Feasibility of deuterium magnetic resonance spectroscopy of 3-O-Methylglucose at 7 Tesla.

Deuterium Magnetic Resonance Spectroscopy (DMRS) is a non-invasive technique that allows the detection of deuterated compounds in vivo. DMRS has a large potential to analyze uptake, perfusion, washout or metabolism, since deuterium is a stable isotope and therefore does not decay during biologic processing of a deuterium labelled substance. Moreover, DMRS allows the distinction between different deuterated substances. In this work, we performed DMRS of deuterated 3-O-Methylglucose (OMG). OMG is a non-metabolizable glucose analog which is transported similar to D-glucose. DMRS of OMG was performed in phantom and in vivo measurements using a preclinical 7 Tesla MRI system. The chemical shift (3.51 ± 0.1 ppm) and relaxation times were determined. OMG was injected intravenously and spectra were acquired over a period of one hour to monitor the time evolution of the deuterium signal in tumor-bearing rats. The increase and washout of OMG could be observed. Three different exponential functions were compared in terms of how well they describe the OMG washout. A mono-exponential model with offset seems to describe the observed time course best with a time constant of 1910 ± 770 s and an offset of 2.5 ± 1.2 mmol/l (mean ± std, N = 3). Chemical shift imaging could be performed with a voxel size of 7.1 mm x 7.1 mm x 7.9 mm. The feasibility of DMRS with deuterium labelled OMG could be demonstrated. These data might serve as basis for future studies that aim to characterize glucose transport using DMRS.

Defining Phenotype, Tropism, and Retinal Gene Therapy Using Adeno-Associated Viral Vectors (AAVs) in New-Born Brown Norway Rats with a Spontaneous Mutation in Crb1.

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.

AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A.

Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures.

Alleviation of diabetes mellitus through the restoration of ß-cell function and lipid metabolism by Aloe vera (L.) Burm. f. extract in obesogenic WNIN/GR-Ob rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aloe vera (L.) Burm. f. extract has been medicinally used for over 5000 years in different cultures for its curative and therapeutic properties ranging from dermatitis to diabetes. It has been demonstrated to alleviate diabetes through its protective effects on pancreatic islets and by improving insulin secretion. AIM OF THE STUDY: To investigate the simultaneous effect of ethanolic A. vera gel extract on diabetes and obesogenic milieu in Streptozotocin-induced WNIN/GR-Ob mutant obese rats. MATERIALS AND METHODS: A total of 30 rats were grouped equally into WNIN/GR-Ob control (received water as a vehicle), WNIN/GR-Ob Diabetic rats (Streptozotocin-35 mg/kg bw), WNIN/GR-Ob Diabetic rats + Sitagliptin (10 mg/kg bw), WNIN/GR-Ob Diabetic rats + A. vera (300 mg/kg bw) and GR-Ob control + A. vera (300 mg/kg bw). After 4 weeks of treatment, fasting blood glucose, serum insulin, Homeostatic Model Assessment - Insulin Resistance and ß-cell function, glucose-stimulated insulin secretion, Dipeptidyl peptidase-IV activity, and lipid profiles were studied. In addition, ultrastructural analysis of isolated islets and dual-energy X-ray absorptiometry analysis for body composition were also carried out. RESULTS: The A. vera treated group showed a significant reduction (p < 0.05) in triglyceride, Very low-density lipoprotein levels, Triglyceride to High-density lipoprotein ratio as well as fasting blood glucose levels and DPP-IV activity with a concomitant increase in the serum insulin levels. The increase in IR was observed in both WNIN/GR-Ob control and diabetic rats with a significant decrease in ß-cell function in the diabetic rats as per Homeostatic Model Assessment values. Oral administration of A. vera was effective in both reducing Homeostatic Model Assessment-Insulin Resistance and increasing Homeostatic Model Assessment-ß values. Also, the treated group demonstrated preservation of islets and a significant increase (p < 0.05) in the diameter of ß-cell as evident through Scanning electron microscope analysis. The increase in lean body mass was manifested in the treated group with a reduction in Fat percent in comparison with other groups. CONCLUSION: The beneficial effects of A. vera in WNIN/GR-Ob strain may be attributed to its ability to lower lipid profile thus improve insulin sensitivity and/or modulating ß-cell function. Thus, it has great therapeutic potential as an herbal remedy for the treatment of diabetes and associated adverse effects such as obesity. The exact mechanism underlying the observation needs to be investigated further to explore the anti-obesity and anti-diabetic properties of A. vera and advocate its potential application as alternative medicine.

