Toll-like receptor 4 signaling in dysfunction of cardiac microvascular endothelial cells under hypoxia/reoxygenation
This study was designed to detect the role of Toll-like receptor 4 (TLR4) signaling in the dysfunction of cardiac microvascular endothelial cells (CMECs) after hypoxia/reoxygenation (H/R).
The cell viability of CMECs was measured by MTT assay. The migration of CMECs was detected by cell scratch wound assay. The expressions of TLR4, nuclear factor-kappa B (NF-κB) and eNOS were analyzed by Western blot. Secretions of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined by NO detection kit and ELISA.
Lipopolysaccharide (LPS) incubation increased the expressions of TLR4, NF-κB, IL-6 and TNF-α in CMECs (P < 0.05 vs. control). The CMECs after H/R injury had impaired cell viability (P < 0.01 vs. control) and migration ability (P < 0.05 vs. control). Moreover, the expressions of TLR4, NF-κB, IL-6 and TNF-α were elevated after H/R in CMECs (P < 0.01 vs. control), while NO and the eNOS expression were significantly decreased. In contrast, administration of the TLR4-neutralizing antibody MTS510 prior to H/R injury down-regulated the expressions of IL-6 and TNF-α and attenuated the dysfunction of CMECs.
TLR4 and its signaling components can be activated by LPS and H/R in CMECs. Blocking the TLR4 signal pathway before H/R injury attenuates CMEC dysfunction.
KeywordsToll-like receptor 4 Cardiac microvascular endothelial cells Lipopolysaccharide Hypoxia/reoxygenation Apoptosis
Restoration of blood flow to the ischemic heart may paradoxically exacerbate tissue injury, which is defined as ischemia/reperfusion (I/R) injury, and remains the major cause of cardiac dysfunction in human cardiovascular pathophysiology . A key to the pathogenesis of myocardium I/R injury is dysfunction of endothelial coronary microvessels, within which cardiac microvascular endothelial cells (CMECs) play an important role . CMECs, which comprise up to one-third of the total heart cells , play an important role in the function of coronary microvessels (diameter <150 μm) and regulate the function of adjacent cardiomyocytes through releasing of bioactive molecules such as nitric oxide (NO), prostacyclin andendothelin-1. Accumulating evidence suggests that I/R injury can induce CMEC malfunction which is one of the most important causes of microcirculation dysfunction . Moreover, in the very early stage of reperfusion, the apoptosis of CMECs actually precedes cardiomyocyte apoptosis. Some soluble inflammation mediators from damaged CMECs have been shown to induce cardiomyocyte apoptosis , but the mechanism of CMEC dysfunction induced by I/R injury is still not clear.
The innate immune response has been recognized as the first line of defense to pathogen motifs, designated pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs), which belong to the pattern recognizing receptors, have since been shown to be critical in the innate immune response. To date, at least 11 human and 13 mouse TLRs have been discovered, among which TLR4 is the most extensively investigated because of its recognition of lipopolysaccharide (LPS) . However, there is evidence that the ligands of TLR4 include LPS as well as some specific endogenous molecules, such as fibronectin, fibrin, extracellular matrix fragments, heat-shock proteins 60 (HSP-60), HSP-70, etc. . TLR4, initially found in monocytes, has been shown to be expressed in other tissues, including cardiomyocyte and endothelial cells. Recently, myocardial I/R injury has been viewed as an innate immune response which is mediated in part through the TLR4 pathway [8, 9, 10, 11]. TLR4-deficient mice sustained significantly smaller infarctions compared with wild-type control mice given similar areas at risk [12, 13, 14]. Moreover, inhibition of the TLR4 signal with eritoran could also attenuate cardiac dysfunction induced by I/R injury . Previous clinical studies also indicated that TLR4 activation plays an important role in the pathological process of atherosclerosis and heart failure [16, 17, 18, 19, 20]. Although several studies have shown that TLR4 takes part in I/R injury of the heart, these researches mostly focused on the relationship between TLR4 and global heart dysfunction or cardiocyte apoptosis. The effect of TLR4 on CMEC I/R injury is still not fully understood.
