Unique and analogous functions of aquaporin 0 for fiber cell architecture and ocular lens transparency

Aquaporin (AQP) 1 and AQP0 water channels are expressed in lens epithelial and fiber cells, respectively, facilitating fluid circulation for nourishing the avascular lens to maintain transparency. Even though AQP0 water permeability is 40-fold less than AQP1, AQP0 is selectively expressed in the fibers. Delimited AQP0 fiber expression is attributed to a unique structural role as an adhesion protein. To validate this notion, we determined if wild type (WT) lens ultrastructure and fiber cell adhesion are different in AQP0^−/−, and TgAQP1^+/+/AQP0^−/− mice that transgenically express AQP1 (TgAQP1) in fiber cells without AQP0 (AQP0^−/−). In WT, lenses were transparent with ‘Y’ sutures. Fibers contained opposite end curvature, lateral interdigitations, hexagonal shape, and were arranged as concentric growth shells. AQP0^−/− lenses were cataractous, lacked ‘Y’ sutures, ordered packing and well-defined lateral interdigitations. TgAQP1^+/+/AQP0^−/− lenses showed improvement in transparency and lateral interdigitations in the outer cortex while inner cortex and nuclear fibers were severely disintegrated. Transmission electron micrographs exhibited tightly packed fiber cells in WT whereas AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses had wide extracellular spaces. Fibers were easily separable by teasing in AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses compared to WT. Our data suggest that the increased water permeability through AQP1 does not compensate for loss of AQP0 expression in TgAQP1^+/+/AQP0^−/− mice. Fiber cell AQP0 expression is required to maintain their organization, which is a requisite for lens transparency. AQP0 appears necessary for cell-to-cell adhesion and thereby to minimize light scattering since in the AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses, fiber cell disorganization was evident.     

Anxiety and increased 5‐HT1A receptor response in NCAM null mutant mice

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety‐like behavior of homozygous (NCAM^−/−) and heterozygous (NCAM^+/−) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety‐like behavior was reduced in both NCAM^+/+ and NCAM^−/− mice by systemic administration of the benzodiazepine agonist diazepam and the 5‐HT_1A receptor agonists buspirone and 8‐OH‐DPAT. However, NCAM^−/− mice showed anxiolytic‐like effects at lower doses of buspirone and 8‐OH‐DPAT than NCAM^+/+ mice. Such increased response to 5‐HT_1A receptor stimulation suggests a functional change in the serotonergic system of NCAM^−/− mice, likely involved in the control of anxiety and aggression. However, 5‐HT_1A receptor binding and tissue content of serotonin and its metabolite 5‐hydroxyindolacetic acid were found unaltered in every brain area of NCAM^−/− mice investigated, indicating that expression of 5‐HT_1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM^−/− mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5‐HT_1A receptors and inwardly rectifying K⁺ channels as the respective effector systems. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 343–355, 1999     

Protection of Vascular Endothelial Growth Factor to Brain Edema Following Intracerebral Hemorrhage and Its Involved Mechanisms: Effect of Aquaporin-4

