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Proteomic analysis of oxidative stress response in human umbilical vein endothelial cells (HUVECs): role of heme oxygenase 1 (HMOX1) in hypoxanthine-induced oxidative stress in HUVECs

Background Erectile dysfunction (ED) is a well-known complication of diabetes, affecting up to 75% of diabetic men. Although the etiology of diabetic ED is multifactorial, endothelial dysfunction is considered to be a pillar of its pathophysiology. Endothelial dysfunction is caused by the harmful effects of high glucose levels and increased oxidative stress on the endothelial cells that comprise the vascular endothelium. The aim of this study was to identify the proteomic changes caused by high glucose-induced oxidative stress and explore the role of heme oxygenase 1 (HMOX1) in it. Methods The cellular proteomic response to hypoxanthine-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were analyzed through Network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Further validation assays was performed to validate the role of HMOX1. Results The results showed that 66 and 76 DEPs were markedly upregulated and downregulated, respectively, for HUVECs oxidative stress. Among these proteins, we verified eight dysregulated genes by quantitative reverse transcription PCR, including nucleolin (NCL), X-ray repair cross-complementing protein 6 (XRCC6), ubiquinol-cytochrome C reductase binding protein (UQCRB), non-POU domain containing octamer binding (NONO), heme oxygenase 1 (HMOX1), nucleobindin 1 (NUCB1), DEK, and chromatin target of prmt1 (CHTOP). Further, using overexpression and genetic knockdown approaches, we found that HMOX1 was critical for the oxidative stress response in HUVECs. Conclusions We found that HMOX1 was closely related to the oxidative stress response induced by hypoxanthine. To the best of our knowledge, this study is the first overview of the responses of HUVECs to oxidative stress. The findings will contribute to analyses of the detailed molecular mechanisms involved in the pathogenesis of endothelial dysfunction and related molecular mechanisms in ED patients.


Introduction
With improvements in the standard of living, the incidence of diabetes is increasing rapidly, and the number of patients diagnosed with diabetes is expected to rise to approximately 300 million worldwide by 2025 (1). Diabetic erectile dysfunction (DED) is a common complication of diabetes, with an incidence of more than 50% in diabetic men (2). Erectile dysfunction (ED) is a common disease in men. It is defined as periodic or persistent ED of the penis, which cannot achieve or maintain erectile function sufficient to satisfy sexual behavior (3)(4)(5). Penile erection is a complex physiological process regulated by the central and peripheral nervous systems, vascular system, and endocrine system (5). It requires the collaboration of multiple factors, including normal vascular endothelial cells (VECs) and the corpus cavernosum; any abnormalities in these processes may lead to ED (6). The vascular endothelium, a simple squamous epithelial layer on the surface of blood vessels, plays a major role in endocrine signaling and has a wide variety of biological functions (7,8), including the process of penile erection (9). Endothelial dysfunction, caused by harmful changes due to high glucose levels and increased oxidative stress in endothelial cells, is critical for the progression of DED (10). In addition to direct damage to endothelial cells, diabetes may hinder the angiogenesis implicated in vascular repair mechanisms, further affecting vasodilation and cavernous blood perfusion, which are essential for normal erectile function.
Hyperglycemia causes various metabolic disorders and promotes endothelial dysfunction and vascular complications (11). Chronic high glucose levels are thought to induce the formation of advanced glycation endproducts (12), as well as the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (13)(14)(15). The increased production of ROS and RNS, especially the free radical superoxide anion (O 2 − ) that reacts with nitric oxide (NO) to form peroxynitrite anion (ONOO − ), has been shown to interfere with endothelial NO bioavailability, p r o p a g a t e e n d o t h e l i a l d y s f u n c t i o n , a n d i m p a i r endothelial and cavernosal smooth muscle reactivity (16).
Hyperglycemia-induced dysregulation of erectile-related signaling pathways in endothelial cells, which mimic the pathological process of DED, provide an effective way to find potential molecular pathologic and therapeutic targets in DED. Thus, the current study aimed to investigate the underlying mechanisms of hyperglycemia-induced endothelial dysfunction using proteomic approaches.

