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. 2020 Translational Andrology and Urology. All rights reserved.

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 (3-5). 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 end-products (12), as well as the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (13-15). The increased production of ROS and RNS, especially the free radical superoxide anion (O2−) that reacts with nitric oxide (NO) to form peroxynitrite anion (ONOO−), has been shown to interfere with endothelial NO bioavailability, propagate endothelial dysfunction, and impair 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.

Methods

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 °C in a humidified atmosphere containing 5% CO2. 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 1.5×105 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 °C and 5% CO2, 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 collected cells for lysis. The lysed cells were sonicated and centrifuged at 14,000 ×g for 40 min. Protein content in the supernatant was then quantified using the bicinchoninic acid (BCA) Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Then, 200 µg of protein was digested with 4 µg trypsin (Promega, Madison, WI, USA) at 37 °C overnight. According to the protocol of the iTRAQ kit (8plex, Applied Biosystems, Foster City, CA, USA), 100 µg of the resulting peptide mixture from each sample was labeled as follows: three normal control samples were labeled with iTRAQ 113, 114, or 115, and three treated samples were labeled with iTRAQ 117, 118, or 119. Then, the labeled samples were mixed and dried with a rotary vacuum concentrator.

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 HCOONH4, 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). The vacuum-dried sample was resuspended in 20 µL high-performance liquid chromatography (HPLC) Buffer A (0.1% formic acid, 2% acetonitrile), injected into a ZORBAX 300-C18 RP column (5 µm, 300 Å, 0.1 mm × 150 mm), and then equilibrated with Buffer B (0.125% formic acid, 95% ACN). The sample was eluted with a concentration gradient of acetonitrile (5–35% in 0.1% formic acid) in 90-µm volumes with a flow rate of 0.3 µL/min. The eluted sample was then analyzed by Q-Orbitrap System first-order mass spectrometry (MS) and tandem mass spectrometry (MS/MS) (Thermo Fisher Scientific, Waltham, MA, USA).

Bioinformatics analysis

ProteinPilot Software 5.0 (ABSciex, Redwood City, CA, USA) was used to identify and quantify differentially expressed proteins (DEPs) from the LC-MS/MS data. To further understand the impact of DEPs on endothelial cells and to investigate relationships between the DEPs, gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) analyses were performed. GO enrichment was performed for biological process, molecular function, and cellular component, and was applied based on Fisher’s exact test considering the whole quantified protein annotations as the background dataset. The Benjamini-Hochberg correction for multiple testing was further applied to adjust the derived P values. In our iTRAQ proteomic analysis, the screening was based on the following criteria: P<0.05 and fold change (FC) ratio ≥1.3 or ≤0.76. GO terms with P<0.05 and false discovery rate (FDR) <0.05 were considered significantly enriched. KEGG pathway enrichment was performed using the clusterProfiler package in R software (version 3.6.0) based on the KEGG pathway database (http://www.kegg.jp/kegg/pathway.html). PPI network analysis was performed according to the STRING database (http://string-db.org/). Then, the results were imported into CytoScape software for visualization.

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

Primers used for RT-qPCR

Gene	Primer sequence
NCL	Forward: 5'-CTGATGAGGGCACCCGTTTGCTAC-3'Reverse: 5'-AAACAGTCCATTTAATCTCTGACCTCACG-3'
XRCC6	Forward: 5'-AAGAATGTCTCCCCTTATTTTGTGG-3'Reverse: 5'-TCTCGAAACTGTCGCTCCTGTATGT-3'
UQCRB	Forward: 5'-ATGTGAATTCATGGCTGGTAAGCAGGCC-3'Reverse: 5'-ATGCCTCGAGCTTCTTTGCCCATTCTTC-3'
NONO	Forward: 5'-AAAGCAGGCGAAGTTTTCATTC-3'Reverse: 5'-ATCCCGCTGACTGTTCCCT-3'
HMOX1	Forward: 5'-CACTTCGTCAGAGGCCTGCTA-3'Reverse: 5'-GTCTGGGATGAGCTAGTGCTGAT-3'
NUCB1	Forward: 5'-CTGCTCAAGGCCAAGATGGA-3'Reverse: 5'-CCTTGAGCATCTCGTAGCGT-3'
DEK	Forward: 5'-GTGGGTCAGTTCAGTGGC-3'Reverse: 5'-AGGACATTTGGTTCGCTTAG-3'
CHTOP	Forward: 5'-AGAGAGGCTTGCCCAGAGG-3'Reverse: 5'-CCGACCTATCATACCCCGAC-3'
β-actin	Forward: 5'-GTTGACATCCGTAAAGACC-3'Reverse: 5'-TAGGAGCCAGGGCAGTAATC-3'

RT-qPCR, reverse transcription PCR.

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-2-phenylindole (DAPI). The cells were mounted with glycerol and observed using a Nikon A1 laser scanning confocal microscope (Tokyo, Japan).

