Absence of cytoglobin promotes multiple organ abnormalities in aged mice

Cytoglobin (Cygb) was identified in hepatic stellate cells (HSCs) and pericytes of all organs; however, the effects of Cygb on cellular functions remain unclear. Here, we report spontaneous and age-dependent malformations in multiple organs of Cygb−/− mice. Twenty-six percent of young Cygb−/− mice (<1 year old) showed heart hypertrophy, cystic disease in the kidney or ovary, loss of balance, liver fibrosis and lymphoma. Furthermore, 71.3% (82/115) of aged Cygb−/− mice (1–2 years old) exhibited abnormalities, such as heart hypertrophy and cancer development in multiple organs; by contrast, 5.8% (4/68) of aged wild-type (WT) mice had abnormalities (p < 0.0001). Interestingly, serum and urine analysis demonstrated that the concentration of nitric oxide metabolites increased significantly in Cygb−/− mice, resulting in an imbalance in the oxidative stress and antioxidant defence system that was reversed by NG-monomethyl-L-arginine treatment. A senescent phenotype and evidence of DNA damage were found in primary HSCs and the liver of aged Cygb−/− mice. Moreover, compared with HSC+/+, HSC−/− showed high expression of Il-6 and chemokine mRNA when cocultured with mouse Hepa 1–6 cells. Thus, the absence of Cygb in pericytes provokes organ abnormalities, possibly via derangement of the nitric oxide and antioxidant defence system and through accelerated cellular senescence.

. The NO-scavenging function of Cygb was found to protect the NO-sensitive aconitase, decrease peroxynitrite (ONOO − ) formation and protect cellular respiration 8 . The Cygb expression patterns in human and rat hippocampus showed co-expression and subsequent upregulation of Cygb and neuronal NO synthase (nNOS) following chronic restraint stress 11 . The high level of Cygb and nNOS co-expression supports the hypothesised involvement of Cygb in NO metabolism. Accumulation of ONOO − and other nitrosative molecules results in nitrosative stress, which might affect protein tyrosine residues, metalloproteins, lipids and nucleic acids 12,13 . Thus, the NO-scavenging function of Cygb seems to be crucial for protecting cells/tissues from NO accumulation.
Finally, the hypoxia and oxidative responses of Cygb have been examined in various tumour cell lines, including sporadic head-and-neck squamous cell carcinoma 14 and human glioblastoma multiform 15 , as well as in animal models, such as models of murine embryogenesis and processes in adult tissues 16 . Furthermore, in vitro and in vivo overexpression of Cygb in rat HSCs protected these cells against oxidative stress and inhibited their differentiation into an activated phenotype 17 . Recently, Latina et al. reported that Cygb is transcriptionally regulated by Δ Np63 in primary epithelial cells (keratinocytes) and in cancer cells (H226, MCF-7) under normal proliferating conditions (normoxia) and following oxidative stress 18 . These reports suggest that in addition to functioning as a gas carrier, Cygb might act as a cytoprotective factor under conditions of hypoxia and oxidative stress.
To study the biological function of Cygb at the tissue level, we generated Cygb-deficient (Cygb −/− ) mice and reported their high susceptibility to tumour development in the liver and lungs when treated with N, N-diethylnitrosamine (DEN) 19 . Furthermore, Cygb −/− mice exhibited increases in liver inflammation, fibrosis and cancer development in a non-alcoholic steatohepatitis (NASH) model induced by a choline-deficient L-amino acid-defined diet via activation of the oxidative stress pathway 20 . Therefore, the absence of Cygb probably promotes fibrotic and carcinogenic processes in chronic liver diseases. However, it remains unclear whether CYGB plays a protective role in various organs under physiological conditions.
During the maintenance and propagation of our Cygb −/− mice, we detected the formation of age-dependent abnormalities in these mice. The main abnormalities in the Cygb −/− mice under 1 year of age (hereafter, called young mice) were heart hypertrophy and cystic diseases in the kidney and ovary, and the less frequent abnormalities included paralysis, loss of balance, liver fibrosis and lymphoma. In contrast, a total of 82 out of 115 (71.3%) Cygb −/− mice ranging from 1-2 years of age (hereafter, referred to as aged mice) displayed multiple organ abnormalities, including heart hypertrophy and tumours in the lung, liver, ovary, small intestine and lymphatic organs. Interestingly, the concentration of NO metabolites was increased significantly in the serum and urine of Cygb −/− mice compared with that in WT counterparts. These data confirmed an imbalance in the oxidative stress and antioxidant defence system with increased expression of oxidative stress-related genes, in contrast to the downregulation of antioxidative genes. Thus, the presence of Cygb in the pericytes of all organs serves an important function in maintaining homeostasis of the antioxidant system.

