Genomic Changes in Regenerated Porcine Coronary Arterial Endothelial Cells

【摘要】  Objective— Genomic changes were defined in cultures of regenerated porcine coronary endothelial cells to explain the alterations that underlie their dysfunction.

Methods and Results— Regeneration of the endothelium was triggered in vivo by endothelial balloon denudation. After 28 days, both left circumflex (native cells) and left anterior descending (regenerated cells) coronary arteries were dissected, their endothelial cells harvested, and primary cultures established. The basal cyclic GMP production was reduced in regenerated cells without significant reduction in the response to bradykinin and A23187. The mRNA expression levels in both native and regenerated cells were measured by microarray and RT-PCR. The comparison revealed genomic changes related to vasomotor control (cyclooxygenase-1, angiotensin II receptor), coagulation (F2 and TFPI), oxidative stress (Mn SOD, GPX3, and GSR), lipid metabolism (PLA2 and HPGD), and extracellular matrix (MMPs). A-FABP and MMP7 were induced by regeneration. RT-PCR revealed upregulation of A-FABP and downregulation of eNOS and TR. The differential gene expression profiles were confirmed at the protein level by Western blotting for eNOS, F2, Mn SOD, MMP7, and TR.

Conclusions— Cultures from regenerated coronary endothelial cells exhibit genomic changes explaining endothelial dysfunction and suggesting facilitation of coagulation, lipid peroxidation, and extracellular matrix remodeling.

Genomic changes were determined in cultured regenerated endothelial cells. cGMP production was reduced. Changes in mRNA expression related to vasomotor control, coagulation, oxidative stress, extracellular matrix, and lipid were confirmed by Western blotting for eNOS, F2, Mn SOD, matrix metalloproteinase 7, and TR. These findings reveal linkages between regeneration and endothelial dysfunction.

【关键词】  endothelial regeneration genomics nitric oxide ROS coagulation extracellular matrix lipids

Introduction

The endothelium produces nitric oxide (NO) and prostacyclin, 1–3 which contribute to the anticoagulant properties of the intima and the inhibition of the adhesion and transmigration of inflammatory cells into the arterial wall. 4,5 Endothelial dysfunction attributable to aging and prolonged exposure to turbulent shear stress, coupled with risk factors, accelerates apoptosis, senescence, and turnover of endothelial cells. 4,5 Denuded areas are relined by regenerated endothelium. However, the latter is dysfunctional and no longer produces sufficient NO in response to thrombin and platelet-derived serotonin, 5–7 losing part of its protective role. 4,8–10 Impairment of NO synthesis favors cell apoptosis, which contributes to endothelial dysfunction and the development of atherosclerosis. 9 Other phenotypic changes in regenerated endothelial cells suggest an accelerated senescence and an accumulation of oxidized forms of modified lipoproteins (LDL). 6,11–13

The cellular mechanisms underlying these phenotypic changes are unknown. The present experiments were designed to define the genomic changes in regenerated endothelial cells, which could explain their phenotypic alterations.

Materials and Methods

The Online Data Supplement gives a detailed description of Materials & Methods (please see http://atvb.ahajournals.org.).

Briefly, female pigs were subjected to endothelial denudation of the left anterior descending coronary artery (LAD) to induce endothelial regeneration. 6 Twenty-eight days later, the animals were euthanized and their hearts removed. Native (left circumflex artery) and regenerated cells were harvested for primary cell culture. Cultures derived from native and regenerated endothelial cells are termed native and regenerated cells, respectively. Confluent cultures at passage zero were studied, except when stated otherwise. Native and regenerated cells derived from the same 6 hearts were compared as regards:

Genomic expression by Microarray (GeneChips Porcine Genome Array, Affymetrix). Fold-changes greater than or equal to 0.8 were considered to indicate genomic differences. 14 Certain changed genes were subjected to computational simulation of biological interactions (PathwayAssist version 3).

Basal and stimulated (bradykinin, A23187 ) levels of cyclic GMP by radioimmunoassay. 11

mRNA expression by RT-PCR. 15

Protein presence by Western blotting. 16

Histochemical staining. 17

Student t test for paired observations was used for the statistical analysis of differences between native and regenerated cells.

