Prevention of kidney fibrosis is an essential requisite for effective therapy in preventing chronic kidney disease (CKD). Here, we identify Old astrocyte specifically induced substance (OASIS)/cAMP ...responsive element‐binding protein 3‐like 1 (CREB3l1), a CREB/ATF family transcription factor, as a candidate profibrotic gene that drives the final common pathological step along the fibrotic pathway in CKD. Although microarray data from diseased patient kidneys and fibrotic mouse model kidneys both exhibit OASIS/Creb3l1 upregulation, the pathophysiological roles of OASIS in CKD remains unknown. Immunohistochemistry revealed that OASIS protein was overexpressed in human fibrotic kidney compared with normal kidney. Moreover, OASIS was upregulated in murine fibrotic kidneys, following unilateral ureteral obstruction (UUO), resulting in an increase in the number of OASIS‐expressing pathological myofibroblasts. In vitro assays revealed exogenous TGF‐β1 increased OASIS expression coincident with fibroblast‐to‐myofibroblast transition and OASIS contributed to TGF‐β1–mediated myofibroblast migration and increased proliferation. Significantly, in vivo kidney fibrosis induced via UUO or ischemia/reperfusion injury was ameliorated by systemic genetic knockout of OASIS, accompanied by reduced myofibroblast proliferation. Microarrays revealed that the transmembrane glycoprotein Bone marrow stromal antigen 2 (Bst2) expression was reduced in OASIS knockout myofibroblasts. Interestingly, a systemic anti‐Bst2 blocking antibody approach attenuated kidney fibrosis in normal mice but not in OASIS knockout mice after UUO, signifying Bst2 functions downstream of OASIS. Finally, myofibroblast‐restricted OASIS conditional knockouts resulted in resistance to kidney fibrosis. Taken together, OASIS in myofibroblasts promotes kidney fibrosis, at least in part, via increased Bst2 expression. Thus, we have identified and demonstrated that OASIS signaling is a novel regulator of kidney fibrosis.
Abstract
Runt-related transcription factor 2 (Runx2), a regulator of osteoblast differentiation, is pathologically involved in vascular calcification; however, the significance of Runx2 in cardiac ...homeostasis remains unclear. Here, we investigated the roles of Runx2 in cardiac remodeling after myocardial infarction (MI). The expression of Runx2 mRNA and protein was upregulated in murine hearts after MI. Runx2 was expressed in heart-infiltrating myeloid cells, especially in macrophages, at the border zone of post-infarct myocardium. To analyze the biological functions of Runx2 in cardiac remodeling, myeloid cell-specific
Runx2
deficient (CKO) mice were exposed to MI. After MI, ventricular weight/tibia length ratio was increased in CKO mice, concomitant with severe cardiac dysfunction. Cardiac fibrosis was exacerbated in CKO mice, consistent with the upregulation of collagen 1a1 expression. Mechanistically, immunohistochemical analysis using anti-CD31 antibody showed that capillary density was decreased in CKO mice. Additionally, conditioned culture media of myeloid cells from Runx2 deficient mice exposed to MI induced the tube formation of vascular endothelial cells to a lesser extent than those from control mice. RNA-sequence showed that the expression of pro-angiogenic or anti-angiogenic factors was altered in macrophages from Runx2-deficient mice. Collectively,
Runx2
+
myeloid cells infiltrate into post-infarct myocardium and prevent adverse cardiac remodeling, at least partially, by regulating endothelial cell function.