Results
Multi-organ abnormalities in Cygb deficient mice. Previously, we generated Cygb-deficient mice by deleting exon 1 of the mouse Cygb gene and backcrossing on the C57BL/6J background 19 . The mice that were homozygous for the disrupted allele appeared normal both morphologically and histopathologically 1 month (M) after birth. However, we found a time-dependent emergence of abnormalities in various organs of Cygb −/− mice. Among 92 Cygb −/− young mice of both sexes, 24 (26.0%) showed abnormalities. Nine mice had heart hypertrophy at 10 M, five mice had kidney cysts at 4 M, five had liver fibrosis and lymphoma from 5-11 M, two displayed loss of balance at 2 M, one mouse each had a cyst in the uterus or ovary at 4 M, and one displayed paralysis of the rear legs at 4 M. Meanwhile, none of the 135 WT young mice showed any abnormalities (p < 0.0001 by the Fisher exact test, two-tailed) (Fig. 1A). Importantly, aged Cygb −/− mice displayed a significantly greater number of abnormalities (82/115; 71.3%) compared with the number observed in aged WT mice (4/68; 5.8%) (p < 0.0001), and some Cygb −/− mice showed multiple organ abnormalities ( Fig. 1A and Table 1). The macroscopic abnormalities in Cygb −/− mice included lung tumour (Fig. 1Ba), liver tumour (Fig. 1Bb), liver cholestasis (Fig. 1Bc), swelling of the mesenteric lymph node (Fig. 1Bd), hepatosplenomegaly (Fig. 1Be), intestinal tumour (Fig. 1Bf), kidney cyst (Fig. 1Bg), kidney deformity and uterine cyst (Fig. 1Bh), mesenteric cyst (Fig. 1Bi), and heart hypertrophy (Fig. 1Bj). Histopathological analysis of lungs from Cygb −/− mice at 18 M revealed that the primary tumour types were adenoma (Fig. 1Ca) and adenocarcinoma (Fig. 1Cb). The livers of Cygb −/− mice at 17 M exhibited hepatocellular carcinoma (HCC) (Fig. 1Cc). Systemic lymphoma in Cygb −/− mice at 11 M occurred in the liver (Fig. 1Cd), spleen (Fig. 1Ce) and mesenteric lymph node (Fig. 1Cf). All lymphoma cases were immunohistochemically stained for CD3 and CD22, markers of the T and B cells, respectively. We found that these lymphomas were derived from T cells but not B cells (see Supplementary Fig. S2). Intestinal adenoma (Fig. 1Cg) was found at 21 M. Intestinal lymphoma (Fig. 1Ch), which was metastatic to the lung (Fig. 1Ci), was found at 24 M. A cyst in the kidney (Fig. 1Cj) was found at 4 M. A potential renal myomatous lesion (Fig. 1Ck) was accompanied by robust collagen fibres, as shown by Sirius red and fast green (SiR-FG) staining (Fig. 1Cl). Fibrosis of the spleen and cyst of uterus (red arrow), 6 M; mesenteric cyst, 10 M (i); heart hypertrophy, 22 M (j). (C) Representative haematoxylin and eosin (H&E)-stained sections of Cygb −/− mice with adenoma (a) and adenocarcinoma (b) of the lung; hepatocellular carcinoma (HCC) (c); lymphoma in the liver (d, arrow), spleen (e), and mesenteric lymph node (f); intestinal adenoma (g); intestine lymphoma (h), which was metastatic to the lung (i, arrows; right inset, × 1200); kidney cysts (j); H&E and Sirius Red and Fast Green (SiR-FG) staining of a renal myomatous lesion (k, l) and spleen fibrosis (m, n; right inset, × 800); hypertrophy of cardiomyocytes in KO mice compared with WT mice (o, WT; p, KO). was demonstrated by haematoxylin and eosin (H&E; Fig. 1Cm) and SiR-FG (Fig. 1Cn) staining. Cardiomyocyte hypertrophy (Fig. 1Co, WT; Fig. 1Cp, KO) was observed. Heart hypertrophy was further demonstrated by the increase in the heart weight (HW)/body weight (BW) ratio and by the increase in the heart size in terms of length and width ( Supplementary Fig. S1A) compared with WT. The HW/BW ratio in young Cygb −/− mice was 16.9% greater than that in WT. However, the HW/BW ratio in aged Cygb −/− mice showed a 41.2% increase (p < 0.0001) compared with WT ( Supplementary Fig. S1A). The length of the heart increased significantly in young Cygb −/− mice, and both the length and width increased significantly in aged mice compared with those in WT mice ( Supplementary Fig. S1B). Microscopic analysis demonstrated enlarged hearts (Supplementary Fig.  S1C) in Cygb −/− mice compared with those in WT. Interstitial fibrosis was observed in Cygb −/− mice, as indicated by SiR-FG staining ( Supplementary Fig. S1D).
