MicroRNA-24 regulates vascularity after myocardial infarction

BACKGROUND: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. METHODS AND RESULTS: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. CONCLUSIONS: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease. [KEYWORDS: Animals, Apoptosis/drug effects, Arterioles/pathology, Capillaries/pathology, Cell Hypoxia, Cells, Cultured/drug effects/metabolism, Collagen, Drug Combinations, Drug Evaluation, Preclinical, Endothelial Cells/ metabolism/pathology, GATA2 Transcription Factor/biosynthesis/genetics, Gene Expression Profiling, Heart Failure/etiology, Heme Oxygenase-1/biosynthesis/genetics, Laminin, Male, Mice, Mice, Inbred C57BL, MicroRNAs/antagonists & inhibitors/genetics/ physiology, Myocardial Infarc

Generation of mice with a conditional allele of the p120 Ras GTPase-activating protein

p120 Ras GTPase-activating protein (RasGAP) encoded by the rasa1 gene in mice is a prototypical member of the RasGAP family of proteins involved in negative-regulation of the p21 Ras proto-oncogene. RasGAP has been implicated in signal transduction through a number of cell surface receptors. In humans, inactivating mutations in the coding region of the RASA1 gene cause capillary malformation arteriovenous malformation. In mice, generalized disruption of the rasa1 gene results in early embryonic lethality associated with defective vasculogenesis and increased apoptosis of neuronal cells. The early lethality in this mouse model precludes its use to further study the importance of RasGAP as a regulator of cell function. Therefore, to circumvent this problem, we have generated a conditional rasa1 knockout mouse. In this mouse, an exon that encodes a part of the RasGAP protein essential for catalytic activity has been flanked by loxP recognition sites. With the use of different constitutive and inducible Cre transgenic mouse lines, we show that deletion of this exon from the rasa1 locus results in effective loss of expression of catalytically-active RasGAP from a variety of adult tissues. The conditional rasa1 mouse will be useful for the analysis of the role of RasGAP in mature cell types. genesis 45:762–767, 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57536/1/20354_ftp.pd

High-Flow Vascular Malformations in Children.

Children can have a variety of intracranial vascular anomalies ranging from small and incidental with no clinical consequences to complex lesions that can cause substantial neurologic deficits, heart failure, or profoundly affect development. In contrast to high-flow lesions with direct arterial-to-venous shunts, low-flow lesions such as cavernous malformations are associated with a lower likelihood of substantial hemorrhage, and a more benign course. Management of vascular anomalies in children has to incorporate an understanding of how treatment strategies may affect the normal development of the central nervous system. In this review, we discuss the etiologies, epidemiology, natural history, and genetic risk factors of three high-flow vascular malformations seen in children: brain arteriovenous malformations, intracranial dural arteriovenous fistulas, and vein of Galen malformations

Parkes Weber syndrome associated with two somatic pathogenic variants in RASA1

Parkes Weber syndrome is associated with autosomal dominant inheritance, caused by germline heterozygous inactivating changes in th

The Genetics Of Vein Of Galen Malformation And Assessment Of Candidate Genes In Xenopus Tropicalis