The number of patients with chronic kidney disease (CKD) is increasing worldwide. When kidneys are exposed to severe injury, tubular cell death occurs and kidney fibrosis progresses by activating ...fibroblasts and myofibroblasts (referred to as (myo)fibroblasts), leading to CKD; however, the pathological and molecular mechanisms underlying CKD, including kidney fibrosis, remain obscure. In the present study, we focused on a transcription factor PBX/Knotted Homeobox 2 (PKNOX2) in kidney fibrosis. The transcript and protein expression of PKNOX2 was upregulated in fibrotic kidneys after unilateral ureteral obstruction (UUO). Importantly, immunofluorescence microscopic analysis revealed that the number of PKNOX2-expressing myofibroblasts was increased, whereas the expression of PKNOX2 was decreased in proximal tubular epithelial cells after UUO. In (myo)fibroblasts, PKNOX2 was induced by TGF-β1. Knockdown of PKNOX2 using shRNA lentiviral system reduced the viability of (myo)fibroblasts either in the presence or absence of TGF-β1, accompanied by increased apoptosis. Moreover, PKNOX2 knockdown decreased TGF-β1-induced migration of myofibroblasts and differentiation of fibroblasts into myofibroblasts. Significantly, knockdown of PKNOX2 also decreased the viability and increased apoptosis of tubular epithelial cells. Collectively, PKNOX2 regulates the function of (myo)fibroblasts and the viability of proximal tubular epithelial cells in progression of kidney fibrosis.
•Myofibroblasts expressing PKNOX2 were increased in fibrotic kidneys.•PKNOX2 expression in tubular epithelial cells was downregulated in fibrotic kidneys.•TGF-β1 induced PKNOX2 expression in (myo)fibroblasts.•PKNOX2 is necessary for the function and viability of myofibroblasts.•PKNOX2 knockdown induced cell death in tubular epithelial cells.
•Plasmid was transferred into murine thigh muscles by PJI with higher efficiency than by needle.•PJI-mediated delivery of FGF2 plasmid reduced skeletal muscle injury in mice.•PJI was also applicable ...to gene transfer into murine hearts.
The angiogenic gene therapy is an attractive approach for the treatment of ischemic muscle diseases, including peripheral arterial disease and ischemic heart diseases. Although a variety of gene transfer methods have been developed, the efficiency of gene transfer is still limited. We have been developing the needleless high-energy bioinjector device, Pyro-drive Jet Injector (PJI), based on pyrotechnics using a combination of ignition powder and gunpowder, however, the utility of PJI in gene transfer into muscle tissues remains unclear. pcDNA3.1 plasmid containing Flag was injected to the thigh muscles of C57BL/6J mice using PJI or needle, as a control. Histological analysis demonstrated that the protein expression of Flag was observed in a wider range in PJI group than in needle group. To assess the validity of PJI for gene therapy, pcDNA3.1-human fibroblast growth factor 2 (FGF2), which has angiogenic activity and tissue protective properties, was injected into the ischemic thigh muscles with PJI or needle. ELISA assay revealed that the protein expression of FGF2 was increased in the thigh muscle tissues by PJI-mediated gene delivery. Significantly, histological analyses revealed that muscle fiber cross-sectional area and the number of endothelial marker CD31 (+) cells was increased in ischemic hind-limb tissues of the PJI-FGF2 group but not in those of needle-FGF2 group. To expand the applicability of the PJI-mediated gene transfer, pcDNA3.1-venus plasmid was injected into murine hearts with PJI or needle. PJI method was successful in gene transfer into murine hearts, especially into cardiomyocytes, with high efficiency when compared to needle method. Collectively, the non-needle, non-liposomal and non-viral gene transfer by PJI could be a novel therapeutic approach for muscle diseases.
BackgroundGene transfer into the target organ is a critical process of gene therapy. Although gene transfer by physicochemical method has relatively safe, the transfer efficiency is low depending on ...the target organ. To develop the effective and efficient gene transfer method, we focused on Pyro-drive Jet Injector (PJI) which was developed based on pyrotechnics. The aim is to explore the effectiveness of PJI in gene transfer into muscle tissues.Methods/Results pcDNA3.1-venus-plasmid was injected to the thigh muscles of C57BL/6J mice with PJI or needle. Immunofluorescence demonstrated the venus was expressed in a wider range in PJI group than needle group. To examine the validity of PJI for gene therapy, pcDNA3.1-humanFGF2 plasmid, an angiogenic factor, was injected into the ischemic thigh muscles with PJI or needle. ELISA showed the FGF2 protein was increased in the thigh muscle tissue in PJI group. Significantly, histological analyses revealed the muscle cell cross-sectional area and endothelial marker CD31 (+) cells was increased in the PJI-FGF2 group compared with needle-FGF2 or PJI-control plasmid. Finally, PJI method was successful in gene transfer into murine heart with high efficiency when compared to needle method. Conclusion Gene transfer by PJI may be a useful approach for the treatment of muscle diseases.
