Understanding molecular mechanisms for regeneration of hair follicles provides new opportunities for developing treatments for hair loss and other skin disorders. Here we show that fibroblast growth factor 9 (Fgf9), initially secreted by γδ T cells, modulates hair follicle regeneration after wounding the skin of adult mice. Reducing Fgf9 expression decreases this wound-induced hair neogenesis (WIHN). Conversely, overexpression of Fgf9 results in a two- to threefold increase in the number of neogenic hair follicles. We found that Fgf9 from γδ T cells triggers Wnt expression and subsequent Wnt activation in wound fibroblasts. Through a unique feedback mechanism, activated fibroblasts then express Fgf9, thus amplifying Wnt activity throughout the wound dermis during a crucial phase of skin regeneration. Notably, humans lack a robust population of resident dermal γδ T cells, potentially explaining their inability to regenerate hair after wounding. These findings highlight the essential relationship between the immune system and tissue regeneration. The importance of Fgf9 in hair follicle regeneration suggests that it could be used therapeutically in humans.
Type 2 diabetes mellitus (T2DM) progresses from compensated insulin resistance to beta ceil failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. The Nlrp3 inflammasome has been implicated in obesity-induced insulin resistance and beta cell failure. Endocannabinoids contribute to insuiin resistance through activation of peripheral CB1 receptors (CB1Rs) and also promote beta cell failure. Here we show that beta cell failure in adult ZDF rats is not associated with CB1R signaling in beta ceils, but rather in M1 macrophages infiltrating into pancreatic islets, and that this leads to activation of the Nlrp3-ASC inflammasome in the macrophages. These effects are replicated in vitro by incubating wild-type human or rodent macrophages, but not macrophages from CB1R-deficient [Cnr1−/−) or Nlrp3−/− mice, with the endocannabinoid anandamide. Peripheral CB1R blockade, in vivo depletion of macrophages or macrophage-specific knockdown of CB1R reverses or prevents these changes and restores normoglycemia and glucose-induced insulin secretion. These findings implicate endocannabinoids and inflammasome activation in beta cell failure and identify macrophage-expressed CB1R as a therapeutic target in T2DM.
All patients with metastatic lung, colorectal, pancreatic or head and neck cancers who initially benefit from epidermal growth factor receptor (EGFR)-targeted therapies eventually develop resistance. An increasing understanding of the number and complexity of resistance mechanisms highlights the Herculean challenge of killing tumors that are resistant to EGFR inhibitors. Our growing knowledge of resistance pathways provides an opportunity to develop new mechanism-based inhibitors and combination therapies to prevent or overcome therapeutic resistance in tumors. We present a comprehensive review of resistance pathways to EGFR-targeted therapies in lung, colorectal and head and neck cancers and discuss therapeutic strategies that are designed to circumvent resistance.
The anticancer efficacy of conventional chemotherapies seems to be due,
in part, to augmentation of the host immune reactivity. However, a new study
reveals that two common chemotherapeutic agents, gemcitabine and 5-fluorouracil,
can also activate immune regulatory cells, which stimulates the emergence of
protumorigenic cytokines via inflammasome pathways, limiting the antitumor
efficacy of the drugs (pages 57–64).
Acute myocardial infarction is a severe ischemic disease responsible for heart failure and sudden death. Here, we show that after acute myocardial infarction in mice, mature B lymphocytes selectively produce Ccl7 and induce Ly6Chi monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Genetic (Baff receptor deficiency) or antibody-mediated (CD20- or Baff-specific antibody) depletion of mature B lymphocytes impeded Ccl7 production and monocyte mobilization, limited myocardial injury and improved heart function. These effects were recapitulated in mice with B cell–selective Ccl7 deficiency. We also show that high circulating concentrations of CCL7 and BAFF in patients with acute myocardial infarction predict increased risk of death or recurrent myocardial infarction. This work identifies a crucial interaction between mature B lymphocytes and monocytes after acute myocardial ischemia and identifies new therapeutic targets for acute myocardial infarction.
