Karen Hirschi - US grants

Blood vessels and nerves develop in parallel and their survival and function in postnatal tissues are interdependent; thus, when one system is damaged, the other degenerates. Ischemic stroke is one example in which vascular damage leads to neurological degeneration and functional deficits; stroke affects 1 in 59 adults annually of whom ~5 million are permanently disabled. While the initial damage from stroke produces neuronal cell loss, the process quickly evolves into loss of other cell types and extracellular matrix, resulting in a cavitational void. Our preliminary animal studies, in a model that mimics human stroke, suggest that transplantation of neural stem cells (NSC) alone may ameliorate functional deficits caused by stroke; however, we found no neural restoration since transplanted cells integrated only into areas that retained tissue architecture. Moreover, engrafted NSC did not persist, limiting repair. We will circumvent these current limitations of cell transplantation by bioengineering a microenvironment that will sustain NSC and enable their propagation ex vivo, as well as in vivo upon transplantation. We laid the experimental groundwork for our project in previous studies in which we established a 3D model of the NSC niche via imaging and quantitative analysis, and developed biomaterials suitable for engineering this microenvironment ex vivo. We also established proof of principle that transplantation of cell-matrix constructs into stroke models is feasible and reduces lesion size. In the proposed studies, we will continue to optimize the design of our engineered niches based on our biological studies of the regulation of neurogenesis and angiogenesis in the brain (Aim 1), and by sequential testing in vitro (Aim 2) and in vivo (Aim 3) in progressively more challenging and realistic models of stroke, which will enable us to move closer to developing neuro- vascular regenerative therapies for human patients. Although our initial clinical target will be stroke-injured tissues, the insights gained, and strategies developed, from our proposed studies will be broadly applicable to repair of other neurovascular injuries such as traumatic brain injury and multiple sclerosis.

? DESCRIPTION (provided by applicant): Hematological malignancies account for ~10% of new cancer diagnoses in the US, and many require transplantation of multipotent hematopoietic stem cells (HSC) derived from allogeneic donors. Although HSC transplantation can greatly improve patient survival, the availability of matched donors is limited, and the procedure can result in life- threatening graft-vs-host disease. Ideally, patients would be transplanted with corrected autologous HSC. The recent discoveries of induced pluripotent stem (iPS) cells, and technology enabling their efficient genetic modification, transform this idea into a realistic therapeutic goal. However, one critical, and currently limiting, step toward this goal is the efficient derivation of HSC with long-term engraftment potential from patient- specific iPS cells. We have learned from murine studies that HSC are generated from specialized hemogenic endothelial cells. Studies of human embryos suggest that hemogenic endothelial cell specification is critical for human hematopoiesis, as well. Thus, understanding the molecular events that specify hemogenic endothelium is critically important for promoting the generation of HSC; its recapitulation in human stem cell systems may generate a source of HSC for clinical therapies. To begin to dissect the molecular regulation of this process, we defined the phenotype of hemogenic endothelial cells in major sites of embryonic blood production - yolk sac and aorta-gonad- mesonephros (AGM). We determined that retinoic acid (RA) signaling is essential for their development; RA deficient mutants exhibit endothelial hyper-proliferation, and do not develop hemogenic endothelium or generate HSC. We found that Notch signaling functions downstream of RA to regulate endothelial cell cycle progression and hemogenic specification in the yolk sac. With these results as a foundation, the current proposal aims to advance our knowledge of the field. Specifically, we will further elucidate the role of Notch signaling in hemogenic endothelial cell specification and define the signaling components involved in this process (Aim 1); determine the role of endothelial cell cycle control in hemogenic specification (Aim 2); and apply insights from our murine developmental studies to the formation of hemogenic endothelial cells, and HSC production, from human iPS-derived primordial endothelium (Aim 3).

