ROLE OF ATYPICAL CHEMOKINE RECEPTOR 2 IN PERIVASCULAR ADIPOSE TISSUE INFLAMMATION IN ANGIOTENSIN II DEPENDENT HYPERTENSION
Mikolajczyk, T;Skiba, D;Vidler, F;Love, S;Justo-Junior, A;Nosalski, R;Graham, D;Maffia, P;Graham, G;Guzik, T;
| DOI: 10.1097/01.hjh.0000744876.53171.8b
Chronic infusion of Ang II caused increased of leukocyte (CD45+) content in PVAT in both WT and ACKR2-/- mice. This increase was particularly evident for T cell subsets in WT but not in ACKR2-/-. However the number of macrophages (F4/80+CD11b+) was increased in both groups. Interestingly, ACKR2-/- revealed decreased T cell number infiltrating PVAT upon Ang II infusion in comparison to WT. Hypertension was associated with increased CCR1, CCR2 and CCR3 expression in PVAT in both WT and ACKR2-/-. However, ACKR2-/- revealed higher expression of CCR1 and CCR2 but not CCR3 in comparison to WT. Interestingly, increased expression of CCR5 in PVAT upon Ang II infusion in WT was not observed in ACKR2-/-. Hypertension resulted in increased RANTES level in aorta and PVAT to the same extent in both WT and ACKR2-/-. However, MCP-1, MIP-1 alpha and MIP-1 beta level was higher in ACKR2-/- aorta than in WT aorta upon Ang II infusion. ACKR2 expression was lower in PVAT than in aorta. Interestingly, Ang II administration decreased the expression of ACKR2 in aorta but increased ACKR2 level in thoracic PVAT. RNAscope analysis revealed ACKR2 expression in vascular smooth muscle cells (VSMC). Vessels isolated from ACKR2-/- were protected from vascular dysfunction and revealed reduced ROS production in comparison to WT upon Ang II administration. ACKR2-/- mice developed similar extent of blood pressure increase as WT in hypertension.
RNA in situ hybridization and expression of related genes regulating the accumulation of triterpenoids in Cyclocarya paliurus
Chen, X;Chen, B;Shang, X;Fang, S;
PMID: 33960380 | DOI: 10.1093/treephys/tpab067
Cyclocarya paliurus, a woody medicinal species in the Juglandaceae, grows extensively in subtropical areas of China. Triterpenoids in the leaves have health-promoting effects, including hypoglycemic and hypolipidemic activities. To understand triterpenoid biosynthesis, transport, and accumulation in C. paliurus during the growing season, gene cloning, gene expression, and RNA in situ hybridization of related genes were used, and accumulation was examined in various organs. The complete CDSs of three genes, CpHMGR, CpDXR, and CpSQS, were obtained from GenBank and RACE. RNA in situ hybridization signals of the three genes mainly occurred in the epidermis, palisade tissue, phloem, and xylem of leaf, shoot, and root, with the signals generally consistent with the accumulation of metabolites in tissues, except in the xylem. Both gene expression and triterpenoid accumulations showed seasonal variations in all organs. However, total triterpenoid content in the leaves was significantly higher than that in the shoots, with the maximum in shoots in August and in leaves in October. According to Pearson correlation analysis, triterpenoid accumulation in the leaves was significantly positively related with the relative expression of CpSQS. However, the relation between gene expression and accumulation was dependent on the role of the gene in the pathway, as well as on the plant organ. The results suggested that most of the intermediates catalyzed by CpHMGR and CpDXR in young shoots and roots were used in growth and flowering in the spring, whereas subsequent triterpenoid biosynthesis in the downstream catalyzed by CpSQS mainly occurred in the leaves by using transferred and in situ intermediates as substrates. Thus, this study provides a reference to improve triterpenoid accumulation in future C. paliurus plantations.
Hayashi, S;Sato, T;Ono, H;Ito, S;Takai, R;Shibuya, K;Sasakawa, C;
PMID: 37087879 | DOI: 10.1016/j.vetmic.2023.109740
Porcine circovirus type 3 (PCV3) is a novel porcine circovirus that has been detected in pigs showing various clinical and pathological conditions, as well as in many asymptomatic pigs. The pathogenesis of PCV3 infection in pigs remains unclear. To evaluate the in vivo growth and pathogenicity of PCV3, we performed two experiments on PCV3 infection in laboratory-grade miniature pigs with strictly controlled genetic backgrounds and microbiological status. A PCV3 passage experiment confirmed PCV3 genome detection in the sera and multiple organs via in vivo serial passage generations. PCV3 was successively passaged in miniature pigs by inoculating tissue homogenates from infected pigs supporting Koch's principles. In the PCV3 infection experiment, viremia was observed in all the inoculated pigs, and transient neurological signs were observed in one of the three pigs. Histopathologically, all three pigs in the PCV3 inoculation group exhibited lung disorders such as interstitial pneumonia and lymphoplasmacytic perivasculitis. In addition, one pig with neurological signs in the PCV3 inoculation group showed focal thrombosis in the meninges of the cerebellum. Vascular lesions in both the lungs and brain suggest that PCV3 may cause injury to vascular tissues. In situ hybridization (ISH)-RNA analysis demonstrated that the PCV3 genome was localized in the lymph nodes of pigs inoculated with PCV3. The PCV3 in vivo passage system in NIBS miniature pigs will help investigate the pathogenicity of PCV3.
Atallah, R;Gindlhuber, J;Platzer, W;Bärnthaler, T;Tatzl, E;Toller, W;Strutz, J;Rittchen, S;Luschnig, P;Birner-Gruenberger, R;Wadsack, C;Heinemann, A;
| DOI: 10.20944/preprints202110.0291.v1
Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that succinate-SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between succinate-SUCNR1 axis and placental angiogenesis. In our in-vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.
Shi Z, Cassaglia PA, Pelletier NE, Brooks VL.
PMID: PMID: 30628058 | DOI: DOI:10.1113/JP277517
KEY POINTS: ICV insulin increased SNA and baroreflex control of SNA and HR dramatically more in obese male rats; in obese females, the responses were abolished. In obese males, the enhanced LSNA responses were associated with reduced tonic inhibition of LSNA by NPY in the PVN. Yet, PVN NPY injection decreased LSNA similarly in OP/OR/CON rats. Collectively, these results suggest that NPY inputs were decreased. In obese females, NPY inhibition in the PVN was maintained. Moreover, NPY neurons in the ArcN became resistant to the inhibitory effects of insulin. A HFD did not alter arcuate NPY neuronal InsR expression in males or females. Obesity-induced "selective sensitization" of the brain to the sympathoexcitatory effects of insulin and leptin may contribute to elevated basal SNA, and therefore hypertension development, in males with obesity. These data may explain in part why obesity increases SNA less in women compared to men. ABSTRACT: Obesity increases sympathetic nerve activity (SNA) in men, but not women; however, the mechanisms are unknown. We tested if intracerebroventricular insulin infusion increases SNA more in obese male than female rats and if sex differences are mediated by changes in tonic inhibition of SNA by Neuropeptide Y (NPY) in the paraventricular nucleus (PVN). When consuming a high fat diet, obesity prone (OP) rats accrued excess fat, whereas obesity resistant (OR) rats maintained adiposity as in rats eating a control (CON) diet. Insulin increased lumbar SNA (LSNA) similarly in CON/OR males and females under urethane-anesthesia. The LSNA response was magnified in OP males, but abolished in OP females. In males, blockade of PVN NPY Y1 receptors with BIBO3304 increased LSNA in CON/OR rats, but not OP rats. Yet, PVN nanoinjections of NPY decreased LSNA similarly between groups. Thus, tonic PVN NPY inhibition of LSNA may be lost in obese males, due to a decrease in NPY inputs. In contrast, in females, PVN BIBO3304 increased LSNA similarly in OP, OR and CON rats. After insulin, PVN BIBO3304 failed to increase LSNA in CON/OR females, but increased LSNA in OP females, suggesting that with obesity NPY neurons become resistant to the inhibitory effects of insulin. These sex differences were not associated with changes in arcuate NPY neuronal insulin receptor expression. Collectively, these data reveal a marked sex difference in the impact of obesity on insulin's sympathoexcitatory actions and implicate sexually dimorphic changes in NPY inhibition of SNA in the PVN as one mechanism.
Shiers, S;Elahi, H;Hennen, S;Price, T;
| DOI: 10.1016/j.ynpai.2021.100081
The anterior cingulate cortex (ACC) is a critical region of the brain for the emotional and affective components of pain in rodents and humans. Hyperactivity in this region has been observed in neuropathic pain states in both patients and animal models and ablation of this region from cingulotomy, or inhibition with genetics or pharmacology can diminish pain and anxiety. Two adenylyl cyclases (AC), AC1 and AC8 play an important role in regulating nociception and anxiety-like behaviors through an action in the ACC, as genetic and pharmacological targeting of these enzymes reduces mechanical hypersensitivity and anxiety-like behavior, respectively. However, the distribution of these ACs in the ACC has not been studied in the context of neuropathic pain. To address this gap in knowledge, we conducted RNAscope in situ hybridization to assess AC1 and AC8 mRNA distribution in mice with spared nerve injury (SNI). Given the key role of AC1 in nociception in neuropathic, inflammatory and visceral pain animal models, we hypothesized that AC1 would be upregulated in the ACC of mice following nerve injury. This hypothesis was also founded on data showing increased AC1 expression in the ACC of mice with zymosan-induced visceral inflammation. We found that AC1 and AC8 are widely expressed in many regions of the mouse brain including the hippocampus, ACC, medial prefrontal cortex and midbrain regions, but AC1 is more highly expressed. Contrary to our hypothesis, SNI causes an increase in AC8 mRNA expression in NMDAR-2B (Nr2b) positive neurons in the contralateral ACC but does not affect AC1 mRNA expression. Our findings show that changes in Adcy1 mRNA expression in the ACC are insufficient to explain the important role of this AC in mechanical hypersensitivity in mice following nerve injury and suggest a potential unappreciated role of AC8 in regulation of ACC synaptic changes after nerve injury.
In Vitro Silencing of lncRNA Expression Using siRNAs
Methods in molecular biology (Clifton, N.J.)
Thijssen, MS;Bintz, J;Arnes, L;
PMID: 34160804 | DOI: 10.1007/978-1-0716-1581-2_9
Recent advances in sequencing technologies have uncovered the existence of thousands of long noncoding RNAs (lncRNAs) with dysregulated expression in cancer. As a result, there is burgeoning interest in understanding their function and biological significance in both homeostasis and disease. RNA interference (RNAi) enables sequence-specific gene silencing and can, in principle, be employed to silence virtually any gene. However, when applied to lncRNAs, it is important to consider current limitations in their annotation and current principles regarding lncRNA regulation and function when assessing their phenotype in cancer cell lines. In this chapter we describe the analysis of lncRNA splicing variant expression, including subcellular localization, transfection of siRNAs in cancer cell lines, and validation of gene silencing by quantitative PCR and single molecule in situ hybridization. All protocols can be performed in a laboratory with essential equipment for cell culture, molecular biology, and imaging.
Schneider JS.
PMID: 29902255 | DOI: 10.1371/journal.pone.0199189
Reduced expression of GM1 and other major brain gangliosides GD1a, GD1b and GT1b have been reported in Parkinson's disease (PD) brain. Mechanisms underlying these changes are unclear but may be due to a deficit in the ganglioside biosynthetic process. The present study examined the extent to which deficits in gene expression of key biosynthetic enzymes involved in synthesis of GM1 and GD1b (B3galt4) and GD1a and GT1b (St3gal2) exist in neuromelanin-containing neurons in the PD substantia nigra (SN). In situ hybridization histochemistry was used to examine gene expression of B3GALT4 and ST3GAL2 in neuromelanin-containing neurons in the SN in 8 normal controls (61-92 yrs.) and 7 PD subjects (77-95 yrs). There was a significant decrease in both B3GALT4 and ST3GAL2 gene expression in residual neuromelanin-containing cells in the SN of PD patients compared to age-matched neurologically normal controls. These changes appeared to be cell-type specific as abundant B3GALT4 and ST3GAL2 gene expression was observed in non-neuromelanin containing neurons located outside of the SN in the PD brain. These data show that residual neuromelanin-containing neurons in the PD SN have decreased expression of the ganglioside biosynthetic genes B3GALT4 and ST3GAL2, consistent with previous reports of decreased levels of gangliosides GM1, GD1a, GD1b and GT1b in the PD SN. These changes may increase the vulnerability of these neurons to degeneration in response to a variety of potential stressors.
Di Cicco E, Ferguson HW, Kaukinen KH, Schulze AD, Li S, Tabata A, Gunther OP, Mordecai G, Suttle CA, Miller KM.
PMID: - | DOI: 10.1139/facets-2018-0008
Piscine orthoreovirus Strain PRV-1 is the causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar). Given its high prevalence in net pen salmon, debate has arisen on whether PRV poses a risk to migratory salmon, especially in British Columbia (BC) where commercially important wild Pacific salmon are in decline. Various strains of PRV have been associated with diseases in Pacific salmon, including erythrocytic inclusion body syndrome (EIBS), HSMI-like disease, and jaundice/anemia in Japan, Norway, Chile and Canada. We examine the developmental pathway of HSMI and jaundice/anemia associated with PRV-1 in farmed Atlantic and Chinook (Oncorhynchus tshawytscha) salmon in BC, respectively. In situ hybridization localized PRV-1 within developing lesions in both diseases. The two diseases showed dissimilar pathological pathways, with inflammatory lesions in heart and skeletal muscle in Atlantic salmon, and degenerative-necrotic lesions in kidney and liver in Chinook salmon, plausibly explained by differences in PRV load tolerance in red blood cells. Viral genome sequencing revealed no consistent differences in PRV-1 variants intimately involved in the development of both diseases, suggesting that migratory Chinook salmon may be at more than a minimal risk of disease from exposure to the high levels of PRV occurring on salmon farms.
Virchows Archiv : an international journal of pathology
Adachi, K;Sakurai, Y;Ichinoe, M;Tadehara, M;Tamaki, A;Kesen, Y;Kato, T;Mii, S;Enomoto, A;Takahashi, M;Koizumi, W;Murakumo, Y;
PMID: 34762199 | DOI: 10.1007/s00428-021-03230-2
CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, whose expression is upregulated in some types of malignant tumors. High levels of CD109 in tumor cells have been reported to correlate with poor prognosis; however, significance of CD109 stromal expression in human malignancy has not been elucidated. In this study, we investigated the tumorigenic properties of CD109 in pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analysis of 92 PDAC surgical specimens revealed that positive CD109 expression in tumor cells was significantly associated with poor prognosis (disease-free survival, p = 0.003; overall survival, p = 0.002), and was an independent prognostic factor (disease-free survival, p = 0.0173; overall survival, p = 0.0104) in PDAC. Furthermore, CD109 expression was detected in the stroma surrounding tumor cells, similar to that of α-smooth muscle actin, a histological marker of cancer-associated fibroblasts. The stromal CD109 expression significantly correlated with tumor progression in PDAC (TNM stage, p = 0.033; N factor, p = 0.024; lymphatic invasion, p = 0.028). In addition, combined assessment of CD109 in tumor cells and stroma could identify the better prognosis group of patients from the entire patient population. In MIA PaCa-2 PDAC cell line, we demonstrated the involvement of CD109 in tumor cell motility, but not in PANC-1. Taken together, CD109 not only in the tumor cells but also in the stroma is involved in the progression and prognosis of PDAC, and may serve as a useful prognostic marker in PDAC.
Expression analysis of neuropeptide FF receptors on neuroendocrine-related neurons in the rat brain using highly sensitive in situ hybridization
Histochemistry and cell biology
Higo, S;Kanaya, M;Ozawa, H;
PMID: 33398437 | DOI: 10.1007/s00418-020-01956-9
RF-amide peptides, a family of peptides characterized by a common carboxy-terminal Arg-Phe-NH2 motif, play various physiological roles in the brain including the modulation of neuroendocrine signaling. Neuropeptide FF (NPFF) receptors exhibit a high affinity for all RF-amide peptides, which suggests that the neurons expressing these NPFF receptors may have multiple functions in the brain. However, the distribution of the neurons expressing NPFF receptors in the rat brain remains poorly understood. This study aimed to determine the detailed histological distribution of mRNA that encodes the neuropeptide FF receptors (Npffr1 and Npffr2) in the rat brain using in situ hybridization. Neurons with strong Npffr1 expression were observed in the lateral septal nucleus and several hypothalamic areas related to neuroendocrine functions, including the paraventricular nucleus (PVN) and arcuate nucleus, whereas Npffr2-expressing neurons were observed mainly in brain regions involved in somatosensory pathways, such as several subnuclei of the thalamus. Npffr1 expression was observed in 70% of corticotropin-releasing hormone neurons, but in only a small population of oxytocin and vasopressin neurons in the PVN. Npffr1 expression was also observed in the dopaminergic neurons in the periventricular nucleus and the dorsal arcuate nucleus, and in the kisspeptin neurons in the anteroventral periventricular nucleus. These results suggest that NPFFR1-mediated signaling may be involved in neuroendocrine functions, such as in reproduction and stress response. In conjunction with a detailed histological map of NPFFRs, this study provides useful data for future neuroendocrine research.
Zhang N, Zhang HY, Bi SA, Moran TH and Bi S
PMID: 30902570 | DOI: 10.1016/j.neulet.2019.03.030
Thyrotropin-releasing hormone (TRH) plays an important role in the regulation of energy balance. While the regulation of TRH in the paraventricular nucleus (PVN) in response to changes of energy balance has been well studied, how TRH is regulated in the dorsomedial hypothalamus (DMH) in maintaining energy homeostasis remains unclear. Here, we assessed the effects of food restriction and exercise on hypothalamic Trh expression using Otsuka Long-Evens Tokushima Fatty (OLETF) rats. Sedentary ad lib fed OLETF rats (OLETF-SED) became hyperphagic and obese. These alterations were prevented in OLETF rats with running wheel access (OLETF-RW) or food restriction in which their food was pair-fed (OLETF-PF) to the intake of lean control rats (LETO-SED). Evaluation of hypothalamic gene expression revealed that Trh mRNA expression was increased in the PVN of OLETF-SED rats and normalized in OLETF-RW and OLETF-PF rats compared to LETO-SED rats. In contrast, the expression of Trh in the DMH was decreased in OLETF-SED rats relative to LETO-SED rats. This alteration was reversed in OLETF-RW rats as seen in LETO-SED rats, but food restriction resulted in a significant increase in DMH Trh expression in OLETF-PF rats compared to LETO-SED rats. Strikingly, while Trh mRNA expression was decreased in the PVN of intact rats in response to acute food deprivation, food deprivation resulted in increased expression of Trh in the DMH. Together, these results demonstrate the differential regulation of Trh expression in the PVN and DMH in OLETF rats and suggest that DMH TRH also contributes to hypothalamic regulation of energy balance.