Results

Microarray Gene Expression

The array used contained 23 937 probe sets that interrogate approximately 23 256 transcripts from 20 201 S. scrofa genes for target hybridization. The average presence of probe sets for native and regenerated endothelial cells of 6 animals was 61.15±0.0% and 60.3±0.01%, respectively, of the 24 123 probe sets present. The distribution profile (supplemental Figure I) of all differentially expressed genes in regenerated endothelial cells consisted of 7.7±1.2% (1860±278) upregulated and 9.4±1.8% (2266±443) downregulated genes.

Of the identified genes 125 were upregulated and 444 downregulated (supplemental Table I). The expression of the genes for adipocyte-fatty acid binding protein (A-FABP) and matrix-metalloproteinase 7 (MMP7) was observed only in regenerated cells ( Figure 1 ). Among the other genes known to be related to endothelial function and vasomotor control, 18 were upregulated ( Figure 1 ) and 25 downregulated ( Figure 2 ) significantly. The Microarray analysis revealed 425 unknown genes which were either up- or downregulated (supplemental Table II).

Figure 1. Upregulation of genes in regenerated cells. A-FABP indicates adipocyte-fatty acid binding protein; MMP7, matrix-metalloproteinase 7; HPGD, hydroxyprostaglandin dehydrogenase 15-(NAD); GADD45A&B, growth arrest and DNA-damage-inducible, alpha and beta; F2, coagulation factor II; CXCL12, stromal cell-derived factor-1; MMP23A&B, matrix-metalloproteinase 23A&B; COL1A1, collagen alpha 1; GM-CSF, granulocyte macrophage-colony stimulating factor; TGF-β1, TGF beta 1; PPAR gamma 1a, peroxisome proliferators-activated receptor gamma 1a; Gp VII PLA2, phospholipase A2. * P <0.05 (with GCOS normalization strategy). P <0.05 (with GeneSpring GX analysis).

Figure 2. Downregulation of genes in regenerated cells. LOX receptor 1 indicates lectin-like oxidized LDL receptor-1; VLCAD, acyl-coenzyme A dehydrogenase (very long chain); TGF-β1R-II, TGF-beta 1 receptor, type II; ACAT, acyl-coenzyme A:cholesterol acyltransferase; PAI-2, plasminogen activator inhibitor-2; GSR, glutathione reductase; Lrp 1&6, LDL receptor-related protein 1 and 6; COX-1/PGHS-1, cyclooxygenase 1; GPX3, glutathione peroxidase 3; TXNIP, thioredoxin interacting protein; MnSOD, superoxide dismutase (Mn type); GPIIIa, glycoprotein IIIa; Apo B, apolipoprotein B; TFPI (Lp-associated), tissue factor pathway inhibitor (lipoprotein-associated); I B, IkappaB; ABCA1, ATP-binding cassette transporter A1; CAV1, caveolin-1. * P <0.05 (with GCOS normalization strategy). P <0.05 (with GeneSpring GX analysis).

The expression of a number of genes known to be related to endothelial function, including eNOS (NOS3), was comparable in native and regenerated cells (supplemental Table III).

The Microarray analysis demonstrated absence or minimal expression, in both native and regenerated cells, of CD14, CD18, CD133, Flk 1 type VEGF receptor VEGFR2 (KDR/Flk), and comparable expression of CD31 and CD34 antigens (supplemental Table III).

Cyclic GMP

The basal level of cyclic GMP (measured as an index of the production of NO, and thus of the activity of eNOS) in regenerated cells at passage one was reduced significantly compared with native cells ( Figure 3 D). Bradykinin (1 µmol/L) and A23187 (1 µmol/L) increased the level of cyclic GMP significantly and to a comparable extent in native and regenerated cells ( Figure 3 E).

Figure 3. mRNA expression (A) and protein presence of eNOS (B) and Akt 1/2 (C) in native and regenerated cells. Cyclic GMP levels in native and regenerated cells at basal level (D) and on stimulation by bradykinin (BK) and A23187 (E). The asterisks indicate statistical significant differences ( P <0.05) from the corresponding control.

Real-Time PCR

Real-time PCR revealed a significant reduction in the expression of eNOS ( Figure 3 A) and thioredoxin reductase ( Figure 4 A) in regenerated cells. It also confirmed the upregulation of the expression of A-FABP gene ( Figure 5 A) in these cells.