Liver abnormalities in Cygb-deficient mice. After discovering Cygb in the HSCs of rat liver, we focused our efforts on examining the liver of Cygb −/− mice. We observed spontaneous development of liver tumours in 22.6% of aged Cygb −/− mice ( Table 1). The liver weight (LW)/BW ratio tended to increase in all female and aged male mice ( Fig. 2A). Although the alanine aminotransferase (ALT) level was similar between Cygb −/− and WT mice, the aspartate aminotransferase (AST) level was significantly increased in aged Cygb −/− mice compared with WT controls (Fig. 2B). In addition, we found other liver abnormalities, such as dilation of the portal vasculature (Fig. 2Ca), and hyperplasia of the bile duct (Fig. 2Cb) and HSCs (Fig. 2Cc,d). In accordance with these observations, SiR-FG staining and quantification revealed fibrosis in the liver of aged Cygb −/− mice, but not WT, in the absence of stimulants (Fig. 2D,F). Cellular retinol binding protein-1 (CRBP-1) immunostaining indicated the presence of HSCs in the absence of Cygb (Fig. 2D), similar to findings for the WT mouse liver. In addition, the expression of α smooth muscle actin (α SMA), a marker of activated HSCs, was increased at both the mRNA and protein levels in Cygb −/− mice (Fig. 2D,E). These data suggest that Cygb deficiency induced mild hepatocyte injury, activated HSCs, and stimulated the development of spontaneous liver fibrosis in an age-dependent manner.

Possible involvement of NO and oxidative stress in the liver damage of Cygb-deficient mice.
Cygb scavenges NO and other reactive oxygen species (ROS) 21 . Therefore, we hypothesised that the liver and the other organs might suffer from high concentrations of NO and ROS in the absence of Cygb. As shown in Fig. 3A (left), the concentration of nitrite + nitrate, oxidised forms of nitrogen, in the serum of Cygb −/− mice was increased significantly compared with that in WT mice, and this difference was observed in both young and aged mice. In addition, the nitrite + nitrate concentration in the urine of Cygb −/− mice was elevated markedly compared with that in WT mice (Fig. 3A, right). These results indicated that the absence of Cygb augments production of NO in the whole body. In addition, we detected robust expression of nitrotyrosine (NT) protein adducts in the liver and liver tumour lesions in aged Cygb −/− mice compared with WT ( Fig. 3B), implying an enhanced reaction of NO with superoxide anion (O 2 − ) to produce ONOO − . It is plausible that long term increases in NO production in the body would induce vasodilation and increased cardiac volume load, which might explain the heart hypertrophy observed in Cygb −/− mice 22 .
With regard to ROS productions, we assessed the level of malondialdehyde (MDA), an end product of lipid peroxidation, in the liver and serum of young mice. As shown in Fig. 3C, MDA changed slightly in the liver but increased significantly in the serum of young Cygb −/− mice compared with WT, indicating that ROS production was augmented in the absence of Cygb.
Extracellular NO reacts and consumes intracellular glutathione 23 , and it also triggers cellular oxidative stress 24 . Therefore, we measured the glutathione (GSH) concentration in the serum and liver. As expected, the total GSH in the serum showed a decreasing tendency in young Cygb −/− mice and a significant decrease in aged Cygb −/− mice compared with WT mice (Fig. 3D, left). The redox status expressed as the GSH:oxidised GSH (GSSG) ratio was lower in the liver tissues from aged Cygb −/− mice compared with WT, suggesting enhanced oxidative stress in Cygb −/− mice (Fig. 3D, right).
We examined the expression of 84 key genes involved in the oxidative stress and antioxidant defence system using a PCR array in Cygb −/− and WT mouse livers. of this array. Livers from 1-month-old Cygb −/− mice showed downregulation of almost all antioxidative genes, including glutathione peroxidase 3 (Gpx3), flavin-containing monooxygenase 2 (Fmo-2), and serine (or cysteine) peptidase inhibitor (Serpinb1b), compared with WT. Such downregulation of antioxidative genes was more prominent in 14-month-old Cygb −/− mice and was accompanied by increased expression of pro-oxidant genes,   3F). Thus, the absence of Cygb induced an imbalance between ROS production and the endogenous antioxidant system. Haem oxygenase-1 (HO-1), also known as heat shock protein 32 (HSP32) 25 , is another component of the cellular defence mechanism against oxidative stress. Here, we detected increased HO-1 expression at the protein and mRNA levels in the livers of aged Cygb −/− mice and in tumour lesions (Fig. 3F). The results obtained from this liver analysis implied that the loss of Cygb, which is dominantly expressed in the pericytes of all organs, induces oxidative stress conditions in the whole body, which consequently promote multiple organ abnormalities.
