Srividya Velagapudi

Plasmalogens of high-density lipoproteins (HDL) are associated with coronary artery disease and anti-apoptotic activity of HDL

OBJECTIVE Low high-density lipoprotein (HDL) cholesterol and loss of atheroprotective functions of HDL are associated with coronary artery disease (CAD). Here, we investigated the associations of HDL phospholipids with acute and stable CAD as well as with the anti-apoptotic activity of HDL. METHODS 49 species of phosphatidylcholines (PCs), lysophosphatidylcholines and sphingomyelins (SMs) as well as three species of sphingosine-1-phosphate (S1P) were quantified by liquid chromatography - mass spectrometry in HDL isolated from 22 healthy subjects as well as 23 and 22 patients with stable CAD and acute coronary syndrome (ACS), respectively. Native HDL and artificially reconstituted HDL (rHDL) were tested for their capacity to inhibit apoptosis of endothelial cells (ECs) induced by serum deprivation. RESULTS HDL of CAD or ACS patients differed from HDL of healthy controls by the content in nine of the 52 quantified phospholipid species as well as reduced anti-apoptotic activity. The capacity of HDL to inhibit EC apoptosis correlated significantly with five of eleven odd-chain PCs (= plasmalogens), two S1Ps, SM42:2, PC34:2, and PC32:0. An orthogonal partial least square - discriminant analysis revealed independent associations of stable CAD with HDL-associated PC34:2, PC33:3 and PC35:2 as well as anti-apoptotic activity of HDL and of ACS with HDL-associated PC33:3, PC35:2, SM42:1, PC34:2 and PC36:2. rHDL reconstituted with apoA-I, PC34:1, and PC35:2 inhibited apoptosis of ECs more effectively than rHDL containing only apoA-I and PC34:1. CONCLUSIONS The inverse association of HDL-plasmalogen levels with both stable and acute CAD may reflect direct anti-apoptotic effects of plasmologens on ECs.

VEGF-A Regulates Cellular Localization of SR-BI as Well as Transendothelial Transport of HDL but Not LDL

Objective— Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. Approach and Results— Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of 125I-HDL but not 125I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells. Conclusions— The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall.

Prevalence and causes of abnormal PSA recovery

Abstract Background: Prostate-specific antigen (PSA) test is of paramount importance as a diagnostic tool for the detection and monitoring of patients with prostate cancer. In the presence of interfering factors such as heterophilic antibodies or anti-PSA antibodies the PSA test can yield significantly falsified results. The prevalence of these factors is unknown. Methods: We determined the recovery of PSA concentrations diluting patient samples with a standard serum of known PSA concentration. Based on the frequency distribution of recoveries in a pre-study on 268 samples, samples with recoveries <80% or >120% were defined as suspect, re-tested and further characterized to identify the cause of interference. Results: A total of 1158 consecutive serum samples were analyzed. Four samples (0.3%) showed reproducibly disturbed recoveries of 10%, 68%, 166% and 4441%. In three samples heterophilic antibodies were identified as the probable cause, in the fourth anti-PSA-autoantibodies. The very low recovery caused by the latter interference was confirmed in serum, as well as heparin- and EDTA plasma of blood samples obtained 6 months later. Analysis by eight different immunoassays showed recoveries ranging between <10% and 80%. In a follow-up study of 212 random plasma samples we found seven samples with autoantibodies against PSA which however did not show any disturbed PSA recovery. Conclusions: About 0.3% of PSA determinations by the electrochemiluminescence assay (ECLIA) of Roche diagnostics are disturbed by heterophilic or anti-PSA autoantibodies. Although they are rare, these interferences can cause relevant misinterpretations of a PSA test result.

Endocytosis of lipoproteins

During their metabolism, all lipoproteins undergo endocytosis, either to be degraded intracellularly, for example in hepatocytes or macrophages, or to be re-secreted, for example in the course of transcytosis by endothelial cells. Moreover, there are several examples of internalized lipoproteins sequestered intracellularly, possibly to exert intracellular functions, for example the cytolysis of trypanosoma. Endocytosis and the subsequent intracellular itinerary of lipoproteins hence are key areas for understanding the regulation of plasma lipid levels as well as the biological functions of lipoproteins. Indeed, the identification of the low-density lipoprotein (LDL)-receptor and the unraveling of its transcriptional regulation led to the elucidation of familial hypercholesterolemia as well as to the development of statins, the most successful therapeutics for lowering of cholesterol levels and risk of atherosclerotic cardiovascular diseases. Novel limiting factors of intracellular trafficking of LDL and the LDL receptor continue to be discovered and to provide drug targets such as PCSK9. Surprisingly, the receptors mediating endocytosis of high-density lipoproteins or lipoprotein(a) are still a matter of controversy or even new discovery. Finally, the receptors and mechanisms, which mediate the uptake of lipoproteins into non-degrading intracellular itineraries for re-secretion (transcytosis, retroendocytosis), storage, or execution of intracellular functions, are largely unknown.