Section of murine colon immunostained with anti-PCNA antibody to show proliferating cells (from Lerner et al.). As the last part of the digestive system, the colon plays a key role in extracting water and salt from solid waste before it is eliminated from the body. In this issue, we have several articles showing how dysfunction in the colon can lead various pathologies from colitis to cancer. Qiu and colleagues (page 1722) investigate how intestinal epithelial apoptosis can lead to ulcerative colitis. Lerner et al. (page 1709), Nguyen et al. (page 1733), and Schiechl et al. (page 1692) all focus on how the inflammation associated with colitis can lead to tumorigenesis. Steine et al. (page 1748) find a causal role for DNA methyltransferase 3b (Dnmt3b) in the aberrant methylation seen in colon cancer.
Human cytomegalovirus (CMV), one of the eight herpesviruses that commonly infect humans, is best known for its propensity to cause disease in immunocompromised patients, especially transplant recipients, patients with advanced AIDS, and congenitally infected newborns. Advances in molecular virology coupled with improvements in diagnostic methods and treatment options have vastly improved our understanding of and ability to manage CMV, but many uncertainties remain, including the mechanisms of persistence and pathogenesis and its hypothesized roles in a variety of human illnesses. Here we review recent advances that are reshaping our view and approach to this fascinating virus.
Michael Boeckh, Adam P. Geballe
Identification of the cell of origin of cancers is a hot topic, and recently no fewer than five groups have addressed this question for basal cell carcinomas (BCCs) of the skin. In this issue of the JCI, Grachtchouk et al. present evidence from engineered mouse models that at least some BCCs can arise from hair follicle tissue stem cells. Although these cells sustain oncogenic mutations while in the stem cell niche, they may have to leave that stem cell niche to become tumor cells.
Ervin H. Epstein Jr.
Proinflammatory stimuli induce inflammation that may progress to sepsis or chronic inflammatory disease. The cytokine TNF-α is an important endotoxin-induced inflammatory glycoprotein produced predominantly by macrophages and lymphocytes. TNF-α plays a major role in initiating signaling pathways and pathophysiological responses after engaging TNF receptors. In this issue of JCI, Rowlands et al. demonstrate that in lung microvessels, soluble TNF-α (sTNF-α) promotes the shedding of the TNF-α receptor 1 ectodomain via increased mitochondrial Ca2+ that leads to release of mitochondrial ROS. Shedding mediated by TNF-α–converting enzyme (TACE) results in an unattached TNF receptor, which participates in the scavenging of sTNF-α, thus limiting the propagation of the inflammatory response. These findings suggest that mitochondrial Ca2+, ROS, and TACE might be therapeutically targeted for treating pulmonary endothelial inflammation.
Laura A. Dada, Jacob I. Sznajder
Most individuals infected with Mycobacterium tuberculosis develop a latent infection, which does not progress to active tuberculosis (TB). This occurs, in part, because infected macrophages recruit immune cells to form a granuloma, isolating the bacteria and preventing its spread. In some individuals, granulomas undergo necrosis and tissue destruction occurs, releasing the bacteria and allowing the development of active disease. In this issue of the JCI, Elkington et al. provide evidence that M. tuberculosis drives the expression of MMP-1, which in turn promotes the collagen breakdown that leads to alveolar destruction in TB. These findings identify putative therapeutic targets for the prevention of TB.
Pelvic organ prolapse (POP) is a disabling disorder in women characterized by a loss of pelvic floor support leading to the herniation of the uterus into or through the vagina. POP is a complex problem that likely involves multiple mechanisms, and available therapies are limited. In this issue of the JCI, Budatha et al. explore the dual role carried out by fibulin-5 in facilitating the assembly of normal elastic fibers and inhibiting MMP-9 activity, revealing a new mechanism critical to the maintenance of pelvic organ support.
Gina M. Northington
Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13–producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11bhighGr1low M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate–induced colitis, F4/80+CD11bhighGr1intermediate macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88–deficient (Myd88–/–) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88–/– mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11bhighGr1intermediate macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.
