Untreated Smooth Muscle Cells Research Articles

Abstract 13040: Insulin-Like Growth Factor-1 Upregulates Larp6 via Translational and Post-Translational Mechanisms in Vascular Smooth Muscle Cells, Leading to Elevated Collagen Production

Introduction: Collagen content in atherosclerotic plaque is a hallmark of plaque stability. We have shown that insulin-like growth factor-1 (IGF1) increased collagen content in atherosclerotic plaques of Apoe -/- mice. We have identified LARP6 (La Ribonucleoprotein Domain Family, Member 6), a type I collagen mRNA-binding protein, as a mediator of IGF1-dependent type I collagen upregulation and increased mature fibril formation by smooth muscle cells (SMC), however specific mechanism has not been elucidated yet. Methods and Results: Micro-RNAs, including miR-1976 have been shown to bind to LARP6 mRNA. We found that SMC treatment with IGF1 significantly downregulated miR-1976 by 41±5% after 3h (P<0.05), consistent with the IGF1-induced LARP6 upregulation. In fact, miR-1976 mimic (20 nM) decreased LARP6 by 67% (P<0.05) and reduced type I procollagen protein levels by 85% (P<0.05). MiR-1976 mimic inhibits IGF1-induced LARP6 upregulation showing miR-1976 involvement in IGF1 effect on LARP6. It has been reported that LARP6-mediated promotion of procollagen synthesis may be regulated by phosphorylation of LARP6, therefore we investigated whether IGF1 induces LARP6 phosphorylation in cultured human aortic SMCs. For this experiment, SMCs were serum-starved and treated for 0-48h with 10 ng/mL IGF1. LARP6 protein was detected by immunoblots as a single 67kDa band in untreated SMC. However, after 6h of IGF1 treatment there was additional phosphorylated LARP6 band detected at 70 kDa. The levels of 70kDa LARP6 time-dependently increased up to 18h of treatment, with a concomitant increase of type I procollagen and collagen expression levels. Conclusions: In summary, our data suggests that (i) IGF1 downregulates miR-1976, thereby increasing LARP6 expression levels and (ii) IGF1 induces phosphorylation of LARP6, thereby promoting LARP6 activity. Our study revealed novel mechanisms underlying IGF1-induced type I collagen upregulation in vascular SMC and potentially in atherosclerotic plaques.

Mir‐1976 Downregulation and Phosphorylation of LARP6 are Associated with Insulin‐like Growth Factor‐1 (IGF1)‐induced Collagen Upregulation in Vascular Smooth Muscle Cells

Collagen content in atherosclerotic plaque is a hallmark of plaque stability. We have shown that insulin‐like growth factor‐1 (IGF1) increased collagen content in atherosclerotic plaques of Apoe−/− mice. We have identified LARP6 (La Ribonucleoprotein Domain Family, Member 6), a collagen mRNA binding protein, as a mediator of IGF1‐dependent collagen I upregulation and increased mature fibril formation in smooth muscle cells (SMCs), however specific mechanisms have not been elucidated yet. A total of six micro RNAs (miRs), including miR‐1976, have been shown to bind to LARP6 mRNA. In fact, miR‐1976 mimic (20 nM) decreased LARP6 by 67% and reduced procollagen I by 85% (both are P<0.05), indicating that miR‐1976 negatively regulates LARP6 expression. We found that IGF1 significantly downregulated miR‐1976 by 41±5% in SMCs after 3h of exposure. Moreover, miR‐1976 mimic inhibited IGF1‐induced LARP6 upregulation, suggesting that the downregulation of miR‐1976 mediates IGF1 upregulation of LARP6.It has been reported that LARP6 phosphorylation is critical for regulating translation of collagen polypeptides. To investigate whether IGF1 induces LARP6 phosphorylation, we assessed LARP6 electrophoretic mobility shift by SDS‐PAGE and immunoblotting. SMCs were serum‐starved and treated for 0–48h either with 10 ng/ml IGF1, or 2 ng/ml IFNy, or 2 ng/ml IL‐1b, or 2 ng/ml TNFa, or 10 ng/ml IL‐6. LARP6 protein was detected on immunoblots as a single 65kDa band in untreated SMCs. However, additional 67kDa LARP6 band was evident after 6h of cell treatment with IGF1 or with IL‐6. The density of 67kDa LARP6 band was gradually increased up to 18h of treatment. IGF1 and IL‐6 also increased expression levels of procollagen I and collagen I in a time‐dependent manner, with a peak at 18h. IFNy, IL‐1b, and TNFa did not induce 67kDa LARP6 band or increase in expression level of collagen in SMCs.Taken together, our data suggest that IGF1 promotes collagen expression by upregulating LARP6 levels via a downregulation of miR‐1976 and also by inducing phosphorylation of LARP6. Suggested mechanisms by which IGF1 induces collagen expression in vascular SMCs and thus potentially in atherosclerotic plaques would provide novel insights for a therapeutic approach increasing plaque stability.Support or Funding InformationNIH/NHLBI R01HL070241 (PD)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Monocytes Induce Apoptosis of Vascular Smooth Muscle Cells via a Monocyte Chemoattractant Protein‐1 (MCP‐1) Mediated Mechanism

