Shahin Rafii - US grants

Thrombocytopenia, is a life threatening complication of treatment of many hematological and oncological malignancies. High dose chemotherapeutic regimens used for treatment of resistent lymphomas, leukemias, and bone marrow transplantation frequently result in profound refractory thrombocytopenia. This complication not only limits further treatment, but also may result in life threatening hemorrhagic complications. Even though many cytokines such as GM-CSF, IL-3, and erythropoietin, that enhance erythroid and myeloid progenitor cell proliferation and maturation are in clinical use, there is no known cytokine(s) that can selectively induce(s) platelet formation. Platelet formation is a complex process that involves sequential maturation of megakaryocytes, the formation of a pseudopod-like projection called the proplatelet, and fragmentation into platelets. The cellular events leading to proplatelet formation and platelet production are complex processes that are poorly understood. Although it is known that mature megakaryocytes are located adjacent to the subluminal surface of bone marrow endothelial cells (BMEC) and extend pseudopods into sinusoidal space, the manner by which pseudopods are formed and fragment into platelets and the role of the BMEC in this process is incompletely understood. The central premise of this proposal is that BMEC is a unique type of endothelium that regulates megakaryocytopoiesis, and thrombopoiesis. In preliminary studies we have developed a technique for isolation and cultivation of BMEC and megakaryocytes. We show that BMEC elaborates cytokine that induces megakaryocyte maturation and induce platelet formation. The specific aim of this proposal is to characterize the cellular and molecular factor(s) mediated by BMEC that is (are) responsible for final stages of megakaryocyte maturation and platelet formation. The known cytokines elaborated by BMEC will be examined by ELISA, Northern analysis, and by functional studies. Platelet-like particle formation and maturation of GPIIb/Illa positive megakaryocytic precursors from pluripotent stem cells are in vitro models that will be used to characterize the functional activity of BMEC post-culture supernatant and will allow for the isolation and purification of putative thrombopoietins. Classical cloning approaches using reagents derived from these studies will lead to cloning of potentially useful cytokines. Functional expression cloning using mammalian (Cos) cells will provide an alternative approach, independent of successful purification, for defining and cloning BMEC elaborated cytokines. These studies may not only result in isolation, purification and cloning of a novel thrombopoietin/platelet maturation factor (PMF), but may also lay the foundation for the in vitro production of platelets.

Engraftment of hematopoietic stem or progenitor cells during bone marrow transplantation is dependent on the preferential homing of progenitors to the bone marrow microenvironment. Within the hematopoietic micro-environment, whether it is embryonic yolk sac, fetal liver, or adult bone marrow, microvascular endothelium not only provides cellular contact and secrete cytokines that allows for the preservation of the steady state hematopoiesis, but also acts as a gatekeeper controlling the trafficking and homing of the HSC. Most studies in the role of human bone marrow endothelial cells (BMEC) in the regulation of hematopoiesis had been stymied by the lack of techniques to isolate homogenous population of BMEC monolayers. We have developed a reproducible technique for isolation and cultivation of BMEC monolayers. We have shown that direct cellular contact between BMEC and CD34+ progenitors is critical for the long-term proliferation, commitment and terminal-differentiation of the hematopoietic stem cells. BMEC monolayers support the adhesion and transmigration of a subset of HSC cells with high proliferative potential. This binding which is dependent on divalent cations, can be partially blocked by antibodies to B1 and B2 integrins. The goal of this project is to define BMEC membrane bound factor(s) that regulate trafficking, expansion and in this proposal we intend to explore the role of membrane bound factors, including CD34/L- selectin, B1, B2 integrins, and membrane bound KL/c-kit receptor that may regulate trafficking as well as proliferation of progenitor cells. We have recently shown that BMEC also express membrane bound delta like protein (DLL1). DLL1 and its receptor human Notch 1/TAN-, which regulate developmental cell fate processes, may also play a major role in adhesion/proliferation of HSC. In this regard, we plan to study the role of these ligand pairs including DLL1/Notch1, by using blocking monoclonal antibodies to either ligand pair in blinding, and transmigration studies as well as in long-term BMEC-CD34+ progenitor coculture studies. In a novel approach, genetically engineered adenoviral vectors to overexpress BMEC membrane bound cytokines and adhesion molecules will be used to examine their function in binding, and long-term coculture studies. We have utilized expression cloning using a BMEC cDNA library to screen for human DLL1 homologue, and as yet unrecognized adhesion/homing receptor that regulates adhesion but also may regulate proliferation of HSC. Disruption of BMEC metabolism within the bone marrow microenvironment may contribute to stem cell dysfunction and progression to aplastic anemias, myelodysplasia, and graft failure during bone marrow transplantation. These studies may culminate in the identification of known or as yet unrecognized adhesion molecule that may regulate selective homing of HSC to the bone marrow.

