A protective role for kidney apolipoprotein E. Regulation of mesangial cell proliferation and matrix expansion.

Mesangial expansion is a key feature in the pathogenesis of numerous renal diseases involving the glomerulus. Studies indicate that mutations in apolipoprotein E (apoE) might independently contribute to kidney dysfunction. Although the role of apoE as an atheroprotective molecule is well established, its role in kidney is unclear. In this study, we sought to explore whether apoE has a protective function in kidney. Northern blotting and reverse transcriptase-polymerase chain reaction showed apoE expression in kidney, and mesangial cell is a major source of apoE in kidney. In the kidneys of 14-16-month-old apoE-null mice, hematoxylin-eosin (HE) staining revealed increased mesangial cell proliferation and matrix formation compared with wild type mice or apoB-overexpressing mice, which have elevated plasma cholesterol and triglycerides. These data suggest that lack of apoE, rather than hyperlipidemia, contributes to increased mesangial expansion. We isolated mesangial cells from mouse kidney and determined the effect of apoE on cell growth. ApoE (E3, 10 microg/ml) completely inhibited serum, platelet-derived growth factor (10 ng/ml), as well as low density lipoprotein-induced mesangial cell proliferation. Among the three isoforms, E3 was found to be most effective in inhibiting mesangial cell proliferation. ApoE did not show any cytotoxic effect, and moreover, inhibited mesangial cell apoptosis induced by oxidized low density lipoprotein. These data suggest that apoE regulates growth as well as survival of mesangial cells. We previously showed that apoE induces matrix heparan sulfate proteoglycan (HSPG) in vascular cells, which has an antiproliferative effect. Similarly, apoE induced the mesangial matrix HSPG. Perlecan is the major HSPG of mesangial matrix and subendothelial space, and consistent with this, blockade of perlecan reversed the antiproliferative effect of apoE. Immunohistochemistry revealed reduced staining of perlecan in kidney from apoE-null mice. Because the loss of anionic HSPG in the basement membrane and mesangial matrix is associated with disruption of filtration barrier, these data suggest a novel role for kidney apoE in preserving the filtration barrier. In summary, apoE has a protective function in kidney as an autocrine regulator of mesangial expansion and kidney function.

Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor.

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of circulating lipoprotein triglyceride molecules, is synthesized in myocytes and adipocytes but functions while bound to heparan sulfate proteoglycans (HSPGs) on the luminal surface of vascular endothelial cells. This requires transfer of LPL from the abluminal side to the luminal side of endothelial cells. Studies were performed to investigate the mechanisms of LPL transcytosis using cultured monolayers of bovine aortic endothelial cells. We tested whether HSPGs and members of the low density lipoprotein (LDL) receptor superfamily were involved in transfer of LPL from the basolateral to the apical side of cultured endothelial cells. Heparinase/heparinitase treatment of the basolateral cell surface or addition of heparin to the basolateral medium decreased the movement of LPL. This suggested a requirement for HSPGs. To assess the role of receptors, we used either receptor-associated protein, the 39-kDa inhibitor of ligand binding to the LDL receptor-related protein and the very low density lipoprotein (VLDL) receptor, or specific receptor antibodies. Receptor-associated protein reduced (125)I-LPL and LPL activity transfer across the monolayers. When the basolateral surface of the cells was treated with antibodies, only anti-VLDL receptor antibodies inhibited transcytosis. Moreover, overexpression of the VLDL receptor using adenoviral-mediated gene transfer increased LPL transcytosis. Thus, movement of active LPL across endothelial cells involves both HSPGs and VLDL receptor.

The heparin-binding proteins apolipoprotein E and lipoprotein lipase enhance cellular proteoglycan production.

Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with (35)[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [(3)H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non-apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.

Perlecan mediates the antiproliferative effect of apolipoprotein E on smooth muscle cells. An underlying mechanism for the modulation of smooth muscle cell growth?

