Phenolic Compounds Prevent Alzheimer’s Pathology through Different Effects on the Amyloid-β Aggregation Pathway

Inhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for Alzheimer’s disease (AD). Certain phenolic compounds have been reported to have anti-Aβ aggregation effects in vitro. This study systematically investigated the effects of phenolic compounds on AD model transgenic mice (Tg2576). Mice were fed five phenolic compounds (curcumin, ferulic acid, myricetin, nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA)) for 10 months from the age of 5 months. Immunohistochemically, in both the NDGA- and RA-treated groups, Aβ deposition was significantly decreased in the brain (P < 0.05). In the RA-treated group, the level of Tris-buffered saline (TBS)-soluble Aβ monomers was increased (P < 0.01), whereas that of oligomers, as probed with the A11 antibody (A11-positive oligomers), was decreased (P < 0.001). However, in the NDGA-treated group, the abundance of A11-positive oligomers was increased (P < 0.05) without any change in the levels of TBS-soluble or TBS-insoluble Aβ. In the curcumin- and myricetin-treated groups, changes in the Aβ profile were similar to those in the RA-treated group, but Aβ plaque deposition was not significantly decreased. In the ferulic acid-treated group, there was no significant difference in the Aβ profile. These results showed that oral administration of phenolic compounds prevented the development of AD pathology by affecting different Aβ aggregation pathways in vivo. Clinical trials with these compounds are necessary to confirm the anti-AD effects and safety in humans.Alzheimer’s disease (AD) is the most common form of dementia, resulting in deterioration of cognitive function and behavioral changes.¹ One of the pathological hallmarks of AD is extracellular deposits of aggregated amyloid-β protein (Aβ) in the brain parenchyma (senile plaques) and cerebral blood vessels (cerebral amyloid angiopathy (CAA)).¹ Deposition of high levels of fibrillar Aβ in the AD brain is associated with loss of synapses, impairment of neuronal functions, and loss of neurons.^2,3,4,5 Aβ was sequenced from meningeal vessels and senile plaques of AD patients and individuals with Down’s syndrome.^6,7,8 The subsequent cloning of the gene encoding the β-amyloid precursor protein and its localization to chromosome 21,^9,10,11,12 coupled with the earlier recognition that trisomy 21 (Down’s syndrome) invariably leads to the neuropathology of AD,¹³ set the stage for the proposal that Aβ accumulation is the primary event in AD pathogenesis. In addition, certain mutations associated with familial AD have been identified within or near the Aβ region of the coding sequence of gene of the amyloid precursor proteins,^14,15 presenilin-1 and presenilin-2,¹⁶ which alter amyloid precursor protein metabolism through a direct effect on γ-secretase.^17,18 These findings set the stage for the proposal that Aβ aggregation is the primary event in AD pathogenesis and leading to the proposal that anti-Aβ aggregation is a strategy for AD therapy.^19,20 Furthermore, there have been recent reports^{21,22,23,24,25} that Aβ fibrils are not the only toxic form of Aβ for developing AD, and smaller species of aggregated Aβ, Aβ oligomers, may represent the primary toxic species in AD. Therefore, it is necessary to consider the inhibition of Aβ oligomer formation as well as Aβ fibrils for the treatment of AD.²⁶To date, it has been reported that various compounds inhibit the formation and extension of Aβ fibrils, as well as destabilizing Aβ fibrils in vitro.^{19,20,27,28,29,30,31,32,33,34,35,36} Among the reported compounds, several phenolic compounds, such as wine-related polyphenols (myricetin (Myr), morin, and tannic acid, and so on), curcumin (Cur), ferulic acid (FA), nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA) had especially strong anti-Aβ aggregation effects in vitro. Furthermore, it was shown recently that a commercially available grape seed polyphenolic extract, MegaNatural-Az, inhibited fibril formation, protofibril formation, and oligomerization of Aβ.³⁷ Moreover, MegaNatural-Az also reduced cerebral amyloid deposition as well as attenuating AD-type cognitive deterioration using transgenic mice.³⁸ In addition to these studies by the current authors, several other researchers have reported similar effects of phenolic compounds.^{26,39,40,41,42,43,44} First, Cur decreased cerebral Aβ plaque burden in vivo,^{39,40,41,42,44} and inhibited the formation of Aβ oligomers in vitro.^26,39 Second, epigallocatechin gallate efficiently inhibited fibril and oligomer formation of Aβ.⁴³ However, a very recent in vitro study²⁶ reported that Cur, Myr, and NDGA inhibited the formation of Aβ oligomers, but Cur and NDGA promoted the formation of Aβ fibrils. This indicated that the effects of these phenolic compounds on Aβ aggregation remain controversial. These different results may reflect different experimental conditions in these studies. To resolve this problem, a systematic in vivo study is required; however, few reports on the effects of phenolic compounds on Aβ aggregation in vivo have been published so far, except for reports about Cur.^{39,40,41,42,44}To elucidate the inhibitory effects of phenolic compounds on Aβ aggregation in vivo, several phenolic compounds, including Cur, FA, Myr, NDGA, and RA, were fed to AD model mice, and the cerebral plaque burden and formation of Aβ oligomers were compared systematically.     

Integrin-Mediated Transforming Growth Factor-β Activation,a Potential Therapeutic Target in Fibrogenic Disorders