Figure 4. mRNA expression of thioredoxin reductase (TR) as analyzed by RT-PCR (A). Protein presence of TR (B) and superoxide dismutase (Mn SOD; C). The asterisks indicate statistical significant differences ( P <0.05) from the corresponding control.

Figure 5. mRNA expression of A-FABP as analyzed by RT-PCR (A); protein presence of MMP7 (B) and coagulation factor II (F2; C). The asterisks indicate statistical significant differences ( P <0.05) from the corresponding control.

Western Blotting

The experiments were performed in cells at passage 1 to 4. The presence of eNOS protein was significantly reduced in regenerated compared with native cells ( Figure 3 B), as was that of Akt 1/2 ( Figure 3 C). The protein levels of thioredoxin reductase ( Figure 4 B) and superoxide dismutase (Mn SOD; Figure 4 C) also were reduced significantly in regenerated cells. The protein level of coagulation factor II (F2) was augmented significantly in regenerated cells ( Figure 5 C). The presence of MMP7 protein was demonstrated in regenerated cells only ( Figure 5 B).

Immunohistochemistry

Cultures of regenerated endothelium contained more multinucleated and enlarged cells (supplemental Figure II). More than 99% of native and regenerated cells stained for von Willebrand factor (vWF; supplemental Figure II). A more intense immunohistochemistry staining for senescence-associated galactosidase beta 1 (SA-β-Gal) was observed in regenerated cells at passage one ( Figure 6 ).

Figure 6. Histochemical staining of SA-β-Galactosidase activity in (A) native and (B) regenerated cells (in blue) in the cytoplasmic region of the cells.

Pathway Analysis

Changed genes for which we believe that they play a role in endothelial function were subjected to computational simulation of biological interactions (supplemental Figure III). This simulation suggested that the downregulation of Mn SOD, GPX3, and GSR should raise the level of oxidative stress, hamper DNA integrity, and reduce the level of nitric oxide, and thus that of cyclic GMP. At the same time, the formation of peroxynitrite should accelerate the lipid peroxidation and promote inflammatory responses. Similarly, the increased level of F2 as well as the elevated level of tissue factor caused by the reduction in TFPI should reduce the anticoagulatory properties of regenerated cells. The reduced expression of plasminogen activator inhibitor (PAI)-2 and the increased levels of MMP7, MMP23, and several subtypes of collagen should facilitate extracellular matrix remodeling leading to vascular thickening.

Discussion

The present study was initiated to identify the genomic changes in regenerated endothelial cells. The pig was selected as experimental animal because the development, morphology, and function of its cardiovascular system closely resemble that of humans. 18 In the present experiments, endothelial regeneration was induced in a minimal area along the LAD to maintain the well-being of the animals. This limits the amount of samples for the analysis both at genomic and proteomic levels. Hence, primary cultures of endothelial cells 11–13 were used to permit harvesting of sufficient RNA to perform the microarray analysis (cells at passage zero) and protein analysis (cells between passage 1 and 4 which provided sufficient proteins, at the risk of introducing changes due to the multiple passaging). To minimize genetic background noise, the comparison was performed systematically in parallel between native and regenerated cells of the same hearts. The microarray data revealed a large number of up- or downregulated genes. This manuscript focuses on the changes that we felt are the most important in terms of endothelial dysfunction and atherosclerosis.

The regeneration of injured endothelium occurs mainly by the proliferation and migration of the neighboring mature endothelial cells to cover the damaged area in order to uphold the homeostasis within the vascular system. 19 Bone marrow–derived stem cells, particularly the circulating endothelial progenitor cells (EPC; CD133 + KDR + CD34 + cells), 19 can differentiate into endothelial phenotype in response to injury. 19 This accelerates reendothelialization and influences the release of cytokines and growth factors (VEGF, M-colony stimulating factor , IGF). 19,20 After 28 days, 70% or more of the previously denuded surface is covered by regenerated endothelium. 20,21 In the present study, upregulation was observed of CXCL12 and GM-CSF, which may reflect the mobilization of progenitor cells by chemokines to site of injury for reendothelialization 22 although the expression of CD34 + was not changed in regenerated cells. The absent or insignificant expression in CD133, KDR, 19 CD14 and CD18 23 demonstrated the purity of endothelial mRNA with minimal contamination by progenitor or mononuclear cells. The endothelial origin of both native and regenerated cells was confirmed by genetic expression of eNOS and CD31, as well as by positive staining for vWF. 24