Apolipoprotein E (apoE) is known to inhibit cell proliferation; however, the mechanism of this inhibition is not clear. We recently showed that apoE stimulates endothelial production of heparan sulfate (HS) enriched in heparin-like sequences. Because heparin and HS are potent inhibitors of smooth muscle cell (SMC) proliferation, in this study we determined apoE effects on SMC HS production and cell growth. In confluent SMCs, apoE (10 microg/ml) increased (35)SO(4) incorporation into PG in media by 25-30%. The increase in the medium was exclusively due to an increase in HSPGs (2.2-fold), and apoE did not alter chondroitin and dermatan sulfate proteoglycans. In proliferating SMCs, apoE inhibited [(3)H]thymidine incorporation into DNA by 50%; however, despite decreasing cell number, apoE increased the ratio of (35)SO(4) to [(3)H]thymidine from 2 to 3.6, suggesting increased HS per cell. Purified HSPGs from apoE-stimulated cells inhibited cell proliferation in the absence of apoE. ApoE did not inhibit proliferation of endothelial cells, which are resistant to heparin inhibition. Analysis of the conditioned medium from apoE-stimulated cells revealed that the HSPG increase was in perlecan and that apoE also stimulated perlecan mRNA expression by >2-fold. The ability of apoE isoforms to inhibit cell proliferation correlated with their ability to stimulate perlecan expression. An anti-perlecan antibody completely abrogated the antiproliferative effect of apoE. Thus, these data show that perlecan is a potent inhibitor of SMC proliferation and is required to mediate the antiproliferative effect of apoE. Because other growth modulators also regulate perlecan expression, this may be a key pathway in the regulation of SMC growth.

Lipolysis-induced iron release from diferric transferrin: Possible role of lipoprotein lipase in LDL oxidation.

Conditions leading to oxidation of LDL in vivo are still unknown. While the occurrence of oxidized lipoproteins and catalytic free iron in advanced atherosclerotic lesions has been demonstrated, the origin of both is unclear. In vivo, iron metabolism is tightly regulated by iron-binding proteins that ensure that virtually no free iron exists. We examined whether physiological events such as lipolysis might reduce pH, facilitate iron release from transferrin (Tf), and promote low density lipoprotein (LDL) oxidation. Lipolysis is brought about by lipoprotein lipase (LpL), a triglyceride hydrolase present on endothelial cell surfaces and in atherosclerotic lesions. LpL hydrolysis of Intralipid lowered pH from 7.40 to 7.00 in 10% human serum and from 7.40 to 6.88 in phosphate-buffered saline. Similar decreases in pH were also observed when very low density lipoproteins were hydrolyzed by LpL. Lipolysis was accompanied by a 2-fold increase in the release of 59Fe from Tf. Tf binding to subendothelial matrix (SEM), a site of key events in atherosclerosis, increased 2-fold as the pH decreased from 7.40 to 6.00. More free iron also bound to SEM as the pH decreased below 7.40. We next tested whether a reduction in pH promotes LDL oxidation. More oxidation products were found in LDL incubated at low pH for 24 h in 10% human serum. Malonaldehyde contents (nmol/mg protein), measured as TBARS, were 7.11 +/- 0.34 at pH 7.40, 7.65 +/- 0.49 at pH 7.00, 9.00 +/- 1.18 at pH 6.50, and 11. 54 +/- 0.63 at pH 6.00. Based on these results, we hypothesize that lipolysis-induced acidic conditions enhance iron release from Tf and increase formation of oxidized LDL.

Apolipoprotein E containing high density lipoprotein stimulates endothelial production of heparan sulfate rich in biologically active heparin-like domains. A potential mechanism for the anti-atherogenic actions of vascular apolipoprotein e.

Reduced heparin and heparan sulfate (HS) proteoglycans (PG) have been observed in both inflammation and atherosclerosis. Methods to increase endogenous heparin and heparan sulfate are not known. We found that incubation of endothelial cells with 500-1,000 micrograms/ml high density lipoprotein (HDL) increased 35SO4 incorporation into PG by 1.5-2.5-fold. A major portion of this increase was in HS and was the result of increased synthesis. Total PG core proteins were not altered by HDL; however, the ratio of 35SO4 to [3H]glucosamine was increased by HDL, suggesting increased sulfation of glycosaminoglycans. In addition, HDL increased the amount of highly sulfated heparin-like HS in the subendothelial matrix. HS from HDL-treated cells bound 40 +/- 5% more 125I-antithrombin III (requires 3-O sulfated HS) and 49 +/- 3% fewer monocytes. Moreover, the HS isolated from HDL-treated cells inhibited smooth muscle cell proliferation (by 83 +/- 5%) better than control HS (56 +/- 6%) and heparin (42 +/- 6%). HDL isolated from apolipoprotein E (apoE)-null mice did not stimulate HS production unless apoE was added. ApoE also stimulated HS production in the absence of HDL. ApoE did not increase 35SO4 incorporation in macrophages and fibroblasts, suggesting that this is an endothelial cell-specific process. Receptor-associated protein inhibited apoE-mediated stimulation of HS only at higher (20 micrograms/ml) doses, suggesting the involvement of a receptor-associated protein-sensitive pathway in mediating apoE actions. In summary, our data identify a novel mechanism by which apoE and apoE-containing HDL can be anti-atherogenic. Identification of specific apoE peptides that stimulate endothelial heparin/HS production may have important therapeutic applications.

