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Identification of genes induced by oxidized phospholipids in human aortic endothelial cells
Vascular Pharmacology 38 (2002) 211 – 218 www.elsevier.com/locate/vph
Identification of genes induced by oxidized phospholipids in human aortic endothelial cells Srinivasa T. Reddy*, Victor Grijalva, Carey Ng, Khaled Hassan, Susan Hama, Rachel Mottahedeh, David J. Wadleigh, Mohamad Navab, Alan M. Fogelman Atherosclerosis Research Unit, University of California, Los Angeles, CA 90095-1679, USA
Abstract Oxidized-L-a-1-Palmitoyl-2-Arachidonoyl-sn-glycero-3-Phosphorylcholine (Ox-PAPC), a component of mildly oxidized/minimally modified low-density lipoprotein (MM-LDL), accounts for many of the biological activities of MM-LDL. Having hypothesized that OxPAPC initiates gene expression changes in endothelial cells that result in enhanced endothelial/monocyte interactions and the subsequent development of atherosclerotic lesions, we used the suppression subtractive hybridization (SSH) procedure to compare mRNA isolated from PAPC-treated human aortic endothelial cells (HAEC) with mRNA isolated from Ox-PAPC-treated cells. Genes induced by Ox-PAPC but not by PAPC in HAEC included genes involved in signal transduction, extracellular matrix, growth factors, chemokines and several genes with unknown functions. The observed pattern of gene induction suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing. D 2002 Elsevier Science Inc. All rights reserved. Keywords: MM-LDL; Ox-PAPC; Gene expression; Endothelial cell function; Atherosclerosis; Suppression subtractive hybridization
1. Introduction The vascular endothelium directs the inflammatory response by regulating the production of adhesion molecules and cytokines in response to various agents (Ross, 1999). The entrapment and oxidation of low-density lipoproteins (LDL) in the subendothelial space results in the seeding of LDL with reactive oxygen species (e.g., hydroperoxyoctadecadienoic acid (HPODE), hydroperoxyeicosatetraenoic acid (HPETE) and cholesterol linoleate hydroperoxide, which may result from the action of HPODE and HPETE on cholesterol linoleate) (Navab et al., 2000a,b). When these
Abbreviations: SSH, Suppression subtractive hybridization; HDL, highdensity lipoprotein; LDL, low-density lipoprotein; MM-LDL, mildly oxidized LDL; Apo, apolipoprotein; PAPC, 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphocholine; Ox-PAPC, oxidized PAPC; MKP-1, mitogenactivated protein kinase phosphatase-1; MCP-1, monocyte chemoattractant protein-1; IL-8, interleukin-8; HPODE, hydroperoxyoctadecadienoic acid; HPETE, hydroperoxyeicosatetraenoic acid. * Corresponding author. Department of Medicine and Department of Molecular and Medical Pharmacology, University of California Los Angeles, 650 Charles E. Young Drive South, A8-131 CHS, Los Angeles, CA 90095, USA. Tel.: +1-310-206-3915; fax: +1-310-206-3605. E-mail address: [email protected] (S.T. Reddy).
reactive oxygen species reach a critical threshold in LDL, a nonenzymatic oxidation of LDL phospholipids that contain arachidonic acid produces biologically active oxidized phospholipids (Navab et al., 2000a,b). Subbanagounder et al. (2000) demonstrated that oxidation of arachidonic acid in the sn-2 position of an intact phospholipid molecule is required for biologic activity. The most common phospholipid that provides the substrate for this oxidation is L-a-1-Palmitoyl2-Arachidonyl-sn-gycero-3-Phosphorylcholine (PAPC). The oxidation of PAPC (Ox-PAPC) has been shown to account for many of the biologic activities of mildly oxidized/ minimally modified LDL (MM-LDL) (Watson et al., 1997). The structures of three of the biologically active components of Ox-PAPC have been identified as 1-palmitoyl-2-oxovaleryl-sn-gycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaryl-sn-gycero-3-phosphorylcholine (PGPC) and 1-palmitoyl-2-(5,6-epoxyisoprostane E 2 )-sn-glycero-3-phosphorylcholine (PEIPC) (Watson et al., 1997, 1999). These structurally similar oxidized phospholipids have been shown to differentially regulate endothelial binding of monocytes and neutrophils (Leitinger et al., 1999), suggesting that oxidized phospholipids may play a general role in inflammation, atherosclerosis and wound healing, and thus influence the expression of a number of important gene families.
1537-1891/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 7 - 1 8 9 1 ( 0 2 ) 0 0 1 7 1 - 4
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It is evident that the biologically active components of Ox-PAPC regulate gene expression in target cells (Lee et al., 2000; Subbanagounder et al., 2001). Ox-PAPC induces inflammatory cytokines and adhesion molecules in human aortic endothelial cells (HAEC), similar to other inflammatory cytokines such as TNF-a and LPS. Both TNF-a and Ox-PAPC activate endothelial cells to bind monocytes partly through the induction of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) (Leitinger et al., 1997). Interestingly, Ox-PAPC, but not TNF-a, enhances monocyte – endothelial binding by mediating activation of b1-integrins resulting in deposition of fibronectin-containing CS-1 on the apical cell surface (Leitinger et al., 1997). On the other hand, TNF-a enhances the endothelial/monocyte interactions through the induction of E-selectin and VCAM-1, which are not affected by OxPAPC (Leitinger et al., 1997). Thus, Ox-PAPC and TNF-a utilize both similar and distinct induction patterns of gene expression to initiate endothelial/monocyte interactions. The molecular mechanisms of cytokine-induced gene expression are well understood, however, the target receptors, the signaling pathways and the molecular mechanisms by which oxidized phospholipids, such as Ox-PAPC, induce gene expression have not been studied in detail. In an effort to understand the molecular mechanisms of Ox-PAPC-induced gene expression and whether similarities in signal transduction pathways exist between inflammatory cytokines and Ox-PAPC, Yeh et al. (2001) recently examined the regulation of IL-8 by Ox-PAPC and TNF-a in HAEC. Transient transfection studies, using reporter constructs containing 1.4 kb of the IL-8 promoter, identified an Ox-PAPC-specific response region between 133 and 1481 bp of the IL-8 promoter. In contrast, induction of the IL-8 promoter by TNF-a is mediated almost entirely through the NF-kB, AP-1 and C/EBP-b response elements located between 70 and 131 bp of the IL-8 promoter. These studies provided the first evidence that the mechanisms of signal transduction utilized by oxidized phospholipids, such as Ox-PAPC, are different from those utilized by pro-inflammatory cytokines such as TNF-a, IL-1b, IL-6 and LPS. Hans et al. (Lee et al., 2000) demonstrated that OxPAPC activates peroxisome – proliferator activator receptor response element (PPRE) and showed that PPAR-a is required for the production of MCP-1 in mouse aortic endothelial cells (Lee et al., 2000). Although functional PPRE sequences have been reported as indispensable cisenhancing elements as far as 2 kb upstream to the transcription initiation site (Barbera et al., 2001), a classical PPRE is not found between 1481 and 133 bp in the human IL-8 promoter. It is clear from these studies that the target DNA sequences, transcription factors and the signaling mechanisms required for oxidized phospholipid-induced gene expression are unique and previously unidentified. Based on their ability to induce endothelial/monocyte interactions, we hypothesized that oxidized phospholipids regulate the expression of a number of important genes. We
also hypothesized, based on the recent studies on Ox-PAPCmediated IL-8 transcription, that oxidized phospholipids induce gene expression in target cells through novel signal transduction mechanisms. We first sought to determine the changes in gene expression in Ox-PAPC-treated HAEC. We exposed HAEC to either PAPC or Ox-PAPC and used the recently described suppression subtractive hybridization (SSH) technique to identify differences in mRNA expression. The SSH technique utilizes subtractive hybridization and polymerase chain reaction (PCR) to generate a population of PCR fragments enriched for gene sequences of high and low abundance that are differentially expressed (Diatchenko et al., 1996). Using the SSH procedure, we cloned sequences, which are (i) induced by Ox-PAPC but not by PAPC and (ii) induced/present in PAPC treated HAEC but inhibited/absent in Ox-PAPC-treated HAEC. In this paper, we report the set of genes induced by OxPAPC in HAEC. The observed pattern of gene induction suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing. Future studies aimed at the transcriptional regulation of this new group of Ox-PAPC-induced genes will allow us to identify the signal transduction pathways utilized by oxidized phospholipids to induce gene expression in target cells.