Cell culture and oxidative stress treatment
Human umbilical vein endothelial cells (HUVECs) were cultured in RPMI-1640 supplemented with 50 U/mL penicillin, 50 U/mL streptomycin (Invitrogen, Carlsbad, CA, USA), and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The cells were incubated at 37 ℃ in a humidified atmosphere containing 5% CO 2 . To establish an in vitro model of HUVECs oxidative stress and endothelial dysfunction, HUVECs were treated with different concentrations of hypoxanthine in order to observe their oxidative damage. HUVECs were prepared as a suspension of 1.5×10 5 cells/mL and then seeded in 24-well plates at 1 mL per well. When the cells reached 80% confluence, they were divided into the normal control group and treatment groups. Specifically, as previously described (17), HUVECs were treated with hypoxanthine (dissolved in PBS) at a final concentration of 500, 1,000, 2,000, or 5,000 ng/mL. Three wells were used for each treatment group. The cells were further incubated at 37 ℃ and 5% CO 2 , and then visualized with an inverted microscope to reveal cell morphology.

Sample preparation, protein extraction, digestion, and isobaric tags for relative and absolute quantitation (iTRAQ) labeling
HUVECs were collected from the normal control and 1,000 ng/mL hypoxanthine-treated groups. All samples were analyzed in triplicate, and each independent sample was analyzed in three technical replicates. Protease inhibitor was added in the mixture of the lysate, and then

Fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS)
The labeled peptides were reconstituted with high-pH reverse-phase (RP) liquid phase (20 mM HCOONH 4 , pH 10) and mixed. According to the peak type and time, 24 components were collected in Eppendorf tubes from a linear gradient, acidified with 50% trifluoroacetic acid, vacuum-dried, and analyzed by two-dimensional liquid chromatography-mass spectrometry (LC-MS

Quantitative reverse transcription PCR (RT-qPCR)
HUVECs were harvested using trypsin and lysed with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and eluted with nuclease-free water. First-strand cDNA was synthesized using PrimeScript RT Master Mix (Thermo Fisher Scientific). The primers used for real-time PCR are listed in Table 1.

Immunofluorescence
HUVECs were plated in 4-well, 35-mm dishes (Greiner Bio-One, Kremsmünster, Austria) at a density of 1,000 cells/well and grown for 48 h. Then, the cells were fixed with 4% paraformaldehyde for 20 min and permeabilized in PBS supplemented with 0.5% Triton X-100. After blocking, the indicated antibodies were added to the cells and incubated for 2 h. The cells were washed in PBS, incubated with tetramethylrhodamine (TRITC)or fluorescein isothiocyanate (FITC)-labeled secondary antibodies (Pierce Biotechnology, Waltham, MA, USA) for 1 h at room temperature, and stained with 4,6-diamidino-2phenylindole (DAPI). The cells were mounted with glycerol and observed using a Nikon A1 laser scanning confocal microscope (Tokyo, Japan).

Gene
Primer sequence buffer, and the supernatants were collected by centrifugation. The protein content in the RIPA lysates is quantified, and the concentration was determined using the BCA Protein Assay reagent kit. Proteins were fractionated using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes; the membranes were blocked for 1 h in 5% skim milk before being incubated with antibodies against heme oxygenase 1 (HMOX1), p-AKT1, and AKT1 (Abcam, UK) at 4 ℃ overnight. The membranes were then washed three times in TBST (Tris-buffered saline, 0.1% Tween 20) for 10 min each. Then, each membrane was incubated with antirabbit IgG H&L for 1 h at room temperature. Finally, each membrane was washed again with TBST and bound protein was visualized using an enhanced chemiluminescence (ECL) Western blotting detection kit (GE Healthcare, Chicago, IL, USA); signals were observed using GeneGnome (Syngene, Bangalore, India). For HMOX1 gene silencing and overexpression, the cells were transfected with plasmids designed by OBiO Technology (Shanghai, China).