Excessive apoptosis of smooth muscle cells (SMCs) contributes to pathogenesis of abdominal aortic aneurysm (AAA). We have previously demonstrated in a murine model of AAA that apoptotic SMCs stimulate vascular inflammation through production of MCP‐1. In the current study, we tested the hypothesis that MCP‐1 further induces cell death by enhancing the pro‐apoptotic capability of infiltrating inflammatory cells. Cultured mouse aortic SMCs and a mouse monocyte/macrophage cell line RAW264.7 were used. MCP‐1 (100ng/ml, 24h) primed RAWs elicited significant apoptosis of SMCs (2.21±0.349 fold increase in percentage of Annexin V‐PE+/7AAD− cells compared to untreated SMCs, n=5, p<0.05, ANOVA), while untreated RAWs or MCP‐1 had no effect. The pro‐apoptotic effect required cell‐cell contact or proximity between SMCs and RAWs because conditioned media made by MCP‐1 primed RAWs had minimal effect on apoptosis. In a CaCl2 induced murine aneurysm model, co‐staining with TUNEL and MOMA2 showed spatial correlation of apoptosis and monocyte/macrophage in aortic wall 7 days after AAA induction. In conclusion, our results suggest MCP‐1 may play a central role in pathogenesis of AAA. Produced by apoptotic SMCs and other cell types in injured vessels, MCP‐1 contributes directly to the recruitment of inflammatory cells and indirectly to induce apoptosis of SMCs.This work was supported by NIH grant R01HL088447‐01A2 (to B. Liu).

Effects of Hypertension on Morphological, Contractile and Mechanical Properties of Rat Aortic Smooth Muscle Cells

We studied the effects of hypertension on morphological, contractile, and tensile properties of smooth muscle cells (SMCs) isolated from rat aortas. Male Wistar rats with induced Goldblatt hypertension had their thoracic aortas excised 16–24 weeks postoperatively to isolate SMCs by enzymatic digestion. Cell shape was measured before and after cell contraction induced by norepinephrine. Tensile properties of untreated SMCs were obtained with a laboratory-made micro tensile tester. The cell diameter was significantly larger (p < 0.01) in hypertensive animals (9.2 μm) than age-matched controls (7.4 μm), while the length was not statistically different (55 μm). Cell contraction was significantly smaller (p < 0.01) in hypertensive animals (7%) than controls (30%). The initial elastic modulus of SMCs was significantly smaller (p < 0.05) in hypertensive animals (4.3 kPa) than controls (10.2 kPa). Wall thickening and reduction of contractility in hypertensive aortas was also observed in tubular segments of the aorta in rats exposed to hypertension for 16 weeks. The results showed that these changes also occurred at the cellular level, and they were accompanied by softening of the cells. Hypertension causes smooth muscle hypertrophy, which may decrease actin filaments as during the phenotypic transformation from a contractile to synthetic state, and thus may cause the inhibition of contractile properties and cell softening.