2. Materials and methods 2.1. Materials All cell culture reagents were purchased from GIBCOBRL (Grand Island, NY). L-a-1-Palmitoyl-2-arachidonoylsn-glycero-3-phosphorylcholine was obtained from Avanti Polar Lipids (Alabaster, AL). Ox-PAPC was prepared as described previously (Watson et al., 1997). Endotoxin levels in all lipid and lipoprotein preparations were below 20 pg/ml. 2.2. Cell culture HAEC were isolated and cultured as described previously (Navab et al., 1991) and were used at Passage 6. HAEC were plated at a density of 2105 cells/cm2 and were allowed to grow, forming a confluent monolayer in 2 days. The day before experiments, HAEC were shifted to M199 medium containing 10% lipoprotein deficient serum (LPDS). PAPC and Ox-PAPC at a concentration of 50 mg/ ml were added to the cultures and total RNA was harvested at 1-, 2- and 4-h time points. 2.3. RNA isolation and northern analysis Total RNA from cell cultures was purified using the RNeasy kit (Qiagen, Valencia, CA). Poly(A)+ RNA was isolated from total RNA using the PolyATtract mRNA
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Schuell, Keene, NH). The filters were placed on freshly prepared LB/ampicillin agar plates and incubated overnight at 37 C. The membranes were removed from the plates, bacteria were lysed and the DNA denatured by incubating the filters (colony side up) in a solution containing 0.5 N NaOH/1.5 M NaCl for 3 min. The DNA was then neutralized by placing the filters in a 0.5 M Tris – HCl (pH 7.5)/3 M NaCl solution for an additional 3 min followed by a final 3-min incubation in 2 SSC. The filters were dried and baked in a vacuum oven for 2 h at 80 C. The filters were hybridized to 32 P-dCTP-labeled cDNA probes from the forward and reverse subtractions. Following identification of the differentially expressed clones (see Section 2.6), the inserts were released from the corresponding plasmid DNA by EcoR1 digestion and used as probes on northern blots. Fig. 1. Ox-PAPC induces IL-8 and Gro-a expression in HAEC. Prior to the subtraction protocol, HAEC were tested for their response to Ox-PAPC. HAEC were treated with Ox-PAPC for 0, 1, 2 and 4 h, and total RNA was isolated and subjected to northern analysis using IL-8, Gro-a and GAPDH cDNA probes.
Isolation System III (Promega, Madison, WI) according to manufacturer’s protocol. For northern analysis, 10 mg of total RNA were subjected to electrophoresis on 1% agarose gel, transferred to Hybond-N membranes (Amersham Pharmacia Biotech, Piscataway, NJ) and hybridized with 32Plabeled DNA of selected clones from the subtracted library. GAPDH was used as a loading control for the northerns. Electrophoresis and hybridization protocols were described previously (Reddy et al., 1997).
2.6. Labeling cDNA and dot blot hybridization The forward and reverse subtracted cDNA were digested with Rsa1 and Sma1 to remove the SSH adapters. The adapter-free cDNA was resolved by electrophoresis on a 2% agarose gel and purified from the agarose gels using ‘‘QIAquick’’ gel extraction kit (Qiagen). A total of 100 ng of each cDNA was labeled with 32P-dCTP (ICN Biochemicals, Costa Mesa, CA) by random priming method. The labeled cDNA was further purified using Nick columns (Amersham) and equal counts were then added to the appropriate filters. For hybridizing the labeled probe, the
2.4. SSH Poly(A)+ RNA from HAEC treated with Ox-PAPC for 1, 2 and 4 h were pooled and similarly poly(A)+ RNA from HAEC treated with PAPC for 1, 2 and 4 h were pooled prior to the SSH procedure. A total of 2 mg each of pooled poly(A)+ RNA from Ox-PAPC- and PAPC-treated HAEC were used as the starting material for each of the SSH procedures. The entire SSH protocol, including reverse transcription of poly(A)+ RNA and generation of subtracted cDNA molecules, was performed using the PCR-Select cDNA subtraction kit (Clonetech, Palo Alto, CA) according to the manufacturer’s protocol. All PCR amplifications were carried out using the Advantage polymerase mix (Clonetech) and the Peltier Thermal Cycler-200 (MJ Research). 2.5. Cloning and analysis of the subtracted cDNA library The products from the secondary PCR (SSH protocol) were cloned into the PCRII-TOPO vector (Invitrogen, Carlsbad, CA). Individual clones, following E. coli transformation, were grown overnight and plasmid DNA was isolated. For colony dot blot screening, identical blots were prepared by arraying 1 ml of overnight bacterial cultures on ‘‘Optitran’’ nitrocellulose transfer membrane filters (Schleicher and
Fig. 2. Subtracted cDNA was obtained following SSH. Following subtraction, the subtracted material (equal volumes, 8 ml) was run on a 2% agarose gel. Lane 1: DNA size markers; Lane 2: forward subtraction, OxPAPC-treated HAEC subtracted from PAPC-treated HAEC; Lane 3: reverse subtraction; PAPC-treated HAEC subtracted from Ox-PAPC-treated HAEC; and Lane 4: positive control subtraction, skeletal muscle cDNA doped with HaeIII digested fX174 DNA subtracted from skeletal muscle cDNA.
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for 45 min. The filters were exposed to Kodak X-ray film at 80 C overnight. 2.7. Sequencing and analysis of selected clones Following dot blot hybridization, the differentially expressed clones were sequenced using ‘‘M13’’ forward and reverse primers and the dye termination procedure, at the UCLA sequencing core facility. Homology searches against GenBank databases were done using BLAST at the WWW server for National Center for Biotechnology Information (Bethesda, MD).
