Published online before print August 19, 2004, doi: 10.1161/01.ATV.0000143097.90868.c8
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© 2004 American Heart Association, Inc.
Vascular Biology |
In Vivo Transcriptional Response of Cardiac Endothelium to Lipopolysaccharide
From Internal Medicine–Cardiology, University of Texas Southwestern Medical Center, Dallas, Tex.
Correspondence to Ralph V. Shohet, MD, Internal Medicine–Cardiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390-8573. E-mail ralph.shohet{at}utsouthwestern.edu
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Abstract |
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Objective— Vascular endothelial cells must integrate stimuli from multiple sources, including plasma, leukocytes, and neighboring components of the vessel. These stimuli are difficult to recapitulate in vitro. We have developed a method to examine the in vivo regulation of gene expression in endothelial cells and have applied it to a model of sepsis.
Methods and Results— We used fluorescent-activated cell sorting to isolate highly purified endothelial cells from the hearts of transgenic mice that express green fluorescent protein driven by the endothelial-specific promoter Tie2. We treated these mice with intraperitoneal lipopolysaccharide and identified those genes within cardiac endothelium that were >3-fold dysregulated 4 and 24 hours later by microarray analysis. These findings were confirmed by real-time polymerase chain reaction and compared with in vitro regulation in a murine endothelial cell line.
Conclusions— The in vivo regulation was distinct and, in general, more robust than that seen in vitro. We identified endothelial-expressed genes not previously recognized to be regulated in response to lipopolysaccharide. This approach provides insight into the cardiac-specific responses of the endothelium that contribute to the specific responses of the heart to sepsis, and can be generalized to the exploration of endothelial responses in any organ.
Fluorescent cell sorting was used to isolate green fluorescent protein expressing endothelial cells from the hearts of mice exposed to lipopolysaccharide. In vivo transcriptional changes were distinct compared with in vitro experiments and identified novel regulated genes. This method can be generalized to the exploration of in vivo endothelial responses in any organ.
Key Words: lipopolysaccharide • microarray • gene expression • endothelial • sepsis
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Introduction |
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The body’s first line of defense when confronted with microbial exposure includes a system of pattern-recognition proteins able to trigger systemic defenses against infection, a response that promotes numerous pathophysiological alterations and can lead to septic shock with a mortality of 30% to 50%.1 This innate immune system, including Toll-like receptors and associated signaling molecules, is activated immediately on cellular exposure to a variety of microbial components produced during infection, including endotoxins such as lipopolysaccharide (LPS), and has been extensively studied in experimental and clinical settings. LPS alone, in the absence of infection, can initiate a series of responses culminating in septic shock. In light of the major vascular effects during sepsis, numerous studies2,3 have examined direct effects of LPS or other endotoxins within cultured endothelial cells and identified several components of the inflammatory response. However, such an experimental strategy may inadequately reflect the clinical scenario, in which inflammatory cells and systemic responses modulate the endothelial response. An examination of the endothelial responses to in vivo LPS exposure should provide a more accurate view of the salient biology and produce new mechanistic insights and therapeutic leads.
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Methods and Materials |
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LPS serotype 0127:B8 (Sigma, St Louis, Mo) was dissolved by sonication in 0.9% saline containing 1% triethylamine and brought to 10 mmol/L Tris·HCl, pH 8, with a final LPS concentration of 200 µg/mL.
Animals
Mice homozygous for Tie2–green fluorescent protein (GFP) transgene (Tg[TIE2GFP]287Sato, stock number 003658) were obtained from Jackson Laboratories (Bar Harbor, Me) and bred for these experiments. Males were used at 8 to 16 weeks of age. Controls were siblings of the treated animals. For LPS exposure, animals were injected intraperitoneally with 1 mL of LPS adjusted to a concentration of 75 µg/mL with 0.9% saline. All procedures were approved by the Institutional Animal Care Committee of the University of Texas Southwestern Medical Center at Dallas.
Endothelial Cell Isolation
After exposure to LPS or saline for periods of 4 or 24 hours, animals were euthanized by CO2 asphyxiation. The hearts were rapidly excised, rinsed with ice cold PBS to remove blood, and diced into 1-mm-sized fragments with a scalpel.
