Endogenously expressed apolipoprotein E has different effects on cell lipid metabolism as compared to exogenous apolipoprotein E carried on triglyceride-rich particles

Apolipoprotein E (apoE) on model triglyceride-rich particles (TGRP) increases triglyceride (TG) utilization and cholesteryl ester (CE) hydrolysis, independent of its effect on enhancing particle uptake. We questioned whether, under physiological concentrations, endogenously expressed apoE has similar effects on cellular lipid metabolism as compared to exogenous apoE. J774 macrophages, which do not express apoE, were engineered to express endogenous apoE by transfection of human apoE3 cDNA expression constructs (E(+)) or control vectors (E(-)) into the cells. To compare the effects of exogenous apoE and endogenous apoE on TGRP uptake, cells were incubated with or without apoE associated with (3)H-cholesteryl ether-labeled TGRP. Exogenous apoE enhanced TGRP uptake in both E(-) and E(+) cells. E(-) cells displayed significantly higher TGRP uptake than E(+) cells. Sodium chlorate, which inhibits cell proteoglycan synthesis, markedly diminished differences in TGRP uptake between E(-) and E(+) cells, suggesting that endogenous apoE-proteoglycan interaction contributes to differences in uptake between the two cell types. Particle uptake by the LDL receptor, by the LDL receptor related protein, or by scavenger receptors were similar between E(-) and E(+) cells indicating that endogenous apoE expression does not have a general effect on endocytic pathways. Exogenous apoE carried on TGRP stimulated TG utilization and CE hydrolysis in both cell types. However, TG utilization and CE hydrolysis were not affected by endogenous apoE expression. In conclusion, macrophage expression of apoE has very different effects on TGRP metabolism than exogenously supplied apoE. The fluorescence microscopy results in this study showing that exogenous apoE and endogenous apoE were confined in separate cellular compartments support the hypothesis that these differences resulted from distinct intracellular trafficking pathways followed by exogenous apoE bound to TGRP as compared to endogenous cell-expressed apoE.     

Adenoviral-mediated overexpression of catalase inhibits endothelial cell proliferation

Although hydrogen peroxide (H(2)O(2)) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding beta-galactosidase (Adbetagal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by beta-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adbetagal-transduced cells (536.8 +/- 31 vs. 1,875.1 +/- 132.9 vs. 1,347.5 +/- 93.7 dpm/well, respectively; P < 0.05 vs. control and Adbetagal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 +/- 1.8% of cell counts in control, P < 0.05; and 45 +/- 2% of cell count in Adbetagal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H(2)O(2) (50 microM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H(2)O(2) is an important modulator of endothelial cell proliferation.     

Apolipoprotein E: A potent inhibitor of endothelial and tumor cell proliferation

Recombinant human apolipoprotein E3 (apoE), purified from E. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose- and time-dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin-binding growth factors. Thus, at low serum concentrations (0–2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF-dependent proliferation was dramatically inhibited by apoE with an IC₅₀ ≈ 50 nM. Under conditions where cell proliferation was mainly serum-dependent, apoE also suppressed growth but required higher concentrations to be effective (IC₅₀ ≈ 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC₅₀ values obtained with these cells were generally 3–5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.     

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin.

The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4-II-E cells overexpressing RC stably. H4-II-E cells were transfected with RC/pCXN2 vector and the multiple neomycin-resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2-transfected cells used in this study was 19.7-fold as compared with that of the parental wild type H4-II-E cells. Wild type H4-II-E cells, pCXN2 vector-transfected cells (mock type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4-II-E cells was significantly suppressed in transfectants with culture for 12-48 h. The presence of anti-RC monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti-RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 microM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4-II-E overexpressing RC stably.     

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line.

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.     

