Inhibitors selective for prostaglandin G/H synthase-2 (PGHS-2) (known colloquially as COX-2) were designed to minimize gastrointestinal complications of traditional NSAIDs — adverse effects attributed to suppression of COX-1–derived PGE2 and prostacyclin (PGI2). Evidence from 2 randomized controlled-outcome trials (RCTs) of 2 structurally distinct selective inhibitors of COX-2 supports this hypothesis. However, 5 RCTs of 3 structurally distinct inhibitors also indicate that such compounds elevate the risk of myocardial infarction and stroke. The clinical information is biologically plausible, as it is compatible with evidence that inhibition of COX-2–derived PGI2 removes a protective constraint on thrombogenesis, hypertension, and atherogenesis in vivo. However, the concept of simply tipping a “balance” between COX-2–derived PGI2 and COX-1–derived platelet thromboxane is misplaced. Among the questions that remain to be addressed are the following: (a) whether this hazard extends to all or some of the traditional NSAIDs; (b) whether adjuvant therapies, such as low-dose aspirin, will mitigate the hazard and if so, at what cost; (c) whether COX-2 inhibitors result in cardiovascular risk transformation during chronic dosing; and (d) how we might identify individuals most likely to benefit or suffer from such drugs in the future.
Tilo Grosser, Susanne Fries, Garret A. FitzGerald
Some forms of hypertrophic cardiomyopathy (HCM) are caused by mutations in cardiac sarcomeric genes, but environmental factors are believed to influence the hypertrophic response. A highly variable but potentially significant environmental factor is diet. Since soy-rich diets have been speculated to confer protection against cardiovascular disease, Stauffer et al. have explored the influence of a soy diet on cardiac growth and function in a transgenic mouse model of HCM. They report that mice fed a soy diet exhibited significantly worse HCM than mice fed a soy-free (milk protein) diet. This study provides the first evidence of an environmental modifier — diet — on the hypertrophic phenotype and has implications for the way in which disease phenotypes are assessed in genetically altered murine models of disease.
Cathy J. Hatcher, Craig T. Basson
Hair follicle stem cells sustain growth and cycling of the hair follicle and are located in the permanent portion of the follicle known as the bulge. In this issue of the JCI, Ohyama et al. report the characterization of global gene expression patterns of human hair follicle stem cells after their isolation using sophisticated laser capture techniques to microdissect out bulge cells. They discovered a panel of cell surface markers useful for isolating living hair follicle stem cells, a finding with potential therapeutic implications since isolated stem cells in mice can generate new hair follicles when transplanted to other mice. The findings of Ohyama et al. validate the use of the mouse for studying hair follicle biology but also underscore critical differences between mouse and human stem cell markers. In particular, CD34, which delineates hair follicle stem cells in the mouse, is not expressed by human hair follicle stem cells, while CD200 is expressed by stem cells in both species. Ultimately, this information will assist efforts to develop cell-based and cell-targeted treatments for skin disease.
Gastric mucosa-associated lymphoid tissue (MALT) lymphomas can arise in a variety of extranodal sites. Interestingly, at least 3 different, apparently site-specific, chromosomal translocations, all affecting the NF-κB pathway, have been implicated in the development and progression of MALT lymphoma. The most common is the translocation t(11;18)(q21;q21), which results in a fusion of the cIAP2 region on chromosome 11q21 with the MALT1 gene on chromosome 18q21 and is present in more than one-third of cases. The frequency of this translocation is site-related: common in the gastrointestinal tract and lung, rare in conjunctiva and orbit, and almost absent in salivary glands, thyroid, liver, and skin. In this issue of the JCI, Hu et al. add to our understanding of the molecular consequences of this translocation, showing that its fusion product, cIAP2-MALT1, may concomitantly contribute to lymphomagenesis both as a tumor suppressor gene and as an oncogene.