The heart fragments pooled from 2 animals were suspended in 5 mL of Dulbecco’s PBS with dextrose. The suspension was combined with 5 mL of prewarmed PBS containing 5 mg/mL type II collagenase (Worthington) and deoxyribonuclease (300 U), agitated continuously at 37°C on a shaking platform, and triturated 10x every 10 minutes for a total digestion period of 40 to 60 minutes to generate a single cell suspension. The suspension was combined with 10 mL of 10% FBS in DMEM, and cells were collected by brief centrifugation and resuspended in PBS. This suspension was filtered through a sterile 40-µm mesh filter to remove tissue fragments. Cells were again isolated by brief centrifugation and resuspended in 1 mL of red-cell lysis buffer (Sigma). This suspension was carefully overlaid on 5 mL of FBS followed by centrifugation at 1000 rpm for 5 minutes to generate a cellular pellet free of erythrocytes and debris. The pellet was then suspended in 3.5 mL of a solution of PBS (Ca2+- and Mg2+-free) containing dextrose EDTA 0.5 mmol/L, 2800 U of deoxyribonuclease, and 3% FBS, and filtered through a 70-µm mesh filter. After the digestion step, cells were maintained at 4°C throughout the isolation, which lasted 3 to 4 hours in aggregate.
Total cardiac cell suspensions were sorted using a MoFlo from Dako Cytomation. Cells were excited by a 488-nm laser and GFP signals collected via the FL1 channel (510 to 550 nm). A pressure of up to 30 psi was used, generating 5000 to 10 000 events per second. Positive cells were collected into ice-cold DMEM. GFP positive cells were pooled and sorted a second time. Approximately 100 000 GFP positive cells were obtained after the second fluorescence-activated cell sorter (FACS) sorting. The final suspension was pelleted by centrifugation for 5 minutes at 1000 rpm and was carefully aspirated to remove all but 200 µL of supernatant. RNA was extracted from these cells with Trizol (Invitrogen) according to the manufacturer’s protocol and recovered in a volume of 30 µL of water. Ten µL was then subjected to 2 cycles of amplification using the Message Amp kit (Ambion) to produce 
30 µg of amplified RNA after 2 rounds of amplification.
Cell Culture
The SV40-transformed mouse endotheial cells (SVEC4–10) murine endothelial cell line (ATCC #CRL-2181) was maintained in DMEM with 10% FBS. Confluent cultures were used for experimental exposure to LPS, added at concentrations ranging from 50 to 7200 ng/mL in media. PMJ2-PC macrophage cells (ATCC# CRL-2457) were cultured in DMEM with 5% FBS and exposed to LPS (50 ng/mL) for 24 hours. After centrifugation to remove cells, conditioned media were stored frozen at –20°C until use. SVEC cells were exposed to macrophage-conditioned medium combined 1:1 with fresh 10% DMEM±LPS at up to 525 ng/mL final concentration.
Probe Preparation and Array Hybridization
Total RNA was extracted with Trizol and amplified as described above for array hybridizations. Linear RNA amplification can generate adequate probes for array comparisons.4 To control for any amplification-related differences between in vitro and in vivo experiments, SVEC cell RNA was amplified in a manner identical to that used for in vivo RNA. Amplified cRNA (2 µg) was directly labeled with Cy3 and Cy5 with the Micromax ASAP RNA Labeling kit (Perkin Elmer) according to the manufacturer’s instructions. In experiments assessing LPS exposure, microarrays containing the 15 000 gene National Institutes of Health cDNA set5 and microarrays containing 2 copies of the 13 000 gene murine long oligo V1 set (Qiagen, Valencia, Calif) on glass slides were used to directly compare experimental and control RNA samples for gene expression. Scanning was performed with a Genepix instrument (Axon Instruments), and Gene Traffic software (Iobion) was used to analyze the data. Cluster analysis of the array data by the method of self-organizing maps was performed with GeneSpring software (Silicon Genetics).
Real-Time Confirmation
Array results were confirmed by real-time polymerase chain reaction (PCR) assays. For cDNA synthesis, 2 µg quantities of amplified RNA from Tie2-GFP–sorted murine endothelial cells or total SVEC cell RNA were primed with random hexamers and reverse-transcribed with Superscript II (Invitrogen). We designed oligonucleotide amplimers from the cDNA sequences that were predicted to cross an intron; if the mouse gene organization was not available, we used homology to the human gene (sequences are available in Table I, available online at http://atvb.ahajournals.org). These primers were used to amplify product from cDNA representing 5 ng of total RNA. PCR was run in triplicate with SYBR green fluorophore (Molecular Probes) in an Opticon device (MJ Research). Expression level was interpolated from a standard curve generated from a series of dilutions at cycle times where threshold intensity was clearly exceeded. Cyclophilin A was used as an internal control. A standard 2-phase reaction (95°C 15 s, 60°C 1 minute) worked for all amplifications.
