Obesity comprises great risks for human health, contributing to the development of other diseases such as metabolic syndrome, type 2 diabetes and cardiovascular disease. Previously, obese patients were found to have elevated serum levels of VEGF-C, which correlated with worsening of lipid parameters. We recently identified that neutralization of VEGF-C and -D in the subcutaneous adipose tissue during the development of obesity improves metabolic parameters and insulin sensitivity in mice. To test the hypothesis that VEGF-C plays a role in the promotion of the metabolic disease, we used K14-VEGF-C mice that overexpress human VEGF-C under control of the keratin-14 promoter in the skin and monitored metabolic parameters over time. K14-VEGF-C mice had high levels of VEGF-C in the subcutaneous adipose tissue and gained more weight than wildtype littermates, became insulin resistant and had increased ectopic lipid accumulation at 20 weeks of age on regular mouse chow. The metabolic differences persisted under high-fat diet induced obesity. These results indicate that elevated VEGF-C levels contribute to metabolic deterioration and the development of insulin resistance, and that blockade of VEGF-C in obesity represents a suitable approach to alleviate the development of insulin resistance.

VEGF-C/VEGFR-3 signaling plays a central role in lymphatic development, regulating the budding of lymphatic progenitor cells from embryonic veins and maintaining the expression of PROX1 during later developmental stages. However, how VEGFR-3 activation translates into target gene expression is still not completely understood. We used cap analysis of gene expression (CAGE) RNA sequencing to characterize the transcriptional changes invoked by VEGF-C in LECs and to identify the transcription factors (TFs) involved. We found that MAFB, a TF involved in differentiation of various cell types, is rapidly induced and activated by VEGF-C. MAFB induced expression of PROX1 as well as other TFs and markers of differentiated LECs, indicating a role in the maintenance of the mature LEC phenotype. Correspondingly, E14.5 Mafb(-/-) embryos showed impaired lymphatic patterning in the skin. This suggests that MAFB is an important TF involved in lymphangiogenesis.

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.

Lymphedema is a common complication that occurs after breast cancer treatment in up to 30% of the patients undergoing surgical lymph node excision. It is associated with tissue swelling, fibrosis, increased risk of infection, and impaired wound healing. Despite the pronounced clinical manifestations of the disease, little is known about the morphological and functional characteristics of the lymphatic vasculature during the course of lymphedema progression. We used an experimental murine tail lymphedema model where sustained fluid stasis was generated on disruption of lymphatic flow, resulting in chronic edema formation with fibrosis and adipose tissue deposition. Morphological analysis of the lymphatic vessels revealed a dramatic expansion during the course of the disease, with active proliferation of lymphatic endothelial cells at the early stages of lymphedema. The lymphatic capillaries exhibited progressively impaired tracer filling and retrograde flow near the surgery site, whereas the collecting lymphatic vessels showed a gradually decreasing contraction amplitude with unchanged contraction frequency, leading to lymphatic contraction arrest at the later stages of the disease. Lymphedema onset was associated with pronounced infiltration by immune cells, predominantly Ly6G(+) and CD4(+) cells, which have been linked to impaired lymphatic vessel function.

Glycine receptors (GlyRs) are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs) have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA) are positive modulators of α(1), α(2) and α(3) GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly) potentiate α(1) GlyRs but inhibit α(2) and α(3). This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM) region 2 and intracellular lysine 385 determine the positive modulation of α(1) GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α(2) converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α(1) GlyRs, without affecting inhibition of α(2) and α(3). Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.

Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain.

The gate control theory of pain proposes that inhibitory neurons of the spinal dorsal horn exert critical control over the relay of nociceptive signals to higher brain areas. Here we investigated how the glycinergic subpopulation of these neurons contributes to modality-specific pain and itch processing. We generated a GlyT2::Cre transgenic mouse line suitable for virus-mediated retrograde tracing studies and for spatially precise ablation, silencing, and activation of glycinergic neurons. We found that these neurons receive sensory input mainly from myelinated primary sensory neurons and that their local toxin-mediated ablation or silencing induces localized mechanical, heat, and cold hyperalgesia; spontaneous flinching behavior; and excessive licking and biting directed toward the corresponding skin territory. Conversely, local pharmacogenetic activation of the same neurons alleviated neuropathic hyperalgesia and chloroquine- and histamine-induced itch. These results establish glycinergic neurons of the spinal dorsal horn as key elements of an inhibitory pain and itch control circuit.