Background Since kidney fibrosis is a common pathway to many kidney diseases, prevention of kidney fibrosis could be a therapeutic strategy for kidney diseases. Previously, we reported that old ...astrocyte specifically induced substance (OASIS), a transcription factor, promotes proliferation of renal myofibroblasts, analyzed by in vitro assay. However, the pathophysiological significance of OASIS in kidney fibrosis in vivo remains to be elucidated.Methods/Results C57BL/6J mice were subjected to unilateral ureteral obstruction (UUO) to cause kidney fibrosis, analyzed by Sirius red staining and hydroxyproline assay. AEBSF, an inhibitor of OASIS, suppressed kidney fibrosis at day 7 after UUO. Moreover, kidney fibrosis was ameliorated in OASIS KO mice compared with WT mice, accompanied by decreased proliferation of myofibroblasts. To explore the downstream of OASIS, DNA microarray was performed using myofibroblasts from OASIS KO mice. Interestingly, it was found that bone marrow stromal antigen 2 (BST2) was a candidate downstream gene of OASIS. Anti-BST2 antibody treatment attenuated UUO-induced kidney fibrosis.Conclusion OASIS contributes to the develop of kidney fibrosis by promoting the proliferation of myofibroblasts, in part, through increased BST2 expression. OASIS could be a therapeutic target against kidney fibrosis.
BackgroundGene transfer into the target organ is a critical process of gene therapy. Although gene transfer by physicochemical method has relatively safe, the transfer efficiency is low depending on ...the target organ. To develop the effective and efficient gene transfer method, we focused on Pyro-drive Jet Injector (PJI) which was developed based on pyrotechnics. The aim is to explore the effectiveness of PJI in gene transfer into muscle tissues.Methods/Results pcDNA3.1-venus-plasmid was injected to the thigh muscles of C57BL/6J mice with PJI or needle. Immunofluorescence demonstrated the venus was expressed in a wider range in PJI group than needle group. To examine the validity of PJI for gene therapy, pcDNA3.1-humanFGF2 plasmid, an angiogenic factor, was injected into the ischemic thigh muscles with PJI or needle. ELISA showed the FGF2 protein was increased in the thigh muscle tissue in PJI group. Significantly, histological analyses revealed the muscle cell cross-sectional area and endothelial marker CD31 (+) cells was increased in the PJI-FGF2 group compared with needle-FGF2 or PJI-control plasmid. Finally, PJI method was successful in gene transfer into murine heart with high efficiency when compared to needle method. Conclusion Gene transfer by PJI may be a useful approach for the treatment of muscle diseases.
Background Since kidney fibrosis is a common pathway to many kidney diseases, prevention of kidney fibrosis could be a therapeutic strategy for kidney diseases. Previously, we reported that old ...astrocyte specifically induced substance (OASIS), a transcription factor, promotes proliferation of renal myofibroblasts, analyzed by in vitro assay. However, the pathophysiological significance of OASIS in kidney fibrosis in vivo remains to be elucidated.Methods/Results C57BL/6J mice were subjected to unilateral ureteral obstruction (UUO) to cause kidney fibrosis, analyzed by Sirius red staining and hydroxyproline assay. AEBSF, an inhibitor of OASIS, suppressed kidney fibrosis at day 7 after UUO. Moreover, kidney fibrosis was ameliorated in OASIS KO mice compared with WT mice, accompanied by decreased proliferation of myofibroblasts. To explore the downstream of OASIS, DNA microarray was performed using myofibroblasts from OASIS KO mice. Interestingly, it was found that bone marrow stromal antigen 2 (BST2) was a candidate downstream gene of OASIS. Anti-BST2 antibody treatment attenuated UUO-induced kidney fibrosis.Conclusion OASIS contributes to the develop of kidney fibrosis by promoting the proliferation of myofibroblasts, in part, through increased BST2 expression. OASIS could be a therapeutic target against kidney fibrosis.
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