The protective role of Sirt1 in renal damage was investigated. Sirt1 in proximal tubules (PT) was downregulated before albuminuria occurred in streptozotocin-induced or obese-type (db/db) diabetic mice. PT-specific Sirt1 transgenic (TG) and knockout (KO) mice showed prevention and aggravation of the glomerular changes occurring in diabetes, respectively, and non-diabetic KO mice exhibited albuminuria, suggesting that Sirt1 in PT affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by Sirt1-mediated epigenetic regulation in podocytes contributed to albuminuria. These phenomena were not observed in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PT to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the auto-fluorescence of photoactivatable NMN, and injection of fluorescence-labeled NMN. In human subjects with diabetes, Sirt1 and Claudin-1 levels were correlated with proteinuria level. Sirt1 in PT protects against albuminuria in diabetes through maintaining NMN concentrations around glomeruli and controlling podocyte function.
Using brain surgery, specific areas in the brain can be stimulated with electrical impulses to reversibly change their activity and alleviate symptoms related to mental illnesses. This so-called deep brain stimulation and other methodological advances that even more selectively activate specific group of neurons can give us clues as to what neural circuitry is involved in a particular mental disorder and whether therapeutic activation of these brain areas and neurons may be effective. In ‘Bedside to Bench’, Eric Nestler discusses two trials of individuals with anorexia nervosa in which deep brain stimulation of different brain areas resulted in improvement of behavioral domains associated with the syndrome. The results and potential of this technique in animals and humans may bring us closer to understanding the neurobiology of anorexia nervosa, which still remains a mystery and poses a challenge for treatment. In ‘Bench to Bedside’, Jennifer Warner-Schmidt peruses recent findings that uncover the functional connectivity of brain regions involved in depression and how activation of cortical regions can result in antidepressant effects that can compensate for the malfunction of other brain circuits that results in depression.
Total anomalous pulmonary venous connection (TAPVC) is a potentially lethal congenital disorder that occurs when the pulmonary veins do not connect normally to the left atrium, allowing mixing of pulmonary and systemic blood1. In contrast to the extensive knowledge of arterial vascular patterning, little is known about the patterning of veins. Here we show that the secreted guidance molecule semaphorin 3d (Sema3d) is crucial for the normal patterning of pulmonary veins. Prevailing models suggest that TAPVC occurs when the midpharyngeal endothelial strand (MES), the precursor of the common pulmonary vein, does not form at the proper location on the dorsal surface of the embryonic common atrium2,3. However, we found that TAPVC occurs in Sema3d mutant mice despite normal formation of the MES. In these embryos, the maturing pulmonary venous plexus does not anastomose uniquely with the properly formed MES. In the absence of Sema3d, endothelial tubes form in a region that is normally avascular, resulting in aberrant connections. Normally, Sema3d provides a repulsive cue to endothelial cells in this area, establishing a boundary. Sequencing of SEMA3D in individuals with anomalous pulmonary veins identified a phenylalanine-to-leucine substitution that adversely affects SEMA3D function. These results identify Sema3d as a crucial pulmonary venous patterning cue and provide experimental evidence for an alternate developmental model to explain abnormal pulmonary venous connections.
Elevated blood pressure (BP) and chronic kidney disease (CKD) are complex traits representing major global health problems1,2. Multiple genome-wide association studies (GWAS) identified common variants giving independent susceptibility for CKD and hypertension in the promoter of the UMOD gene3-9, encoding uromodulin, the major protein secreted in the normal urine. Despite compelling genetic evidence, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants directly increase UMOD expression in vitro and in vivo. We modeled this effect in transgenic mice and showed that uromodulin overexpression leads to salt-sensitive hypertension and to age-dependent renal lesions that are similarly observed in elderly subjects homozygous for UMOD risk variants. We demonstrate that the link between uromodulin and hypertension is caused by activation of the renal sodium co-transporter NKCC2. This very mechanism is relevant in humans, as pharmacological inhibition of NKCC2 is more effective in lowering BP in hypertensive patients homozygous for UMOD risk variants. Our findings establish a link between the genetic susceptibility to hypertension and CKD, the control of uromodulin expression and its role in a salt-reabsorbing tubular segment of the kidney. These data point to uromodulin as a novel therapeutic target to lower BP and preserve renal function.
Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. We report that Pdgfrb-Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the αv integrin subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Depletion of the αv integrin subunit in HSCs protected mice from CCl4-induced hepatic fibrosis, whereas global loss of αvβ3, αvβ5 or αvβ6 or conditional loss of αvβ8 on HSCs did not. Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of αv integrins using this system was also protective in models of pulmonary and renal fibrosis. Critically, pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. These data identify a core pathway that regulates fibrosis, and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.
We describe a quantitative neuroimaging method to estimate the macromolecular tissue volume (MTV), a fundamental measure of brain anatomy. By making measurements over a range of field strengths and scan parameters, we tested the key assumptions and the robustness of the method. The measurements confirm that a consistent, quantitative estimate of macromolecular volume can be obtained across a range of scanners. MTV estimates are sufficiently precise to enable a comparison between data obtained from an individual subject with control population data. We describe two applications. First, we show that MTV estimates can be combined with T1 and diffusion measurements to augment our understanding of the tissue properties. Second we show that MTV provides a sensitive measure of disease status in individual patients with multiple sclerosis. The MTV maps are obtained using short clinically appropriate scans that can reveal how tissue changes influence behavior and cognition.
Myelin; Demyelinating disorders; Multiple sclerosis; T1 relaxometry; Proton density; diffusion; macromolecular tissue volume
Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. PAR4 thrombin receptor induced platelet aggregation and calcium mobilization were significantly greater in black subjects. Numerous differentially expressed (DE) RNAs were associated with both race and PAR4 reactivity, including phosphatidylcholine transfer protein (PCTP), and platelets from blacks expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked activation of platelets or megakaryocytic cell lines through PAR4 but not PAR1. MiR-376c levels were DE by race and PAR4 reactivity, and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. MiR-376c regulated expression of PC-TP in human megakaryocytes. A disproportionately high number of miRNAs DE by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus, and were lower in platelets from blacks. These results support PC-TP as a regulator of the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race, and emphasize a need to consider race effects when developing anti-thrombotic drugs.
Podocytes are critical in the maintenance of a healthy glomerular filter, however they have been difficult to study in the intact kidney due to technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PEC) in vivo. In Podocin-GFP mice podocytes formed sporadic multi-cellular clusters after unilateral ureteral ligation (UUO) and migrated into the parietal Bowman’s capsule. The tracking of single cells in Podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP, or RFP revealed the simultaneous migration of multiple podocytes. In PEPCK-GFP mice serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support the highly dynamic rather than static nature of the glomerular environment and cellular composition. Future application of this new approach promises to advance our understanding of the mechanisms of glomerular injury and regeneration.
Tumor recurrence represents a major clinical challenge. Our data show that emergent recurrent tumors acquire a phenotype radically different from that of their originating primary tumors. This phenotype allows them to evade a host-derived innate immune response elicited by the progression from minimal residual disease (MRD) to actively growing recurrence. Screening for this innate response predicted accurately in which mice recurrence would occur. Premature induction of recurrence resensitized MRD to the primary therapy, suggesting a possible paradigm shift for clinical treatment of dormant disease in which the current expectant approach is replaced with active attempts to uncover MRD before evolution of the escape phenotype is complete. By combining screening with second-line treatments targeting innate insensitivity, up to 100% of mice that would have otherwise relapsed were cured. These data may open new avenues for early detection and appropriately timed, highly targeted treatment of tumor recurrence irrespective of tumor type or frontline treatment.