3. Results 3.1. Selection of HAEC for the SSH procedure
Fig. 3. Monitoring the expression of a positive control gene, IL-8 and a constitutive gene, GAPDH, validated the subtraction procedure. (A) Equal amounts of cDNA from PAPC-treated HAEC (PAPC), Ox-PAPC-treated HAEC (Ox-PAPC), the reverse subtraction (PAPC – Ox-PAPC) and the forward subtraction (Ox-PAPC – PAPC) were subjected to southern analysis with an IL-8 probe as described in Section 2. (B) PCR amplification was performed (18 cycles) on equal amounts of cDNA from Ox-PAPC-treated HAEC (Ox-PAPC), PAPC-treated HAEC (PAPC), the reverse subtraction (PAPC – Ox-PAPC) and the forward subtraction (Ox-PAPC – PAPC) with GAPDH 50 and 30 primers provided in the PCR select kit (Clonetech).
filters were first prehybridized at 65 C for 4 h in a solution containing 5 SSC/5 Denhardt’s/0.5% SDS/ single stranded salmon sperm DNA (100 mg/ml). The hybridization was performed at 65 C overnight, and the filters were washed three times at room temperature in 2 SSC/0.5% SDS and once at 65 C in 0.1 SSC/0.5% SDS
HAEC in our laboratory are derived from primary cultures, and therefore can have batch-specific quantitative variations in their response to a variety of agents including Ox-PAPC. We previously reported that MM-LDL and OxPAPC induce chemokines such as IL-8, MCP-1 and Gro-a (Lee et al., 2000; Navab et al., 1991) in HAEC. We chose several batches of HAEC, which had been frozen at an early passage (Passages 2 and 3) to identify the HAEC, which responded best to Ox-PAPC. Eight different batches of HAEC were treated with Ox-PAPC (50 mg/ml) or PAPC (50 mg/ml) for 4 h, and total RNA were analyzed by northern analysis for the expression of IL-8 and Gro-a. In all of the HAEC tested, Ox-PAPC induced the expression of both IL-8 and Gro-a, however, as expected, the degree of induction varied between batches (data not shown). We chose the batch of HAEC, which had the highest OxPAPC-mediated induction of IL-8 and Gro-a (approxi-
Fig. 4. Dot blot analysis of individual clones from the subtracted library. Colony dot blot analysis of differentially expressed genes. Two identical dot blots were prepared using overnight cultures of 80 bacterial colonies, which were randomly picked following TA cloning protocol of the forward subtraction. Blot A was hybridized with 32P-labeled cDNA prepared from Ox-PAPC-treated HAEC and Blot B was hybridized with 32P-labeled cDNA prepared from PAPC-treated HAEC. Colonies that hybridized with the forward subtraction probe but not the reverse subtraction probe were selected for further analysis.
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ment, HAEC had to be put through additional passages. We therefore tested whether the batch of HAEC chosen for the SSH experiments were responsive to Ox-PAPC at Passage 6. These cultures were treated with Ox-PAPC (50 mg/ml), and total RNA was isolated at 1, 2 and 4 h later. A total of 10 mg of total RNA was subjected to northern analysis using IL-8 and Gro-a cDNA probes. Both IL-8 and Gro-a were rapidly induced by Ox-PAPC (Fig. 1). 3.2. Subtraction hybridization
Fig. 5. Confirmation of clones, selected by colony dot blot analysis, by northern analysis. Representative northern blot analysis of genes selected following colony dot blot analysis. A total of 10 mg of total RNA was loaded in each lane. The positive clones following northern analysis were sequenced.
mately 50- and 20-fold induction, respectively) for our SSH experiments. The SSH procedure requires a minimum of 2 mg of poly(A+) RNA from each of the starting population of cells. Based on our calculation, we needed a minimum of 40 confluent 10-cm dishes of HAEC for the SSH experiment. We have routinely used early passages of primary HAEC in our experiments (Passages 2 and 3) (Navab et al., 1988). In order to obtain enough cells for the SSH experi-
Subtractive hybridization was performed between OxPAPC- and PAPC-treated HAEC (forward subtraction) and between PAPC- and Ox-PAPC-treated HAEC (reverse subtraction). As a control for the SSH procedure, skeletal muscle cDNA doped with HaeIII digested fX174 bacteriophage DNA was subtracted from skeletal muscle cDNA. In both forward and reverse subtractions, we obtained the characteristic smear of subtracted DNA and more importantly the control subtraction amplified the HaeIII digested fX174 bacteriophage DNA fragments at the expected sizes (Fig. 2). 3.3. Subtraction efficiency To determine the success of subtraction protocol, we first tested whether differentially expressed sequences such as IL-8 and Gro-a were enriched following forward subtraction and whether constitutively expressed housekeeping genes such as GAPDH were eliminated in both the forward and reverse subtractions. IL-8 was highly enriched in the cDNA from the forward subtraction suggesting that this SSH procedure enriched for genes, which
Table 1 Identity and characteristics of known Ox-PAPC-induced genes Gene
Gene product and function
Reference
MKP-1
Keyse, 2000
IL-8 HBGF-1 Fe – H chain uPAR
Mitogen-activated protein kinase phosphatase-1; nuclear, nonreceptor dual-specificity protein phosphatase, inactivates MAPKs Annexins are a family of proteins with calcium-dependent phospholipid binding properties. Annexin II is a coreceptor for tissue plasminogen activator (t-PA) and plasminogen Interleukin-8, a potent neutrophil chemotactic factor Heparin-binding growth factor-1, also known as acidic-FGF, mitogen and chemoattractant for EC Ferritin heavy chain, iron homeostasis Urokinase plasminogen activator receptor, cell migration and adhesion
ThB4 TSP-1
Thymosin-b4, is an actin sequestering protein and has angiogenic properties Thrombospondin-1, an ECM protein and a ligand for the scavenger receptor CD36
CD59
CD59 is also known as homologous restriction factor 20 or protectin, maintains vascular integrity during coagulation Transcription factor Lamin B2 Translation elongation factor-1 Putative tumor suppressor ST13 Oxidase (cytochrome c) assembly 1-like protein
Annexin II
FosB LAMB2 EF-1 ST13 OXA1L
Kang et al., 1999; Raynal and Pollard, 1994 Mukaida, 2000 Winkles et al., 1987 Percy et al., 1998 Lundgren et al., 1994; Preissner et al., 1999 Malinda et al., 1999 Dawson et al., 1997; Tuszynski and Nicosia, 1996 Lidington et al., 2000 Tulchinsky, 2000 Wilson et al., 2001 Negrutskii and El’skaya, 1998 Zhang et al., 1998 Molina-Gomes et al., 1995
Genes induced by Ox-PAPC in HAEC. Summary of the known genes differentially expressed in Ox-PAPC-treated HAEC. Candidate clones chosen following northern blot analysis were sequenced. The sequences were analyzed using the BLAST program in the NCBI databases and the corresponding genes were identified.
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are differentially expressed after exposure to Ox-PAPC (Fig. 3A). Furthermore, GAPDH was practically eliminated in both forward and reverse subtractions as compared to the starting cDNA from both Ox-PAPC- and PAPCtreated HAEC (Fig. 3B), further confirming that the SSH procedure was successful. 3.4. Identification of differentially expressed genes by colony dot blot analysis To determine the clones that were differentially expressed in Ox-PAPC-treated HAEC, the forward subtraction products were first shotgun cloned into the TA vector. Eighty individual TA vector clones with distinct insert sizes were chosen for colony dot blot analysis. Two identical dot blots (A and B) were prepared using overnight cultures of the 80 bacterial colonies. Blot A was hybridized with 32Plabeled cDNA prepared from Ox-PAPC-treated HAEC and Blot B was hybridized with 32P-labeled cDNA prepared from PAPC-treated HAEC. Several colonies hybridized with the forward subtraction probe but not the reverse subtraction probe (Fig. 4). 3.5. Clones selected by colony dot blot analysis were confirmed by northern analysis To further confirm the results from the colony dot blot analysis, inserts were isolated from the positive clones and used as probes on northern blots containing total RNA from Ox-PAPC- and PAPC-treated HAEC (a representative northern is shown in Fig. 5). Twenty-nine percent (23 out of 80) of the clones obtained from the shotgun cloning approach turned out to be differentially expressed genes. The positive clones were sequenced, and the identity of the genes was obtained by searching NCBI databases (Table 1).