Vasomotor Control

eNOS and Akt

The basal production of cyclic GMP was reduced in regenerated cells by approximately 30% compared with native cells, which is indicative of endothelial dysfunction. 11 This interpretation is supported by the reduced genetic expression of eNOS, as detected by RT-PCR (but not by microarray), and by the reduced protein presence of the enzyme. The reduced expression of flow-sensitive caveolin-1, which in the caveolae along the cell membrane can activate resident eNOS to produce nitric oxide, may suggest a reduced response to physiological stimulations such as shear stress. 25 The reduced protein presence of Akt also is consistent with a diminished production of NO. 26 The observed downregulation of arginase (type II) implies that competition by this enzyme for substrate arginine is not likely to contribute to reduced production of NO. 27

The cyclic GMP response to endothelium-dependent vasodilators was not reduced. This is in line with earlier studies demonstrating normal endothelium-dependent relaxations to bradykinin and A23187 in arteries covered with regenerated endothelium. 6 By contrast, earlier work has shown that the endothelium-dependent relaxations to serotonin (and thrombin) are impaired by the regeneration process. 6,7,13,28

Other Enzymes and GPCRs

The present data did not reveal significant changes in expression level of the detected serotonin (5-HT1D and 5-HT2B receptors) and endothelin-1 receptors in regenerated cells, whereas that of angiotensin II and oxytocin receptors was augmented. The observed upregulated gene expression of the angiotensin II (Ang II) type 1 receptor may result in inactivation of the bradykinin-nitric oxide pathway and thus endothelial dysfunction on exposure to angiotensin II. 29 It also could facilitate inflammatory reactions attributable to oxidative stress resulting from inactivation of antioxidant enzymes such as superoxide dismutase which would favor lipid oxidation and damage proteins and nucleic acids in the endothelial cells. 29,30 The expression of the 5-HT1D subtype was minimal in cultures derived from both native and regenerated cells. The minimal expression of serotonin receptors observed in the present study helps to explain earlier work demonstrating a loss in ability to release endothelium-derived relaxing factor in cultured cells in response to monoamine. 28 The downregulation of endothelin-converting enzyme 1 (ECE 1) observed in the present studies in regenerated cells may imply that despite the unchanged expression of endothelin receptors, endothelin-1 does not contribute to the dysfunction of these cells, although obviously a reduced bioavailability of NO could favor the production and the action of the peptide. 31

G-Proteins

The selective loss of the response to serotonin and thrombin has been attributed to a reduced activity of pertussin-toxin sensitive Gi-proteins, whereas that of Gq proteins (which mediate the response to bradykinin) is maintained much longer. 6,7,13,28 The absence of differences in the expression of Gi-protein family genes in regenerated cells supports that interpretation and is in line with the earlier demonstration, by immunostaining, of a comparable presence of these proteins in native and regenerated cells. 13 Endothelium-dependent vasodilatation may also be dampened further by a reduced production of prostacyclin by the main producing enzyme, COX-1/PGHS-1, 32 the expression of which is reduced in regenerated cells.

Oxidative Stress

Endothelial reactive oxygen species (ROS) can originate from various sources (eg, the mitochondrial oxidative respiratory chain, xanthine oxidase, uncoupled NOS, cytochrome P-450 enzymes, cyclooxygenases and NADPH oxidases). 33 ROS alter gene transcription and enzyme activities, reduce the production of nitric oxide, and cause oxidative damage to lipids, proteins, and DNA. 34,35 The expression of the subunit complexes of oxidative phosphorylation were not changed in regenerated cells. The present experiments predict an elevated level of hydrogen peroxide, originating from the mitochondria in regenerated cells attributable to downregulation of several antioxidant enzymes (Mn SOD , GPX3, GSR, TR, 35 and thioredoxin-interacting protein). High levels of ROS, including hydrogen peroxide, as a result of the reduced activities of Mn SOD in the mitochondria diffuse and exert an oxidative effect in cytosol to catalyze the conversion of nitric oxide into peroxynitrite. 35 This could reduce eNOS level in regenerated cells, bioavailability of NO, 35 and augment the production of endothelium-derived vasoconstrictor substances. 36 The protective role of adenosine on oxidant injury 37 could also be diminished as a result of upregulation of adenosine deaminase, the major enzyme that catabolizes this nucleoside. Furthermore, an augmented production of ROS would also inhibit other mitochondrial enzymes including aconitase and pyruvate dehydrogenase kinase, 33 explaining the reduced expression of those enzymes revealed by the present microarray experiments.