Cell counting assay
The effect of shRNA-HMOX1 on HUVEC proliferation was examined using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). Briefly, the cells were incubated in 100 μL medium in 96-well plates at 2,000 cells/well. Then, 10 μL CCK-8 reagent was added to each well and incubated for 2 h at 37 ℃, and a microplate reader was used to determine the absorbance at 450 nm.

Statistical analysis
Data are reported as the means ± SD, and statistical analyses were performed using Graphpad Prism version 7.0 (GraphPad Software, San Diego, California, USA). Comparisons were performed by one-way ANOVA or independent Student's t-tests. A value of P<0.05 was considered statistically significant.

Oxidative stress model of cultured HUVECs
Endothelial dysfunction is caused by the harmful effects of increased glucose and oxidative stress on endothelial cells (2).
To further investigate the underlying mechanism of oxidative stress-induced endothelial dysfunction in DED patients, we stimulated HUVECs with the ROS generator hypoxanthine to mimic an oxidative stress model (3,18,19). Four different   Figure 1A, cultured HUVECs in the control group formed a flattened monolayer and displayed typical cobblestone-like morphology at confluence. Upon hypoxanthine treatment, the HUVECs were rearranged and interconnected sparsely; damaged and floating cells were apparent, and the cell density decreased significantly in a hypoxanthine concentrationdependent manner ( Figure 1A). Furthermore, the levels of LDH ( Figure 1B) and MDA ( Figure 1C) were increased, while SOD activity was decreased ( Figure 1D) by hypoxanthine treatment in a time-and concentration-dependent manner.
For subsequent experiments, we chose 1,000 ng/mL hypoxanthine treatment for HUVECs; at this concentration, no visible morphological damage occurred, but significant changes were observed in LDH, MDA, and SOD levels.

General information on iTRAQ analysis
Cell samples were collected and prepared for iTRAQ analysis. iTRAQ-coupled LC-MS/MS analysis identified 40,868 peptides and 4,769 proteins ( Figure 2). Most peptides were 6-21 amino acids in length, with the majority being 9 amino acids in length (Figure 2A). Among the 4,796 identified proteins, most contained fewer than ten peptides, and the protein amount decreased as the number of matched peptides increased ( Figure 2B). Next, the distribution of protein sequence coverage was analyzed ( Figure 2C). The majority of the identified proteins showed good peptide coverage; 50.8% of the identified proteins had more than 10% peptide coverage, and 23.6% had more than 20% coverage. The matching error distribution of the peptides is shown in Figure 2D. These results suggested that the protein isolation and identification were successful, and the data were subjected to further analysis.

Identification of DEPs in oxidatively stressed HUVECs
Three groups of normal control samples were labeled with iTRAQ 113, 114, and 115, respectively, and three groups of oxidized samples were labeled with iTRAQ 117, 118, and 119, respectively. We used principal component analysis (PCA) to test the correlation between the values of the three biological replicates, and found that a proportion of the analyzed proteins was responsible for hypoxanthineinduced oxidative stress in HUVECs ( Figure 3A). In our iTRAQ proteomic analysis, the screening was based on the following criteria: P<0.05 and FC ratio ≥1.3 or ≤0.76. Among the DEPs, 66 proteins were significantly upregulated and 76 were significantly downregulated ( Figure 3B). The top 20 DEPs are shown in Table 2. The   7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Figure S1.