In Situ Hybridization
To generate cardiac tissues for analysis, mice were exposed to intraperitoneal LPS (75 µg/animal) or saline and euthanized 4 hours later. Hearts were rapidly prepared for analysis by whole animal perfusion fixation through the left ventricle using ice-cold 4% paraformaldehyde in PBS. Riboprobe templates were generated by PCR-addition of T-7 promoters to the 5' end of the PCR primers listed in Table I. The Maxiscript kit (Ambion) with T-7 RNA polymerase was used to generate 35S-UTP-labeled antisense RNA probes for Lcn2, Cxcl1, Jak1, Igtp, Cycs, Ctsl, B2m, Tbp, Rps4X, and Isg20. RNEasy (Qiagen) column purification was used to purify probes free of unincorporated label. Next, probes were hybridized to LPS or control cardiac tissue sections as described by Zhao et al.6
LPS Determinations
The concentration of LPS in the plasma of mice exposed to intraperitoneal injection of LPS was measured by the limulus amebocyte lysate method (BioWhittaker) according to manufacturer’s instructions. After injection of 75 µg of LPS, mice were euthanized by CO2 asphyxiation at LPS exposure times of 30 minutes to 24 hours. Plasma was obtained from blood samples obtained by cardiac puncture.
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Results |
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Cell Sorting and RNA Amplification
Two rounds of FACS cell sorting of proteolytically digested heart tissue from 2 Tie-2 GFP transgenic mice yielded
100 000 cells. As shown in Figure 1A, these populations were determined by FACS analysis to have >95% GFP-positive cells. In results not shown, qualitative epifluorescence studies of sorted Tie-2 GFP cells indicated 50% of the sorted cells died during isolation. Total RNA from these cells was subsequently amplified by 2 cycles of amplification. As an assessment of the purity of the GFP-positive cells, real-time PCR indicated the relative expression levels of the endothelial-specific gene Tie-2 was 5-fold higher in twice-sorted GFP-positive compared with once-sorted GFP-negative cells (Figure 1B).
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Microarray Results
This study was performed to measure in vivo gene expression alterations within endothelial cells on intraperitoneal LPS exposure, a standard experimental model of sepsis. We compared these in vivo responses to those of a murine endothelial cell line exposed to LPS at 50 ng/mL in culture. The complete microarray data from these studies may be found in the Gene Expression Omnibus database7 in Supplement I. We identified a generalized augmentation of the transcriptional response in vivo, as shown by the cluster analysis presented in Figure 2, which represents dendrograms for the highly regulated genes from individual microarray comparisons. There are substantially more highly regulated genes in RNA from in vivo treated mice, and for those genes that are highly regulated in both models, the in vivo regulation is higher than that obtained in vitro.
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In Vivo LPS Measurements
As determined by the limulus amebocyte lysate assay (Figure I, available online at http://atvb.ahajournals.org), plasma concentrations of LPS reached in excess of 3000 ng/mL within several hours and then rapidly declined after intraperitoneal injection of 75 µg of LPS.
Real-Time PCR Confirmation
Our examination of the cardiac endothelial response to in vivo LPS exposure has revealed several genes undergoing dramatic regulation as assessed by microarray and confirmed by real-time PCR. Microarray experiments identified candidate genes responsive to 75 µg LPS injected intraperitoneally in vivo. Subsequent real-time PCR confirmed several genes dysregulated after 4 hours in vivo LPS treatment (Table). Of those confirmed LPS-responsive genes from in vivo cardiac endothelium, several also exhibit an LPS response within cultured SVEC cells (Figure 3). Lipocalin-2 (Lcn2), glial cell line-derived neurotrophic factor receptor 
-1 (Gfra1), and an unknown gene (BG083535, having some similarity to molybdopterin synthase sulfurylase), were strongly regulated in vivo but only relatively modestly regulated in vitro. The Lipocalin-2 response to LPS at 50 ng/mL in SVEC cells, measured by real-time PCR, was identical whether the source cDNA was derived from total RNA or amplified RNA (results not shown).
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The in vitro dose response to LPS, as assessed by the real-time PCR of Lipocalin-2, was not increased by doses of LPS as high as 7000 ng/mL after 4 or 24 hours (Figure II, available online at http://atvb.ahajournals.org). Also, microarray experiments directly comparing the transcriptional response within RNA from cultured SVEC cells exposed to LPS added at 50 versus 5000 ng/mL revealed no substantial differences after 4 and 24 hours of LPS exposure (results not shown), indicating that the 50 ng/mL concentration is sufficient to evoke a maximal in vitro LPS-response.
Macrophage Humoral Factors
The in vitro response of several genes examined (including Lcn2, Gfra1, and BG083535) could be dramatically increased by combining conditioned culture medium of the LPS-stimulated macrophage cell line PMJ2 with LPS (Figure 4), an effect that partially recapitulates the in vivo response presented here. We attribute this response to multiple cytokines (tumor necrosis factor , interleukins, interferon [IFN]) and other inflammatory mediators known to be secreted into culture media by PMJ2 cells responding to LPS.8
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In Situ Hybridization
To further define the location of in vivo endothelial gene regulation, the 10 most highly regulated genes were further analyzed by in situ hybridization of cardiac tissue from LPS-exposed versus control animals. As shown in Figure 5, transcripts for the genes Lcn2 and Cxcl1 increase on LPS treatment as revealed by increased silver grains over cardiac endothelial cells of coronary arteries. These 2 genes were the most highly regulated genes as determined by real-time PCR, as shown in Figure 3. In situ hybridizations of the other tested transcripts had low or diffuse signal and were not compelling for differences between treated and control tissue (data not shown).
