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The A10 cells, rat aortic smooth muscle cell line, were obtained from American type culture collection (American type culture collection, USA) and grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma Aldrich, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37^oC in a humidified atmosphere containing 5% CO₂/95% air.

For all experiments, cells were seeded at a density of 4 × 10⁴/well into 12-well microplates with DMEM containing 10% FBS and supplements. Cells were allowed to adhere in dish overnight, and then the culture medium was replaced with fresh DMEM (0.5% FBS) containing HNE after pre-incubated for 30 min with various inhibitors. A commercial HNE (purity > 98%) was obtained from Cayman Chemical Inc. (Cayman Chemical Inc, USA), and working solutions of HNE were made in phosphate buffered saline (PBS) immediately before use. Working solutions of HNE (<0.1% ethanol) were made in PBS immediately before use.

The 3,3'-dihexyloxacarbocyanine iodide, DiOC₆(3) (Sigma Aldrich, USA), was used as a measure of ΔΨm according to methods described previously. ^[19,20] In brief, cells grown to sub-confluence on glass cover slips were incubated with HNE. Following stimulation for 3 hr, cells were incubated with 100 nM of the dye for an additional 30 min. Then, cells were washed with PBS. Cellular fluorescence was analyzed flow cytometric analysis(BD FACSLyric, BD Biosciences, USA ).

Intracellular reactive oxygen species (ROS) formation was measured by fluorescenceusing 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma Aldrich, USA), Invitrogen, Molecular probes, a dye that permeates cells where it is trapped as the deesterified free acid, which can then react with ROS to form a highly fluorescent 2,7-dichlorofluorescein (DCF). ^[21] The cells grown in 12-well plates were loaded with 10M DCFH-DA for 30 min, and then incubated with HNE for 30 min at 37^oC. Reactions were stopped by aspiration and cells were trypsinized, collected on ice, and analyzed on flow cytometry. Fluorescence intensity was measured for 10,000 cells in each sample. Results were obtained as histogram plots of cell number versus fluorescence intensity (FL-1), and the mean fluorescence for each sample within an experiment was analyzed as % of total cells.

Cells were harvested, washed twice with ice-cold NaCl/P i, and lysed in a TNN buffer (50 m M Tris/HCl, pH 8.0, 120 mM sodium chloride, 0.5% Nonidet P-40) supplemented with protease inhibitors (2 mg/mL^-1 aprotinin, 2 mg/mL^-1 leupeptin, 100 mg/mL^-1 phenylmethanesulfonyl fluoride, 5 lg/mL^-1 pepstatin, 1 m M dithiothreitol) and phosphates inhibitors (20 mM NaF, 2 mM Na₃VO₄)for 1 h on ice, vortexing every 10 min. Lysates were centrifuged at 8000 g for 30 min to remove insoluble material. Protein concentration was determined by the Lowry method using BSA as standard. Equal amounts of protein were separated on 10-15% SDS/PAGE gels. The gels were subsequently transferred onto a nitrocellulose membrane (BIO-RAD, Korea).

Lipid peroxidation and its reactive byproducts, like HNE are reported to cause redox-related degenerative processes, including vascular dysfunction. ^[13,22] 4-HNE has been suggested to mediate a number of oxidative stress-linked pathological events such as cell proliferation and apoptosis.^[23] To assess the HNE-induced VSMC activation contained oxidative stress, we analyzed depolarization of mitochondrial membrane ΔΨm is an early event of apoptosis. We monitored changes in ΔΨm by loading mitochondrial with DiOC₆(3). The fluorescence intensity analyzed using FACS. As shown in Fig. 1, the quantity of fluorescent profiles decreased as a result of the loss of ΔΨm (see the M2 in Fig. 1) in the cells treated with 30 mM HNE. These observations indicate that HNE induced loss of ΔΨm that may result in apoptosis.

Fig. 1. 
Induction of VSMC mitochondrial depolarization by HNE. Representative histogram plots of cell number versus fluorescence intensity (FL-1). The cells were incubated with HNE for 4 h and then treated with DiOC₆(3) for 30 min. The fluorescence intensity was analyzed using FACS. The results were expressed as the percentage of total cells. The quantity of fluorescent profiles decreased due to the loss of ΔΨm in cells treated with 30 μM HNE (M2 indicated by arrowhead).

Consistent with the reports that lipid oxidation products including HNE and other reactive aldehydes stimulate reactive species (RS) formation in various types of cells. ^[24] Furthermore, it is well documented and widely known that oxidative stress is a precursor to elevated levels of ROS. ^[25] Recently, it has become evident that mitochondria are a major source of ROS, and so contribute to redox cell signaling. ^[26] Furthermore, mitochondria have been considered as an important source of HNE-induced ROS generation since the literatures describing experiments with isolated mitochondria suggest that lipid electrophiles, such as HNE, can modify mitochondrial function. ^[27]

We monitored protection of LO inhibitors against HNE-induced ROS formation. As shown in Fig. 2, LO inhibitors protected the ROS generation in the cells treated with 30 mM HNE. Pretreatment with LO inhibitor, nordihydroguaiaretic acid (NDGA), AA 861 (5-LO inhibitor), and baicalin (12-LO inhibitor) prevented HNE-induced ROS generation. These observations indicate that HNE induced ROS generation mediated with LO.