Premature senescence of HSCs in Cygb −/− mice. Various cellular stresses, such as oncogene activation, oxidative stress and DNA damage, can induce cellular senescence 26 . The senescence-associated secretory phenotype (SASP), which includes various inflammatory and tumour-promoting factors in HSCs, has crucial roles in promoting obesity-associated HCC development in mice 27 . Therefore, we examined whether the loss of Cygb induces HSC senescence in our model. We detected cells positive for p16 and p21 (two senescence-related genes or senescence inducers) in the sinusoidal cells but not the hepatocytes of aged Cygb −/− mice. WT mouse liver showed negligible expression of these proteins (Fig. 4A). Consistent with these results, qRT-PCR analysis showed elevated mRNA expression of p16, p21 and p27 in the liver and liver tumours of aged Cygb −/− mice compared with WT controls (Fig. 4B). Double immunofluorescence staining of p21 and desmin, a marker of HSCs, showed localization of p21 in the nucleus and desmin in the cytoplasm of HSCs (Fig. 4C). In addition, positive staining was observed for a marker of oxidative stress induced-DNA double strand breaks, phosphorylated γ H2AX (pSer139), in a non-tumourous area of both aged WT and Cygb −/− mice, but the level was markedly elevated in Cygb-deficient mice (Fig. 4A). In a 400× field, 42.3 ± 10.9 cells were positive for phosphorylated γ H2AX in the liver of aged Cygb −/− mice, which differed significantly from the 15.3 ± 4.57 positive cells observed in the WT counterparts (p = 0.0037 by a two-tailed t-test). We further examined the expression of phosphorylated γ H2AX in HSCs isolated from 12-week-old WT and Cygb −/− mice (Fig. 4D). A significantly greater number of  phosphorylated γ H2AX-positive cells was observed in HSC −/− (28.4 ± 6.9%) than in HSC +/+ mice (9.9 ± 3.4%, p = 0.014 by a two-tailed t-test).
Previously, mRNA profiling in HSCs isolated from Cygb −/− mice showed important features of priming conditions with elevated expression of interleukin (Il)-6, tumour necrosis factor α (Tnfα ), Il-1β , C-X-C motif chemokine ligand (Cxcl)-1, Cxcl-2, Cxcl-7, C-C motif chemokine ligand (Ccl)-2, Ccl-3, and Ccl-4 mRNA levels compared with those from WT 20 . These conditions were probably similar to the SASP of cells in which proinflammatory factors-such as ILs, chemokines and other inflammatory mediators, e.g., matrix metalloproteinases (Mmps) and NO-are the major secreted components 28 . Moreover, the expression of these cytokines and chemokines was increased in the liver and liver tumours of aged Cygb −/− mice compared with WT liver (Fig. 4E). These data suggest that loss of Cygb induced HSC senescence and SASP formation, thus affecting the tissue microenvironment for the promotion of tumour growth.

Increased expression of chemokines in cocultures of hepatocytes and Cygb-deficient
HSCs. The senescent phenotype of HSCs is believed to be involved in HCC development and propagation in mouse liver 27 . Therefore, we examined the interaction between hepatoma cells and HSCs in the presence or absence of Cygb. Accordingly, we cocultured mouse hepatoma Hepa 1-6 cells with HSCs +/+ or HSCs −/− cells using a transwell insert. The Ccl-2 mRNA level in Hepa 1-6 cells was increased two-fold when cocultured for 48 h with HSCs −/− cells compared with HSCs +/+ (Fig. 5A). The other genes tested showed no significant changes in expression (data not shown). In contrast, when HSCs were cocultured with Hepa 1-6 cells for 48 h, the gene expression profile of HSCs −/− showed upregulation of a variety of cytokine and chemokine mRNAs, including Il-6, Ccl-2, Ccl-3, Ccl-4, Cxcl-2, and vascular endothelial cell growth factor α (Vegfα ), compared with those of HSCs +/+ (Fig. 5B). These results implied that the soluble products excreted from Hepa 1-6 cells stimulated the senescent HSCs −/− to produce more soluble signalling factors.
Inhibition of NO synthesis reversed the phenotype of Cygb −/− mice. To evaluate the potential of NO depletion to reverse the phenotype observed in Cygb −/− mice, we examined young (12-week-old) mice exposed to 9 weeks of 0.01 mg/mL L-NAME in their drinking water. The NO level (total nitrite and nitrate) in serum was decreased significantly in both WT (6-fold compared with the untreated control) and Cygb −/− mice (4-fold) following L-NAME treatment (Fig. 6A). Subsequently, all the changes in young Cygb −/− mice, i.e., elevated expression of α Sma, Tnfα , and Ccl-2, were decreased to the same level as that of WT following L-NAME treatment (Fig. 6B). The liver of young age Cygb −/− mice still showed elevated expression of antioxidant genes, such as Sod-2 and Cat-1, as shown in Fig. 3E; the expression decreased in the aged mice (Fig. 3E). Here, after L-NAME treatment, the expression levels of both antioxidant and oxidative stress-related genes were reduced to the WT level (Fig. 6C). Interestingly, we observed an undetectable level of Mpo transcript gene (depicted as 0) together with very low expression of Ho-1 in both WT and KO mice under L-NAME treatment (Fig. 6C). Thus, L-NAME treatment in vivo can reverse the phenotype observed in Cygb −/− mice at least at the young age.