A subset of integrins function as cell surface receptors for the profibrotic cytokine transforming growth factor-β (TGF-β). TGF-β is expressed in an inactive or latent form, and activation of TGF-β is a major mechanism that regulates TGF-β function. Indeed, important TGF-β activation mechanisms involve several of the TGF-β binding integrins. Knockout mice suggest essential roles for integrin-mediated TGF-β activation in vessel and craniofacial morphogenesis during development and in immune homeostasis and the fibrotic wound healing response in the adult. Amplification of integrin-mediated TGF-β activation in fibrotic disorders and data from preclinical models suggest that integrins may therefore represent novel targets for antifibrotic therapies.The multifunctional cytokine transforming growth factor-β (TGF-β) plays major roles in the biology of immune, endothelial, epithelial, and mesenchymal cells during development and adult life in invertebrate and vertebrate species.^1,2 In mammals, these functions are mediated by three isoforms, TGF-β1, 2, and 3, which are each widely expressed.³ All three isoforms interact with the same cell surface receptors (TGFBR2 and ALK5) and signal through the same intracellular signaling pathways, which involve either canonical (ie, SMADs) or noncanonical (ie, MAPK, JUN, PI3K, PP2A, Rho, PAR6) signaling effectors.^4,5 The canonical TGF-β signaling pathway, whereby TGF-β signaling is propagated from the TGF-β receptor apparatus through phosphorylation of cytoplasmic SMAD-2/3, complex formation with SMAD-4, nuclear translocation of the SMAD-2/3/4 complex, and binding to SMAD response elements located in the promoter regions of many genes involved in the fibrogenic response, has been the most intensively studied.⁶ However, despite having similar signaling partners, each isoform serves individual biological functions, perhaps due to differences in binding affinity to TGF-β receptors, activation mechanism, signaling intensity or duration, or spatial and/or temporal distribution.⁷Knockout and conditional deletion models of TGF-β isoforms, receptors, and signaling mediators, as well as function-blocking reagents targeting all TGF-β isoforms, have revealed essential roles for TGF-β in T-cell, cardiac, lung, vascular, and palate development.^{8,9,10,11,12,13,14,15} For instance, mice deficient in TGF-β1 either die in utero owing to defects in yolk sac vasculogenesis or are born and survive into adult life but develop severe multiorgan autoimmunity.¹² Genetic deletion of TGF-β signaling mediators has shown an essential role for Smad2 in early patterning and mesodermal formation,^16,17 and mice lacking Smad3 are viable and fertile, but exhibit limb malformations,¹⁸ immune dysregulation, colitis,¹⁹ colon carcinomas,²⁰ and alveolar enlargement.²¹In adult tissues, the TGF-β pathway is thought to regulate the dynamic interactions among immune, mesenchymal, and epithelial cells to maintain homeostasis in response to environmental stress.²² The normal homeostatic pathways mediated by TGF-β are perturbed in response in chronic repetitive injury. In cases of injury, TGF-β becomes a major profibrogenic cytokine, delaying epithelial wound healing by inhibiting epithelial proliferation and migration and promoting apoptosis and expanding the mesenchymal compartment by inducing fibroblast recruitment, fibroblast contractility, and extracellular matrix deposition.²³ Indeed, intratracheal transfer of adenoviral recombinant TGF-β1 to the rodent lung dramatically increases fibroblast accumulation and expression of type I and type III collagen around airways and in the pulmonary interstitium,^24,25 and neutralizing anti-TGF-β antibodies can block experimental bleomycin or radiation-induced pulmonary fibrosis.^26,27 Increased activity of the TGF-β pathway has also been implicated in fibrotic lung disease, glomerulosclerosis, and restenosis of cardiac vessels.^23,28,29,30 Most TGF-β-mediated pathological changes appear to be attributed to the TGF-β1 isoform.³¹The complexity of TGF-β1 function in humans is illuminated by hereditary disorders with generalized or cell type-specific enhancement or deficiency in either TGF-β1 itself or its signaling effectors. Mutations that increase the activity of the TGF-β pathway lead to defects in bone metabolism (ie, Camurati-Engelmann disease) and in connective tissue (ie, Marfan syndrome), and in aortic aneurysms (ie, Loeys-Dietz syndrome), whereas mutations that lead to decreased activity of the TGF-β pathway correlate with cancer occurrence and prognosis.³² The role of TGF-β as a tumor suppressor in cancer is not straightforward, however, because TGF-β can also enhance tumor growth and metastasis, perhaps through its roles in immune suppression, cell invasion, epithelial-mesenchymal transition, or angiogenesis.^19,33,34,35Despite the multiple essential functions of TGF-β, a single dose or short-term administration of a pan-TGF-β neutralizing antibody is reportedly well tolerated at doses that inhibit organ fibrosis or experimental carcinoma cell growth and metastasis, with no reported side effects in adult mice and rats. This treatment has shown therapeutic efficacy in inhibiting experimental fibrosis.^{27,28,36,37,38,39,40} Because of these promising results, single-dose phase I/II clinical trials using neutralizing pan-TGF-β antibodies have been performed or are ongoing for metastatic renal cell carcinoma, melanoma, focal segmental glomerulosclerosis, and idiopathic pulmonary fibrosis (Genzyme Corporation, http://www.genzymeclinicalresearch.com, last accessed August 27, 2009). However, it is likely that long-term global inhibition of TGF-β will have undesirable side effects, because targeted deletion of TGF-β signaling in various cell types may lead to accelerated atherosclerosis, autoimmunity, or carcinoma development.^9,12,41 Clearly, careful targeting of the TGF-β pathway to minimize systemic effects is a highly desirable goal.     

A Key Role for the Integrin α2β1 in Experimental and Developmental Angiogenesis

The α2β1 integrin receptor plays a key role in angiogenesis. Here we investigated the effects of small molecule inhibitors (SMIs) designed to disrupt integrin α2 I or β1 I-like domain function on angiogenesis. In unchallenged endothelial cells, fibrillar collagen induced robust capillary morphogenesis. In contrast, tube formation was significantly reduced by SMI496, a β1 I-like domain inhibitor and by function-blocking anti-α2β1 but not -α1β1 antibodies. Endothelial cells bound fluorescein-labeled collagen I fibrils, an interaction specifically inhibited by SMI496. Moreover, SMI496 caused cell retraction and cytoskeletal collapse of endothelial cells as well as delayed endothelial cell wound healing. SMI activities were examined in vivo by supplementing the growth medium of zebrafish embryos expressing green fluorescent protein under the control of the vascular endothelial growth factor receptor-2 promoter. SMI496, but not a control compound, interfered with angiogenesis in vivo by reversibly inhibiting sprouting from the axial vessels. We further characterized zebrafish α2 integrin and discovered that this integrin is highly conserved, especially the I domain. Notably, a similar vascular phenotype was induced by morpholino-mediated knockdown of the integrin α2 subunit. By live videomicroscopy, we confirmed that the vessels were largely nonfunctional in the absence of α2β1 integrin. Collectively, our results provide strong biochemical and genetic evidence of a central role for α2β1 integrin in experimental and developmental angiogenesis.Angiogenesis is the formation of new capillaries from pre-existing blood vessels and is essential for human development, wound healing, and tissue regeneration.¹ Angiogenesis is dependent on interactions of endothelial cells with growth factors and extracellular matrix components.^2,3 Endothelial cell-collagen interactions are thought to play a role in angiogenesis in vivo and in vitro and require the function of the α1β1 and α2β1 integrins,³ two receptors known to cross talk.⁴ Thus, vascular endothelial growth factor (VEGF)-induced angiogenesis in Matrigel plugs implanted in mice is markedly inhibited by anti-α1β1 and -α2β1 integrin antibodies.^5,6 Studies using various collagen-induced angiogenesis assays also suggest a critical role for endothelial cell α2β1 integrin^2,7,8 binding to the GFPGER_502–507 sequence of the collagen triple helix.⁹ Consistent with these findings, endorepellin, a potent anti-angiogenic molecule derived from the C terminus of perlecan^10,11 disrupts α2β1 integrin function,^{12,13,14,15,16} and some of the affected gene products have been associated with the integrin-mediated angiogenesis.¹⁷ Endothelial cell-collagen interactions may also contribute to tumor-associated angiogenesis.¹⁸ For example, gene products up-regulated in tumor-associated endothelial cells include types I, III, and VI collagens,¹⁹ and tumor-associated angiogenesis is sensitive to endorepellin treatment.^15,20,21Interestingly, α2β1 integrin-null mice show no overt alteration in either vasculogenesis or angiogenesis but display only a mild platelet dysfunction phenotype and altered branching morphogenesis of the mammary glands.^22,23 This observation suggests that in mammals, there is functional compensation during development, but that α2β1 integrin might be required for postnatal angiogenesis. Indeed, when adult α2β1-null mice are experimentally challenged, they show an enhanced angiogenic response during wound healing²⁴ and tumor xenograft development.^15,25The α1β1 and α2β1 integrins include inserted domains (I domains) in their α subunits that mediate ligand binding.^26,27 The α2 I domain is composed of a Rossman fold and a metal ion coordination site (MIDAS), proposed to ligate the GFPGER_502–507 sequence of collagen, thereby inducing receptor activation.^26,28 Other integrin domains may also play a role in ligand binding and receptor activation. For example, the β1 I-like domain seems to allosterically modulate collagen ligation by the α2 I domain, and, intracellularly, the cytoplasmic sequence of the α2 subunit functions as a hinge, locking the receptor in an inactive conformation, and membrane-soluble peptide mimetics of this sequence were shown to promote α2β1 receptor activation.²⁹ Recently, a family of small molecule inhibitors (SMIs)² targeting the function of the α2β1 integrin were designed.³⁰ Specifically, inhibitors of α2β1 integrin function were prepared using modular synthesis, enabling substitutions of arylamide scaffold backbones with various functional groups, creating SMIs targeted to the I domain or the intact integrin.^30,31,32 In this study, we tested the activities of a group of SMIs on endothelial cell-collagen interactions and angiogenesis in vitro and in vivo. We provide evidence that SMI496, which binds between the I domains of β1 and α2 subunits,³² interferes with α2β1 integrin activity on endothelial cells both in vitro and in vivo, suggesting a potential therapeutic modality to interfere with angiogenesis. Moreover, interference with α2 integrin expression in embryonic zebrafish caused a vascular phenotype characterized by abnormal angiogenesis.     