Vascular endothelial growth factor (VEGF) has protective effects on many neurological diseases. However, whether VEGF acts on brain edema following intracerebral hemorrhage (ICH) is largely unknown. Our previous study has shown aquaporin-4 (AQP4) plays an important role in brain edema elimination following ICH. Meanwhile, there is close relationship between VEGF and AQP4. In this study, we aimed to test effects of VEGF on brain edema following ICH and examine whether they were AQP4 dependent. Recombinant human VEGF165 (rhVEGF165) was injected intracerebroventricularly 1 d after ICH induced by microinjecting autologous whole blood into striatum. We detected perihemotomal AQP4 protein expression, then examined the effects of rhVEGF165 on perihemotomal brain edema at 1 d, 3 d, and 7 d after injection in wild type (AQP4^+/+) and AQP4 knock-out (AQP4^−/−) mice. Furthermore, we assessed the possible signal transduction pathways activated by VEGF to regulate AQP4 expression via astrocyte cultures. We found perihemotomal AQP4 protein expression was highly increased by rhVEGF165. RhVEGF165 alleviated perihemotomal brain edema in AQP4^+/+ mice at each time point, but had no effect on AQP4^−/− mice. Perihemotomal EB extravasation was increased by rhVEGF165 in AQP4^−/− mice, but not AQP4^+/+ mice. RhVEGF165 reduced neurological deficits and increased Nissl’s staining cells surrounding hemotoma in both types of mice and these effects were related to AQP4. RhVEGF165 up-regulated phospharylation of C-Jun amino-terminal kinase (p-JNK) and extracellular signal-regulated kinase (p-ERK) and AQP4 protein in cultured astrocytes. The latter was inhibited by JNK and ERK inhibitors. In conclusion, VEGF reduces neurological deficits, brain edema, and neuronal death surrounding hemotoma but has no influence on BBB permeability. These effects are closely related to AQP4 up-regulation, possibly through activating JNK and ERK pathways. The current study may present new insights to treatment of brain edema following ICH.     

The circadian gene Cryptochrome 2 influences stress-induced brain activity and depressive-like behavior in mice

Cryptochrome 2 (Cry2) is a core clock gene important for circadian regulation. It has also been associated with anxiety and depressive-like behaviors in mice, but the previous findings have been conflicting in terms of the direction of the effect. To begin to elucidate the molecular mechanisms of this association, we carried out behavioral testing, PET imaging, and gene expression analysis of Cry2^−/− and Cry2^+/+ mice. Compared to Cry2^+/+ mice, we found that Cry2^−/− mice spent less time immobile in the forced swim test, suggesting reduced despair-like behavior. Moreover, Cry2^−/− mice had lower saccharin preference, indicative of increased anhedonia. In contrast, we observed no group differences in anxiety-like behavior. The behavioral changes were accompanied by lower metabolic activity of the ventro-medial hypothalamus, suprachiasmatic nuclei, ventral tegmental area, anterior and medial striatum, substantia nigra, and habenula after cold stress as measured by PET imaging with a glucose analog. Although the expression of many depression-associated and metabolic genes was upregulated or downregulated by cold stress, we observed no differences between Cry2^−/− and Cry2^+/+ mice. These findings are consistent with other studies showing that Cry2 is required for normal emotional behavior. Our findings confirm previous roles of Cry2 in behavior and extend them by showing that the effects on behavior may be mediated by changes in brain metabolism.     

Browning deficiency and low mobilization of fatty acids in gonadal white adipose tissue leads to decreased cold-tolerance of transglutaminase 2 knock-out mice

During cold-exposure ‘beige’ adipocytes with increased mitochondrial content are activated in white adipose tissue (WAT). These cells, similarly to brown adipose tissue (BAT), dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). We investigated the effect of tissue transglutaminase (TG2) ablation on the function of ATs in mice. Although TG2^+/+ and TG2^−/− mice had the same amount of WAT and BAT, we found that TG2^+/+ animals could tolerate acute cold exposure for 4 h, whereas TG2^−/− mice only for 3 h. Both TG2^−/− and TG2^+/+ animals used up half of the triacylglycerol content of subcutaneous WAT (SCAT) after 3 h treatment; however, TG2^−/− mice still possessed markedly whiter and higher amount of gonadal WAT (GONAT) as reflected in the larger size of adipocytes and lower free fatty acid levels in serum. Furthermore, lower expression of ‘beige’ marker genes such as UCP1, TBX1 and TNFRFS9 was observed after cold exposure in GONAT of TG2^−/− mice, paralleled with a lower level of UCP1 protein and a decreased mitochondrial content. The detected changes in gene expression of Resistin and Adiponectin did not provoke glucose intolerance in the investigated TG2^−/− mice, and TG2 deletion did not influence adrenaline, noradrenaline, glucagon and insulin production. Our data suggest that TG2 has a tissue-specific role in GONAT function and browning, which becomes apparent under acute cold exposure.     

