This Article Abstract Full Text (PDF) All Versions of this Article: 91/7/570    most recent 01.RES.0000036607.05037.DAv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parhami, F. Articles by Demer, L. L. Search for Related Content PubMed PubMed Citation Articles by Parhami, F. Articles by Demer, L. L. Related Collections Pathophysiology Cell signalling/signal transduction Growth factors/cytokines Mechanism of atherosclerosis/growth factors

(Circulation Research. 2002;91:570.)
© 2002 American Heart Association, Inc.

Molecular Medicine

High-Density Lipoprotein Regulates Calcification of Vascular Cells

Farhad Parhami, Benjamin Basseri, Jason Hwang, Yin Tintut, Linda L. Demer

From the Departments of Medicine (F.P., B.B., J.H., Y.T., L.L.D.) and Physiology (L.L.D.), University of California, Los Angeles.

Correspondence to Farhad Parhami, PhD, Associate Professor of Medicine, UCLA Division of Cardiology, CHS 47-123, 10833 Le Conte Ave, Los Angeles, CA 90095. E-mail fparhami{at}mednet.ucla.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Accumulating evidence has suggested the protective role of HDL in cardiovascular disease processes. Calcification is a common feature of atherosclerotic lesions and contributes to cardiovascular complications due to the loss of aortic resilience and function. Recent studies have suggested that vascular calcification shares several features with skeletal bone formation at the cellular and molecular levels. These include the presence of osteoblast-like calcifying vascular cells in the artery wall that undergo osteoblastic differentiation and calcification in vitro. We hypothesized that HDL may also protect against vascular calcification by regulating the osteogenic activity of these calcifying vascular cells. When treated with HDL, alkaline phosphatase activity, a marker of osteogenic differentiation of osteoblastic cells, was significantly reduced in those cells. Prolonged treatment with HDL also inhibited calcification of these cells, further supporting the antiosteogenic differentiation property of HDL when applied to vascular cells. Furthermore, HDL inhibited the osteogenic activity that was induced by inflammatory cytokines interleukin (IL)-1ß and IL-6 as well as by minimally oxidized LDL. HDL also partially inhibited the IL-6-induced activation of signal transducer and activator of transcription 3 in calcifying vascular cells, suggesting that HDL may inhibit cytokine-induced signal transduction pathways. The inhibitory effects of HDL were mimicked by lipids extracted from HDL but not by HDL-associated apolipoproteins or reconstituted HDL. Furthermore, oxidation of HDL rendered it pro-osteogenic. Taken together, these results suggest that HDL regulates the osteoblastic differentiation and calcification of vascular cells and that vascular calcification may be another target of HDL action in the artery wall.

Key Words: vascular calcification • calcifying vascular cells • high-density lipoproteins • cytokines

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular calcification is a hallmark of atherosclerosis and has been linked to increased cardiovascular disease and mortality.^1–4 Atherosclerotic calcification contains numerous factors associated with bone mineralization, including matrix vesicles, the bone-liver-kidney isozyme of alkaline phosphatase, bone morphogenetic protein-2, osteopontin, osteocalcin, and collagen I.⁵ In addition, our laboratory and others have isolated osteoblast-like cells from the artery wall. These calcifying vascular cells (CVCs) spontaneously calcify in vitro and express osteoblastic differentiation markers such as alkaline phosphatase, osteopontin, osteocalcin, osteonectin, and collagen I.⁶ We have previously found that osteoblastic differentiation and mineralization of these cells are enhanced by inflammatory factors such as minimally oxidized LDL (MM-LDL), tumor necrosis factor (TNF)-, and leptin.^7–9 Therefore, CVCs and factors that regulate their behavior may also regulate vascular calcification in vivo.

HDL plasma levels are inversely correlated with coronary artery disease.^10–12 However, the mechanisms by which HDL exerts its protective effects are diverse. HDL is responsible for reverse cholesterol transport, ie, the removal of free cholesterol from blood vessels to the liver.^13,14 It also possesses antioxidant and antiinflammatory properties.¹⁵ Several studies have demonstrated the ability of HDL to inhibit cytokine-induced responses in endothelial cells.^16,17 Furthermore, studies by Navab et al¹⁸ and Parthasarathy et al¹⁹ have shown that HDL inhibits LDL oxidation and production of monocyte chemotactic proteins in an in vitro coculture model of artery wall cells. The antiinflammatory effects of HDL appear to be mediated in part by associated proteins, such as apolipoprotein (apo)A-I, which has lipid-binding properties, and the enzymes platelet factor acetyl hydrolase and serum paraoxonase (PON-1), which can eliminate the bioactivity of oxidized lipids.^15,20

However, HDL can be modified to lose its protective abilities and to become proinflammatory.²⁰ During the acute-phase response, a systematic reaction to tissue injury, HDL is converted to a proinflammatory molecule.^21,22 In addition, overexpression of apoA-II in mice elicits a reduction in PON-1 levels and converts HDL to a proinflammatory molecule.²³ HDL is susceptible to oxidation, and oxidized HDL has proinflammatory characteristics²⁴ and has been localized to atherosclerotic plaques.²⁵