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

Removal of synaptically-released glutamate by astrocytes is necessary to spatially and temporally limit neuronal activation. Recent evidence suggests that astrocytes may have specialized functions in specific circuits, but the extent and significance of such specialization are unclear. By performing direct patch-clamp recordings and two-photon glutamate imaging, we report that in the somatosensory cortex, glutamate uptake by astrocytes is slower during sustained synaptic stimulation when compared to lower stimulation frequencies. Conversely, glutamate uptake capacity is increased in the frontal cortex during higher frequency synaptic stimulation, thereby limiting extracellular buildup of glutamate and NMDA receptor activation in layer 5 pyramidal neurons. This efficient glutamate clearance relies on Na+/K+-ATPase function and both GLT-1 and non-GLT-1 transporters. Thus, by enhancing their glutamate uptake capacity, astrocytes in the frontal cortex may prevent excessive neuronal excitation during intense synaptic activity. These results may explain why diseases associated with network hyperexcitability differentially affect individual brain areas.

Inactivation of transcription factor Foxa1 in mice results in neonatal mortality of unknown cause. Here, we report that ablation of Foxa1 causes impaired development and loss of the subthalamic nucleus (STN). Functional deficits in the STN have been implicated in the etiology of Huntington's and Parkinson's disease. We show that neuronal ablation by Synapsin1-Cre-mediated Foxa1 deletion is sufficient to induce hyperlocomotion in mice. Transcriptome profiling of STN neurons in conditional Foxa1 knockout mice revealed changes in gene expression reminiscent of those in neurodegenerative diseases. We identified Ppargc1a, a transcriptional co-activator that is implicated in neurodegeneration, as a Foxa1 target. These findings were substantiated by the observation of Foxa1-dependent demise of STN neurons in conditional models of Foxa1 mutant mice. Finally, we show that the spontaneous firing activity of Foxa1-deficient STN neurons is profoundly impaired. Our data reveal so far elusive roles of Foxa1 in the development and maintenance of STN function.

Cancer stem cells (CSCs) play major roles in cancer initiation, metastasis, recurrence and therapeutic resistance. Targeting CSCs represents a promising strategy for cancer treatment. The purpose of this study was to identify selective inhibitors of breast CSCs (BCSCs). We carried out a cell-based phenotypic screening with cell viability as a primary endpoint, using a collection of 2,546 FDA-approved drugs and drug-like molecules in spheres formed by malignant human breast gland-derived cells (HMLER-shEcad cells, representing BCSCs) and control immortalized non-tumorigenic human mammary cells (HMLE cells, representing normal stem cells). 19 compounds were identified from screening. The chemically related molecules benztropine mesylate and deptropine citrate were selected for further validation and both potently inhibited sphere formation and self-renewal of BCSCs in vitro. Benztropine mesylate treatment decreased cell subpopulations with high ALDH activity and with a CD44+/CD24- phenotype. In vivo, benztropine mesylate inhibited tumor-initiating potential in a 4T1 mouse model. Functional studies indicated that benztropine mesylate inhibits functions of CSCs via the acetylcholine receptors, dopamine transporters/receptors, and/or histamine receptors. In summary, our findings identify benztropine mesylate as an inhibitor of BCSCs in vitro and in vivo. This study also provides a screening platform for identification of additional anti-CSC agents.

Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a > 20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.

The role of lymphangiogenesis in inflammation has remained unclear. To investigate the role of lymphatic versus blood vasculature in chronic skin inflammation, we inhibited vascular endothelial growth factor (VEGF) receptor (VEGFR) signaling by function-blocking antibodies in the established keratin 14 (K14)-VEGF-A transgenic (Tg) mouse model of chronic cutaneous inflammation. Although treatment with an anti-VEGFR-2 antibody inhibited skin inflammation, epidermal hyperplasia, inflammatory infiltration, and angiogenesis, systemic inhibition of VEGFR-3, surprisingly, increased inflammatory edema formation and inflammatory cell accumulation despite inhibition of lymphangiogenesis. Importantly, chronic Tg delivery of the lymphangiogenic factor VEGF-C to the skin of K14-VEGF-A mice completely inhibited development of chronic skin inflammation, epidermal hyperplasia and abnormal differentiation, and accumulation of CD8 T cells. Similar results were found after Tg delivery of mouse VEGF-D that only activates VEGFR-3 but not VEGFR-2. Moreover, intracutaneous injection of recombinant VEGF-C156S, which only activates VEGFR-3, significantly reduced inflammation. Although lymphatic drainage was inhibited in chronic skin inflammation, it was enhanced by Tg VEGF-C delivery. Together, these results reveal an unanticipated active role of lymphatic vessels in controlling chronic inflammation. Stimulation of functional lymphangiogenesis via VEGFR-3, in addition to antiangiogenic therapy, might therefore serve as a novel strategy to treat chronic inflammatory disorders of the skin and possibly also other organs.