Activation of Bovine Lymphocyte Subpopulations by Staphylococcal Enterotoxin C

Staphylococcus aureus is a major mastitis-causing pathogen in cattle. The chronic nature of bovine staphylococcal mastitis suggests that some products or components of S. aureus may interfere with the development of protective immunity. One class of molecules that could be involved are superantigens (SAgs). Although a significant number of mastitis isolates produce SAgs, the effect of these molecules on the bovine immune system is unresolved. To determine if immunosuppression caused by SAgs could play a role in pathogenesis, we monitored bovine lymphocytes exposed to staphylococcal enterotoxin C1 (SEC1). Activation of bovine lymphocytes by either SEC1 or concanavalin A (ConA) was influenced by the γδ/αβ T-cell ratio in the culture. Compared to ConA-induced stimulation, cultures stimulated with SEC1 generated small numbers of CD4⁺ αβ T cells expressing high levels of interleukin-2 receptor α chain (IL-2Rα) and major histocompatibility complex class II (MHCII), suggesting that SAg exposure does not lead to full activation of these cells. This state of partial activation was most pronounced in cultures with a high γδ/αβ ratio. In contrast, significant numbers of CD8⁺ αβ T cells expressed high levels of IL-2Rα and MHCII, regardless of the γδ/αβ ratio and the stimulant used. CD8⁺ blasts in cultures stimulated with SEC1 also expressed another activation marker, ACT3, previously detected predominantly on thymocytes and CD4⁺ T cells. Although γδ CD2⁻ and CD2⁺ T cells expressed MHCII and IL-2Rα following stimulation with SEC1, only a few cells increased to blast size, suggesting that they were only partially activated. The results suggest ways in which SAgs might facilitate immunosuppression that promotes the persistence of bacteria in cattle and contributes to chronic intramammary infection.Staphylococcus aureus is a prominent pathogen in bovine mastitis (24). This organism is frequently isolated from milk (2, 16, 40) and from cows with intramammary infection (IMI) (17). IMI caused by S. aureus tends to become chronic and may resist antibiotic therapy (49). It has been postulated that persistent infection with S. aureus is associated with an impairment of the immune response, mediated by factors produced by S. aureus (34). Thus far, however, no single factor has been clearly implicated.Bovine isolates of S. aureus frequently produce one or more pyrogenic toxins (PTs), especially types C and D staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin (24). The staphylococcal PTs are prototype microbial superantigens (SAgs), characterized by the ability to bind to major histocompatibility complex class II (MHCII) molecules and to specific Vβ segments of αβ T-cell receptor (TCR) outside the binding groove associated with MHC-restricted immune system recognition of processed peptides. By bypassing antigenic specificity, SAgs stimulate abnormally large numbers of T cells and are able, at nanomolar concentrations, to induce T-cell proliferation (33). Few studies have been performed to investigate the effects of SAgs on the bovine immune system (53); most studies have involved other animals. In several species, SAgs exert wide-ranging and deleterious effects, including induction of shock (4), T-cell unresponsiveness and deletion (23), differential stimulation of CD4⁺ and CD8⁺ T-cell subsets (47), and B-cell differentiation (46). Thus, although largely unconfirmed, there is a clear potential for SEs, and other SAgs, to modulate immune responses and contribute to the virulence and persistence of S. aureus in cattle.The T-cell population consists of cells expressing either the αβ TCR (TCR2) or the γδ TCR (TCR1). While the roles of αβ T cells in immune responses of many species have been well characterized, the function of γδ T cells is less well understood (22). This is especially true in ruminants. Recent investigations have shown that the ruminant γδ T cells comprise two disparate subpopulations, characterized by constitutive expression of cell surface molecules. One subpopulation, similar in composition and tissue distribution to γδ T cells from other species, consists of cells that express CD2, CD5, and CD6 and are positive or negative for CD8 (10). These cells are present in low concentrations (3 to 5%) in peripheral blood and in high concentrations (35 to 40%) in spleen, gut epithelium, and mammary gland secretions (41). The second subpopulation, negative for CD2, CD6, and CD8, is unique and has been identified in only one other member of the Artiodactyla, swine (3, 28). This subpopulation is positive for CD5 and two lineage-restricted molecules, workshop cluster 1 (WC1) (32, 36, 50) and GD3.5 (21). The concentration of WC1⁺ GD3.5⁺ CD2⁻ CD6⁻ γδ T cells is high (30 to 50%) in the peripheral blood in young ruminants, decreasing with age, and is low (3 to 8%) in secondary lymphoid tissues and mammary gland secretions (41, 52). Definitive data on the function of either of these major subpopulations of γδ T cells have not been obtained. However, previous studies have suggested that they may be involved in regulating the proliferative response of CD4⁺ T cells to antigens (7).Park et al. (42) identified a subset of T cells, positive for CD2 and CD8, in mammary gland secretions of cows infected with S. aureus. These cells had the ability to inhibit the proliferative response of bovine CD4⁺ cells to staphylococcal antigens (42). Although the mechanisms by which these cells were induced and mediated their effect were not determined, they clearly have the potential to contribute to the pathogenesis of staphylococcal IMI. Although not all bovine staphylococcal isolates produce known SAgs, it is important to determine whether SAg production could induce these or other immunosuppressive subpopulations in cows and promote the development of some infections such as IMI. The objective of this study was to extend these initial observations. We examined the effect of a representative SAg (SEC1) on the major subpopulations of bovine αβ and γδ T cells.     

Toll-Like Receptor 3 Ligand Dampens Liver Inflammation by Stimulating Vα14 Invariant Natural Killer T Cells to Negatively Regulate γδT Cells