Proinflammatory cytokines contribute to early atherogenesis.^26–28 Interleukin (IL)-1, one of the most potent proinflammatory cytokines, acts on endothelial cells and smooth muscle cells and is involved in atherogenesis.²⁹ Recently, Subbanagounder et al³⁰ have demonstrated the accumulation of atherogenic oxidized phospholipids in cultures of human aortic endothelial cells treated with IL-1ß. IL-6 is another proinflammatory cytokine expressed in atherosclerotic plaques, and it mediates at least some of the inflammatory effects of atherogenic oxidized phospholipids.^31,32 Reports suggest that the protective effects of HDL may encompass an ability to inhibit cytokine-induced inflammatory responses, such as the induction of cell adhesion molecule expression by IL-1ß.³³

In the present study, we present the first evidence that HDL may also play an important role in regulating vascular calcification associated with atherosclerotic lesions. We demonstrate that human HDL inhibits the spontaneous osteogenic differentiation and mineralization of CVCs in vitro. In addition, HDL inhibits the osteogenic differentiation of CVCs induced by MM-LDL, IL-1ß, and IL-6. Furthermore, we found that minimally oxidized HDL (MM-HDL) enhances the osteogenic behavior of CVCs. Altogether, these results suggest that HDL may regulate vascular calcification by directly inhibiting the osteogenic differentiation of vascular cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
CVC clones were isolated from cultures of bovine smooth muscle cells harvested from bovine aortic medial explants and cultured as previously described.⁶ Recombinant human IL-1ß was obtained from R&D Systems, Inc, and recombinant human IL-6 and recombinant human interferon- were obtained from Biosource International. Human apoA-I, apoA-II, and apoE were from Sigma Chemical Co, purified human PON-1 was a generous gift from Dr Bert La Du (University of Michigan Medical School, Ann Arbor), and reconstituted HDL was a generous gift from Dr Geeta Datta (University of Alabama at Birmingham). HDL pretreatment of CVCs was performed in DMEM containing 5% FBS (Hyclone) for 24 hours, followed by the addition of fresh HDL and media and the addition of cytokines or MM-LDL.

Lipoprotein Preparation and Oxidation
Human HDL and LDL were isolated by density-gradient centrifugation of serum and stored in phosphate-buffered 0.15 mol/L NaCl containing 0.01% EDTA as previously described. MM-LDL was prepared by iron oxidation of human LDL as previously described.⁷ The concentrations of lipoproteins used in the present study are reported in micrograms of protein. HDL oxidation was also performed by following the same protocol that was used for the preparation of MM-LDL. Total lipid extracts of HDL were prepared by chloroform/methanol extraction in the presence of antioxidant butylated hydroxytoluene (0.01%). Extracted lipids were dried under argon gas and resuspended in DMEM containing 5% FBS for application to cells. All lipoproteins were tested before and after oxidation for lipopolysaccharide levels and found to have <30 pg lipopolysaccharide/mL medium.

Alkaline Phosphatase Activity Assay
A cell-associated alkaline phosphatase activity assay was performed as previously described.⁷

⁴⁵Ca Incorporation
⁴⁵Ca incorporation into a matrix of CVC cultures was performed as previously described.⁷

Western Blot Analysis
At the end of treatments, CVC cultures were rinsed twice with PBS and lysed in cold lysis buffer consisting of 50 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 µmol/L Na₃VO₄, 0.1% Triton X-100, and 10 mmol/L HEPES, pH 7.4, along with protease inhibitor cocktail (Calbiochem). The lysate was then sonicated for 10 seconds and microcentrifuged, and the supernatant was used for Western blotting. Lysate (20 µg) was electrophoresed on 8% Tris-glycine gels (Novex). This was followed by transfer to nitrocellulose membranes, as previously described.³⁴ The blots were probed with either antibody to native or phosphorylated signal transducer and activator of transcription 3 (STAT3, Santa Cruz Biotechnology) according to the manufacturer’s instructions. Detection of immunoreactive proteins was performed by chemiluminescence (Amersham), and signals were quantified by densitometric analysis.