Transferrin receptor 2 mitigates periodontitis-driven alveolar bone loss

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2^−/−) and wild-type (Tfr2^+/+) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2^+/+ and Tfr2^−/− mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2^+/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2^+/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2^+/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss.     

Phenotypic consequences of transforming growth factor β1 gene ablation in murine embryonic fibroblasts: Autocrine control of cell proliferation and extracellular matrix biosynthesis

The profound effects of transforming growth factor β1 (TGF-β1) on the immune system, cardiogenesis, in yolk sac hematopoeisis and in differentiation of endothelium have been demonstrated by detailed analyses of TGF-β1 knockout mice during embryogenesis. We have systematically examined the autocrine and paracrine roles of TGF-β1 in cell proliferation and in its ability to modulate the gene expression of selected components of extracellular matrix (ECM) using embryonic fibroblasts from TGF-β1 null mice (TGF-β1^−/−). The rates of cell proliferation of embryonic fibroblasts from normal mice (TGF-β1^+/+) and TGF-β1 null mice were compared by cell counting, by ³H thymidine incorporation, and by measuring the fraction of cells in the G1, S, and G2/M phases of the cell cycle by fluorescent activated cell sorting (FACS). Concurrently, the expression of pro-α1(I) collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) was also quantified by hybridization of total mRNA from TGF-β1^+/+ and TGF-β1^−/− embryonic fibroblasts. We report that TGF-β1^−/− cells proliferated at about twice the rate of TGF-β1^+/+ cells. Further, TGF-β1 null fibroblasts accumulated and synthesized lower constitutive levels of pro-α1(I) collagen, fibronectin, and PAI-1 mRNA. The quantitative differences in the rates of cell proliferation and ECM gene expression between TGF-β1^+/+ and TGF-β1^−/− cells could be eliminated by treatment of TGF-β1^+/+ cells with a neutralizing antibody of TGF-β1. Thus, our results are consistent with the hypothesis that TGF-β1 acts as a negative autocrine regulator of growth and a positive autocrine regulator of ECM biosynthesis in embryonic fibroblasts. 176:67–75, 1998. Published 1998 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

Remarkable induction of UV-signature mutations at the 3′-cytosine of dipyrimidine sites except at 5′-TCG-3′ in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh^−/−) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh^−/− epidermis and dermis than in the wild-type (Polh^+/+) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh^−/− mice were determined and compared with mutant sequences from irradiated Polh^+/+ mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C → T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5′-TCG-3′ sequence context than at the other dipyrimidine contexts in the Polh^+/+ epidermis, whereas the mutations were induced remarkably and exclusively at the 3′-cytosine of almost all dipyrimidine contexts with no preference for 5′-TCG-3′ in the Polh^−/− epidermis. In addition, in Polh^−/− mice, a small but remarkable fraction of G → T transversions was also observed exclusively at the 3′-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh^+/+ genotype.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

Secretion of zona pellucida glycoprotein mZP2 by growing oocytes from mZP3+/+ and mZP3−/− mice