4. Discussion Understanding the biology of multifactorial physiological and pathophysiological processes requires identification of sequential and quantitative changes in gene expression. DNA arrays, although expensive and not easily accessible to researchers at the present time, have greatly simplified the process of molecular dissection of changes in gene expression. However, the lack of a complete set of human genes prevents the identification of important unknown genes when using array technology. Several new advances as well as recent improvements to molecular biology techniques allow the high throughput identification of differentially expressed genes in any experimental context. SSH, a PCR-based subtraction technique, has been successfully used to identify differentially expressed sequences in diseased cells as well as tissues (Fang et al., 2000; Pitzer et al., 1999; Diatchenko et al., 1999). In this paper, we have successfully used the SSH procedure to
identify a number of genes whose expression is induced by Ox-PAPC in HAEC. Some of the genes identified after exposure to Ox-PAPC were induced as early as 15 min (data not shown). All of the genes were induced strongly within 4 h of exposure of the HAEC to Ox-PAPC. IL-8 and Gro-a were used as markers to select the cells that would maximally respond to OxPAPC (Fig. 1) and they were detected in the SSH screen as would be expected. One of the genes identified by this screen was mitogen-activated protein kinase phosphatase-1 (MKP1). We have recently shown that inhibition of MKP-1 using either the phosphatase inhibitor sodium orthovanadate or antisense oligonucleotides prevents the accumulation of monocyte chemotactic activity in Ox-PAPC treated HAEC supernatants (Reddy et al., 2001). These results suggest a key role for MKP-1 in oxidized lipid-mediated induction of monocyte/endothelial cell interactions. Our laboratory is currently studying the role of MKP-1 in animal models of atherosclerosis. The known genes (Table 1) identified in the SSH screen to be differentially induced by oxidized phospholipids encode for proteins involved in a multitude of physiological and pathophysiological phenomena including angiogenesis, atherosclerosis, inflammation and wound healing. To our knowledge, this is the first report of regulation at the level of message induction by oxidized phospholipids for all of the genes we identified. Four of the genes identified as Ox-PAPC-induced genes encode for proteins that are associated with functions related to plasma membrane and extracellular matrix. These include urokinase plasminogen activator receptor (uPAR), annexin II, thrombospondin 1 and thymosin-b4. uPAR is critical for cell migration (Lundgren et al., 1994), as well as in cell adhesion (Preissner et al., 1999), and plays an important role in cardiovascular biology. Annexins are a family of proteins with calcium-dependent phospholipid-binding properties. Annexin II, which is a coreceptor for tissue plasminogen activator (t-PA) and plasminogen (Kang et al., 1999), plays a role in thrombolysis and cell migration. Thrombospondin1 (TSP-1) is an extracellular matrix protein ligand for the CD36 receptor (Dawson et al., 1997) and is a potent negative regulator of angiogenesis, inhibiting endothelial cell proliferation and migration (Tuszynski and Nicosia, 1996). TSP-1 also plays a role in the mitogen-dependent proliferation of vascular smooth muscle cells (Roth et al., 1998). Thymosin-b4 is an actin sequestering protein that is angiogenic and promotes reepithelialization and wound healing (Malinda et al., 1999). Heparin-binding growth factor-I (HBGF-I or FGF-1 or acidic FGF) was identified by the SSH screen as being induced by Ox-PAPC. This protein is a potent mitogen and chemoattractant for endothelial cells in vitro and induces angiogenesis in vivo (Winkles et al., 1987). Moreover, oxidized LDL was previously shown to mediate the release of FGF-1 protein in endothelial cells (Ananyeva et al., 1997). Thus, three of the genes identified in
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this screen play a role in angiogenesis: TSP-1, thymosinb4 and FGF-1. Ferritin heavy chain (H-chain) was also induced by Ox-PAPC. This protein was identified previously by a comparative hybridization screen of cDNA isolated from atherosclerotic vs. normal human aorta (Pang et al., 1996), and is known to be induced in artery wall cells as a consequence of LDL oxidation (Van Lenten et al., 1995). CD59 is expressed on endothelial cells and is thought to help maintain vascular integrity during coagulation associated with complement activation (Lidington et al., 2000). CD59 is thought to protect against complement C5b-9 attack in human atherosclerotic lesions (Seifert et al., 1992). Loss of CD59 from cells compromised by ischemia/reperfusion may contribute to direct damage of the coronary vascular bed by the terminal complement complex (Chakraborti et al., 2000). The SSH screen also revealed that the putative tumor suppressor ST13 (Zhang et al., 1998), FOSB, lamin B2, translation elongation factor and an oxidase (cytochrome c) assembly 1-like protein were all induced by Ox-PAPC in HAEC. Future studies will determine whether these genes play a role in Ox-PAPC-mediated functional changes in endothelial cells. Additionally, nine other genes that are not in the databases were also induced by Ox-PAPC. Taking into account the fact that 9 out of 14 (Table 1) known genes isolated by SSH are associated with the biology of angiogenesis and inflammation, we are confident that characterization of the 9 unknown genes will result in important findings. The nature of the known genes induced by OxPAPC suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing. Finally, understanding the transcriptional regulation of this new group of Ox-PAPC-induced genes will allow the identification of (i) Ox-PAPC response elements (OPREs), (ii) transcription factors that participate in oxidized phospholipid-mediated gene expression and (iii) signaling pathways utilized by oxidized phospholipids to transduce their effects to the nucleus.
Acknowledgments The authors thank Dr. Jeff Smith (UCLA) for his advice on the SSH protocols, Greg Hough, Alan Wagner and Linda Jin for technical support. This work was supported by USPHS grant HL 30568, the Laubisch, Castera and M.K. Grey Fund at UCLA.