In the present study, we tested the hypothesis that TLR4 mediates the early inflammatory response in the setting of simulated hypoxia/reperfusion (H/R) in cardiac microvascular endothelial cells.
Materials and methods
Sprague–Dawley (SD) rats aged 4 weeks, weight 80–100 g, were purchased from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). All procedures were conducted in conformity with the National Institutes of Health Guideline on the Use of Laboratory Animals and all experiments were performed in accordance with the Helsinki declaration.
Isolation, maintenance and identification of CMECs
Cardiac microvascular endothelial cells were isolated from adult Sprague–Dawley rat hearts by the enzyme dissociation method as described by Nishida et al. . Briefly, hearts were removed from adult male SD rats (80–100 g) under sterile conditions. After removing the atria, visible connective tissue, right ventricle and valvular tissue, the left ventricle was immersed in 75% ethanol for 10 s to devitalize epicardial and endocardial endothelial cells. After peeling away the outer one-third of the ventricular wall, the remaining tissue was washed in PBS. After mincing and digesting the tissue (0.02% collagenase type II for 10 min and 0.025% trypsin for 10 min at 37°C in a shaking bath), the solution was filtered through an 100 μm nylon mesh to remove undigested tissue. The dissociated cells were resuspended in DMEM supplemented with 15% FBS and then were seeded on dishes.
Cultured CMECs were tested for their purity using morphological and functional characteristics : (1) the phenotypic profile showed that the CMECs displayed a uniform ‘cobblestone’ morphology; (2) immunofluorescence assay of vWF and factor VIII demonstrated positive; (3) uptake of acetylated low-density lipoprotein.
LPS treatment of CMECs
CMECs were incubated with Escherichia coli LPS (100 ng/ml) at 37°C for 2, 12, and 24 h, respectively, and then TLR4 and NF-κB expressions were measured by Western blot assay and the secretions of IL-6 and TNF-α were detected by ELISA. After blocking the TLR4 signal with the neutralizing antibody MST510 (10 µg/ml) (eBioscience) for 60 min, or administration of mouse IgG2a (10 µg/ml) (eBioscience) which was used as an isotype control antibody, CMECs were stimulated with LPS (100 ng/ml) for 2, 12 and 24 h and the secretions of IL-6 and TNF-α were determined.
Hypoxia/reoxygenation injury of CMECs
H/R injury was performed as described previously . Briefly, CMECs were divided into four groups: (1) control group; (2) H/R group; (3) TLR4-blocking group; (4) TLR4-blocking plus H/R group. In the control group, CMECs were maintained at normoxia (95% air–5% CO2). In the H/R group, after being replaced with Hanks buffer, CMECs were exposed to hypoxia (94% N2–5% CO2–1% O2) in an anaerobic system (Thermo Forma) at 37°C for equivalent periods. In the TLR4-blocking group, CMECs were pretreated with TLR4-neutralizing antibody MTS510 (10 µg/ml) (eBioscience) for 60 min and 10 µg/ml mouse IgG2a was used as an isotype control antibody. After blocking, cells were maintained at normoxia. Moreover, for the last group, after blocking the TLR4 signal with the neutralizing antibody MST510 (10 µg/ml), cells were placed in an anaerobic system as described above. After a total of 6 h of hypoxia, all cells were removed from the chamber and then maintained in a regular incubator (95% air–5% CO2) for reoxygenation.
Measurement of inflammatory cytokines
The levels of IL-6 and TNF-α in the cell culture supernatant were measured using ELISA kits (R&D Systems). According to the manufacturers’ instructions, recombinant peptides were used to construct standard curves. Absorbance of standards and samples was determined spectrophotometrically at 450 nm using a microplate reader. Results were plotted according to the linear portion of the standard curve.
MTT cell viability assay
The cell viability of CMECs was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described in Ref. . Cells were plated onto 96-well plates at 1 × 104/ml. After different H/R treatments, cells were administered with MTT (5 g/L, Sigma) for 4 h at 37°C. The medium was then removed and 200 μl dimethyl sulphoxide (DMSO) was added to each well. After incubation at room temperature for 30 min, the absorbance was photometrically determined at a wavelength of 490 nm. Experiments were carried out in triplicate and repeated three times.