Syndecan-4 Is Required for Thrombin-induced Migration and Proliferation in Human Vascular Smooth Muscle Cells

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration.

Fas and Fas-associated death domain protein regulate monocyte chemoattractant protein-1 expression by human smooth muscle cells through caspase- and calpain-dependent release of interleukin-1alpha.

We previously reported that treatment of human vascular smooth muscle cells (SMCs) with proapoptotic stimuli, including Fas ligand plus cycloheximide (FasL/Chx), or overexpression of Fas-associated death domain protein (FADD) result in increased expression of monocyte chemoattractant protein-1 (MCP-1) and other proinflammatory genes. In this study, we demonstrate that Fas/FADD-induced MCP-1 upregulation is driven by an autocrine/paracrine signaling loop in which interleukin (IL)-1alpha synthesis and release are activated through caspase- and calpain-dependent processes. Untreated SMCs contain very little IL-1alpha protein or transcript. Both were increased greatly in response to Fas/FADD activation, primarily through an autocrine/paracrine pathway in which secreted IL-1alpha stimulated additional IL-1alpha synthesis and release. Caspase 8 (Csp8) activity increased in response to FasL/Chx treatment, and Csp8 inhibitors markedly reduced IL-1alpha release and MCP-1 upregulation. In contrast, Csp8 activity was not significantly increased in response to FADD overexpression and caspase inhibitors did not effect FADD-induced MCP-1 upregulation. Both FasL/Chx treatment and FADD overexpression increased the activity of calpains. Calpain inhibitors reduced IL-1alpha release and MCP-1 upregulation in both FADD-overexpressing SMCs and FasL/Chx-treated SMCs without blocking Csp8 activity. This indicates that calpains are not required for activation of caspases and that caspase activation is not sufficient for IL-1alpha release and MCP-1 upregulation. These data suggest that calpains play a dominant role in Fas/FADD-induced IL-1alpha release and MCP-1 upregulation and that caspase activation may function to amplify the effects of calpain activation.

Pitavastatin alters the expression of thrombotic and fibrinolytic proteins in human vascular cells.

In addition to lowering blood lipids, clinical benefits of 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A; EC 1.1.1.34) reductase inhibitors may derive from altered vascular function favoring fibrinolysis over thrombosis. We examined effects of pitavastatin (NK-104), a relatively novel and long acting statin, on expression of tissue factor (TF) in human monocytes (U-937), plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (t-PA) in human aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC). In monocytes, pitavastatin reduced expression of TF protein induced by lipopolysaccharide (LPS) and oxidized low-density lipoprotein (OxLDL). Similarly, pitavastatin also reduced expression of TF mRNA induced by LPS. Pitavastatin reduced PAI-1 antigen released from HUVEC under basal, OxLDL-, or tumor necrosis factor-alpha (TNF-alpha)-stimulated conditions. Reductions of PAI-1 mRNA expression correlated with decreased PAI-1 antigen secretion and PAI-1 activity as assessed by fibrin-agarose zymography. In addition, pitavastatin decreased PAI-1 antigen released from OxLDL-treated and untreated SMC. Conversely, pitavastatin enhanced t-PA mRNA expression and t-PA antigen secretion in untreated OxLDL-, and TNF-alpha-treated HUVEC and untreated SMC. Finally, pitavastatin increased t-PA activity as assessed by fibrin-agarose zymography. Our findings demonstrate that pitavastatin may alter arterial homeostasis favoring fibrinolysis over thrombosis, thereby reducing risk for thrombi at sites of unstable plaques.