Cholesterol/Fatty Acid Metabolism

Another aspect related to endothelial function is the metabolism of lipids. The gene expression of A-FABP was induced in regenerated cells, whereas the gene for this protein was not present in native cells. Activation of A-FABP is dependent on ox-LDL and PPAR gamma and essential for transformation of macrophages to foam cells in the subendothelial layer. 38 It is also involved in the development of atherosclerosis by promoting accumulation of cholesterol esters and production of inflammatory mediators. 39 The gene for Gp VII PLA2 (which releases proinflammatory eicosanoids as well as platelet-activating factor 40 ) and HPGD (which minimizes the availability of lipoxin 41 ) were also upregulated. This, together with elevated levels of ROS, would provide a genomic explanation for increased acetylated LDL uptake 11 and intracellular accumulation of ox-LDL 12 without changes in number of LOX receptors in regenerated cells. 12 An increase in acetylated and oxidized LDL as well as in oxidized ApoB-100 in cells decreases the production of nitric oxide, which contributes to vascular disease. 4,5 The observed downregulated expression of acyl-coA oxidase and VLCAD should reduce the β-oxidation of long chain fatty acids in mitochondria 42 and increase lipid accumulation in the cells, 43 whereas the reduced expression of ACAT and ApoB should diminish inflammation in vascular cells. 44 A reduced presence of HDL binding protein, ABCA1 in regenerated cells, would limit the efflux of lipids from endothelial cells 45 and pose further cardiovascular risk by facilitating the accumulation of cholesterol.

Coagulation

The endothelium becomes prothrombotic after the regeneration process. 8 Indeed, the reduced expression of endogenous lipoprotein-associated TFPI gene in regenerated cells could lead to augmented levels of tissue factor. 46 Similarly, lipid peroxidation can accelerate the oxidative degradation of TFPI in endothelial cells which becomes a marker for endothelial dysfunction. 47 Upregulation of genes like F2 in regenerated cells should facilitate thrombin formation. 48

Extracellular Matrix, Vascular Smooth Muscle Proliferation, and Neointimal Formation

The immediate vascular response after the injury leads to elastic recoil of the media and the adventitia 49 and initiates several subsequent events including recruitment and migration of inflammatory cells, proliferation of vascular smooth cells, and deposition of extracellular matrix at site of injury with the formation of neointima. 50 The reduced expression of PAI-2 in regenerated cells may increase the proteolytic power of proteinases and hence the upregulation of MMP genes in regenerated cells 51 including MMP7, MMP23A&B. MMP7 at both the genomic and proteomic levels was expressed only in regenerated cells. This proteinase may promote plaque rupture. 52 The altered gene expression of other extracellular matrix proteins in particular the collagens could also accelerate the susceptibility toward cardiovascular disease. 53 Although intima-media thickening was not observed significantly in the present experiments, the augmented transforming growth factor (TGF) beta 1 but downregulated expression of TGF beta 1 receptor II in association with altered expression in thrombospondins in regenerated cells may have pathophysiological consequences. 54

Inflammation and Apoptosis

The gene expression data indicate that inflammatory, tumor necrosis factor (TNF)-related and apoptotic events were downregulated. This probably reflects complete reendothelialization of denuded area to maintain vascular homeostasis after injury. These observations then imply that inflammatory reaction, which initiates atherosclerotic process 55 follows rather than precedes regeneration.

Growth Regulation and Senescence

The present genomic and histochemical data demonstrate that β-galactosidase, an established marker for cellular senescence during aging and after chronic oxidative stress, 17 is upregulated in regenerated cells. The exact mechanism causing increase of this enzyme during aging is unclear. However, its upregulation is in line with that of genes such as GADD45A and GADD45B as well as growth arrest specific 6. This could cause endoplasmic reticulum stress and prevent DNA synthesis. 56 That early senescence occurs in regenerated cells is suggested also by morphological changes (larger, multinucleated cells) observed, confirming earlier observations. 13

Angiogenesis

The expression of angiogenic factors for endothelial cell proliferation like VEGF and angiopoietin-2 57 was not changed, presumably reflecting the fact that completed reendothelialization has occurred after the injury.

