HHcy (hyperhomocysteinaemia) is one of the major risk factors for cardiovascular diseases. A high concentration of Hcy (homocysteine) induces endothelial dysfunction by activating endothelial oxidative stress. LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) plays a vital role in regulating the progression of atherosclerotic lesions. LOX-1 activation causes endothelial apoptosis and inflammation. The mechanism is still unclear as to whether Hcy affects human endothelial LOX-1 expression. LOX-1 expression level was confirmed by Western blotting assay in Hcy-treated endothelial cells. L-Methionine was used for HHcy induction in animals. Our results suggested that Hcy increased PKCβ (protein kinase Cβ) activation to enhance the LOX-1 expression level. The up-regulation of PKCβ phosphorylation subsequently causes ROS (reactive oxygen species) formation and SIRT1 (sirtuin 1) degradation through a proteasome-dependent mechanism, thereby mitigating the activity of SIRT1 by deacetylating HSF1 (heat-shock transcription factor 1). We also found that NOX2 is a key NAPDH oxidase isoform responsible for the Hcy-caused ROS formation. The overexpression of SIRT1 and HSF1 reduced the Hcy-induced LOX-1 activation. Silencing PKCβ function also reduced LOX-1 activation and endothelial apoptosis caused by Hcy. Our hypothesis was supported by analysing the data from methionine-induced HHcy-affected animals. Our data indicate a new direction for LOX-1 regulation by the modulation of the PKCβ/NAPDH oxidase/SIRT1/HSF1 mechanism. Our findings might provide a novel route for developing new therapeutic treatments for HHcy.
Hyperhomocysteinaemia (HHcy) is known as a main risk factor for cardiovascular diseases through increasing the incidence rate of atherosclerosis in humans. In the present study, we clearly confirmed that homocysteine (Hcy) caused up-regulation of LOX-1 by the modulation of an elaborate mechanism, related to PKCβ up-regulation and NADPH oxidase activation, thereby degrading SIRT1.
We also found that HSF1 acetylation was enhanced through Hcy-caused degradation of SIRT1, thereby inhibiting its function to reduce Hcy-induced LOX-1 activation.
Moreover, we have validated that PKCβ inhibition and SIRT1 and HSF1 overexpression protects against endothelial cells from Hcy-facilitated apoptosis. In summary, our findings from the present study suggest a well-distinguished pathway that might guide new therapeutic approaches for treatment.
The chronic inflammation of vascular cells has been shown to cause atherosclerosis. Pathological properties, including endothelial cellular dysfunction, the migration of lipid particles, foam cell generation and the migration of macrophages, have been identified in atherosclerotic lesions . HHcy (hyperhomocysteinaemia) is one of the main risk factors for cardiovascular diseases by increasing the incidence rate of atherosclerosis . HHcy is able to promote pro-inflammatory responses and oxidative injuries, remodelling smooth muscle cells, which may cause a progression of the atherosclerotic lesion in humans . LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) was traditionally described as a receptor for oxLDL (oxidized low-density lipoprotein) . In general, LOX-1 could be located in human smooth muscle cells, macrophages and vascular endothelial cells, which play vital roles in regulating the progression of atherosclerotic lesions . Holven et al.  suggested that HHcy up-regulated the level of LOX-1, indicating that HHcy causes atherosclerotic lesions possibly through LOX-1 activation. The activation of LOX-1 expression promptly leads to ROS (reactive oxygen species) generation  and the up-regulation of pro-apoptotic signal transduction and nitric oxide (NO) catabolism, thereby facilitating oxidative injuries and endothelial cell death .
PKC (protein kinase C) family isoforms play important roles in regulating cellular functions. Several pro-atherosclerotic factors, such as apolipoprotein CIII, up-regulate the expression level of PKCβ in human endothelial cells . In addition, oxLDL activates the LOX-1 expression level and PKCβ phosphorylation . As a result, PKCβ becomes a key mediator of atherosclerosis.
Hcy (homocysteine) promotes LOX-1 activation in human mononuclear cells . It is still unknown whether Hcy induces LOX-1 activation in human endothelial cells. In fact, the detail of the mechanism of LOX-1 gene control and the activation of LOX-1 levels by Hcy remain unclear.
