Transcriptomic analysis to identify genes associated with selective hippocampal vulnerability in Alzheimer's disease.

Selective vulnerability of different brain regions is seen in many neurodegenerative disorders. The hippocampus and cortex are selectively vulnerable in Alzheimer's disease (AD), however the degree of involvement of the different brain regions differs among patients. We classified corticolimbic patterns of neurofibrillary tangles in postmortem tissue to capture extreme and representative phenotypes. We combined bulk RNA sequencing with digital pathology to examine hippocampal vulnerability in AD. We identified hippocampal gene expression changes associated with hippocampal vulnerability and used machine learning to identify genes that were associated with AD neuropathology, including SERPINA5, RYBP, SLC38A2, FEM1B, and PYDC1. Further histologic and biochemical analyses suggested SERPINA5 expression is associated with tau expression in the brain. Our study highlights the importance of embracing heterogeneity of the human brain in disease to identify disease-relevant gene expression.

Annexin A3 is necessary for parallel artery-vein alignment in the mouse retina.

BACKGROUND: Annexin A3 (Anxa3) is a member of the calcium-regulated, cell membrane-binding family of annexin proteins. We previously confirmed that Anxa3 is expressed in the endothelial lineage in vertebrates and that loss of anxa3 in Xenopus laevis leads to embryonic blood vessel defects. However, the biological function of Anxa3 in mammals is completely unknown. In order to investigate Anxa3 vascular function in mammals, we generated an endothelial cell-specific Anxa3 conditional knockout mouse model (Anxa3f/f ;Tie2-Cre). RESULTS: Anxa3f/f ;Tie2-Cre mice are born at Mendelian ratios and display morphologically normal blood vessels during development. However, loss of Anxa3 leads to artery-vein (AV) misalignment characterized by atypical AV crossovers in the postnatal and adult retina. CONCLUSIONS: Anxa3 is not essential for embryonic blood vessel formation but is required for proper parallel AV alignment in the murine retina. AV crossovers associated with Anxa3f/f ;Tie2-Cre mice are similar to AV intersections observed in patients with branch retinal vein occlusion (BRVO), although we did not observe occluded vessels. This new Anxa3 mouse model may provide a basis for understanding AV crossover formation associated with BRVO.

Angiopoietin-2 Inhibition Rescues Arteriovenous Malformation in a Smad4 Hereditary Hemorrhagic Telangiectasia Mouse Model.

BACKGROUND: Hereditary hemorrhagic telangiectasia is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in 4 transforming growth factor beta (TGFß) pathway members, including the central transcriptional mediator of the TGFß pathway, Smad4. Loss of Smad4 causes the formation of inappropriate, fragile connections between arteries and veins called arteriovenous malformations (AVMs), which can hemorrhage leading to stroke, aneurysm, or death. Unfortunately, the molecular mechanisms underlying AVM pathogenesis remain poorly understood, and the TGFß downstream effectors responsible for hereditary hemorrhagic telangiectasia-associated AVM formation are currently unknown. METHODS: To identify potential biological targets of the TGFß pathway involved in AVM formation, we performed RNA- and chromatin immunoprecipitation-sequencing experiments on BMP9 (bone morphogenetic protein 9)-stimulated endothelial cells (ECs) and isolated ECs from a Smad4-inducible, EC-specific knockout ( Smad4-iECKO) mouse model that develops retinal AVMs. These sequencing studies identified the angiopoietin-Tek signaling pathway as a downstream target of SMAD4. We used monoclonal blocking antibodies to target a specific component in this pathway and assess its effects on AVM development. RESULTS: Sequencing studies uncovered 212 potential biological targets involved in AVM formation, including the EC surface receptor, TEK (TEK receptor tyrosine kinase) and its antagonistic ligand, ANGPT2 (angiopoietin-2). In Smad4-iECKO mice, Angpt2 expression is robustly increased, whereas Tek levels are decreased, resulting in an overall reduction in angiopoietin-Tek signaling. We provide evidence that SMAD4 directly represses Angpt2 transcription in ECs. Inhibition of ANGPT2 function in Smad4-deficient mice, either before or after AVMs form, prevents and alleviates AVM formation and normalizes vessel diameters. These rescue effects are attributed to a reversion in EC morphological changes, such as cell size and shape that are altered in the absence of Smad4. CONCLUSIONS: Our studies provide a novel mechanism whereby the loss of Smad4 causes increased Angpt2 transcription in ECs leading to AVM formation, increased blood vessel calibers, and changes in EC morphology in the retina. Blockade of ANGPT2 function in an in vivo Smad4 model of hereditary hemorrhagic telangiectasia alleviated these vascular phenotypes, further implicating ANGPT2 as an important TGFß downstream mediator of AVM formation. Therefore, alternative approaches that target ANGPT2 function may have therapeutic value for the alleviation of hereditary hemorrhagic telangiectasia symptoms, such as AVMs.