Gabriela Schiechl ... Warren Strober, Stefan Fichtner-Feigl
Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Expression of the enzyme heparanase is clearly linked to colon carcinoma progression, but its role in UC is unknown. Here we demonstrate for what we believe to be the first time the importance of heparanase in sustaining the immune-epithelial crosstalk underlying colitis-associated tumorigenesis. Using histological specimens from UC patients and a mouse model of dextran sodium sulfate–induced colitis, we found that heparanase was constantly overexpressed and activated throughout the disease. We demonstrate, using heparanase-overexpressing transgenic mice, that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors. We found that highly coordinated interactions between the epithelial compartment (contributing heparanase) and mucosal macrophages preserved chronic inflammatory conditions and created a tumor-promoting microenvironment characterized by enhanced NF-κB signaling and induction of STAT3. Our results indicate that heparanase generates a vicious cycle that powers colitis and the associated tumorigenesis: heparanase, acting synergistically with the intestinal flora, stimulates macrophage activation, while macrophages induce production (via TNF-α–dependent mechanisms) and activation (via secretion of cathepsin L) of heparanase contributed by the colon epithelium. Thus, disruption of the heparanase-driven chronic inflammatory circuit is highly relevant to the design of therapeutic interventions in colitis and the associated cancer.
Immanuel Lerner ... Israel Vlodavsky, Michael Elkin
Intestinal epithelial cell (IEC) apoptosis contributes to the development of ulcerative colitis (UC), an inflammatory bowel disease (IBD) that affects the colon and rectum. Therapies that target the inflammatory cytokine TNF have been found to inhibit IEC apoptosis in patients with IBD, although the mechanism of IEC apoptosis remains unclear. We therefore investigated the role of p53-upregulated modulator of apoptosis (PUMA), a p53 target and proapoptotic BH3-only protein, in colitis and IEC apoptosis, using patient samples and mouse models of UC. In UC patient samples, PUMA expression was elevated in colitis tissues relative to that in uninvolved tissues, and the degree of elevation of PUMA expression correlated with the severity of colitis and the degree of apoptosis induction. In mice, PUMA was markedly induced in colonic epithelial cells following induction of colitis by either dextran sulfate sodium salt (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). The induction of PUMA was p53-independent but required NF-κB. Absence of PUMA, but neither absence of p53 nor that of another BH3-only protein (Bid), relieved DSS- and TNBS-induced colitis and inhibited IEC apoptosis. Furthermore, treating mice with infliximab (Remicade), a clinically used TNF-specific antibody, suppressed DSS- and TNBS-induced PUMA expression and colitis. These results indicate that PUMA induction contributes to the pathogenesis of colitis by promoting IEC apoptosis and suggest that PUMA inhibition may be an effective strategy to promote mucosal healing in patients with UC.
Wei Qiu ... Jian Yu, Lin Zhang
Expression of the transmembrane glycoprotein CD98 (encoded by SLC3A2) is increased in intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), and in various carcinomas, yet its pathogenetic role remains unknown. By generating gain- and loss-of-function mouse models with genetically manipulated CD98 expression specifically in intestinal epithelial cells (IECs), we explored the role of CD98 in intestinal homeostasis, inflammation, and colitis-associated tumorigenesis. IEC-specific CD98 overexpression induced gut homeostatic defects and increased inflammatory responses to DSS-induced colitis, promoting colitis-associated tumorigenesis in mice. Further analysis indicated that the ability of IEC-specific CD98 overexpression to induce tumorigenesis was linked to its capacity to induce barrier dysfunction and to stimulate cell proliferation and production of proinflammatory mediators. To validate these results, we constructed mice carrying conditional floxed Slc3a2 alleles and crossed them with Villin-Cre mice such that CD98 was downregulated only in IECs. These mice exhibited attenuated inflammatory responses and resistance to both DSS-induced colitis and colitis-associated tumorigenesis. Together, our data show that intestinal CD98 expression has a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in IECs therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as IBD and colitis-associated cancer.
Hang Thi Thu Nguyen ... Shanthi V. Sitaraman, Didier Merlin
Human cancer cells frequently have regions of their DNA hypermethylated, which results in transcriptional silencing of affected genes and promotion of tumor formation. However, it is still unknown whether cancer-associated aberrant DNA methylation is targeted to specific genomic regions, whether this methylation also occurs in noncancerous cells, and whether these epigenetic events are maintained in the absence of the initiating cause. Here we have addressed some of these issues by demonstrating that transgenic expression of DNA methyltransferase 3b (Dnmt3b) in the mouse colon initiates de novo DNA methylation of genes that are similar to genes that become methylated in human colon cancer. This is consistent with the notion that aberrant methylation in cancer may be attributable to targeting of specific sequences by Dnmt3b rather than to random methylation followed by clonal selection. We also showed that Dnmt3b-induced aberrant DNA methylation was maintained in regenerating tissue, even in the absence of continuous Dnmt3b expression. This supports the concept that transient stressors can cause permanent epigenetic changes in somatic stem cells and that these accumulate over the lifetime of an organism in analogy to DNA mutations.