In our innovative study, we found that deacetylated HSF1 (heat-shock transcription factor 1) attenuated LOX-1 gene expression. However, Hcy increased the LOX-1 level via the up-regulation of PKCβ and NADPH oxidase activation. This oxidative signalling downgraded the HSF1 deacetylase SIRT1 (sirtuin 1). Then, the inhibition of SIRT1 caused HSF1 acetylation, which repressed the interactions of HSF1 and the LOX-1 promoter and mitigated the LOX-1 expression level. Clearly, the overexpression of SIRT1 and HSF1 mitigates Hcy-induced LOX-1 activation and reduces Hyc-induced endothelial apoptosis.
MATERIALS AND METHODS
Cell cultures and reagents
HUVECs (human umbilical vein endothelial cells) were bought from the A.T.C.C. (Manassas, VA, U.S.A.). HUVECs was cultured with M199 basal medium that was supplemented with a low-serum growth supplement and penicillin (50 IU/ml)/streptomycin (50 μg/ml). Trypsin/EDTA was used for the passage of cells. M199 and trypsin/EDTA were bought from Gibco. Low-serum growth supplement was purchased from Cascade. LY333531, dPPA (12-deoxyphorbol 13-phenylacetate 20-acetate), MG132, apocynin, NAC (N-acetylcysteine), penicillin and streptomycin were purchased from Sigma.
Investigation of apoptosis
Apoptotic cells as assessed using the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay were visualized under a fluorescence microscope or analysed by flow cytometry. After treatment, HUVECs were rinsed twice in PBS before fixation for 30 min at room temperature with 4% (w/v) paraformaldehyde. Next, the slides were washed in PBS before incubation in the prepared solution (0.1% Triton X-100 and 0.1% sodium citrate) for 5 min. The slides were then incubated with 100 μl of TUNEL reaction mixture in a humidified atmosphere for 1 h at 37°C in the dark. Active caspase 3 levels were detected by flow cytometry using a commercial fluorescein active caspase kit (BioVision K105-100).
A total of 12 C57BL mice (six in each group) were used in the present study. The animals were fed with or without 1% (w/w) L-methionine in water for 4 months to induce HHcy. All of the animal studies followed the guidelines that were required for the care and use of laboratory animals and were approved by the Animal Center of National Cheng Kung University, Tainan, Taiwan. The Hcy levels in the serum were tested to confirm that HHcy was induced.
Anti-LOX-1, anti-β-actin, anti-PKCβ, anti-p-PKCβ, anti-gp91, anti-p22phox, and anti-HSF1 were bought from Santa Cruz Biotechnology. Anti-FLAG, anti-acetylated lysine, anti-Rac-1 and anti-p47phox were obtained from BD Biosciences.
SIRT1 and HSF1 were obtained from Addgene. The LOX-1 luciferase promoter constructs were produced as described previously .
Promoter activity assay
The Dual Luciferase Reporter Assay System (Promega) was used to test luciferase activity. The luciferase intensity was measured using a luminometer. The relative intensities were measured by normalizing with a Renilla luciferase activity for the efficiency of transfection.
Western blot analysis
RIPA, which was purchased from Millipore, was used to extract lysate. The proteins were transferred on to a PVDF membrane after the proteins were separated by SDS/PAGE. The membranes were blocked by buffer for 1 h at 37°C. Then, the membranes were incubated with primary antibodies overnight at 4°C, followed by hybridization with HRP (horseradish peroxidase)-conjugated secondary antibody for 1 h. The intensities were quantified by densitometric analysis.
The PureProteome Protein G Magnetic Bead System from Millipore was used to perform immunoprecipitation. An aliquot of 300 μg of protein was hybridized with primary antibodies (2 μg) overnight at 4°C. The binding proteins were denatured at 95°C for 10 min. The samples were analysed by Western blot assay.
SOD and MDA levels
Serum was obtained through blood collection. The SOD (superoxide dismutase) and MDA (malondialdehyde) levels in the serum were tested via an enzymatic assay method using commercial kits (Sigma) according to the manufacturer's instructions.
NAPDH oxidase activity assay
The lucigenin method was used to determine NAPDH oxidase activity in HUVECs. The crude membrane fraction of HUVECs was obtained as described above. Total protein concentration was adjusted to 1 mg/ml. An aliquot of 200 μl of protein (100 μg) was incubated in the presence of 5 μl of lucigenin and 100 μM NADPH. Luminescence was assessed after 10 min using a plate reader (VICTOR3; PerkinElmer) to determine the relative changes in NAPDH oxidase activity.