Inhibition of PARP activity improves therapeutic effect of ARPE-19 transplantation in RCS rats through decreasing photoreceptor death.

Photoreceptor (PR) dysfunction or death is the key pathological change in retinal degeneration (RD). The death of PRs might be due to a primary change in PRs themselves or secondary to the dysfunction of the retinal pigment epithelium (RPE). Poly(ADP-ribose) polymerase (PARP) was reported to be involved in primary PR death, but whether it plays a role in PR death secondary to RPE dysfunction has not been determined. To clarify this question and develop a new therapeutic approach, we studied the changes in PAR/PARP in the RCS rat, a RD model, and tested the effect of PARP intervention when given alone or in combination with RPE cell transplantation. The results showed that poly(ADP-ribosyl)ation of proteins was increased in PRs undergoing secondary death in RCS rats, and this result was confirmed by the observation of similar changes in sodium iodate (SI)-induced secondary RD in SD rats. The increase in PAR/PARP was highly associated with increased apoptotic PRs and decreased visual function, as represented by lowered b-wave amplitudes on electroretinogram (ERG). Then, as we expected, when the RCS rats were treated with subretinal injection of the PARP inhibitor PJ34, the RD process was delayed. Furthermore, when PJ34 was given simultaneously with subretinal ARPE-19 cell transplantation, the therapeutic effects were significantly improved and lasted longer than those of ARPE-19 or PJ34 treatment alone. These results provide a potential new approach for treating RD.

17ß-Estradiol and estrogen receptor α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17ß-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-ß-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Estrogen receptor-α prevents right ventricular diastolic dysfunction and fibrosis in female rats.

Although women are more susceptible to pulmonary arterial hypertension (PAH) than men, their right ventricular (RV) function is better preserved. Estrogen receptor-α (ERα) has been identified as a likely mediator for estrogen protection in the RV. However, the role of ERα in preserving RV function and remodeling during pressure overload remains poorly understood. We hypothesized that loss of functional ERα removes female protection from adverse remodeling and is permissive for the development of a maladapted RV phenotype. Male and female rats with a loss-of-function mutation in ERα (ERαMut) and wild-type (WT) littermates underwent RV pressure overload by pulmonary artery banding (PAB). At 10 wk post-PAB, WT and ERαMut demonstrated RV hypertrophy. Analysis of RV pressure waveforms demonstrated RV-pulmonary vascular uncoupling and diastolic dysfunction in female, but not male, ERαMut PAB rats. Similarly, female, but not male, ERαMut exhibited increased RV fibrosis, comprised primarily of thick collagen fibers. There was an increased protein expression ratio of TIMP metallopeptidase inhibitor 1 (Timp1) to matrix metalloproteinase 9 (Mmp9) in female ERαMut compared with WT PAB rats, suggesting less collagen degradation. RNA-sequencing in female WT and ERαMut RV revealed kallikrein-related peptidase 10 (Klk10) and Jun Proto-Oncogene (Jun) as possible mediators of female RV protection during PAB. In summary, ERα in females is protective against RV-pulmonary vascular uncoupling, diastolic dysfunction, and fibrosis in response to pressure overload. ERα appears to be dispensable for RV adaptation in males. ERα may be a mediator of superior RV adaptation in female patients with PAH.NEW & NOTEWORTHY Using a novel loss-of-function mutation in estrogen receptor-α (ERα), we demonstrate that female, but not male, ERα mutant rats display right ventricular (RV)-vascular uncoupling, diastolic dysfunction, and fibrosis following pressure overload, indicating a sex-dependent role of ERα in protecting against adverse RV remodeling. TIMP metallopeptidase inhibitor 1 (Timp1), matrix metalloproteinase 9 (Mmp9), kallikrein-related peptidase 10 (Klk10), and Jun Proto-Oncogene (Jun) were identified as potential mediators in ERα-regulated pathways in RV pressure overload.

Retina Organoid Transplants Develop Photoreceptors and Improve Visual Function in RCS Rats With RPE Dysfunction.