Eveline J. Steine ... Rudolf Jaenisch, Heinz G. Linhart
Trefoil factor 1 (TFF1) is a tumor suppressor gene that encodes a peptide belonging to the trefoil factor family of protease-resistant peptides. Although TFF1 expression is frequently lost in gastric carcinomas, the tumorigenic pathways this affects have not been determined. Here we show that Tff1-knockout mice exhibit age-dependent carcinogenic histological changes in the pyloric antrum of the gastric mucosa, progressing from gastritis to hyperplasia, low-grade dysplasia, high-grade dysplasia, and ultimately malignant adenocarcinoma. The histology and molecular signatures of gastric lesions in the Tff1-knockout mice were consistent with an inflammatory phenotype. In vivo, ex-vivo, and in vitro studies showed that TFF1 expression suppressed TNF-α–mediated NF-κB activation through the TNF receptor 1 (TNFR1)/IκB kinase (IKK) pathway. Consistent with these mouse data, human gastric tissue samples displayed a progressive decrease in TFF1 expression and an increase in NF-κB activation along the multi-step carcinogenesis cascade. Collectively, these results provide evidence that loss of TFF1 leads to activation of IKK complex–regulated NF-κB transcription factors and is an important event in shaping the NF-κB–mediated inflammatory response during the progression to gastric tumorigenesis.
Mohammed Soutto ... Richard M. Peek Jr., Wael El-Rifai
Uncontrolled Hedgehog (Hh) signaling leads to the development of basal cell carcinoma (BCC), the most common human cancer, but the cell of origin for BCC is unclear. While Hh pathway dysregulation is common to essentially all BCCs, there exist multiple histological subtypes, including superficial and nodular variants, raising the possibility that morphologically distinct BCCs may arise from different cellular compartments in skin. Here we have shown that induction of a major mediator of Hh signaling, GLI2 activator (GLI2ΔN), selectively in stem cells of resting hair follicles in mice, induced nodular BCC development from a small subset of cells in the lower bulge and secondary hair germ compartments. Tumorigenesis was markedly accelerated when GLI2ΔN was induced in growing hair follicles. In contrast, induction of GLI2ΔN in epidermis led to the formation of superficial BCCs. Expression of GLI2ΔN at reduced levels in mice yielded lesions resembling basaloid follicular hamartomas, which have previously been linked to low-level Hh signaling in both mice and humans. Our data show that the cell of origin, tissue context (quiescent versus growing hair follicles), and level of oncogenic signaling can determine the phenotype of Hh/Gli-driven skin tumors, with high-level signaling required for development of superficial BCC-like tumors from interfollicular epidermis and nodular BCC-like tumors from hair follicle stem cells.
Marina Grachtchouk ... Monique Verhaegen, Andrzej A. Dlugosz
The success of a non-live vaccine requires improved formulation and adjuvant selection to generate robust T cell immunity following immunization. Here, using protein linked to a TLR7/8 agonist (conjugate vaccine), we investigated the functional properties of vaccine formulation, the cytokines, and the DC subsets required to induce protective multifunctional T cell immunity in vivo. The conjugate vaccine required aggregation of the protein to elicit potent Th1 CD4+ and CD8+ T cell responses. Remarkably, the conjugate vaccine, through aggregation of the protein and activation of TLR7 in vivo, led to an influx of migratory DCs to the LN and increased antigen uptake by several resident and migratory DC subsets, with the latter effect strongly influenced by vaccine-induced type I IFN. Ex vivo migratory CD8–DEC205+CD103–CD326– langerin-negative dermal DCs were as potent in cross-presenting antigen to naive CD8+ T cells as CD11c+CD8+ DCs. Moreover, these cells also influenced Th1 CD4+ T cell priming. In summary, we propose a model in which broad-based T cell–mediated responses upon vaccination can be maximized by codelivery of aggregated protein and TLR7/8 agonist, which together promote optimal antigen acquisition and presentation by multiple DC subsets in the context of critical proinflammatory cytokines.
