((b) and (c)) ROS level in nonischemic and ischemic muscle tissue on day 7 was determined by an in situ detection of superoxide with dihydroethidium (DHE) fluorescence

((b) and (c)) ROS level in nonischemic and ischemic muscle tissue on day 7 was determined by an in situ detection of superoxide with dihydroethidium (DHE) fluorescence. (HMVECs). Bach1-induced apoptosis was largely abolished when the cells were cultured with N-acetyl-l-cysteine (NAC), a ROS scavenger. Exogenous expression of VD3-D6 Bach1 inhibited the cell proliferation and the expression of cyclin D1, induced an S-phase arrest, and increased the expression of cyclin E2, which were partially blocked by NAC. Taken together, our results suggest that Bach1 suppresses cell proliferation and induces cell-cycle arrest and apoptosis by increasing mitochondrial ROS production, suggesting that Bach1 may be a promising treatment target for the treatment of vascular diseases. 1. Introduction The transcription factor BTB and CNC homology 1 (Bach1) regulates genes involved in apoptosis, the oxidative stress response, mitotic chromatin dynamics, and the cell-cycle [1C5]. Previous studies suggest that Bach1 deficiency may protect against oxidative tissue damage in murine models of lung, liver, intestine, pancreas, and cardiovascular disease [6C11], and we have shown that Bach1 suppresses angiogenesis in mice with surgically induced hindlimb ischemia (HLI) [2]. Our results also indicate that Bach1 disrupts Wnt/= 12 per experimental group). Injections were administered in the gastrocnemius muscle and the adductor muscle immediately after HLI induction. Blood flow measurements were performed at the indicated time points with a MoorLDI2-2 laser Doppler imaging system (Moor Instruments, Devon, UK); the mice were euthanized with sodium pentobarbital (50?mg/kg i.p.) and maintained at 37C on a heating plate to minimize temperature variation. Measurements in the ischemic limb were normalized to measurements in the nonischemic, contralateral limb. Mice were sacrificed on day seven or 14 postsurgery and adductor muscles were harvested and snap frozen in OCT compound for cryosectioning. ROS levels were determined via DHE fluorescence. Briefly, the unfixed tissues were cut into 10?t< 0.05. Analyses were performed using GRAPHPAD Prism Version 5.0 (GraphPad Software, La Jolla, VD3-D6 CA). 3. Results 3.1. Bach1 Overexpression Promotes ROS Production and Apoptosis in the Ischemic Limbs of Mice Recently, we have shown that Bach1 disrupts Wnt/= 12; < 0.05, < 0.01 versus Ad-GFP). ((b) and (c)) ROS level in nonischemic and ischemic muscle tissue on day 7 was determined by an in situ detection of superoxide with dihydroethidium (DHE) fluorescence. Bars, 100?= 6 for each group ((c), < 0.05 versus Ad-GFP). (d) Seven days after HLI, cleaved caspase 3 and cyclin D1 protein levels were evaluated in HLI and non-HLI limbs via Western blot (= 6; < 0.05, < 0.01 versus Ad-GFP). 3.2. Bach1 Promotes Mitochondrial ROS Production and Apoptosis in Cultured ECs The results from our previous investigation suggested that when Ad-Bach1 was delivered to the ischemic limbs of mice, the vectors tended to be expressed by ECs [12]. Thus, we performed a series of in vitro experiments to determine whether ROS levels, apoptosis, and cell-cycle progression can be altered in ECs by manipulating Bach1 expression. The effect of Bach1 upregulation was evaluated by comparing assessments Clec1a in Ad-Bach1 infected and Ad-GFP infected HMVECs, while Bach1 downregulation was evaluated by performing experiments in HMVECs that had been transfected with Bach1 siRNA (Bach1siRNA) or a control siRNA (Con siRNA). Bach 1 overexpression appeared to promote apoptotic nuclear condensation (Figure 2(a)) and cell apoptosis (Figure 2(b)), and cleaved caspase 3 levels (Figure 2(c)) and intracellular ROS levels (Figure 3(a)) were significantly higher in populations of Ad-Bach1 HMVECs than in Ad-GFP HMVECs. MitoSOX Red, a redox fluorophore detecting selectively mitochondrial superoxide, was used to evaluate mitochondrial ROS generation in HMVECs. The fluorescence intensity of MitoSOX Red was significantly higher in Ad-Bach1 HMVECs than that in Ad-GFP HMVECs (Figure VD3-D6 3(b)), indicating that Bach1 increases mitochondrial ROS levels. The higher levels of Bach1 expression was also associated with declines in expression of the apoptosis inhibitors Bcl2, Bcl-xL, and heme oxygenase 1 (HO-1) (Figure 2(c)). However, when Ad-Bach1 HMVECs were cultured with the ROS scavenger N-acetyl-L-cysteine (NAC; 10?mM), mitochondrial ROS levels (Figure 3(b)), cell apoptosis (Figure 3(c)), and cleaved caspase 3 levels (Figure 3(d)) declined significantly. Open in a separate window Figure 2 Bach1 promotes apoptosis in cultured HMVECs. (a) HMVECs were infected with the adenoviruses (Ad-GFP or Ad-Bach1); then cells were fixed at 72 hours after infection, and nuclei were visualized by Hoechst 33342. Cells with nuclear condensation are indicated by white arrows. Bars, 100?= 3; < 0.05 versus Ad-GFP, upper panel). Bach1 protein levels were evaluated via Western blot (lower panel). (c) Cleaved caspase 3, Bcl2, Bcl-xL, HO-1, and Bach1 protein levels were determined via Western blot in HMVECs that had been infected with Ad-GFP or Ad-Bach1 (= 3; < 0.05 versus Ad-GFP). Open in a separate window Figure 3 Bach1 induces apoptosis through mitochondrial ROS VD3-D6 production. Ad-GFP- and Ad-Bach1-infected HMVECs were incubated with or without NAC (10?mM) for 48 hours, ROS production was then determined by the detection of dihydroethidium (DHE) fluorescence (a), or mitochondrial ROS production.