Purpose: To study if human embryonic stem cell-derived photoreceptors could survive and function without the support of retinal pigment epithelium (RPE) after transplantation into Royal College of Surgeons rats, a rat model of retinal degeneration caused by RPE dysfunction. Methods: CSC14 human embryonic stem cells were differentiated into primordial eye structures called retinal organoids. Retinal organoids were analyzed by quantitative PCR and immunofluorescence and compared with human fetal retina. Retinal organoid sheets (30-70 day of differentiation) were transplanted into immunodeficient RCS rats, aged 44 to 56 days. The development of transplant organoids in vivo in relation to the host was examined by optical coherence tomography. Visual function was assessed by optokinetic testing, electroretinogram, and superior colliculus electrophysiologic recording. Cryostat sections were analyzed for various retinal, synaptic, and donor markers. Results: Retinal organoids showed similar gene expression to human fetal retina transplanted rats demonstrated significant improvement in visual function compared with RCS nonsurgery and sham surgery controls by ERGs at 2 months after surgery (but not later), optokinetic testing (up to 6 months after surgery) and electrophysiologic superior colliculus recordings (6-8 months after surgery). The transplanted organoids survived more than 7 months; developed photoreceptors with inner and outer segments, and other retinal cells; and were well-integrated within the host. Conclusions: This study, to our knowledge, is the first to show that transplanted photoreceptors survive and function even with host's dysfunctional RPE. Our findings suggest that transplantation of organoid sheets from stem cells may be a promising approach/therapeutic for blinding diseases.

Diabetes-Induced Inflammation and Vascular Alterations in the Goto-Kakizaki Rat Retina.

PURPOSE: Diabetic retinopathy is characterized by multiple microcirculatory dysfunctions and angiogenesis resulting from hyperglycemia, oxidative stress, and inflammation. In this study, the retina and retinal pigmented epithelium of non-insulin-dependent diabetic Goto-Kakizaki (GK) rats were examined to detect microvascular alterations, gliosis, macrophage infiltration, lipid deposits, and fibrosis. Emphasis was given to the distribution of kinin B1 receptor (B1R) and vascular endothelial growth factor (VEGF), two major factors in inflammation and angiogenesis. MATERIALS AND METHODS: 30-week-old male GK rats and age-matched Wistar rats were used. The retinal vascular bed was examined using ADPase staining. The level of lipid accumulation was graded using triglyceride staining with Oil red O. Macrophage and retinal microglia activation, as well as other markers, were revealed by immunohistochemistry and studied with confocal laser scanning microscopy. RESULTS: Abundant lipid deposits were observed in the Bruch's membrane of GK rats. Immunohistochemistry and quantitative analysis showed significantly higher B1R, VEGF, Iba1 (microglia), CD11 (macrophages), fibronectin, and collagen I labeling in the diabetic retina. B1R immunolabeling was detected in the vascular layers of the GK retina. A strong VEGF staining within different retinal cell processes was detected and a pattern of GFAP staining suggested strong Müller cells/astrocytes reactivity. Microgliosis was apparent in the GK retina. A greater tortuosity of the retinal microvessels (an index of endothelial dysfunction) and their increased number were also observed in GK retinas. CONCLUSIONS: Data suggest retinal vascular bed alterations in spontaneous type 2 diabetic retinas at 30 weeks. Lipid and collagen accumulation in the retina and choroid, in addition to retinal upregulation of VEGF and B1R, microgliosis, and Müller cell reactivity, may contribute to vascular alterations and inflammatory processes.

Effective Differentiation and Biological Characterization of Retinal Pigment Epithelium Derived from Human Induced Pluripotent Stem Cells.

PURPOSE: Human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelium (RPE) cells are therapeutic cells that have been shown to be promising in the rescue of lost photoreceptors. In this study, we generated hiPSC from human epidermal keratinocytes and subsequently differentiated them into RPE cells to investigate their ability to influence the retinal functions of the Royal College of Surgeon (RCS) rats. METHODS: Keratinocytes were reprogrammed to hiPSC using a non-integrating Sendai reprogramming system. Established hiPSCs were differentiated into RPE cells, and complete characterization was performed. Next, the suspension of hiPSC-RPE cells was transplanted into the subretinal space of 3-week-old RCS rats (n = 12). Posttransplantation evaluations were performed using optical coherence tomography (OCT), electroretinography, and immunohistochemical analysis. RESULTS: The hiPSC colonies were identical to embryonic stem-like cells that revealed the expression of pluripotency markers and retention of the normal genome. These cells exhibited the ability to differentiate into an amalgam of germ layers and produce RPE cells. The differentiated RPE cells exhibited an identical pigmented morphology that expressed RPE-specific markers, such as CRALBP, BESTROPHIN, RPE65, and MERTK. At 8 weeks of longitudinal culture, the RPE cells exhibited maximum pigmentation with in vitro phagocytotic activity. Furthermore, transplantation data showed improved retinal function till week 12 post-transplantation and a significantly higher number of rod/cone ratios in transplanted eyes compared to non-surgery control eyes. CONCLUSION: hiPSC-derived RPE cells exhibited naïve RPE cell properties and functionality that provided trophic support and the transient rescue of photoreceptor cells.