Francesco Bertoni, Emanuele Zucca
The low molecular weight region of the serum peptidome contains protein fragments derived from 2 sources: (a) high-abundance endogenous circulating proteins and (b) cell and tissue proteins. While some researchers have dismissed the serum peptidome as biological trash, recent work using mass spectrometry–based (MS-based) profiling has indicated that the peptidome may reflect biological events and contain diagnostic biomarkers. In this issue of the JCI, Villanueva et al. report on MS-based peptide profiling of serum samples from patients with advanced prostate, bladder, or breast cancer as well as from healthy controls. Surprisingly, the peptides identified as cancer-type–specific markers proved to be products of enzymatic breakdown generated after patient blood collection. The impact of these results on cancer biomarker discovery efforts is significant because it is widely believed that proteolysis occurring ex vivo should be suppressed because it destroys endogenous biomarkers. Villanueva et al. now suggest that this suppression may in fact be preventing biomarker generation.
Lance A. Liotta, Emanuel F. Petricoin
In the last 5 years, global gene expression profiling has allowed for the subclassification of the heterogeneous disease of breast cancer into new subgroups with prognostic significance. However, for most subgroups, the nature of the contributions of individual genes to the clinical phenotypes remains largely unknown. In this issue of the JCI, Moyano and colleagues further examine the oncogenic potential of the small heat shock protein α-basic–crystallin, commonly expressed in tumors of the basal-like breast cancer subtype associated with poor prognosis, and show that it is an oncogenic protein in the breast.
Sofia K. Gruvberger Saal , Ramon Parsons
Obesity is associated with increased macrophage infiltration of adipose tissue, and these macrophages may be an important component of the chronic inflammatory response playing a crucial role in the development of insulin resistance. This prompts the question as to how macrophages infiltrate obese adipose tissue. In this issue of the JCI, Weisberg et al. show the importance of C-C motif chemokine receptor 2 (CCR2) in macrophage recruitment to adipose tissue and the development of obesity and its complications.
Jaap G. Neels, Jerrold M. Olefsky
Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.
Oliver G. McDonald, Brian R. Wamhoff, Mark H. Hoofnagle, Gary K. Owens
In the face of systemic risk factors, certain regions of the arterial vasculature remain relatively resistant to the development of atherosclerotic lesions. The biomechanically distinct environments in these arterial geometries exert a protective influence via certain key functions of the endothelial lining; however, the mechanisms underlying the coordinated regulation of specific mechano-activated transcriptional programs leading to distinct endothelial functional phenotypes have remained elusive. Here, we show that the transcription factor Kruppel-like factor 2 (KLF2) is selectively induced in endothelial cells exposed to a biomechanical stimulus characteristic of atheroprotected regions of the human carotid and that this flow-mediated increase in expression occurs via a MEK5/ERK5/MEF2 signaling pathway. Overexpression and silencing of KLF2 in the context of flow, combined with findings from genome-wide analyses of gene expression, demonstrate that the induction of KLF2 results in the orchestrated regulation of endothelial transcriptional programs controlling inflammation, thrombosis/hemostasis, vascular tone, and blood vessel development. Our data also indicate that KLF2 expression globally modulates IL-1β–mediated endothelial activation. KLF2 therefore serves as a mechano-activated transcription factor important in the integration of multiple endothelial functions associated with regions of the arterial vasculature that are relatively resistant to atherogenesis.
Kush M. Parmar ... Michael A. Gimbrone Jr., Guillermo García-Cardeña
The majority of acute clinical manifestations of atherosclerosis are due to the physical rupture of advanced atherosclerotic plaques. It has been hypothesized that macrophages play a key role in inducing plaque rupture by secreting proteases that destroy the extracellular matrix that provides physical strength to the fibrous cap. Despite reports detailing the expression of multiple proteases by macrophages in rupture-prone regions, there is no direct proof that macrophage-mediated matrix degradation can induce plaque rupture. We aimed to test this hypothesis by retrovirally overexpressing the candidate enzyme MMP-9 in macrophages of advanced atherosclerotic lesions of apoE–/– mice. Despite a greater than 10-fold increase in the expression of MMP-9 by macrophages, there was only a minor increase in the incidence of plaque fissuring. Subsequent analysis revealed that macrophages secrete MMP-9 predominantly as a proform, and this form is unable to degrade the matrix component elastin. Expression of an autoactivating form of MMP-9 in macrophages in vitro greatly enhances elastin degradation and induces significant plaque disruption when overexpressed by macrophages in advanced atherosclerotic lesions of apoE–/– mice in vivo. These data show that enhanced macrophage proteolytic activity can induce acute plaque disruption and highlight MMP-9 as a potential therapeutic target for stabilizing rupture-prone plaques.