Estrogen-dependent DNA synthesis and parenchymal cell proliferation in the liver of adult male Xenopus frogs

Estradiol-17 beta treatment of adult male Xenopus laevis induces liver parenchymal cells to synthesize DNA and proliferate. DNA synthesis begins 3 to 4 days after estrogen treatment and continues for approximately 10 days. Over this 2-week period, the total number of liver parenchymal cells increases fourfold, the wet weight of the liver remains constant, and there is a 50% reduction in cell volume. The elevated number of cells persists for several months and then returns to the control value. The extent of proliferation is hormone dose dependent. Pulse-chase experiments demonstrate that as a result of hormone treatment a minority of the parenchymal cells in the initial population enter the cell cycle, and via repeated divisions become the majority (79%) of the population by Day 14. The implications of this phenomenon for estrogen-induced liver cell differentiation and vitellogenin gene function are discussed.     

Exogenous HSP70 becomes cell associated,but not internalized,by stressed arterial smooth muscle cells

Summary Cell death within atherosclerotic plaques leads to necrosis and rupture, resulting in vascular occlusion. We have previously demonstrated that addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSMCs or acts from outside cells to preserve viability, cultured rabbit aSMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or¹²⁵I-radiolabeled HSP70. Cell-associated HSP70 was analyzed using Western blotting, fluoresence spectroscopy, and gamma counting/autoradiogarphy. Surface binding of HSP70 to aSMCs was differentiated from uptake by using trypsin treatment to degrade non-internalized HSP70. Specificity of HSP70 binding was tested by inhibiting uptake of¹²⁵I-HSP70 with excess unlabeled HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogenous HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels present in unstressed aSMCs. However, <5% of the exogenous HSP70 was trypsin-insensitive, indicating that bound HSP70 was not internalized. Binding of¹²⁵I-HSP70 was inhibited by both unlabeled HSP70 and BSA, implying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSMCs, but it did not specifically inhibit synthesis of endogenous HSPs after heat shock. The results indicate that exogenous HSP70 protects viability of stressed aSMCs through interactions with the cell surface rather than via internalization.     

Inhibition of cell proliferation by apolipoprotein E isoform expression

The anti-atherogenic effects of human apolipoprotein E3 (apoE3) have been partially attributed to its anti-proliferation properties. We studied if endogenously expressed apoE elicits isoform-dependent effects on cell proliferation. Rat F111 fibroblasts without native expression of apoE were used to establish cell lines with stable expression of the three human apoE isoforms. Cell growth curve studies showed that expression of apoE isoforms prolonged cell population doubling time in an isoform-dependent manner with apoE3 showing the most potent effect followed by apoE2 and apoE4 exhibiting comparable effects. Interestingly, saturation density of cell population was significantly reduced by the expression of apoE4 isofom. Further analyses revealed that all three apoE isoforms significantly lengthened G0/G1 phase (p < 0.05) of the cell cycle and were associated with the suppression of ERK1/2 activities. However, these changes were not sufficient to explain the isoform-dependent effects of apoE expression on cell population doubling time and saturation density.     

Gangliosides stimulate astroglial cell proliferation in the absence of serum

Previous studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 X 10(-5)M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12-24 hr. The ganglioside-induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10(-5)M--while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.     

Exogenous GM1 ganglioside caused G1-arrest of human diploid fibroblasts. Flow cytometric studies

Exogenous GM1 ganglioside (II3 NeuAc-Gg0se4-Cer) inhibited growth and DNA synthesis of human diploid fibroblasts, TIG-1 cells. We examined the effect of exogenous GM1 on their cell cycle traverse by flow cytometry. When the cells were partially synchronized by serum deprivation, addition of GM1 at the time of refeeding caused about 70% reduction of their reentry into S phase from the level observed in the control culture untreated with the ganglioside. However, the addition of GM1 6 h later caused only about 30% reduction of the reentry from the control level. These results suggest that the exogenous ganglioside blocks the cell cycle traverse in an early G1 period. This is consistent with the fact that GM1-treated cells showed a high level of histone H1(0) similar to that observed in G1-arrested cells in confluent culture.     