Ca(2+)-permeable AMPA receptors are densely expressed in the spinal dorsal horn, but their functional significance in pain processing is not understood. By disrupting the genes encoding GluR-A or GluR-B, we generated mice exhibiting increased or decreased numbers of Ca(2+)-permeable AMPA receptors, respectively. Here, we demonstrate that AMPA receptors are critical determinants of nociceptive plasticity and inflammatory pain. A reduction in the number of Ca(2+)-permeable AMPA receptors and density of AMPA channel currents in spinal neurons of GluR-A-deficient mice is accompanied by a loss of nociceptive plasticity in vitro and a reduction in acute inflammatory hyperalgesia in vivo. In contrast, an increase in spinal Ca(2+)-permeable AMPA receptors in GluR-B-deficient mice facilitated nociceptive plasticity and enhanced long-lasting inflammatory hyperalgesia. Thus, AMPA receptors are not mere determinants of fast synaptic transmission underlying basal pain sensitivity as previously thought, but are critically involved in activity-dependent changes in synaptic processing of nociceptive inputs.

Thrombospondin-2 (TSP2) is an anti-angiogenic matricellular protein that inhibits tumor growth and angiogenesis. Tumor-associated blood vascular endothelial cells (BECs) were isolated from human invasive bladder cancers and from matched normal bladder tissue by immuno-laser capture microdissection. Exon expression profiling analyses revealed a particularly high expression of a short TSP2 transcript containing only the last 9 (3') exons of the full-length TSP2 transcript. Using 5' and 3' RACE (rapid amplification of cDNA ends) and Sanger sequencing, we confirmed the existence of the shorter transcript of TSP2 (sTSP2) and determined its sequence which completely lacked the anti-angiogenic thrombospondin type 1 repeats domain. The largest open reading frame predicted within the transcript comprises 209 amino acids and matches almost completely the C-terminal lectin domain of full-length TSP2. We produced recombinant sTSP2 and found that unlike the full-length TSP2, sTSP2 did not inhibit vascular endothelial growth factor-A-induced proliferation of cultured human BECs, but in contrast when combined with TSP2 blocked the inhibitory effects of TSP2 on BEC proliferation. In vivo studies with stably transfected A431 squamous cell carcinoma cells revealed that full-length TSP2, but not sTSP2, inhibited tumor growth and angiogenesis. This study reveals that the transcriptional program of tumor stromal cells can change to transcribe a new version of an endogenous angiogenesis inhibitor that has lost its anti-angiogenic activity.

GABAA receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2-null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission.

To evaluate the protein expression of connexin 43 (Cx43) in primary urothelial bladder cancer and test its association with the histopathological characteristics and clinical outcome.

Tumor-associated lymphatic vessels (LVs) play multiple roles during tumor progression, including promotion of metastasis and regulation of antitumor immune responses by delivering antigen from the tumor bed to draining lymph nodes (LNs). Under steady-state conditions, LN resident lymphatic endothelial cells (LECs) have been found to maintain peripheral tolerance by directly inhibiting autoreactive T-cells. Similarly, tumor-associated lymphatic endothelium has been suggested to reduce antitumor T-cell responses, but the mechanisms that mediate this effect have not been clarified. Using two distinct experimental tumor models, we found that tumor-associated LVs gain expression of the T-cell inhibitory molecule PDL1, similar to LN resident LECs, whereas tumor-associated blood vessels downregulate PDL1. The observed lymphatic upregulation of PDL1 was likely due to IFN-g released by stromal cells in the tumor microenvironment. Furthermore, we found that blocking PDL1 results in increased T-cell stimulation by antigen-presenting LECs in vitro. Taken together, our data suggest that peripheral, tumor-associated lymphatic endothelium contributes to T-cell inhibition, by a mechanism similar to peripheral tolerance maintenance described for LN resident LECs. These findings may have clinical implications for cancer therapy, as lymphatic expression of PDL1 could represent a new biomarker to select patients for immunotherapy with PD1 or PDL1 inhibitors.

Tumor-associated lymphangiogenesis and lymphatic invasion of tumor cells correlate with poor outcome in many tumor types, including breast cancer. Various explanations for this correlation have been suggested in the past, including the promotion of lymphatic metastasis and an immune-inhibitory function of lymphatic endothelial cells (LECs). However, the molecular features of tumor-associated lymphatic vessels and their implications for tumor progression have been poorly characterized. Here, we report the first transcriptional analysis of tumor-associated LECs directly isolated from the primary tumor in an orthotopic mouse model of triple negative breast cancer (4T1). Gene expression analysis showed a strong upregulation of inflammation-associated genes, including endothelial adhesion molecules such as VCAM-1, in comparison to LECs derived from control tissue. In vitro experiments demonstrated that VCAM-1 is not involved in the adhesion of tumor cells to LECs but unexpectedly promoted lymphatic permeability by weakening of lymphatic junctions, most likely through a mechanism triggered by interactions with integrin α4 which was also induced in tumor-associated LECs. In line with this, in vivo blockade of VCAM-1 reduced lymphatic invasion of 4T1 cells. Taken together, our findings suggest that disruption of lymphatic junctions and increased permeability via tumor-induced lymphatic VCAM-1 expression may represent a new target to block lymphatic invasion and metastasis.