Vascular deficiency of Smad4 causes arteriovenous malformations: a mouse model of Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). Mutations in TGFß pathway members ALK1, ENG and SMAD4 lead to HHT. However, a Smad4 mouse model of HHT does not currently exist. We aimed to create and characterize a Smad4 endothelial cell (EC)-specific, inducible knockout mouse (Smad4f/f;Cdh5-CreERT2) that could be used to study AVM development in HHT. We found that postnatal ablation of Smad4 caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery-vein gene expression are associated with Smad4 deficiency in the vasculature. Furthermore, we show that depletion of Smad4 leads to decreased Vegfr2 expression, and concurrent loss of endothelial Smad4 and Vegfr2 in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFß and VEGF signaling pathways in AVM pathogenesis.

Increased expression of deleted in malignant brain tumors (DMBT1) gene in precancerous gastric lesions: Findings from human and animal studies.

Helicobacter pylori infection triggers a cascade of inflammatory stages that may lead to the appearance of non-atrophic gastritis, multifocal atrophic, intestinal metaplasia, dysplasia, and cancer. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by binding to pathogens. Initial studies showed its deletion and loss of expression in a variety of tumors but the role of this gene in tumor development is not completely understood. Here, we examined the role of DMBT1 in gastric precancerous lesions in Caucasian, African American and Hispanic individuals as well as in the development of gastric pathology in a mouse model of H. pylori infection. We found that in 3 different populations, mucosal DMBT1 expression was significantly increased (2.5 fold) in individuals with dysplasia compared to multifocal atrophic gastritis without intestinal metaplasia; the increase was also observed in individuals with advanced gastritis and positive H. pylori infection. In our animal model, H. pylori infection of Dmbt1-/- mice resulted in significantly higher levels of gastritis, more extensive mucous metaplasia and reduced Il33 expression levels in the gastric mucosa compared to H. pylori-infected wild type mice. Our data in the animal model suggest that in response to H. pylori infection DMBT1 may mediate mucosal protection reducing the risk of developing gastric precancerous lesions. However, the increased expression in human gastric precancerous lesions points to a more complex role of DMBT1 in gastric carcinogenesis.

Characterization of arteriovenous identity in the developing neonate mouse retina.

The murine retina has become an ideal model to study blood vessel formation. Blood vessels in the retina undergo various processes, including remodeling and differentiation, to form a stereotypical network that consists of precisely patterned arteries and veins. This model presents a powerful tool for understanding many different aspects of angiogenesis including artery and vein (AV) cell fate acquisition and differentiation. However, characterization of AV differentiation has been largely unexplored in the mouse retinal model. In this study, we describe the expression of previously established AV markers and assess arteriovenous acquisition and identity in the murine neonatal retina. Using in situ hybridization and immunofluorescent antibody staining techniques, we analyzed numerous AV differentiation markers such as EphB4-EphrinB2 and members of the Notch pathway. We find that at postnatal day 3 (P3), when blood vessels are beginning to populate the retina, AV identity is not immediately established. However, by P5 expression of many molecular identifiers of arteries and veins become restricted to their respective vessel types. This molecular distinction is more obvious at P7 and remains unchanged through P9. Overall, these studies indicate that, similar to the embryo, acquisition of AV identity occurs in a step-wise process and is largely established by P7 during retina development.