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Discussion |
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Numerous in vivo LPS effects are cell-type specific and, in the case of endothelial responses, involve a host of endogenous pro- and antiinflammatory mediators produced by nonendothelial cell types. The net response to sepsis of the endothelium involves the actions of activated immune cells and increased levels of cytokines produced systemically in response to LPS as well as the paracrine responses of other neighboring vascular components.
Several of the regulated genes that we detected in our in vivo experiments have been recognized in previous studies of responses to LPS or other inflammatory mediators known to be generated during sepsis (Table). Most genes were previously examined in cell culture, peripheral leukocytes, or other ex vivo models.
The mRNA for Lcn2 and Cxcl1 genes was >100-fold more abundant in cardiac endothelial cells from LPS-treated animals, a finding confirmed by in situ analysis. Lcn2 (also called Ngal) is found within neutrophil granules,9 and plasma levels increase during bacterial infection.10 We speculate that endothelial upregulation of Lcn2 on LPS-exposure may contribute to increases in vascular permeability11 and neutrophil extravasation12 observed during sepsis. Our observed increase of Cxcl1 within the coronary artery endothelium on in vivo LPS exposure is reminiscent of the prominent and localized increase of a related chemokine, fractalkine, within coronary arterial endothelium of rats exposed to in vivo LPS.13 (The microarrays used in this report did not contain fractalkine.)
The robust endothelial response to in vivo LPS exposure contrasts with the relatively mild response of SVEC cells to LPS in culture, a difference which can be explained by the absence of key immune cells that facilitate this response, including natural killer cells, natural killer T cells, and macrophages,14 which are known to produce IFN-
15 and possibly other cytokines during the LPS response. In the present study, we partially recapitulate the dramatic in vivo response by costimulation of SVEC cells with LPS and conditioned media from a macrophage PMJ-2 cell line exposed to LPS.
In our study, cytochrome c and Jak1, molecules previously not reported to be transcriptionally regulated by LPS, were identified and confirmed by real-time PCR to undergo 5- to 6-fold upregulation in vivo (versus 2-fold in vitro). In agreement with our in vitro results, published investigation of the LPS responses within endothelia did not uncover transcriptional regulation of these genes. There are potentially important biochemical relationships between these proteins and the LPS response. Jak1 and Jak2 (IFN receptor-associated Janus tyrosine kinase 1 and 2) protein phosphorylation levels have been reported to respond to LPS and IFN signals,16 and the Jak-Stat signaling is a critical component of IFN and other cytokine signal transduction pathways.17 Cytochrome c has been shown to translocate out of mitochondria on LPS stimulation of endothelium18 where it serves as an integral component of the apoptosome.19
BG083535 (dramatically downregulated by LPS in vivo) is an intron-less gene with 80% similarity to human molybdopterin synthase sulfurylase. Genetic deficiencies within the human molybdopterin synthesis pathway result in abnormal neurological development;20 the role of the murine homologue in heart is unknown.
The importance of the targeted approach taken here is underscored by the organ-specific characteristics of endothelia, consistent with the diversity of endothelial types determined in comparisons using gene array technology21,22 and diversity assessed by morphological and cell-biological analysis.23 Recently, FACS sorting has been used to sort endothelial cells from lung, exogenously labeled with fluorescent lipid, with subsequent gene expression analysis,24 an approach which also identified novel gene regulation and supports the more easily generalized strategy described here. One of the limitations of these techniques is the potential for changes in gene expression during the isolation procedure. To address this concern we directly isolated RNA from the hearts of LPS animals. This approach suffers from a distinct type of confounding (from the presence of nonendothelial cells), but can be performed without opportunity for postmortem changes in RNA expression. And indeed, for the same 2 highly regulated genes examined, Lcn2 and Cxcl1, regulation was consistent with that seen in the purified endothelial preparation (results not shown). Moreover, the transcriptional changes from sorted cells correlated with in situ studies, providing an even more compelling demonstration of the authenticity of the results.
Our findings of gene expression changes within cardiac endothelial cells on systemic LPS exposure are interesting in light of the importance of cardiac effects associated with sepsis and advance a new strategy for a realistic assessment of gene regulation in endothelial cells. This methodology can be applied to examinations of organ-specific endothelial gene regulation in response to a wide variety of pathophysiological stimuli, including those that contribute to hypertension and atherosclerosis.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health/National Heart, Lung, and Blood Institute Program in Genomic Applications (to R.V.S.).
Received February 27, 2004; accepted July 30, 2004.
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