Statistical Analysis
Data are presented as mean±SD and were analyzed by using the Statview 4.5 program. All probability values were calculated by ANOVA and the Fisher protected least significant difference test. A value of P<0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
HDL Inhibits Spontaneous Osteogenic Activity of CVCs
CVCs undergo an in vitro differentiation process similar to that of preosteoblasts.⁶ This process involves nodule formation and the sequential upregulation of several osteoblast-specific genes, such as osteocalcin and Cbfa-1.³⁴ Alkaline phosphatase, an early marker of osteoblastic differentiation, provides phosphate from organic sources for calcium phosphate mineral formation.³⁵ HDL treatment caused a dose-dependent inhibition of alkaline phosphatase activity in CVCs at 4 days (Figure 1A). Inhibition was also achieved even when HDL was present for only the first 24 hours of incubation, although to a lesser extent than when HDL was continuously present during the 4 days of incubation (Figure 1B). Furthermore, continuous incubation with HDL (200 µg/mL) also dramatically inhibited spontaneous calcification of CVC cultures, as shown by a ⁴⁵Ca incorporation assay (Figure 1C). However, HDL did not inhibit nodule formation in the CVC cultures (data not shown). These results suggest that HDL inhibits the spontaneous osteoblastic differentiation of CVCs.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of HDL on alkaline phosphatase activity and calcification in CVC cultures. A, CVCs were incubated for 4 days with HDL at the indicated concentrations in DMEM containing 5% FBS. Alkaline phosphatase activity was measured in total cell homogenates, and results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.005 for all HDL-treated vs control (0) samples. B, CVCs were incubated for the first 24 hours only (HDL-Pre) or continuously (HDL) for 4 days with HDL (200 µg/mL) in DMEM containing 5% FBS. At the end of the 4-day incubation period, alkaline phosphatase activity was determined in total cell homogenates, and the results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.05 for HDL-Pre and HDL-treated samples vs untreated control sample. C, CVCs were treated for 10 days with HDL (200 µg/mL) as described above, and then a 45Ca incorporation assay was performed. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.01 for HDL-treated vs untreated control samples.

HDL Inhibits Cytokine-Induced and MM-LDL-Induced Osteogenic Activity of CVCs
We previously found that CVC differentiation and mineralization are enhanced by several inflammatory agents such as TNF- and oxidized lipids and lipoproteins.^7,8 More recently, we have found that CVC differentiation is also dose-dependently enhanced by IL-1ß and IL-6. Treatment of CVCs with IL-1ß or IL-6 for 2 or 4 days, respectively, caused a dose-dependent increase in alkaline phosphatase activity (Figures 2A and 2B). In contrast, treatment of CVCs with interferon- (1 to 10 ng/mL) for 4 days did not significantly affect alkaline phosphatase activity (data not shown). Mineralization was also enhanced in CVC cultures treated for 10 days with IL-1ß or IL-6 (data not shown). Twenty-four-hour pretreatment with HDL (200 µg/mL) significantly inhibited alkaline phosphatase and calcification induced by IL-1ß (Figures 3A and 3B) and IL-6 (Figures 4A and 4B). Furthermore, the previously reported enhancement of alkaline phosphatase activity by the atherogenic lipoprotein MM-LDL was also blocked in the presence of HDL (Figure 5). Altogether, these results demonstrate that HDL can prevent the pro-osteogenic differentiation effects of several inflammatory agents on CVCs.

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of IL-1ß and IL-6 on alkaline phosphatase activity in CVC cultures. CVCs were incubated with IL-1ß (A) or IL-6 (B) for 2 or 4 days, respectively, at the indicated concentrations in DMEM containing 5% FBS. Alkaline phosphatase activity was measured in total cell homogenates, and results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.001 for all IL-1ß- and IL-6-treated samples vs control untreated (0) samples.

View larger version (24K): [in this window] [in a new window]   Figure 3. Effect of HDL on CVC responses to IL-1ß (IL-1). A, CVC cultures were pretreated for 24 hours with HDL (200 µg/mL) in DMEM containing 5% FBS, followed by the addition of fresh HDL with or without IL-1 (10 ng/mL). After 2 days, alkaline phosphatase activity was measured as previously described. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.005 for untreated control vs IL-1-treated samples, for IL-1- vs IL-1+HDL-treated samples, and for untreated control vs HDL-treated samples. B, Cells were treated as described above. After 10 days, a 45Ca incorporation assay was performed. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.005 for untreated control vs IL-1-treated samples and for IL-1- vs IL-1+HDL-treated samples.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of HDL on CVC responses to IL-6. A, CVC cultures were pretreated for 24 hours with HDL (200 µg/mL) in DMEM containing 5% FBS, followed by the addition of fresh HDL with or without IL-6 (50 ng/mL). After 4 days, alkaline phosphatase activity was measured as previously described. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.001 for control vs IL-6-treated samples and for IL-6- vs IL-6+HDL-treated samples; P<0.05 for control vs HDL-treated samples. B, Cells were treated as described above. After 10 days, a 45Ca incorporation assay was performed. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.001 for control vs IL-6-treated samples and for IL-6- vs IL-6+HDL-treated samples.

View larger version (39K): [in this window] [in a new window]   Figure 5. Effect of HDL on CVC response to MM-LDL. CVC cultures were pretreated for 24 hours with HDL (200 µg/mL) in DMEM containing 5% FBS, followed by the addition of HDL (50 µg/mL) with or without MM-LDL (200 µg/mL) or control buffer. After 4 days, alkaline phosphatase activity was measured as previously described. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.001 for control vs MM-LDL-treated samples and for MM-LDL- vs MM-LDL+HDL-treated samples; P<0.005 for control vs HDL-treated samples.