Kathrin Kastenmüller ... Ronald N. Germain, Robert A. Seder
A link between autoimmunity and improved antitumor immunity has long been recognized, although the exact mechanistic relationship between these two phenomena remains unclear. In the present study we have found that vitiligo, the autoimmune destruction of melanocytes, generates self antigen required for mounting persistent and protective memory CD8+ T cell responses to melanoma. Vitiligo developed in approximately 60% of mice that were depleted of regulatory CD4+ T cells and then subjected to surgical excision of large established B16 melanomas. Mice with vitiligo generated 10-fold larger populations of CD8+ memory T cells specific for shared melanoma/melanocyte antigens. CD8+ T cells in mice with vitiligo acquired phenotypic and functional characteristics of effector memory, suggesting that they were supported by ongoing antigen stimulation. Such responses were not generated in melanocyte-deficient mice, indicating a requirement for melanocyte destruction in maintaining CD8+ T cell immunity to melanoma. Vitiligo-associated memory CD8+ T cells provided durable tumor protection, were capable of mounting a rapid recall response to melanoma, and did not demonstrate phenotypic or functional signs of exhaustion even after many months of exposure to antigen. This work establishes melanocyte destruction as a key determinant of lasting melanoma-reactive immune responses, thus illustrating that immune-mediated destruction of normal tissues can perpetuate adaptive immune responses to cancer.
Katelyn T. Byrne ... Edward J. Usherwood, Mary Jo Turk
Type 1 diabetes is a chronic autoimmune disease in which genetic predispositions affect the immune system, leading to a loss of T cell tolerance to β cells and consequent T cell–mediated destruction of insulin-producing islet cells. Genetic studies have suggested that PRSS16 is linked to a diabetes susceptibility locus of the extended HLA class I region in humans. PRSS16 encodes what we believe to be a novel protease, thymus-specific serine protease (TSSP), which shows predominant expression in thymic epithelial cells and is suspected to have a restricted role in the class II presentation pathway. Consistently, Tssp is necessary for the intrathymic selection of few class II–restricted T cell receptor specificities in B6 mice. To directly assess the role of Tssp in autoimmune diabetes, we generated Tssp-deficient (Tssp°) NOD mice. While remaining immunocompetent, Tssp° NOD mice were protected from diabetes and severe insulitis. Diabetes resistance of Tssp° NOD mice was a property of the CD4 T cell compartment that is acquired during thymic selection and correlated with an impaired selection of CD4 T cells specific for islet antigens. Hence, in the NOD mouse, Tssp is a critical regulator of diabetes development through the selection of the autoreactive CD4 T cell repertoire.
Christophe Viret ... Alice Carrier, Sylvie Guerder
Targeted T cell immunotherapies using engineered T lymphocytes expressing tumor-directed chimeric antigen receptors (CARs) are designed to benefit patients with cancer. Although incorporation of costimulatory endodomains within these CARs increases the proliferation of CAR-redirected T lymphocytes, it has proven difficult to draw definitive conclusions about the specific effects of costimulatory endodomains on the expansion, persistence, and antitumor effectiveness of CAR-redirected T cells in human subjects, owing to the lack of side-by-side comparisons with T cells bearing only a single signaling domain. We therefore designed a study that allowed us to directly measure the consequences of adding a costimulatory endodomain to CAR-redirected T cells. Patients with B cell lymphomas were simultaneously infused with 2 autologous T cell products expressing CARs with the same specificity for the CD19 antigen, present on most B cell malignancies. One CAR encoded both the costimulatory CD28 and the ζ-endodomains, while the other encoded only the ζ-endodomain. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced expansion and persistence compared with CAR+ T cells lacking this endodomain. These results demonstrate the superiority of CARs with dual signal domains and confirm a method of comparing CAR-modified T cells within individual patients, thereby avoiding patient-to-patient variability and accelerating the development of optimal T cell immunotherapies.
Barbara Savoldo ... Malcolm K. Brenner, Gianpietro Dotti
Mycobacterium tuberculosis can cause lung tissue damage to spread, but the mechanisms driving this immunopathology are poorly understood. The breakdown of lung matrix involves MMPs, which have a unique ability to degrade fibrillar collagens at neutral pH. To determine whether MMPs play a role in the immunopathology of tuberculosis (TB), we profiled MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), in sputum and bronchoalveolar lavage fluid from patients with TB and symptomatic controls. MMP-1 concentrations were significantly increased in both HIV-negative and HIV-positive patients with TB, while TIMP concentrations were lower in HIV-negative TB patients. In primary human monocytes, M. tuberculosis infection selectively upregulated MMP1 gene expression and secretion, and Ro32-3555, a specific MMP inhibitor, suppressed M. tuberculosis–driven MMP-1 activity. Since the mouse MMP-1 ortholog is not expressed in the lung and mice infected with M. tuberculosis do not develop tissue destruction equivalent to humans, we infected transgenic mice expressing human MMP-1 with M. tuberculosis to investigate whether MMP-1 caused lung immunopathology. In the MMP-1 transgenic mice, M. tuberculosis infection increased MMP-1 expression, resulting in alveolar destruction in lung granulomas and significantly greater collagen breakdown. In summary, MMP-1 may drive tissue destruction in TB and represents a therapeutic target to limit immunopathology.
