Kim M. Hansen

Inflammation-induced dysfunction of the low-density lipoprotein receptor-related protein-1 at the blood–brain barrier: Protection by the antioxidant N-acetylcysteine

Impairment in two blood-brain barrier (BBB) efflux transporters, p-glycoprotein (Pgp) and low-density lipoprotein receptor-related protein-1 (LRP-1) are thought to contribute to the progression of Alzheimers disease (AD) by resulting in the brain accumulation of their substrate amyloid beta peptide (Aβ). The initial cause of impaired efflux, however, is unknown. We have shown that induction of systemic inflammation by intraperitoneal administration of lipopolysaccharide impairs the efflux of Aβ from the brain, suggesting that systemic inflammation could be one such initiator. In this study, we determined whether pre-administration of the antioxidant N-aceytlcysteine (Nac) has a protective effect against LPS-induced Aβ transporter dysfunction. Our findings were that Nac protected against LPS-induced Aβ transport dysfunction at the BBB through an LRP-1-dependent and Pgp-independent mechanism. This was associated with Nac exerting antioxidant effects in the periphery but not the brain, despite an increased rate of entry of Nac into the brain following LPS. We also found that Nac pre-administration resulted in lower blood levels of the cytokines and chemokines interferon-γ, interleukin-10, CCL2, CCL4, and CCL5, but only lowered CCL4 in the cerebral cortex and hippocampus. Finally, we observed that hippocampal cytokine responses to LPS were decreased compared to cortex. These findings demonstrate a novel mechanism by which antioxidants prevent Aβ accumulation in the brain caused by inflammation, and therefore protect against AD.

Laminin α4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

Each extracellular matrix compartment in the kidney has a unique composition, with regional specificity in the expression of various laminin isoforms. Although null mutations in the majority of laminin chains lead to specific developmental abnormalities in the kidney, Lama4-/- mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor beta in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to alpha4-containing laminins, but not other isoforms, results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. Given the known role of overexpression of PDGF-BB and PDGF-DD on glomerular and tubulointerstitial fibrosis, these data suggest that failure of laminin alpha4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin alpha4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans.

Differential expression of laminin chains and anti-laminin autoantibodies in experimental lupus nephritis.

Mice with chronic graft‐versus‐host disease (GvHD) develop a lupus‐like disease with severe immune complex glomerulonephritis. Previous studies with this model have shown that anti‐laminin autoantibodies are involved in immune complex formation and that glomerular laminin expression alters qualitatively. The present study investigated glomerular laminin chain expression and autoantibody reactivity with matrix antigens during disease development in mice with chronic GvHD, killed before and 6, 8, 10, and 11 weeks after disease induction, using antibodies raised against laminin chain peptides, in immunofluorescence and western blotting studies. Decreased glomerular expression of the laminin β1 chain, unaltered expression of the laminin β2 and γ1 chains, and increased expression of the laminin α1 chain and filamin/actin‐binding protein 280 (ABP 280) were found during disease progression. Furthermore, 4 weeks after disease induction, autoantibodies appeared which were reactive with laminin α1, β1, β2, and γ1 chains, and filamin in rat mesangial cell matrix. Ten weeks after disease induction, autoantibodies reacted with filamin, and β2 and γ1 laminin chains. Autoantibodies reacted with laminin chains only and not with other proteins in matrices extracted from glomeruli of normal and diseased mice. Staining with H50, an anti‐laminin α1 chain/anti‐filamin monoclonal autoantibody derived from an MRL/lpr mouse with spontaneous lupus nephritis, confirmed these observations and showed identical anti‐laminin/anti‐filamin autoantibody reactivity in two different models for lupus nephritis. In summary, differential glomerular expression of laminin chains was found during the development of chronic GvHD. Concomitantly with expression of the laminin α1 chain and/or filamin in the glomerulus, anti‐laminin α1 and/or anti‐filamin reactivity was present, pointing towards a role for (neo) antigen expression in the epitope spreading of the immune response. Furthermore, glomerular expression of laminin β1 decreased in conjunction with decreased presence of anti‐laminin β1 reactivity, presumably due to antigen masking or shedding of immune complexes into the urine. These changes in anti‐laminin chain autoantibodies, with concomitant alterations in the glomerular expression of laminin chains, may aggravate progressive immune injury in this model for lupus nephritis. Copyright

RNA-binding protein IGF2BP2/IMP2 is required for laminin-β2 mRNA translation and is modulated by glucose concentration