A ChIP assay was performed using lysates from HUVECs that were exposed to Hcy. The investigation was carried out using EZ ChIP Chromatin (Millipore) according to the manufacturer's instructions. HUVECs were washed twice in PBS and incubated with 1% (w/v) formaldehyde for 10 min. After quenching with 0.1 M glycine, the cross-linked material was sonicated into chromatin fragments with an average length of 500–800 bp. The chromatin solution (100 ml of chromatin sample and 900 ml of dilution buffer) was pre-cleared by adding Protein G–agarose for 2 h at 4°C, and immunoprecipitation was then performed with Protein G–agarose and 1–10 mg of the indicated antibodies overnight at 4°C on a rotating wheel. The immunoprecipitated material was washed five times with ice-cold washing buffer. The cross-links were reversed by incubating the samples with 8 ml of 5 M NaCl for 5 h at 65°C, and 10 mg of RNase A was added to eliminate the RNA. The recovered material was treated with proteinase K, placed in spin columns and precipitated. The pellets were resuspended in 50 ml of double-distilled water and analysed using PCR using sense primer 5′-GAGTGGGTACAATATCTCTC-3′ and antisense primer 5′-AAGCAGTCACGAACTTCAAC-3′.
Isolation of mRNA and quantitative real-time PCR
The total RNA was isolated using an RNeasy kit (Qiagen). The oligonucleotides for LOX-1 and β-actin were designed using the computer software package Primer Express 2.0 (Applied Biosystems). The oligonucleotide specificity was determined by a homology search within the human genome (NCBI BLAST) and was confirmed by dissociation curve analysis. The oligonucleotide sequences were as follows: LOX-1 sense primer 5′-GATGCCCCACTTGTTCAGAT-3′ and antisense primer 5′-CAGAGTTCGCACCTACGTCA-3′, and β-actin sense primer 5′-AGGTCATCACTATTGGCAACGA-3′ and antisense primer 5′-CACTTCATGATGGAATTGAATGTAGTT-3′. PCR was performed with SYBR Green in an ABI 7000 sequence detection system (Applied Biosystems) according to the manufacturer's instructions.
HUVECs were stimulated with Hcy for 24 h. After stimulation, HUVECs were incubated with 10 μM DHE (dihydroethidium) for 30 min. The fluorescence intensity was measured by flow cytometry.
Data are expressed as means±S.D. A one-way ANOVA followed by Student's t test was used to analyse the differences between the groups. Pearson's correlation coefficients were used to analyse the correlation between Hcy and genes. P<0.05 was accepted as significant.
Hcy induces LOX-1 up-regulation through PKCβ activation
LOX-1 is expressed in components of the human circulatory system, such as monocytes, endothelial cells and smooth muscle cells . Up-regulated LOX-1 could be detected in the atherosclerotic lesions and ischaemic hearts in humans, therefore the promotion of LOX-1 has been thought to correlate with the pathological responses in the cardiovascular system . Most of the clinical findings are highly related to pro-inflammatory and pro-apoptotic events. LOX-1 is activated by oxLDL . However, it is still unclear whether Hcy mediates LOX-1 expression levels. As revealed in Figure 1(A), we found that the LOX-1 mRNA level was significantly increased by treatment with 100 and 150 μM Hcy in human endothelial cells. We selected 100 μM Hyc to induce endothelial cell dysfunction. We also confirmed that 200 μM cysteine and 200 μM methionine did not increase LOX-1 expression in endothelial cells (results not shown). A previous study suggested that HHcy damages endothelial cell function through the activation of PKC . Siow et al.  also reported that Hcy promotes PKCβ activation, thereby activating NAPDH oxidase and ROS formation. Therefore PKCβ activation is involved in the Hcy-induced oxidative injuries of the circulatory system. In Figures 1(B) and (C), we confirm that the treatment of Hcy promotes the activation of both LOX-1 and PKCβ. In addition, dPPA, an activator of PKCβ, was used to confirm the role of PKCβ in the Hcy-induced up-regulation of LOX-1 expression. As shown in Figures 1(D) and 1(E), treatment with dPPA results in the activation of LOX-1. However, this finding was reversed by silencing PKCβ. The inhibition of PKCβ by the PKCβ-specific inhibitor LY333531 inhibited Hcy-induced LOX-1 activation. Altogether, PKCβ activation is an essential part of the up-regulation of Hcy-induced LOX-1 up-regulation.
