteries, and from 12 non-insulin-dependent diabetic patients. The degree of lipoprotein aggregation was evaluated by the method based on the analysis of light transmission fluctuations in LDL suspension.29 The relative dispersion of the optical density fluctuations caused by random changes in the number of particles in the optical channel reflects the deviations in their average size, i.e., the degree of aggregation. The optical density fluctuations were measured using a semiconductor laser with collimating optics (wavelength, 860 nm). The aggregate size was determined by methods of quasielastic laser scattering on an Autosizer 2 (Malvern Instrument, UK).For the analysis of lipoprotein aggregation, native and modified lipoproteins were passed through a Sepharose CL-2B column (25 x 0.6 cm) at a flow rate of 0.15 ml/mnin. Fractions (0.30 ml) were collected, and total cholesterol content was determined in each fraction. Examination of Lipoprotein-Lipoprotein InteractionsNinety-six-well microtiter plates were precoated with freshly prepared native and modified lipoproteins (1 ,ug LDL protein per well) and incubated for 1 hour at 37°C. Then the wells were washed with 0.2% bovine serum albumin in PBS, and 0.01-100 ,g/ml`PI-LDL was added to each well. After a 1-hour incubation at 370C, the wells were washed thoroughly with PBS, and radioactivity was...