In conclusion, the present study outlines genomic and proteomic changes that accompany endothelial regeneration and presumably vascular changes related to intima-media thickening (supplemental Figures III and IV). These findings provide new information on genomic changes which help to understand the phenotypic alterations of regenerated endothelial cells.

Acknowledgments

The authors thank Professor H. Shimokawa (Tohoku University, Japan) for the demonstration of angioplasty.

Sources of Funding

This work was supported in part by grant HKU7490/06M of the Research Grant Council of Hong Kong and by the Research Centre of Heart, Brain, Hormone and Healthy Aging of the University of Hong Kong.

Disclosures

None.

【参考文献】
  Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980; 288: 373–376.

Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J. 1989; 3: 2007–2018.

Moncada S, Vane JR. Prostacyclin and its clinical applications. Ann Clin Res. 1984; 16: 241–252.

Vanhoutte PM. Endothelial dysfunction and vascular disease. In Endothelium, Nitric Oxide and Atherosclerosis. 1999. Panza JA, Cannon RO III. New York: Futura Publishing Co, Inc, Armonk. 79–95.

Vanhoutte PM. Endothelial control of vasomotor function: from health to coronary disease. Circ J. 2003; 67: 572–575.

Shimokawa H, Aarhus LL, Vanhoutte PM. Porcine coronary arteries with regenerated endothelium have a reduced endothelium-dependent responsiveness to aggregating platelets and serotonin. Circ Res. 1987; 61: 256–270.

Shimokawa H, Flavahan NA, Vanhoutte PM. Natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries. Possible dysfunction of a pertussis toxin-sensitive G protein. Circ Res. 1989; 65: 740–753.

Bombeli T, Karsan A, Tait JF, Harlan JM. Apoptotic vascular endothelial cells become procoagulant. Blood. 1997; 89: 2429–2442.

Lüscher TF, Noll G. The pathogenesis of cardiovascular disease: role of the endothelium as a target and mediator. Atherosclerosis. 1995; 118 (suppl): S81–S90.

Tschudi MR, Barton M, Bersinger NA, Moreau P, Cosentino F, Noll G, Malinski T, Luscher TF. Effect of age on kinetics of nitric oxide release in rat aorta and pulmonary artery. J Clin Invest. 1996; 98: 899–905.

Fournet-Bourguignon MP, Castedo-Delrieu M, Bidouard JP, Leonce S, Saboureau D, Delescluse I, Vilaine JP, Vanhoutte PM. Phenotypic and functional changes in regenerated porcine coronary endothelial cells: increased uptake of modified LDL and reduced production of NO. Circ Res. 2000; 86: 854–861.

Kennedy S, Fournet-Bourguignon MP, Breugnot C, Castedo-Delrieu M, Lesage L, Reure H, Briant C, Leonce S, Vilaine JP, Vanhoutte PM. Cells derived from regenerated endothelium of the porcine coronary artery contain more oxidized forms of apolipoprotein-B-100 without a modification in the uptake of oxidized LDL. J Vasc Res. 2003; 40: 389–398.

Borg-Capra C, Fournet-Bourguignon MP, Janiak P, Villeneuve N, Bidouard JP, Vilaine JP, Vanhoutte PM. Morphological heterogeneity with normal expression but altered function of G proteins in porcine cultured regenerated coronary endothelial cells. Br J Pharmacol. 1997; 122: 999–1008.

Braam B, de Roos R, Dijk A, Boer P, Post JA, Kemmeren PP, Holstege FC, Bluysen HA, Koomans HA. Nitric oxide donor induces temporal and dose-dependent reduction of gene expression in human endothelial cells. Am J Physiol Heart Circ Physiol. 2004; 287: H1977–H1986.

Rose?Meyer RB, Mellick AS, Garnham BG, Harrison GJ, Massa HM, Griffiths LR. The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis. Brain Res Brain Res Protoc. 2003; 11: 9–18.

Fisslthaler B, Benzing T, Busse R, Fleming I. Insulin enhances the expression of the endothelial nitric oxide synthase in native endothelial cells: a dual role for Akt and AP-1. Nitric Oxide. 2003; 8: 253–261.