Postnatal morphological lung development of wild type and CD26/DPP4 deficient rat pups in dependency of LPS exposure.

BACKGROUND: Rodents are born with morphological immature lungs and an intact surfactant system. CD26/DPP4 is a multifactorial transmembrane integral type II protein, which is involved in physiological and pathophysiological processes and is already expressed during development. CD26/DPP4, called CD26 in the following, is able to enhance or dampen differently triggered inflammation. LPS exposure often used to simulate perinatal infection delays lung development. OBJECTIVE: A perinatal LPS rat model was used to test the hypothesis that CD26 deficiency modulates LPS-induced retardation in morphological lung development. METHODS: New born Fischer CD26 positive (CD26+) and deficient (CD26-) rats were exposed to LPS on postnatal day (day post partum, dpp) 3 and 5. Morphological parameters of lung development were determined stereologically. Lung development was analysed in 7, 10 14 and 21day old rats. RESULTS: Compared to controls LPS application resulted (1) in a mild inflammation independent of the strain, (2) in significantly lower total surface and volume of alveolar septa combined with significantly higher total volume of airspaces and alveolar size on dpp 7 in both substrains. However, compared to controls in LPS treated CD26- rats significant lower values of total septal surface and volume combined with higher values of total parenchymal airspaces and alveolar size were found until the end of classical alveolarization (dpp14). In LPS treated CD26+ rat pups the retardation was abolished already on dpp 10. CONCLUSION: In absence of CD26, LPS enhances the delay of morphological lung development. Morphological recovery was slower after the end of LPS exposure in CD26 deficient lungs.

Porous gold layer coated silver nanoplates with efficient antimicrobial activity.

Although silver nanoparticles are considered as promising antibacterial agents because of their antibacterial activity, the acute cytotoxicity of Ag+ released from Ag nanoparticles restricts their potential practical applications. Herein, porous Ag@Au nanoplates, which could balance the Ag+ release and the toxicity of Ag naoparticles, were fabricated by stepwise seed-mediated growth and oxidation. Laser irradiation further boosted their antimicrobial activity, and significantly accelerated the curing rate of wound. Comparing with Ag nanoplates, the irradiated porous Ag@Au nanoplates showed the similar antibiotic ability against S. aureus strains and lower cytotoxicity in vitro. When the porous Ag@Au nanoplates were applied to treat S. aureus-infected wound, they had the best curing effect. Thus, these porous Ag@Au nanoplates could act as promising antibacterial agents for wound healing applications.

Oncolytic Activity of the Vaccine Strain of Type 3 Poliovirus on the Model of Rat Glioma C6 Cells.

Rat glioma cell line C6 expressing human poliovirus receptor (PVR) and susceptible to polioviruses (C6-PVR-BFP) was used to produce a clone with knockout of IFNα/ß (Ifnar1) receptor subunit 1 gene (Ifnar1). The sensitivity of C6-PVR-BFP cells to the vaccine strain of poliovirus type 3 (PV3) depended on the signaling pathways of the cell response to type 1 IFN. Using the model of subcutaneous tumor xenografts, we demonstrated oncolytic activity of PV3 against C6-PVR-BFP cells that depended on the expression of PVR and increased considerably upon disturbances in IFN response pathways.

Metabolic and oncogenic adaptations to pyruvate dehydrogenase inactivation in fibroblasts.