Paul Elkington ... Jeanine D’Armiento, Jon S. Friedland
Nonthyroidal illness syndrome (NTIS) is a state of low serum 3,5,3′ triiodothyronine (T3) that occurs in chronically ill patients; the degree of reduction in T3 is associated with overall prognosis and survival. Iodothyronine deiodinases are enzymes that catalyze iodine removal from thyroid hormones; type I and II deiodinase (D1 and D2, respectively) convert the prohormone thyroxine T4 to active T3, whereas the type III enzyme (D3) inactivates T4 and T3. Increased production of cytokines, including IL-6, is a hallmark of the acute phase of NTIS, but the role of cytokines in altered thyroid hormone metabolism is poorly understood. Here, we measured the effect of IL-6 on both endogenous cofactor–mediated and dithiothreitol-stimulated (DTT-stimulated) cell sonicate deiodinase activities in human cell lines. Active T3 generation by D1 and D2 in intact cells was suppressed by IL-6, despite an increase in sonicate deiodinases (and mRNAs). N-acetyl-cysteine (NAC), an antioxidant that restores intracellular glutathione (GSH) concentrations, prevented the IL-6–induced inhibitory effect on D1- and D2-mediated T3 production, which suggests that IL-6 might function by depleting an intracellular thiol cofactor, perhaps GSH. In contrast, IL-6 stimulated endogenous D3–mediated inactivation of T3. Taken together, these results identify a single pathway by which IL-6–induced oxidative stress can reduce D1- and D2-mediated T4-to-T3 conversion as well as increasing D3-mediated T3 (and T4) inactivation, thus mimicking events during illness.
Simone Magagnin Wajner, Iuri Martin Goemann, Ana Laura Bueno, P. Reed Larsen, Ana Luiza Maia
The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) have emerged as key mediators in the pathophysiology of several mood disorders, including anxiety and depression. However, therapeutic compounds that interact with TrkB receptors have been difficult to develop. Using a combination of structure-based in silico screening and high-capacity functional assays in recombinant and neuronal cells, we identified a low–molecular weight TrkB ligand (ANA-12) that prevented activation of the receptor by BDNF with a high potency. ANA-12 showed direct and selective binding to TrkB and inhibited processes downstream of TrkB without altering TrkA and TrkC functions. KIRA-ELISA analysis demonstrated that systemic administration of ANA-12 to adult mice decreased TrkB activity in the brain without affecting neuronal survival. Mice administered ANA-12 demonstrated reduced anxiety- and depression-related behaviors on a variety of tests predictive of anxiolytic and antidepressant properties in humans. This study demonstrates that structure-based virtual screening strategy can be an efficient method for discovering potent TrkB-selective ligands that are active in vivo. We further propose that ANA-12 may be a valuable tool for studying BDNF/TrkB signaling and may constitute a lead compound for developing the next generation of therapeutic agents for the treatment of mood disorders.
Maxime Cazorla, Joël Prémont, Andre Mann, Nicolas Girard, Christoph Kellendonk, Didier Rognan
Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid–induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.
William L. Holland ... Philipp E. Scherer, Scott A. Summers
Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.
Aubrey C. Chan ... Kevin J. Whitehead, Dean Y. Li
Neovessel formation is a complex process governed by the orchestrated action of multiple factors that regulate EC specification and dynamics within a growing vascular tree. These factors have been widely exploited to develop therapies for angiogenesis-related diseases such as diabetic retinopathy and tumor growth and metastasis. WNT signaling has been implicated in the regulation and development of the vascular system, but the detailed mechanism of this process remains unclear. Here, we report that Dickkopf1 (DKK1) and Dickkopf2 (DKK2), originally known as WNT antagonists, play opposite functional roles in regulating angiogenesis. DKK2 induced during EC morphogenesis promoted angiogenesis in cultured human endothelial cells and in in vivo assays using mice. Its structural homolog, DKK1, suppressed angiogenesis and was repressed upon induction of morphogenesis. Importantly, local injection of DKK2 protein significantly improved tissue repair, with enhanced neovascularization in animal models of both hind limb ischemia and myocardial infarction. We further showed that DKK2 stimulated filopodial dynamics and angiogenic sprouting of ECs via a signaling cascade involving LRP6-mediated APC/Asef2/Cdc42 activation. Thus, our findings demonstrate the distinct functions of DKK1 and DKK2 in controlling angiogenesis and suggest that DKK2 may be a viable therapeutic target in the treatment of ischemic vascular diseases.
