Imaging mass spectrometry (IMS) provides untargeted, highly multiplexed maps of molecular distributions in tissue. Ion images are routinely presented as heatmaps and can be overlaid onto complementary microscopy images that provide greater context. However, heatmaps use transparency blending to visualize both images, obscuring subtle quantitative differences and distribution gradients. Here, we developed a contour mapping approach that combines information from IMS ion intensity distributions with that of stained microscopy. As a case study, we applied this approach to imaging data from Staphylococcus aureus-infected murine kidney. In a univariate, or single molecular species, use-case of the contour map representation of IMS data, certain lipids colocalizing with regions of infection were selected using Pearson's correlation coefficient. Contour maps of these lipids overlaid with stained microscopy showed enhanced visualization of lipid distributions and spatial gradients in and around the bacterial abscess as compared to traditional heatmaps. The full IMS data set comprising hundreds of individual ion images was then grouped into a smaller subset of representative patterns using non-negative matrix factorization (NMF). Contour maps of these multivariate NMF images revealed distinct molecular profiles of the major abscesses and surrounding immune response. This contour mapping workflow also enabled a molecular visualization of the transition zone at the host–pathogen interface, providing potential clues about the spatial molecular dynamics beyond what histological staining alone provides. In summary, we developed a new IMS-based contour mapping approach to augment classical stained microscopy images, providing an enhanced and more interpretable visualization of IMS-microscopy multimodal molecular imaging data sets.
The early mechanisms and genetic risk factors driving the pathogenesis of preeclampsia (PE), a cardiovascular disorder of pregnancy, remain largely unclear. Various hormone activators of Gα q second-messenger signaling pathways have been implicated in PE. Regulator of G-protein Signaling 2 (RGS2) acts as an endogenous terminator of Gα q signaling and previous studies identified a SNP (rs4606), which results in reduced RGS2 mRNA, as a risk factor for development of PE and its sequelae. We hypothesized reduced placental expression of RGS2 may precipitate the development of PE due to disinhibited Gα q signaling. In silico reanalysis of publically available dataset GSE75010 revealed RGS2 mRNA is reduced in placentas from pre-term PE pregnancies compared to normal pre-term pregnancies (con: 8.73 ± 0.07 n=35, PE: 8.37 ± 0.055 n=49, p<0.05). Using human placental tissue samples from the University of Iowa Maternal-Fetal Tissue Bank, we confirmed RGS2 mRNA is reduced in PE placentas (19% of control, p<0.05), despite a lack of correlation between the rs4606 SNP and PE. Additionally, in further reanalysis of other datasets, RGS2 mRNA is among the highest-expressed RGS member in normal human placenta, and appears to be selectively reduced in syncytio- and invasive cytotrophoblasts during PE (GSE93839, -26.3%, -23.3% of control). We next examined RGS2 expression in mouse placenta by FISH and found RGS2 mRNA colocalizes with markers of syncytiotrophoblast II (GCMA) and spongiotrophoblast (Tpbpα) layers. To test the effect of RGS2 loss during pregnancy, wildtype C57BL/6J female mice were mated with RGS2-deficient sires and developed diastolic hypertension, placental hypoxia by HIF1α ELISA (con 0.144±0.004, RGS2-KO 0.155±0.004 AU, p<0.05, n=5 each), and reduced placental PlGF mRNA (fold; con=1.0 n=7, RGS2-KO=0.23 n=12, p<0.05), compared to females mated with RGS2 littermate sires. These data support the concept that loss of RGS2 may contribute to the pathogenesis of PE rather than simply correlating with the disorder. Taken together, we have shown placental RGS2 is suppressed in PE, RGS2 is present in cytoplasm of specific layers of trophoblasts, and loss of feto-placental RGS2 is sufficient to cause placental hypoxia and maternal diastolic hypertension.
Front cover: The cover image, by Erin M. Fricke et al., is based on the Original Article LPS-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood, DOI 10.1111/aji.12816 Front cover: The cover image, by Erin M. Fricke et al., is based on the Original Article LPS-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood, DOI 10.1111/aji.12816
The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor α-chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions including asthma. STAT6 binds poly(ADP-ribose) polymerase (PARP)14, an ADP-ribosyl monotransferase. Elimination of PARP14 by gene targeting led to attenuation of OVA-specific allergic lung inflammation. However, PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation, and it is not clear whether inhibition of the catalytic function has any biological consequence. Using BALB/c mice sensitized to the allergen Alternaria alternata, we show that peroral administration of RBN012759, a highly selective inhibitor of ADP-ribosylation by PARP14 with negligible impact on other members of the PARP gene family, achieved biologically active plasma concentrations and altered several responses to the Ag. Specifically, the pharmaceutical compound decreased mucus after allergen challenge, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that PARP14 catalytic activity can contribute to pathogenesis in allergic or atopic processes and propose that other biological endpoints dependent on ADP-ribosylation by PARP14 can be targeted using selective inhibition.