ABSTRACT Self-renewing, multipotent hematopoietic stem and progenitor cells (HSPCs) are essential for the foundation and lifetime maintenance of the adult blood system. HSPCs are born during embryonic development when a subset of vascular endothelial cells (ECs), called hemogenic endothelium (hemECs), acquire hematopoietic potential and give rise to HSPC that bud from the ventral wall of the dorsal aorta. Unfortunately, the regulators of hemogenic endothelial cell specification and HSPC formation from endothelium are largely undefined. Recently, we identified miR-223 as novel regulator of hemogenic endothelial cells in zebrafish. miR-223 mutant zebrafish embryos had an increased number hemogenic endothelial cells, resulting in mature HSPC expansion from the onset and into later stages of hematopoiesis. We also found that miR-223 deficiency in mouse embryos lead to increased hemogenic endothelial cell formation. While our studies establish miR-223 as a novel regulatory factor of hemogenic endothelial cell development and HSPC generation, the specific cellular events and direct molecular targets regulated by miR-223 in these processes remain unknown. Transcriptome analysis of wild type and miR-223 mutant endothelial cells from zebrafish and mouse embryos, at stages when definitive hematopoiesis is occurring, revealed that miR-223 target-genes were enriched for genes relating to protein N- glycosylation. Interestingly, miRNAs are known to target glycosylation enzymes in processes analogous to EHT, such as endothelial-to-mesenchymal transition and cancer metastasis. However, whether miRNA-dependent glycosylation regulation extends to EHT and HSPC induction remains uninvestigated. Here, we will test the hypothesis that miR-223 fine tunes N-glycosylation levels to control endothelial to hematopoietic cell fate transition. Ultimately, we will combine mouse genetics with the optical clarity and high accessibility of the zebrafish model to discover novel mechanisms of hemogenic endothelial cell specification and HSPC formation. Discovery of new molecular pathways is imperative to overcoming current obstacles to mass-production of HSPCs from human endothelial cells for blood regenerative therapies

Blood vessels and nerves develop in parallel and their survival and function in postnatal tissues are interdependent; thus, when one system is damaged, the other degenerates. Ischemic stroke is one example in which vascular damage leads to neurological degeneration and functional deficits; stroke affects 1 in 59 adults annually of whom ~5 million are permanently disabled. While the initial damage from stroke produces neuronal cell loss, the process quickly evolves into loss of other cell types and extracellular matrix, resulting in a cavitational void. Our preliminary animal studies, in a model that mimics human stroke, suggest that transplantation of neural stem cells (NSC) alone may ameliorate functional deficits caused by stroke; however, we found no neural restoration since transplanted cells integrated only into areas that retained tissue architecture. Moreover, engrafted NSC did not persist, limiting repair. We will circumvent these current limitations of cell transplantation by bioengineering a microenvironment that will sustain NSC and enable their propagation ex vivo, as well as in vivo upon transplantation. We laid the experimental groundwork for our project in previous studies in which we established a 3D model of the NSC niche via imaging and quantitative analysis, and developed biomaterials suitable for engineering this microenvironment ex vivo. We also established proof of principle that transplantation of cell-matrix constructs into stroke models is feasible and reduces lesion size. In the proposed studies, we will continue to optimize the design of our engineered niches based on our biological studies of the regulation of neurogenesis and angiogenesis in the brain (Aim 1), and by sequential testing in vitro (Aim 2) and in vivo (Aim 3) in progressively more challenging and realistic models of stroke, which will enable us to move closer to developing neurovascular regenerative therapies for human patients. Although our initial clinical target will be stroke-injured tissues, the insights gained, and strategies developed, from our proposed studies will be broadly applicable to repair of other neurovascular injuries such as traumatic brain injury and multiple sclerosis.

