A systemic inflammatory response leads to widespread organ dysfunction, such as kidney dysfunction. Plasminogen activator inhibitor-1 (PAI-1) is involved in the pathogenesis of inflammatory kidney ...injury; however, the regulatory mechanism of PAI-1 in injured kidneys remains unclear. PAI-1 is induced by interleukin (IL)-6 in patients with sepsis. In addition, the stabilization of IL-6 is regulated by the adenine–thymine-rich interactive domain-containing protein 5a (Arid5a). Therefore, the aim of the present study was to examine the involvement of Arid5a/IL-6/PAI-1 signaling in lipopolysaccharide (LPS)-induced inflammatory kidney injury. LPS treatment to C57BL/6J mice upregulated Pai-1 mRNA in the kidneys. Enzyme-linked immunosorbent assay (ELISA) revealed that PAI-1 expression was induced in the culture supernatants of LPS-treated human umbilical vein endothelial cells, but not in those of LPS-treated human kidney 2 (HK-2) cells, a tubular cell line. Combined with single-cell analysis, endothelial cells were found to be responsible for PAI-1 elevation in LPS-treated kidneys. Administration of TM5441, a PAI-1 inhibitor, reduced the urinary albumin/creatinine ratio, concomitant with downregulation of Il-6 and Arid5a mRNA expressions. IL-6 treatment in LPS model mice further upregulated Pai-1 mRNA expression compared with LPS alone, accompanied by renal impairment. Furthermore, the expression of Il-6 and Pai-1 mRNA was lower in Arid5a knockout mice than in wild-type mice after LPS treatment. Taken together, the vicious cycle of Arid5a/IL-6/PAI-1 signaling is involved in LPS-induced kidney injury.
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.
Tumor suppressor protein p53 plays crucial roles in the onset of heart failure. p53 activation results in cardiac dysfunction, at least partially by suppressing angiogenesis. Though p53 has been ...reported to reduce VEGF production by inhibiting hypoxia‐inducible factor, the anti‐angiogenic property of p53 remains to be fully elucidated in cardiomyocytes. To explore the molecular signals downstream of p53 that regulate vascular function, especially under normoxic conditions, DNA microarray was performed using p53‐overexpressing rat neonatal cardiomyocytes. Among genes induced by more than 2‐fold, we focused on CXCL10, an anti‐angiogenic chemokine. Real‐time PCR revealed that p53 upregulated the CXCL10 expression as well as p21, a well‐known downstream target of p53. Since p53 is known to be activated by doxorubicin (Doxo), we examined the effects of Doxo on the expression of CXCL10 and found that Doxo enhanced the CXCL10 expression, accompanied by p53 induction. Importantly, Doxo‐induced CXCL10 was abrogated by siRNA knockdown of p53, indicating that p53 activation is necessary for Doxo‐induced CXCL10. Next, we examined the effect of hypoxic condition on p53‐mediated induction of CXCL10. Interestingly, CXCL10 was induced by hypoxia and its induction was potentiated by the overexpression of p53. Finally, the conditioned media from cultured cardiomyocytes expressing p53 decreased the tube formation of endothelial cells compared with control, analyzed by angiogenesis assay. However, the inhibition of CXCR3, the receptor of CXCL10, restored the tube formation. These data indicate that CXCL10 is a novel anti‐angiogenic factor downstream of p53 in cardiomyocytes and could contribute to the suppression of vascular function by p53.
The conditioned media from p53‐expressing cardiomyocytes decreased the tube formation of Rat Aortic Endothelial Cells. The blockade of CXCR3, a receptor of CXCL10, restored the angiogenic activity of the conditioned media from p53‐expressing cardiomyocytes.
After myocardial infarction (MI), various kinds of cytokines are produced from pro- and/or anti- inflammatory cells that infiltrated into myocardium and contribute to scar formation and/or tissue ...repair. Among these cytokines, it is widely accepted that TGFβ family performs multiple functions, such as cell proliferation and fibrosis; however, it remains to be fully clarified whether there are functional differences among TGFβ1, 2, and 3. The aim of this study is to elucidate the pathophysiological significance of TGFβ3 after MI. MI was generated by coronary ligation. We measured the expression of TGFβ3 over time after MI by quantitative RT-PCR, and found that the expression of TGFβ3 mRNA peaked 7 days after MI (17.8±12.9 fold v.s non-MI). Quantitative RT-PCR and immunohistological staining showed that TGFβ3 was mainly expressed at the border region of infarction. To examine the effects of TGFβ3 on post-infarct remodeling, we administered TGFβ3 neutralizing antibody (TGFβ3 nAb) intravenously after MI. Echocardiographic analysis revealed that TGFβ3 nAb reduced cardiac dysfunction (fractional shortening: control IgG;29.8±7.3%, TGFβ3 nAb; 36.9±5.8%). In addition, Masson trichrome staining showed that neutralizing antibodies inhibited cardiac fibrosis after MI. TGFβ3 could promote adverse cardiac remodeling after MI as a novel therapeutic target.
［Background］Though the importance of myeloid cells in the cardiac remodeling after myocardial infarction (MI) is widely accepted, it remains to be fully elucidated how myeloid cells regulate ...post-infarct inflammation, at least partially, because subpopulation-specific cell knock-out methods are not available.［Methods and Results］We generated transgenic mice expressing diphtheria toxin receptor (DTR)/GFP fusion protein under the control of CD11b promoter in a Cre recombinase-expressing cell-specific manner (CD11b-DTR TG mice). Double TG mice (DTG mice) were generated by crossing CD11b-DTR TG mice with LysM-Cre mice that express Cre recombinase preferentially in monocytes/macrophages. The MI model was created in DTG mice by ligation of the left anterior descending branch. Flow cytometry analysis revealed that monocytes were labeled with GFP in the peripheral blood 4 days after MI. Consistently, immunofluorescent microscopic analysis showed that GFP⁺ cells infiltrated into the infarcted heart. Importantly, the administration of diphtheria toxin resulted in the depletion of GFP⁺ cells in peripheral blood and post-infarct myocardium. ［Conclusion］CD11b-DTR TG mice are useful for labeling and/or depleting subpopulation of myeloid cells in MI model.