Cell scratch wound healing assay
Cell migration through a wound was determined as described in Ref. . Briefly, when CMECs became confluent in 24-well plates, a wound was made using a sterile cell scraper. Cell debris was removed by PBS and images of the wound were acquired. After further incubated for 24 h in DMEM with 1% FBS (which allows endothelial cell survival but not proliferation), the wound images were acquired again.
NO release assay
The amount of nitric oxide (NO) production released by CMECs was determined by measuring the concentration of nitrite, a metabolite of nitric oxide, with a modified Griess reaction method. Briefly, culture media (100 μl) were harvested after different H/R treatments and mixed with an equal volume of modified Griess reagent for the colorimetric assay. After 10 min of incubation at room temperature, the concentration of resultant chromophore was measured spectrophotometrically at 550 nm after enzymatic conversion of the supernatant nitrate to nitrite by nitrate reductase. The nitrite concentrations in the samples were calculated from nitrite standard curves made from sodium nitrite with the same culture medium. Experiments were repeated three times.
Apoptosis of CMECs was determined by Hoechst staining. Briefly, after different H/R injury, CMECs were stained with Hoechst 33258 (5 µg/ml, Sigma), and then the proportion of apoptotic CMECs was determined by manually counting pyknotic nuclei. Each group was assessed at least in triplicate.
Western blot analysis for TLR4 signaling pathway
After different treatments, CMECs were washed three times with cold PBS and then scraped using an ice-cold lysis buffer. The insoluble material was removed by centrifugation at 4°C (10,000 rpm, 10 min). Equal amounts of proteins were run on 12% SDS-PAGE gels and electroblotted onto nitrocellulose membrane. Equal rates of transfer among lanes were confirmed by reversible staining with Ponceau S. After blocking with 5% skim milk, the membranes were subjected to immunoblotting with appropriate primary antibody overnight at 4°C. After washing and further incubation with appropriate secondary antibody at 37°C for 60 min, bands were visualized using an enhanced chemiluminescence system (ECL; Amersham). Densitometric analysis of Western blots was carried out using VisionWorks LS, version 6.7.1.
The following primary antibodies were used: goat anti-rat TLR4 (M-16, Santa Cruz Laboratories, 1:200), rabbit anti-rat NF-κB (C-20, Santa Cruz Laboratories, 1:200), mouse anti-rat β-actin (Cell Signaling, 1:2,000) and rabbit anti-rat eNOS (Santa Cruz Laboratories, 1:200). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG, rabbit anti-goat (Santa Cruz Laboratories) and rabbit anti-mice (Santa Cruz Laboratories) at 1:5,000 dilution.
Data analysis was performed with Prism 5.0 (GraphPad Software Inc, San Diego, CA, USA). The results are presented as mean ± SEM. Differences were tested using one-way analysis of variance (ANOVA) or LSD t test where appropriate. P values <0.05 were considered statistically significant.
LPS activated the TLR4/NF-κB signal pathway
H/R injury activated the TLR4/NF-κB signal pathway
To detect the impact of H/R injury on the TLR4/NF-κB signal pathway, the expression of TLR4 and NF-κB and the levels of IL-6 and TNF-α were measured at 2, 12 and 24 h of reoxygenation after ischemia for 6 h. As shown in Fig. 1a and b, peptide levels of TLR4 after 2 h (expressed as a fraction of β-actin blot) and 12 h of reoxygenation significantly increased compared with the control group (P < 0.05). However, the peptide level of TLR4 was not significantly different from the control group after 24 h of reoxygenation (P > 0.05). Moreover, H/R injury also increased NF-κB expression at 2 and 24 h after reoxygenation compared with control (P < 0.05).
The levels of IL-6 and TNF-α were detected by ELISA. As shown in Fig. 1c and d, the levels of IL-6 in CMECs increased significantly after 2, 12 and 24 h reoxygenation as compared with control (P < 0.05), while MTS510 (10 µg/ml) administration impaired the up-regulation of IL-6 levels (P < 0.05 vs. IgG2a). Similar to the IL-6 results, the TNF-α levels increased significantly after 2, 12 and 24 h reoxygenation as compared with control (P < 0.05), and blocking TLR4 also decreased TNF-α secretion (P < 0.05).