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Seifert, P.S., Roth, I., Schmiedt, W., Oelert, H., Okada, N., Okada, H., Bhakdi, S., 1992. CD59 (homologous restriction factor 20), a plasma membrane protein that protects against complement C5b-9 attack, in human atherosclerotic lesions. Atherosclerosis 96, 135 – 145. Subbanagounder, G., Leitinger, N., Schwenke, D.C., Wong, J.W., Lee, H., Rizza, C., Watson, A.D., Faull, K.F., Fogelman, A.M., Berliner, J.A., 2000. Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position. Arterioscler., Thromb. Vasc. Biol. 20, 2248 – 2254. Subbanagounder, G., Wong, J.W., Lee, H., Faull, K.F., Miller, E., Witztum, J.L., Berliner, J.A., 2001. Epoxyisoprostane and epoxycyclopentenone phospholipids regulate monocyte chemotactic protein-1 and interleukin8 synthesis: formation of these oxidized phospholipids in response to interleukin-1 {beta}. J. Biol. Chem. 277, 7271 – 7281. Tulchinsky, E., 2000. Fos family members: regulation, structure and role in oncogenic transformation. Histol. Histopathol. 15, 921 – 928. Tuszynski, G.P., Nicosia, R.F., 1996. The role of thrombospondin-1 in tumor progression and angiogenesis. BioEssays 18, 71 – 76. Van Lenten, B.J., Prieve, J., Navab, M., Hama, S., Lusis, A.J., Fogelman, A.M., 1995. Lipid-induced changes in intracellular iron homeostasis in vitro and in vivo. J. Clin. Invest. 95, 2104 – 2110. Watson, A.D., Leitinger, N., Navab, M., Faull, K.F., Horkko, S., Witztum, J.L., Palinski, W., Schwenke, D., Salomon, R.G., Sha, W., Subbanagounder, G., Fogelman, A.M., Berliner, J.A., 1997. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo. J. Biol. Chem. 272, 13597 – 13607. Watson, A.D., Subbanagounder, G., Welsbie, D.S., Faull, K.F., Navab, M., Jung, M.E., Fogelman, A.M., Berliner, J.A., 1999. Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein. J. Biol. Chem. 274, 24787 – 24798. Wilson, K.L., Zastrow, M.S., Lee, K.K., 2001. Lamins and disease: insights into nuclear infrastructure. Cell 104, 647 – 650. Winkles, J.A., Friesel, R., Burgess, W.H., Howk, R., Mehlman, T., Weinstein, R., Maciag, T., 1987. Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor). Proc. Natl. Acad. Sci. U.S.A. 84, 7124 – 7128. Yeh, M., Leitinger, N., de Martin, R., Onai, N., Matsushima, K., Vora, D.K., Berliner, J.A., Reddy, S.T., 2001. Increased transcription of IL8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids. Arterioscler., Thromb. Vasc. Biol. 21, 1585 – 1591. Zhang, Y., Cai, X., Schlegelberger, B., Zheng, S., 1998. Assignment of human putative tumor suppressor genes ST13 (alias SNC6) and ST14 (alias SNC19) to human chromosome bands 22q13 and 11q24!q25 by in situ hybridization. Cytogenet. Cell Genet. 83, 56 – 57.
Identification of genes induced by oxidized phospholipids in human aortic endothelial cells Srinivasa T. Reddy*, Victor Grijalva, Carey Ng, Khaled Hassan, Susan Hama, Rachel Mottahedeh, David J. Wadleigh, Mohamad Navab, Alan M. Fogelman Atherosclerosis Research Unit, University of California, Los Angeles, CA 90095-1679, USA
Abstract Oxidized-L-a-1-Palmitoyl-2-Arachidonoyl-sn-glycero-3-Phosphorylcholine (Ox-PAPC), a component of mildly oxidized/minimally modified low-density lipoprotein (MM-LDL), accounts for many of the biological activities of MM-LDL. Having hypothesized that OxPAPC initiates gene expression changes in endothelial cells that result in enhanced endothelial/monocyte interactions and the subsequent development of atherosclerotic lesions, we used the suppression subtractive hybridization (SSH) procedure to compare mRNA isolated from PAPC-treated human aortic endothelial cells (HAEC) with mRNA isolated from Ox-PAPC-treated cells. Genes induced by Ox-PAPC but not by PAPC in HAEC included genes involved in signal transduction, extracellular matrix, growth factors, chemokines and several genes with unknown functions. The observed pattern of gene induction suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing. D 2002 Elsevier Science Inc. All rights reserved. Keywords: MM-LDL; Ox-PAPC; Gene expression; Endothelial cell function; Atherosclerosis; Suppression subtractive hybridization
1. Introduction The vascular endothelium directs the inflammatory response by regulating the production of adhesion molecules and cytokines in response to various agents (Ross, 1999). The entrapment and oxidation of low-density lipoproteins (LDL) in the subendothelial space results in the seeding of LDL with reactive oxygen species (e.g., hydroperoxyoctadecadienoic acid (HPODE), hydroperoxyeicosatetraenoic acid (HPETE) and cholesterol linoleate hydroperoxide, which may result from the action of HPODE and HPETE on cholesterol linoleate) (Navab et al., 2000a,b). When these
Abbreviations: SSH, Suppression subtractive hybridization; HDL, highdensity lipoprotein; LDL, low-density lipoprotein; MM-LDL, mildly oxidized LDL; Apo, apolipoprotein; PAPC, 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphocholine; Ox-PAPC, oxidized PAPC; MKP-1, mitogenactivated protein kinase phosphatase-1; MCP-1, monocyte chemoattractant protein-1; IL-8, interleukin-8; HPODE, hydroperoxyoctadecadienoic acid; HPETE, hydroperoxyeicosatetraenoic acid. * Corresponding author. Department of Medicine and Department of Molecular and Medical Pharmacology, University of California Los Angeles, 650 Charles E. Young Drive South, A8-131 CHS, Los Angeles, CA 90095, USA. Tel.: +1-310-206-3915; fax: +1-310-206-3605. E-mail address: [email protected] (S.T. Reddy).
reactive oxygen species reach a critical threshold in LDL, a nonenzymatic oxidation of LDL phospholipids that contain arachidonic acid produces biologically active oxidized phospholipids (Navab et al., 2000a,b). Subbanagounder et al. (2000) demonstrated that oxidation of arachidonic acid in the sn-2 position of an intact phospholipid molecule is required for biologic activity. The most common phospholipid that provides the substrate for this oxidation is L-a-1-Palmitoyl2-Arachidonyl-sn-gycero-3-Phosphorylcholine (PAPC). The oxidation of PAPC (Ox-PAPC) has been shown to account for many of the biologic activities of mildly oxidized/ minimally modified LDL (MM-LDL) (Watson et al., 1997). The structures of three of the biologically active components of Ox-PAPC have been identified as 1-palmitoyl-2-oxovaleryl-sn-gycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaryl-sn-gycero-3-phosphorylcholine (PGPC) and 1-palmitoyl-2-(5,6-epoxyisoprostane E 2 )-sn-glycero-3-phosphorylcholine (PEIPC) (Watson et al., 1997, 1999). These structurally similar oxidized phospholipids have been shown to differentially regulate endothelial binding of monocytes and neutrophils (Leitinger et al., 1999), suggesting that oxidized phospholipids may play a general role in inflammation, atherosclerosis and wound healing, and thus influence the expression of a number of important gene families.