Hcy induces PKCβ and LOX-1 up-regulation in HUVECs
Hcy promotes SIRT1 degradation by post-translational modification
SIRT1 modulates cellular physiological function and metabolism. SIRT1 inhibits the degeneration of human endothelial cells through the regulation of anti-apoptotic genes, thereby reducing endothelial inflammation and promoting survival . In addition, antioxidant enzymes, such as SOD, are up-regulated and oxidative stress was inhibited by SIRT1 activation. Thus the facilitation of SIRT1 function is considered an effective approach to mitigate human endothelial dysfunction. Evidence demonstrating that Hcy regulates SIRT1 expression has not yet been found. Therefore we tested SIRT1 levels under Hcy induction using Western blot analysis. In Figures 2(A) and 2(B), both the Hcy and dPPA treatments reduced SIRT1 expression. However, this result was reversed by the PKCβ antagonist. The up-regulation of Hcy-induced LOX-1 was inhibited by the overexpression of SIRT1 (Figure 2C). Silencing endogenous SIRT1 enhanced the LOX-1 level (Figures 2E and 2F). We conducted a time course study to identify the sequence of Hcy-caused PKCβ activation and SIRT1 repression, as well as LOX-1 up-regulation. We found 4 h of treatment with Hcy caused PKCβ activation, but not LOX-1 up-regulation and SIRT1 repression. We also found that 8 h of treatment with Hcy caused both PKCβ activation and SIRT1 repression, but not LOX-1 up-regulation. We suggest that Hcy causes PKCβ activation at first, and then SIRT1 inhibition and LOX-1 up-regulation (Figures 2G–2I). Furthermore, Hcy increased the LOX-1 expression level through PKCβ activation and SIRT1 degradation. An intervention for the proteasome inhibitor MG132 reduced Hcy- or dPPA-induced SIRT1 degradation, suggesting that SIRT1 downgrading is proteasome-dependent (Figures 2J–2M). In order to confirm this assumption, an immunoprecipitation assay was used to confirm whether Hcy degrades SIRT1 by ubiquitination. Our data indicate that Hcy promoted the ubiquitination of SIRT1 (Figure 2N). The time course results indicate that Hcy caused the ubiquitination of SIRT1 after 8 h of treatment (Figure 2O).
Hcy impairment of the SIRT1 level is proteasome-dependent
NAPDH oxidase is critical in Hcy-induced SIRT1 degradation
Hcy-induced endothelial dysfunction facilitates oxidative stress. In human endothelial cells, most of the ROS are generated by increasing the activity of NADPH oxidase. The membrane translocation of the NADPH subunits and membrane assembly are reflected by PKC activation, thereby facilitating ROS formation . We demonstrated that Hcy significantly increases the activity of NAPDH oxidase (Figure 3A) and assembles the NADPH oxidase subunits (Figures 3B–3D). LY333531, the selective and potent inhibitor of PKCβ, reduces the Hcy-induced NADPH oxidase activation and ROS generation (Figure 3E), suggesting that Hcy-induced NADPH oxidase is activated through the up-regulation of PKCβ. We also found that treatment with SOD or glutathione (GSH) reduces Hcy-induced ROS formation, this finding clearly suggests that Hcy induces oxidative stress through enhancement of superoxide anion radical in HUVECs. Because our data suggested that NOX2 was increased in membrane fractions of Hcy-treated HUVECs. We silenced NOX2 function using NOX2 siRNA. In Figure 3(E), silencing of NOX2 reduced Hcy-caused ROS generation in HUVECs, indicating that NOX2 is most critical in Hcy-induced ROS generation. Moreover, The pharmacological inhibitor of NADPH oxidase (apocynin) and the free radical scavenger (NAC) not only inhibited Hyc-induced ROS formation, but also reversed the Hyc-impaired SIRT1 expression level (Figures 3F and 3G), indicating that they caused endothelial SIRT1 degradation by increasing ROS formation via NADPH oxidase activation.