DESCRIPTION (Adapted from applicant's abstract) Thrombocytopenia is one of the life threatening hematologic disorders that may occur as a result of autoimmune process or during the asymptomatic and clinical stage of HIV-1 infection. It is hypothesized that disruption of chemokine network, and adhesive interactions between megakaryocytes (MKs) and bone marrow endothelium (BMEC) as it may occur during HIV infection, plays a seminal role in the failure of MK transmigration and platelet release. The exact mechanism and site of platelet formation is not well defined. Studies have shown that transmigration of MK through BMEC, may be critical for platelet formation. The investigators have discovered that mature polypoid MKs express the chemokine receptor (HIV co-receptor): CXCR4. Stromal Derived Factor 1 (SDF1) which is the ligand for the CXCR4, promotes transmigration of MKs through BMEC monolayers. They have also identified a novel endothelial cell derived factor (ECDF1) that selectively induce migration of MKs through BMEC. Transendothelial migration of MKs in response to SDF1 or ECDF1 enhances formation of functional platelets. Interaction of migration MK with adhesion molecules expressed on MBEC such as E-selectin and PECAM is critical for MK migration and optimal platelet formation. They have also discovered that HIV can inject MKs through CXCR receptor, interfering with transendothelial migration of Mks, and platelet release. In this proposal they plan to 1) Define the mechanism whereby SDF1 and ECDF1 modulate adhesion molecule adhesion molecule expression of MK and BMEC cells. 2) Characterize cellular signaling pathways such as apoptotic pathways that may be induced by transmigration of Mks. 3) Take advantage of the availability of MK and BMEC derived from E-selectin knockout mice to study the role of these factors in regulation of CXCR expression. Both MKs and BMEC express CD4 and CXCR4, and are therefore susceptible to HIV infection. Therefore, it is planned to define the mechanism whereby HIV infection of either Mks and BMEC may influence chemokine receptor, and adhesion molecule expression resulting in dysfunction or platelet formation. Whether HIV-1 gp120 or other factors including megakaryopoietins that interact with CXCR4 may also influence platelet formation will also be explored. They plan to over-express CXCR4 and SDF1 within the milieu of marrow microenvironment by adenoviral vectors to explore the possibility of augmenting platelet production. This project should lead to the definition of the role of chemokines and adhesion molecules expressed by BMEC that regulate platelet production. Identification of chemokine receptors that may regulate platelet production may elucidate pathogenesis of thrombocytopenia in HIV or other thrombocytopenic states and suggest potential pharmacological interventions. Modulation of chemokine receptors expression by adenoviral vectors overexpressing SDF1, ECDF1 or their receptors may allow for developing therapies to ameliorate thrombocytopenia in vivo.

Hematopoietic stem cells (HSC) and endothelial precursor cells (EPC) provide an invaluable source of cells for autologous and allogeneic transplantation as well as gene therapy for congenital hematological and vascular disorders. The focus of this proposal is the challenge to identify and deliver factors that will allow for efficient in vivo expansion and mobilization for adequate pluripotent HSC and EPC that could be used for transplantation and gene therapy. The strategy is to capitalize on the robust, albeit transient expression mediated by adenovirus (Ad) gene transfer to express stem-cell active chemokines and angiogenic factors that promote extramedullary mobilization of both HSC and EPC. The preliminary data shows that Ad vector mediated in vivo expression of stem cell active cytokines and angiogenic factors with chemotactic potential such as vascular endothelial growth factor (VEGF), stromal derived factor-1 (SDF-1) and Angiopoietin-1 in the peripheral circulation can induce mobilization of HSCs and EPCs. Based on these studies, we hypothesize that regional and temporal expression of secreted and membrane bound angiogenic factors and stem cell active chemocytokines by Ad gene delivery will promote in vivo expansion and mobilization of marrow derived EPCs and HSCs to the peripheral circulation. These mobilized puripotent stem cells may be used for autologous or allogeneic transplantation or gene therapy. On the basis, this project seeks this strategy in the context of moving in to human application. First, we plan to determine whether regional delivery of Ad vectors expressing stem active chemocytokines induced in vivo