Vα14 invariant natural killer T (Vα14iNKT) cells are at the interface between the innate and adaptive immune responses and are thus critical for providing full engagement of host defense. We investigated the role of polyriboinosinic:polycytidylic acid (poly I:C), a replication-competent viral double-stranded RNA mimic and a specific agonist that recognizes the cellular sensor Toll-like receptor 3 (TLR3), in regulating Vα14iNKT cell activation. We established for the first time that hepatic Vα14iNKT cells up-regulate TLR3 extracellularly after poly I:C treatment. Notably, activation of TLR3-expressing hepatic Vα14iNKT cells by a TLR3 ligand was suppressed by TLR3 deficiency. Our studies also revealed that Vα14iNKT cell activation in response to poly I:C administration uniquely suppressed the accumulation and activation of intrahepatic γδT cells (but not natural killer cells) by inducing apoptosis. Furthermore, we established that activated hepatic Vα14iNKT cells (via cytokines and possibly reactive oxygen species) influenced the frequency and absolute number of intrahepatic γδT cells, as evidenced by increased hepatic γδT cell accumulation in Vα14iNKT cell-deficient mice after poly I:C treatment relative to wild-type mice. Thus, hepatic Vα14iNKT cells and intrahepatic γδT cells are functionally linked on application of TLR3 agonist. Overall, our results demonstrate a novel and previously unrecognized anti-inflammatory role for activated hepatic Vα14iNKT cells in negatively regulating intrahepatic γδT cell accumulation (probably through TLR3 signaling) and thereby preventing potentially harmful activation of intrahepatic γδT cells.Vα14 invariant natural killer T (Vα14iNKT) cells are thymic-derived innate murine T lymphocytes with significant immunoregulatory effects in cardiovascular, infectious, and autoimmune diseases as well as in tumors.^{1,2,3,4,5,6,7,8,9,10} In contrast to conventional T cells, which recognize peptide antigens presented by major histocompatability class I and II molecules, Vα14iNKT cells respond to glycolipid antigen presented by CD1d expressed on antigen-presenting cells.^11,12 In the last decade, several potential mechanisms underlying Vα14iNKT cell activation during immune responses have been revealed. Vα14iNKT cells are activated by lipids presented by CD1d.^5,13 The established dogma is that the lipid tail of glycolipid antigen (including α-galactosylceramide or exogenous antigens from pathogens) is buried in CD1d, whereas the sugar head group of glycolipid antigen protrudes out of the CD1d to activate the T-cell receptor (TCR) α on the Vα14iNKT cell.^2,13,14 After activation, Vα14iNKT cells exert multiple effects including the production of several cytokines (such as interferon [IFN]-γ, interleukin [IL]-4, and tumor necrosis factor [TNF]-α), chemokines (regulated on activation normal T cell expressed and secreted/CCL5, monocyte chemotactic protein-1/CCL2, and macrophage inflammatory protein-1α/CCL3)^15,16,17,18 and cytotoxic proteins (such as tumor necrosis factor-related apoptosis-inducing ligand and Fas/Fas ligand).^13,19 Through these mediators, activated Vα14iNKT cells can interact with and transactivate other immune cells.^15,20,21 Thus, Vα14iNKT cells act as a “bridge” between the innate and adaptive immune systems.Vα14iNKT cells are also activated by a TCR-independent mechanism involving Toll like receptors (TLRs). TLRs are pathogen recognition receptors that identify molecular patterns of components specific to microbes and play a critical role in initiating the innate immune response to microbes.²² To date, more than 10 TLRs have been reported in humans and mice, and each recognizes different microbial components.²² TLRs are located on the plasma membrane and in endosomal compartments of cells.²² Among the TLRs, TLR2, TLR4, and TLR5 recognize the bacterial signals peptidoglycan, lipopolysaccharide, and flagellin, respectively,²² whereas TLR3, TLR7, TLR8, and TLR9 play fundamental roles in detecting viral signals.^23,24,25,26 An additional mechanism for activation of Vα14iNKT cells (in the absence of foreign antigen for their TCRs) is by IL-12 and/or IL-18 derived from antigen-presenting cells that have been activated via a TLR (4,7,8,9)-dependent pathway.^13,14,27,28 The precise pathway of activation may depend on the pathogen. For example, TLR4 traditionally recognizes the bacterial signal lipopolysaccharide,²² whereas TLR7, TLR8, and TLR9 all sense viral signals.^23,24,25,26 The potential contribution of the viral sensor TLR3 to Vα14iNKT cell activation has not yet been determined. Poly I:C is the specific TLR3 agonist and a replication-competent viral double-stranded RNA (dsRNA) mimic.^23,29 dsRNA is a structure found in the genome of some viruses and is produced as a replication intermediate by viruses.^23,29,30 Therefore, poly I:C is routinely used in experimental studies to assess the functional activity of TLR3 during immune responses.^23,29,30,31In the present study, we evaluated the potential role of TLR3 in promoting Vα14iNKT cell activation by examining the response of hepatic Vα14iNKT cells after treatment with the TLR3 ligand, poly I:C. In addition, we assessed the functional consequences of Vα14iNKT cell activation on the hepatic innate immune response after poly I:C treatment. We demonstrate that a functional consequence of hepatic Vα14iNKT cell activation in response to poly I:C administration is the subsequent induction of apoptotic death of hepatic γδT cells. Overall, our findings demonstrate a novel role for activated hepatic Vα14iNKT cells in negatively regulating the recruitment, activation, and potentially harmful effector function(s) of intrahepatic γδT cells on application of the TLR3 ligand, poly I:C.     

Lysophosphatidic Acid Induces αvβ6 Integrin-Mediated TGF-β Activation via the LPA2 Receptor and the Small G Protein Gαq