Hcy induces NADPH oxidase activation by PKCβ
HSF-1 represses LOX-1 expression by binding to the LOX-1 promoter
Because Hcy stimulation of LOX-1 gene up-regulation was confirmed in Figure 1, we investigated whether Hcy affects the LOX-1 promoter. In Figure 4(A), we treated human endothelial cells with the LOX-1 promoter–luciferase plasmid, then exposed the cells to Hcy for 24 h and measured the luciferase intensity to the pGL3 basic vector. The LOX-1 promoter was largely up-regulated by Hcy treatment. Hsp (heat-shock protein) is increased in patients with coronary artery diseases . In addition, Kubo et al.  reported that HSF1 contributes to ischaemia-induced angiogenesis by affecting the mobilization of endothelial cells in ischaemic mice . These studies indicate that HSF1 may play a key role in regulating cardiovascular disease. We identified a binding site for HSF1 to bind to the LOX-1 promoter using TFSEARCH. In order to examine the importance of HSF1, we silenced HSF1 using siRNA. The Western blot results revealed that the inhibition of HSF1 increased LOX-1 expression without Hcy treatment. As expected, the inductive effects of Hcy on LOX-1 activation were increased by siHSF1 (Figures 4B and 4C). The overexpression of HSF1 mitigates the Hcy-increased LOX-1 expression, which was also confirmed in Figures 4(D) and 4(E). These results demonstrate that HSF1 functions as a transcriptional inhibitor to mitigate LOX-1 expression in human endothelial cells. Moreover, our data suggest that HSF1 forfeits its capacity to bind to the LOX-1 promoter after being exposed to Hcy. We therefore assume that HSF1 mitigating the DNA-binding ability might undergo post-transcriptional modification. The decision was made to examine whether HSF1 acetylation controls LOX-1 expression. The overexpression of HSF1 and immunoprecipitation were used to confirm our hypothesis. In Figure 4(F), we revealed that the Hcy increased HSF1 deacetylation. Similarly, the same results were found in immunoprecipitation with a definite antibody to the endogenous HSF1 and the acetylated HSF-1 (Figure 4G), indicating that Hcy induced the acetylation of HSF1. Finally, ChIP assay confirmed that HSF-1 represses LOX-1 expression by binding to the LOX-1 promoter (Figure 4I).
HSF1 represses the LOX-1 level through acetylation
Hcy increases the LOX-1 expression level involving the PKCβ/SIRT1/HSF1 axis
In Figure 2(E), we demonstrate that SIRT1 inhibition enhances the LOX-1 expression level, indicating that the SIRT1 protects against oxidative stress-induced endothelial dysfunction through LOX-1 repression. In order to confirm whether HSF1 participates in the SIRT1-inhibited LOX-1 level, we overexpressed HSF1 in SIRT1-silenced endothelial cells. As shown in Figures 5(A) and 5(B), the LOX-1 expression level was largely mitigated by the overexpression of HSF1 in SIRT1-silenced human endothelial cells. This finding indicates that SIRT1 inhibits the Hcy-increased LOX-1 level through the regulation of HSF1. Moreover, we revealed previously that Hcy promotes SIRT1 degradation mainly by PKCβ activation. We therefore we hypothesized that Hcy facilitates PKCβ activation, thereby reducing the SIRT1 expression level and further promoting HSF1 acetylation. Thus we used PKCβ and SIRT1 siRNAs to eliminate PKCβ and SIRT1 functions. As predicted, silencing the SIRT1 increased Hcy-induced LOX-1 activation. However, this silencing was inhibited by PKCβ inhibition (Figure 5C). This statement was supported further by the LOX-1 promoter assay (Figure 5E). Oxidative stress induces atherosclerotic events by modulating different responses, such as endothelial apoptosis or inflammation . Promoting pro-apoptotic signalling might inhibit LOX-1 activation . We therefore investigated whether the PKCβ/SIRT1/HSF1 pathway was involved in LOX-1 inhibition under Hcy stimulation. In Figure 6(A), Hcy obviously induced apoptosis in human endothelial cells as detected by flow cytometry. However, these TUNEL-positive cells were reduced by PKC inhibition and by SIRT1 and HSF1 up-regulation. Silencing of LOX-1 also reduced Hcy-induced endothelial apoptosis (Figure 6A). This was confirmed by caspase 3 activity assay (Figure 6B), and these findings suggest that the PKCβ/SIRT1/HSF1 mechanism is important in LOX-1 mitigation. In order to verify our assumption in vivo, we fed C75BL/6 mice with 1% (w/w) L-methionine in water for 3 months to induce HHcy in animals. As shown in Figure 6(C), the serum Hcy levels increased by methionine feeding, suggesting that the animal model of HHcy was successful. Additionally, the serum MDA levels increased and the SOD activity decreased in HHcy mice (Figures 6D and 6E). To test further the molecular signalling in HHcy, we isolated the tissue of the endothelial vessels and tested the protein expression level using Western blot analysis in HHcy mice. Through the induction of methionine, our data revealed that LOX-1, p-PKCβ and cleaved caspase 3 were up-regulated. However, SIRT1 was down-regulated (Figures 6F and 6G).