The mouse egg extracellular coat, or zona pellucida (ZP), is composed of three glycoproteins, called mZP1–3, which are synthesized and secreted concomitantly by growing oocytes. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous nulls (mZP3^−/−). Growing oocytes from mZP3^−/− mice do not synthesize mZP3 mRNA or protein and, as a result, do not assemble a ZP. Here, we examined secretion of mZP2 by growing oocytes and eggs from mZP3^−/− mice, as well as incorporation of mZP2 into the ZP of oocytes from mZP3^+/+ mice. Laser scanning confocal microscopy (LSCM) of antibody‐labeled samples showed that, indeed, mZP2 was synthesized and secreted by oocytes isolated from mZP3^−/− mice and cultured in vitro. Nascent mZP2 was found in the culture medium, associated with the surface of the plasma membrane of growing oocytes, and in the oocyte cytoplasm. By contrast, mZP2 was barely detectable at any of these sites when ovulated eggs from mZP3^−/− mice were examined. Examination of oocytes from wild‐type (mZP3^+/+) mice showed that, while a portion of nascent mZP2 was assembled into the ZP (approximately 40%), here too a significant fraction was secreted into the culture medium (approximately 60%). Similar results also were obtained when intact pre‐antral follicles were isolated from mZP3^+/+ mice and cultured in vitro. Several of these observations are consistent with previous results obtained with oocytes from heterozygous null mice (mZP3^+/−). Furthermore, the results suggest that ZP assembly from nascent glycoproteins may be a stochastic process that requires the presence of both mZP2 and mZP3 and occurs completely outside the growing oocyte. Dev. Genet. 25:95–102, 1999. © 1999 Wiley‐Liss, Inc.     

Bmi1 regulate tooth and mandible development by inhibiting p16 signal pathway

To determine whether the deletion of p16 can correct tooth and mandible growth retardation caused by Bmi1 deficiency, we compared the tooth and mandible phenotypes of homozygous p16-deficient (p16^−/−) mice, homozygous Bmi1-deficient (Bmi1^−/−) mice, double homozygous Bmi1 and p16-deficient (Bmi1^−/−p16^−/−) mice to those of their wild-type littermates at 4 weeks of age by radiograph, histochemistry and immunohistochemistry. Results showed that compared to Bmi1^−/− mice, the dental mineral density, dental volume and dentin sialoprotein immunopositive areas were increased, whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in Bmi1^−/−p16^−/− mice. These results indicate that the deletion of p16 can improve tooth development in Bmi1 knockout mice. Compared to Bmi1^−/− mice, the mandible mineral density, cortical thickness, alveolar bone volume, osteoblast number and activity, alkaline phosphatase positive area were all increased significantly in Bmi1^−/−p16^−/− mice. These results indicate that the deletion of p16 can improve mandible growth in Bmi1 knockout mice. Furthermore, the protein expression levels of cyclin D, CDK4 and p53 were increased significantly in p16^−/− mice compared with those from wild-type mice; the protein expression levels of cyclin D and CDK4 were decreased significantly, whereas those of p27 and p53 were increased significantly in Bmi1^−/− mice; these parameters were partly rescued in Bmi1^−/−p16^−/− mice compared with those from Bmi1^−/− mice. Therefore, our results indicate that Bmi1 plays roles in regulating tooth and mandible development by inhibiting p16 signal pathway which initiated entry into cell cycle.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Gnpat does not play an essential role in systemic iron homeostasis in murine model

The GNPAT variant rs11558492 (p.D519G) was identified as a novel genetic factor that modifies the iron-overload phenotype in homozygous carriers of the HFE p.C282Y variant. However, the reported effects of the GNPAT p.D519G variant vary among study populations. Here, we investigated the role of GNPAT in iron metabolism using Gnpat-knockout (Gnpat^−/−), Gnpat/Hfe double-knockout (Gnpat^−/−Hfe^−/− or DKO) mice and hepatocyte-specific Gnpat-knockout mice (Gnpat^fl/fl;Alb-Cre). Our analysis revealed no significant difference between wild-type (Gnpat^+/+) and Gnpat^−/− mice, between Hfe^−/− and DKO mice, or between Gnpat^fl/fl and Gnpat^fl/fl;Alb-Cre with respect to serum iron and tissue iron. In addition, the expression of hepcidin was not affected by deleting Gnpat expression in the presence or absence of Hfe. Feeding Gnpat^−/− and DKO mice a high-iron diet had no effect on tissue iron levels compared with wild-type and Hfe^−/− mice, respectively. Gnpat knockdown in primary hepatocytes from wild-type or Hfe^−/− mice did not alter hepcidin expression, but it repressed BMP6-induced hepcidin expression. Taken together, these results support the hypothesis that deleting Gnpat expression has no effect on either systemic iron metabolism or the iron-overload phenotype that develops in Hfe^−/− mice, suggesting that GNPAT does not directly mediate iron homeostasis under normal or high-iron dietary conditions.     