Peter J. Gough, Ivan G. Gomez, Paul T. Wille, Elaine W. Raines
Dialysis dependency is one of the leading causes of morbidity and mortality in the world, and once end-stage renal disease develops, it cannot be reversed by currently available therapy. Although administration of large doses of bone morphogenetic protein–7 (BMP-7) has been shown to repair established renal injury and improve renal function, the pathophysiological role of endogenous BMP-7 and regulatory mechanism of its activities remain elusive. Here we show that the product of uterine sensitization-associated gene–1 (USAG1), a novel BMP antagonist abundantly expressed in the kidney, is the central negative regulator of BMP function in the kidney and that mice lacking USAG-1 (USAG1–/– mice) are resistant to renal injury. USAG1–/– mice exhibited prolonged survival and preserved renal function in acute and chronic renal injury models. Renal BMP signaling, assessed by phosphorylation of Smad proteins, was significantly enhanced in USAG1–/– mice with renal injury, indicating that the preservation of renal function is attributable to enhancement of endogenous BMP signaling. Furthermore, the administration of neutralizing antibody against BMP-7 abolished renoprotection in USAG1–/– mice, indicating that USAG-1 plays a critical role in the modulation of renoprotective action of BMP and that inhibition of USAG-1 is a promising means of development of novel treatment for renal diseases.
Motoko Yanagita ... Toru Kita, Takeshi Sakurai
Topoisomerase II (Topo II) inhibitors are cell cycle–specific DNA-damaging agents and often correlate with secondary leukemia with chromosomal translocations involving the mixed-lineage leukemia/myeloid lymphoid leukemia (MLL) gene on chromosome 11 band q23 (11q23). In spite of the clinical importance, the molecular mechanism for this chromosomal translocation has yet to be elucidated. In this study, we employed 2-color FISH and detected intracellular chromosomal translocations induced by etoposide treatment. Cells such as ataxia-telangiectasia mutated–deficient fibroblasts and U2OS cells, in which the early G2/M checkpoint after treatment with low concentrations of etoposide has been lost, executed mitosis with etoposide-induced DNA double-strand breaks, and 2-color FISH signals located on either side of the MLL gene were segregated in the postmitotic G1 phase. Long-term culture of cells that had executed mitosis under etoposide treatment showed frequent structural abnormalities of chromosome 11. These findings provide convincing evidence for Topo II inhibitor–induced 11q23 translocation. Our study also suggests an important role of the early G2/M checkpoint in preventing fixation of chromosomal abnormalities and reveals environmental and genetic risk factors for the development of chromosome 11 translocations, namely, low concentrations of Topo II inhibitors and dysfunctional early G2/M checkpoint control.
Shinichiro Nakada ... Johji Inazawa, Shuki Mizutani
DC-based tumor vaccine research has largely focused on enhancing DC maturation/costimulation and antigen presentation in order to break tolerance against self tumor-associated antigens. DC immunization can activate autoreactive T cells but rarely causes autoimmune pathologies, indicating that self tolerance at the host level is still maintained in the vaccinated hosts. This study in mice reveals a novel regulatory mechanism for the control of self tolerance at the host level by DCs through the restriction of positive cytokine feedback loops by cytokine signaling inhibitor SOCS1. The study further finds the requirement of persistent antigen presentation by DCs for inducing pathological autoimmune responses against normal tissues and tumor, which can be achieved by silencing SOCS1 to unleash the unbridled signaling of IL-12 and the downstream cytokine cascade. However, the use of higher-affinity self peptides, enhancement of DC maturation, and persistent stimulation with cytokines or TLR agonists fail to break tolerance and induce pathological antitumor immunity. Thus, this study indicates the necessity of inhibiting SOCS1, an antigen presentation attenuator, to break self tolerance and induce effective antitumor responses.