Transfection and Western blotting

HUVECs were harvested using trypsin, lysed with RIPA 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 °C 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 anti-rabbit 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 °C, 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.

Results

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 concentrations of hypoxanthine were added to HUVECs for 2, 4, 8, and 12 h. Lactate dehydrogenase (LDH) activity, malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity were measured by ELISA. As shown in , 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 concentration-dependent manner (). Furthermore, the levels of LDH () and MDA () were increased, while SOD activity was decreased () 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.

Figure 1

Hypoxanthine treatment induces oxidative stress response in human umbilical vein endothelial cells (HUVECs). HUVECs were treated without or with hypoxanthine at 500, 1,000, 2,000, or 5,000 ng/mL for 2, 4, 8, or 12 h. (A) Representative images of HUVECs under each treatment. Scale bar, 20 μm; (B,C,D) the levels of lactate dehydrogenase (LDH) (B), malondialdehyde (MDA) (C), and superoxide dismutase (SOD) (D) were measured. Data represent the means ± SD from three independent experiments in triplicate. *, P<0.05 1,000 ng/mL versus the normal group; **, P<0.01 1,000 ng/mL versus the normal group by one-way ANOVA.

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 (). Most peptides were 6–21 amino acids in length, with the majority being 9 amino acids in length (). Among the 4,796 identified proteins, most contained fewer than ten peptides, and the protein amount decreased as the number of matched peptides increased (). Next, the distribution of protein sequence coverage was analyzed (). 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 . These results suggested that the protein isolation and identification were successful, and the data were subjected to further analysis.

Figure 2

Results of isobaric tags for relative and absolute quantitation analysis. (A) Distribution of peptide lengths; (B) unique peptides in the detected proteins; (C) coverage of proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS); (D) error distribution of spectrogram quality matching.

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 hypoxanthine-induced oxidative stress in HUVECs (). 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 (). The top 20 DEPs are shown in . The detail information of the DEPs is shown as a heatmap in Figure S1.

Figure 3

Analysis of differentially expressed proteins (DEPs) between hypoxanthine-treated and untreated human umbilical vein endothelial cells (HUVECs). (A) Principal component analysis plot showing the corrected correlation values of the three biological replicates; (B) volcano plot of DEPs. FC, fold change.

Table 2

Top 20 differentially expressed proteins in HUVECs under hypoxanthine-induced oxidative stress

Accession	Gene symbol	FC ratio (model/control)
Upregulated
sp|Q86WR0	CCD25	2.104782303
sp|P27695	APEX1	1.946251088
sp|O60784	TOM1	1.848857416
sp|Q9Y399	RT02	1.758804427
tr|I3L504	I3L504	1.752057433
sp|Q16543	CDC37	1.714400662
sp|Q96K37	S35E1	1.623314752
sp|Q5SNV9	CA167	1.623209715
sp|P04818	TYSY	1.607742018
sp|P53999	TCP4	1.594754683
sp|Q96QC0	PP1RA	1.586030126
sp|Q06830	PRDX1	1.584336493
sp|Q16706	MA2A1	1.583226217
sp|Q8IU81	I2BP1	1.578175558
sp|Q9UH65	SWP70	1.573015438
sp|Q9BVG9	PTSS2	1.521094534
sp|O15511	ARPC5	1.518378933
sp|O15446	RPA34	1.495074259
sp|P62979	RS27A	1.490537537
sp|Q4VCS5	AMOT	1.474095782
Downregulated
sp|P49588	SYAC	0.353742354
sp|P62805	H4	0.395187226
sp|Q9BV57	MTND	0.404758182
sp|P63261	ACTG	0.414486246
sp|P63244	RACK1	0.444610609
sp|P35527	K1C9	0.446953376
sp|P09132	SRP19	0.507089072
sp|Q9NV92	NFIP2	0.512578726
sp|Q9Y3Y2	CHTOP	0.5417456
sp|P62888	RL30	0.552873929
sp|P09601	HMOX1	0.560592135
sp|Q9BW92	SYTM	0.573749512
tr|A0A087WWE2	A0A087WWE2	0.591824353
sp|Q92896	GSLG1	0.599185434
sp|P35914	HMGCL	0.612094243
sp|P04264	K2C1	0.623481936
sp|P04406	G3P	0.629085359
sp|Q9BY77	PDIP3	0.631970955
sp|O94925	GLSK	0.633027938
sp|Q02818	NUCB1	0.63618553

HUVECs, human umbilical vein endothelial cells; FC, fold change.

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 (). Ligase activity, formation of carbon-oxygen bonds, aminoacyl-tRNA ligase activity, and telomeric DNA binding were the most highly enriched under molecular function (). Endoplasmic reticulum Sec complex, cortical microtubule cytoskeleton, and CCAAT-binding factor complex were the most highly enriched under cellular component ().

Figure 4

Gene ontology (GO) term enrichment of the identified dysregulated proteins. **, P<0.01; ***, P<0.001; #, false discovery rate (FDR) <0.05; ##, FDR <0.01; ###, FDR <0.001.

