Armc5 deletion causes developmental defects and compromises T-cell immune responses.

Armadillo repeat containing 5 (ARMC5) is a cytosolic protein with no enzymatic activities. Little is known about its function and mechanisms of action, except that gene mutations are associated with risks of primary macronodular adrenal gland hyperplasia. Here we map Armc5 expression by in situ hybridization, and generate Armc5 knockout mice, which are small in body size. Armc5 knockout mice have compromised T-cell proliferation and differentiation into Th1 and Th17 cells, increased T-cell apoptosis, reduced severity of experimental autoimmune encephalitis, and defective immune responses to lymphocytic choriomeningitis virus infection. These mice also develop adrenal gland hyperplasia in old age. Yeast 2-hybrid assays identify 16 ARMC5-binding partners. Together these data indicate that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signalling pathways.

Multimodal detection of GM2 and GM3 lipid species in the brain of mucopolysaccharidosis type II mouse by serial imaging mass spectrometry and immunohistochemistry.

Mucopolysaccharidosis type II (Hunter's disease) mouse model (IdS-KO) was investigated by both imaging mass spectrometry (IMS) and immunohistochemistry (IHC) performed on the same tissue sections. For this purpose, IdS-KO mice brain sections were coated with sublimated 1,5-diaminonaphtalene and analyzed by high spatial resolution IMS (5 µm) and anti-GM3 IHC on the same tissue sections to characterize the ganglioside monosialated ganglioside (GM) deposits found in Hunter's disease. IMS analysis have found that two species of GM3 and GM2 that are only different due to the length of their fatty acid residue (stearic or arachidic residue) were overexpressed in the IdS-KO mice compared to a control mouse. GM3 and GM2 were characterized by on-tissue exact mass and MS/MS compared to a GM3 standard. Realignment of both IMS and IHC data sets further confirmed the observed regioselective signal previously detected by providing direct correlation of the IMS image for the two GM3 overly expressed MS signals with the anti-GM3 IHC image. Furthermore, these regioselective GM MS signals were also found to have highly heterogeneous distributions within the GM3-IHC staining. Some deposits showed high content in GM3 and GM2 stearic species (r = 0.74) and others had more abundant GM3 and GM2 arachidic species (r = 0.76). Same-section analysis of Hunter's disease mouse model by both high spatial resolution IMS and IHC provides a more in-depth analysis of the composition of the GM aggregates while providing spatial distribution of the observed molecular species. Graphical Abstract Ganglioside imaging mass spectrometry followed by immunohistochemistry performed on the same tissue section.

Assessment of pathological response to therapy using lipid mass spectrometry imaging.

In many cancers, the establishment of a patient's future treatment regime often relies on histopathological assessment of tumor tissue specimens in order to determine the extent of the 'pathological response' to a given therapy. However, histopathological assessment of pathological response remains subjective. Here we use MALDI mass spectrometry imaging to generate lipid signatures from colorectal cancer liver metastasis specimens resected from patients preoperatively treated with chemotherapy. Using these signatures we obtained a unique pathological response score that correlates with prognosis. In addition, we identify single lipid moieties that are overexpressed in different histopathological features of the tumor, which have potential as new biomarkers for assessing response to therapy. These data show that computational methods, focusing on the lipidome, can be used to determine prognostic markers for response to chemotherapy and may potentially improve risk assessment and patient care.

Oxytocin in cardiac ontogeny.

Previous studies demonstrated the presence of oxytocin (OT) and oxytocin receptors (OTRs) in the heart. The present work provides results supporting a potential role of OT in cardiomyogenesis. Here, we show a maximal OT and OTR protein level in the developing rat heart at day 21 of gestation and postnatal days 1-4, when cardiac myocytes are at a stage of intense hyperplasia. Between postnatal days 1 and 66, OT decreased linearly in all heart chambers (4.1- to 6.6-fold). Correspondingly, immunocytochemistry demonstrated that OTRs, which were eminent in postnatal cardiomyocytes, declined with age to low levels in adults. Interestingly, in coronary vasculature, OTRs developed in endothelial cells at postnatal days 12 and 22 and achieved a plateau in adult rats. These findings suggest that OT can be involved in developmental formation of the coronary vessels. In vivo, the OT/OTR system in the fetal heart was sensitive to the actions of retinoic acid (RA), recognized as a major cardiac morphogen. RA treatment produced a significant increase (2- to 3-fold) both in the OT concentration and in the OT mRNA levels. Ex vivo, an OT antagonist inhibited RA-mediated cardiomyocyte differentiation of P19 embryonic stem cells. The decline of cardiac OT expression from infancy to adulthood of the rat and changes in cell types expressing OTR indicate a dynamic regulation of the OT system in the heart rather than constitutive expression. The results support the hypothesis that RA induces cardiomyogenesis by activation of the cardiac OT system.