Hyc-activated LOX-1 expression by the inhibition of the SIRT1/HSF1 axis
Hcy induces endothelial apoptosis through PKCβ/SIRT1/HSF1
The up-regulation of the LOX-1 expression level had been identified in atherosclerotic lesions. However, the probable mechanism and molecular regulation remain unclear. In the present study, we clearly demonstrated that the Hcy-induced LOX-1 up-regulation through an elaborate pathway relates to the PKCβ up-regulation and NADPH oxidase activation for inducing SIRT1 ubiquitination. We also confirmed that HSF1 acetylation was enriched by the Hcy-induced degradation of SIRT1, thereby mitigating its function to attenuate Hcy-induced LOX-1 up-regulation. Moreover, the PKCβ inhibition and SIRT1/HSF1 overexpression protected the endothelial cells from Hcy-induced apoptosis as determined by the TUNEL assay. Altogether, our data suggest a well-distinguished pathway that might guide new therapeutic approaches for managing HHcy-induced endothelial dysfunction via the PKCβ/SIRT1/HSF1/LOX-1 mechanism (Figure 7).
Proposed mechanisms of the regulation of LOX-1 induced by the interaction of PKCβ, SIRT1 and HSF1
The expression level of LOX-1 in healthy human endothelial vessels is suppressed. However, activated LOX-1 was identified in atherosclerotic vessels, suggesting that LOX-1 up-regulation is important in regulating atherosclerosis. Another main regulator, oxLDL, angiotensin II or other pro-inflammatory events activating LOX-1 expression have been reported . Specifically, further studies are recommended to examine whether Hcy affects LOX-1 expression. Moreover, HHcy is a well-recognised risk factor for cardiovascular diseases, affecting the thickness of human blood vessels and the disintegration of vascular elastin and leading to high blood pressure . Hcy causes and triggers inflammation in human endothelial vessels via the regulation of pro-inflammatory responses, such as TNFα (tumour necrosis factor α) and NF-κB (nuclear factor κB) . Hcy also induces DNA damage and reduces the bioviability of NO through eNOS (endothelial nitric oxide synthase) inhibition, thereby facilitating endothelial apoptosis and dysfunction . So far, very few studies have reported a link between LOX-1 and Hcy-induced endothelial dysfunction. In Figures 1(A)–1(C), we show that Hcy increases LOX-1 mRNA and protein expression. Our results agree with the previous findings suggesting that Hcy causes the global hypomethylation of blood vessels through LOX-1 .
The normal Hcy level in human plasma is approximately 13 μM. Concentrations between 13 μM and 60 μM are moderately elevated, and concentrations between 60 μM and 100 μM are due to severe HHcy in humans . In the present study, we used 100 μM Hcy to induce endothelial dysfunction, this concentration might reflect physiological HHcy in humans. HHcy is an independent atherosclerotic event that causes cardiovascular disease. HHcy not only causes coronary artery disease, but also promotes peripheral vascular pathological responses that are attributed to Hcy-induced oxidative stress . Despite Hcy automatic oxidation, other sources of Hcy-facilitated ROS formation have been identified, such as eNOS uncoupling and the impairment of antioxidant enzymes . In fact, previous studies have suggested that Hcy obviously enhances the activity of NADPH oxidase . However, the probable mechanism of Hcy-induced NADPH oxidase activation is still controversial. In the present study, we confirmed that Hcy increases NADPH oxidase activation through the mobilization of p47phox and Rac-1 from the cytosolic fraction to the membrane fraction, which interact with gp91 (NOX2) and p22phox (Figures 3A–3D). We identified that NOX2 might be the most important molecule to facilitate ROS formation. In addition, PKCβ inhibition reduces Hcy-induced NADPH oxidase activation and ROS formation, suggesting that Hcy-induced endothelial oxidative stress mainly modulates the PKCβ and NADPH oxidase axis. This finding is compatible with a previous study that was conducted by Siow et al.  that reported that Hcy facilitates p47phox and p67phox phosphorylation by PKCβ up-regulation in monocytes .