Activation of latent transforming growth factor β (TGF-β) by αvβ6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes αvβ6 integrin-mediated TGF-β activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce αvβ6 integrin-mediated TGF-β activation is unknown; in addition, the αvβ6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces αvβ6-mediated TGF-β activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced αvβ6 integrin-mediated TGF-β activity is mediated via the LPA2 receptor, which signals via Gα_q. Finally, we show that the expression levels of both the LPA2 receptor and αvβ6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and αvβ6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces αvβ6 integrin-mediated TGF-β activation in epithelial cells via LPA2, Gα_q, RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.Transforming growth factor (TGF)-β includes a pleiotropic group of cytokines that exist in three mammalian isoforms (TGF-β1, -β2, and -β3) that are all secreted as latent complexes. This latent complex needs to be activated for TGF-β family members to exert their biological effect. The small latent complex contains the latency associated peptide (LAP), which, in TGF-β1 and TGF-β3, contains an arginine-glycine-aspartate (RGD) motif. This RGD motif can bind integrins, facilitating TGF-β activation. The LAP of TGF-β2 does not contain an RGD motif and no role for integrin mediated TGF-β2 activation has been described. TGF-β1 exerts profound effects on matrix deposition and is a central mediator of lung injury and fibrosis. There are several mechanisms by which TGF-β1 may be activated, including extremes of heat, oxidation, proteolytic cleavage, deglycosylation, and activation by thrombospondin-1.^{1,2,3,4,5,6,7,8} In vivo, activation by integrins appears to play a major role in activating TGF-β1 during development⁹ and in various disease models.^{10,11,12,13,14}Integrins are heterodimeric transmembrane proteins made up of α and β subunits. Six, of the 24 currently described integrins are able to bind the RGD motif in the LAP of TGF-β. Four of these integrins (αvβ3, αvβ5, αvβ6, and αvβ8) are thought to be able to activate TGF-β1.^13,14,15 The role of integrin-mediated TGF-β activation in vivo has only been confirmed for the αvβ6 and αvβ8 integrins.^13,14 Mice in which the aspartic acid in the RGD site of TGF-β1 is replaced by glutamic acid, preventing integrin-mediated TGF-β1 activation, completely phenocopy TGF-β1 null mice, highlighting the importance of TGF-β1 interactions with integrins.⁹ Furthermore, activation of TGF-β1 by the epithelially restricted αvβ6 integrin is central to the pathogenesis of acute lung injury and pulmonary fibrosis.^12,14Further regulation of TGF-β bioavailability is afforded by interaction of the small latent complex with the latent TGF-β binding proteins (LTBPs). There are four LTBPs (1, 2, 3, and 4) that belong to the LTBP/fibrillin family of extracellular glycoproteins. Of these, three, LTBP-1, -3, and -4, associate with the small latent complex through covalent attachment with the LAP, forming the large latent complex.¹⁶ The LTBPs are required to ensure correct post-translational modification of the small latent complex,¹⁷ and they target storage of TGF-β in the extracellular matrix by crosslinking the large latent complex to the matrix via the actions of tissue transglutaminase.^18,19 The LTBPs are also likely to determine, at least in part, the specificity of TGF-β activation. LTBPs-1 and -3 can bind all isoforms of TGF-β, whereas LTBP-4 can only bind TGF-β1.^16,20 There is further evidence of the importance of LTBP modulating TGF-β activation from in vivo studies using mice null for various LTBPs. LTBP-1 null mice have reduced TGF-β activity and are protected from hepatic fibrosis.²¹ LTBP-3 null mice have phenotypic features consistent with reduced TGF-β activity in the bones.²² Mice with a gene trap disruption of LTBP-4 show reduced epithelial Smad2 phosphorylation and abnormal cardiopulmonary development and develop colonic tumors similar to those seen in Smad3 null mice.²³Overexpression of the αvβ6 integrin is not sufficient to promote fibrosis and the αvβ6 integrin itself must be activated during injury to promote TGF-β1 activation.^12,24 αvβ6-dependent TGF-β activation also requires an intact actin cytoskeleton¹⁴ and is critically dependent on association of latent complexes with the specific LTBP family member, LTBP-1.²⁵ In cells lacking LTBP-1, αvβ6 cannot activate TGF-β, but this response can be rescued by expression of a short fusion protein composed of the region of LTBP-1 that forms a disulfide bond to TGF-β1 LAP and the region required to cross-link LTBP-1 to the extracellular matrix protein fibronectin.²⁵ These findings are consistent with a model by which the integrin-expressing cell activates latent TGF-β by exerting traction on the tethered latent complex. Such a mechanism received further support from a recent report that myofibroblasts can activate latent TGF-β using the αvβ5 integrin and cell contraction.²⁶ It is thought that cellular injury may induce contractile forces through the cytoskeleton and integrins to the latent TGF-β1 molecule, which is itself tethered by the LTBP-1 to either the cell surface or the extracellular matrix.^25,26 We identified that agonists of the seven-transmembrane domain, G protein-coupled receptor (GPCR), PAR1, can promote αvβ6 integrin-mediated TGF-β activation via RhoA, known to modulate cell contraction, and that this pathway is important in acute lung injury.¹² However, whether other GPCRs are able to contribute to αvβ6 integrin-mediated, TGF-β activation is not known. Furthermore, the G protein involved in mediating injury-induced αvβ6 integrin-mediated, TGF-β activation has not yet been identified.Lysophosphatidic acid (LPA), is a bioactive phospholipid known to mediate contraction in a number of cell types.²⁷ It is released from activated platelets at sites of injury,²⁸ contributing to wound repair. LPA is present in bronchoalveolar lavage fluid and increased during inflammation,²⁹ and can mediate pro-inflammatory effects on several cells types within the lung.^30,31,32 Furthermore, LPA is increased in patients with pulmonary fibrosis.³³ LPA mediates its cellular effects via the LPA class of GPCRs. Currently five subtypes have been identified (LPA1-5), and like PAR1, these receptors couple to the small G proteins Gα_i, Gα_q, and Gα_12/13.^34,35 It has recently been shown that LPA can induce fibroblast chemotaxis through an LPA1-dependent mechanism, and this could be important in the pathogenesis of pulmonary fibrosis.³³The purpose of the present study was to investigate whether LPA can activate TGF-β in epithelial cells via the αvβ6 integrin and to dissect the proximal signaling pathway from the relevant LPA receptor to RhoA. We demonstrate that the LPA2 receptor is the predominant receptor for transducing LPA-induced, αvβ6 integrin-mediated, TGF-β activation and that this pathway is coupled to the G protein, Gα_q. Finally we provide evidence that both the αvβ6 integrin and LPA2 receptor are induced in epithelial cells overlying areas of pulmonary fibrosis in the lungs of mice treated with intratracheal bleomycin and in samples from patients with idiopathic pulmonary fibrosis.     

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 β in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to α4-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 α4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin α4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans.Laminin (LN) is a large, heterotrimeric, cruciform molecule composed of α, β, and γ subunits.¹ Five distinct α (LAMA1-5), 3 β (LAMB1-3), and 3 γ (LAMC1-3) chains^1,2 variably assemble to create distinct isoforms³ that are temporally and spatially regulated, and each conveys a variety of biological functions.^{4,5,6,7,8,9,10,11} The LNα4-containing isoforms, LN411 (α4β1γ1) and LN421 (α4β2γ1), are abundant in microvessels. Studies of LNα4-deficient mutant mice (lama4−/−) reveal that although α4-LNs are not required for blood vessel formation, they play important roles in blood vessel maturation, and in stabilization of vessels that form with injury, inflammation and tumor growth.^7,12,13 In vitro studies indicate that α4LNs directly regulate endothelial cell proliferation and inhibit apoptosis.¹⁴ α4-LNs are produced by endothelial cells in most microvessels; however, endothelial cells in the renal glomerulus do not express LNα4-containing isoforms.¹⁵ Instead, LN411 and LN421 at the endothelial-mesangial interface are produced by the mesangial cells (MCs).¹⁵ Platelet-derived growth factor (PDGF) is the primary growth factor responsible for MC proliferation and migration during glomerulogenesis,¹⁶ and we have shown that PDGF-induced MC migration requires LNα4.¹⁵ This function could not be replaced by LN111 or LN511/521.¹⁵ Together these observations suggested the possibility that deficiency of LNα4 might impair the ability of the kidney microvasculature to mature or be repaired in lama4−/− adult mice, resulting in kidney disease despite normal initial development.Previous reports have documented a spectrum of developmental defects and tissue maintenance defects in lama4−/− mice. Early postal-natal hemorrhage from birth-related trauma to fragile blood vessels occurs in lama4−/− mice; yet, by three-weeks of age, accumulation of LNα5 stabilizes vessels, although they remain dilated.¹² Vessel fragility recurs when new vessels form in response to injury.¹² The heart forms normally, but lama4−/− mice develop cardiomyopathy with time.¹⁷ Neurological dysfunction occurs in lama4−/− mice, through independent defects in organizing presynaptic specializations at neuromuscular synapses,¹⁸ and in the ability of developing Schwann cells to properly sort and myelinate.^19,20 This report details the characteristics of kidney abnormalities, including the development of glomerulosclerosis and tubulointerstitial fibrosis over time in lama4−/− mice.     

ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing

Wound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1^−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1^−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1^−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium.Intercellular adhesion molecule-1 (ICAM-1, CD54)¹ is a conserved member of the immunoglobulin supergene family² and is expressed by many cell types in response to stimuli such as cytokines,^3,4 and oxidative and physical stress.^5,6 It has been extensively studied in the context of adhesion and transmigration of leukocytes through endothelium⁷ and epithelium,^8,9 and it also serves as an adhesive ligand for leukocyte-mediated cytotoxic activity.^9,10,11 ICAM-1 is recognized by members of the β2 (CD18) integrin family, especially lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18),¹² and this adhesion is critical to many of the migratory and cytotoxic events in which ICAM-1 participates.^7,10,11 ICAM-1 also functions as a signaling molecule, dependent on its cytoplasmic tail interacting with cytoskeletal elements.⁷ This capability influences functions such as leukocyte transendothelial migration⁷ and vascular permeability.¹³Of importance to the current study is the fact that ICAM-1 can be expressed by corneal epithelial cells and limbal vessel endothelial cells.^{14,15,16,17,18,19,20,21} It appears to be expressed in conditions associated with inflammation, but its role in this context is poorly understood, especially its expression by the epithelial cells. Using a murine model of central corneal epithelial abrasion, we observed ICAM-1 on corneal epithelial cells in the periphery of the cornea, a region not directly injured by the abrasion.¹⁴ Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,^22,23 it was of interest to determine whether ICAM-1 is necessary for these processes. To this end we studied wound healing in mice that do not express ICAM-1.^24,25As a part of this evaluation, we focused attention on γδ T cells. We observed in earlier studies that epithelial expression of ICAM-1 occurred at a time when γδ T cells increased within the corneal epithelium,^14,26 and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,²⁷ and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,^20,27 it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1.     

