CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation.

Myeloid immune cells are abundant in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. We hypothesize that CNS resident macrophages enhance bAVM development and hemorrhage. RNA sequencing using cultured endothelial cells (ECs) and mouse bAVM samples revealed that downregulation of two bAVM causative genes, activin-like kinase 1 (ALK1) or endoglin, increased inflammation and innate immune signaling. To understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor inhibitor to bAVM mice with brain focal Alk1 deletion. Transient depletion of CNS resident macrophages at an early stage of bAVM development mitigated the phenotype severity of bAVM, including a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages increased EC tight junction protein expression, reduced the number of dysplasia vessels and severe hemorrhage in established bAVMs. Thus, EC AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophage could be a therapeutic target to mitigate the development and severity of bAVMs.

Bisphenol S stimulates Leydig cell proliferation but inhibits differentiation in pubertal male rats through multiple mechanisms.

Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 µM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 µM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17ß-hydroxysteroid dehydrogenase 3 activity at 10-100 µM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17ß-hydroxysteroid dehydrogenase 3 activity.

CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation.

Myeloid immune cells present abundantly in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. RNA sequencing using cultured cells and bAVM samples revealed that downregulation of activin-like kinase 1 (ALK1) or endoglin (two bAVM causative genes) increased pro-angiogenic, endothelial inflammation and innate immune signaling, which provided endogenous underpinnings of the active inflammation in bAVM. To further understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor (CSF1R) inhibitor to bAVM mice with endothelial Alk1 deletion. Transient depletion of CNS resident macrophages at early stage of bAVM development remarkably mitigated the subsequent phenotype severity of bAVM. This therapeutic effect exhibited a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages also reduced the dysplasia vessels and improved the integrity of endothelial tight junctions in established bAVMs. Administration of CSF1R inhibitor also prevented severe hemorrhage of bAVMs. Thus, endothelial AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophages could be specific targets to mitigate the development and severity of bAVMs.

Characterization of Recruited Mononuclear Phagocytes following Corneal Chemical Injury.

Mononuclear phagocytes (MP) have central importance in innate immunity, inflammation, and fibrosis. Recruited MPs, such as macrophages, are plastic cells and can switch from an inflammatory to a restorative phenotype during the healing process. However, the role of the MPs in corneal wound healing is not completely understood. The purpose of this study is to characterize the kinetics of recruited MPs and evaluate the role of macrophage metalloelastase (MMP12) in the healing process, using an in vivo corneal chemical injury model. Unwounded and wounded corneas of wild-type (WT) and Mmp12-/- mice were collected at 1, 3, and 6 days after chemical injury and processed for flow cytometry analysis. Corneal MP phenotype significantly changed over time with recruited Ly6Chigh (proinflammatory) cells being most abundant at 1 day post-injury. Ly6Cint cells were highly expressed at 3 days post-injury and Ly6Cneg (patrolling) cells became the predominant cell type at 6 days post-injury. CD11c+ dendritic cells were abundant in corneas from Mmp12-/- mice at 6 days post-injury. These findings show the temporal phenotypic plasticity of recruited MPs and provide valuable insight into the role of the MPs in the corneal repair response, which may help guide the future development of MP-targeted therapies.

Bone Marrow-Derived Alk1 Mutant Endothelial Cells and Clonally Expanded Somatic Alk1 Mutant Endothelial Cells Contribute to the Development of Brain Arteriovenous Malformations in Mice.

We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1- mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1- ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk12f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk12f/2f;confetti+/- mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk12f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1- ECs at early stage of bAVM development, suggesting that Alk1- ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1- ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.

Review of treatment and therapeutic targets in brain arteriovenous malformation.

Brain arteriovenous malformations (bAVM) are an important cause of intracranial hemorrhage (ICH), especially in younger patients. The pathogenesis of bAVM are largely unknown. Current understanding of bAVM etiology is based on studying genetic syndromes, animal models, and surgically resected specimens from patients. The identification of activating somatic mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene and other mitogen-activated protein kinase (MAPK) pathway genes has opened up new avenues for bAVM study, leading to a paradigm shift to search for somatic, de novo mutations in sporadic bAVMs instead of focusing on inherited genetic mutations. Through the development of new models and understanding of pathways involved in maintaining normal vascular structure and functions, promising therapeutic targets have been identified and safety and efficacy studies are underway in animal models and in patients. The goal of this paper is to provide a thorough review or current diagnostic and treatment tools, known genes and key pathways involved in bAVM pathogenesis to summarize current treatment options and potential therapeutic targets uncovered by recent discoveries.

Tebuconazole exposure disrupts placental function and causes fetal low birth weight in rats.