Recent studies from our laboratory have demonstrated that angiotensin II (ANG) type 1A receptors (AT1A) specifically localize to the subset of neurons which express both the leptin receptor (LepR) and agouti‐related peptide (AgRP). These are critically involved in the control of thermogenic adipose sympathetic nerve activity (SNA) and thereby resting metabolic rate (RMR).This mechanism appears to involve AT1A‐mediated suppression of gamma‐aminobutyric acid (GABA) synthesis and packaging in AgRP neurons of the arcuate nucleus (ARC). It remains unclear, however, how LepR signaling results in AT1A activation within AgRP neurons. We hypothesize that activation of LepR in POMC and/or AgRP neurons results in de novo synthesis and local release of angiotensinogen (AGT) within the ARC, and consequently increased autocrine or paracrine ANG signaling within the ARC. To test this hypothesis, we examined the localization of AGT mRNA expression within the ARC of wildtype C57BL/6J mice by fluorescent in situ hybridization methods (RNAscope) and confocal fluorescent microscopy. Consistent with our working hypothesis, cells within the ARC expressing AGT mRNA colocalize with both AgRP and POMC. Cells within the ARC expressing insulin II (INS2) also expressed AGT. Finally, as expected, AGT was also expressed in astrocytes, colocalizing with glial fibrillary acidic protein (GFAP). We and others have previously demonstrated that LepR activation increases phosphorylation of the signal transduction and activator of transcription‐3 (STAT3) transcription factor, and that p‐STAT3 appears to stimulate AGT expression in other cell types including cardiac myocytes and hepatocytes. We therefore hypothesize that the phosphorylation of STAT3 by LepR activation is mechanistically involved in the regulation of AGT expression by leptin in POMC and AgRP neurons. Ongoing studies are aimed at (i) examining AGT expression control by pSTAT3 in POMC and AgRP neurons of the ARC, and (ii) using Cre‐lox recombination methods to selectively disrupt the AGT gene in LepR, POMC and AgRP neurons to study the functional significance of AGT in these cells in the control of SNA and RMR, and thereby energy homeostasis.
Background Vision loss in inherited retinal diseases such as age‐related macular degeneration or retinitis pigmentosa result from death of the photoreceptor cells of the outer retina. Subretinal transplantation of induced pluripotent stem cell‐derived retinal progenitor cells (iPSC‐RPCs) has been found somewhat effective in restoring vision in blind animals, although survival of transplanted cells is poor. Polymeric support scaffolds have been shown to greatly enhance cellular survival and integration post‐transplantation, however attempts to materialize this concept have been largely unsuccessful for two reasons: 1) mechanical mis‐matching between material and the host retina and 2) improper cell packing . The goal of this study was to determine the ideal polymer fabrication parameters that promote optimal cell packing and biocompatibility. Methods Methacrylate‐functionalized PCL scaffolds were polymerized with either Irgacure 651 or Irgacure 369 using two‐photon polymerization. Compressive modulus was measured using dynamic mechanical analysis. Four‐month‐old Yucatan mini pigs (n=10, 5 with Irgacure 651 and 5 with Irgacure 369) received 5mm diameter scaffolds containing iPSC‐RPCs via subretinal transplantation. Animals were examined clinically at 1 month post‐surgery and were subsequently sacrificed for histological analysis of the retina. Results PCL scaffolds were successfully generated using both photoinitiators. Scaffolds generated using Irgacure 651 cured rapidly and had a tendency to curl. Both Irgacure 651‐ and Irgacure 369‐generated scaffolds supported attachment and survival of human iPSC‐RPCs and both were successfully transplanted into the porcine subretinal space. At one month post‐transplantation, 50% of the retinas that received the Irgacure 651 scaffolds were attached. In the animals that received Irgacure 369 scaffolds all of the retinas reattached with no signs of ocular inflammation. In these animals retinal integrity, including maintenance of photoreceptor outer segments, was maintained. Conclusion PCL scaffolds support the maintenance of human iPSC‐RPCs and were biocompatible in the pig retina. These scaffolds may be ideal for retinal transplantation. Support or Funding Information Wynn Institute for Vision Research
Preeclampsia is a disease of pregnancy and the postpartum period, affecting 5-7% of all U.S. pregnancies. We recently determined that elevated secretion of arginine vasopressin (AVP) in humans is detected as early as the 6th week of pregnancy in the women that subsequently develop preeclampsia. Further, we have determined that in C57BL/6 mice, chronic AVP infusion serves as a clinically-relevant and pregnancy-specific model of preeclampsia. Although the pathogenesis of preeclampsia is still poorly understood, an inflammatory immune response is clearly involved. AVP secretion is stimulated by proinflammatory cytokines and AVP in turn can act upon receptors present on lymphocytes. We hypothesize that hypersecretion of AVP results in maternal-fetal immune alterations in preeclamptics. Immune responses were evaluated in wild-type C57BL/6 female mice chronically infused with AVP (24 ng/hr or saline s.c.) throughout pregnancy. We observed an increase in the frequency of IL-12 (saline N=7: 1.1±0.2, vs AVP N=9: 2.2±0.4, P<0.05), IL-17 (saline N=7: 1.2±0.17, vs AVP N=9: 2.6±0.4, P<0.05) and TNFa (saline N=8: 0.9±0.1, vs AVP N=9: 1.6±0.1, P<0.005) expressing lymphocytes isolated from AVP mice. Additionally, the frequency of IFNg expressing lymphocytes was increased in AVP mice, approaching statistical significance (saline N=8: 2.2±0.9, vs AVP N=9: 3.0±0.4, P=0.059). The frequency of IL-6, IL-10, and LAP expressing splenic lymphocytes were similar between saline vs. AVP mice. Interestingly, maternal plasma revealed a significant reduction in IL-17 (saline N=4: 1.0e+7±3.8e+6, vs AVP N=5: 3.1e=5±1.3e+5 ng/g, P<0.05) in AVP mice. Neither IL-4 nor IFNg were detectable in maternal plasma of either group. Additionally, IL-17 trended higher in the amnionic fluid and lower in the placenta of AVP mice. IL-4 production trended lower in both the placenta and amnionic fluid of AVP-infused mice, while IFNg was similar between groups. These data support our hypothesis that AVP hypersecretion results in the maternal-fetal immune alterations associated with the development of preeclampsia. Ongoing experiments are aimed at identifying the lymphocytes and cytokines involved as well as local vs. systemic immune dysfunction associated with AVP-induced preeclampsia.