1537-1891/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 7 - 1 8 9 1 ( 0 2 ) 0 0 1 7 1 - 4
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It is evident that the biologically active components of Ox-PAPC regulate gene expression in target cells (Lee et al., 2000; Subbanagounder et al., 2001). Ox-PAPC induces inflammatory cytokines and adhesion molecules in human aortic endothelial cells (HAEC), similar to other inflammatory cytokines such as TNF-a and LPS. Both TNF-a and Ox-PAPC activate endothelial cells to bind monocytes partly through the induction of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) (Leitinger et al., 1997). Interestingly, Ox-PAPC, but not TNF-a, enhances monocyte – endothelial binding by mediating activation of b1-integrins resulting in deposition of fibronectin-containing CS-1 on the apical cell surface (Leitinger et al., 1997). On the other hand, TNF-a enhances the endothelial/monocyte interactions through the induction of E-selectin and VCAM-1, which are not affected by OxPAPC (Leitinger et al., 1997). Thus, Ox-PAPC and TNF-a utilize both similar and distinct induction patterns of gene expression to initiate endothelial/monocyte interactions. The molecular mechanisms of cytokine-induced gene expression are well understood, however, the target receptors, the signaling pathways and the molecular mechanisms by which oxidized phospholipids, such as Ox-PAPC, induce gene expression have not been studied in detail. In an effort to understand the molecular mechanisms of Ox-PAPC-induced gene expression and whether similarities in signal transduction pathways exist between inflammatory cytokines and Ox-PAPC, Yeh et al. (2001) recently examined the regulation of IL-8 by Ox-PAPC and TNF-a in HAEC. Transient transfection studies, using reporter constructs containing 1.4 kb of the IL-8 promoter, identified an Ox-PAPC-specific response region between 133 and 1481 bp of the IL-8 promoter. In contrast, induction of the IL-8 promoter by TNF-a is mediated almost entirely through the NF-kB, AP-1 and C/EBP-b response elements located between 70 and 131 bp of the IL-8 promoter. These studies provided the first evidence that the mechanisms of signal transduction utilized by oxidized phospholipids, such as Ox-PAPC, are different from those utilized by pro-inflammatory cytokines such as TNF-a, IL-1b, IL-6 and LPS. Hans et al. (Lee et al., 2000) demonstrated that OxPAPC activates peroxisome – proliferator activator receptor response element (PPRE) and showed that PPAR-a is required for the production of MCP-1 in mouse aortic endothelial cells (Lee et al., 2000). Although functional PPRE sequences have been reported as indispensable cisenhancing elements as far as 2 kb upstream to the transcription initiation site (Barbera et al., 2001), a classical PPRE is not found between 1481 and 133 bp in the human IL-8 promoter. It is clear from these studies that the target DNA sequences, transcription factors and the signaling mechanisms required for oxidized phospholipid-induced gene expression are unique and previously unidentified. Based on their ability to induce endothelial/monocyte interactions, we hypothesized that oxidized phospholipids regulate the expression of a number of important genes. We
also hypothesized, based on the recent studies on Ox-PAPCmediated IL-8 transcription, that oxidized phospholipids induce gene expression in target cells through novel signal transduction mechanisms. We first sought to determine the changes in gene expression in Ox-PAPC-treated HAEC. We exposed HAEC to either PAPC or Ox-PAPC and used the recently described suppression subtractive hybridization (SSH) technique to identify differences in mRNA expression. The SSH technique utilizes subtractive hybridization and polymerase chain reaction (PCR) to generate a population of PCR fragments enriched for gene sequences of high and low abundance that are differentially expressed (Diatchenko et al., 1996). Using the SSH procedure, we cloned sequences, which are (i) induced by Ox-PAPC but not by PAPC and (ii) induced/present in PAPC treated HAEC but inhibited/absent in Ox-PAPC-treated HAEC. In this paper, we report the set of genes induced by OxPAPC in HAEC. The observed pattern of gene induction suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing. Future studies aimed at the transcriptional regulation of this new group of Ox-PAPC-induced genes will allow us to identify the signal transduction pathways utilized by oxidized phospholipids to induce gene expression in target cells.
2. Materials and methods 2.1. Materials All cell culture reagents were purchased from GIBCOBRL (Grand Island, NY). L-a-1-Palmitoyl-2-arachidonoylsn-glycero-3-phosphorylcholine was obtained from Avanti Polar Lipids (Alabaster, AL). Ox-PAPC was prepared as described previously (Watson et al., 1997). Endotoxin levels in all lipid and lipoprotein preparations were below 20 pg/ml. 2.2. Cell culture HAEC were isolated and cultured as described previously (Navab et al., 1991) and were used at Passage 6. HAEC were plated at a density of 2105 cells/cm2 and were allowed to grow, forming a confluent monolayer in 2 days. The day before experiments, HAEC were shifted to M199 medium containing 10% lipoprotein deficient serum (LPDS). PAPC and Ox-PAPC at a concentration of 50 mg/ ml were added to the cultures and total RNA was harvested at 1-, 2- and 4-h time points. 2.3. RNA isolation and northern analysis Total RNA from cell cultures was purified using the RNeasy kit (Qiagen, Valencia, CA). Poly(A)+ RNA was isolated from total RNA using the PolyATtract mRNA
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Schuell, Keene, NH). The filters were placed on freshly prepared LB/ampicillin agar plates and incubated overnight at 37 C. The membranes were removed from the plates, bacteria were lysed and the DNA denatured by incubating the filters (colony side up) in a solution containing 0.5 N NaOH/1.5 M NaCl for 3 min. The DNA was then neutralized by placing the filters in a 0.5 M Tris – HCl (pH 7.5)/3 M NaCl solution for an additional 3 min followed by a final 3-min incubation in 2 SSC. The filters were dried and baked in a vacuum oven for 2 h at 80 C. The filters were hybridized to 32 P-dCTP-labeled cDNA probes from the forward and reverse subtractions. Following identification of the differentially expressed clones (see Section 2.6), the inserts were released from the corresponding plasmid DNA by EcoR1 digestion and used as probes on northern blots. Fig. 1. Ox-PAPC induces IL-8 and Gro-a expression in HAEC. Prior to the subtraction protocol, HAEC were tested for their response to Ox-PAPC. HAEC were treated with Ox-PAPC for 0, 1, 2 and 4 h, and total RNA was isolated and subjected to northern analysis using IL-8, Gro-a and GAPDH cDNA probes.
Isolation System III (Promega, Madison, WI) according to manufacturer’s protocol. For northern analysis, 10 mg of total RNA were subjected to electrophoresis on 1% agarose gel, transferred to Hybond-N membranes (Amersham Pharmacia Biotech, Piscataway, NJ) and hybridized with 32Plabeled DNA of selected clones from the subtracted library. GAPDH was used as a loading control for the northerns. Electrophoresis and hybridization protocols were described previously (Reddy et al., 1997).
2.6. Labeling cDNA and dot blot hybridization The forward and reverse subtracted cDNA were digested with Rsa1 and Sma1 to remove the SSH adapters. The adapter-free cDNA was resolved by electrophoresis on a 2% agarose gel and purified from the agarose gels using ‘‘QIAquick’’ gel extraction kit (Qiagen). A total of 100 ng of each cDNA was labeled with 32P-dCTP (ICN Biochemicals, Costa Mesa, CA) by random priming method. The labeled cDNA was further purified using Nick columns (Amersham) and equal counts were then added to the appropriate filters. For hybridizing the labeled probe, the
2.4. SSH Poly(A)+ RNA from HAEC treated with Ox-PAPC for 1, 2 and 4 h were pooled and similarly poly(A)+ RNA from HAEC treated with PAPC for 1, 2 and 4 h were pooled prior to the SSH procedure. A total of 2 mg each of pooled poly(A)+ RNA from Ox-PAPC- and PAPC-treated HAEC were used as the starting material for each of the SSH procedures. The entire SSH protocol, including reverse transcription of poly(A)+ RNA and generation of subtracted cDNA molecules, was performed using the PCR-Select cDNA subtraction kit (Clonetech, Palo Alto, CA) according to the manufacturer’s protocol. All PCR amplifications were carried out using the Advantage polymerase mix (Clonetech) and the Peltier Thermal Cycler-200 (MJ Research). 2.5. Cloning and analysis of the subtracted cDNA library The products from the secondary PCR (SSH protocol) were cloned into the PCRII-TOPO vector (Invitrogen, Carlsbad, CA). Individual clones, following E. coli transformation, were grown overnight and plasmid DNA was isolated. For colony dot blot screening, identical blots were prepared by arraying 1 ml of overnight bacterial cultures on ‘‘Optitran’’ nitrocellulose transfer membrane filters (Schleicher and
Fig. 2. Subtracted cDNA was obtained following SSH. Following subtraction, the subtracted material (equal volumes, 8 ml) was run on a 2% agarose gel. Lane 1: DNA size markers; Lane 2: forward subtraction, OxPAPC-treated HAEC subtracted from PAPC-treated HAEC; Lane 3: reverse subtraction; PAPC-treated HAEC subtracted from Ox-PAPC-treated HAEC; and Lane 4: positive control subtraction, skeletal muscle cDNA doped with HaeIII digested fX174 DNA subtracted from skeletal muscle cDNA.