Tebuconazole (TEB) is one of the widely used broad-spectrum triazole fungicides. Its accumulation in mammals leads to various endocrine disruptions. However, it is unclear whether the exposure of TEB during pregnancy affects the growth and development of fetus and placenta. Here, TEB was exposed to pregnant Sprague-Dawley female rats from gestational days 12-21 of 0, 25, 50 or 100 mg/kg for 10 days. TEB reduced placental estradiol levels. TEB disrupted the structure and function of the placenta, leading to hypertrophy, fibrin exudation, edema, calcification, arterial fibroblast proliferation, and trophoblastic infarction. RNA-seq analysis showed that TEB mainly down-regulated the expression of iron transport genes and up-regulated the expression of genes for immune/inflammatory responses. Further qPCR showed that TEB down-regulated Tfrc, Hamp, Eif2ak2 and up-regulated the expression of Cd34, Cd36, Jag1, Pln, Cyp1a1, Esrra, and Aqp1 at 50 and 100 mg/kg. Western blot and semi-quantitative immunohistochemical staining also demonstrated that TEB lowered the levels of TFRC and EIF2AK2 and increased the levels of CD34, CD36, JAG1, CYP1A1, and ESRRA at 50 and 100 mg/kg. In conclusion, TEB severely damages the structure and function of the placenta, leading to hypertrophy of the placenta, low birth weight and feminization of the male fetus possibly via several pathways including iron transport and TNF signaling.

UBE2N Regulates Paclitaxel Sensitivity of Ovarian Cancer via Fos/P53 Axis.

BACKGROUND: Chemo-resistance is still considered one of the key factors in the mortality of ovarian cancer. In this work, we found that ubiquitin-conjugating enzyme E2 N (UBE2N) is downregulated in paclitaxel-resistant ovarian cancer cells. It suggests UBE2N to be critical in the regulation of paclitaxel sensitivity in ovarian cancer. MATERIALS AND METHODS: Ovarian cancer cells with stably overexpressed UBE2N were injected into nude mice to assess tumor growth and paclitaxel sensitivity in vivo. The MTT assay was applied to observe the effect of UBE2N expression on paclitaxel sensitivity. A real-time PCR array, specific for human cancer drug resistance, was used to examine the potential downstream target genes of UBE2N. The expression of UBE2N and potential downstream target genes was determined by Western blotting. The analysis of Gene Ontology and protein-protein interactions of these differentially expressed genes (DEGs) was performed using online tools. To evaluate the prognostic value of hub genes expression for ovarian cancer patients treated with paclitaxel, we applied the online survival analysis tool. RESULTS: Overexpressed UBE2N enhanced the paclitaxel sensitivity of ovarian cancer cells in vitro and in vivo. Thirteen upregulated DEGs and 11 downregulated DEGs were identified when we knockdown UBE2N. Meanwhile, 9 hub genes with a high degree of connectivity were selected. Only Fos proto-oncogene, AP-1 transcription factor subunit (Fos), was overexpressed upon decreasing UBE2N levels, indicating a poor outcome for patients treated with paclitaxel. Moreover, reduced UBE2N could increase Fos expression and reduce P53. Furthermore, reversed regulation of Fos and P53 based on UBE2N reduction could reverse paclitaxel sensitivity, respectively. CONCLUSION: Our study suggests that UBE2N could be used as a therapeutic agent for paclitaxel-resistant ovarian cancer through Fos/P53 pathway. Further studies are needed to elucidate the specific mechanism.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Food components and environmental chemicals of inhibiting human placental aromatase.

Human placental CYP19A1 catalyzes the estrogen synthesis from androgens. The enzyme is encoded by CYP19A1 gene located in chromosome 15q21. This enzyme is a monooxygenase in the smooth endoplasmic reticulum. The various promoters of the CYP19A1 gene determine its expression in different tissues and the distal promoter I.1 controls its expression in the placenta and retinoids can regulate the expression. Many food components and environmental chemicals inhibit CYP19A1 activity via different modes of action. These chemicals include gossypol, flavones, flavanones, chalconoids, resveratrol, and tobacco alkaloids derived from foods as well as phthalates, insecticides, fungicides, and biocides in the contaminated foods. The inhibition of placental CYP19A1 could impair pregnancy.

Effects of chlorpyrifos and trichloropyridinol on HEK 293 human embryonic kidney cells.

Chlorpyrifos (CPF) [O, O-diethyl -O-3, 5, 6-trichloro-2-pyridyl phosphorothioate] is an organophosphate insecticide widely used for agricultural and urban pest control. Trichloropyridinol (TCP; 3,5,6-trichloro-2-pyridinol), the primary metabolite of CPF, is often used as a generic biomarker of exposure for CPF and related compounds. Human embryonic kidney 293 (HEK 293) cells were exposed to CPF and TCP with varying concentrations and exposure periods. Cell cultures enable the cost-effective study of specific biomarkers to help determine toxicity pathways to predict the effects of chemical exposures without relying on whole animals. Both CPF and TCP were found to induce cytotoxic effects with CPF being more toxic than TCP with EC50 values of 68.82 µg/mL and 146.87 µg·ml-1 respectively. Cell flow cytometric analyses revealed that exposure to either CPF or TCP leads to an initial burst of apoptotic induction followed by a slow recruitment of cells leading towards further apoptosis. CPF produced a strong induction of IL6, while TCP exposure resulted in a strong induction of IL1α. Importantly, the concentrations of CPF and TCP required for these cytokine inductions were higher than those required to induce apoptosis. These data suggest CPF and TCP are cytotoxic to HEK 293 cells but that the mechanism may not be related to an inflammatory response. CPF and TCP also varied in their effects on the HEK 293 proteome with 5 unique proteins detected after exposure to CPF and 31 unique proteins after TCP exposure.