Ammonia is a ubiquitous waste product of protein metabolism that can accumulate in numerous metabolic disorders, causing neurological dysfunction ranging from cognitive impairment to tremor, ataxia, seizures, coma and death1. The brain is especially vulnerable to ammonia as it readily crosses the blood-brain barrier in its gaseous form, NH3, and rapidly saturates its principal removal pathway located in astrocytes2. Thus, we wanted to determine how astrocytes contribute to the initial deterioration of neurological functions characteristic of hyperammonemia in vivo. Using a combination of two-photon imaging and electrophysiology in awake head-restrained mice, we show that ammonia rapidly compromises astrocyte potassium buffering, increasing extracellular potassium concentration and overactivating the Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) in neurons. The consequent depolarization of the neuronal GABA reversal potential (EGABA) selectively impairs cortical inhibitory networks. Genetic deletion of NKCC1 or inhibition of it with the clinically used diuretic bumetanide potently suppresses ammonia-induced neurological dysfunction. We did not observe astrocyte swelling or brain edema in the acute phase, calling into question current concepts regarding the neurotoxic effects of ammonia3,4. Instead, our findings identify failure of potassium buffering in astrocytes as a crucial mechanism in ammonia neurotoxicity and demonstrate the therapeutic potential of blocking this pathway by inhibiting NKCC1.
Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the coinhibitory receptor CTLA-4 develop fatal autoimmunity characterized by massive lymphocytic invasion into non-lymphoid tissues. Here we demonstrate that the CD28 costimulatory pathway regulates the trafficking of self-reactive Ctla4−/− T cells to tissues. Co-ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation, but instead causes self-reactive Ctla4−/− T cells to accumulate in secondary lymphoid organs. Despite a fulminant autoimmune process in the lymphoid compartment, Itk−/−Ctla4−/− mice are otherwise healthy and exhibit a long lifespan. We propose that ITK licenses autoreactive T cells to enter tissues to mount destructive immune responses. Importantly, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of Type I diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.
Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear1–6. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.
A long-standing question in the HIV field is why HIV-1 fails to replicate
in resting CD4+ T cells. A new study shows that the host
deoxynucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) sterile alpha
motif and histidine/aspartic domain-containing protein 1 (SAMHD1), previously
shown to block HIV infection in myeloid cells, also restricts HIV replication in
resting CD4+ T cells by hydrolyzing dNTPs, which are needed for
reverse transcription of the virus (aaa-bbb).
The adipocyte fatty-acid–binding protein, aP2, has an important role in regulating systemic insulin resistance and lipid metabolism. Here we demonstrate that aP2 is also expressed in macrophages, has a significant role in their biological responses and contributes to the development of atherosclerosis. Apolipoprotein E (ApoE)-deficient mice also deficient for aP2 showed protection from atherosclerosis in the absence of significant differences in serum lipids or insulin sensitivity. aP2-deficient macrophages showed alterations in inflammatory cytokine production and a reduced ability to accumulate cholesterol esters when exposed to modified lipoproteins. Apoe–/– mice with Ap2+/+ adipocytes and Ap2–/– macrophages generated by bone-marrow transplantation showed a comparable reduction in atherosclerotic lesions to those with total aP2 deficiency, indicating an independent role for macrophage aP2 in athero-genesis. Through its distinct actions in adipocytes and macrophages, aP2 provides a link between features of the metabolic syndrome and could be a new therapeutic target for the prevention of atherosclerosis.