Mechanism of HDL Effects on CVCs
Several different mechanisms have been proposed for the antiinflammatory effects of HDL in different experimental systems.¹⁵ We found that spontaneous differentiation and calcification of CVCs, which correspond to the growth stages of preconfluence, confluence, condensing, and calcification,³⁴ were associated with increased levels of STAT3 and its activated phosphorylated form, pSTAT3 (Figure 6A). Furthermore, the effects of IL-6 on CVC differentiation were associated with STAT3 activation. Treatment of CVCs for 15 minutes with IL-6 (50 ng/mL) caused a significant increase in the levels of pSTAT3 in those cells (Figure 6B). Pretreatment of CVCs for 24 hours with HDL (200 µg/mL) caused a 50% inhibition of IL-6-induced STAT3 activation (Figure 6B). This suggests that inhibition of cytokine-induced signaling in CVCs may be at least in part responsible for the inhibitory effects of HDL.

View larger version (26K): [in this window] [in a new window]   Figure 6. STAT3 and pSTAT3 levels in CVCs. A, CVC cultures were grown in DMEM containing 15% FBS to allow for rapid growth conditions. Cells were lysed and analyzed by Western blotting for levels of pSTAT3 or native STAT3, as described in the Materials and Methods section, at the following stages of growth: preconfluence (Pre), confluence (Con), condensing (Cond), and calcification (Calc). A representative of 2 experiments is shown, with each lane corresponding to a separate sample. B, CVC cultures were pretreated for 24 hours with HDL (200 µg/mL) in DMEM containing 0.1% BSA, followed by the addition of IL-6 (50 ng/mL). After 15 minutes, cells were lysed and analyzed by Western blotting for levels of pSTAT3 and STAT3. Bar graph shows densitometric analysis of the scanned autoradiograph normalized to STAT3 levels, expressed as the mean of duplicate samples.

To determine which component(s) of HDL exerts its inhibitory effects on CVC differentiation, we examined the effects of lipids extracted from HDL. Results showed that total lipids extracted from HDL had effects similar to those of intact HDL, inhibiting both spontaneous and IL-1ß-induced alkaline phosphatase activity in CVCs (Figure 7). Furthermore, reconstituted HDL and purified apoA-I, apoA-II, apoE, and PON-1 did not have effects similar to those of HDL. Treatment of CVCs with up to 200 µg/mL reconstituted HDL, 100 µg/mL purified apoA-I or apoA-II, 10 µg/mL apoE, or 4 µg/mL purified PON-1 did not inhibit the spontaneously increased alkaline phosphatase activity in those cells (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 7. Effect of HDL and HDL lipids on CVC responses to IL-1ß (IL-1). CVC cultures were pretreated for 24 hours with HDL (200 µg/mL) or total lipids extracted from HDL (Lip, 200 µg/mL) in DMEM containing 5% FBS, followed by the addition of fresh HDL or fresh HDL lipids with or without IL-1 (10 ng/mL). After 2 days, alkaline phosphatase activity was measured as previously described. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.005 for untreated control vs IL-1-treated samples and for IL-1- vs IL-1+HDL- and IL-1+Lip-treated samples.

Minimally Oxidized HDL Enhances CVC Osteogenic Activity
HDL can lose its protective capacity after modification by oxidation or after an acute-phase response.^21,24 To examine the effect of oxidation on the antiosteogenic effects of HDL, HDL was oxidized by dialysis against iron by the same approach that was used for minimal oxidation of LDL to form MM-LDL. We found that oxidized HDL was no longer antiosteogenic and was able to induce alkaline phosphatase activity (Figure 8A) and calcification (Figure 8B) in CVC cultures after 4 and 10 days of treatment, respectively.

View larger version (30K): [in this window] [in a new window]   Figure 8. Effect of oxidized HDL on CVCs. A, CVC cultures were treated with control buffer (0) or MM-HDL at the indicated concentrations in DMEM containing 5% FBS. After 4 days, alkaline phosphatase activity was measured as previously described. Results from a representative of 3 experiments are shown as mean±SD of quadruplicate determinations. P<0.001 for all MM-HDL- vs control buffer-treated samples. B, CVC cultures were treated with control buffer or MM-HDL (200 µg/mL) as described above. After 10 days, a 45Ca incorporation assay was performed. Results from a representative of 2 experiments are shown as mean±SD of quadruplicate determinations. P<0.05 for MM-HDL- vs control buffer-treated samples.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present report suggests that HDL regulates calcification of vascular cells. CVCs treated with HDL had significantly lower alkaline phosphatase activity, an established marker of osteoblastic differentiation in osteoprogenitor cells, including CVCs, suggesting a block in the spontaneous osteoblastic differentiation process as these cells mature into osteoblast-like cells. HDL also blocked matrix calcification in CVC cultures, the late marker of differentiation, suggesting that HDL regulates early and late events in osteogenic differentiation. The inhibition by HDL did not affect nodule formation. In previous studies, we demonstrated that calcification and differentiation of CVCs appear to be under regulatory mechanisms different from those of nodule formation. This was evidenced by the decrease in nodule formation in face of increased differentiation and calcification when CVCs were treated with prodifferentiation agents such as dibutyryl cAMP or TNF-.^8,34 Furthermore, HDL blocked the calcification induced by inflammatory cytokines IL-1ß and IL-6 as well as that induced by MM-LDL. In contrast, after oxidation, HDL not only lost its antiosteogenic effect but also induced calcification of CVCs. Altogether, these results demonstrate the ability of HDL to negatively regulate the calcification of vascular cells and may be an important determinant of vascular calcification in vivo. Interestingly, our data demonstrate that lipid components of HDL are responsible for its inhibitory effects on CVCs, whereas reconstituted HDL and purified apoA-I, apoA-II, apoE, and PON-1 did not have similar inhibitory effects on CVCs. Previous reports by other investigators have also demonstrated potentially antiatherogenic biological activity of HDL-associated lipids, including inhibitory effects of lysosphingolipids on endothelial cell apoptosis³⁶ and inhibitory effects of specific phospholipids on endothelial cell adhesion molecule expression.³⁷ These data suggest that HDL contains important lipids as well as proteins that may regulate cellular responses to inflammatory factors.