Jeong-Ki Min ... Ki-Chul Hwang, Young-Guen Kwon
The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.
Bin Zhou ... Francis X. McGowan, William T. Pu
Hypoxia inducible factor–1 (HIF-1) is the master transcriptional regulator of the cellular response to altered oxygen levels. HIF-1α protein is elevated in most solid tumors and contributes to poor disease outcome by promoting tumor progression, metastasis, and resistance to chemotherapy. To date, the relationship between HIF-1 and these processes, particularly chemoresistance, has remained largely unexplored. Here, we show that expression of the MAPK-specific phosphatase dual-specificity phosphatase–2 (DUSP2) is markedly reduced or completely absent in many human cancers and that its level of expression inversely correlates with that of HIF-1α and with cancer malignancy. Analysis of human cancer cell lines indicated that HIF-1α inhibited DUSP2 transcription, which resulted in prolonged phosphorylation of ERK and, hence, increased chemoresistance. Knockdown of DUSP2 increased drug resistance under normoxia, while forced expression of DUSP2 abolished hypoxia-induced chemoresistance. Further, reexpression of DUSP2 during cancer progression caused tumor regression and markedly increased drug sensitivity in mice xenografted with human tumor cell lines. Furthermore, a variety of genes involved in drug response, angiogenesis, cell survival, and apoptosis were found to be downregulated by DUSP2. Our results demonstrate that DUSP2 is a key downstream regulator of HIF-1–mediated tumor progression and chemoresistance. DUSP2 therefore may represent a novel drug target of particular relevance in tumors resistant to conventional chemotherapy.
Shih-Chieh Lin ... Shao-Chieh Lin, Shaw-Jenq Tsai
Disordered glucagon secretion contributes to the symptoms of diabetes, and reduced glucagon action is known to improve glucose homeostasis. In mice, genetic deletion of the glucagon receptor (Gcgr) results in increased levels of the insulinotropic hormone glucagon-like peptide 1 (GLP-1), which may contribute to the alterations in glucose homeostasis observed in Gcgr–/– mice. Here, we assessed the contribution of GLP-1 receptor (GLP-1R) signaling to the phenotype of Gcgr–/– mice by generating Gcgr–/–Glp1r–/– mice. Although insulin sensitivity was similar in all genotypes, fasting glucose was increased in Gcgr–/–Glp1r–/– mice. Elimination of the Glp1r normalized gastric emptying and impaired intraperitoneal glucose tolerance in Gcgr–/– mice. Unexpectedly, deletion of Glp1r in Gcgr–/– mice did not alter the improved oral glucose tolerance and increased insulin secretion characteristic of that genotype. Although Gcgr–/–Glp1r–/– islets exhibited increased sensitivity to the incretin glucose-dependent insulinotropic polypeptide (GIP), mice lacking both Glp1r and the GIP receptor (Gipr) maintained preservation of the enteroinsular axis following reduction of Gcgr signaling. Moreover, Gcgr–/–Glp1r–/– islets expressed increased levels of the cholecystokinin A receptor (Cckar) and G protein–coupled receptor 119 (Gpr119) mRNA transcripts, and Gcgr–/–Glp1r–/– mice exhibited increased sensitivity to exogenous CCK and the GPR119 agonist AR231453. Our data reveal extensive functional plasticity in the enteroinsular axis via induction of compensatory mechanisms that control nutrient-dependent regulation of insulin secretion.
Safina Ali, Benjamin J. Lamont, Maureen J. Charron, Daniel J. Drucker
α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.