We report that breast cancer cells that infiltrate the lungs support their own metastasis-initiating ability by expressing tenascin C (TNC). We find that the expression of TNC, an extracellular matrix protein of stem cell niches, is associated with the aggressiveness of pulmonary metastasis. Cancer cell–derived TNC promotes the survival and outgrowth of pulmonary micrometastases. TNC enhances the expression of stem cell signaling components, musashi homolog 1 (MSI1) and leucine-rich repeat–containing G protein– coupled receptor 5 (LGR5). MSI1 is a positive regulator of NOTCH signaling, whereas LGR5 is a target gene of the WNT pathway. TNC modulation of stem cell signaling occurs without affecting the expression of transcriptional enforcers of the stem cell phenotype and pluripotency, namely nanog homeobox (NANOG), POU class 5 homeobox 1 (POU5F1), also known as OCT4, and SRY-box 2 (SOX2). TNC protects MSI1-dependent NOTCH signaling from inhibition by signal transducer and activator of transcription 5 (STAT5), and selectively enhances the expression of LGR5 as a WNT target gene. Cancer cell– derived TNC remains essential for metastasis outgrowth until the tumor stroma takes over as a source of TNC. These findings link TNC to pathways that support the fitness of metastasis-initiating breast cancer cells and highlight the relevance of TNC as an extracellular matrix component of the metastatic niche.
Targeting the mammalian target of rapamycin (mTOR) is a promising strategy for cancer therapy. However, the mTOR kinase functions in two complexes, TORC1 and TORC2, neither of which is fully inhibited by the allosteric inhibitor rapamycin or analogs. We compared rapamycin with the active-site TORC1/2 inhibitor PP242, in acute leukemia models harboring the Philadelphia chromosome (Ph) translocation. We demonstrate that PP242, but not rapamycin, causes death of mouse and human leukemia cells. In vivo, PP242 delays leukemia onset and augments the effects of current front-line tyrosine kinase inhibitors, more effectively than rapamycin. Surprisingly, PP242 has much weaker effects than rapamycin on proliferation and function of normal lymphocytes. PI-103, a less selective TORC1/2 inhibitor that also targets phosphoinositide 3-kinase, is more immunosuppressive than PP242. These findings establish that Ph+ transformed cells are more sensitive than normal lymphocytes to selective TORC1/2 inhibitors, and support the development of such inhibitors for leukemia therapy.
A common feature of many neurodegenerative diseases is the deposition of β-sheet-rich amyloid aggregates formed by proteins specific to these diseases. These protein aggregates are thought to cause neuronal dysfunction, directly or indirectly. Recent studies have strongly implicated cell-to-cell transmission of misfolded proteins as a common mechanism for the onset and progression of various neurodegenerative disorders. Emerging evidence also suggests the presence of conformationally diverse ‘strains’ of each type of disease protein, which may be another shared feature of amyloid aggregates, accounting for the tremendous heterogeneity within each type of neurodegenerative disease. Although there are many more questions to be answered, these studies have opened up new avenues for therapeutic interventions in neurodegenerative disorders.
Current methods of protein detection are insensitive to detecting subtle changes in oncoprotein activation that underlie critical cancer signaling processes. The requirement for large numbers of cells precludes serial tumor sampling for assessing a response to therapeutics. Therefore, we have developed a nano-fluidic proteomic immunoassay (NIA) to quantify total and low abundance protein isoforms in 4 nanoliters of lysate. Our method could quantify levels of MYC and BCL2 proteins in Burkitt’s versus follicular lymphoma; identify changes in activation of ERK1/2, MEK1, STAT3/5, JNK and caspase 3 in imatinib-treated chronic myelogeneous leukemia (CML) cells; measure a novel change in phosphorylation of an ERK2 isomer in CML patients who responded to imatinib; and detect a decrease in STAT3/5 phosphorylation in lymphoma patients treated with atorvastatin. Therefore, we have described a novel and highly sensitive method for interrogating oncoprotein expression and phosphorylation in clinical specimens for the development of new therapeutics for cancer.
Fragile X Syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of Fmr1, which encodes the translational repressor protein FMRP. FMRP and CPEB, an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs, and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1-/y Cpeb−/− double knockout mice displayed significant amelioration of biochemical, morphological, electrophysiological, and behavioral phenotypes associated with FXS. Acute depletion of CPEB in the hippocampus of Fmr1 -/y mice rescued working memory deficits, demonstrating reversal of this FXS phenotype in adults. Finally, we find that FMRP and CPEB balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS, and that the maintenance of this homeostasis by FMRP and CPEB is necessary for normal neurologic function.