These results indicate that H/R injury activated the TLR4/NF-κB signal pathway in CMECs. MTS510, a neutralizing antibody of TLR4, effectively reduced IL-6 and TNF-α production after H/R injury.
H/R injury impaired the proliferation ability and increased the apoptosis index of CMECs
Moreover, Hoechst staining was performed to examine the effect of TLR4 on the apoptosis index of CMECs caused by H/R injury. The proportion of apoptotic cells was determined by pyknotic nuclei counting. Typical apoptosis led to the cells shrinking and the nuclei condensing (Fig. 2b). The percentage of apoptotic CMECs in the H/R group was approximately three times greater than that in the control group (P < 0.05). In contrast, pretreatment with MTS510 (10 µg/ml) decreased the apoptosis index of CMECs after H/R injury (P < 0.05) (Fig. 2c).
These results suggest that H/R injury impaired the cell viability and increased the apoptosis index of CMECs through the TLR4/NF-κB pathway.
H/R injury impaired CMECs’ migration ability
H/R injury decreased NO secretion and the expression of eNOS in CMECs
It is well recognized that I/R injury occurs in a wide variety of ischemic cardiovascular disorders, leading to loss of cardiomyocytes and endothelial cells and impaired heart function . Previous studies have suggested that endothelial cell dysfunction is an important pathophysiological event in I/R injury, which includes microvascular endothelial cell dysfunction, swelling and apoptosis, microcirculation low-perfusion and local ischemia . Although the importance of CMECs’ I/R injury or dysfunction in the overall myocardial I/R process is established without doubt , the underlying mechanism for CMECs’ I/R injury is still not fully understood.
Studies have shown that TLR4/NF-κB signaling plays an important role in I/R injury of heart, but to the best of our knowledge the relationship between TLR4/NF-κB signaling and CMEC dysfunction has not been fully clarified. In the present study, for the first time, we investigated the effect of TLR4/NF-κB signal on CMEC dysfunction using an in vitro cell model of H/R injury which simulated I/R in vivo. Our study demonstrated a correlation between CMEC dysfunction and TLR4/NF-κB signaling. On the one hand, H/R injury induced CMEC apoptosis and impaired the proliferation, migration and NO secretion of CMECs by activation of the TLR4/NF-κB signaling pathway; on the other hand, blocking the TLR4 pathway with neutralizing antibody attenuated CMEC dysfunction and decreased IL-6 and TNF-α levels after H/R injury. Thus, our data demonstrated an important role of TLR4/NF-κB signaling in H/R-induced inflammation and injury to CMECs.
Endothelial cells were proved to express predominantly TLR4 which is regulated by inflammatory molecules such as LPS, TNF-α or IFN-γ. In addition, ECs, like macrophages, are critical targets for LPS and many cytokines. Activation of vascular endothelium by LPS results in increased production of various cytokines .
Previous studies have shown that LPS induces TLR4 mRNA expression in rat coronary EC but with no accompanying increase in TLR4 protein . However, in our study, a significant increase in TLR4 protein expression was found in CMECs after LPS incubation for 2, 12 or 24 h. This divergence most likely reflects differences in cell types and differentiation stages. Moreover, NF-κB expression and levels of IL-6, TNF-α were also up-regulated by LPS, which was inhibited by TLR4 blocking. Our results demonstrate that LPS can active the TLR4/NF-κB signal pathway of CMECs.
Previous studies on models of regional myocardial I/R have suggested that I/R can induce endothelial cell apoptosis, impair endothelium-dependent vasodilation by decreasing NO secretion, and increase the adherence of circulating leukocytes to endothelial cells by up-regulating the expression of cell adhesion molecules [23, 29, 30]. In concordance with these findings, we further observed CMEC dysfunction in proliferation, migration and secretion after H/R injury.
Studies have reported that TLR4 mutation or inhibition reduces infarct size and improves cardiac function [12, 15], which indicates that TLR4/NF-κB signaling plays a critical role in myocardial I/R injury. In view of the crucial role CMEC plays in I/R injury process, we set out to explore the relationship between TLR4 signaling and CMEC injury. Our study demonstrated that TLR4 also played a critical role in CMEC dysfunction after H/R injury. Cha et al.  have provided evidence that TLR4 exerts its influence in I/R injury through TNF-α and IL-1 production. However, we found that TLR4 induces inflammation and injury in CMECs by TNF-α and IL-6 production.