The NH2-terminal region of apolipoprotein B is sufficient for lipoprotein association with glycosaminoglycans.

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. The co-localization of apolipoprotein (apo) B and proteoglycans within lesions has suggested that retention is due to lipoprotein interaction with these highly electronegative glycoconjugates. Both apoB100- and apoB48-containing lipoproteins, i.e. low density lipoproteins (LDLs) and chylomicron remnants, are atherogenic. This suggests that retention is due to determinants in the initial 48% of apoB. To test this, the interaction of an apoB fragment (apoB17), and apoB48- and apoB100- containing lipoproteins with heparin, subendothelial matrix, and artery wall purified proteoglycans was studied. ApoB100-containing LDL from humans and human apoB transgenic mice and apoB48-containing LDLs from apoE knockout mice were used. Despite the lack of the carboxyl-terminal 52% of apoB, the apoB48-LDL bound to heparin-affinity gel as well as did apoB100-LDL. An NH2-terminal fragment containing 17% of full-length apoB was made using a recombinant adenovirus; apoB17 bound to heparin as well as did LDL. Monoclonal antibodies against the NH2-terminal region of apoB decreased apoB100 LDL binding to heparin, whereas antibodies against the LDL receptor-binding region did not alter LDL-heparin interaction. The role of the NH2-terminal region of apoB in LDL interaction with matrix molecules was also assessed. Media containing apoB17 decreased LDL binding to subendothelial matrix by 42%. Moreover, removal of the apoB17 by immunoprecipitation abrogated the inhibitory effect of these media. Antibodies to the NH2-terminal region decreased LDL binding to matrix and dermatan sulfate proteoglycans. Purified apoB17 effectively competed for binding of LDL to artery derived decorin and to subendothelial matrix. Thus, despite the presence of multiple basic amino acids near the LDL receptor-binding domain of LDL, the NH2-terminal region of apoB is sufficient for the interaction of lipoproteins with glycoconjugates produced by endothelial and smooth muscle cells. The presence of a proteoglycan-binding site in the NH2-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.

Subendothelial retention of lipoprotein (a). Evidence that reduced heparan sulfate promotes lipoprotein binding to subendothelial matrix.

Vessel wall subendothelial extracellular matrix, a dense mesh formed of collagens, fibronectin, laminin, and proteoglycans, has important roles in lipid and lipoprotein retention and cell adhesion. In atherosclerosis, vessel wall heparan sulfate proteoglycans (HSPG) are decreased and we therefore tested whether selective loss of HSPG affects lipoprotein retention. A matrix synthesized by aortic endothelial cells and a commercially available matrix (Matrigel; , Rutherford, NJ) were used. Treatment of matrix with heparinase/heparitinase (1 U/ml each) increased LDL binding by approximately 1.5-fold. Binding of lipoprotein (a) [Lp(a)] to both subendothelial matrix and Matrigel(R) increased 2-10-fold when the HSPG were removed by heparinase treatment. Incubation of endothelial cells with oxidized LDL (OxLDL) or lysolecithin resulted in decreased matrix proteoglycans and increased Lp(a) retention by matrix. The effect of OxLDL or lysolecithin on endothelial PG was abolished in the presence of HDL. The decrease in matrix HSPG was associated with production of a heparanase-like activity by OxLDL-stimulated endothelial cells. To test whether removal of HSPG exposes fibronectin, a candidate Lp(a) binding protein in the matrix, antifibronectin antibodies were used. The increased Lp(a) binding after HSPG removal was inhibited 60% by antifibronectin antibodies. Similarly, the increased Lp(a) binding to matrix from OxLDL-treated endothelial cells was inhibited by antifibronectin antibodies. We hypothesize that atherogenic lipoproteins stimulate endothelial cell production of heparanase. This enzyme reduces HSPG which in turn promotes Lp(a) retention.