Discussion
Organ abnormalities induced by Cygb deficiency. This study demonstrated that the absence of Cygb augments oxidative stress, induces DNA damage and cellular senescence, and triggers HSC activation, implying the importance of Cygb in pericytes for the homeostasis of individual organs and tissue microenvironment. In particular, this study suggested a role for Cygb in NO metabolism, as indicated by the increased concentration of NO metabolites (nitrite and nitrate) in both the serum and urine in Cygb −/− mice compared with WT (Fig. 3). Importantly, all the phenotype observed in young Cygb −/− mice including the increased expression of fibrosis, inflammation and oxidative stress regulated genes was reversed when the mice were treated with NO inhibitor, L-NAME (Fig. 6). NO is an important gas radical that causes dilation of blood vessels in the body and acts as a cytoprotective agent under physiological conditions. However, in pathological conditions, such as inflammation, NO binds to O 2 − to form peroxynitrite and nitrotyrosine, which, in turn, cause DNA nucleotide modifications and induce the dysfunction and degradation of many functional proteins in the body 29 . Persistent nitration of proteins and DNA is potentially carcinogenic; significantly higher levels of nitrated proteins and/or iNos expression have been reported in lung 30 , breast 31 , head and neck 32 and ovarian 33 cancers in human, as well as in liver and lung cancers 19,20 in mice. Thus, tumour development via Cygb deficiency involving deregulation of NO metabolism potentially represents a novel target in cancer research.
Young Cygb −/− mice frequently exhibited cyst formation, especially cystic kidney diseases, which involve a dilation of tubules 34 and renal myomatous lesions, as shown in Fig. 1Ck,l. NO controls nephron transport at the proximal tubule and thick ascending limb, and it affects both the stimulation and inhibition of net fluid and bicarbonate 35 . Studies in patients 36 and in rats 37 suggested a role for NO in the pathogenesis of hypertension and cyst development in autosomal dominant polycystic kidney disease (ADPKD).
We found two mice that showed severe loss of balance, and they did not survive past 8 months. These mice first exhibited distinct head and body tilt and then ran in circles until becoming moribund. This phenomenon was reported in head slant mice, which contained inactivated NADPH oxidase organiser 1 (Noxo1) due to a deoxyadenosine insertion in exon 1 of Noxo1, resulting in the truncation of the 349-amino acid Noxo1 protein to a peptide of 34 amino acids 38 . Interestingly, such downregulated expression of Noxo1 mRNA was also found in 1-month-old Cygb −/− mice ( Table 2). (C) Effect of L-NAME in the expression of oxidative stress regulated genes in WT and KO mouse livers. Note the undetectable level of the Mpo transcript gene (depict as 0) together with the very low expression of Ho-1 in both WT and KO mice under L-NAME treatment. Data represent the mean ± SD; n = 5-10 for each group; *p < 0.05; **p < 0.01; ***p < 0.001; a, p < 0.05 for comparisons of the WT and KO control with the L-NAME treatment using one sample t-test with a hypothetical mean value of 0. Although a number of events at the molecular, cellular, and physiologic levels were changed in young Cygb-deficient mice, the most severe abnormalities were found in the aged mice. It is well documented that the incidence of malignant tumours increases progressively with age, in both animals and humans 39,40 . Three major hypotheses have been proposed to explain the association between cancer and age, including (1) the duration of carcinogenesis 41 ; (2) age-related progressive changes in the internal milieu of the organism, including proliferative senescence 42,43 ; and (3) the combined effects of a cumulative mutational load, increased epigenetic gene silencing, telomere dysfunction, and altered stromal milieu 44 . In this study, the age-related presence of tumours in multiple organs and fibrosis in Cygb −/− mice might be related to the second hypothesis because the primary HSCs from Cygb −/− mice exhibited the senescent phenotype.
