Blake Pouch: It Is Time to Revisit Classical Descriptions on the Development of the Roof of the Fourth Ventricle.

Objectives: Lipid-lowering agents have been shown to be associated with decreased abdominal aortic aneurysm (AAA) growth and rupture. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that binds and degrades the LDL receptor to reduce LDL uptake. It is hypothesized that PCSK9 inhibition reduces aortic inflammation and vascular remodeling to mitigate AAA growth. Methods: AAAs were induced in 8–12-week-old male C57BL6 (WT) mice with 0.4U/ml topical porcine pancreatic or heat-inactivated elastase with or without Evolocumab (10mg/kg) given i.p. on days 1 and 7. Aortic diameter was measured and tissue harvested for histology and cytokine analysis on day 14. A model of chronic AAA with elastase+BAPN treatment was also used with or without administration of Evolocumab on day 14 and 21 following aneurysm formation, with harvest on day 28. Finally, a retrospective chart review was performed on AAA patients from 2015-2023 taking PCSK9 inhibitor alone (n=54), statins (n=3664), or neither (n=645) to determine survival. Statistical analyses were performed using GraphPad Prism with two-tailed t-tests, ANOVA, or non-parametric tests and Kaplan-Meier analysis was used to estimate survival. Data is shown as mean+/-S.E. with p<0.05 being significant. Results: An increase in aortic diameter was observed in elastase-treated WT mice which was attenuated by Evolocumab on day 14 (147.5±7.2% vs. 103.4±5.6%, n=19-20/group, p<0.0001). Similarly, the elastase+BAPN-induced increase in aortic diameter was mitigated with Evolocumab on day 28 (368.0±62.3% vs. 206.0±32.1%, n=8-10/group, p<0.05). Evolocumab treatment significantly decreased proinflammatory cytokine expression (IL-1β, HMGB1, IL-6,&TNFα) in aortic tissue compared to untreated controls in both models and was associated with less macrophage infiltration and elastin disruption on VVG staining, and increased SMa-actin expression. In the human data, freedom from all-cause mortality in patients with AAAs at the time of last follow up was lowest in the PCSK9i group (85%) compared to statins (72%) and no treatment groups (69%; log-rank p<0.001). Conclusion: Our results suggest that PCSK9 inhibition attenuates aortic inflammation and aneurysm formation in murine models of AAA. Moreover, PCSK9 inhibition was associated with reduced mortality in AAA patients. Further studies are underway to elucidate the molecular pathways of PCSK9 inhibition and decipher their role in the pathogenesis of AAAs and aortic rupture.

WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the aggregation of misfolded α-synuclein and progressive spreading of the aggregates from a few discrete regions to wider brain regions. Although PD has been classically considered a movement disorder, a large body of clinical evidence has revealed the progressive occurrence of non-motor symptoms. Patients present visual symptoms in the initial stages of the disease, and accumulation of phospho-α-synuclein, dopaminergic neuronal loss, and retinal thinning has been observed in the retinas of PD patients. Based on such human data, we hypothesized that α-synuclein aggregation can initiate in the retina and spread to the brain through the visual pathway. Here, we demonstrate accumulation of α-synuclein in the retinas and brains of naive mice after intravitreal injection of α-synuclein preformed fibrils (PFFs). Histological analyses showed deposition of phospho-α-synuclein inclusions within the retina 2 months after injection, with increased oxidative stress leading to loss of retinal ganglion cells and dopaminergic dysfunction. In addition, we found accumulation of phospho-α-synuclein in cortical areas with accompanying neuroinflammation after 5 months. Collectively, our findings suggest that retinal synucleinopathy lesions initiated by intravitreal injection of α-synuclein PFFs spread to various brain regions through the visual pathway in mice.