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. As shown in , 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 hypoxanthine-induced oxidative stress in HUVECs.

Table 3

KEGG pathway analysis of dysregulated proteins in HUVECs under oxidative stress

Pathway ID	Description	Genes in this pathway (gene ID)	P value
hsa03060	Protein export	23478, 6734, 10952, 6728	1.71×10−8
hsa05131	Shigellosis	1399, 10092, 960, 4793, 71	1.28×10−7
hsa00970	Aminoacyl-tRNA biosynthesis	8565, 4677, 16, 57505, 80222	1.41×10−7
hsa05100	Bacterial invasion of epithelial cells	1399, 1212, 1211, 10092, 71	3.84×10−7
hsa01100	Metabolic pathways	3945, 6888, 7381, 7298, 521, 522, 4124, 81490, 2597, 4719, 5033, 2744, 6241, 3155, 283871, 5106, 55256	4.82×10−7
hsa05016	Huntington’s disease	7381, 1212, 1211, 522, 4719, 160, 293	5.06×10−7
hsa03010	Ribosome	6233, 51116, 6156, 6138, 6218, 6224	6.59×10−7
hsa05130	Pathogenic Escherichia coli infection	4691, 10971, 10092, 71	1.62×10−6
hsa04666	Fc gamma R-mediated phagocytosis	1399, 4082, 65108, 10092	2.17×10−5
hsa04961	Endocrine and other factor-regulated calcium reabsorption	1212, 1211, 160	2.45×10−5

KEGG, Kyoto Encyclopedia of Genes and Genomes; HUVECs, human umbilical vein endothelial cells.

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 (3-5). Excessive ROS accumulation can activate factors such as nuclear factor E2-related factor 2(Nrf2), nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) to regulate the expression of oxidant and antioxidant factors (1,2). Thus, in this study, we selected 20 DEPs involved in oxidative stress, cell apoptosis, and DNA damage (XRCC6, LDHB, TALDO1, RPS27A, UQCRB, GLRX3, APEX1, PPP1R10, SUB1, TOP2A, RIF1, GAPDH, NONO, SLC25A6, CPD, HMOX1, NUCB1, DEK, CHTOP, and ING2) through literature research, and then identified sixteen impotence-related proteins among them, namely NOS1, PRL, PDE5A, KLK3, VIP, SHBG, NOS3, KCNMA1, NOS2, EDN1, PDE3A, CYP3A4, ALDH7A1, KNG1, SRD5A1, ARG2, and SEPT3 using the MalaCards database (http://www.malacards.org/). These proteins were imported into STRING and further analyzed by CytoScape ().

Figure 5

Protein-protein interaction network analysis. (A) Network of twenty differentially expressed proteins (DEPs) involved in the oxidative stress response, cell apoptosis, and DNA damage, as well as erectile dysfunction-related proteins; (B) network of twenty DEPs associated with the Keap1-Nrf2-ARE, PI3K/AKT, and toll-like receptor (TLR) signaling pathways. Red and green boxes indicate upregulated and downregulated proteins, respectively. Blue, light blue, and pink boxes indicate proteins associated with the Keap1-Nrf2-ARE, PI3K/AKT, and TLR signaling pathways, respectively.

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 , 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.

Figure 6

Role of heme oxygenase 1 (HMOX1) in the hypoxanthine-induced oxidative stress response in human umbilical vein endothelial cells (HUVECs). (A) Cultured HUVECs were treated with 1,000 ng/mL hypoxanthine or left untreated (control, Con), and the cells were subjected to RT-qPCR to verify the results of iTRAQ. The relative mRNA expression levels of the indicated genes are shown. ***, P<0.001 versus the control group. (B) HUVECs were treated with (HX) or without (Con) hypoxanthine, or together with HMOX1 control (HX + pcDNA-Con) or HMOX1 overexpression plasmids (HX + pcDNA-HMOX1); thereafter, cell viability was determined in each group. (C) Western blot validation of the plasmid transfection, with β-actin as a loading control. (D,E,F) Levels of superoxide dismutase (SOD) (D), catalase (CAT) (E), and NO (F) in each group. (G) Cultured HUVECs were treated with or without hypoxanthine together with the AKT-pathway activator IGF-1 or HMOX1 knockdown plasmids. Western blot validation and quantification of the plasmid transfection, with β-actin as a loading control. (H) The production of NO was analyzed. ***, P<0.001 versus the control group. ###, P<0.001 versus the HX + IGF-1 + shRNA-Con group.

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 , 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 overexpression plasmid had the opposite effect (). SOD, catalase (CAT), and NO levels were decreased upon hypoxanthine treatment, and could be restored by HMOX1 overexpression (). 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 () and increased NO production (). These data suggest that HMOX1 is important in the hypoxanthine-induced 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 (29-31). 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 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.

Heatmap Analysis of differentially expressed proteins (DEPs) between hypoxanthine-treated and untreated human umbilical vein endothelial cells (HUVECs).

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