A Protein Kinase Cδ-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cδ-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCδ−/− mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCδ-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCδ, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCδ-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.Asbestos is a group of naturally occurring mineral fibers that are linked to the development of lung cancer, mesothelioma, and pleural and pulmonary fibrosis, ie, asbestosis.^1,2 The mechanisms leading to asbestos-related diseases are still unclear, but oxidative stress due to phagocytosis of longer fibers, iron-driven generation of oxidants from fiber surfaces, and depletion of cellular antioxidants are linked to cell injury and inflammation.^3,4,5,6Bronchiolar and alveolar type II epithelial cells, which first encounter asbestos fibers after inhalation, are key cell types in asbestos-associated inflammation and fibroproliferation.² Initial cell reactions to asbestos include epithelial cell injury, ie, apoptosis and necrosis,^5,6 which may lead to compensatory cell proliferation^7,8 and the production of inflammatory and fibrogenic cytokines.^8,9,10 Asbestos-induced signaling mechanisms governing these cell responses appear to involve a broad variety of cascades including the mitogen-activated protein kinases (MAPK),^3,7,11,12 nuclear factor-κB (NF-κB),^9,13,14 and the protein kinase (PK)C^10,12,15,16 and A families.¹⁷A critical signaling protein involved in asbestos signaling is PKCδ, which is known to be activated in bronchiolar and alveolar epithelial cells in vivo and in vitro^10,12,16 via increased formation of diacylglycerol.¹⁸ We have shown that PKCδ governs apoptosis via an oxidant-dependent mitochondrial pathway after exposure of lung epithelial cells to asbestos fibers.¹⁶ Recent studies comparing PKCδ +/+ and PKCδ −/− mice also reveal an important role of PKCδ in metalloproteinase expression as well as cytokine production in vitro and in vivo.^10,15 A variety of other studies also link PKCδ to either pro-apoptotic or anti-apoptotic events depending on the stimulus and cell type.^19,20In this study, we focused on PKD as a potential link between PKCδ, activation of MAPKs and downstream repercussions such as expression of fos/jun proto-oncogenes and apoptosis in asbestos-exposed lung epithelium. PKD is a serine/threonine protein kinase classified as a subfamily of the Ca²⁺/calmodulin-dependent kinase superfamily.²¹ PKD1, which includes mouse PKD and its human homolog PKCμ, is the most extensively studied PKD.²² The other two members of this family include PKD2²³ and PKD3, (originally PKCν).²⁴ Conserved regions of PKDs include a phosphorylation-dependent catalytic domain, a pleckstrin-homology domain that inhibits the catalytic activity, and cysteine-rich motifs that recruit PKD to the plasma membrane. PKCδ is proposed to interact with the pleckstrin-homology domain of PKD, transphosphorylating its activation loop at Ser744 and Ser748, and leading to PKD activation.²⁵ In addition, PKD can be activated through the Src-Abl pathway by tyrosine phosphorylation of Tyr463 (T463) in the pleckstrin-homology domain after oxidative stress,²⁶ as well as by caspase-mediated proteolytic cleavage 27 and by bone morphogenetic protein 2.²⁸ Downstream targets of PKD signaling include several important signaling molecules such as ERK1/2, JNK1/2, and NF-κB,^21,26,29,30 but how these affect functional ramifications of carcinogens, such as asbestos, are unclear.The BH3-only protein, Bim, is a pro-apoptotic member of the Bcl-2 family that links stress-induced signals to the core apoptotic machinery.^31,32 There are three different splice variants of the Bim gene encoding short, long, and extra-long Bim proteins (BimS, BimL, and BimEL).³³ BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptosis proteins,³⁴ whereas BimL is bound to microtubules and is less cytotoxic.³⁵ Disruption of BimL binding to microtubules via JNK-dependent phosphorylation can cause its redistribution to the mitochondria and induction of pro-apoptotic machinery.³⁶ BimEL is post-translationally regulated by ERK1/2, which promotes its phosphorylation and rapid dissociation from Mcl-1 and Bcl-x(L)³⁷ and proteasomal degradation.³⁸We reveal here that PKD is involved in multiple signaling events after asbestos inhalation and in vitro. Specifically, PKD is a downstream effector of PKCδ and modulates phosphorylation of both ERK1/2 and JNK1/2 in lung epithelial cells after asbestos exposure. Our data also suggest that PKD inhibits apoptosis through an ERK1/2-mediated destabilization of the pro-apoptotic BH3-only protein, BimEL. The fact that PKD is an important signaling molecule in MAPK signaling and survival after cell injury by asbestos may have important therapeutic implications in asbestos-related diseases.     

Amyloid-Peptide Vaccinations Reduce β-Amyloid Plaques but Exacerbate Vascular Deposition and Inflammation in the Retina of Alzheimer’s Transgenic Mice

Alzheimer’s disease (AD) is pathologically characterized by accumulation of β-amyloid (Aβ) protein deposits and/or neurofibrillary tangles in association with progressive cognitive deficits. Although numerous studies have demonstrated a relationship between brain pathology and AD progression, the Alzheimer’s pathological hallmarks have not been found in the AD retina. A recent report showed Aβ plaques in the retinas of APPswe/PS1ΔE9 transgenic mice. We now report the detection of Aβ plaques with increased retinal microvascular deposition of Aβ and neuroinflammation in Tg2576 mouse retinas. The majority of Aβ-immunoreactive plaques were detected from the ganglion cell layer to the inner plexiform layer, and some plaques were observed in the outer nuclear layer, photoreceptor outer segment, and optic nerve. Hyperphosphorylated tau was labeled in the corresponding areas of the Aβ plaques in adjacent sections. Although Aβ vaccinations reduced retinal Aβ deposits, there was a marked increase in retinal microvascular Aβ deposition as well as local neuroinflammation manifested by microglial infiltration and astrogliosis linked with disruption of the retinal organization. These results provide evidence to support further investigation of the use of retinal imaging to diagnose AD and to monitor disease activity.Cerebral abnormalities including neuronal loss, neurofibrillary tangles, senile plaques with aggregated β-amyloid protein (Aβ) deposits, microvascular deposition of Aβ, and inflammation are well-known pathological hallmarks of Alzheimer’s disease (AD).^1,2,3 Despite the controversial evidence about the contribution of Aβ to the development of AD-related cognitive deficits, accumulation of toxic, aggregated forms of Aβ plays a crucial role in the pathogenesis of familial types of AD.^4,5 Overexpression of amyloid precursor protein (APP) in trisomy 21, altered APP processing resulting from mutations in APP, presenilin 1 (PS1), or 2 (PS2), and, as-of-yet unidentified other familial AD, related mutations, lead to Aβ deposition and Aβ plaques in the brain as well as cognitive abnormalities.^6,7 Therefore, to understand the molecular basis of amyloid protein deposition and to detect Aβ plaques in brain, parenchyma ante-mortem are currently among the most active areas of research in AD.Besides cognitive abnormalities, patients with AD commonly complain of visual anomalies, in particular, related to color vision,^8,9 spatial contrast sensitivity,¹⁰ backward masking,¹¹ visual fields,¹² and other visual performance tasks.^13,14,15,16 In addition to the damage and malfunction in the central visual pathways, retinal abnormalities such as ganglion cell degeneration,¹⁷ decreased thickness of the retinal nerve fiber layer,^18,19 and optic nerve degeneration^20,21 may, in part, account for AD-related visual dysfunction. Although intracellular Aβ deposition has been detected in both ganglion and lens fiber cells of patients with glaucoma, AD, or Down’s syndrome,^22,23,24,25 other typical hallmarks of AD have not yet been demonstrated. Interestingly, thioflavine-S-positive Aβ plaques were recently found in the retinal strata of APPswe/PS1ΔE9 transgenic mice²⁶ but not in the other animal models of AD. The current study used Tg2576 mice that constitutively overexpress APPswe and develop robust Aβ deposits in brain as well as cognitive abnormalities with aging.²⁷ We assessed the pathological changes in the retina of aged mice following different immunization schemes. We immunized Tg2576 with fibrillar Aβ42 and with a prefibrillar oligomer mimetic that gives rise to a prefibrillar oligomer-specific immune response. Both types of immunogens have been shown to be equally effective in reducing plaque deposition and inflammation in Tg2576 mouse brains.²⁸ In this study, we also used another prefibrillar oligomer mimetic antigen that uses the islet amyloid polypeptide (IAPP) instead of Aβ, but which gives rise to the same generic prefibrillar oligomer-specific immune response that also recognizes Aβ prefibrillar oligomers.²⁹ Aβ plaques and microvascular Aβ deposition were observed in the control Tg2576 mouse retinas. In contrast, Aβ and IAPP prefibrillar oligomer vaccinations differentially removed retinal Aβ deposits but exacerbated retinal amyloid angiopathy and inflammation as demonstrated by a significantly enhanced microglial infiltration and astrogliosis.     