Functional characterization of the DEPs
To study the biological functions of the 142 DEPs, GO enrichment was performed. GO terms with P<0.05 and FDR <0.05 were considered significantly enriched. Negative regulation of microtubule polymerization, cytoplasmic translation, and regulation of microtubule polymerization were the most highly enriched under biological process ( Figure 4A). Ligase activity, formation of carbon-oxygen bonds, aminoacyl-tRNA ligase activity, and telomeric DNA binding were the most highly enriched under molecular function ( Figure 4B). Endoplasmic reticulum Sec complex, cortical microtubule cytoskeleton, and CCAAT-binding factor complex were the most highly enriched under cellular component ( Figure 4C).

KEGG pathway annotation of DEPs
In order to further study and screen the pathways related to DEPs, we conducted KEGG pathway analysis. P<0.05 was defined as significant KEGG pathway enrichment.  Table 3, pathways such as protein export (hsa03060), Shigellosis (hsa05131), aminoacyl-tRNA biosynthesis (hsa00970), bacterial invasion of epithelial cells (hsa05100), metabolic pathways (hsa01100), and Huntington's disease (hsa05016) were the most significantly enriched KEGG pathways associated with hypoxanthineinduced oxidative stress in HUVECs.

PPI network analysis of the DEPs
In the animal body, when cells are stimulated by internal and external oxidative factors, the production of ROS is increased, disrupting the balance between the oxidation and anti-oxidation systems, leading to oxidative stress (3)(4)(5).

Role of HMOX1 in hypoxanthine-induced oxidative stress in HUVECs
To verify the results of iTRAQ-MS, we selected the significant DEPs NCL, XRCC6, UQCRB, NONO, HMOX1, NUCB1, DEK, and CHTOP for RT-qPCR verification. As shown in Figure 6A, compared to those in the normal control group, the mRNA levels of NCL and NUCB1 were significantly upregulated, while that of HMOX1 was downregulated in hypoxanthine-treated cells. Thus, our real-time PCR results corroborated the iTRAQ-MS results.
The iTRAQ results showed that the expression of HMOX1 was downregulated in oxidative stress-exposed HUVECs, indicating that this gene may play an important role in the oxidative stress response in HUVECs. To test this hypothesis, we constructed an HMOX1 overexpression plasmid (pcDNA-HMOX1) and its control plasmid (pcDNA-Con). Cells treated with hypoxanthine were transfected with the two plasmids. As indicated in Figure 6B, the transfection did not alter cell viability compared with that of the normal control. As shown by Western blotting, the expression of HMOX1 was downregulated in hypoxanthine-treated HUVECs, while transfection with the Transl Androl Urol 2020 | http://dx.doi.org/10.21037/tau.2020.03.11 © Translational Andrology and Urology. All rights reserved. overexpression plasmid had the opposite effect ( Figure 6C). SOD, catalase (CAT), and NO levels were decreased upon hypoxanthine treatment, and could be restored by HMOX1 overexpression (Figure 6D,E,F). HMOX1 is known to be a downstream target of AKT (20). We activated the AKT pathway using insulin-like growth factor 1 (IGF-1), and this stimulation increased the expression of HMOX1 ( Figure 6G) and increased NO production ( Figure 6H). These data suggest that HMOX1 is important in the hypoxanthineinduced oxidative stress response of HUVECs.