expansion and mobilization of HSC and EPCs. Studies will be carried out to: 1) investigate whether sufficient, soluble and membrane bound Kit-ligand (Skl, Mkl) AND Flk- 2, alone or in combination can be delivered to marrow using Ad vectors to promote expansion and mobilization of HSCs; and 2) to determine whether sufficient angiogenic factors including soluble VEGF/121, VEGF/165, and matrix bound VEGF/189, placental growth factor (PLGF), Angiopoietin-1 and Angiopoietin-2 could be delivered and produced by Ad vectors to induce proliferation and mobilization of EPCs. Second, we plan to define the mechanism whereby chemocytokines induce mobilization of HSC and EPCs. The studies are planned to 1) evaluate the significance of chemokine-induced metalloproteinase (MMP) activation in the mobilization of stem cells by Ad vectors expressing SDF-1, VEGF isoforms in MMP (MMP-9) knock out mice; and 2) examine the role of endothelial specific adhesion molecules in the regulation of stem cell mobilization by Ad vectors expressing SDF-1, VEGF and angiopoietins in ICAM1, E-selectin and P-selectin knock out mice; and 3) assess the role of chemocytokine modulation of stem cell cycle in the mobilization of HSC and EPCs. Third, assess the efficacy of a novel approach of transplantation ex vivo AD vector transduced hematopoietic cells over-expressing chemocytokines, in mobilization HSC and EPCs. To evaluate this, studies have vector transduced hematopoietic cells over-expressing chemocytokines in mobilization HSC and EPCs. To evaluate this, studies have been designed to: 1) examine whether sufficient hematopoietic cells over-expressing Mkl, Flk-2, SDF- 1, VEGF/165 and VEGF/189 can be transplanted and delivered to the marrow to induce expansion of HSC and EPCs; and 2) assess whether sufficient chemokines can be delivered to the marrow environment by transduced hematopoietic cells to induce mobilization of HSC and EPCs.

The broad, long-term objective of this proposal is to examine the role of bone marrow (BM)-derived circulating endothelial precursor cells (CEPs) in the regulation of post-natal angiogenic physiological processes. In particular, we plan to define the mechanism(s) whereby expression of vascular endothelial growth factor receptors, VEGFR2 (Flk-1, KDR) and VEGFR-1 (Flt-1) regulate survival, proliferation, mobilization and recruitment of CEPs during physiological processes such as wound healing or vascular trauma. Despite recent identification of angiogenic factors that regulate embryonic development, the role of angiogenic factors and effector cells that modulate post-natal angiogenesis is not well known. We have identified a population of circulating AC133+VEGFR2+ CEPs that have the capacity to be recruited from BM to the injured vascular tissue, accelerating the angiogenic processes. We have shown that signaling through VEGFR2 is essential for the proliferation and differentiation of CEPs. In addition, VEGF induces mobilization angiogenesis. We have also shown that the angiogenic defect (tumor growth, Matrigel vascularization) in Id1 and Id3 knock out mice (Id1+/-Id3-/-) can be reversed by BM transplantation, suggesting that BM derived CEPs and/or HCs, may play an essential role in the promotion of post-natal angiogenesis. In addition, we speculate that the primary angiogenic defect in (Id1+/-Id3-/-) mice is due to dysregulated VEGF/VEGFR2 and possibly VEGF/VEGFR1 signaling, resulting in a failure of mobilization and proper recruitment of the CEPs and/or HCs to the sites of active neo-angiogenesis. Based on these studies, we hypothesize that VEGF42 and VEGFR1 signaling are essential for the survival, proliferation and mobilization of marrow-derived VEGF2+ CEPs and VEGFR1+ hematopoietic cells (HCs) to the angiogenic vascular bed. Recruitment of CEPs and VEGF-responsive HCs facilitate rapid temporal, spatial and regional expression pattern of VEGFR2 and VEGFFR1 on pre-existing endothelial and recruited BM derived CEPs and HCs during post-natal angiogenesis, 2) Define the role of VEGFR2 and VEGFR1 signaling in the regulation of mobilization, survival and recruitment of CEPs, 3) Assess the physiological significance and contribution of BM-derived VEGFR2+ CEPs and VEGFR1+ HCs to post-natal angiogenesis. These experiments will lead to understanding the mechanism(s) whereby CEPs contribute to the post-natal physiological angiogenic processes. Identifying factors that regulate VEGFR2 and VEGFR1 expression will allow for optimal mobilization, and facilitate recovery of CEPs. These studies will set forth the stage for ex vivo expansion of this rare population of cells that may ultimately be used clinically to accelerate vascular healing.