Kevin Evel-Kabler, Xiao-Tong Song, Melissa Aldrich, Xue F. Huang, Si-Yi Chen
Insulin receptor substrates, including Irs1 and Irs2, integrate insulin and IGF receptor signals with heterologous pathways to coordinate growth and metabolism. Since Irs2 is thought to be especially important in hepatic nutrient homeostasis, we deleted Irs1 from hepatocytes of WT mice (called LKO) or genetically insulin-resistant Irs1–/– mice (called LKO::Irs1–/–). Viable LKO::Irs1–/– mice were 70% smaller than WT or LKO mice, and 40% smaller than Irs1–/– mice. Hepatic insulin receptors were functional in all the mice, but insulin signaling via the Akt—FoxO1 pathway was reduced in Irs1–/– and LKO liver, and undetected in LKO::Irs1–/– liver; however, Gsk3β phosphorylation (Ser9) and hepatic glycogen stores were nearly normal in all of the mice. LKO and Irs1–/– mice developed insulin resistance and glucose intolerance that never progressed to diabetes, whereas LKO::Irs1–/– mice developed hyperglycemia and hyperinsulinemia immediately after birth. Regardless, few hepatic genes changed expression significantly in Irs1–/– or LKO mice, whereas hundreds of genes changed in LKO::Irs1–/– mice — including elevated levels of Pck1, G6pc, Ppargc1, Pparg, and Igfbp1. Thus, signals delivered by Irs1 or Irs2 regulate hepatic gene expression that coordinates glucose homeostasis and systemic growth.
Xiaocheng Dong, Sunmin Park, Xueying Lin, Kyle Copps, Xianjin Yi, Morris F. White
The C-C motif chemokine receptor–2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.
Stuart P. Weisberg ... Rudolph L. Leibel, Anthony W. Ferrante Jr.
Using an siRNA-based screen, we identified the transcriptional corepressor RIP140 as a negative regulator of insulin-responsive hexose uptake and oxidative metabolism in 3T3-L1 adipocytes. Affymetrix GeneChip profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, TCA cycle, fatty acid oxidation, mitochondrial biogenesis, and oxidative phosphorylation in these cells. Conversely, we show that reexpression of RIP140 in mouse embryonic fibroblasts derived from RIP140-null mice downregulates expression of many of these same genes. Consistent with these microarray data, RIP140 gene silencing in cultured adipocytes increased both conversion of [14C]glucose to CO2 and mitochondrial oxygen consumption. RIP140-null mice, previously reported to resist weight gain on a high-fat diet, are shown here to display enhanced glucose tolerance and enhanced responsiveness to insulin compared with matched wild-type mice upon high-fat feeding. Mechanistically, RIP140 was found to require the nuclear receptor ERRα to regulate hexose uptake and mitochondrial proteins SDHB and CoxVb, although it likely acts through other nuclear receptors as well. We conclude that RIP140 is a major suppressor of adipocyte oxidative metabolism and mitochondrial biogenesis, as well as a negative regulator of whole-body glucose tolerance and energy expenditure in mice.
Aimee M. Powelka ... Malcolm G. Parker, Michael P. Czech
V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T–B–NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T–B–NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T–B–NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual DH-JH junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed DH-JH ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.
Mirjam van der Burg ... Jacques J.M. van Dongen, Dik C. van Gent
We previously reported that human CD4+ Tregs secrete high levels of IL-10 when stimulated in the presence of dexamethasone and calcitriol (vitamin D3). We now show that following stimulation by allergen, IL-10–secreting Tregs inhibit cytokine secretion by allergen-specific Th2 cells in an IL-10–dependent manner. A proportion of patients with severe asthma fail to demonstrate clinical improvement upon glucocorticoid therapy, and their asthma is characterized as glucocorticoid resistant (SR, abbreviation derived from “steroid resistant”). Dexamethasone does not enhance secretion of IL-10 by their CD4+ T cells. Addition of vitamin D3 with dexamethasone to cultures of SR CD4+ T cells enhanced IL-10 synthesis to levels observed in cells from glucocorticoid-sensitive patients cultured with dexamethasone alone. Furthermore, pretreatment with IL-10 fully restored IL-10 synthesis in these cells in response to dexamethasone. Vitamin D3 significantly overcame the inhibition of glucocorticoid-receptor expression by dexamethasone while IL-10 upregulated glucocorticoid-receptor expression by CD4+ T cells, suggesting potential mechanisms whereby these treatments may overcome poor glucocorticoid responsiveness. We show here that administration of vitamin D3 to healthy individuals and SR asthmatic patients enhanced subsequent responsiveness to dexamethasone for induction of IL-10. This strongly suggests that vitamin D3 could potentially increase the therapeutic response to glucocorticoids in SR patients.