Jianqiang Ding ... Ira J. Fox, Jayanta Roy-Chowdhury
Although mutations in Kras are present in 21% of lung tumors, there is a high level of heterogeneity in phenotype and outcome among patients with lung cancer bearing similar mutations, suggesting that other pathways are important. Wnt/β-catenin signaling is a known oncogenic pathway that plays a well-defined role in colon and skin cancer; however, its role in lung cancer is unclear. We have shown here that activation of Wnt/β-catenin in the bronchiolar epithelium of the adult mouse lung does not itself promote tumor development. However, concurrent activation of Wnt/β-catenin signaling and expression of a constitutively active Kras mutant (KrasG12D) led to a dramatic increase in both overall tumor number and size compared with KrasG12D alone. Activation of Wnt/β-catenin signaling altered the KrasG12D tumor phenotype, resulting in a phenotypic switch from bronchiolar epithelium to the highly proliferative distal progenitors found in the embryonic lung. This was associated with decreased E-cadherin expression at the cell surface, which may underlie the increased metastasis of tumors with active Wnt/β-catenin signaling. Together, these data suggest that activation of Wnt/β-catenin signaling can combine with other oncogenic pathways in lung epithelium to produce a more aggressive tumor phenotype by imposing an embryonic distal progenitor phenotype and by decreasing E-cadherin expression.
Eugenia C. Pacheco-Pinedo ... Francesco J. DeMayo, Edward E. Morrisey
Mucosal antibody responses play a major role in mediating homeostasis with the intestinal flora. It has been suggested that imbalance in the IgA+ and IgG+ intestinal B cell repertoire may be associated with the development of diseases such as inflammatory bowel disease. Despite this, little is known about the antibody specificity of human intestinal plasmablasts. Here, we have determined the reactivity profile of single isolated IgA+ and IgG+ plasmablasts from human terminal ileum using antibody cloning and in vitro expression. We found that approximately 25% of intestinal IgA and IgG plasmablast antibodies were polyreactive; the majority were antigen-specific. Antigen specificity was not only directed against enteropathogenic microbes but also against commensal microbes and self antigens. Regardless of their reactivity, all intestinal antibodies were somatically mutated and showed signs of antigen-mediated selection, suggesting that they developed from antigen-specific B cell responses. Together, our data indicate that antigen-specific immune responses to intestinal microbes are largely responsible for the maintenance of intestinal homeostasis and thus provide a basis for understanding the deregulated immune responses observed in patients with inflammatory bowel disease.
Julia Benckert ... Bertram Wiedenmann, Hedda Wardemann
Infection with different strains of the same species of bacteria often results in vastly different clinical outcomes. Despite extensive investigation, the genetic basis of microbial strain-specific virulence remains poorly understood. Recent whole-genome sequencing has revealed that SNPs are the most prevalent form of genetic diversity among different strains of the same species of bacteria. For invasive serotype M3 group A streptococci (GAS) strains, the gene encoding regulator of proteinase B (RopB) has the highest frequency of SNPs. Here, we have determined that ropB polymorphisms alter RopB function and modulate GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically divergent effects on GAS global gene expression. Additionally, generation of isoallelic GAS strains differing only by a single amino acid in RopB confirmed that variant proteins affected transcript levels of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that ropB polymorphisms influence GAS virulence and disease manifestations. These data detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections.
Ronan K. Carroll ... Anthony R. Flores, James M. Musser
Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies.
Abigail F. Welford ... Gillian M. Tozer, Claire E. Lewis
During development, the retinal vasculature grows toward hypoxic areas in an organized fashion. By contrast, in ischemic retinopathies, new blood vessels grow out of the retinal surfaces without ameliorating retinal hypoxia. Restoration of proper angiogenic directionality would be of great benefit to reoxygenize the ischemic retina and resolve disease pathogenesis. Here, we show that binding of the semaphorin 3E (Sema3E) ligand to the transmembrane PlexinD1 receptor initiates a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In developing mouse retinas, inhibition of VEGF signaling resulted in downregulation of endothelial PlexinD1 expression, suggesting that astrocyte-derived VEGF normally promotes PlexinD1 expression in growing blood vessels. Neuron-derived Sema3E signaled to PlexinD1 and activated the small GTPase RhoJ in ECs, thereby counteracting VEGF-induced filopodia projections and defining the retinal vascular pathfinding. In a mouse model of ischemic retinopathy, enhanced expression of PlexinD1 and RhoJ in extraretinal vessels prevented VEGF-induced disoriented projections of the endothelial filopodia. Remarkably, intravitreal administration of Sema3E protein selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Our study suggests a new paradigm for vascular regeneration therapy that guides angiogenesis precisely toward the ischemic retina.