Heart hypertrophy. The most dominant phenotype found in aged Cygb −/− mice was heart hypertrophy, as demonstrated by the increased size of cardiomyocytes and the HW/BW ratio. These results may be caused by the chronically slower metabolism of NO and the higher NO concentration in the aorta and vascular system, leading to an increased circulating volume in Cygb −/− mice 45 . In general, cardiac hypertrophy occurs in response to long-term increases in haemodynamic load related to a variety of physiological and pathological conditions. The process of cardiac hypertrophy is characterised by structural changes in the cardiomyocytes that are translated into alterations in chamber size and geometry, collectively called remodelling. NO has emerged as an important regulator of cardiac hypertrophy, apoptosis and remodelling 46 . The diverse cardiac effects of NO depend on its source and concentration and on the local scavenging activity of radicals, including NO 47 . Exogenous NO exerted inhibitory effects on cardiomyocyte hypertrophy in vivo 48 and in vitro 49 , which differed from our results. However, Moreau et al. showed that NO might be a necessary factor for cardiac hypertrophy in a rat model in which the concomitant administration of L-NAME, an inhibitor of nitric oxide synthesis, together with angiotensin II prevented the vascular hypertrophy induced by treatment with angiotensin II only 50 . Moreover, Mungrue et al. showed that conditional overexpression of iNos on cardiomyocytes was associated with peroxynitrite generation, cardiac fibrosis, myocyte death, increased cardiac mass and, ultimately, cardiac dilatation 51 . These data suggested that increased myocardial iNos activity initiates a process of cardiac remodelling that is characterised by ventricular dilatation, hypertrophy, and sudden cardiac death 51 . Consistent with our study, Mungrue et al. showed that upregulation of iNos led to increased formation of − O 2 and ONOOin the heart 51 . ROS have been recognised as prohypertrophic signalling intermediates in cardiomyocytes 52,53 . Therefore, Cygb might be a key molecule in metabolizing NO and balancing the antioxidant defence in the heart.

Fibrosis of the liver and other organs.
HSCs play a critical role in extracellular matrix remodelling and fibrosis progression in chronic liver diseases 54 . Reactive oxygen intermediates, apoptotic bodies from hepatocytes, and paracrine stimuli from Kupffer cells trigger HSC activation 55 . Cygb was initially found in activated HSCs with increased expression 1 ; thus, it was hypothesised that Cygb expression might protect HSCs from exposure to endogenous and exogenous ROS during liver injury. The ROS scavenger function of Cygb is evidenced by its ability to detoxify radicals via reaction with its haem 56 . Xu et al. demonstrated that forced overexpression of Cygb significantly increased the total oxy-radical scavenging capacity compared with that for the expression of control eGFP 17 and that overexpression of Cygb protected primary rat HSCs against oxidative stress, as indicated by reduced production of MDA and 4-hydroxy-2-nonenal (4-HNE), biomarkers of lipid peroxidation. Furthermore, Cygb overexpression reduced tissue fibrosis in both toxic and cholestatic models of liver injury 17 .
Previously, we found that loss of Cygb was associated with the priming of HSCs, which amplified the expression of fibrogenesis-related genes, cytokines and a variety of chemokines 20 . The priming of HSCs probably contributes to the chronic progression of fibrosis in the Cygb −/− mice in this study. Moreover, when Cygb −/−−/− mice were administered a high-fat diet, such as the choline-deficient L-amino acid defined diet (CDAA), to induce steatohepatitis, they rapidly developed serious liver inflammation and fibrosis at an early time point 20 . Similarly, as patients with NASH developed more fibrosis, Cygb expression decreased in HSCs 20 . A study in human liver tissues damaged by hepatitis C virus (HCV) infection at various fibrosis stages revealed that the number of Cygb-positive cells decreased with fibrosis progression 5 .
Interestingly, a number of Cygb −/− mice developed fibrosis in the kidney (Fig. 1Ck,l). Renal fibrosis, characterised by glomerulosclerosis and tubulointerstitial fibrosis, is the final common manifestation of a wide variety of chronic kidney diseases, similar to the wound-healing response in chronic liver injuries 57 . In line with our results, decreased kidney fibrosis induced by subtotal nephrectomy (remnant kidney) was demonstrated using transgenic rats overexpressing Cygb 58 . Therefore, Cygb supplementation might serve as a potential therapy for suppressing fibrosis in various organs. Cellular senescence and cancer development. Stromal fibroblasts from humans and mice have been studied extensively with respect to cellular senescence. Upon senescence, such cells show striking changes in gene expression 59 , some of which relate to the growth arrest and senescent morphology. Senescent fibroblasts secrete growth factors, cytokines, extracellular matrix and Mmps, all of which can alter tissue microenvironments and affect the function of nearby epithelial cells 60,61 . Recently, it was reported that obesity-associated HCC development is promoted by senescent HSCs 27 . Here we found that in the absence of Cygb, HSCs exhibited a senescent-like phenotype with expression of p16, p21, and p27 and production of SASP, which might contribute to liver tumour formation. Such senescence of mesenchymal fibroblast-like cells could occur in virtually all organs because tumour formation occurred in the lung, liver, intestine, and lymphoid system. Cygb might be an interesting tumour suppressor gene candidate not only in the liver but also in other organs due to its control of the senescence of pericytes, such as HSCs. Numerous investigations on the tumour-suppressing activity of Cygb have been reported since 2005; the studies showed that most cancer cells and tissues have reduced expression of Cygb and/or loss of heterozygosity, in addition to promoter hypermethylation both in vitro and in vivo 14,18,19,62 .