Discussion
ED has become a worldwide issue affecting the health of men; its incidence has risen sharply not only in Europe and North America, but also in other countries (21). Penile erection is a vascular phenomenon, and blood flow plays a central role in the erectile mechanism (22,23). Hyperglycemia affects the formation of capillary basement membranes and macromolecular polysaccharides, and it induces endothelial cell membrane glycation (24,25). Vascular endothelial injury is a key factor in the occurrence and development of vascular diseases in diabetic patients (26). There is known to be a close relationship between vascular disease and diabetic ED (2). Diabetic patients with macrovascular diseases are vulnerable to internal iliac artery and sponge spiral atherosclerosis, which can lead to reduced blood pressure, blood perfusion to the cavernous sinus, and penile erection hardness (27). Furthermore, capillary microcirculation in diabetic patients with organic impotence shows significant pathological changes, including poor microvascular filling and a significant reduction in the number of blood vessels (27,28).
Corpus cavernosum endothelial cells, endothelial cells arranged on the inner surface of the cavernous sinus, are one of the basic components of penile vessels; their major function is the synthesis of vasodilatory factors and contractile factors (such as NO and endothelin), which play important roles in penile erection. However, the molecular mechanisms involved in the oxidative stress response of endothelial cells have not been fully determined. In particular, no studies have yet performed a differential proteome analysis of endothelial cells in response to oxidative stress. Thus, in the current study, we utilized iTRAQ to identify DEPs and further explore the pathologic mechanisms of the oxidative stress response in HUVECs. In total, we identified 66 upregulated and 76 downregulated proteins; these proteins are thought to play a role in the oxidative stress response in HUVECs. Among them, we verified the mRNA expression of several dysregulated proteins using RT-qPCR. Further, using gene overexpression and knockdown, we found that HMOX1 was critical for the oxidative stress response in HUVECs.
HMOX1 is critical in the defense response against oxidant-induced injury in many pathological conditions (29)(30)(31). HMOX1 is known to be regulated by oxidative stress-promoting stimuli including hypoxia, hyperoxia, heat shock, excess heme accumulation, NO, and endotoxins (32,33). HMOX1 shows anti-inflammatory, antioxidant, antiapoptotic and antiproliferative effects (34,35), and is B A thus considered a cytoprotective enzyme (36). HMOX1 was previously found to modulate oxidative stress and inflammation and regulate cell cycle progression to prevent apoptosis in acute kidney injury (37). NO stimulates HMOX1 expression through the Nrf2/antioxidant responsive element (ARE) complex and promotes the survival of vascular smooth muscle cells (38). Additionally, we found that hypoxanthine treatment downregulated HMOX1 in HUVECs, indicating that HMOX1 regulates a variety of processes related to penile function in vascular smooth muscle cells and endothelial cells. Furthermore, HMOX1 is known to attenuate lipogenesis disorders, as its upregulation reduces visceral and subcutaneous fat accumulation and improves insulin sensitivity (39,40); thus, HMOX1 plays a major role in DED. HMOX1 has been reported to be the downstream target of AKT (20); in the current study, we demonstrated that AKT activity affected HMOX1 expression. Furthermore, reduced phosphoinositide 3-kinase (PI3K)/AKT/endothelial nitric oxide synthase (eNOS) activity was previously shown to be closely related to metabolic syndrome in a rat model of ED (41). Our data showed that activation of the AKT pathway increased NO production, consistent with previous reports (41,42).
Although the pathogenesis of DED is clear, the intracellular mechanism of the injury of penile endothelial cells is not. In this experiment, we used proteomics to analyze the difference in protein expression in endothelial cell injury induced by high glucose, which provided a basis for understanding the occurrence, development, and prognosis of DED. According to the function and location of these proteins, we can further understand the mechanism of dynamic changes in cells when DED occurs. These DEPs are mainly related to oxidative stress and energy metabolism, suggesting that the high glucose environment of penile epithelial cells in diabetic patients is closely related to inflammatory responses. It is confirmed that the damage of high glucose load can be regulated by reducing oxidative stress damage and affecting energy metabolism. The limitation of this experiment is that the functions of other molecules beyond that of HMOX1 have yet to be tested, and these are expected to represent other potential markers of DED. Although our in vitro study advances understanding of the role of HMOX1 in DED, it is necessary to confirm our findings in multiple cell lines and in vivo experiments in the future.

Conclusions
The present study provides the first overview of protein alterations in HUVECs under oxidative stress. Our identification of DEPs by iTRAQ analysis revealed a comprehensive interaction network in HUVECs during their oxidative stress response. Several significantly dysregulated proteins were identified to be associated with the pathological response of VECs under ED. Further functional exploration should be implemented to reveal pathologic mechanisms and identify new therapeutic targets for preventing ED.