Photodynamic therapy generates a matrix barrier to invasive vascular cell migration.

Photodynamic therapy (PDT) inhibits experimental intimal hyperplasia. PDT results in complete vascular wall cell eradication with subsequent adventitia but minimal media repopulation. This study was designed to test the hypothesis that PDT alters the vascular wall matrix thereby inhibiting invasive cell migration, and as such, provides an important barrier mechanism to favorably alter the vascular injury response. Untreated smooth muscle cells (SMCs) and fibroblasts were seeded on control and PDT-treated (100 J/cm(2); photosensitizer was chloroaluminum-sulfonated phthalocyanine, 5 microg/mL) 3-dimensional collagen matrix gels. Invasive cell migration was temporally quantified by calibrated microscopy. Zymography and ELISA assessed SMC matrix metalloproteinase levels. Molecular changes of gel proteins and their susceptibility to collagenase were analyzed by SDS-PAGE and Western blot. Limited pepsin digestion and histology were used to assess the in vivo relevance of the model, using an established rat carotid artery model at 1 and 4 weeks after balloon injury and PDT. PDT of 3-dimensional matrix of gels led to a 52% reduction of invasive SMCs and to a 59% reduction of fibroblast migration (P<0.001) but did not significantly affect secretion of matrix metalloproteinases. PDT induced collagen matrix changes, including cross-linking, which resulted in resistance to protease digestion. PDT led to a durable 45% reduction in pepsin digestion susceptibility of treated arteries (P<0.001) and inhibition of periadventitial cell migration into the media. These data suggest that PDT of matrix gels generates a barrier to invasive cellular migration. This newly identified effect on matrix proteins underscores its pleiotropic actions on the vessel wall, and as such, PDT may be of considerable potential therapeutic value to inhibit restenosis.

Photodynamic Therapy Inhibits the Injury-induced Fibrotic Response of Vascular Smooth Muscle Cells

Objectives: excessive deposition of extracellular matrix (ECM) proteins plays a key role in the intervention-related vascular fibroproliferative response, resulting in intimal hyperplasia (IH). Cytokines, such as Platelet-derived growth factor (PDGF), released after vascular injury and deposited in the ECM, are known to stimulate production of matrix proteins. Photodynamic therapy (PDT), the combination of light and a photosensitive dye to produce free radicals, is a novel approach to inhibit experimental IH by the local eradication of smooth-muscle cells (SMC) and alteration of ECM. This in vitro study examined whether PDT can inhibit the fibrotic response of vascular SMC. Materials and methods: the effect of PDT on important pro-fibrotic factors was determined by performing PDT of isolated ECM, injured SMC and pure PDGF. SMC production of collagen was monitored by cellular [3H]-proline incorporation. Results: untreated SMC seeded on ECM demonstrated an increase of 50% in collagen production ( p <0.0001) as compared to SMC on an empty plate. This increase was also seen when SMC was incubated with the conditioned media of mechanically injured SMC, or pure PDGF. However, after PDT of ECM, injured SMC or PDGF, there was an inhibition of 40% ( p <0.05) in SMC-collagen production.Conclusions: these findings indicate that PDT can interfere with factors that lead to the vascular fibrotic response. In this way, PDT, with its cytotoxic and extracellular effects, can promote healing of the vessel wall without the stimulus of fibrosis that can lead to restenosis.

Fas is expressed in human atherosclerotic intima and promotes apoptosis of cytokine-primed human vascular smooth muscle cells.