In conclusion, the absence of Cygb in pericytes provokes organ abnormalities and tumour formation due to possible derangement of ROS, including the NO and antioxidant system. Induced cellular senescence might be involved in the initiation of local inflammatory reactions and microenvironment modifications, leading to organ fibrosis. Further studies using targeted Cygb overexpression and knockdown in in vivo and cultured pericytes are needed to further clarify the molecular function of Cygb.

Materials and Methods
Animal and histopathological analysis. C57BL/6 Cygb knockout (Cygb −/− ) mice were generated in our laboratory as described previously 19 . Cygb heterozygous mice were backcrossed to the C57BL/6J background for more than nine generations. To assess the role of Cygb in development, we intercrossed Cygb heterozygous mice. The homozygotes appeared normal morphologically and histopathologically at 1 M. Mice were genotyped for the absence of Cygb at the DNA, RNA, and protein levels as described previously 19 . Both males and females were kept for observation and sacrificed at the designated age: 1 to 6 months old (1-6 M group), 7 to 12 months old (7-12 M group), 13 to 18 months old (13-18 M group), and 19 to 24 months old (19-24 M group). Each group of males or females contained 15 to 56 WT or Cygb −/− mice. The BW, LW, and HW were recorded. Heart sizes, including length and width, were measured. A complete necropsy was performed in each mouse. The blood was collected for the assays. All tissues were harvested, and abnormalities were noted. Tissue portions were kept at − 80 °C for further analysis, while other portions were fixed in neutral buffered formalin, embedded in paraffin, sectioned and stained with H&E. Mouse neoplastic and non-neoplastic lesions were diagnosed according to standard criteria 63,64 , and all abnormal histologic changes were recorded using a computerised autopsy data system. Records of morbidity or mortality were assigned subjectively to each mouse that died or to those that were sacrificed while moribund after review of the autopsy and histopathologic findings 65 . Selected tissues from mice with lymphoma were subjected to immunohistochemistry using CD3 for T cells or CD22 for B cells (Supplementary Table 1).
A subgroup of WT and Cygb −/− mice at 12 weeks of age, n = 10 each group, received L-NAME (Sigma-Aldrich, Tokyo, Japan) treatment at the dose of 0.01 mg/mL in drinking water. Untreated mice were used as control. After 9 weeks of L-NAME treatment, the mice were sacrificed for further analysis. All mice were housed in a facility with a 12-h light/dark cycle and allowed free access to food and water. All protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee of Osaka City University and performed in accordance with the guidelines of the National Institutes of Health for the use of animals in research.
Immunohistochemistry and immunofluorescence analysis. H&E staining, immunohistochemistry and immunofluorescence analysis were performed as described previously 19 . The primary antibodies used are listed in Supplementary Table 1. Polyclonal antibodies against Cygb were generated in our laboratory 1,5,19 . To compare the size of the heart, images representing the whole vertical section were acquired at 100× magnification and digitalised. These separately captured and digitalised images were consolidated to create one large image using the e-Tiling system (Mitani Corporation, Tokyo, Japan). To quantitate liver fibrosis, 5-μ m sections were cut, stained with Picrosirius red (Sigma-Aldrich, Tokyo, Japan) and counterstained with Fast Green dye (Sigma-Aldrich). Sirius red-positive areas were quantified in whole liver lobes using a BZ-X700 microscope and its BZ-X Analyser analysis software (Keyence, Osaka, Japan). For quantification, phosphorylated γ H2AX-positive cells in immunohistochemical/immunofluorescence stained mouse liver (n = 5 each group) and primary HSCs sections (n = 3 each group) were counted in at least 10 high-power fields (400× ) per one section.
Nitric oxide assay. The concentrations of total nitrate and nitrite in the serum and urine were measured by colourimetric methods using a Nitric Oxide Assay Kit (Abcam, Cambridge, UK) according to the manufacturer's protocol. Briefly, two-step process was performed, in which first step converted nitrate to nitrite utilizing nitrate reductase. The second step used Griess reagents to convert nitrite to a deep purple azo compound recorded at 540 nm. The amount of the azochromophore accurately reflects nitric oxide amount in samples.
Urine from individual mice was collected over 24 h using silicon wafers that covered the bottom of the mouse cage. Collections occurred at 3 h intervals, including 6:00-9:00, 9:00-12:00, 12:00-15:00, 15:00-18:00, 18:00-21:00, and one 9-h interval from 21:00 until the next day at 6:00 AM. A fresh silicon wafer was placed at each collection time. The total urine volume in a 24 h period was measured and used to calculate the total nmol of nitrate + nitrite in urine per day.