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for 45 min. The filters were exposed to Kodak X-ray film at 80 C overnight. 2.7. Sequencing and analysis of selected clones Following dot blot hybridization, the differentially expressed clones were sequenced using ‘‘M13’’ forward and reverse primers and the dye termination procedure, at the UCLA sequencing core facility. Homology searches against GenBank databases were done using BLAST at the WWW server for National Center for Biotechnology Information (Bethesda, MD).
3. Results 3.1. Selection of HAEC for the SSH procedure
Fig. 3. Monitoring the expression of a positive control gene, IL-8 and a constitutive gene, GAPDH, validated the subtraction procedure. (A) Equal amounts of cDNA from PAPC-treated HAEC (PAPC), Ox-PAPC-treated HAEC (Ox-PAPC), the reverse subtraction (PAPC – Ox-PAPC) and the forward subtraction (Ox-PAPC – PAPC) were subjected to southern analysis with an IL-8 probe as described in Section 2. (B) PCR amplification was performed (18 cycles) on equal amounts of cDNA from Ox-PAPC-treated HAEC (Ox-PAPC), PAPC-treated HAEC (PAPC), the reverse subtraction (PAPC – Ox-PAPC) and the forward subtraction (Ox-PAPC – PAPC) with GAPDH 50 and 30 primers provided in the PCR select kit (Clonetech).
filters were first prehybridized at 65 C for 4 h in a solution containing 5 SSC/5 Denhardt’s/0.5% SDS/ single stranded salmon sperm DNA (100 mg/ml). The hybridization was performed at 65 C overnight, and the filters were washed three times at room temperature in 2 SSC/0.5% SDS and once at 65 C in 0.1 SSC/0.5% SDS
HAEC in our laboratory are derived from primary cultures, and therefore can have batch-specific quantitative variations in their response to a variety of agents including Ox-PAPC. We previously reported that MM-LDL and OxPAPC induce chemokines such as IL-8, MCP-1 and Gro-a (Lee et al., 2000; Navab et al., 1991) in HAEC. We chose several batches of HAEC, which had been frozen at an early passage (Passages 2 and 3) to identify the HAEC, which responded best to Ox-PAPC. Eight different batches of HAEC were treated with Ox-PAPC (50 mg/ml) or PAPC (50 mg/ml) for 4 h, and total RNA were analyzed by northern analysis for the expression of IL-8 and Gro-a. In all of the HAEC tested, Ox-PAPC induced the expression of both IL-8 and Gro-a, however, as expected, the degree of induction varied between batches (data not shown). We chose the batch of HAEC, which had the highest OxPAPC-mediated induction of IL-8 and Gro-a (approxi-
Fig. 4. Dot blot analysis of individual clones from the subtracted library. Colony dot blot analysis of differentially expressed genes. Two identical dot blots were prepared using overnight cultures of 80 bacterial colonies, which were randomly picked following TA cloning protocol of the forward subtraction. Blot A was hybridized with 32P-labeled cDNA prepared from Ox-PAPC-treated HAEC and Blot B was hybridized with 32P-labeled cDNA prepared from PAPC-treated HAEC. Colonies that hybridized with the forward subtraction probe but not the reverse subtraction probe were selected for further analysis.
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ment, HAEC had to be put through additional passages. We therefore tested whether the batch of HAEC chosen for the SSH experiments were responsive to Ox-PAPC at Passage 6. These cultures were treated with Ox-PAPC (50 mg/ml), and total RNA was isolated at 1, 2 and 4 h later. A total of 10 mg of total RNA was subjected to northern analysis using IL-8 and Gro-a cDNA probes. Both IL-8 and Gro-a were rapidly induced by Ox-PAPC (Fig. 1). 3.2. Subtraction hybridization
Fig. 5. Confirmation of clones, selected by colony dot blot analysis, by northern analysis. Representative northern blot analysis of genes selected following colony dot blot analysis. A total of 10 mg of total RNA was loaded in each lane. The positive clones following northern analysis were sequenced.
mately 50- and 20-fold induction, respectively) for our SSH experiments. The SSH procedure requires a minimum of 2 mg of poly(A+) RNA from each of the starting population of cells. Based on our calculation, we needed a minimum of 40 confluent 10-cm dishes of HAEC for the SSH experiment. We have routinely used early passages of primary HAEC in our experiments (Passages 2 and 3) (Navab et al., 1988). In order to obtain enough cells for the SSH experi-
Subtractive hybridization was performed between OxPAPC- and PAPC-treated HAEC (forward subtraction) and between PAPC- and Ox-PAPC-treated HAEC (reverse subtraction). As a control for the SSH procedure, skeletal muscle cDNA doped with HaeIII digested fX174 bacteriophage DNA was subtracted from skeletal muscle cDNA. In both forward and reverse subtractions, we obtained the characteristic smear of subtracted DNA and more importantly the control subtraction amplified the HaeIII digested fX174 bacteriophage DNA fragments at the expected sizes (Fig. 2). 3.3. Subtraction efficiency To determine the success of subtraction protocol, we first tested whether differentially expressed sequences such as IL-8 and Gro-a were enriched following forward subtraction and whether constitutively expressed housekeeping genes such as GAPDH were eliminated in both the forward and reverse subtractions. IL-8 was highly enriched in the cDNA from the forward subtraction suggesting that this SSH procedure enriched for genes, which
Table 1 Identity and characteristics of known Ox-PAPC-induced genes Gene
Gene product and function
Reference
MKP-1
Keyse, 2000
IL-8 HBGF-1 Fe – H chain uPAR
Mitogen-activated protein kinase phosphatase-1; nuclear, nonreceptor dual-specificity protein phosphatase, inactivates MAPKs Annexins are a family of proteins with calcium-dependent phospholipid binding properties. Annexin II is a coreceptor for tissue plasminogen activator (t-PA) and plasminogen Interleukin-8, a potent neutrophil chemotactic factor Heparin-binding growth factor-1, also known as acidic-FGF, mitogen and chemoattractant for EC Ferritin heavy chain, iron homeostasis Urokinase plasminogen activator receptor, cell migration and adhesion
ThB4 TSP-1
Thymosin-b4, is an actin sequestering protein and has angiogenic properties Thrombospondin-1, an ECM protein and a ligand for the scavenger receptor CD36
CD59
CD59 is also known as homologous restriction factor 20 or protectin, maintains vascular integrity during coagulation Transcription factor Lamin B2 Translation elongation factor-1 Putative tumor suppressor ST13 Oxidase (cytochrome c) assembly 1-like protein
Annexin II
FosB LAMB2 EF-1 ST13 OXA1L
Kang et al., 1999; Raynal and Pollard, 1994 Mukaida, 2000 Winkles et al., 1987 Percy et al., 1998 Lundgren et al., 1994; Preissner et al., 1999 Malinda et al., 1999 Dawson et al., 1997; Tuszynski and Nicosia, 1996 Lidington et al., 2000 Tulchinsky, 2000 Wilson et al., 2001 Negrutskii and El’skaya, 1998 Zhang et al., 1998 Molina-Gomes et al., 1995
Genes induced by Ox-PAPC in HAEC. Summary of the known genes differentially expressed in Ox-PAPC-treated HAEC. Candidate clones chosen following northern blot analysis were sequenced. The sequences were analyzed using the BLAST program in the NCBI databases and the corresponding genes were identified.