DESCRIPTION (provided by applicant): Endothelial and hematopoietic cells are ideal targets for gene therapy because are readily accessible to gene therapy vectors via the circulation and play a critical role in the progression of disease processes including inflammation and tumor angiogenesis. Adenoviral (Ad) vectors which could infect quiescent vascular cells provide ideal vectors to introduce genes into vascular endothelium as well as hematopoietic stem and progenitor cells with high efficiency and low toxicity. However, expression of genes by Ad vectors has been hampered by infiltration of inflammatory cells and intravascular activation of neo-intimal cells. Despite numerous studies defining the role of immune response to Ad vectors the exact mechanism whereby Ad vectors modulate activation status of ECs and hematopoietic cells and subsequent inflammatory response is not know. We have shown that introduction of E1-E4+, but not E1-E4-Ad vectors into primary ECs results in profound alteration in the proliferation and inflammatory status of the ECs. Infection of ECs with E4+ Ad vectors result in the generation of unique state where ECs do not proliferate or undergo apoptosis. This state is also associated with the upregulation of inflammatory adhesion molecules including ICAM, VCAM, and CD34. Moreover, E4 mediated activation of ECs enhance transendothelial migration of hematopoietic precursor cells and proinflammatory cells. These data clearly demonstrate that one or a combination of E4 gene products encoded by seven distinct E4 open reading frames (ORFs) regions may play a critical role in modulation of angiogenic and inflammatory potential of ECs. Based on these data, we hypothesize that gene products encoded by the E4 region of Ad vectors pirate the molecular machinery of ECs resulting in transformation of ECs into a unique pro-angiogenic and proinflammatory state. Identification of E4ORF genes that modulate angiogenic and inflammatory potential of ECs hematopoietic cells will facilitate designing strategies to attenuate vascular toxicity associated with Ad vector gene therapy. The mechanism by which E4ORFs gene products modulate angiogenic and inflammatory potential will be investigated through studying the following experiments: 1) Identifying specific E4ORF genes that alone or in combination modulate angiogenic and inflammatory potential of ECs in in vitro and in vivo models. 2) Assess the effect of E4ORFs gene products in the modulation of angiogenic potential of circulating VEGFR2+AC133+endothelial precursor cells (EPCs) and hematopoietic stem and progenitor cells. 3) Define the mechanism(s) whereby E4ORFs modulate the motility and mitogenic potential of ECs and hematopoietic progenitor and stem cells. 4) Define the role of E4ORFs in physiological models of angiogenesis and inflammation. These experiments may culminate in identification of E4ORFs that promote or inhibit angiogenesis, and diminish E4 mediated vascular toxicity. Incorporation of pro-angiogenic E4ORFs in conjunction with transgenes expressing VEGF, will facilitate developing clinical strategies to enhance collateral formation in ischemic myocardium or limbs. Conversely, E4ORFs with anti-angiogeni properties may be used in clinical strategies that are targeted at inhibiting tumor angiogenesis or blocking aberrant angiogenesis in clinical scenarios, such as diabetic retinopathy.

