BCL6 Attenuates Proliferation and Oxidative Stress of Vascular Smooth Muscle Cells in Hypertension

Proliferation and oxidative stress of vascular smooth muscle cells (VSMCs) contribute to vascular remodeling in hypertension and several major vascular diseases. B-cell lymphoma 6 (BCL6) functions as a transcriptional repressor. The present study is designed to determine the roles of BCL6 in VSMC proliferation and oxidative stress and underlying mechanism. Angiotensin (Ang) II was used to induce VSMC proliferation and oxidative stress in human VSMCs. Effects of BCL6 overexpression and knockdown were, respectively, investigated in Ang II-treated human VSMCs. Therapeutical effects of BCL6 overexpression on vascular remodeling, oxidative stress, and proliferation were determined in the aorta of spontaneously hypertensive rats (SHR). Ang II reduced BCL6 expression in human VSMCs. BCL6 overexpression attenuated while BCL6 knockdown enhanced the Ang II-induced upregulation of NADPH oxidase 4 (NOX4), production of reactive oxygen species (ROS), and proliferation of VSMCs. BCL6 expression was downregulated in SHR. BCL6 overexpression in SHR reduced NOX4 expression, ROS production, and proliferation of the aortic media of SHR. Moreover, BCL6 overexpression attenuated vascular remodeling and hypertension in SHR. However, BCL6 overexpression had no significant effects on NOX2 expression in human VSMCs or in SHR. We conclude that BCL6 attenuates proliferation and oxidative stress of VSMCs in hypertension.


Introduction
Vascular smooth muscle cells (VSMCs) are dominant cellular constituent of arteries [1,2]. VSMC proliferation is closely linked with vascular remodeling and stiffening in the initiation and progression of vascular diseases such as hypertension, atherosclerosis, myocardial hypertrophy, myocardial infarction, stroke, dementia, and renal failure [3][4][5]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a major source of reactive oxygen species (ROS) in VSMCs and is essential to cell proliferation [6]. It is known that Ang II binds to Ang II type 1 receptor (AT 1 R) and consequently activates NOX and promotes ROS generation and VSMC proliferation [7][8][9]. Angiotensin II (Ang II) is associated with the pathogeneses of hypertension and cardiovascular remodeling [10][11][12]. Intervention of reninangiotensin system in hypertensive patients lowers morbidity and mortality [13,14].
B-cell lymphoma 6 (BCL6) is initially discovered as an oncogene in B-cell lymphomas. It drives the malignant phenotype by repressing proliferation and DNA damage checkpoints and blocking B-cell terminal differentiation, and BCL6 inhibitors yield dramatic antitumor effects [15]. BCL6 protein is an evolutionarily conserved zinc finger transcription factor with an N-terminal POZ/BTB domain. This protein interacts with several corepressor complexes to inhibit transcription and functions as a sequence-specific transcriptional repressor [16]. BCL6 member B (BCL6B), a BCL6 homologous gene, suppresses proliferation of colorectal carcinoma cells through inhibition of the PI3K signaling pathway [17]. BCL6 is considered as a therapeutic target for autoimmune diseases and cancer treatment [15]. BCL6 overexpression inhibits ROS generation and apoptosis induced by chemotherapy in B-cell lymphoma cells [18]. However, it is unknown whether BCL6 plays a role in vascular remodeling in hypertension. This study reveals the role of BCL6 in inhibiting Ang II-induced VSMC proliferation and oxidative stress and attenuating vascular remodeling in hypertensive rats.

Materials and Methods
2.1. Animals. Thirteen-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were obtained from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) and housed in a temperaturecontrolled room with a 12 h light/dark cycle and free access to standard chow and tap water. Experiments were approved by the Experimental Animal Care and Use Committee of Nanjing Medical University and the Guide for the Care and Use of Laboratory Animal published by the US National Institutes of Health (NIH publication, 8th edition, 2011). At the end of experiments, each rat was euthanized with an overdose of pentobarbital sodium (150 mg/kg, iv), and the aorta was harvested for histological and molecular biological analyses.

Cell
Culture. Human aortic VSMCs were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured as described previously [3]. Simply, the VSMCs were cultured in F12K Kaighn's modification medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 . The medium was replaced at intervals of 3-4 days. The cells were starved for 24 h in a serum-free medium before use [19].
2.6. CCK-8 Assay. Cell counting kit-8 kits (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China) were used for evaluating VSMC proliferation according to the manufacturer's instructions. As we reported previously [3], 10 μL of CCK-8 solution was added into each well and incubated for 2 h at 37°C. The absorbance was measured at 450 nm with a microplate reader (ELX800, BioTek, Vermont, USA).