Eukaryotic cell metabolism consists of processes that generate available energy, such as glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (Oxphos), and those that consume it, including macromolecular synthesis, the maintenance of ionic gradients, and cellular detoxification. By converting pyruvate to acetyl-CoA (AcCoA), the pyruvate dehydrogenase (PDH) complex (PDC) links glycolysis and the TCA cycle. Surprisingly, disrupting the connection between glycolysis and the TCA cycle by inactivation of PDC has only minor effects on cell replication. However, the molecular basis for this metabolic re-equilibration is unclear. We report here that CRISPR/Cas9-generated PDH-knockout (PDH-KO) rat fibroblasts reprogrammed their metabolism and their response to short-term c-Myc (Myc) oncoprotein overexpression. PDH-KO cells replicated normally but produced surprisingly little lactate. They also exhibited higher rates of glycolysis and Oxphos. In addition, PDH-KO cells showed altered cytoplasmic and mitochondrial pH, redox states, and mitochondrial membrane potential (ΔΨM). Conditionally activated Myc expression affected some of these parameters in a PDH-dependent manner. PDH-KO cells had increased oxygen consumption rates in response to glutamate, but not to malate, and were depleted in all TCA cycle substrates between α-ketoglutarate and malate despite high rates of glutaminolysis, as determined by flux studies with isotopically labeled glutamine. Malate and pyruvate were diverted to produce aspartate, thereby potentially explaining the failure to accumulate lactate. We conclude that PDH-KO cells maintain proliferative capacity by utilizing glutamine to supply high rates of AcCoA-independent flux through the bottom portion of the TCA cycle while accumulating pyruvate and aspartate that rescue their redox defects.

Transplantation of rat embryonic stem cell-derived retinal cells restores visual function in the Royal College of Surgeons rats.

AIM OF STUDY: To evaluate the feasibility of transplantation of embryonic stem cell (ESC)-derived retinal cells in the treatment of retinal degeneration. MATERIALS AND METHODS: Rat ESCs were isolated and induced into retinal progenitor cells (RPCs) in vitro, which were subsequently induced into retinal pigment epithelium cells (RPEs) and photoreceptors (PRCs). All cells were identified by Western blot detection of their specific markers. RPEs and PRCs were, respectively, injected into the retina of Royal College of Surgeons (RCSs) rats. Control group was injected with PBS. Post-transplantation visual function was determined by electroretinography (ERG). The histology of the whole eye was compared by H&E staining. RESULTS: RPEs and PRCs were successfully derived from rat ESCs through the two-step differentiation as indicated by the presence of ESC- (Oct-3/4, Nanog, TRA-1-60 and TRA-1-81), RPC- (Rx, Mitf, Pax6 and Chx10), RPE- (RPE65 and keratin) and PRC-specific markers (blue opsin, red/green opsin, recoverin and rhodopsin) in Western blot. The amplitude of ERG a- and b-wave in RPE- and PRC-transplanted groups at week 2 and 10 after transplantation was markedly higher compared with PBS controls. Retinal injury and vascular stress response was not detected in any of the RCS rats after transplantation. CONCLUSION: The developed stepwise protocol can derive retinal cells from ESCs. Transplantation of these retinal cells can restore visual function of RCS rats. Our study provides evidence for potential clinical application of ESC-based cell therapy for retinal degeneration.

Vasodilator-stimulated phosphoprotein (VASP) is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and antiplatelet effect of prasugrel in rats.

Vasodilator-stimulated phosphoprotein (VASP) is a member of actin regulatory proteins implicated in platelet adhesion. In addition, phosphorylation of VASP is utilised for the assessment of platelet reactivity in patients treated with P2Y12 receptor antagonists, a class of antiplatelet agents. However, the role of VASP in platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of P2Y12 receptor antagonists remains unclear. We investigated these effects using heterozygous and homozygous VASP knockout rats generated with a CRISPR/Cas9 system. Baseline characteristics, such as haematology and other biochemical parameters, were comparable among the genotypes. In vitro platelet aggregation stimulated by adenosine diphosphate (ADP) or collagen, P-selectin expression of rat platelets treated with ADP, and in vivo thrombocytopenia induced by collagen were also comparable among the genotypes. In addition, in vivo thrombogenesis in a ferric chloride-induced arterial thrombosis model and bleeding time were also comparable among the genotypes. Furthermore, the in vitro antiplatelet effect of prasugrel, a third-generation P2Y12 receptor antagonist, was unaffected by VASP knockout. Although phosphorylated VASP is still an important surrogate marker specific for P2Y12 antagonists, our findings demonstrate that VASP is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of prasugrel in rats.

Endocrine disruptor exposure during development increases incidence of uterine fibroids by altering DNA repair in myometrial stem cells.