Fig. 2. 
Protection of LO inhibitors against HNE-induced ROS formation. The cells were incubated with 30 μM HNE for 30 min after pretreatment with the antioxidant N-acetylcysteine (NAC, 500 μM) and lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 1 μM, nonspecific), AA 861 (1 μM, 5-LO), and baicalin (1 μM, 12-LO), respectively, with DCF-DA (10 μM) and analyzed using FACS. Data represent the percentage of DCF fluorescence intensity relative to the control. The results are presented as the mean±SEM of three independent experiments. Statistical significance: *p<0.05 vs. control; ##p<0.01 vs. vehicle

In order to study the action of LO on mitochondrial depolarization induced by HNE, inhibitors were tested to block mitochondrial depolarization. The results showed that HNE (high level, 30 mM) induces ROS formation and alteration of mitochondrial membrane potential (ΔΨm), ultimately leading to VSMC apoptosis(Fig 3). Nonspecific LO inhibitor, NDGA and 12-LO inhibitor bicalin did not prevent HNE-induced depolarization of mitochondrial membrane. A AA 861 also blocked loss of ΔΨm indicating that 5-LO is closely involved in mitochondria-derived ROS production, thereby leading to apoptosis. Based on these results, it is considered that mitochondrial depolarization by HNE is closely related 5-LO.

Fig. 3. 
Inhibitory effects of AA 861 on HNE-induced mitochondrial depolarization. Representative histogram plots of cell number versus fluorescence intensity (FL-1). The cells were incubated with 30 μM HNE for 4 h after pretreatment with inhibitors and loaded with DiOC₆(3) for 30 min. The fluorescence intensity was analyzed using FACS. The results were expressed as the percentage of total cells. AA 861 (5-LO inhibitor) blocked mitochondrial depolarization induced by HNE (arrowhead).

In our study, HNE-induced mitochondrial depolarization was found to be associated with LO. Therefore, it was tested whether HNE actually induced expression of 5LO. At 1 mM HNE (Low level), 5-LO gene expression was increased(Fig. 4). These findings suggest that 5-LO mediates HNE-induced VSMC apoptosis by triggering mitochondrial dysfunction and activation by ROS excessive generation leading to the deterioration of vasculature homeostasis and subsequent vascular dysfunction including retinal neovascularization.

Fig. 4. 
Upregulation of VSMC 5-LO expression induced by HNE. The cells were incubated with HNE for 24 h. 5-LO proteins were analyzed by western blotting. A. Upregulation of 5-LO gene expression by HNE. The data are representative of at least three separate experiments. B. Quantification of 5-LO expression was performed by densitometric analysis. The results are presented as the mean±SEM of three independent experiments. Statistical significance: ^*p<0.05, ^**p<0.01, vs. control

HNE is end-products and remnants of lipid peroxidation processes that may also act as mediators for the primary free radicals that initiated lipid peroxidation. ^[28] HNE produced with relatively large amounts is believed to be responsible for the key mediator of oxidative stress-induced cell death.^[10,12] These reactive aldehydes are known to cause redox disturbances and various degenerative processes, including the vascular dysfunction associated with aging.^[29] It seems logical that HNE is the subjects of recent intensified studies investigating their roles in the modulation of cell signaling pathways and the induction of apoptosis. ^[13,23]

Mitochondrial dysfunction caused by chronic exposure to oxidants and increased activation of mitochondrial permeability transition was found to involve mitochondrial depolarization in human aortic endothelial cell apoptosis.^[16] Recent study showed that mitochondrial dysfunction in age-related macular degeneration.^[30] The enzyme lipoxygenase (LO), which participated in mediating the inflammatory reaction there by retinal change in diabetic retinopathy.^[18,31]

Ocular vascular diseases such as diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration, whose population increases along with aging, have become leading causes of severe visual disturbance. HNE is a substance closely related to aging, which increases with aging. Retinopathy is induced by oxygen exposure induced by HNE in several retinal diseases including retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration and central vein occlusion.

However, ROS-dependent HNE’s ability to induce VSMC dysfunction through the regulation of mitochondrial apoptosis is not yet well described. Thus, it is important to define the role of HNE in VSMC apoptosis in relation to mitochondrial dysfunction. The author established that HNE induced the apoptosis of vascular smooth muscle cells through by mitochondrial depolarization. Furthermore, our study show that in VSMC HNE triggers 5-LO gene expression. These findings strongly suggest that 5-LO mediate VSMC apoptosis induced by HNE, leading to vascular dysfunction such as NV.