Kurz DJ, Decary S, Hong Y, Trivier E, Akhmedov A, Erusalimsky JD. Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells. J Cell Sci. 2004; 117: 2417–2426.

de Smet BJ, van der Zande J, van der Helm YJ, Kuntz RE, Borst C, Post MJ. The atherosclerotic Yucatan animal model to study the arterial response after balloon angioplasty: the natural history of remodeling. Cardiovasc Res. 1998; 39: 224–232.

Urbich C, Dimmeler S. Endothelial progenitor cells: Characterization and role in vascular biology. Circ Res. 2004; 95: 343–353.

Kong D, Melo LG, Gnecchi M, Zhang L, Mostoslavsky G, Liew CC, Pratt RE, Dzau VJ. Cytokine-induced mobilization of circulating endothelial progenitor cells enhances repair of injured arteries. Circulation. 2004; 110: 2039–2046.

Fonseca FA, Izar MC, Fuster V, Gallo R, Padurean A, Fallon JT, Schachter EN, Chesebro JH, Badimon JJ. Chronic endothelial dysfunction after oversized coronary balloon angioplasty in pigs: a 12-week follow-up of coronary vasoreactivity in vivo and in vitro. Atherosclerosis. 2001; 154: 61–69.

Schober A, Karshovska E, Zernecke A, Weber C. SDF-1 -mediated tissue repair by stem cells: a promising tool in cardiovascular medicine? Trends Cardiovasc Med. 2006; 16: 103–108.

Serrano CV Jr, Rocha Giraldez R, de Lara Fernandes J, Nicolau JC, Zweier JL, Ramires JA. Platelet and leukocyte adhesion and activation in unstable angina and post-PTCA. Int J Cardiol. 2005; 99: 423–428.

Hunting CB, Noort WA, Zwaginga JJ. Circulating endothelial (progenitor) cells reflect the state of the endothelium: vascular injury, repair and neovascularization. Vox Sang. 2005; 88: 1–9.

Yu J, Bergaya S, Murata T, Alp IF, Bauer MP, Lin MI, Drab M, Kurzchalia TV, Stan RV, Sessa WC. Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels. J Clin Invest. 2006; 116: 1284–1291.

Hartell NA, Archer HE, Bailey CJ. Insulin-stimulated endothelial nitric oxide release is calcium independent and mediated via protein kinase B. Biochem Pharmacol. 2005; 69: 781–790.

Ryoo S, Lemmon CA, Soucy KG, Gupta G, White AR, Nyhan D, Shoukas A, Romer LH, Berkowitz DE. Oxidized low-density lipoprotein-dependent endothelial arginase II activation contributes to impaired nitric oxide signaling. Circ Res. 2006; 99: 951–960.

Boulanger CM, Vanhoutte PM. G proteins and endothelium-dependent relaxations. J Vasc Res. 1997; 34: 175–185.

De Gennaro Colonna V, Fioretti S, Rigamonti A, Bonomo S, Manfredi B, Muller EE, Berti F, Rossoni G. Angiotensin II type 1 receptor antagonism improves endothelial vasodilator function in L-NAME-induced hypertensive rats by a kinin-dependent mechanism. J Hypertens. 2006; 24: 95–102.

Dandona P, Dhindsa S, Ghanim H, Chaudhuri A. Angiotensin II and inflammation: the effect of angiotensin-converting enzyme inhibition and angiotensin II receptor blockade. J Hum Hypertens. 2007; 21: 20–27.

Vanhoutte PM. Say NO to ET. J Auton Nerv Syst. 2000; 81: 271–277.

Vane JR, Botting RM Pharmacodynamic profile of prostacyclin. Am J Cardiol. 1995; 75: 3A–10A.

Madamanchi NR, Runge MS. Mitochondrial dysfunction in atherosclerosis. Circ Res. 2007; 100: 460–473.

Droge W. Free radicals in the physiological control of cell functions. PhysiolRev. 2001; 82: 47–95.

Li JM, Shah AM. Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology. Am J Physiol Regul Integr Comp Physiol. 2004; 287: R1014–R1030.

Vanhoutte PM, Feletou M, Taddei S. Endothelium-dependent contractions in hypertension. Br J Pharmacol. 2005; 144: 449–458.

Zhang Y, Handy DE, Loscalzo J. Adenosine-dependent induction of glutathione peroxidase 1 in human primary endothelial cells and protection against oxidative stress. Circ Res. 2005; 96: 831–837.