Despite the major negative impact uterine fibroids (UFs) have on female reproductive health, little is known about early events that initiate development of these tumors. Somatic fibroid-causing mutations in mediator complex subunit 12 (MED12), the most frequent genetic alterations in UFs (up to 85% of tumors), are implicated in transforming normal myometrial stem cells (MSCs) into tumor-forming cells, though the underlying mechanism(s) leading to these mutations remains unknown. It is well accepted that defective DNA repair increases the risk of acquiring tumor-driving mutations, though defects in DNA repair have not been explored in UF tumorigenesis. In the Eker rat UF model, a germline mutation in the Tsc2 tumor suppressor gene predisposes to UFs, which arise due to "second hits" in the normal allele of this gene. Risk for developing these tumors is significantly increased by early-life exposure to endocrine-disrupting chemicals (EDCs), suggesting increased UF penetrance is modulated by early drivers for these tumors. We analyzed DNA repair capacity using analyses of related gene and protein expression and DNA repair function in MSCs from adult rats exposed during uterine development to the model EDC diethylstilbestrol. Adult MSCs isolated from developmentally exposed rats demonstrated decreased DNA end-joining ability, higher levels of DNA damage, and impaired ability to repair DNA double-strand breaks relative to MSCs from age-matched, vehicle-exposed rats. These data suggest that early-life developmental EDC exposure alters these MSCs' ability to repair and reverse DNA damage, providing a driver for acquisition of mutations that may promote the development of these tumors in adult life.

Probiotics Affect One-Carbon Metabolites and Catecholamines in a Genetic Rat Model of Depression.

SCOPE: Probiotics may influence one-carbon (C1) metabolism, neurotransmitters, liver function markers, or behavior. METHODS AND RESULTS: Male adult Flinders Sensitive Line rats (model of depression, FSL; n = 22) received Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 (109 or 1010 colony-forming units per day) or vehicle for 10 weeks. The controls, Flinders Resistant Line rats (FRL, n = 8), only received vehicle. C1-related metabolites were measured in plasma, urine, and different tissues. Monoamine concentrations were measured in plasma, hippocampus, and prefrontal cortex. Vehicle-treated FSL rats had higher plasma concentrations of betaine, choline, and dimethylglycine, but lower plasma homocysteine and liver S-adenosylmethionine (SAM) than FRLs. FSL rats receiving high-dose probiotics had lower plasma betaine and higher liver SAM compared to vehicle-treated FSL rats. FSLs had higher concentrations of norepinephrine, dopamine, and serotonin than FRLs across various brain regions. Probiotics decreased plasma dopamine in FSLs in a dose-dependent manner. There were no detectable changes in liver function markers or behavior. CONCLUSIONS: Probiotics reduced the flow of methyl groups via betaine, increased liver SAM, and decreased plasma dopamine and norepinephrine. Since these changes in methylation and catecholamine pathways are known to be involved in several diseases, future investigation of the effect of probiotics is warranted.

Characterization of neuroendocrine tumors in heterozygous mutant MENX rats: a novel model of invasive medullary thyroid carcinoma.

Rats affected by the MENX syndrome spontaneously develop multiple neuroendocrine tumors (NETs) including adrenal, pituitary and thyroid gland neoplasms. MENX was initially reported to be inherited as a recessive trait and affected rats were found to be homozygous for the predisposing Cdkn1b mutation encoding p27. We here report that heterozygous MENX-mutant rats (p27+/mut) develop the same spectrum of NETs seen in the homozygous (p27mut/mut) animals but with slower progression. Consequently, p27+/mut rats have a significantly shorter lifespan compared with their wild-type (p27+/+) littermates. In the tumors of p27+/mut rats, the wild-type Cdkn1b allele is neither lost nor silenced, implying that p27 is haploinsufficient for tumor suppression in this model. Transcriptome profiling of rat adrenal (pheochromocytoma) and pituitary tumors having different p27 dosages revealed a tissue-specific, dose-dependent effect of p27 on gene expression. In p27+/mut rats, thyroid neoplasms progress to invasive and metastatic medullary thyroid carcinomas (MTCs) accompanied by increased calcitonin levels, as in humans. Comparison of expression signatures of late-stage vs early-stage MTCs from p27+/mut rats identified genes potentially involved in tumor aggressiveness. The expression of a subset of these genes was evaluated in human MTCs and found to be associated with aggressive RET-M918T-positive tumors. Altogether, p27 haploinsufficiency in MENX rats uncovered a novel, representative model of invasive and metastatic MTC exploitable for translational studies of this often aggressive and incurable cancer.