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to identify angiogenic pathways that are involved in selective mobilization and recruitment of bone marrow (BM)-derived endothelial and hematopoietic stem and progenitor cells thereby dictating heterogeneity of organ-specific vasculature. In particular, we plan to determine the mechanism by which the expression of Vascular Endothelial Growth Factor-receptors, VEGFR2 (Flk-1, KDR), VEGFR1 (Flt-1) and VEGFR3 (Flt-4) orchestrate proliferation, mobilization and incorporation of BM-derived progenitors into organ-specific neo-vasculature during regenerating processes, including lung regeneration and BM hemangiogenic reconstitution. We have shown that VEGF family of angiogenic factors promote recruitment of CD133+VEGFR2+ endothelial progenitor cells (EPCs) from BM to the angiogenic neo-vessels. We have also demonstrated that functional VEGFR1 is expressed on the subsets of hematopoietic stem and progenitors cells (HSPCs) supporting mobilization of these cells from BM. Co-recruitment of angio-competent VEGFR1+HSPCs to the neo-angiogenic vessels facilitate incorporation of VEGFR2+EPCs into functional neo-vessels. Mobilization of BM-derived progenitor cellsl is a dynamic process and requires recruitment of these cells from unique BM niches. Angiogenic factors, induce expression of metalloproteinase-9 (MMP-9), which in turn promote the release of soluble Kit-ligand (sKitL). Increase in bio-available sKitL enhance cycling and proliferation of HSPCs, setting up the stage for mobilization to the circulation. BM also contains a population of CD133+VEGFR3+ lymphatic EPCs that could possibly contribute to lymphangiogenesis. Based on these studies, we hypothesize that regenerating lung and BM provide for a pro-hemangiogenic microenvironment that is permissive for recruitment and incorporation of angio-competent BM-derived progenitors. Organ-specific angiogenic factors promote mobilization and recruitment of VEGFR2+ and VEGFR3+ EPCs to the neo-vessels. Co-recruitment of the VEGFR1+ hematopoietic cells facilitate functional incorporation of vascular progenitors and dictate vascular heterogeneity in the initial phases of organ regeneration. This hypothesis through studying the following specific aims: We plan to determine temporal, spatial and regional recruitment patterns of BM-derived progenitors during tissue revascularization-remodeling and compare their incorporation pattern in transgenic mice with diminished hemangiogenic potential including, VEGF164/164, VEGF189/189, PIGF-/-, and Id1+/-Id3-/- mice. Define the role of VEGFR1, VEGFR2 and VEGFR3 signaling in the regulation of mobilization, homing and recruitment of progenitors to the pulmonary and BM vasculature. Assess the physiological significance and contribution of BM-derived CD133+VEGFR2+, CD133+VEGFR3+EPCs and VEGFR1+HSPCs to revascularization during organ regeneration. These studies will lay the foundation for using BM-marrow derived cells for therapeutic cell therapy to enhance organ (i.e. lung, marrow) revascularization.

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to identify angiogenic pathways that are involved in selective mobilization and recruitment of bone marrow (BM)-derived endothelial and hematopoietic stem and progenitor cells thereby dictating heterogeneity of organ-specific vasculature. In particular, we plan to determine the mechanism by which the expression of Vascular Endothelial Growth Factor-receptors, VEGFR2 (Flk-1, KDR), VEGFR1 (Flt-1) and VEGFR3 (Flt-4) orchestrate proliferation, mobilization and incorporation of BM-derived progenitors into organ-specific neo-vasculature during regenerating processes, including lung regeneration and BM hemangiogenic reconstitution. We have shown that VEGF family of angiogenic factors promote recruitment of CD133+VEGFR2+ endothelial progenitor cells (EPCs) from BM to the angiogenic neo-vessels. We have also demonstrated that functional VEGFR1 is expressed on the subsets of hematopoietic stem and progenitors cells (HSPCs) supporting mobilization of these cells from BM. Co-recruitment of angio-competent VEGFR1+HSPCs to the neo-angiogenic vessels facilitate incorporation of VEGFR2+EPCs into functional neo-vessels. Mobilization of BM-derived progenitor cellsl is a dynamic process and requires recruitment of these cells from unique BM niches. Angiogenic factors, induce expression of metalloproteinase-9 (MMP-9), which in turn promote the release of soluble Kit-ligand (sKitL). Increase in bio-available sKitL enhance cycling and proliferation of HSPCs, setting up the stage for mobilization to the circulation. BM also contains a population of CD133+VEGFR3+ lymphatic EPCs that could possibly contribute to lymphangiogenesis. Based on these studies, we hypothesize that regenerating lung and BM provide for a pro-hemangiogenic microenvironment that is permissive for recruitment and incorporation of angio-competent BM-derived progenitors. Organ-specific angiogenic factors promote mobilization and recruitment of VEGFR2+ and VEGFR3+ EPCs to the neo-vessels. Co-recruitment of the VEGFR1+ hematopoietic cells facilitate functional incorporation of vascular progenitors and dictate vascular heterogeneity in the initial phases of organ regeneration. This hypothesis through studying the following specific aims: We plan to determine temporal, spatial and regional recruitment patterns of BM-derived progenitors during tissue revascularization-remodeling and compare their incorporation pattern in transgenic mice with diminished hemangiogenic potential including, VEGF164/164, VEGF189/189, PIGF-/-, and Id1+/-Id3-/- mice. Define the role of VEGFR1, VEGFR2 and VEGFR3 signaling in the regulation of mobilization, homing and recruitment of progenitors to the pulmonary and BM vasculature. Assess the physiological significance and contribution of BM-derived CD133+VEGFR2+, CD133+VEGFR3+EPCs and VEGFR1+HSPCs to revascularization during organ regeneration. These studies will lay the foundation for using BM-marrow derived cells for therapeutic cell therapy to enhance organ (i.e. lung, marrow) revascularization.