Emmanuel Xystrakis ... Christopher Corrigan, Catherine M. Hawrylowicz
Rare cases of stable allograft acceptance after discontinuation of immunosuppression are often accompanied by macrochimerism (> 1% donor cells in blood) or microchimerism (< 1% donor cells in blood). Here, we have investigated whether persistence of donor cells is the cause or the consequence of long-lasting CTL unresponsiveness. We found that engraftment of splenocytes bearing a single foreign MHC class I–restricted epitope resulted in lifelong donor cell microchimerism and specific CTL unresponsiveness. This status was reversed in a strictly time- and thymus-dependent fashion when the engrafted cells were experimentally removed. The results presented herein show that microchimerism actively maintains CTL unresponsiveness toward a minor histocompatibility antigen by deleting the specific repertoire and thus excluding dominant, T cell extrinsic mechanisms of CTL unresponsiveness independent of systemically persisting donor cell antigen.
Weldy V. Bonilla ... Hans Hengartner, Rolf M. Zinkernagel
IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13–induced inflammation and alveolar remodeling. There were associated decreases in IL-13–induced chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of α1-antitrypsin. IL-13–induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13–induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13–induced diseases and disorders.
Patty J. Lee ... Brooke T. Mossman, Jack A. Elias
The pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphomas is associated with independent chromosomal translocations that lead to the upregulation of either BCL10 or MALT1 or the generation of a fusion protein, cIAP2-MALT1. While both BCL10 and MALT1 are critically involved in antigen receptor–mediated NF-κB activation, the role of cIAP2 is not clear. Here we show that cIAP2 is a ubiquitin ligase (E3) of BCL10 and targets it for degradation, inhibiting antigen receptor–mediated cytokine production. cIAP2-MALT1 lacks E3 activity, and concomitantly, the BCL10 protein is stabilized in MALT lymphomas harboring this fusion. Furthermore, BCL10 and cIAP2-MALT1 synergistically activate NF-κB. These results reveal cIAP2 as an inhibitor of antigenic signaling and implicate its dysfunction in MALT lymphomas.
Shimin Hu ... James L. Riley, Xiaolu Yang
Missense mutations in perforin, a critical effector of lymphocyte cytotoxicity, lead to a spectrum of diseases, from familial hemophagocytic lymphohistiocytosis to an increased risk of tumorigenesis. Understanding of the impact of mutations has been limited by an inability to express human perforin in vitro. We have shown, for the first time to our knowledge, that recombinant human perforin is expressed, processed appropriately, and functional in rat basophilic leukemia (RBL) cells following retroviral transduction. Subsequently, we have addressed how perforin missense mutations lead to absent perforin detection and impaired cytotoxicity by analyzing 21 missense mutations by flow cytometry, immunohistochemistry, and immunoblot. We identified perforin missense mutations with partial maturation (class 1), no apparent proteolytic maturation (class 2), and no recognizable forms of perforin (class 3). Class 1 mutations exhibit lytic function when expressed in RBL cells and are associated with residual protein detection and variable cytotoxic function in affected individuals, suggesting that carriers of class 1 alleles may exhibit more subtle immune defects. By contrast, class 3 mutations cause severely diminished perforin detection and cytotoxicity, while class 2 mutations have an intermediate phenotype. Thus, the pathologic mechanism of perforin missense mutation likely involves a protein dosage effect of the mature protein.