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are differentially expressed after exposure to Ox-PAPC (Fig. 3A). Furthermore, GAPDH was practically eliminated in both forward and reverse subtractions as compared to the starting cDNA from both Ox-PAPC- and PAPCtreated HAEC (Fig. 3B), further confirming that the SSH procedure was successful. 3.4. Identification of differentially expressed genes by colony dot blot analysis To determine the clones that were differentially expressed in Ox-PAPC-treated HAEC, the forward subtraction products were first shotgun cloned into the TA vector. Eighty individual TA vector clones with distinct insert sizes were chosen for colony dot blot analysis. Two identical dot blots (A and B) were prepared using overnight cultures of the 80 bacterial colonies. Blot A was hybridized with 32Plabeled cDNA prepared from Ox-PAPC-treated HAEC and Blot B was hybridized with 32P-labeled cDNA prepared from PAPC-treated HAEC. Several colonies hybridized with the forward subtraction probe but not the reverse subtraction probe (Fig. 4). 3.5. Clones selected by colony dot blot analysis were confirmed by northern analysis To further confirm the results from the colony dot blot analysis, inserts were isolated from the positive clones and used as probes on northern blots containing total RNA from Ox-PAPC- and PAPC-treated HAEC (a representative northern is shown in Fig. 5). Twenty-nine percent (23 out of 80) of the clones obtained from the shotgun cloning approach turned out to be differentially expressed genes. The positive clones were sequenced, and the identity of the genes was obtained by searching NCBI databases (Table 1).
4. Discussion Understanding the biology of multifactorial physiological and pathophysiological processes requires identification of sequential and quantitative changes in gene expression. DNA arrays, although expensive and not easily accessible to researchers at the present time, have greatly simplified the process of molecular dissection of changes in gene expression. However, the lack of a complete set of human genes prevents the identification of important unknown genes when using array technology. Several new advances as well as recent improvements to molecular biology techniques allow the high throughput identification of differentially expressed genes in any experimental context. SSH, a PCR-based subtraction technique, has been successfully used to identify differentially expressed sequences in diseased cells as well as tissues (Fang et al., 2000; Pitzer et al., 1999; Diatchenko et al., 1999). In this paper, we have successfully used the SSH procedure to
identify a number of genes whose expression is induced by Ox-PAPC in HAEC. Some of the genes identified after exposure to Ox-PAPC were induced as early as 15 min (data not shown). All of the genes were induced strongly within 4 h of exposure of the HAEC to Ox-PAPC. IL-8 and Gro-a were used as markers to select the cells that would maximally respond to OxPAPC (Fig. 1) and they were detected in the SSH screen as would be expected. One of the genes identified by this screen was mitogen-activated protein kinase phosphatase-1 (MKP1). We have recently shown that inhibition of MKP-1 using either the phosphatase inhibitor sodium orthovanadate or antisense oligonucleotides prevents the accumulation of monocyte chemotactic activity in Ox-PAPC treated HAEC supernatants (Reddy et al., 2001). These results suggest a key role for MKP-1 in oxidized lipid-mediated induction of monocyte/endothelial cell interactions. Our laboratory is currently studying the role of MKP-1 in animal models of atherosclerosis. The known genes (Table 1) identified in the SSH screen to be differentially induced by oxidized phospholipids encode for proteins involved in a multitude of physiological and pathophysiological phenomena including angiogenesis, atherosclerosis, inflammation and wound healing. To our knowledge, this is the first report of regulation at the level of message induction by oxidized phospholipids for all of the genes we identified. Four of the genes identified as Ox-PAPC-induced genes encode for proteins that are associated with functions related to plasma membrane and extracellular matrix. These include urokinase plasminogen activator receptor (uPAR), annexin II, thrombospondin 1 and thymosin-b4. uPAR is critical for cell migration (Lundgren et al., 1994), as well as in cell adhesion (Preissner et al., 1999), and plays an important role in cardiovascular biology. Annexins are a family of proteins with calcium-dependent phospholipid-binding properties. Annexin II, which is a coreceptor for tissue plasminogen activator (t-PA) and plasminogen (Kang et al., 1999), plays a role in thrombolysis and cell migration. Thrombospondin1 (TSP-1) is an extracellular matrix protein ligand for the CD36 receptor (Dawson et al., 1997) and is a potent negative regulator of angiogenesis, inhibiting endothelial cell proliferation and migration (Tuszynski and Nicosia, 1996). TSP-1 also plays a role in the mitogen-dependent proliferation of vascular smooth muscle cells (Roth et al., 1998). Thymosin-b4 is an actin sequestering protein that is angiogenic and promotes reepithelialization and wound healing (Malinda et al., 1999). Heparin-binding growth factor-I (HBGF-I or FGF-1 or acidic FGF) was identified by the SSH screen as being induced by Ox-PAPC. This protein is a potent mitogen and chemoattractant for endothelial cells in vitro and induces angiogenesis in vivo (Winkles et al., 1987). Moreover, oxidized LDL was previously shown to mediate the release of FGF-1 protein in endothelial cells (Ananyeva et al., 1997). Thus, three of the genes identified in
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this screen play a role in angiogenesis: TSP-1, thymosinb4 and FGF-1. Ferritin heavy chain (H-chain) was also induced by Ox-PAPC. This protein was identified previously by a comparative hybridization screen of cDNA isolated from atherosclerotic vs. normal human aorta (Pang et al., 1996), and is known to be induced in artery wall cells as a consequence of LDL oxidation (Van Lenten et al., 1995). CD59 is expressed on endothelial cells and is thought to help maintain vascular integrity during coagulation associated with complement activation (Lidington et al., 2000). CD59 is thought to protect against complement C5b-9 attack in human atherosclerotic lesions (Seifert et al., 1992). Loss of CD59 from cells compromised by ischemia/reperfusion may contribute to direct damage of the coronary vascular bed by the terminal complement complex (Chakraborti et al., 2000). The SSH screen also revealed that the putative tumor suppressor ST13 (Zhang et al., 1998), FOSB, lamin B2, translation elongation factor and an oxidase (cytochrome c) assembly 1-like protein were all induced by Ox-PAPC in HAEC. Future studies will determine whether these genes play a role in Ox-PAPC-mediated functional changes in endothelial cells. Additionally, nine other genes that are not in the databases were also induced by Ox-PAPC. Taking into account the fact that 9 out of 14 (Table 1) known genes isolated by SSH are associated with the biology of angiogenesis and inflammation, we are confident that characterization of the 9 unknown genes will result in important findings. The nature of the known genes induced by OxPAPC suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing. Finally, understanding the transcriptional regulation of this new group of Ox-PAPC-induced genes will allow the identification of (i) Ox-PAPC response elements (OPREs), (ii) transcription factors that participate in oxidized phospholipid-mediated gene expression and (iii) signaling pathways utilized by oxidized phospholipids to transduce their effects to the nucleus.
Acknowledgments The authors thank Dr. Jeff Smith (UCLA) for his advice on the SSH protocols, Greg Hough, Alan Wagner and Linda Jin for technical support. This work was supported by USPHS grant HL 30568, the Laubisch, Castera and M.K. Grey Fund at UCLA.
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