TLR4, like pattern recognition receptors (PRRs), is expressed and functional in cells of myeloid lineage, which are central to innate immune responses. However, TLR4 is also detectable in nonprofessional immunocyte cell types, such as cardiomyocytes and microvascular endothelial cells . We detected the expression of TLR4 in normal CMECs using Western blot analysis, which confirmed this view.
The signaling of activated TLR4 is mediated through myeloid differentiation protein 88 (MyD88)-dependent and MyD88-independent pathways . The classical MyD88-dependent pathway originates from the Toll/IL-1 receptor (TIR) domain. Subsequently, activated TIR promotes NF-κB translocation to the nucleus and induces proinflammatory cytokine gene transcription such as TNF-α and IL-6 [6, 15]. The MyD88-independent pathway involves the adaptor proteins TRIF or TRAM. Some studies indicated that in endothelial cells TLR4 signaling was restricted to the MyD88-dependent pathway caused by lacking of TRAM. We observed that the protein level of TLR4 increased significantly at 2 and 6 h reoxygenation and decreased to normal at 24 h. However, the expression of NF-κB has two peaks at 2 and 24 h. In many other cell types such as cardiocytes, endothelial cells and astrocytes, NF-κB activation has also been detected in a biphasic manner consisting of an early, rapid phase and a delayed phase. Thompson et al.  have suggested that the biphasic NF-κB activation is implicated in differential degradation of the inhibitor of κB (IκB) which is phosphorylated by IκB kinase (IκK). The transient phase is mediated through IκBα degradation, and the persistent phase is regulated by both IκBα and IκBβ degradation. However, Schmidt et al.  have revealed a novel mechanism in which different MAP3K and IκB subtypes are involved in biphasic NF-κB activation. MEKK3 is essential in IκBα formation, which regulates the rapid activation of NF-κB, whereas MEKK2 participates in assembling IκBβ which is important in controlling the delayed activation phase.
The mechanism by which H/R activates the TLR4/NF-κB pathway is not well understood. TLR4 recognizes LPS as well as certain host molecules, including fipath, fibrinogen, HSP-60, HSP-70 and components of the extracellular matrix . In our in vitro cell culture system, LPS did not exist. Therefore, it is likely that endogenous agents in H/R injury activate the TLR4/NF-κB pathway. Intracellular HSP-60 and HSP-70 endogenous agents which are released from injured cells during H/R may activate myocardial TLR4/NF-κB signaling and mediate the inflammatory response.
In this study we used an in vitro cell model to determine the effect of TLR4/NF-κB signaling on CMEC dysfunction. Although the model is useful for determining the effect of H/R injury on CMEC function and it eliminated well the contribution of endotoxin (LPS) to TLR4 activation, it also has some limitations: the H/R model is an artificial experimental protocol which cannot simulate the in vivo I/R injury perfectly; it also cannot detect the proinflammatory factors from blood or other tissues. Moreover, the mechanism by which H/R activates the TLR4/NF-κB signal was not elucidated in this research. Therefore, further research is needed to explore these pathways and mechanisms.
In summary, to the best of our knowledge, this is the first study to demonstrate the effect of TLR4/NF-κB signaling in CMEC dysfunction after H/R injury. In the present study, we found that H/R injury activated the TLR4/NF-κB signaling which mediated the inflammatory effect on CMECs through production of TNF-α and IL-6 peptides, and blocking TLR4 with neutralizing antibody attenuated CMEC dysfunction after H/R injury. These findings indicate that TLR4 can initiate an exaggerated proinflammatory response early after H/R injury, contributing to CMEC dysfunction. Targeted pharmacological inhibition of TLR4 may be a therapeutic option suitable for myocardial ischemia and reperfusion.
This study was supported by grants from the National Natural Science Foundation of China (NSFC, No. 30970845, 30770784) and Xijing Research Boosting Program (No. XJZT08Z04 XJZT07Z05).
Conflict of interest
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