Kimberly A. Risma, Robert W. Frayer, Alexandra H. Filipovich, Janos Sumegi
Accumulation and aggregation of amyloid β peptide 1–42 (Aβ42) in the brain has been hypothesized as triggering a pathological cascade that causes Alzheimer disease (AD). To determine whether selective targeting of Aβ42 versus Aβ40 or total Aβ is an effective way to prevent or treat AD, we compared the effects of passive immunization with an anti-Aβ42 mAb, an anti-Aβ40 mAb, and multiple Aβ1–16 mAbs. We established in vivo binding selectivity of the anti-Aβ42 and anti-Aβ40 mAbs using novel TgBRI-Aβ mice. We then conducted a prevention study in which the anti-Aβ mAbs were administered to young Tg2576 mice, which have no significant Aβ deposition, and therapeutic studies in which mAbs were administered to Tg2576 or CRND8 mice with modest levels of preexisting Aβ deposits. Anti-Aβ42, anti-Aβ40, and anti-Aβ1–16 mAbs attenuated plaque deposition in the prevention study. In contrast, anti-Aβ42 and anti-Aβ40 mAbs were less effective in attenuating Aβ deposition in the therapeutic studies and were not effective in clearing diffuse plaques following direct injection into the cortex. These data suggest that selective targeting of Aβ42 or Aβ40 may be an effective strategy to prevent amyloid deposition, but may have limited benefit in a therapeutic setting.
Yona Levites ... Michael P. Murphy, Todd E. Golde
Degeneration of peripheral motor axons is a common feature of several debilitating diseases including complicated forms of hereditary spastic paraplegia. One such form is caused by loss of the mitochondrial energy-dependent protease paraplegin. Paraplegin-deficient mice display a progressive degeneration in several axonal tracts, characterized by the accumulation of morphological abnormal mitochondria. We show that adenoassociated virus–mediated (AAV-mediated) intramuscular delivery of paraplegin halted the progression of neuropathological changes and rescued mitochondrial morphology in the peripheral nerves of paraplegin-deficient mice. One single injection before onset of symptoms improved the motor performance of paraplegin-deficient mice for up to 10 months, indicating that the peripheral neuropathy contributes to the clinical phenotype. This study provides a proof of principle that gene transfer may be an effective therapeutic option for patients with paraplegin deficiency and demonstrates that AAV vectors can be successfully employed for retrograde delivery of an intracellular protein to spinal motor neurons, opening new perspectives for several hereditary axonal neuropathies of the peripheral nerves.
Marinella Pirozzi ... Alberto Auricchio, Elena I. Rugarli
We report that dietary modification from a soy-based diet to a casein-based diet radically improves disease indicators and cardiac function in a transgenic mouse model of hypertrophic cardiomyopathy. On a soy diet, males with a mutation in the α-myosin heavy chain gene progress to dilation and heart failure. However, males fed a casein diet no longer deteriorate to severe, dilated cardiomyopathy. Remarkably, their LV size and contractile function are preserved. Further, this diet prevents a number of pathologic indicators in males, including fibrosis, induction of β-myosin heavy chain, inactivation of glycogen synthase kinase 3β (GSK3β), and caspase-3 activation.
Brian L. Stauffer, John P. Konhilas, Elizabeth D. Luczak, Leslie A. Leinwand
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a major role in integrin signaling pathways. Although cardiovascular defects were observed in FAK total KO mice, the embryonic lethality prevented investigation of FAK function in the hearts of adult animals. To circumvent these problems, we created mice in which FAK is selectively inactivated in cardiomyocytes (CFKO mice). We found that CFKO mice develop eccentric cardiac hypertrophy (normal LV wall thickness and increased left chamber dimension) upon stimulation with angiotensin II or pressure overload by transverse aortic constriction as measured by echocardiography. We also found increased heart/body weight ratios, elevated markers of cardiac hypertrophy, multifocal interstitial fibrosis, and increased collagen I and VI expression in CFKO mice compared with control littermates. Spontaneous cardiac chamber dilation and increased expression of hypertrophy markers were found in the older CFKO mice. Analysis of cardiomyocytes isolated from CFKO mice showed increased length but not width. The myocardium of CFKO mice exhibited disorganized myofibrils with increased nonmyofibrillar space filled with swelled mitochondria. Last, decreased tyrosine phosphorylation of FAK substrates p130Cas and paxillin were observed in CFKO mice compared with the control littermates. Together, these results provide strong evidence for a role of FAK in the regulation of heart hypertrophy in vivo.