Yoko Fukushima ... Shin-Ichi Nishikawa, Akiyoshi Uemura
Shedding of the extracellular domain of cytokine receptors allows the diffusion of soluble receptors into the extracellular space; these then bind and neutralize their cytokine ligands, thus dampening inflammatory responses. The molecular mechanisms that control this process, and the extent to which shedding regulates cytokine-induced microvascular inflammation, are not well defined. Here, we used real-time confocal microscopy of mouse lung microvascular endothelium to demonstrate that mitochondria are key regulators of this process. The proinflammatory cytokine soluble TNF-α (sTNF-α) increased mitochondrial Ca2+, and the purinergic receptor P2Y2 prolonged the response. Concomitantly, the proinflammatory receptor TNF-α receptor–1 (TNFR1) was shed from the endothelial surface. Inhibiting the mitochondrial Ca2+ increase blocked the shedding and augmented inflammation, as denoted by increases in endothelial expression of the leukocyte adhesion receptor E-selectin and in microvascular leukocyte recruitment. The shedding was also blocked in microvessels after knockdown of a complex III component and after mitochondria-targeted catalase overexpression. Endothelial deletion of the TNF-α converting enzyme (TACE) prevented the TNF-α receptor shedding response, which suggests that exposure of microvascular endothelium to sTNF-α induced a Ca2+-dependent increase of mitochondrial H2O2 that caused TNFR1 shedding through TACE activation. These findings provide what we believe to be the first evidence that endothelial mitochondria regulate TNFR1 shedding and thereby determine the severity of sTNF-α–induced microvascular inflammation.
David J. Rowlands ... Sunita Bhattacharya, Jahar Bhattacharya
In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.
Dontscho Kerjaschki ... Dieter Steinhilber, Georg Krupitza
Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10–related forms of SRNS and hearing loss can be molecularly identified and potentially treated.
Saskia F. Heeringa ... Christian Faul, Friedhelm Hildebrandt
Experimental models of atherosclerosis suggest that recruitment of monocytes into plaques drives the progression of this chronic inflammatory condition. Cholesterol-lowering therapy leads to plaque stabilization or regression in human atherosclerosis, characterized by reduced macrophage content, but the mechanisms that underlie this reduction are incompletely understood. Mice lacking the gene Apoe (Apoe–/– mice) have high levels of cholesterol and spontaneously develop atherosclerotic lesions. Here, we treated Apoe–/– mice with apoE-encoding adenoviral vectors that induce plaque regression, and investigated whether macrophage removal from plaques during this regression resulted from quantitative alterations in the ability of monocytes to either enter or exit plaques. Within 2 days after apoE complementation, plasma cholesterol was normalized to wild-type levels, and HDL levels were increased 4-fold. Oil red O staining and quantitative mass spectroscopy revealed that esterified cholesterol content was markedly reduced. Plaque macrophage content decreased gradually and was 72% lower than baseline 4 weeks after apoE complementation. Importantly, this reduction in macrophages did not involve migratory egress from plaques or CCR7, a mediator of leukocyte emigration. Instead, marked suppression of monocyte recruitment coupled with a stable rate of apoptosis accounted for loss of plaque macrophages. These data suggest that therapies to inhibit monocyte recruitment to plaques may constitute a more viable strategy to reduce plaque macrophage burden than attempts to promote migratory egress.
Stephane Potteaux ... Mary G. Sorci-Thomas, Gwendalyn J. Randolph
Pannexin-1 (Panx1) plays a role in the release of ATP and glutamate in neurons and astrocytes. Panx1 can be opened at the resting membrane potential by extracellular ATP via the P2X7 receptor (P2X7R). Panx1 opening has been shown to induce neuronal death and aberrant firing, but its role in neuronal activity has not been established. Here, we report the role of the P2X7R-Panx1 complex in regulating muscarinic acetylcholine 1 (M1) receptor function. P2X7R knockout (P2X7–/–) mice showed greater susceptibility to seizures induced by pilocarpine (PILO), an M1 receptor agonist, than their WT littermates, despite having similar levels of hippocampal M1 receptor expression. This hypersensitivity to PILO in the P2X7–/– mice did not involve the GABA or glutamate system. Both administration of P2X7R antagonists and gene silencing of P2X7R or Panx1 in WT mice increased PILO-induced seizure susceptibility in a process mediated by PKC via intracellular Ca2+ release. Therefore, we suggest that the P2X7R-Panx1 complex may play an important role as a negative modulator of M1 receptor–mediated seizure activity in vivo.
Ji-Eun Kim, Tae-Cheon Kang
Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, fibulin-5 inhibited the β1 integrin–dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5RGE/RGE) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to fibulin-5 knockouts (Fbln5–/–), Fbln5RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with β-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5–/– mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre- and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women.
Madhusudhan Budatha ... R. Ann Word, Hiromi Yanagisawa
Chaoshe Guo ... Anne Moon, Xue Li
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