The ability of HDL to protect against vascular disease has been the subject of intense research. Epidemiological data suggest a protective effect of HDL against atherosclerotic disease. As a result, HDL-based intervention is considered a potential strategy against cardiovascular disease in humans. HDL may act by mediating reverse cholesterol transport and thereby preventing accumulation and oxidation of lipids and lipoproteins in the artery wall. Enzymes associated with HDL, platelet-activating factor acetyl hydrolase, and PON are able to directly act on oxidized phospholipids and lipoproteins and, hence, destroy their proinflammatory activity. HDL may also exert its antiinflammatory effects by impairing the response of vascular cells to inflammatory cytokines. Cockerill et al¹⁶ have shown that HDL inhibits IL-1ß- or TNF--induced expression of endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. HDL may also facilitate the production of protective molecules such as NO by activating endothelial NO synthase and by preventing the inhibitory effects of oxidized LDL on that enzyme.^38,39 Our data also suggest that HDL may prevent calcification of vascular cells and, hence, prevent calcification-induced vascular complications that would impair the proper functioning of the vessel wall.

The finding that inflammatory cytokines such as IL-1ß, IL-6, and TNF- induce osteogenic differentiation and mineralization of vascular cells suggests that inflammatory cytokines initiate or promote vascular calcification associated with atherosclerotic lesions by regulating the differentiation of CVCs. Hence, strategies that downregulate inflammation or upregulate antiinflammatory agents may prove beneficial in controlling vascular calcification.

One mechanism by which HDL may inhibit cytokine-induced osteogenic activity of vascular cells is interference with osteogenic signal transduction pathways. In the present studies, HDL partially inhibited STAT3 activation by IL-6. STAT3 and pSTAT3 levels increased in association with the differentiation and calcification of CVCs, suggesting that this signaling pathway may be involved in their osteogenic differentiation. The ability of HDL to interfere with other signaling molecules has been demonstrated. Xia et al¹⁷ reported that HDL interrupts the sphingosine kinase signaling pathway that regulates TNF-induced responses in endothelial cells. Similarly, Nofer et al⁴⁰ showed that HDL inhibits thrombin-induced platelet aggregation and fibrinogen binding by decreasing the production of phosphoinositide-derived second messengers. Therefore, HDL may exert its multitude of antiinflammatory effects by blocking cellular responses to inflammatory agents. Such widespread effects of HDL would likely require more than one downstream effector. One mechanism would be a generalized change in plasma membrane characteristics affecting structure and function of multiple membrane bound signaling receptors and effectors. Evidence for this concept comes from studies showing that HDL affects the structure and function of caveolae, which are important in facilitating and integrating a multitude of cellular responses and activities.⁴¹

The present results also support the notion that HDL is itself susceptible to conversion from an antiinflammatory to a proinflammatory state. Van Lenten et al²¹ showed that HDL obtained during an acute-phase response lacks antiinflammatory properties and promotes endothelial-monocyte interactions in an in vitro assay. Similarly, Sharma et al²⁴ reported that oxidized HDL induces proinflammatory effects on monocytes, including an increase in their binding to aortic endothelial cells. In the present study, oxidized HDL was no longer antiosteogenic and was able to induce alkaline phosphatase activity and calcification in cultures of CVCs.

In conclusion, the present study demonstrates the ability of HDL to inhibit the calcification of vascular cells. This effect appears to occur at least in part through blocking the effects of inflammatory factors such as cytokines and oxidized lipids. Future studies should further evaluate the effects of HDL on the complete inflammatory spectrum that regulates vascular calcification associated with atherosclerosis.

	Acknowledgments

 
This study was supported by NIH grant HL-30568 and the Laubisch and Sam Nassi Funds. We thank Drs Bert La Du and Dragomir Draganov for providing PON-1, Dr Geeta Datta for providing reconstituted HDL, Dr Mohamad Navab for insightful discussions, the UCLA Atherosclerosis Research Unit Core Laboratory for providing lipoproteins, and the UCLA Biomedical Technology Research and Instructional Production Facility for assistance with graphics.