Xu Peng ... Hua Gu, Jun-Lin Guan
Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.
Allison D. Fryer ... Erin Fitch, David B. Jacoby
Endothelial cells can protect cardiomyocytes from injury, but the mechanism of this protection is incompletely described. Here we demonstrate that protection of cardiomyocytes by endothelial cells occurs through PDGF-BB signaling. PDGF-BB induced cardiomyocyte Akt phosphorylation in a time- and dose-dependent manner and prevented apoptosis via PI3K/Akt signaling. Using injectable self-assembling peptide nanofibers, which bound PDGF-BB in vitro, sustained delivery of PDGF-BB to the myocardium at the injected sites for 14 days was achieved. A blinded and randomized study in 96 rats showed that injecting nanofibers with PDGF-BB, but not nanofibers or PDGF-BB alone, decreased cardiomyocyte death and preserved systolic function after myocardial infarction. A separate blinded and randomized study in 52 rats showed that PDGF-BB delivered with nanofibers decreased infarct size after ischemia/reperfusion. PDGF-BB with nanofibers induced PDGFR-β and Akt phosphorylation in cardiomyocytes in vivo. These data demonstrate that endothelial cells protect cardiomyocytes via PDGF-BB signaling and that this in vitro finding can be translated into an effective in vivo method of protecting myocardium after infarction. Furthermore, this study shows that injectable nanofibers allow precise and sustained delivery of proteins to the myocardium with potential therapeutic benefits.
Patrick C.H. Hsieh, Michael E. Davis, Joseph Gannon, Catherine MacGillivray, Richard T. Lee
The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs. In this study, we determined the distribution of label-retaining cells to define the human anagen bulge. Using navigated laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and activin/bone morphogenic protein signaling were overrepresented in the bulge, while genes responsible for cell proliferation were underrepresented, consistent with the existence of quiescent noncycling KSCs in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1, while CD24, CD34, CD71, and CD146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hiCD24loCD34loCD71loCD146lo) obtained from hair follicle suspensions demonstrated high colony-forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. The stem cell behavior of enriched bulge cells and their utility for gene therapy and hair regeneration will need to be assessed in in vivo assays.
Manabu Ohyama ... Mark C. Udey, Jonathan C. Vogel
Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein α-basic–crystallin (αB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of αB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, αB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing αB-crystallin by RNA interference inhibited these abnormalities. αB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by αB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing αB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that αB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.
Jose V. Moyano ... Charles M. Perou, Vincent L. Cryns
Recent studies have established distinctive serum polypeptide patterns through mass spectrometry (MS) that reportedly correlate with clinically relevant outcomes. Wider acceptance of these signatures as valid biomarkers for disease may follow sequence characterization of the components and elucidation of the mechanisms by which they are generated. Using a highly optimized peptide extraction and matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) MS–based approach, we now show that a limited subset of serum peptides (a signature) provides accurate class discrimination between patients with 3 types of solid tumors and controls without cancer. Targeted sequence identification of 61 signature peptides revealed that they fall into several tight clusters and that most are generated by exopeptidase activities that confer cancer type–specific differences superimposed on the proteolytic events of the ex vivo coagulation and complement degradation pathways. This small but robust set of marker peptides then enabled highly accurate class prediction for an external validation set of prostate cancer samples. In sum, this study provides a direct link between peptide marker profiles of disease and differential protease activity, and the patterns we describe may have clinical utility as surrogate markers for detection and classification of cancer. Our findings also have important implications for future peptide biomarker discovery efforts.
Josep Villanueva ... Howard I. Scher, Paul Tempst
Copyright © 2014 American Society for Clinical Investigation