Establishing a functional vascular network is a rate-limiting step in embryonic development, the repair of injured tissues, and the engineering of tissue replacements. Although we have made progress in identifying factors that promote endothelial cell proliferation and sprouting, we lack understanding of how to properly control endothelial cell growth and phenotypic specialization during vascular remodeling, which has created a significant roadblock for clinical therapies, tissue engineering and regenerative medicine. Although multiple signaling pathways have been implicated in the regulation of arterial-venous network formation, including flow-induced mechanotransduction and Notch signaling, the mechanisms by which these signals coordinately regulate endothelial cell growth suppression and identity were unclear. Our recent studies revealed that remodeling vascular plexi are subject to systemic blood circulation, and that shear stress of different magnitudes promotes differential growth responses and gene expression. That is, arterial/arteriolar shear stress levels promote Notch signaling, and downstream p27-induced late G1 phase arrest that enables arterial gene expression (Fang 2017). Conversely, flow magnitudes typical of veins/venules induce early G1 arrest, and enables upregulation of venous genes. Interestingly, distinct endothelial cell cycle states appear to be maintained in arteries vs. veins postnatally. We know very little about the role of cell cycle control in endothelial cell fate decisions, or the differential signaling pathways induced by vessel-specific flow magnitudes, and how they may coordinately induce and maintain endothelial cell cycle state and identity. The scientific premise of our research is that endothelial cell cycle control is required for proper arterial and venous specification, such that when endothelial cells are in different cell cycle states, they exhibit different propensity for arterial vs. venous gene expression. Support for this idea comes from studies in embryonic stem cells that show cells in early vs. late G1 phase have a propensity for mesoderm/endoderm vs. ectoderm fate, respectively (Paulkin 2014). Thus, our hypothesis is that differential flow forces in arteries and veins induce different intracellular signaling pathways that promote distinct endothelial cell cycle states, creating distinct windows of opportunity for the regulation of arterial vs. venous gene expression. To ensure scientific rigor, we will test this hypothesis in vivo in models of arterial- venous network formation and repair, and in vitro in human endothelial cell culture systems that allow flow manipulation. We will define mechanisms by which vessel-specific flow magnitudes modulate endothelial cell cycle state, determine how distinct endothelial cell cycle states enable differential phenotypic specialization (artery vs. vein), and determine whether manipulation of endothelial cell cycle state can prevent or correct arterial-venous malformations and enhance post-injury vascular repair. Evaluation of this hypothesis will yield novel fundamental insights into blood vessel formation and regeneration that can be used to create human microvasculature ex vivo and treat vascular pathologies.

PROJECT SUMMARY This application seeks support for UVA's Biotechnology Training Program (BTP). The BTP is a highly interactive, multidisciplinary and >50% diverse community of PhD trainees drawn selectively from an annual May competition open to PhD students from all science and engineering departments university-wide for which we request 10 predoctoral slots. Trainee funding is for 2 years. Our emphasis is on scientific rigor and communication using the latest tools available with award winning and collaborative dual mentoring giving rise to a breadth of multidisciplinary and academic/industrial training unmatched at UVA. Our advocacy for trainee exposure to a wide variety of careers has been transformational - most importantly for trainees but also for the university with which we share many of our offerings. We believe in trainee career preparedness that is personalized, evolving and built on trust, and that relationships do not necessarily end at graduation. Indeed, our graduates welcome the opportunity to return as BTP seminar or industrial panel speakers with talks focused on, or interlaced with, career reflections; or to help in a trainee advisory/advocate role as members of the BTP Board of Corporate Advisors. They are the ultimate measure of our success. Their outcomes are updated at least quarterly on our grads web page with information drawn from LinkedIn. BTP students in training (21, including 11 minorities) or graduated (65) entered with an average undergraduate GPA of 3.7, are currently hosted by 8 different departments. BTP trainees and graduates have received multiple awards, experienced 73 different externships from 51 different companies, and after graduation are now employed in industry (37), academia (15; including an HBCU Dean), government (3), medicine (3), or foundation (2). Since '05, 164 first author articles have been published in journals with impact factors as high as 24. Trainees develop leadership and teamwork skills by taking direct responsibility for programmatic features of the BTP including: BTP Symposia and Seminars, BTP Day of Caring, BTP Industrial Q&A Panels and BTP company tours. Mentoring our trainees is a highly engaged, collaborative and well-funded faculty of 55 individuals from 14 departments. Institutional support has been essential for our success, including Vice President for Research help with externship and Symposia funding, and an additional training slot funded by the School of Engineering. Our overall goal is to develop the next generation of exceptionally rigorous, creative, talented and diverse scientists who are socialized in biotechnology themes and practiced in teamwork. Our aims are to: (1) cement a foundation of rigor in experimental planning, data organization and transparency built on the Nature and eLife recognized 'Open Science Framework' in a collaboration with Charlottesville's 'Center for Open Science', (2) to energetically nurture our trainees love and inquisitiveness for science as they acquire new skills to solve important scientific problems, and (3) to overlay this training with various forms of biotechnology exposure, including required externships ? all coupled with a strong and constant commitment to trainee diversity.