The microtubule associated protein tau plays a critical role in the pathogenesis of neurodegenerative disease. Recent studies suggest that tau also plays a role in disorders of neuronal connectivity, including epilepsy and post-traumatic stress disorder. Animal studies have shown that the MAPT gene, which codes for the tau protein, undergoes complex pre-mRNA alternative splicing to produce multiple isoforms during brain development. Human data, particularly on temporal and regional variation in tau splicing during development are however lacking. In this study, we present the first detailed examination of the temporal and regional sequence of MAPT alternative splicing in the developing human brain. We used a novel computational analysis of large transcriptomic datasets (total n = 502 patients), quantitative polymerase chain reaction (qPCR) and western blotting to examine tau expression and splicing in post-mortem human fetal, pediatric and adult brains. We found that MAPT exons 2 and 10 undergo abrupt shifts in expression during the perinatal period that are unique in the canonical human microtubule-associated protein family, while exon 3 showed small but significant temporal variation. Tau isoform expression may be a marker of neuronal maturation, temporally correlated with the onset of axonal growth. Immature brain regions such as the ganglionic eminence and rhombic lip had very low tau expression, but within more mature regions, there was little variation in tau expression or splicing. We thus demonstrate an abrupt, evolutionarily conserved shift in tau isoform expression during the human perinatal period that may be due to tau expression in maturing neurons. Alternative splicing of the MAPT pre-mRNA may play a vital role in normal brain development across multiple species and provides a basis for future investigations into the developmental and pathological functions of the tau protein.

The order of appearance of the carpals, tarsals and secondary epiphyseal centers in the hand-wrist and foot-ankle regions of the fetal and infant pigtail macaque (Macaca nemestrina) was reconstructed using a cross-sectional sample of 127 animals and a longitudinal one of 14 animals. Comparison of the most common sequence in the monkey with that described in the human studies showed major interspecific differences in the order of appearance of several groups of centers, particularly those involving the thumb and great toe. The monkey displays a degree of sequence variability equivalent to that documented by the human longitudinal data, with certain centers markedly variable in timing and/or order of appearance. The evidence from related animals suggests a strong genetic component underlying some of the variable sequences observed, as has been reported in the human studies. The demonstrated variability in both timing and sequence precludes the use of specific centers in the determination of gestational age in fetuses of unknown conception date.

BackgroundLittle is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.ObjectiveTo investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.MethodsAsthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.ResultsIn total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.ConclusionsOur data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.

Early sites (ca. 12000–10400 cal. BP) located at the Imilac and Punta Negra high Andean basin in the southern Puna de Atacama (24.5°) appear as open sites with little stratigraphic potential. Most of the record corresponds to abundant lithic material as opposed to a few bone remains. Nevertheless, the presence of numerous projectile points and artefacts related to the processing of fauna allows us to infer the importance of this resource. The scarce remains recovered are generally in a very poor state of preservation, fragmented and very fragile. To understand the taphonomic trajectories and the absence of bone remains in the records, we have implemented a line of histotaphonomic actualistic analysis in two ecological floors corresponding to the high ravine zone (4,000 masl) and the current borders of the Salar de Punta Negra (3,000 masl), where the sites are located. Through the sampling of modern skeletal remains, mostly from camelids in both sectors, we sought to explore diagenetic processes by means of histological analysis. Preliminary results based on transmitted light microscopy show that diagenetic modification conditions seem to be more dynamic and variable in the salt-flat sector than in the surrounding ravine environments associated with watercourses that come from the Llullaillaco volcano.

Temporomandibular joint (TMJ) disorders are often associated with development of osteoarthritis-like changes in the mandibular condyle. Discoidin domain receptor 2 (DDR2), a collagen receptor preferentially activated by type I and III collagen found in the TMJ and other fibrocartilages, has been associated with TMJ degeneration, but its role in normal joint development has not been previously examined. Using Ddr2 LacZ-tagged mice and immunohistochemistry, we found that DDR2 is preferentially expressed and activated in the articular zone of TMJs but not knee joints. To assess the requirement for Ddr2 in TMJ development, studies were undertaken to compare wild-type and smallie (slie) mice, which contain a spontaneous deletion in Ddr2 to produce an effective null allele. Analysis of TMJs from newborn Ddr2slie/slie mice revealed a developmental delay in condyle mineralization, as measured by micro–computed tomography and histologic analysis. In marked contrast, knee joints of Ddr2slie/slie mice were normal. Analysis of older Ddr2slie/slie mice (3 and 10 mo) revealed that the early developmental delay led to a dramatic and progressive loss of TMJ articular integrity and osteoarthritis-like changes. Mutant condyles had a rough and flattened bone surface, accompanied by a dramatic loss of bone mineral density. Mankin scores showed significantly greater degenerative changes in the TMJs of 3- and 10-mo-old Ddr2slie/slie mice as compared with wild-type controls. No DDR2-dependent degenerative changes were seen in knees. Analysis of primary cultures of TMJ articular chondrocytes from wild-type and Ddr2slie/slie mice showed defects in chondrocyte maturation and mineralization in the absence of Ddr2. These studies demonstrate that DDR2 is necessary for normal TMJ condyle development and homeostasis and that these DDR2 functions are restricted to TMJ fibrocartilage and not seen in the hyaline cartilage of the knee.

