Subramanian Ranganathan

LOX-1 Mediates Oxidized Low-Density Lipoprotein-Induced Expression of Matrix Metalloproteinases in Human Coronary Artery Endothelial Cells

Background—Oxidized LDL (ox-LDL) accumulation in the atherosclerotic region may enhance plaque instability. Both accumulation of ox-LDL and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. This study was designed to examine the role of LOX-1 in the modulation of metalloproteinases (MMP-1 and MMP-3) in human coronary artery endothelial cells (HCAECs). Methods and Results—HCAECs were incubated with ox-LDL (10 to 80 &mgr;g/mL) for 1 to 24 hours. Ox-LDL increased the expression of MMP-1 (collagenase) and MMP-3 (stromelysin-1) in a concentration- and time-dependent manner. Ox-LDL also increased collagenase activity. Ox-LDL did not significantly affect the expression of tissue inhibitors of metalloproteinases. Native LDL had no effect on the expression of MMPs. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1, because pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 &mgr;g/mL) prevented the expression of MMPs in response to ox-LDL (P <0.01). In parallel experiments, ox-LDL caused the activation of protein kinase C (PKC), which was inhibited by LOX-1 antibody. The PKC-&bgr; isoform played a critical role in the expression of MMPs, because the PKC-&bgr; inhibitor hispidin reduced ox-LDL-induced activation of PKC and the expression of MMPs. Other PKC subunits (&agr;, &ggr;, and &egr;) did not affect the expression of MMPs. Conclusions—These findings indicate that ox-LDL, via LOX-1 activation, modulates the expression and activity of MMPs in HCAECs. In this process, activation of the PKC-&bgr; subunit plays an important signaling role.

Thiazolidinediones Inhibit Lipoprotein Lipase Activity in Adipocytes

The thiazolidinediones troglitazone and BRL 49653 improve insulin sensitivity in humans and animals with insulin resistance. Adipose tissue lipoprotein lipase is an insulin-sensitive enzyme. We examined the effects of thiazolidinediones on lipoprotein lipase expression in adipocytes. When added to 3T3-F442A, 3T3-L1, and rat adipocytes in culture, troglitazone and BRL 49653 inhibited lipoprotein lipase activity. This inhibition was observed at concentrations as low as 0.1 μm and within 2 h after addition of the drug. Lipoprotein lipase activity was inhibited in differentiated adipocytes as well as the differentiating cells. Despite this decrease in enzyme activity, these drugs increased mRNA levels in undifferentiated 3T3-F442A and 3T3-L1 cells and had no effect on mRNA expression or synthesis of lipoprotein lipase in differentiated cells. Western blot analysis showed that these drugs did not affect the mass of the enzyme protein. Lipoprotein lipase activity in cultured Chinese hamster ovary cells was not inhibited by troglitazone. Glucose transport, biosynthesis of lipids from glucose or the biosynthesis of proteins were unaffected by thiazolidinediones in differentiated cells, whereas glucose transport and lipid biosynthesis were increased when these drugs were added during differentiation. These results show that troglitazone and BRL 49653 have a specific, post-translational inhibitory effect on lipoprotein lipase in adipocytes, yet they promote lipid accumulation and differentiation in preadipocytes.

Aspirin inhibits human coronary artery endothelial cell proliferation by upregulation of p53.

Aspirin (acetylsalicylic acid, ASA) is effective in the primary and secondary prevention of vascular events. This effect is mediated in large part by platelet inhibition; however, non-platelet-mediated effects may also be relevant in the overall efficacy of ASA. We determined the effect of ASA on the synthesis of DNA and total proteins in cultured human coronary endothelial cells (HCAECs). Fourth generation HCAECs were cultured and treated with ASA and rate of synthesis of DNA and total proteins was determined by incorporation of [3H]thymidine and [3H]proline, respectively. ASA inhibited DNA synthesis by 50% at a concentration of 1mM and protein synthesis by 50% at a concentration of 2mM. The inhibitory effect of ASA was observed as early as 2h after treatment of HCAECs. The inhibition of DNA and protein synthesis could be reversed within 24h after removal of the drug from the culture medium. Indomethacin also inhibited DNA and protein synthesis. Western blot analysis revealed that the expression of p53 protein was increased after treatment of the cells with ASA. These observations indicate that ASA decreases endothelial cell proliferation through cell cycle arrest mediated by enhanced p53 expression. Arrest of endothelial proliferation and activation may be an important mechanism of the beneficial effect of ASA in acute coronary syndromes.

Low density lipoproteins modulate collagen metabolism in fibroblasts.

High levels of low-density lipoprotein are associated with atherosclerosis, and myocardial and arterial remodeling. We postulated that low-density lipoprotein influences collagen synthesis and degradation in fibroblasts as a potential mechanism of tissue remodeling. Incubation of cultured human skin fibroblasts with low-density lipoproteins resulted in a time-dependent and dose-dependent increase in the secretion of matrix metalloproteinase activity measured by gelatin zymography. Western blot analysis showed a concomitant increase in matrix metalloproteinase-1 protein. Northern blot analysis demonstrated an increase in collagen I messenger RNA after treatment with low-density lipoprotein. The matrix metalloproteinase-1 secretory response of fibroblasts to low-density lipoprotein was attenuated by heparin, which inhibits low-density lipoprotein uptake through the low-density lipoprotein-receptor. These observations suggest that low-density lipoprotein has a regulatory effect on collagen metabolism in fibroblasts.