The membrane protein Fas/Apo-1/CD95 signals programmed cell death or apoptosis in activated T lymphocytes. Vascular smooth muscle cells (SMCs) bear markers of programmed cell death or apoptosis in advanced atherosclerotic plaques that contain immune cells e.g., macrophages and T lymphocytes. This study tested the hypothesis that the Fas death-signaling pathway contributes to apoptosis of SMCs exposed to proinflammatory cytokines produced by these immune cells during atherogenesis. All atherosclerotic plaques examined (n = 14) contained immunoreactive Fas. The majority of the Fas+ SMCs localized in the intima of the plaques, whereas the medial SMCs expressed Fas antigen less prominently. Double staining for DNA fragments (TUNEL) and Fas or cell identification markers colocalized Fas with TUNEL+ SMCs in the areas that contained CD3+ T cells and CD68+ macrophages, suggesting a role for Fas in the induction of SMC apoptosis by activated T cells during atherogenesis. In culture, stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta increased expression of Fas in SMCs. Incubation with an activating anti-Fas antibody triggered apoptosis of the cytokine-primed but not the untreated SMCs, as demonstrated by TUNEL and electrophoresis of oligonucleosomal DNA fragments. These data suggest that activation of the Fas death-signaling pathway contributes to the induction of SMC apoptosis during atherogenesis and furnish a mechanism whereby immune cells and their cytokines promote this cell death process related to vascular remodeling and plaque rupture.

Plasmin potentiates induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells.

Experiments were performed to examine the effect of the major fibrinolytic protease, plasmin, on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured human and rat aortic smooth muscle cells. Incubation of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite and nitrate in the culture media. Plasmin, either added exogenously or generated by the reaction of tissue plasminogen activator with plasminogen, potentiated the IL-1 beta-mediated release of nitrite and nitrate from smooth muscle cells in a concentration-dependent manner, without affecting the production of nitrite and nitrate from cells untreated with IL-1 beta. This potentiating effect was abolished when plasmin was incubated with the protease inhibitor, alpha 2-antiplasmin. The perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium, and the addition of IL-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated platelets inhibited their aggregation. Untreated smooth muscle cells or cells treated with plasmin alone did not have such effects. However, the simultaneous treatment of smooth muscle cells with IL-1 beta and plasmin markedly enhanced both the relaxing activities of the perfusates and the inhibition of platelet aggregation. Treatment of smooth muscle cells with NG-nitro-L-arginine inhibited the cytokine-mediated effects as well as the potentiating effect of plasmin. These results demonstrate that the plasmin can enhance the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells.

Thrombin inhibits induction of nitric oxide synthase in vascular smooth muscle cells

Experiments were designed to examine whether thrombin affects the production of nitric oxide-like factor(s) evoked by interleukin-1 beta (IL-1 beta) in cultured smooth muscle cells from the rat aorta. IL-1 beta stimulated the release of nitrite (a stable oxidation product of nitric oxide) from cultured smooth muscle cells. Thrombin inhibited in a concentration-dependent manner the release of nitrite caused by IL-1 beta. The inhibition was prevented by hirudin (a thrombin inhibitor) and required the presence of thrombin before or during the induction period. Under bioassay conditions, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector rat aortic rings without endothelium. The addition of IL-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated platelets inhibited their aggregation. Control untreated smooth muscle cells or cells treated with thrombin alone did not have such effects. The treatment of smooth muscle cells with IL-1 beta in combination with thrombin blunted both the relaxing activities of the perfusates under bioassay conditions and the inhibition of platelet aggregation. These observations indicate that thrombin inhibits the production of nitric oxide-like factor(s) evoked by the inducible nitric oxide synthase in cultured smooth muscle cells from rat aorta.

The inducible nitric oxide synthase is impaired by thrombin in vascular smooth muscle cells.

The present study investigated whether or not thrombin may affect the induction of nitric oxide (NO) synthase caused by interleukin-1 beta in cultured smooth muscle cells (SMCs) from the rat aorta. The release of nitrite, an oxidation product of NO, from interleukin-1 beta-activated SMCs was inhibited by thrombin. The inhibitory effect of thrombin was prevented by hirudin, a thrombin inhibitor, and required the presence of thrombin during the induction period. Under bioassay conditions, the perfusate from interleukin-1 beta-activated SMCs relaxed endothelium-denuded rat aortic rings precontracted with phenylephrine. The perfusate from untreated SMCs or cells exposed to thrombin alone or to interleukin-1 beta in combination with thrombin relaxed only minimally the detector blood vessel. These observations demonstrate that thrombin inhibits the production of NO by the inducible NO synthase in cultured vascular SMCs.