Laminin-β2 (LAMB2) is a critical component of the glomerular basement membrane as content of LAMB2 in part determines glomerular barrier permeability. Previously, we reported that high concentrations of glucose reduce expression of this laminin subunit at the translational level. The present studies were undertaken to further define systems that control Lamb2 translation and the effect of high glucose on those systems. Complementary studies were performed using in vitro differentiation of cultured podocytes and mesangial cells exposed to normal and elevated concentrations of glucose, and tissues from control and diabetic rats. Together, these studies provide evidence for regulation of Lamb2 translation by IMP2, an RNA binding protein that targets Lamb2 mRNA to the actin cytoskeleton. Expression of Imp2 itself is regulated by the transcription factor HMGA2, which in turn is regulated by the microRNA let-7b. Elevated concentrations of glucose increase let-7b, which reduces HMGA2 expression, in turn reducing IMP2 and LAMB2. Correlative changes in kidney tissues from control and streptozotocin-induced diabetic rats suggest these control mechanisms are operative in vivo and may contribute to proteinuria in diabetic nephropathy. To our knowledge, this is the first time that translation of Lamb2 mRNA has been linked to the actin cytoskeleton, as well as to specific RNA-binding proteins. These translational control points may provide new targets for therapy in proteinuric disorders such as diabetic nephropathy where LAMB2 levels are reduced.

Role of laminin isoforms in glomerular structure

Laminin along with collagen type IV, proteoglycans, and entactin are major components of basement membranes. Basement membrane components are synthesized at high levels during development. The formation of specialized basement membranes may play important roles in cell and tissue function by influencing cell proliferation, phenotype, migration and gene expression as well as tissue architecture. The growing diversity of laminin isoforms influences the formation of distinct basement membranes. Many of the laminin chains sequenced to date are expressed during glomerular development under strict temporal control. Also, some studies suggest that additional laminin chains exist and contribute to unique isoforms expressed within the renal glomerulus. This article will review the status of characterization of laminin isoforms expressed by glomerular cells, point out possible differences in isoforms expressed by different species, and discuss the implications of the complexity of glomerular laminins. In order to fully understand the nature of the glomerular laminins and their importance, information from studies of cells in culture, whole tissue, and those that use molecular and protein analysis must be integrated.

Molecular Hydrogen in Drinking Water Protects against Neurodegenerative Changes Induced by Traumatic Brain Injury

Traumatic brain injury (TBI) in its various forms has emerged as a major problem for modern society. Acute TBI can transform into a chronic condition and be a risk factor for neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases, probably through induction of oxidative stress and neuroinflammation. Here, we examined the ability of the antioxidant molecular hydrogen given in drinking water (molecular hydrogen water; mHW) to alter the acute changes induced by controlled cortical impact (CCI), a commonly used experimental model of TBI. We found that mHW reversed CCI-induced edema by about half, completely blocked pathological tau expression, accentuated an early increase seen in several cytokines but attenuated that increase by day 7, reversed changes seen in the protein levels of aquaporin-4, HIF-1, MMP-2, and MMP-9, but not for amyloid beta peptide 1–40 or 1–42. Treatment with mHW also reversed the increase seen 4 h after CCI in gene expression related to oxidation/carbohydrate metabolism, cytokine release, leukocyte or cell migration, cytokine transport, ATP and nucleotide binding. Finally, we found that mHW preserved or increased ATP levels and propose a new mechanism for mHW, that of ATP production through the Jagendorf reaction. These results show that molecular hydrogen given in drinking water reverses many of the sequelae of CCI and suggests that it could be an easily administered, highly effective treatment for TBI.

Sleep fragmentation and sepsis differentially impact blood-brain barrier integrity and transport of tumor necrosis factor-α in aging