Neuronal and Axonal Loss Are Selectively Linked to Fibrillar Amyloid-β within Plaques of the Aged Primate Cerebral Cortex

The amyloid-β peptide (Aβ) deposited in plaques in Alzheimer’s disease has been shown to cause degeneration of neurons in experimental paradigms in vivo and in vitro. However, it has been difficult to convincingly demonstrate toxicity of native amyloid deposits in the aged and Alzheimer brains. Here we provide evidence that the fibrillar conformation of Aβ (fAβ) deposited in compact plaques is associated with the pathologies observed in Alzheimer brains. fAβ containing compact but not diffuse plaques in the aged rhesus cortex contained activated microglia and clusters of phosphorylated tau-positive swollen neurites. Scholl’s quantitative analysis revealed that the area adjacent to fAβ, containing compact but not diffuse plaques in aged rhesus, aged human, and Alzheimer’s disease cortex, displays significant loss of neurons and small but statistically significant reduction in the density of cholinergic axons. These observations suggest that fAβ toxicity may not be restricted to cultured cells and animal injection models. Rather, fAβ deposited in native compact plaques in aged and AD brains may exert selective toxic effects on its surrounding neural environment.A large body of evidence supports a central role for the amyloid-β peptide (Aβ) in the pathogenesis of Alzheimer’s disease (AD).¹ Deposition of Aβ in plaques represents a signature pathological hallmark of AD. Aβ can exist in various physical conformations, including soluble oligomers (oAβ), protofibrils (pfAβ), nonfibrillar insoluble oligomers, and fibrillar (fAβ) forms.^2,3,4,5,6 Aβ immunoreactive plaques are of two primary types: diffuse and compact.^7,8 Diffuse plaques contain primarily nonfibrillar insoluble oligomers of Aβ. Compact plaques, on the other hand, are composed of fAβ and have consistently been found to contain significantly larger numbers of activated microglia than adjacent brain regions and to be surrounded by astrocytes.^{9,10,11,12,13} They also contain abnormal neurites (neuritic plaques), which represent dystrophic processes of neurons and are composed of abnormally phosphorylated tau.^9,14 Importantly, the presence of the compact neuritic variety of plaques that contain fAβ and activated microglia appears to be a relatively specific feature of AD, and its density is used for the pathological diagnosis of the disease.^9,15 Moreover, a strong correlation has been reported between the density of neuritic plaques and severity of dementia.^16,17The neuritic pathology associated with fAβ is suggestive of the toxic effects of this conformation of the peptide on neurons and their processes. In fact, in vitro and in vivo evidence indicates that fAβ exerts powerful toxic effects on neurons.^18,19,20 We have shown that the aged primate cerebral cortex is selectively vulnerable to fAβ toxicity.²¹ Injections of plaque equivalent concentrations (200 pg) of fAβ into the cerebral cortex of aged rhesus or mormoset monkeys produced significant neuronal loss and induced hyperphosphorylation of tau, both features of the AD brain. Recent in vitro evidence indicates that oAβ, which are believed to form before deposition of Aβ in plaques and are likely to interfere with synaptic function^22,23 and inhibit fast axonal transport,²⁴ may also lead to neuronal degeneration.^25,26,27,28 However, although there is abundant evidence demonstrating that oligomeric Aβ interferes with neuronal function, direct in vivo demonstration that this conformation of Aβ causes neuronal death is lacking. An exception is the demonstration of synaptic degeneration associated with intracellular accumulation of Aβ, most likely of the soluble oligomeric variety.^29,30The experimentally observed toxic effects of injected Aβ imply that deposition of this peptide in plaques should also be associated with neuronal damage. However, it has been difficult to convincingly demonstrate neuronal and axonal loss associated with plaques in aged human or AD brains. A number of isolated studies have reported on limited aspects of neuronal and axonal damage in plaques,^31,32,33 sometimes using very small numbers of specimens.³⁴ Furthermore, none of these studies has addressed the potential differences in fAβ and large oligomeric Aβ in inducing toxic effects in plaques. Recently, neuronal loss has been demonstrated within the region occupied by fAβ in plaques.³⁵ However, this phenomenon could be attributed to physical damage to neurons by the space occupying amyloid. If Aβ exerts toxic effects on neurons, neuronal and axonal loss should be observable in the area surrounding the plaque.In the present set of experiments, we provide evidence suggesting that fAβ exerts toxic effects on neurons and axons in the immediate area next to plaques. We first used plaques in the aged rhesus cortex for this purpose, because such plaques exist in an otherwise intact cortical architecture and only a fraction of them contain fAβ. We then extended our observations to normal human brains and AD cases. We report activated microglia, and for the first time the existence of phosphorylated tau in swollen neurites, exclusively associated with fAβ in plaques in the aged rhesus cortex. We also demonstrate significant loss of neurons and of cholinergic axons, which are selectively vulnerable to degeneration in AD,³⁶ in the immediate vicinity of compact plaques containing fAβ but not next to diffuse plaques. The loss of neurons and axons becomes progressively smaller with distance away from such plaques. Additionally, we demonstrate that fAβ containing compact plaques in the aged human and AD brains display significant neuronal loss in their immediate vicinity.     