The Vein of Galen Malformation (VOGM) is a specific subtype of arteriovenous malformation (AVM) that becomes evident in weeks 6-11 of embryonic development. VOGM comprise less than 1% of all vascular malformations, yet represent 30% of all pediatric intracranial vascular malformations. Depending on their specific characteristics, i.e. its feeding vessels, it can present clinically as devastating congestive heart failure in neonates, hydrocephalus in children, or seizures and headaches in young adults. Advances in treatment provide improved survival, primarily through endovascular surgery. The genetic and molecular etiology of VOGM remains relatively unknown, with the only associated genes being in the context of other syndromes, including seven mutations in p120-RasGAP (RASA1) in Cutaneous Malformations-Arteriovenous Malformations (CM-AVM), as well as one mutation each in activin A receptor type II-like 1 (ACVRL1) mutation and Endoglin (ENG) in Hereditary Hemorraghic Teleangiectasia. Our limited knowledge of the molecular genetics of VOGM has hindered the development of novel therapies. We hypothesized that the apparent sporadic occurrence of VOGM may frequently be attributable to damaging de novo mutation events or incomplete penetrance of rare transmitted variants. Unbiased whole-exome sequencing (WES) can overcome these barriers for gene discovery. We recruited 55 patients, including 52 parent-offspring trios. WES revealed statistically significant rare, damaging de novo mutations in chromatin modifier genes involved in brain and vascular development (p=8.9 x 10-4). VOGM probands also had inherited, missense deleterious and loss of function, in ephrin signaling genes, specifically a whole exome significant mutation burden in EPHB4 (p=7.47 x 10−10). Finally, we observed a whole exome significant inherited mutation in Claudin 14 (p= 6.44 x 10−7). Inherited mutations demonstrated incomplete penetrance and variable expressivity with mutation carriers often exhibiting cutaneous vascular abnormalities suggesting a two-hit mechanism. The identified mutations account for 30% of studied VOGM cases. To functionally validate candidate mutations and establish causality, we developed a screening platform using Xenopus tropicalis by using CRISP/Cas9 gene editing and observing the effects of candidate gene knockdown on vasculogenesis and brain vasculature. CRISPR/Cas9 knockdown of EPHB4 and CLDN14 yielded significantly abnormal vasculogenesis (p=0.0028 and p=0.0001 respectively) observed by in situ hybridization. We implemented and modified established clearing techniques to allow visualization of the full thickness of the Xenopus brain vasculature. The evolutionary precursor to the vein of Galen, the posterior vascular plexus (PVP), and the mesencephalic veins (MSV), were imaged. EPHB4 and CLDN14 CRISPR/Cas9 knockdown revealed significant decreases in MSV length (p Together these findings are the first step in better understanding the mechanism and pathogenesis of VOGM and potential novel therapeutic targets. While Xenopus is not a perfect model system, it does show promise as a tool to assess candidate VOGM that merit further study in mammalian systems harboring a true vein of Galen

The Ras GTPase Activating Protein p120 RasGAP as a Regulator of Cardiovascular System Development, Lymphatic System Maintenance, and a T Cell Lineage Tumor Suppressor.

Ras signaling activates multiple pathways that drive cell growth, proliferation, survival, and differentiation. Ras is activated downstream of multiple receptors by the recruitment and interaction with Ras guanine nucleotide exchange factors (RasGEFs). Efficient regulation of this signaling is essential for the avoidance of developmental defects and cancer, and cessation of Ras signaling is dependent on the recruitment of Ras GTPase activating proteins (RasGAPs) for juxtaposition with Ras. RasGAPs physically interact with Ras to allow for Ras hydrolization of bound GTP. There are multiple RasGAPs, which have varied tissue expression patterns. The RasGAP p120 RasGAP (RASA1) has been shown to be important in the mouse embryonic cardiovasculature and adult lymphatic system. However, RASA1 has other roles than regulation of Ras, including interactions with p190 RhoGAP for directed cell migration. To address this, we generated a mouse carrying a point mutation in its GAP domain that ablates its GAP activity while retaining all other functions. With the use of these mice we show that it is the loss of RASA1’s ability to regulate Ras that results in failed cardiovascular development in embryos and the instability and outgrowth of lymphatic vessels in adult mice. Although RASA1 plays an important role in many tissues, it has a minor role in the regulation of Ras during T cell development. Another RasGAP, Neurofibromin 1 (NF1) also appears to have a largely dispensable function in the T cell compartment. Since loss of both RASA1 and NF1 in developing embryos results in earlier embryonic lethality than loss of either RasGAP alone, we hypothesized that these RasGAPs may have overlapping functions in T cells. Indeed, concurrent deletion of RASA1 and NF1 in the T cell compartment resulted in in a randomly occurring T cell acute lymphoblastic leukemia (T-ALL) in mice associated with acquisition of somatic activating mutations in the Notch1 receptor previously implicated in T-ALL pathogenesis.PHDImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111615/1/balubeck_1.pd

An approach for the identification of targets specific to bone metastasis using cancer genes interactome and gene ontology analysis

Metastasis is one of the most enigmatic aspects of cancer pathogenesis and is a major cause of cancer-associated mortality. Secondary bone cancer (SBC) is a complex disease caused by metastasis of tumor cells from their primary site and is characterized by intricate interplay of molecular interactions. Identification of targets for multifactorial diseases such as SBC, the most frequent complication of breast and prostate cancers, is a challenge. Towards achieving our aim of identification of targets specific to SBC, we constructed a 'Cancer Genes Network', a representative protein interactome of cancer genes. Using graph theoretical methods, we obtained a set of key genes that are relevant for generic mechanisms of cancers and have a role in biological essentiality. We also compiled a curated dataset of 391 SBC genes from published literature which serves as a basis of ontological correlates of secondary bone cancer. Building on these results, we implement a strategy based on generic cancer genes, SBC genes and gene ontology enrichment method, to obtain a set of targets that are specific to bone metastasis. Through this study, we present an approach for probing one of the major complications in cancers, namely, metastasis. The results on genes that play generic roles in cancer phenotype, obtained by network analysis of 'Cancer Genes Network', have broader implications in understanding the role of molecular regulators in mechanisms of cancers. Specifically, our study provides a set of potential targets that are of ontological and regulatory relevance to secondary bone cancer.Comment: 54 pages (19 pages main text; 11 Figures; 26 pages of supplementary information). Revised after critical reviews. Accepted for Publication in PLoS ON