EdU Incorporation
Assay. EdU incorporation assay was used to determine VSMC proliferation with In Vitro Imaging Kit (Guangzhou RiboBio, Guangzhou, China). The DNA synthesis of VSMCs was measured using a Cell-Light™ EdU Apollo®567. The EdU-positive cells were counted and normalized by the total number of Hoechst 33342 stained cells [3].
2.8. RT-PCR. Total RNA was isolated with Trizol reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer's instruction. Reverse transcriptase reactions were performed using the PrimeScript RT reagent Kits. RT-PCR was performed using Quantitative PCR with SYBR Premix Ex Taq TM (Takara, Otsu, Shiga, Japan) and ABI PRISM 7500 sequence detection PCR system (Applied Biosystems, Foster City, CA, USA). The expressions of mRNA were calculated using the comparative cycle threshold (Ct) method where the relative quantization of target transcript levels was determined by subtracting Ct values of target genes from Ct values of GAPDH. The sequences of primers for humans and rats were listed in Tables 1 and 2.     data were expressed as mean ± SE. A value of P < 0 05 was considered statistically significant.

BCL6 Overexpression Inhibits Ang II-Induced VSMC
Proliferation. VSMC proliferation was evaluated with CCK-8 kit, EdU assay, and proliferating cell nuclear antigen (PCNA) expression. CCK-8 assay showed that Ang II-induced VSMC proliferation was inhibited by BCL6 overexpression (Figure 1(a)). EdU incorporation assay detects DNA synthesis in proliferating cells. BCL6 overexpression inhibited the increase in the number of EdU-positive cells caused by Ang II in VSMCs (Figures 1(b) and 1(c)). PCNA is an essential component at the DNA replication and a key marker involved in DNA replication and cell proliferation. Ang II increased PCNA expression in VSMCs, which was attenuated by BCL6 overexpression (Figure 1(d)). On the other hand, Ang II reduced BCL6 expression in VSMCs. The effectiveness of the BCL6 overexpression was confirmed by the increased BCL6 protein level in VSMCs (Figure 1(e)).

BCL6 Knockdown Intensified Effects of Ang II in VSMCs.
Knockdown of BCL6 with shRNA in VSMCs aggravated Ang II-induced VSMC proliferation evidenced by the increased absorbance measured with CCK-8 assay (Figure 3(a)) and the upregulation of PCNA protein expression (Figure 3(b)). BCL6 knockdown had no significant effect on NOX2 protein expression but aggravated Ang II-induced upregulation of NOX4 protein in VSMCs (Figure 3)(c). Moreover, Ang II-induced ROS production was increased by BCL6 knockdown (Figures 3(d) and 3(e)). The effectiveness of BCL6 knockdown was confirmed by the downregulation of BCL6 protein in VSMCs (Figure 3(f)).

BCL6 Overexpression Attenuates Vascular Remodeling in SHR.
Recombinant lentivirus expressing BCL6 was administrated intravenously to induce BCL6 overexpression in WKY and SHR. The effectiveness of BCL6 overexpression was confirmed by the upregulation of BCL6 protein expression in the aortic media of both WKY and SHR. Moreover, the BCL6 protein expression in the aortic media was downregulated in SHR (Figure 4(a)). BCL6 overexpression attenuated the upregulation of PCNA protein expression in the aortic media of SHR (Figure 4(b)). Masson's staining showed that BCL6 overexpression attenuated vascular remodeling in SHR (Figure 4(c)). Media thickness (M), lumen diameter (L), and their ratio of artery were used as indexes of vascular remodeling [22]. Increased M and M/L in the aorta of SHR were prevented by BCL6 overexpression (Figure 4(d)).

BCL6 Overexpression Attenuates
Oxidative Stress in the Aorta of SHR. Both NOX2 and NOX4 mRNA and protein expressions in the aortic media were increased in SHR compared with those in WKY. BCL6 overexpression attenuated the NOX4 mRNA and protein upregulation in SHR rather than the NOX2 mRNA and protein upregulation (Figures 5(a) and 5(b)). DHE staining revealed that the ROS level in the aorta was greatly increased in SHR, which was attenuated by BCL6 overexpression (Figures 5(c) and 5(d)).