Age-Related Changes in Pericellular Hyaluronan Organization Leads to Impaired Dermal Fibroblast to Myofibroblast Differentiation

We have previously demonstrated that transforming growth factor-β1 (TGF-β1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-β1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-α-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-β1-dependent induction of α-smooth muscle actin in “young” cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1β stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-β1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-β1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.Chronic skin wounds represent a major, often unrecognized, cause of distress and disability in the elderly population and have been estimated to affect 4% of the UK population older than 65. The morbidity associated with this impaired wound healing is estimated to cost the health service in excess of £1 billion annually in the UK¹ and $9 billion in the USA.² This amount will grow with the increasing age of the population.Wound healing regardless of the etiology of the wound involves overlapping patterns of events including coagulation, inflammation, epithelialization, formation of granulation tissue, and remodeling of the matrix and tissue. Fibroblasts are central to the wound-healing process and when activated, they undergo a number of phenotypic transitions and eventually acquire a contractile “myofibroblastic” phenotype characterized by the expression of α-smooth muscle actin (α-SMA).³ These myofibroblasts are responsible for closure of wounds and for the formation of the collagen-rich scar. In addition, their presence in tissues has been established as a marker of progressive fibrosis.^4,5 The cytokine transforming growth factor-β1 (TGF-β1) is recognized as a mediator of wound healing and its aberrant expression has also been widely implicated in progressive tissue fibrosis.^4,6,7 In addition to its direct effect on extracellular matrix turnover, it is known to drive fibroblast-myofibroblast differentiation and is capable of up-regulating α-SMA in fibroblasts both in vitro and in vivo.^8,9 Deficits in the signal transduction responsiveness to TGF-β1 have been postulated to explain the age-related defects in wound healing seen in the elderly population.¹⁰Hyaluronan (HA) is a ubiquitous connective tissue glycosaminoglycan synthesized by HA synthase (HAS) enzymes, for which three vertebrate genes have been isolated and characterized (HAS1, HAS2, and HAS3).^11,12 It has a role in maintaining matrix stability and tissue hydration. It is known to play a major role in regulating cell-cell adhesion,¹³ migration,^14,15,16 differentiation,¹⁷ and proliferation^18,19 and therefore plays an important role in wound healing. In addition, it is involved in mediating cellular responses to TGF-β1. For example, our recent studies in epithelial cells have demonstrated that HA modulates TGF-β1 signaling after interaction with its receptor, CD44.^20,21We have demonstrated previously that phenotypic conversion of fibroblasts to myofibroblasts is associated with major changes in the production and metabolism of HA.²² Specifically they have been shown to accumulate larger amounts of intracellular and extracellular HA and assemble larger HA pericellular matrices. Furthermore, we demonstrated that HA plays a pivotal role in regulating TGF-β1-driven cellular differentiation in that it facilitates the fibroblast-myofibroblast transition.^23,24Fibroblasts have distinct phenotypes depending on the site from which they are isolated. Using a library of patient-matched oral and dermal fibroblasts we previously demonstrated the relationship between generation of HA and site-specific phenotypic variation.^23,24 An in vitro aging model based on cell senescence was described previously and validated as a model of age-related alterations in human aortic smooth muscle cell function.^25,26 Similarly alterations in fibroblast function in an in vitro model of aging have demonstrated the validity of this model in terms of in vivo age-related alterations in fibroblast motility and mitogenesis, which are associated with age-dependent impaired wound healing.^27,28 The aim of the work in this article was to understand the age-related regulation of HA generation, using this validated in vitro aging model, and determine how this regulation may contribute to age-related impaired wound healing.     

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7^−/−), or dystrophin and utrophin (mdx/utr^−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr^−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr^−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr^−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease that affects 1 in every 3500 live male births. Patients with DMD have impaired mobility, are restricted to a wheelchair by their teens, and die from cardiopulmonary failure in their early twenties.^1,2 Currently, there is no cure or effective treatment for this devastating disease. Mutations in the dystrophin gene resulting in loss of the dystrophin protein are the cause of disease in DMD patients and the mdx mouse model.^3,4,5,6,7The dystrophin glycoprotein complex links laminin in the extracellular matrix to the actin cytoskeleton. The N-terminal region of dystrophin interacts with cytoskeletal F-actin⁸ and the C-terminal region associates with the dystrophin-associated protein complex, which include α- and β-dystroglycan, α- and β-syntrophin, the sarcoglycans, and sarcospan.⁹ In DMD, the absence of dystrophin leads to disruption of the dystrophin glycoprotein complex, resulting in increased muscle fragility and altered cell signaling.⁹ Loss of this critical transmembrane linkage complex in DMD patients and mdx mice results in progressive muscle damage and weakness, inflammation, necrosis, and fibrosis. Lack of dystrophin also leads to abnormalities at myotendinous and neuromuscular junctions (MTJ and NMJ), which further contribute to skeletal muscle damage.^{10,11,12,13,14,15,16,17} In addition, defective muscle repair in DMD patients eventually results in muscle degeneration exceeding the rate of regeneration.¹⁸ Overall, dystrophin is critical for muscle function, structure, and stability, and its absence results in progressive muscle wasting and severe muscular dystrophy. In the absence of dystrophin two additional laminin-binding receptors, the α7β1 integrin and utrophin, are up-regulated in the skeletal muscle of DMD patients and mdx mice, which may compensate for the loss of the dystrophin glycoprotein complex.^19,20,21The α7β1 integrin is a heterodimeric laminin receptor involved in bidirectional cell signaling and is localized at junctional and extrajunctional sites in skeletal muscle.^22,23 At least six α7 integrin isoforms produced by developmentally regulated RNA splicing are expressed in skeletal muscle.²⁴ Mutations in the α7 integrin gene (ITGA7) cause myopathy in humans.²⁵ Mice lacking the α7 integrin develop myopathy, exhibit vascular smooth muscle defects and have altered extracellular matrix deposition.^{26,27,28,29,30} The observation that the α7β1 integrin is elevated in the muscle of DMD patients and mdx mice led to the hypothesis that the α7β1 integrin may compensate for the loss of dystrophin.¹⁹ Enhanced expression of the α7 integrin in the skeletal muscle of severely dystrophic mice reduced muscle pathology and increased lifespan by threefold.^10,11 In contrast, loss of both dystrophin and α7 integrin in mice results in severe muscular dystrophy and premature death by 4 weeks of age.^28,31 The α7β1 integrin is therefore a major modifier of disease progression in DMD.The utrophin glycoprotein complex is a third major laminin receptor in skeletal muscle. Utrophin has significant sequence homology to dystrophin.^32,33 In normal adult muscle utrophin is restricted to neuromuscular and myotendinous junctions.³⁴ During development or in damaged or diseased muscle, utrophin expression is increased and becomes localized at extrajunctional sites.^35,36 Utrophin interacts with the same proteins as dystrophin, but binds to actin filaments at different sites.³⁷ In mice, loss of utrophin results in a mild form of myasthenia with reduced sarcolemmal folding at the postsynaptic membrane of the neuromuscular junction.^12,15 Transgenic overexpression of utrophin has been shown to rescue mdx mice.³⁸ Mice that lack both dystrophin and utrophin exhibit severe muscular dystrophy and die by 14 weeks of age.^13,14 Thus, utrophin is also a major laminin receptor that modifies disease progression in DMD.To understand the functional overlap between the α7β1 integrin and utrophin in skeletal muscle, we produced mice that lack both α7 integrin and utrophin (α7/utr^−/−). Since both complexes are highly enriched at the MTJ and NMJ, we hypothesized that α7/utr^−/− mice may have severe abnormalities at these critical junctional sites. Our study demonstrates α7/utr^−/− mice exhibit partial embryonic lethality comparable with that observed in α7^−/− mice. Dystrophin is increased in these animals and enriched at the NMJ but not the MTJ. α7/utr^−/− mice display ultrastructural defects in their MTJ and compromised force transmission. Together, these results indicate that the α7β1 integrin, dystrophin and utrophin laminin binding complexes provide continuity between laminin in the extracellular matrix and the cell cytoskeleton, which are necessary for the normal structural and functional properties of skeletal muscle.