**3. Discussion**

BAFF binds to the BAFF receptor to control B-cell differentiation into plasma cells and promote B-cell survival by activating the NF-κB and phosphoinositol 3-kinase/AKT pathways [16]. BAFF is induced by the nongenomic signaling of dioxin in the livers of C57BL/6 mice and HepG2 human hepatoma cells, and BAFF expression significantly contributes to early stress response reaction [17]. In this study, we observed that BAFF was upregulated in the remaining liver tissues after partial hepatectomy, which suggests that BAFF expression might be an important signal for liver regeneration. Although serum BAFF levels were not significantly increased, the local effects of BAFF in the liver tissue may be sufficient to affect regeneration signaling.

The survival of mature resting B cells in the periphery depends on signaling from the B-cell receptor and BAFF of the TNF receptor family. BCL10 promotes NF-κB activity, which contributes to B-cell survival through activation of the inhibitor of NF-κB kinase complex via Carma1 and mucosa-associated lymphoid tissue lymphoma translocation gene 1 and increases the expression of survival genes by directly modifying the chromatin of NF-κB target gene promoters. [18,19] Increased NF-κB activity and elevated cyclin D1 expression are critical for hepatocyte proliferation. [20] In this study, the CL-48 cells were treated with recombinant BAFF, and BAFF/BCL10 signaling was assessed by the detection of BCL10 nuclear translocation. However, there were no significant changes in CL-48 cell proliferation in response to BAFF treatment. In contrast, when CL-48 cells were transfected with BCL10 siRNA, cell growth retardation and cell cycle arrest at G2/M phase were observed. Thus, BCL10 might trigger cell proliferation signaling without BAFF activation. Consistent with this, BCL10 controls the growth of cervical cancer cells via NF-κB-dependent cyclin D1 regulation in cervical cancer cells [11].

Serum BAFF levels have been shown to correlate with parameters of disease activity, such as bone marrow microvascular density and proliferating cell nuclear antigen expression, in patients with myeloma [21]. The inhibition of BAFF expression might have therapeutic applications because of its effects on angiogenesis in human multiple myeloma [22]. In MH7A synovial cells, TNF-α-induced BAFF expression controls VEGF-mediated angiogenesis by increasing the transcription and activity of VEGF [23]. In this study, we observed that CM from BAFF-stimulated CL-48 cells promoted angiogenesis, a process that is essential for liver regeneration. sFlt-1, which is a soluble receptor for VEGF, acts as a dominant-negative receptor and it has been shown to suppress sinusoidal endothelial cell growth and reduce remnant hepatic weight [24]. Immune cells also have important roles in liver regeneration. Indeed, in mice lacking the monocyte adhesion molecule CD11b, partial hepatectomy resulted in severe reduction in angiogenesis and the development of unstable, leaky vessels, eventually producing an aberrant hepatic vascular network and Küpffer cell distribution [25].

In this study, by using an angiogenesis-related protein array, we identified the BAFF-regulated angiogenesis factors MMP-9, FGF4, and IL-8. We also confirmed the transcriptional and translational regulation of MMP-9 and IL-8 through BAFF/BCL10/NF-kB signaling in hepatocytes. Importantly, the angiogenic role of MMP-9 was first identified in homozygous mice with a null mutation in the gene encoding MMP-9/gelatinase B, which revealed an abnormal pattern of skeletal growth plate vascularization and ossification. Additionally, growth plates from gelatinase B-null mice in culture showed a delayed release of angiogenic activators, demonstrating a role of MMP-9 in angiogenesis control [26]. A stress-induced increase in MMP-9 expression are critical for recruitment of human CD34+ progenitors with hematopoietic and/or hepatic-like potential to the livers of NOD/SCID mice [27]. In MMP-9-knockout mice, a delayed hepatic regenerative response after partial hepatectomy was observed [28], highlighting the importance of MMP-9 in liver regeneration. IL-8 is a cytokine that acts as a chemoattractant for lymphocytes and neutrophils. The role of IL-8 in angiogenesis was first demonstrated in a rabbit corneal pocket model, where IL-8 induced neovascularization [29]. The angiogenic role of IL-8 is also evident by its ability to induce proliferation and chemotaxis in HUVECs [30]. In humans, after liver surgery, IL-8 is produced in the remaining liver [31]. Notably, NF-κB regulates the expression of both MMP-9 and IL-8 [32,33]. Consistent with this, in the current study, we also confirmed the role of NF-κB in BAFF/BCL10 signaling by promoter assays and chemical inhibition.

The results demonstrated that the BAFF/BCL10/NF-κB signaling pathway was active in hepatocytes and it was involved in modulating the expression of angiogenesis-related factors. We also found that the inhibition of BAFF expression reduced angiogenesis and hepatocyte proliferation in a liver regeneration model. Thus, these results again confirmed the role of BAFF in liver regeneration and suggested that drugs targeting the BAFF/BCL10/NF-κB signaling pathway should be carefully used in patients with liver regeneration-related conditions.

Importantly, one limitation of this study was the lack of human tissue validation in regenerating liver based on the critical roles of BAFF/BCL10 in angiogenesis in animal models. Thus, further studies are required to investigate the potential interactions between BAFF/BCL10 and IL-6 signaling pathways, both of which involve activation of NF-κB.

In this study, although the HUVECs model demonstrated the role of BAFF in angiogenesis in hepatocytes, HUVECs are quite di fferent than liver sinusoidal endothelial cells (LSECs). LSECs represent a permeable barrier which representing the interface between blood cells on the one side and hepatocytes and hepatic stellate cells on the other side are highly specialized endothelial cells. Furthermore, the absence of diaphragm and lack of basement membrane make LSECs the most permeable endothelial cells of the mammalian body [34]. It is worthy to investigate the BAFF promoted hepatocyte-driven angiogenesis in LSECs.

In conclusion, the increased expression of BAFF and activation of BCL10/NF-κB signaling were critically involved in hepatocyte-driven angiogenesis and survival during liver regeneration.