Negative regulation of cell proliferation by mevalonate or one of the mevalonate phosphates

The role of mevalonate and its products in the regulation of cellular proliferation was examined using 6-fluoromevalonate (Fmev), a compound that blocks the conversion of mevalonate pyrophosphate to isopentenyl pyrophosphate. Fmev suppressed DNA synthesis by a variety of transformed and malignant T cell, B cell, and myeloid cell lines. In contrast to results previously reported with mitogen-stimulated human peripheral blood T cell DNA synthesis, low concentrations of low density lipoprotein (LDL) alone could not restore proliferation to these cell lines. The same concentrations of LDL were able to provide sufficient cholesterol and support the growth of all cell lines when mevalonate synthesis was blocked with a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin. Fmev-mediated inhibition was totally prevented in some but not all cell lines when the concentration of exogenous LDL was increased 5-10-fold above that required to permit proliferation of lovastatin-blocked cells. Residual HMG-CoA reductase activity of cells cultured with LDL inversely correlated with the restoration of growth to Fmev-blocked cultures. Confirmation of the critical role of HMG-CoA reductase activity and mevalonate synthesis in the inhibition of cellular proliferation by Fmev was obtained by demonstrating that the specific inhibitor of this enzyme, lovastatin, restored proliferation of Fmev-blocked cells. Furthermore, supplementation of cultures with mevalonate, the product of HMG-CoA reductase activity, markedly inhibited proliferation of Fmev-blocked cells. These findings indicate that mevalonate or one of the mevalonate phosphates, which accumulates in Fmev-blocked cells, is a critical negative regulator of cellular proliferation.     

Cell density downregulates DNA synthesis and proliferation during osteogenesis in vitro

Abstract. The classical models of in vitro cell culture comprise fibroblasts and epithelial cells. Osteogenic cells represent another interesting cell model; however, it is not known whether during osteogenesis cell density regulates cell growth as seen in cultures of fibroblasts and epithelial cells. We selected MC3T3-E1 cells for study because they are an osteogenic cell line that, when subcultured, grow to confluence and form multilayers of cells in conventional cultures by continued proliferation, as do fibroblasts. Once maximum cell density is obtained, proliferation is down regulated resulting in a mixed population of quiescent and dividing cells. We used this model to determine whether downregulation of proliferation as expressed by cell number and DNA synthesis is cell density-dependent. MC3T3-E1 cells were cultured over a period of 34 days to determine their kinetics, viability, ability to synthesize DNA, distribution within phases of the cell cycle and cell number-response relationships. Our results show that (1) viability ranged between 92% and 96% and the cell number 2.5 x 10⁵ per cm² once cultures reached steady state, (2) most cells entered the G₀/G₁ phase of the cell cycle on day 7, (3) there was no correlation between the proportion of cells in S phase and downregulation of DNA synthesis, (4) a direct relationship exists between cell density and downregulation of DNA synthesis on day 8, (5) the minimum time for cells to be cultured before downregulation of DNA synthesis begins is independent of cell number, and (6) downregulation of DNA synthesis is reversible. These results suggest that density-dependent downregulation of DNA synthesis may be a mechanism of growth control for osteogenic cells in vitro that operates more like density-dependent growth control in cultures of fibroblasts rather than epithelial cells.     

CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation

We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.     

Inhibition of rat fibroblast cell proliferation at specific cell cycle stages by cocaine

We have analyzed the role of cocaine in the control of the rat fibroblast (EL2) cell proliferation. Our data show a dose-related effect on the inhibition of DNA synthesis and cell growth when cocaine is added with serum or with a pure growth factor [Epidermal Growth Factor (EGF)]. Pretreatment by drug did not appreciably enhance the inhibition of S-phase entry above that obtained when cocaine and mitogen were added simultaneously. On the contrary, exposure of quiescent EL2 cells to cocaine has little or no effect on DNA synthesis, when drug is removed before the mitogenic stimulus. Moreover, even when cocaine is added after EGF, an exposure only within 1–8 hours is required in order to inhibit stimulation of DNA synthesis. Cocaine also suppressed the general increase in protein synthesis that occurs during the first hour after EGF addition. The combined data suggest that cocaine inhibits the traverse of mitogen-stimulated quiescent EL2 cells from Go to S phase by acting on processes that take place during the initial phase of the cell cycle.     