Endothelial cell heparanase modulation of lipoprotein lipase activity. Evidence that heparan sulfate oligosaccharide is an extracellular chaperone.

A unique feature of lipoprotein lipase (LpL), the rate-limiting enzyme in the hydrolysis of circulating triglycerides, is its movement from its cell of synthesis, adipocyte or myocyte, to its site of action, the luminal endothelial surface. This involves processes that allow LpL to be released from the adipocyte cell surface and transferred against the flow of interstitial fluid to the luminal surface of endothelial cells. LpL, an unstable enzyme, must retain its activity during this process. Whether a chaperone-like molecule is involved in LpL stabilization and transport is unclear. In the present study, we tested the hypothesis that endothelial cells secrete factors that release LpL and promote its transfer to the luminal endothelial surface. Incubation of adipocytes with endothelial cell conditioned medium (ECCM) led to release of about 2-fold more LpL activity than control medium. Medium from endothelial cells exposed to lysophosphatidylcholine (lyso-ECCM), a product of LpL lipolysis of lipoproteins, released approximately 3-fold more LpL than ECCM. Concomitant with the release of LpL, adipocyte cell surface heparan sulfate (HS) proteoglycans were degraded suggesting that lyso-ECCM contained a heparanase-like activity. More heparanase was found in media from the basolateral than the apical side of lysolecithin-stimulated polarized endothelial cells. In coculture experiments, lipolysis and lysolecithin stimulation of endothelial cells increased LpL release from adipocytes. LpL released by lyso-ECCM remained stable and did not lose enzymatic activity at 37 degrees C for 1 h. LpL activity was also stabilized by heparanase-digested fragments of HS (HS oligosaccharide) and by purified LpL binding decasaccharide. Moreover, LpL.HS oligosaccharide complexes crossed endothelial cell monolayers and bound to the apical side of the cells. Thus, an endothelial heparanase may play a critical role in releasing subendothelial HS bound proteins, and specific HS oligosaccharides produced by this enzyme may serve as extracellular chaperones.

Lipoprotein lipase degradation by adipocytes: receptor-associated protein (RAP)-sensitive and proteoglycan-mediated pathways.

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of triglycerides, is primarily synthesized by adipocytes and myocytes. In addition to synthesis, degradation of cell surface-associated LPL is thought to be important in regulating production of the enzyme. We studied LPL metabolism in the LPL synthesizing adipocyte cell line BFC-1 beta and assessed the contributions of cell surface heparan sulfate proteoglycans (HSPG), low density lipoprotein receptor related protein (LRP), and glycosylphosphatidylinositol (GPI)-linked proteins to LPL uptake and degradation by these cells. Adipocytes degraded 10-12% of total cell surface I-labeled LPL in 2 h and 23-28% in 4 h. In 1 h, 30-54% of the degradation was inhibited by the 39 kDa receptor associated protein (RAP), an inhibitor of ligand binding to LRP. At 4 h, only 19-23% of the LPL degradation was RAP inhibitable. This suggested that two pathways with different kinetics were important for LPL degradation. Heparinase/heparitinase treatment of cells showed that most LPL degradation required the presence of HSPG. Treatment with phosphatidylinositol-specific phospholipase C (PIPLC) inhibited 125I-labeled LPL degradation by 13%. However, neither RAP nor PIPLC treatment of adipocytes significantly increased the amount of endogenously produced LPL activity in the media. To determine whether direct uptake of LPL bound to HSPG could account for the non-RAP sensitive LPL uptake and degradation, proteoglycan metabolism was assessed by labeling cells with 35SO4. Of the total pericellular proteoglycans, 14% were PIPLC releasable; surprisingly, 30% were dissociated from the cells with heparin. The amount of labeled pericellular proteoglycans decreased 26% in 2 h and 50% in 8 h, rapid enough to account for at least half of the degradation of cell surface LPL. We conclude that adipocytes degrade a fraction of the cell surface LPL, and that this process is mediated by both proteoglycans and RAP-sensitive receptors.