Parkes Weber Syndrome

Introduction: Parkes Weber Syndrome (PWS) is a traditional eponymous denomination of a certain type of angiodysplasia. It is a congenital vascular disease which consists of capillary malformation (CM), venous malformation (VM), lymphatic malformation (LM), congenital arteriovenous malformation (AVM) and multiple arteriovenous fistulas (AVFs). There is a soft-tissue and skeletal hypertrophy of the affected extremity (usually a lower extremity). Moreover the affected limb is warmer and longer than the other side. Objective: The aim of this article is to summarize the current state of knowledge about Parkes Weber Syndrome: the pathophysiology, genetic inheritance, the main symptoms, the diagnosis especially differential diagnosis and the current treatment. Brief descriptions of the state of knowledge: Despite many years, physicians still have difficulties with diagnosing PWS correctly. Although the aetiology is unknown, it is claimed that PWS is caused by mutations of the RASA1, gene located on chromosome 5q13.1, which are inherited in an autosomal dominant manner. This gene is responsible for mediating cellular growth, differentiation and proliferation. No efficacious pharmacological treatment has been found. Nowadays Tranexamic Acid, Sirolimus, Everolimus and Miconazole are used in medical practice. The most frequently utilized invasive treatment methods are amputation, surgical AVM resection and occasionally stent-graft implantation. Furthermore it is thought that embolization, alone or combined with surgical resection leads to clinical improvement. Summary: Despite the existence of many clinical trials, long term observations and scientific speculations, PWS can still be challenging for clinicians. There is a need for further scientific, molecular and genetic research to diagnose this phenomenon correctly, because despite fact, that its symptoms are similar to other syndromes or entities, therapeutic strategies differ significantly. It is important to increase the awareness of inheritance in an autosomal dominant manner in generation of patients with PWS

Master of Science

thesisPort wine stains are capillary malformations that are typically located in the dermis of the head and neck. They affect 0.3% of the population. Current theories suggest that port wine stains are caused by somatic mutations that disrupt vascular development. I hypothesize that port wine stains are multifactorial and understanding their genetic determinants could provide insight into novel treatments. This study used a custom next generation sequencing panel and digital polymerase chain reaction to investigate genetic variants in 12 isolated port wine stain cases (i.e., individuals with port wine stain alone). Novel variants were identified in GNAQ, SOX10, and RASA1. The previously identified GNAQ c.548G>A, p.Arg183Gln mutation was confirmed in 9 of 12 cases with an allele frequency ranging from 1.73 to 7.42%. The custom next generation sequencing panel allowed for detection of low level mosaicism found in 10 of 12 isolated port wine stain cases. A novel GNAQ c.547C>G, p.Arg183Gly mutation was identified in one case. Digital polymerase chain reaction confirmed novel variants detected by next generation sequencing. The results from both methods were highly concordant providing an effective way to detect low level mosaicism in port wine stains. Importantly, the identification of novel genetic mutations in port wine stains may facilitate the development of novel treatments

Independent stratum formation on the avian sex chromosomes reveals inter-chromosomal gene conversion and predominance of purifying selection on the w chromosome

We used a comparative approach spanning three species and 90 million years to study the evolutionary history of the avian sex chromosomes. Using whole transcriptomes, we assembled the largest cross-species dataset of W-linked coding content to date. Our results show that recombination suppression in large portions of the avian sex chromosomes has evolved independently, and that long-term sex chromosome divergence is consistent with repeated and independent inversions spreading progressively to restrict recombination. In contrast, over short-term periods we observe heterogeneous and locus-specific divergence. We also uncover four instances of gene conversion between both highly diverged and recently evolved gametologs, suggesting a complex mosaic of recombination suppression across the sex chromosomes. Lastly, evidence from 16 gametologs reveal that the W chromosome is evolving with a significant contribution of purifying selection, consistent with previous findings that W-linked genes play an important role in encoding sex-specific fitness