Cleavage of apolipoprotein E by membrane-type matrix metalloproteinase-1 abrogates suppression of cell proliferation

Apolipoprotein E (apoE) in a human fetal brain cDNA library was identified, using the expression cloning method, as a gene product that formed a complex with latent matrix metalloproteinase (MMP)-2. Co-expression of membrane-type MMP-1 (MT1-MMP) with apoE in HEK293T cells reduced the amount of apoE secreted into the culture medium, whereas cell-associated apoE core protein was not affected. Incubation of native apoE protein with recombinant MT1-MMP resulted in the cleavage of apoE. Recombinant apoE protein fused to glutathione S-transferase (apoE-GST) was cleaved by MT1-MMP at the following peptide bonds; T(85)-M(86), K(93)-S(94), R(246)-L(247), A(255)-E(256) and G(296)-L(297). HT1080 cells transfected with the apoE gene, which express endogenous MT1-MMP, secreted a low level of apoE protein and its cleaved fragments, and treatment with MMP inhibitor BB94 induced accumulation of apoE and retardation of cell proliferation. Addition of apoE-GST protein to the culture of HEK293T cells suppressed cell proliferation, and stable transfection of the MT1-MMP gene partly abrogated the suppression. These results suggest that cleavage of apoE protein by MT1-MMP abrogates apoE-mediated suppression of cell proliferation.     

Exogenous,basal, and flow-induced nitric oxide production and endothelial cell proliferation

The role of nitric oxide (NO) from endogenous and exogenous sources in regulating large vessel and microvascular endothelial cell proliferation was investigated. Exogenous NO liberated from five different chemical donors inhibited bovine aortic, bovine retinal microvascular, and human umbilical vein endothelial cell proliferation in a dose-dependent manner as determined by ³H-thymidine incorporation. The potency of the donors varied as a function of the donors' half-lives. Donors with half-lives greater than 30 min were more effective than donors with significantly shorter half-lives. Coincubation of endothelial cells with 0.4 mM deoxyadenosine and 0.4 mM deoxyguanosine reduced the percentage of inhibition due to an NO donor. These data are consistent with a ribonucleotide reductase-dependent mechanism of inhibition. Inhibition of basal NO production with four different inhibitors of nitric oxide synthase (NOS) did not modify proliferation. Laminar flow with a wall shear stress of 22 dyn/cm²inhibited the proliferation of subconfluent bovine aortic endothelial cells. The addition of a NOS inhibitor did not abrogate the flow-induced inhibition of proliferation, suggesting that flow-stimulated release of NO from endothelial cells did not account for flow-induced inhibition of proliferation. Taken together, these data suggest that relatively large concentrations of exogenous NO inhibit endothelial cell proliferation, while endogenous levels of NO are inadequate to inhibit proliferation. J. Cell. Physiol. 171:252–258, 1997. © 1997 Wiley-Liss, Inc.     

IL-7 administration alters the CD4:CD8 ratio, increases T cell numbers, and increases T cell function in the absence of activation

IL-7 is vital for the development of the immune system and profoundly enhances the function of mature T cells. Chronic administration of IL-7 to mice markedly increases T cell numbers, especially CD8(+) T cells, and enhances T cell functional potential. However, the mechanism by which these effects occur remains unclear. This report demonstrates that only 2 days of IL-7 treatment is needed for maximal enhancement of T cell function, as measured by proliferation, with a 6- to 12-fold increase in the proportion of CD4(+) and CD8(+) T cells in cell cycle by 18 h of ex vivo stimulation. Moreover, a 2-day administration of IL-7 in vivo increases basal proliferation by 4- and 14-fold in CD4(+) and CD8(+) T cells, respectively. These effects occur in the absence of cytokine production, increases in most activation markers, and changes in memory markers. This enhanced basal proliferation is the basis for the increase in T cell numbers in that IL-7 induces an additional 60% and 85% of resting CD4(+) and CD8(+) T cells, respectively, to enter cell cycle in mice given IL-7 for 7 days. These results demonstrate that in vivo administration of IL-7 increases T cell numbers and functional potential via a homeostatic, nonactivating process. These findings may suggest a unique clinical niche for IL-7 in that IL-7 therapy may increase T cell numbers and enhance responses to specific antigenic targets while avoiding a general, nonspecific activation of the T cell population.     

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.