【Background】p53 is reported to play important roles in the onset and progression of heart failure (HF) after myocardial injury. However, it remains to be fully elucidated how p53 works in ...cardiomyocytes.【Methods & Results】Neonatal rat cardiomyocytes were infected with adenoviral vectors expressing p53 and cultured for 48hr. DNA microarray analysis revealed that p53 overexpression resulted in the increased expression of inflammation-related genes, including CXCL10, an anti-angiogenic factor. RT-qPCR experiments showed that the expression of CXCL10 was upregulated by p53, while that of VEGF was not influenced. Next, we examined whether CXCL10 increased in HF animal models. In myocardial infarction (MI) model, the expression of CXCL10 was upregulated after MI both at acute and chronic stages, accompanied by the induction of p53. Moreover, in experimental autoimmune myocarditis models, CXCL10 expression was upregulated at chronic phase in myocardium specific STAT3 knockout mice which exhibited severe HF. Consistent with the previous studies, CXCL10 suppressed the migration of endothelial cells. Finally, since p53 is reported to exhibit anti-angiogenic property by interacting with HIF-1, we examined the effects of hypoxia on CXCL10 expression and found that hypoxia synergistically elevated the mRNA expression of CXCL10 with p53.【Conclusion】CXCL10, anti-angiogenic chemokine, is induced by p53, and this effect is enhanced by hypoxia pathways. Considering that impaired angiogenesis causes HF, CXCL10 could be a novel therapeutic target of HF.
【Background】Maresin1 (MaR1), a lipid mediator biosynthesized from docosahexaenoic acid (DHA), has both anti-inflammatory and proresolving activities. Much attention has been paid to the functional ...regulation by MaR1 in inflammatory cells, but not in tissue component cells. Since inflammatory reactions are involved in cardiovascular diseases, we addressed the effects of MaR1 on cardiomyocytes.【Methods & Results】Neonatal rat cardiomyocytes (NRCMs) were cultured with MaR1 for 48 hours. Immunofluorescent microscopic analyses using anti-sarcomeric α-actinin revealed that MaR1 increased cell surface area in a dose-dependent manner. Real time RT-PCR analyses demonstrated that the expression of the pathological hypertrophy markers, such as BNP and skeletal-actin, was not upregulated in NRCMs cultured with MaR1, indicating that MaR1-induced hypertrophy is physiological. Finally we treated NRCMs with SR3335, an ROR alpha inhibitor, because MaR1 was previously reported to utilize ROR alpha as a receptor. Importantly, SR3335 prevented the increase in cell surface area induced by MaR1. 【Conclusion】MaR1 induces physiological hypertrophy of neonatal rat CMs through stimulating ROR alpha. MaR1 could play an important role in the tissue repair after myocardial injury.
After myocardial infarction (MI), various kinds of cytokines are produced from pro- and/or anti- inflammatory cells that infiltrated into myocardium and contribute to scar formation and/or tissue ...repair. Among these cytokines, it is widely accepted that TGFβ family performs multiple functions, such as cell proliferation and fibrosis; however, it remains to be fully clarified whether there are functional differences among TGFβ1, 2, and 3. The aim of this study is to elucidate the pathophysiological significance of TGFβ3 after MI. MI was generated by coronary ligation. We measured the expression of TGFβ3 over time after MI by quantitative RT-PCR, and found that the expression of TGFβ3 mRNA peaked 7 days after MI (17.8±12.9 fold v.s non-MI). Quantitative RT-PCR and immunohistological staining showed that TGFβ3 was mainly expressed at the border region of infarction. To examine the effects of TGFβ3 on post-infarct remodeling, we administered TGFβ3 neutralizing antibody (TGFβ3 nAb) intravenously after MI. Echocardiographic analysis revealed that TGFβ3 nAb reduced cardiac dysfunction (fractional shortening: control IgG;29.8±7.3%, TGFβ3 nAb; 36.9±5.8%). In addition, Masson trichrome staining showed that neutralizing antibodies inhibited cardiac fibrosis after MI. TGFβ3 could promote adverse cardiac remodeling after MI as a novel therapeutic target.
［Background］Though the importance of myeloid cells in the cardiac remodeling after myocardial infarction (MI) is widely accepted, it remains to be fully elucidated how myeloid cells regulate ...post-infarct inflammation, at least partially, because subpopulation-specific cell knock-out methods are not available.［Methods and Results］We generated transgenic mice expressing diphtheria toxin receptor (DTR)/GFP fusion protein under the control of CD11b promoter in a Cre recombinase-expressing cell-specific manner (CD11b-DTR TG mice). Double TG mice (DTG mice) were generated by crossing CD11b-DTR TG mice with LysM-Cre mice that express Cre recombinase preferentially in monocytes/macrophages. The MI model was created in DTG mice by ligation of the left anterior descending branch. Flow cytometry analysis revealed that monocytes were labeled with GFP in the peripheral blood 4 days after MI. Consistently, immunofluorescent microscopic analysis showed that GFP⁺ cells infiltrated into the infarcted heart. Importantly, the administration of diphtheria toxin resulted in the depletion of GFP⁺ cells in peripheral blood and post-infarct myocardium. ［Conclusion］CD11b-DTR TG mice are useful for labeling and/or depleting subpopulation of myeloid cells in MI model.
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