The factors by which aging predisposes to critical illness are varied, complex, and not well understood. Sepsis is considered a quintessential disease of old age because the incidence and mortality of severe sepsis increases in old and the oldest old individuals. Aging is associated with dramatic changes in sleep quality and quantity and sleep increasingly becomes fragmented with age. In healthy adults, sleep disruption induces inflammation. Multiple aspects of aging and of sleep dysregulation interact via neuroimmune mechanisms. Tumor necrosis factor-α (TNF), a cytokine involved in sleep regulation and neuroimmune processes, exerts some of its effects on the CNS by crossing the blood-brain barrier (BBB). In this study we examined the impact of sepsis, sleep fragmentation, and aging on BBB disruption and TNF transport into brain. We used the cecal ligation and puncture (CLP) model of sepsis in young and aged mice that were either undisturbed or had their sleep disrupted. There was a dichotomous effect of sepsis and sleep disruption with age: sepsis disrupted the BBB and increased TNF transport in young mice but not in aged mice, whereas sleep fragmentation disrupted the BBB and increased TNF transport in aged mice, but not in young mice. Combining sleep fragmentation and CLP did not produce a greater effect on either of these BBB parameters than did either of these manipulations alone. These results suggest that the mechanisms by which sleep fragmentation and sepsis alter BBB functions are fundamentally different from one another and that a major change in the organisms responses to those insults occurs with aging.

Alterations in chromatin are associated with increases in collagen III expression in aging nephropathy

Aging nephropathy is a slowly progressive fibrotic process that affects all compartments of the kidney and eventually impairs kidney function; however, little is known about the mechanisms that contribute to this process. These studies examined the epigenetic control of expression of collagen III (Col3a1), a matrix protein that contributes to kidney fibrosis. Using real-time PCR, Western blotting, and chromatin immunoprecipitation assay of kidneys harvested from 4- and 24-mo-old ad libitum-fed F344 rats, we found increased transcription of Col3a1 that was associated with increased RNA polymerase II recruitment despite elevated posttranslational histone modification (H3K27me3) normally associated with gene silencing. A reduction in the density of another repressive modification (H3K9me3) at the Col3a1 locus in aged rats suggests that cooperation between Polycomb- and heterochromatin-mediated systems are required to maintain repression of the Col3a1 gene. These findings demonstrate alterations in epigenetic control of gene expression in association with the fibrosis of aging nephropathy.

Insulin-like Growth Factor-binding Protein-5-induced Laminin γ1 Transcription Requires Filamin A

Insulin-like growth factor-binding protein-5 (IGFBP-5) has IGF-1-independent intranuclear effects that are poorly defined. Treatment of cells with IGFBP-5 induces migration, prevents apoptosis, and leads to increased laminin subunit transcription. Similarly, filamin A (FLNa), an actin-binding protein that participates in cell attachment, plays important additional roles in signal transduction and modulation of transcriptional responses. In this report, we show that IGFBP-5 leads to dephosphorylation of FLNa with subsequent FLNa cleavage. Following cleavage, there is enhanced recruitment of Smad3/4 to a C-terminal FLNa fragment with nuclear translocation and subsequent binding to the promoter region of the laminin γ1 (lamc1) gene. FLNa knockdown prevents IGFBP-5-mediated increases in lamc1 transcription. These data indicate that IGFBP-5 induces formation of a FLNa-based nuclear shuttle that recruits transcription factors and regulates transcription of IGFBP-5 target genes. These studies provide new insights into the mechanisms whereby IGFBP-5 and FLNa exert intranuclear effects.

Reductions in laminin β2 mRNA translation are responsible for impaired IGFBP-5-mediated mesangial cell migration in the presence of high glucose

Insulin-like growth factor binding protein-5 (IGFBP-5) mediates mesangial cell migration through activation of cdc42, and laminin421 binding to alpha(6)beta(1)-integrin (Berfield AK, Hansen KM, Abrass CK. Am J Physiol Cell Physiol 291: C589-C599, 2006). Because glomerular expression of laminin beta(2) is reduced in diabetic rats (Abrass CK, Spicer D, Berfield AK, St. John PL, Abrahamson DR. Am J Pathol 151: 1131-1140, 1997), we directly examined the effect of hyperglycemia on mesangial cell migration and laminin beta2 expression. Migration mediated by IGFBP-5 is impaired in the presence of 25 mM glucose. This reduction in migration was found to result from a loss in mesangial cell synthesis of laminin421, and IGFBP-5-induced migration could be restored by replacing laminin421. Additional studies showed that there was selective reduction in mRNA translation of laminin beta2 in the presence of high glucose. Preserved synthesis of laminin beta1 indicates that not all proteins are reduced by high glucose and confirms prior data showing that laminin411 cannot substitute for laminin421 in IGFBP-5-mediated migration. Given the importance of mesangial migration in the reparative response to diabetes-associated mesangiolysis, these findings provide new insights into abnormalities associated with diabetic nephropathy and the potential importance of differential control of protein translation in determination of alterations of protein expression.