Received March 28, 2002; revision received August 27, 2002; accepted August 28, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Wayhs R, Zelinger A, Raggi P. High coronary artery calcium scores pose an extremely elevated risk for hard events. J Am Coll Cardiol. 2002; 39: 225–230.[Abstract/Free Full Text]

2. Wallin R, Nadeem W, Greenwood GT, Sane DC. Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy. Med Res Rev. 2001; 21: 274–301.[CrossRef][Medline] [Order article via Infotrieve]

3. Tintut Y, Demer LL. Recent advances in multifactorial regulation of vascular calcification. Curr Opin Lipidol. 2001; 12: 555–560.[CrossRef][Medline] [Order article via Infotrieve]

4. Stanford W, Thompson BH, Weiss RM. Coronary artery calcification: clinical significance and current methods of detection. Am J Roentgenol. 1993; 161: 1139–1146.[Abstract/Free Full Text]

5. Parhami F, Demer LL. New concepts in regulation of vascular calcification.In: Fuster V, ed. The Vulnerable Plaque: Understanding, Identification, and Modification. Armonk, NY: Futura Publishing Co; 1999: 383–391.

6. Watson KE, Bostrom K, Ravindranath R, Lam T, Norton B, Demer LL. TGF-ß1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 1994; 93: 2106–2113.[Medline] [Order article via Infotrieve]

7. Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. Arterioscler Thromb Vasc Biol. 1997; 17: 680–687.[Abstract/Free Full Text]

8. Tintut Y, Patel J, Parhami F, Demer LL. Tumor necrosis factor- promotes in vitro calcification of vascular cells via cAMP pathway. Circulation. 2000; 102: 2636–2642.[Abstract/Free Full Text]

9. Parhami F, Tintut Y, Ballard A, Fogelman AM, Demer LL. Leptin enhances the calcification of vascular cells: artery wall as a target of leptin. Circ Res. 2001; 88: 954–960.[Abstract/Free Full Text]

10. Tribble DL, Krauss RM. HDL and coronary artery disease. Adv Intern Med. 1993; 38: 1–29.[Medline] [Order article via Infotrieve]

11. Libby P. Managing the risk of atherosclerosis: the role of high-density lipoprotein. Am J Cardiol. 2001; 88: 3N–8N.[Medline] [Order article via Infotrieve]

12. Franceschini G. Epidemiologic evidence for high-density lipoprotein cholesterol as a risk factor for coronary artery disease. Am J Cardiol. 2001; 88: 9N–13N.[Medline] [Order article via Infotrieve]

13. Pieters MN, Schouten D, Van Berkel TJC. In vitro and in vivo evidence for the role of HDL in reverse cholesterol transport. Biochim Biophys Acta. 1994; 1225: 125–134.[Medline] [Order article via Infotrieve]

14. von Eckardstein A, Nofer JR, Assmann G. High density lipoproteins and arteriosclerosis: role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol. 2001; 21: 13–27.[Abstract/Free Full Text]

15. Shah PK, Kaul S, Nilsson J, Cercek B. Exploiting the vascular protective effects of high-density lipoprotein and its apolipoproteins: an idea whose time for testing is coming, part I. Circulation. 2001; 104: 2376–2383.[Free Full Text]

16. Cockerill GW, Rye K, Gamble JR, Vadas MA, Barter PJ. High-density lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb Vasc Biol. 1995; 15: 1987–1994.[Abstract/Free Full Text]

17. Xia P, Vadas MA, Rye K, Barter PJ, Gamble JR. High density lipoproteins (HDL) interrupt the sphingosine kinase signaling pathway. J Biol Chem. 1999; 274: 33143–33147.[Abstract/Free Full Text]

18. Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA, Drinkwater DC, Laks H, Fogelman AM. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest. 1991; 88: 2036–2046.

19. Parthasarathy S, Barnett J, Fong LG. High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein. Biochim Biophys Acta. 1990; 1044: 275–283.[Medline] [Order article via Infotrieve]

20. Navab M, Berliner JA, Subbanagounder G, Hama S, Lusis AJ, Castellani LW, Reddy S, Shih D, Shi W, Watson AD, Van Lenten BJ, Vora D, Fogelman AM. HDL and the inflammatory response induced by LDL-derived oxidized phospholipids. Arterioscler Thromb Vasc Biol. 2001; 21: 481–488.[Abstract/Free Full Text]

21. Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, Prescott SM, La Du BN, Fogelman AM, Navab M. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response: loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures. J Clin Invest. 1995; 96: 2758–2767.[Medline] [Order article via Infotrieve]

22. Van Lenten BJ, Wagner AC, Nayak DP, Hama S, Navab M, Fogelman AM. High-density lipoprotein loses its anti-inflammatory properties during acute influenza A infection. Circulation. 2001; 103: 2283–2288.[Abstract/Free Full Text]

23. Castellani LW, Lusis AJ. ApoA-II versus apoA-I: two for one is not always a good deal. Arterioscler Thromb Vasc Biol. 2001; 21: 1870–1872.[Free Full Text]

