• 4-HBA and VA could interact with HSA. • 4-HBA and VA decreased K562 and K562/Dox cells viability. • These current studies could provide useful information about the nature of 4-HBA or VA binding to HSA.

Transforming growth factor beta (TGF-β) is ubiquitously found in bone and plays a key role in bone turnover. Mice expressing constitutively active TGF-β receptor type I (Mx1;TβRI^CA mice) are osteopenic. Here, we identified the candidate genes involved in bone turnover in Mx1;TβRI^CA mice using RNA sequencing analysis. A total of 285 genes, including 87 upregulated and 198 downregulated genes, were differentially expressed. According to the KEGG analysis, some genes were involved in osteoclast differentiation (Fcgr4, Lilrb4a), B cell receptor signaling (Cd72, Lilrb4a), and neutrophil extracellular trap formation (Hdac7, Padi4). Lilrb4 is related to osteoclast inhibition protein, whereas Hdac7 is a Runx2 corepressor that regulates osteoblast differentiation. Silencing Lilrb4 increased the number of osteoclasts and osteoclast marker genes. The knocking down of Hdac7 increased alkaline phosphatase activity, mineralization, and osteoblast marker genes. Therefore, our present study may provide an innovative idea for potential therapeutic targets and pathways in TβRI-associated bone loss.

BACKGROUND: Brain arteriovenous malformations (bAVMs) are a major risk factor for intracerebral hemorrhages in young patients. Recent clinical studies have reported that the majority of human bAVMs harbor somatic KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations. In our previous study, we confirmed the causal role of endothelial KRAS ^G12V mutation in bAVM development in mice through hyperactivation of the MEK/ERK (mitogen-activated protein kinase kinase/extracellular signal–regulated kinase) pathway. However, the treatment efficacy of targeting MEK/ERK signaling in bAVMs, regarding clinical and biological outcomes, remains unclear. METHODS: In this study, we used the US Food and Drug Administration–approved MEK inhibitor, trametinib, to treat mice with bAVMs induced by brain endothelial cell (EC)–specific KRAS ^G12V overexpression (KRAS ^G12V/bEC mice). We treated KRAS ^G12V/bEC mice with either vehicle or trametinib and evaluated outcomes on mortality and the alteration of bAVMs, and the hemorrhages. We also determined the effect of trametinib on the endothelial-to-mesenchymal transition, which has recently been explored in human bAVMs and implicated in bAVM pathology, in bAVMs of KRAS ^G12V/bEC mice and cultured mouse ECs transfected with KRAS ^G12V (EC ^G12V ). RESULTS: We observed that trametinib significantly improved survivability, potentially through improved vessel integrity and reduction in the severity of bAVM ruptures. Our longitudinal observation of mice bAVMs using noninvasive magnetic resonance imaging revealed that trametinib decreased the total hemorrhagic volume at 8 weeks of treatment when administered at 1 mg/kg compared with the vehicle group, which was confirmed by histological analyses. In addition, trametinib altered endothelial-to-mesenchymal transition by reducing the expression levels of mesenchymal markers (N-Cad [N-cadherin], Slug, CD44 [cluster of differentiation 44]) and increasing an EC marker (VE-Cad [vascular endothelial cadherin]) in bAVMs of KRAS ^G12V/bEC mice. Trametinib also decreased proliferation (Ki-67) and angiogenic (CD34 and pVEGFR2 [phosphor-vascular endothelial growth factor receptor 2]) markers in KRAS ^G12V/bEC mice’s bAVMs. We also confirmed that trametinib normalized the endothelial-to-mesenchymal transition-associated marker expression and functions in EC ^G12V . CONCLUSIONS: Our data demonstrate that trametinib improves bAVM pathology by reducing hemorrhagic risk and normalizing endothelial-to-mesenchymal transition. These findings suggest that trametinib is a promising agent to treat patients with bAVM.

Transforming growth factor beta (TGF-β), a master regulator of renal fibrosis, is the hallmark of chronic kidney disease (CKD) progression, and CKD worsens bone remodeling. However, the effects of the dysregulation of TGF-β signaling on bone remodeling during CKD have not been investigated. Here, we determined the effects of TGF-β receptor I (TβRI) overexpression under the control of Mx1-Cre on bone remodeling in CKD mice (Mx1;TβRI^CA-CKD mice). Our results demonstrated that kidney fibrosis and serum urea nitrogen levels were elevated in Mx1;TβRI^CA-CKD mice compared to WT-CKD, indicating that TβRI overexpression exacerbated renal injury during CKD. Serum calcium was decreased, while PTH was enhanced, in Mx1;TβRI^CA-CKD mice. Mx1;TβRI^CA-CKD mice displayed severe osteopenia as assessed by uCT in both femurs and mandibles. An histomorphometric analysis showed that tibial cancellous bone volume was decreased in Mx1;TβRI^CA-CKD. Likewise, mRNA expression levels of an osteoclastogenesis marker, Tnfsf11/Tnfrsf11b, was increased, and osteoblast marker genes Runx2 and Sp7 were decreased in Mx1;TβRI^CA-CKD mice. Mx1;TβRI^CA-CKD mice displayed increased inflammatory cytokines levels. Together, our results indicated that in the setting of CKD, TβRI overexpression induced both CKD progression and the dysregulation of bone remodeling, leading to severe bone loss. As such, these data provide an avenue for the future development of therapeutics for CKD-induced osteoporosis.