The neuropeptide-degrading enzyme NL1 is expressed in specific neurons of mouse brain.

Metalloendopeptidases of the M13 family were shown to play critical roles in normal physiological processes such as pain control, hypertension and phosphate metabolism, and in pathological states such as Alzheimer's disease. Recently, NL1, a novel member of the family, has been identified and shown to be expressed in several tissues both as a membrane-bound and a secreted protein. As a further step to understand the physiological role(s) of NL1 in mouse, we mapped NL1 mRNA expression pattern in embryos and in young animals at postnatal days p1 and p3, and in adult nervous tissue, using in situ hybridization at the cellular level. No expression could be detected in embryos and young animals. In contrast, NL1 expression was evident in adult brain, pituitary gland and spinal cord. In the central nervous system (CNS), NL1 mRNA was predominantly found in the ventro-posterior regions, which are mostly associated with vegetative functions. At the cellular level, NL1 mRNA was non-uniformly distributed within subpopulations of neurons. In the spinal cord, specific signal was observed in the gray matter. Then, in order to identify putative relevant substrates for NL1, we studied its enzymatic activity towards peptides known to be co-expressed in the NL1-positive domains. Our study showed that NL1 degrades several of these peptides in vitro, the most readily degraded peptides being Bradykinin and Substance P. These results suggest that NL1 is likely to play a critical role in the central nervous system.

Coordinated regulation and colocalization of alphav integrin and its activating enzyme proprotein convertase PC5 in vivo.

Integrin alphav is involved in intracellular-extracellular signaling important for cytoskeleton alterations and control of cell movement. In vitro experiments indicate that the integrin alphav-subunit undergoes post-translational endoproteolytic cleavage. This type of activation requires the presence of suitable kexin/subtilisin-like proprotein convertases. In vitro experiments have demonstrated that, among several proprotein convertases, PC5A, and to a threefold lesser extent furin, can activate alphav integrin. The biological significance of these in vitro data would be further supported by a coexpression and coordinated regulation of the gene expression of alphav integrin and its activating enzyme PC5 in vivo. In the present study we investigated the regulation of alphav integrin and PC5 following balloon injury in vivo. Comparative immunocytochemistry revealed a coordinated regulation of alphav integrin and PC5 during vascular remodeling in rodents. Integrin alphav was found to be upregulated in PCNA-positive, proliferating vascular smooth muscle cells. Northern blots revealed no significant regulation of furin mRNA, whereas PC5A mRNA increased during vascular remodeling, suggesting that PC5 is the major convertase during neointima formation in vivo. Incubation of vascular smooth muscle cells with the Golgi-disturbing agent brefeldin A inhibited alphav integrin maturation, indicating that endoproteolytic cleavage occurs in the trans-Golgi network, were PC5 is localized. Thus, the present study further supports the concept that activation of alphav integrin occurs in the trans-Golgi network in vascular smooth muscle cells and involves PC5.

Genistein supplementation stimulates the oxytocin system in the aorta of ovariectomized rats.

OBJECTIVE: In the present study, we localized oxytocin (OT) and its receptor (OTR) in the rat aorta, and investigated whether genistein, an isoflavonic phytoestrogen, influences their expression in ovariectomized (OVX) rats deficient in estrogen. METHODS AND RESULTS: OVX Sprague-Dawley rats were randomized to the following groups: genistein (from 0.02 to 5 microg/g/day, s.c. for 10 days), estradiol (E(2,) 0.1 microg/g/day, s.c. for 10 days) or their respective vehicles. OT and OTR immunostaining was concentrated in the aortic tunica intima, suggesting their paracrine/autocrine action within endothelial cells. Reverse transcription-polymerase chain reaction analysis showed that 1 and 5 microg/g but not 0.1 microg/g genistein elevated OT mRNA (2-fold P<0.05), OTR mRNA (2.5-fold, P<0.05) and endothelial nitric oxide synthase (eNOS) mRNA (2-fold, P<0.05) in the aorta of OVX rats. In addition, genistein treatment increased estrogen receptor alpha (ERalpha) (2- to 3-fold, P<0.05) but resulted in a 50% decrease of ERbeta (P<0.05). These genistein effects were neutralized by treatment of OVX rats with the ER antagonist ICI 182,780 (1.5 microg/g/day, s.c. for 10 days). Similarly, Western blot analysis revealed an increase of 67-kDa OTR, 140-kDa eNOS, 62-kDa ERalpha and a decrease of 55-kDa ERbeta (P<0.05) in the aorta of OVX rats treated with genistein. In contrast, the treatment of OVX rats with E(2) elevated ERbeta mRNA (1.5 fold, P<0.05) but similarly to genistein increased OT, OTR, eNOS and ERalpha mRNA. CONCLUSION: These results provide the first evidence of OT and OTR co-localization in endothelial cells. The response to genistein via ER activation can be regarded as a recovery from endothelial dysfunction induced by ovariectomy.

Declining expression of neprilysin in Alzheimer disease vasculature: possible involvement in cerebral amyloid angiopathy.

Molecular, genetic, and pharmacological studies have shown that neprilysin (also called NEP) catabolizes amyloid beta peptides (A beta) in healthy conditions. However, in Alzheimer disease (AD), A beta accumulates forming senile plaques in brain parenchyma and amyloid deposition around blood vessels. In this study, we tested at cellular level the relationship between neprilysin and A beta in human healthy and AD brain. Our results provided evidence for declining levels of neprilysin in AD brains as compared to healthy controls in parallel with increasing deposition of A beta. In hippocampus of AD individuals we observed a significant down-regulation of neprilysin expression in pyramidal neurons, consistent with the possibility that neprilysin controls the level of A beta accumulation and plaque formation in this area. In the cortex and leptomeninges, neprilysin was expressed in the smooth muscle cells of blood vessels. In sections from AD patients we observed a clear inverse relationship between neprilysin and A beta peptide levels in the vasculature, implicating neprilysin in cerebral amyloid angiopathy.

BetaAPP and furin mRNA concentrates in immature senile plaques in the brain of Alzheimer patients.

This study examined the possibility that in Alzheimer disease (AD) beta-amyloid precursor protein (betaAPP) mRNA is delivered to senile plaques (SPs) via dendritic processes. BetaAPP mRNA was detected in SPs by in situ hybridization, using a 1.4-kb cRNA in which both [35S]-UTP and [35S]-CTP were incorporated together. The betaAPP mRNA was compared with that of furin, a proteolytic enzyme putatively involved in betaAPP processing, and its orthologue proprotein convertase PCI served as a control. Human presenile AD cases with mostly immature SPs and AD cases generally with mature SPs were analyzed. To decrypt SPs after hybridization, brain sections were stained with thioflavin S. To establish relationships between the density of dystrophic fibers, the degree of plaque maturation, and the concentration of mRNA in SPs, the plaque maturity markers Abeta(1-42) and Abeta(1-40) peptides were co-localized with neurofilament protein 200 and compared with microtubule-associated protein 2 (MAP 2). The results suggest that immature, Abeta(1-42)- and dystrophic dendrite-containing SPs (but not mature SPs containing Abeta(1-40) and missing dystrophic dendrites) are capable of concentrating specific mRNAs. Dystrophic dendrites may thus serve as a route for the transport of specific mRNAs from the cell bodies to SPs.

Proprotein convertase PC5 regulation by PDGF-BB involves PI3-kinase/p70(s6)-kinase activation in vascular smooth muscle cells.

We have recently demonstrated that furin, PC5, and PC7, members of the subtilisin/kexin-like mammalian proprotein convertases (PCs), are found in rodent aorta. These PCs have been identified to activate several growth factors, adhesion molecules and extracellular matrix compounds by endoproteolytic cleavage. In the present study, we investigated the regulation of PC5 in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Stimulation of rat aortic VSMCs with platelet-derived growth factor (PDGF)-BB (20 ng/mL), angiotensin II (Ang II, 1 micromol/L), or 10% fetal calf serum (FCS) for 48 hours increased DNA synthesis, as assessed by proliferating cell nuclear antigen (PCNA) immunoblotting. PC5 was strongly upregulated by PDGF-BB and 10% FCS (both 8-fold, P<0.05), whereas Ang II had no effect on PC5 protein levels compared with controls. The PCs furin and PC7, which display a comparable subcellular localization and cleavage activity, were found in VSMCs, but their levels did not increase following PDGF-BB, Ang II, or FCS stimulation. Time-course analysis revealed a rapid increase in PC5 levels after 30 minutes of PDGF-stimulation of VSMCs. PDGF-stimulated PC5 induction was inhibited by the PI3-kinase inhibitor wortmannin, and by rapamycin, an inhibitor of mTOR/p70(s6)-kinase (both P<0.05). In contrast, the mitogen-activated protein kinase (MAPK)-pathway inhibitor PD98059 did not inhibit PDGF-stimulated PC5 induction. Immunocytochemistry and in situ hybridization revealed low PC5 protein and mRNA levels in intact rat aorta in vivo. After balloon injury, PC5 protein and mRNA levels were strongly increased in proliferating PCNA-positive VSMCs. The present data demonstrate that PC5 is upregulated during proliferation of VSMCs in vivo and in vitro. We show that PDGF-induced PC5 expression is PI3-kinase/p70(s6)-kinase dependent. Thus, growth factors regulate the proprotein convertase PC5, which may play an important role during VSMC growth.