PKCβ is critically important in developing atherosclerosis and oxidative damage . The repression of PKCβ function suppresses MAPK (mitogen-activated protein kinase) phosphorylation, mitochondrial dysfunction and free radical formation under oxLDL treatment. PKCβ deregulation has been identified in clinical patients with metabolic diseases. Activated PKCβ was later found in patients with diabetes mellitus except that PKCβ inhibitor treatment enhances eNOS phosphorylation and becomes sensitive to insulin in endothelial cells from clinical patients with diabetes mellitus . Clearly, atherosclerosis has three important characteristics in eNOS dephosphorylation and PKCβ activation . In this study, we demonstrated the relationship between LOX-1 and PKCβ. Moreover, we also confirmed that PKCβ positively regulates LOX-1 expression by modulating NADPH oxidase.
Another main controller, SIRT1, is a principal protein for maintaining the health of the human cardiovascular system. SIRT1 acts as a cytoprotective regulator that protects the cardiovascular system from degeneration and oxidative injuries . Moreover, owing to the inhibitory ability of inflammation and apoptosis, SIRT1 is an anti-atherosclerosis molecule . A previous study suggested that SIRT1 deletion increased foam cell accumulation and oxLDL uptake . In addition, SIRT1 decreased the LOX-1 expression level and NF-κB activation, thereby reducing the uptake of oxLDL . The relationship between Hcy and SIRT1 is unclear. However, up-regulation of SIRT1 by resveratrol reduces the Hcy-induced oxidative injuries in neuronal cells . This result suggests that SIRT1 activation is an effective approach to manage Hcy-induced oxidative stress. Previous reports have suggested that the SIRT1 expression level was influenced by PCK activity . This statement is similar to our findings in the present study. We confirmed that the activation of PKCβ by its agonist increased SIRT1 ubiquitination and LOX-1 up-regulation. The data from the present study demonstrate that Hcy induces LOX-1 up-regulation by the activation of PKCβ and by the ubiquitination of SIRT1 (Figures 1 and 2).
HSF1 not only affects the function of Hsps, but also reduces oxidative stress, which induces damage in the cardiovascular system . Although stimulated by stress, HSF1 translocates from the cytosolic fraction into the nucleus and controls the downstream signalling. Ghemrawi et al.  suggested recently that reduced vitamin B12 availability causes ER stress by decreasing the SIRT1 deacetylation of HSF1, indicating that HSF1 is a new deacetylation target of SIRT1. Moreover, Ding et al.  suggested that the inhibition of PKC markedly reduced HSF1 phosphorylation. In the present study, we confirmed that the Hcy treatment induced PKCβ activation, thereby degrading SIRT1 expression and promoting HSF1 acetylation.
Hsps control the function of human genes as found in many studies. HSF1 activates the eNOS level and decreases the level of ET-1 (endothelin 1). Therefore HSF1 improves the health of endothelial cells . We found in the present study that HSF1 inhibited Hcy-activated LOX-1 expression and endothelial apoptosis (Figures 5 and 6).
The limitation of the present study is that we only used HUVECs for in vitro assay. We need to confirm our findings in human arterial endothelial cells.
In conclusion, the present study demonstrates a new direction for LOX-1 regulation by modulating the PKCβ/NADPH oxidase/SIRT1/HSF1 pathway, which affects HHcy-induced endothelial cell dysfunction and apoptosis. Hcy increases PKCβ and NADPH oxidase activity, resulting in ROS formation and SIRT1 degradation. The dysfunction of SIRT1 then promotes the acetylation of HSF1, which then up-regulates the LOX-1 expression level and endothelial apoptosis. Thus our findings might provide a novel route for developing new therapeutic treatments for HHcy.
Kun-Ling Tsai was involved in conception and design of the study. Ching-Hsia Hung and Kun-Ling Tsai wrote the paper. Ching-Hsia Hung and Kun-Ling Tsai performed experiments. Shih-Hung Chan and Pei-Ming Chu analysed data and prepared Figures.
This work was supported by the National Science Council (NSC) [grant numbers 100-2314-B-039-017-MY3] and the National Cheng Kung University [grant number D103-35A13].
12-deoxyphorbol 13-phenylacetate 20-acetate
endothelial nitric oxide synthase
heat-shock transcription factor 1
human umbilical vein endothelial cell
lectin-like oxidized low-density lipoprotein receptor 1
nuclear factor κB
oxidized low-density lipoprotein
protein kinase C
reactive oxygen species
terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling