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.
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.
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.
Cellular and Molecular Gastroenterology and Hepatology
Xu H, Li J, Chen H, Ghishan FK.
PMID: - | DOI: 10.1016/j.jcmgh.2018.08.005
Abstract
Background and Aims
Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal sodium/hydrogen exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in ulcerative colitis (UC)-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors.
Methods
We assessed NHE8 expression in human CRCs by IHC and studied tumor burden in NHE8KO mice using an AOM/DSS colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NSG mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by PCR and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of β-catenin and c-Myc were also analyzed to evaluate Wnt/β-catenin activation.
Results
NHE8 was undetectable in human CRC tissues. Whereas only 9% of NHE8WT mice exhibited tumorigenesis in the AOM/DSS colon cancer model, almost ten times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared to HT29NHE8WT cells. Furthermore, NHE8 deficiency resulted in elevated Lgr5 expression in the colon, in HT29 derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/β-catenin activation.
Conclusions
NHE8 might be an intrinsic factor that regulates Wnt/β-catenin in the intestine.
Synergistic roles of Wnt modulators R-spondin2 and R-spondin3 in craniofacial morphogenesis and dental development
Alhazmi, N;Carroll, SH;Kawasaki, K;Woronowicz, KC;Hallett, SA;Macias Trevino, C;Li, EB;Baron, R;Gori, F;Yelick, PC;Harris, MP;Liao, EC;
PMID: 33712657 | DOI: 10.1038/s41598-021-85415-y
Wnt signaling plays a critical role in craniofacial patterning, as well as tooth and bone development. Rspo2 and Rspo3 are key regulators of Wnt signaling. However, their coordinated function and relative requirement in craniofacial development and odontogensis are poorly understood. We showed that in zebrafish rspo2 and rspo3 are both expressed in osteoprogenitors in the embryonic craniofacial skeleton. This is in contrast to mouse development, where Rspo3 is expressed in osteoprogenitors while Rspo2 expression is not observed. In zebrafish, rspo2 and rspo3 are broadly expressed in the pulp, odontoblasts and epithelial crypts. However, in the developing molars of the mouse, Rspo3 is largely expressed in the dental follicle and alveolar mesenchyme while Rspo2 expression is restricted to the tooth germ. While Rspo3 ablation in the mouse is embryonic lethal, zebrafish rspo3-/- mutants are viable with modest decrease in Meckel's cartilage rostral length. However, compound disruption of rspo3 and rspo2 revealed synergistic roles of these genes in cartilage morphogenesis, fin development, and pharyngeal tooth development. Adult rspo3-/- zebrafish mutants exhibit a dysmorphic cranial skeleton and decreased average tooth number. This study highlights the differential functions of Rspo2 and Rspo3 in dentocranial morphogenesis in zebrafish and in mouse.
AgRP signalling negatively regulates bone mass
Journal of neuroendocrinology
Enriquez, RF;Lee, NJ;Herzog, H;
PMID: 33913541 | DOI: 10.1111/jne.12978
The central nervous system is an active and major regulator of bone structure and remodelling. Specifically, signalling within the hypothalamus has been shown to be critical to ensuring that skeletal functions align with whole body metabolic supply and demand. Here, we identify agouti-related peptide (AgRP), an orexigenic peptide exclusively co-expressed with neuropeptide Y (NPY) in the arcuate nucleus (ARC) of the hypothalamus, as another critical player in the central control of bone homeostasis. Using novel mouse models, we show that AgRP deletion leads to an increase in cortical and trabecular bone mass as a result of an increase in bone thickness despite a lean phenotype, particularly in male mice. Interestingly, male AgRP deficient mice display a significant decrease in pro-opiomelanocortin (POMC) expression in the ARC, but no change in NPY or CART expression, suggesting that the increase in bone mass in AgRP-deficient mice is unlikely to be a result of altered NPY signalling. This is consistent with the observation that bone mass is unchanged in response to the specific deletion of NPY from AgRP expressing neurones. By contrast, POMC expression in the ARC is significantly increased in female AgRP deficient mice, although AgRP deletion results in altered respiratory exchange ratio regulation in response to re-feeding after a fast in both sexes. Taken together, the present study identifies AgRP as being directly involved in the regulation of bone mass and highlights the complexity intrinsic to the neuropeptide regulation of the skeleton.
Renal AAV2-Mediated Overexpression of Long Non-Coding RNA H19 Attenuates Ischemic Acute Kidney Injury Through Sponging of microRNA-30a-5p
Journal of the American Society of Nephrology : JASN
Haddad, G;Kölling, M;Wegmann, UA;Dettling, A;Seeger, H;Schmitt, R;Soerensen-Zender, I;Haller, H;Kistler, AD;Dueck, A;Engelhardt, S;Thum, T;Mueller, TF;Wüthrich, RP;Lorenzen, JM;
PMID: 33478972 | DOI: 10.1681/ASN.2020060775
Renal ischemia-reperfusion (I/R) injury is a major cause of AKI. Noncoding RNAs are intricately involved in the pathophysiology of this form of AKI. Transcription of hypoxia-induced, long noncoding RNA H19, which shows high embryonic expression and is silenced in adults, is upregulated in renal I/R injury. Lentivirus-mediated overexpression, as well as antisense oligonucleotide-based silencing, modulated H19 in vitro. In vivo analyses used constitutive H19 knockout mice. In addition, renal vein injection of adeno-associated virus 2 (AAV2) carrying H19 caused overexpression in the kidney. Expression of H19 in kidney transplant patients with I/R injury was investigated. H19 is upregulated in kidney biopsies of patients with AKI, in murine ischemic kidney tissue, and in cultured and ex vivo sorted hypoxic endothelial cells (ECs) and tubular epithelial cells (TECs). Transcription factors hypoxia-inducible factor 1-α, LHX8, and SPI1 activate H19 in ECs and TECs. H19 overexpression promotes angiogenesis in vitro and in vivo. In vivo, transient AAV2-mediated H19 overexpression significantly improved kidney function, reduced apoptosis, and reduced inflammation, as well as preserving capillary density and tubular epithelial integrity. Sponging of miR-30a-5p mediated the effects, which, in turn, led to target regulation of Dll4, ATG5, and Snai1. H19 overexpression confers protection against renal injury by stimulating proangiogenic signaling. H19 overexpression may be a promising future therapeutic option in the treatment of patients with ischemic AKI.
Luo, Y;Bao, H;Crowther, A;Asrican, B;Li, Y;Tart, D;Deng, F;Wan, J;Zhang, L;Patel, A;Li, Y;Song, J;
| DOI: 10.2139/ssrn.4017894
The serotonin (5HT) system mediates pathophysiology of stress responses and influences adult hippocampal neurogenesis from radial neural stem cells (rNSCs) in a sex-dependent manner. However, the mechanisms underlying sex differences in serotonergic regulation and stress vulnerability of rNSCs remain elusive. Here we report sex-dependent expression of 5HT1ARs in rNSCs of adult mouse hippocampus, with higher levels of 5HT1AR expression in rNSCs of females. Functionally, selective deletion of 5HT1ARs decreases rNSC production through decreased symmetric self-renewal in females. Mechanistically, 5HT1AR deletion in females results in 5HT-induced depolarization in rNSCs mediated by 5HT7R upregulation. Interestingly, stress exerts sex-dependent effects on 5HT release in the neurogenic niche and interacts with 5HT1ARs to regulate rNSC production in females through 5HT7R-mediated calcium signaling. These findings reveal a sex-dependent role of 5HT1ARs in regulating expansion and stress vulnerability of adult hippocampal rNSCs.
Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers
Lee, HJ;Myung, JK;Kim, HS;Lee, DH;Go, HS;Choi, JH;Koh, HM;Lee, SJ;Jang, B;
PMID: 34493772 | DOI: 10.1038/s41598-021-97351-y
Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.