Platelet inhibition by an L-arginine-derived substance released by IL-1 beta-treated vascular smooth muscle cells.

Experiments were performed to examine whether stimulation of cultured vascular smooth muscle cells by interleukin (IL)-1 beta would induce platelet inhibitory properties of these cells. Incubation of platelets with untreated rat aortic smooth muscle cells had no effect on thrombin-induced platelet aggregation. In contrast, incubation of platelets with IL-1 beta-pretreated smooth muscle cells or the perfusate from such cells resulted in the inhibition of thrombin-induced platelet aggregation. This effect was potentiated by superoxide dismutase and reversed by incubating the IL-1 beta-treated smooth muscle cells with NG-nitro-L-arginine (L-NNA) or by treating the platelets with methylene blue. Cytokine-treated smooth muscle cells inhibited thrombin-stimulated changes in platelet cytosolic ionized calcium, whereas untreated cells were without effect. Incubating platelets with IL-1 beta-treated smooth muscle cells resulted in a 10-fold increase in platelet guanosine 3',5'-cyclic monophosphate (cGMP) levels, whereas untreated smooth muscle cells had no effect. The elevation of platelet cGMP induced by the IL-1 beta-treated smooth muscle cells was prevented by exposing the cytokine-treated cells to L-NNA or by treating platelets with methylene blue. Treatment of smooth muscle cells with IL-1 beta also resulted in an eightfold increase in nitrite production, which was blocked when the cells were incubated with L-NNA. The addition of cycloheximide to smooth muscle cells during their incubation with IL-1 beta completely inhibited smooth muscle cell nitrite production, the effects of the smooth muscle cells on platelet cGMP levels, and platelet responses to thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-density lipoprotein endocytosis: II. Influence of the multivalent ligand cationized ferritin on acetylated low-density lipoprotein endocytosis in cultured cells

Interactions of the basic multivalent ligand cationized ferritin (CF) with cultured cells markedly alter their endocytic function. In this study, the influence of CF treatment on the binding, internalization, and degradation of chemically modified (acetylated) low-density lipoproteins (Ac-LDL) was examined in aortic smooth muscle cells (SMC); and in normal and FH mutant LDL receptor-negative human skin fibroblasts, which lack the Ac-LDL (scavenger) receptor; and in vascular endothelial cells, which normally express the receptor. Although CF treatment of all three cell types at 37°C resulted in the induction of Pronasesensitive, high-capacity, high-affinity binding ( K d = 12.0 ± 2.0 n M at 4°C) of labeled Ac-LDL, which at 37°C was accompanied by significant internalization and degradation, these processes were not receptor-mediated. CF-induced high-affinity binding was inhibited by unlabeled Ac-LDL, fucoidan, carrageenan, and dextran sulfate but was unaffected by native LDL and albumin and only partially inhibited by acetylated albumin. However, analysis of membrane preparations of the cells for “scavenger” receptor protein by solid-phase filtration assay and Western blotting identified the receptor in endothelial cells and in granuloma (positive control) macrophages, but not in either CF-treated or untreated SMC. In addition, studies with both glutaraldehyde-fixed cells and CF bound to culture dishes indicated that Ac-LDL avidly binds to CF. Further, ultrastructural studies using colloidal gold-conjugated Ac-LDL showed Ac-LDL preferentially binding to CF aggregates on the cell surface. Thus, these studies indicate that treatment of cells with CF induces an endocytic process which, although remarkably similar to the scavenger pathway, is mediated by Ac-LDL binding to membrane-associated CF. These observations have implications in terms of mechanisms that might regulate the endocytosis of modified low-density lipoproteins.