From in vitro evaluation to human postmortem pre-validation of a radiopaque and resorbable internal biliary stent for liver transplantation applications

A histological analysis of a high voltage electric current injury to an upper limb

In both human and mouse, the Igf2 gene, localized on chromosomes 11 and 7, respectively, is expressed from the paternally inherited chromosome in the majority of tissues. Insulin-like growth factor-II (IGF-II) plays an important role in embryonic growth, and aberrant IGF2 expression has been documented in several human pathologies, such as Beckwith-Wiedemann syndrome (BWS), and a wide variety of tumors. Human and mouse genetic data strongly implicate another gene, CDKN1C (p57(kip2)), located in the same imprinted gene cluster on human chromosome II, in BWS. p57(KIP2) is a cyclin-dependent kinase inhibitor and is required for normal mouse embryonic development. Mutations in CDKN1C (p57(kip2)) have been identified in a small proportion of patients with BWS, and removal of the gene from mice by targeted mutagenesis produces a phenotype with elements in common with this overgrowth syndrome. Patients with BWS with biallelic expression of IGF2 or with a CDKN1C (p57(kip2)) mutation, as well as overlapping phenotypes observed in two types of mutant mice, the p57(kip2) knockout and IGF-II-overexpressing mice, strongly suggest that the genes may act in a common pathway of growth control in situations where Igf2 expression is abnormal. Herein, we show that p57(kip2) expression is reduced on IGF-II treatment of primary embryo fibroblasts in a dose-dependent manner. In addition, p57(kip2) expression is down-regulated in mice with high serum levels of IGF-II. These data suggest that the effects of increased IGF-II in BWS may, in part, be mediated through a decrease in p57(kip2) gene expression.

BackgroundSphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1−/−) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array.ResultsTwo-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1−/− mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1−/− neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45.ConclusionUsing SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

Development of the motivational system during adolescence, and its sensitivity to disruption by nicotine

We studied the interaction between VEGF and BMP2 during bone formation and bone healing. Results indicate that VEGF antagonist inhibited BMP2-elicited bone formation, whereas the delivery of exogenous VEGF enhanced BMP2-induced bone formation and bone healing through modulation of angiogenesis. Angiogenesis is closely associated with bone formation during normal bone development and is important for the bone formation elicited by BMP4. However, it remains unknown whether vascular endothelial growth factor (VEGF) also interacts with other BMPs, especially BMP2, in bone formation and bone healing. For this study, mouse muscle-derived stem cells were transduced to express BMP2, VEGF, or VEGF antagonist (sFlt1). We studied the angiogenic process during endochondral bone formation elicited by BMP2, a prototypical osteogenic BMP. Using radiographic and histologic analyses, we also evaluated the interaction between VEGF and BMP2 during bone formation and bone healing. Our results indicate that BMP2-elicited bone formation comprises two phases of angiogenesis, with an early phase occurring before the appearance of hypertrophic cartilage, followed by a late phase coupled with the appearance of hypertrophic cartilage. Our finding that the administration of sFlt1, a specific antagonist of VEGF, significantly inhibited BMP2-induced bone formation and the associated angiogenesis indicates that endogenous VEGF activity is important for bone formation. Furthermore, we found that the delivery of exogenous VEGF enhanced BMP2-induced bone formation and bone healing by improving angiogenesis, which in turn led to accelerated cartilage resorption and enhanced mineralized bone formation. Our findings also indicate that the ratio between VEGF and BMP2 influences their synergistic interaction, with a higher proportion of VEGF leading to decreased synergism. Our study also revealed unique VEGF-BMP2 interactions that differ from the VEGF-BMP4 interactions that we have described previously. This study, along with previously published work, shows that VEGF interacts synergistically with both BMP4 and BMP2 but elicits substantially different effects with these two BMPs.