24. Sharma N, Desigan B, Ghosh S, Sanyal SN, Ganguly NK, Majumdar S. The role of oxidized HDL in monocyte/macrophage functions in the pathogenesis of atherosclerosis in Rhesus monkeys. Scand J Clin Lab Invest. 1999; 59: 215–225.[CrossRef][Medline] [Order article via Infotrieve]

25. Nakajima T, Origuchi N, Matsunaga T, Kawai S, Hokari S, Nakamura H, Inoue I, Katayama S, Nagata A, Komoda T. Localization of oxidized HDL in atheromatous plaques and oxidized HDL binding sites on human aortic endothelial cells. Ann Clin Biochem. 2000; 37: 179–186.[Abstract/Free Full Text]

26. Hansson GK. Regulation of immune mechanisms in atherosclerosis. Ann N Y Acad Sci. 2001; 947: 157–165.[CrossRef][Medline] [Order article via Infotrieve]

27. Fazio S, Linton MF. The inflamed plaque: cytokine production and cellular cholesterol balance in the vessel wall. Am J Cardiol. 2001; 88: 12E–15E.[CrossRef][Medline] [Order article via Infotrieve]

28. Kaul D. Molecular link between cholesterol, cytokines, and atherosclerosis. Mol Cell Biochem. 2001; 219: 65–71.[CrossRef][Medline] [Order article via Infotrieve]

29. Tedgui A, Mallat Z. Anti-inflammatory mechanisms in the vascular wall. Circ Res. 2001; 88: 877–887.[Abstract/Free Full Text]

30. Subbanagounder G, Wong JW, Lee H, Faull KF, Miller E, Witztum JL, Berliner JA. Epoxyisoprostane and epoxycyclopentenone phospholipids regulate monocyte chemotactic protein-1 and interleukin-8 synthesis: formation of these oxidized phospholipids in response to interleukin-1ß. J Biol Chem. 2002; 277: 7271–7281.[Abstract/Free Full Text]

31. Sukovich DA, Kauser K, Shirley FD, DelVecchio V, Halks-Miller M, Rubanyi GM. Expression of interleukin-6 in atherosclerotic lesions of male apoE-knockout mice: inhibition by 17ß-estradiol. Arterioscler Thromb Vasc Biol. 1998; 18: 1498–1505.[Abstract/Free Full Text]

32. Van Lenten BJ, Wagner AC, Navab M, Fogelman AM. Oxidized phospholipids induce changes in hepatic paraoxonase and apo-J but not monocyte chemotactic protein-1 via interleukin-6. J Biol Chem. 2001; 276: 1923–1929.[Abstract/Free Full Text]

33. Cockerill GW, Huehns TY, Weerasinghe A, Stocker C, Lerch PG, Miller NE, Haskard DO. Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation. Circulation. 2001; 103: 108–112.[Abstract/Free Full Text]

34. Tintut Y, Parhami F, Bostrom K, Jackson SM, Demer LL. cAMP stimulates osteoblast-like differentiation of calcifying vascular cells. J Biol Chem. 1998; 273: 7547–7553.[Abstract/Free Full Text]

35. Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev. 1993; 14: 424–442.[Abstract/Free Full Text]

36. Nofer JR, Levkau B, Wolinska I, Junker R, Fobker M, Von Eckardstein A, Seedorf U, Assmann G. Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids. J Biol Chem. 2001; 276: 34480–34485.[Abstract/Free Full Text]

37. Baker PW, Rye KA, Gamble JR, Vadas MA, Barter PJ. Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression. J Lipid Res. 2000; 41: 1261–1267.[Abstract/Free Full Text]

38. Yuhanna IS, Zhu Y, Cox BE, Hahner LD, Osborne-Lawrence S, Lu P, Marcel YL, Anderson RGW, Mendelsohn ME, Hobbs HH, Shaul PW. High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat Med. 2001; 7: 853–857.[CrossRef][Medline] [Order article via Infotrieve]

39. Uittenbogaard A, Shaul PW, Yuhanna IS, Blair A, Smart EJ. High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae. J Biol Chem. 2000; 275: 11278–11283.[Abstract/Free Full Text]

40. Nofer JR, Walter M, Kehrel B, Wierwille S, Tepel M, Seedorf U, Assman G. HDL3-mediated inhibition of thrombin-induced platelet aggregation and fibrinogen binding occurs via decreased production of phosphoinositide-derived second messengers 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Arterioscler Thromb Vasc Biol. 1998; 18: 861–869.[Abstract/Free Full Text]

41. Everson WV, Smart EJ. Influence of caveolin, cholesterol, and lipoproteins on nitric oxide synthase: implications for vascular disease. Trends Cardiovasc Med. 2001; 11: 246–250.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				M. A. Frias, R. W. James, C. Gerber-Wicht, and U. Lang Native and reconstituted HDL activate Stat3 in ventricular cardiomyocytes via ERK1/2: Role of sphingosine-1-phosphate Cardiovasc Res, May 1, 2009; 82(2): 313 - 323. [Abstract] [Full Text] [PDF]