Fu Y, Luo N, Lopes-Virella MF, Garvey WT. The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages. Atherosclerosis. 2002; 165: 259–269.

Furuhashi M, Tuncman G, Gorgun CZ, Makowski L, Atsumi G, Vaillancourt E, Kono K, Babaev VR, Fazio S, Linton MF, Sulsky R, Robl JA, Parker RA, Hotamisligil GS. Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2. Nature. 2007; 447: 959–965.

Kadl A, Leitinger N. The role of endothelial cells in the resolution of acute inflammation. Antioxid Redox Signal. 2005; 7: 1744–1754.

McMahon B, Godson C. Lipoxins: endogenous regulators of inflammation. Am J Physiol Renal Physiol. 2004; 286: F189–F201.

Exil VJ, Roberts RL, Sims H, McLaughlin JE, Malkin RA, Gardner CD, Ni G, Rottman JN, Strauss AW. Very-long-chain acyl-coenzyme a dehydrogenase deficiency in mice. Circ Res. 2003; 93: 448–455.

Bostrom P, Magnusson B, Svensson PA, Wiklund O, Boren J, Carlsson LM, Stahlman M, Olofsson SO, Hulten LM. Hypoxia converts human macrophages into triglyceride-loaded foam cells. Arterioscler Thromb Vasc Biol. 2006; 26: 1871–1876.

Kharbanda RK, Wallace S, Walton B, Donald A, Cross JM, Deanfield J. Systemic Acyl-CoA:cholesterol acyltransferase inhibition reduces inflammation and improves vascular function in hypercholesterolemia. Circulation. 2005; 111: 804–807.

Cavelier C, Rohrer L, von Eckardstein A. ATP-binding cassette transporter A1 modulates apolipoprotein A-1 transcytosis through aortic endothelial cells. Circ Res. 2006; 99: 1060–1066.

Steffel J, Luscher TF, Tanner FC. Tissue factor in cardiovascular diseases: molecular mechanisms and clinical implications. Circulation. 2006; 113: 722–731.

Horie S, Hiraishi S, Hirata Y, Kazama M, Matsuda J. Oxidized low-density lipoprotein impairs the anti-coagulant function of tissue-factor-pathway inhibitor through oxidative modification by its high association and accelerated degradation in cultured human endothelial cells. Biochem J. 2000; 352: 277–285.

Kirchhofer D, Tschopp TB, Hadvary P, Baumgartner HR. Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors. J Clin Invest. 1994; 93: 2073–2083.

Yang YM, Moussa I. Percutaneous coronary intervention and drug-eluting stents. CMAJ. 2005; 172: 323–325.

Nakatani M, Takeyama Y, Shibata M, Yorozuya M, Suzuki H, Koba S, Katagiri T. Mechanisms of restenosis after coronary intervention: difference between plain old balloon angioplasty and stenting. Cardiovasc Pathol. 2003; 12: 40–48.

Creemers EE, Cleutjens JP, Smits JF, Daemen MJ. Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure? Circ Res. 2001; 89: 201–210.

Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci U S A. 1996; 93: 9748–9753.

Garcia-Touchard A, Henry TD, Sangiorgi G, Spagnoli LG, Mauriello A, Conover C, Schwartz RS. Extracellular Proteases in Atherosclerosis and Restenosis. Arterioscler Thromb Vasc Biol. 2005; 25: 1119–1127.

Khan R, Agrotis A, Bobik A. Understanding the role of transforming growth factor-β1 in intimal thickening after vascular injury. Cardiovasc Res. 2007; 74: 223–234.

Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999; 340: 115–126.

Outinen PA, Sood SK, Pfeifer SI, Pamidi S, Podor TJ, Li J, Weitz JI, Austin RC. Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells. Blood. 1999; 94: 959–967.

Chong AY, Caine GJ, Lip GYH. Angiopoietin/tie-2 as mediators of angiogenesis: a role in congestive heart failure? Eur J Clin Invest. 2004; 34: 9–13.

作者单位：Department of Pharmacology (M.Y.K.L., R.Y.K.M., P.M.V.) and the Cardiology Division, Department of Medicine (H.F.T., C.W.S., S.G.Z.), Li Ka Shing Faculty of Medicine, The University of Hong Kong.