Discussion
VSMC proliferation contributes to vascular remodeling in several major vascular diseases including hypertension, pulmonary arterial hypertension, atherosclerosis, vascular restenosis, transplantation arteriopathy, and diabetic vascular complications [2]. Oxidative stress is a critical factor in promoting cell proliferation [23]. Primary novel findings in the present study are that BCL6 inhibits Ang II-induced oxidative stress and proliferation in human VSMCs, and BCL6 overexpression attenuates oxidative stress and vascular remodeling in the aorta of SHR. BCL6 reduces NOX4  expression, which may contribute to its antioxidative effect in Ang II-treated VSMCs or the aorta of SHR. Ang II plays a crucial role in hypertension and its associated organ damage [14]. Blockade of AT 1 R or inhibition of Ang II generation is an important strategy for the treatment of hypertension [13]. Ang II is widely used to induce hypertension and vascular remodeling as an animal model of hypertension [24][25][26]. It is well known that Ang II induces VSMC proliferation [27], which is widely used as a model of VSMC proliferation in vitro [28][29][30]. We found that BCL6 overexpression attenuated while BCL6 knockdown enhanced the Ang II-induced VSMC proliferation, indicating that BCL6 is an important protective factor in inhibiting Ang II-induced VSMC proliferation. More importantly, BCL6 overexpression in SHR attenuated vascular remodeling and reduced PCNA expression in the aortic media of SHR. The in vivo study provides evidence that BCL6 is important in attenuating vascular remodeling and VSMC proliferation in SHR. The inhibitory effect of BCL6 on VSMC proliferation at least partially contributed to its role in attenuating vascular remodeling in SHR.
It is noted that Ang II reduced BCL6 expression in human VSMCs, and BCL6 expression was downregulated in the aortic media of SHR, suggesting that the reduced BCL6 levels may be involved in the enhanced VSMC proliferation in both Ang II-treated VSMCs and aortic media of SHR. However, the mechanisms of the BCL6 downregulation are not known, which need further investigation. On the other hand, BCL6 overexpression had no significant effect on blood pressure in normotensive WKY but reduced blood pressure in SHR. The attenuated vascular remodeling caused by BCL6 in hypertension may partially contributes to its antihypertensive effect. We recently have shown that BCL6 attenuates renal inflammation via negative regulation of NLRP3 transcription in SHR, which may be also involved in its antihypertension effect [20].
Oxidative stress is associated with vascular defects leading to hypertension and atherosclerosis [31,32]. Removal of excess ROS is a therapeutic strategy for oxidative stress-related cardiovascular diseases [33][34][35]. Accumulating evidences have shown that Ang II activates NOXs mediated by AT 1 R, which promotes ROS generation and subsequent VSMC proliferation [7][8][9]. BCL6 knockdown increased the transcription of NADPH oxidase subunits P67 and gp91 in normal cardiomyocytes, and hypoxia-induced P67 and gp91 upregulation in cardiomyocytes was enhanced by Bcl6 knockdown [36]. We found that Ang II-induced NAD(P)H oxidase activation, ROS generation, and NOX4 upregulation were attenuated by BCL6 overexpression but exacerbated by BCL6 knockdown in human VSMCs. However, Ang II-induced NOX2 upregulation was not significantly affected by BCL6 overexpression or knockdown. The findings were supported by the in vivo study that BCL6 overexpression in SHR attenuated the increased ROS production and NOX4 upregulation but had no significant effect on the NOX2 upregulation in the aortic media of SHR. These results indicate that BCL6 attenuates oxidative stress in Ang II-treated VSMCs and aortic media of SHR, which may be related to the downregulation of NOX4. The antioxidative effects of BCL6 in VSMCs were supported by previous findings that BCL6 overexpression inhibited oxidative stress response to etoposide and other chemotherapeutic reagents in B-cell lymphoma cells [18] and that BCL6 knockdown augmented the hypoxia-induced oxidative stress in cardiomyocytes [36]. The limitations in the present study were that the effect of specific inhibitor of NOX-2 or NOX-4 on DHE staining was not examined and that DHE staining was used to evaluate ROS production for in vivo experiments instead of a widely accepted marker of oxidative stress. In summary, BCL6 is downregulated in Ang II-treated human VSMCs and aortic media of SHR. BCL6 overexpression attenuates while BCL6 knockdown exacerbates Ang II-induced oxidative stress and proliferation of human VSMCs. BCL6 overexpression inhibits oxidative stress and proliferation of aortic media in SHR. BCL6 inhibits NOX4 expression, which may contribute to its antioxidative effect in Ang II-treated VSMCs and in the aortic media of SHR.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
None of the authors declares any conflict of interest in this work.