				L. L. Demer and Y. Tintut Vascular Calcification: Pathobiology of a Multifaceted Disease Circulation, June 3, 2008; 117(22): 2938 - 2948. [Full Text] [PDF]

				C. M. Shanahan Inflammation Ushers in Calcification: A Cycle of Damage and Protection? Circulation, December 11, 2007; 116(24): 2782 - 2785. [Full Text] [PDF]

				E. Aikawa, M. Nahrendorf, J.-L. Figueiredo, F. K. Swirski, T. Shtatland, R. H. Kohler, F. A. Jaffer, M. Aikawa, and R. Weissleder Osteogenesis Associates With Inflammation in Early-Stage Atherosclerosis Evaluated by Molecular Imaging In Vivo Circulation, December 11, 2007; 116(24): 2841 - 2850. [Abstract] [Full Text] [PDF]

				J. N. Bella, W. Tang, A. Kraja, D. C. Rao, S. C. Hunt, M. B. Miller, V. Palmieri, M. J. Roman, D. W. Kitzman, A. Oberman, et al. Genome-Wide Linkage Mapping for Valve Calcification Susceptibility Loci in Hypertensive Sibships: The Hypertension Genetic Epidemiology Network Study Hypertension, March 1, 2007; 49(3): 453 - 460. [Abstract] [Full Text] [PDF]

				M. Abedin, J. Lim, T.B. Tang, D. Park, L.L. Demer, and Y. Tintut N-3 Fatty Acids Inhibit Vascular Calcification Via the p38-Mitogen-Activated Protein Kinase and Peroxisome Proliferator-Activated Receptor-{gamma} Pathways Circ. Res., March 31, 2006; 98(6): 727 - 729. [Abstract] [Full Text] [PDF]

				M. Rattazzi, B. J. Bennett, F. Bea, E. A. Kirk, J. L. Ricks, M. Speer, S. M. Schwartz, C. M. Giachelli, and M. E. Rosenfeld Calcification of Advanced Atherosclerotic Lesions in the Innominate Arteries of ApoE-Deficient Mice: Potential Role of Chondrocyte-Like Cells Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1420 - 1425. [Abstract] [Full Text] [PDF]

				M. Litwin, E. Wuhl, C. Jourdan, J. Trelewicz, A. Niemirska, K. Fahr, K. Jobs, R. Grenda, Z. T. Wawer, P. Rajszys, et al. Altered Morphologic Properties of Large Arteries in Children with Chronic Renal Failure and after Renal Transplantation J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1494 - 1500. [Abstract] [Full Text] [PDF]

				F. J. Schoen and R. J. Levy Calcification of Tissue Heart Valve Substitutes: Progress Toward Understanding and Prevention Ann. Thorac. Surg., March 1, 2005; 79(3): 1072 - 1080. [Abstract] [Full Text] [PDF]

				B. Wu, S. Elmariah, F. S. Kaplan, G. Cheng, and E. R. Mohler III Paradoxical Effects of Statins on Aortic Valve Myofibroblasts and Osteoblasts: Implications for End-Stage Valvular Heart Disease Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 592 - 597. [Abstract] [Full Text] [PDF]

				C. M. Giachelli Vascular Calcification Mechanisms J. Am. Soc. Nephrol., December 1, 2004; 15(12): 2959 - 2964. [Abstract] [Full Text] [PDF]

				S. M. Moe and N. X. Chen Pathophysiology of Vascular Calcification in Chronic Kidney Disease Circ. Res., September 17, 2004; 95(6): 560 - 567. [Abstract] [Full Text] [PDF]

				M. R. Rubin and S. J. Silverberg Vascular Calcification and Osteoporosis--The Nature of the Nexus J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4243 - 4245. [Full Text] [PDF]

				T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification Endocr. Rev., August 1, 2004; 25(4): 629 - 672. [Abstract] [Full Text] [PDF]

				A. Mazzone, M. C. Epistolato, R. De Caterina, S. Storti, S. Vittorini, S. Sbrana, J. Gianetti, S. Bevilacqua, M. Glauber, A. Biagini, et al. Neoangiogenesis, T-lymphocyte infiltration, and heat shock protein-60 are biological hallmarks of an immunomediated inflammatory process in end-stage calcified aortic valve stenosis J. Am. Coll. Cardiol., May 5, 2004; 43(9): 1670 - 1676. [Abstract] [Full Text] [PDF]

				R. Vattikuti and D. A. Towler Osteogenic regulation of vascular calcification: an early perspective Am J Physiol Endocrinol Metab, May 1, 2004; 286(5): E686 - E696. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 91/7/570    most recent 01.RES.0000036607.05037.DAv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parhami, F. Articles by Demer, L. L. Search for Related Content PubMed PubMed Citation Articles by Parhami, F. Articles by Demer, L. L. Related Collections Pathophysiology Cell signalling/signal transduction Growth factors/cytokines Mechanism of atherosclerosis/growth factors