Perilipin 5 deficiency promotes atherosclerosis progression through accelerating inflammation,apoptosis, and oxidative stress

Excessive plasma triglyceride (TG) and cholesterol levels promote the progression of several prevalent cardiovascular risk factors, including atherosclerosis, which is a leading death cause. Perilipin 5 (Plin5), an important perilipin protein, is abundant in tissues with very active lipid catabolism and is involved in the regulation of oxidative stress. Although inflammation and oxidative stress play a critical role in atherosclerosis development, the underlying mechanisms are complex and not completely understood. In the present study, we demonstrated the role of Plin5 in high-fat-diet-induced atherosclerosis in apolipoprotein E null (ApoE^−/−) mice. Our results suggested that Plin5 expressions increased in the artery tissues of ApoE^−/− mice. ApoE/Plin5 double knockout (ApoE^−/−Plin5^−/−) exacerbated severer atherogenesis, accompanied with significantly disturbed plasma metabolic profiles, such as elevated TG, total cholesterol, and low-density lipoprotein cholesterol levels and reduced high-density lipoprotein cholesterol contents. ApoE^−/−Plin5^−/− exhibited a higher number of inflammatory monocytes and neutrophils, as well as overexpression of cytokines and chemokines linked with an inflammatory response. Consistently, the IκBα/nuclear factor kappa B pathway was strongly activated in ApoE^−/−Plin5^−/−. Notably, apoptosis was dramatically induced by ApoE^−/−Plin5^−/−, as evidenced by increased cleavage of Caspase-3 and Poly (ADP-ribose) polymerase-2. In addition, ApoE^−/−Plin5^−/− contributed to oxidative stress generation in the aortic tissues, which was linked with the activation of phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinases pathways. In vitro, oxidized low-density lipoprotein (ox-LDL) increased Plin5 expression in RAW264.7 cells. Its knockdown enhanced inflammation, apoptosis, oxidative stress, and lipid accumulation, while promotion of Plin5 markedly reduced all the effects induced by ox-LDL in cells. These studies strongly supported that Plin5 could be a new regulator against atherosclerosis, providing new insights on therapeutic solutions.     

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3^−/−/ITGA2^−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3^−/−/ITGA2^+/− and COL4A3^−/−/ITGA2^+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3^−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3^−/−/ITGA2^+/+ Alport mice, but not in COL4A3^+/+/ITGA2^−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.     

Enhancing effect of taurohyodeoxycholate on ABCB4-mediated phospholipid efflux

Hydrophilic bile salts, ursodeoxycholate and hyodeoxycholate, have choleretic effects. ABCB4, a member of the ABC transporter family, is essential for the secretion of phospholipids from hepatocytes into bile. In this study, we assessed the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate and hyodeoxycholate on the ABCB4-mediated phosphatidylcholine (PC) efflux using Abcb4 knockout mice and HEK293 cells stably expressing ABCB4. To evaluate the effects of bile salts on bile formation in Abcb4^+/+ or Abcb4^−/− mice, the bile was collected during intravenous infusion of saline or bile salts. The biliary PC secretion in Abcb4^+/+ mice was significantly increased by the infusions of all tested bile salts, especially taurohyodeoxycholate. On the other hand, Abcb4^−/− mice exhibited extremely low secretion of PC into bile, which was not altered by bile salt infusions. We also showed that the PC efflux from ABCB4-expressing HEK293 cells was stimulated by taurohyodeoxycholate much more strongly than the other tested bile salts. However, taurohyodeoxycholate did not restore the activities of ABCB4 mutants. Furthermore, light scattering measurements demonstrated a remarkable ability of taurohyodeoxycholate to form mixed micelles with PC. Therefore, the enhancing effect of taurohyodeoxycholate on the ABCB4-mediated PC efflux may be due to the strong mixed micelle formation ability.     