Glutathione assay. GSH, the major endogenous antioxidant produced by the cells, participates directly in the neutralization of free radicals and reactive oxygen compounds. GSH was measured in the serum (reduced GSH) and homogenised liver lysates. Both the reduced GSH and oxidised glutathione (GSSG) were determined using a glutathione assay kit (Cayman, Ann Arbor, MI) according to the manufacturer's protocol. Briefly, a carefully optimized enzymatic recycling method using glutathione reductase was utilized. The sulfhydryl group of GSH reacts with 5,5′ -dithio-bis-2-nitrobenzoic acid (DNTB) and produces a yellow coloured 5-thio-2-nitrobenzoic acid (TNB) which was recorded at 405 nm. The oxidized glutathione (GSSG) was measured after its reduction by glutathione reductase.
Malondialdehyde assay. To assess the oxidative stress status in Cygb −/− mice, lipid peroxidation in the serum and homogenised liver lysates was quantified by measuring its end product, MDA (BioVision, CA, USA) according to the manufacturer's protocol. Briefly, MDA in the sample is reacted with Thiobarbituric acid (TBA) to generate the MDA-TBA adduct which was quantified colorimetrically at 530 nm.
Scientific RepoRts | 6:24990 | DOI: 10.1038/srep24990 ALT and AST measurement. ALT and AST activities were measured in serum using a commercially available kit (Wako, Osaka, Japan) according to the manufacturer's protocol.
Quantitative real-time PCR. Total RNA was extracted from cells and tissues using a miRNeasy Mini Kit (Qiagen, Valencia, CA). cDNAs were synthesised using total RNA, a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) and oligo(dT) [12][13][14][15][16][17][18] primers according to the manufacturer's instructions. Gene expression was measured by real-time PCR using the cDNAs, SYBR qPCR Mix reagents (Toyobo) and gene-specific oligonucleotide primers (Supplementary Table 2) with an ABI Prism 7500 Fast Real-Time PCR System (Applied Biosystems, Foster, CA). The Gapdh level was used to normalise the relative abundance of mRNAs.
Gene expression profile for a specific pathway. An RT 2 Profiler ™ PCR Array for the Mouse Oxidative Stress and Antioxidant Defense (SA Biosciences, Frederick, Maryland; cat # PAMM-065) was used to examine the expression of 84 genes related to oxidative stress according to the manufacturer's protocol. Briefly, 1 μ g of total RNA from 1-and 14-month-old WT or Cygb −/− mice was used to make first strand complementary DNA (cDNA) using the RT 2 First Strand Kit (SA Biosciences). The PCR mixture containing cDNA, distilled water, and SYBR Green master mix (SA Biosciences) was loaded onto each well of 96-well plates containing the pre-dispensed gene-specific primer sets. PCR was performed with an ABI Prism 7500 Fast Real-Time PCR System (Applied Biosystems). PCR was performed in 96-well plates with 84 genes related to oxidative stress. Five housekeeping genes (actin B, Gapdh, Hsp90ab1, Hprt1, and Gusb) were used for normalizing the PCR array data, one negative control was used to verify genomic DNA contamination, and three wells of reverse transcription controls (RTC) were employed to verify the efficiency of the RT reaction. Excel-based PCR array data analysis (SA Biosciences) was used to calculate the threshold cycle (Ct) values for all genes in the array. Then, fold-changes in gene expression for pairwise comparisons using the Δ Δ Ct method were used to determine the relative expression levels of genes of interest for each sample.
Coculture experiments. Mouse hepatoma Hepa 1-6 cells, or HSCs isolated from Cygb +/+ (HSC +/+ ) and Cygb −/− mice (HSC −/− ) were cultured alone or cocultured in serum-free William's E medium (Invitrogen) using 6-well plates and transwell inserts with 1-μ m pore size, which allowed diffusion of medium components but prevented cell migration (BD Biosciences). Co-cultured of Hepa 1-6 cells with HSC +/+ or HSC −/− cells were prepared as follows: Hepa 1-6 cells were plated on the bottom of the six-well transwell cell culture system in serum-free William's E medium and cultured at 37 °C in a 5% CO 2 /95% room air. HSC +/+ or HSC −/− cells were cultured onto the membrane of transwell cell culture inserts. All cells were allowed to grow overnight using the above mentioned condition. The next day the all cells were washed with the serum-free media and membrane transwell inserts containing HSC +/+ or HSC −/− cells were placed into the six-well plates cultured containing the Hepa 1-6 cells to initiate the co-cultured experiment. Similar pattern was used for HSC +/+ or HSC −/− cells co-cultured with Hepa 1-6 cells. After 48 h, the cells were harvested for RNA isolation.
Statistical analysis. All data are expressed as the mean ± standard error of the mean. Two groups were compared using an unpaired Student t-test (two-tailed). P values less than 0.05 were considered statistically significant.