Protein deglycase DJ-1 deficiency induces phenotypic switching in vascular smooth muscle cells and exacerbates atherosclerotic plaque instability

Protein deglycase DJ-1 (DJ-1) is a multifunctional protein involved in various biological processes. However, it is unclear whether DJ-1 influences atherosclerosis development and plaque stability. Accordingly, we evaluated the influence of DJ-1 deletion on the progression of atherosclerosis and elucidate the underlying mechanisms. We examine the expression of DJ-1 in atherosclerotic plaques of human and mouse models which showed that DJ-1 expression was significantly decreased in human plaques compared with that in healthy vessels. Consistent with this, the DJ-1 levels were persistently reduced in atherosclerotic lesions of ApoE^−/− mice with the increasing time fed by western diet. Furthermore, exposure of vascular smooth muscle cells (VSMCs) to oxidized low-density lipoprotein down-regulated DJ-1 in vitro. The canonical markers of plaque stability and VSMC phenotypes were evaluated in vivo and in vitro. DJ-1 deficiency in Apoe^−/− mice promoted the progression of atherosclerosis and exaggerated plaque instability. Moreover, isolated VSMCs from Apoe^−/−DJ-1^−/− mice showed lower expression of contractile markers (α-smooth muscle actin and calponin) and higher expression of synthetic indicators (osteopontin, vimentin and tropoelastin) and Kruppel-like factor 4 (KLF4) by comparison with Apoe^−/−DJ-1^+/+ mice. Furthermore, genetic inhibition of KLF4 counteracted the adverse effects of DJ-1 deletion. Therefore, our results showed that DJ-1 deletion caused phenotype switching of VSMCs and exacerbated atherosclerotic plaque instability in a KLF4-dependent manner.     

Hormone-sensitive lipase deficiency affects the expression of SR-BI,LDLr, and ABCA1 receptors/transporters involved in cellular cholesterol uptake and efflux and disturbs fertility in mouse testis

Hormone-sensitive lipase (HSL) hydrolyse acylglycerols, cholesteryl and retinyl esters. HSL is a key lipase in mice testis, as HSL deficiency results in male sterility. The present work study the effects of the deficiency and lack of HSL on the localization and expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in uptake and efflux of cholesterol in mice testis, to determine the impact of HSL gene dosage on testis morphology, lipid homeostasis and fertility. The results of this work show that the lack of HSL in mice alters testis morphology and spermatogenesis, decreasing sperm counts, sperm motility and increasing the amount of Leydig cells and lipid droplets. They also show that there are differences in the localization of HSL, SR-BI, LDLr and ABCA1 in HSL^+/+, HSL^+/− and HSL^−/− mice. The deficiency or lack of HSL has effects on protein and mRNA expression of genes involved in lipid metabolisms in mouse testis. HSL^−/− testis have augmented expression of SR-BI, LDLr, ABCA1 and LXRβ, a critical sterol sensor that regulate multiple genes involved in lipid metabolism; whereas LDLr expression decreased in HSL^+/− mice. Plin2, Abca1 and Ldlr mRNA levels increased; and LXRα (Nr1h3) and LXRβ (Nr1h2) decreased in testis from HSL^−/− compared with HSL^+/+; with no differences in Scarb1. Together these data suggest that HSL deficiency or lack in mice testis induces lipid homeostasis alterations that affect the cellular localization and expression of key receptors/transporter involved in cellular cholesterol uptake and efflux (SR-BI, LDRr, ABCA1); alters normal cellular function and impact fertility.