[unreadable] DESCRIPTION (provided by applicant): Lineage-specific cytokines, including thrombopoietin (TPO), fail to reconstitute early phases of myelosuppression induced by chemotherapy or irradiation (chemo-radiation), leading to prolonged thrombocytopenia and bleeding complications. The long-term objective of this proposal is to demonstrate that stem cell active chemokines, including Stromal Derived Factor-1 (SDF-1), SDF-1 analogue (CTCE-0214, Chemokine Therapeutics), FGF-4 and FGF-20 (CG53135, CuraGen), by rapid recruitment of stem and progenitor cells could restore tri-lineage hematopoiesis and thrombopoiesis after myelosuppression induced by chemo-radiation. Within the bone marrow (BM), hematopoietic stem and progenitor cells reside in defined "niches" where they receive molecular and cellular instructions for proliferation, differentiation and mobilization to the circulation. The majority of stem cells are localized to the periendosteal region referred to as the osteoblastic niche. We have shown that another dynamic BM microenvironment, demarcated by the sinusoidal BM endothelial cells, identified as the "vascular niche", supports differentiation of progenitors and their mobilization to the circulation. Chemo-radiation exposure to the BM not only induces rapid apoptosis of subsets of hematopoietic stem and progenitor cells, but also damages the BM's vascular microenvironment. This leads to long-lasting life-threatening thrombocytopenia and engraftment failure after BM transplantation. Based on these studies, we hypothesize that SDF-1, FGF-4 and their analogues by rapid recruitment of stem and progenitor cells from the osteoblastic niche to the vascular niche support timely reconstitution of hematopoiesis and thrombopoiesis thereby protecting against life-threatening chemo-radiation induced myelosuppression. In addition, FGFs and angiogenic factors, such as VEGF-A, by accelerating the regeneration of the BM's vascular niche contribute to the reconstitution of thrombopoiesis after high doses of chemo-radiation. This hypothesis will be tested through studying the following specific aims: 1) Assess the potential of FGF-4, FGF-20, SDF-1 and SDF-1 peptide analogue (CTCE-0214) in restoring thrombopoietic reconstitution after chemo-radiation induced myelosuppression: 2) Determine the role of the FGFs and angiogenic factors in reconstruction and regeneration of BM's vascular niche to protect against chemo-radiation induced thrombocytopenia: 3) Evaluate the potential of pre-conditioning the BM microenvironment with chemokines to enhance engraftment after BM transplantation into the chemo-irradiated treated BM, thereby accelerating thrombopoietic recovery. We anticipate that clinical availability of the SDF-1 analogue, CTCE-0214 (Chemokine Therapeutics), and FGF-20 (CuraGen, CGS3135) that are currently being evaluated in phase I clinical trials, would provide the platform for assessing the role of these agents in amelioration and rapid restoration of thrombocytopenia Induced by chemotherapy and irradiation [unreadable] [unreadable]

The broad, long-term objective of this proposal is to define the mechanism by which the chemokine SDF1 through activation of its receptor CXCR4, contributes to the revascularization of the regenerating lungs. In particular, we plan to determine as to how left lung pnueomonectomy through upregulation of SDF-1 supports the mobilization and incorporation of pro-angiogenic CXCR4+VEGFR1+ hematopoietic cells thereby promoting neo-angiogenesis of the regenerating right lung. We have shown that VEGF family of angiogenic factors promote recruitment of CD133+VEGFR2+ vascular progenitor cells from bone marrow (BM) to the neo-vessels. Signaling through VEGFR2 is essential for the proliferation and differentiation of vascular progenitors. We have also demonstrated that functional VEGFR1 and CXCR4 are co-expressed on the subsets of hematopoietic progenitors cells, supporting mobilization of these cells from the BM's microenvironment. Recruitment of pro-angiogenic CXCR4+VEGFR1+ cells to the regenerating vessels facilitates incorporation of VEGFR2 into functional vasculature. Mobilization and incorporation of BM-derived progenitor cells is a dynamic process and requires activation of a cascade of molecular events that drives recruitment of these cells from unique BM niches. Angiogenic factors, including Placental Growth Factor (P1GF), which exclusively signals through VEGFR1, induce expression of metalloproteinase-9 (MMP-9), which in turn promote the release of soluble Kit-ligand (sKitL, stem cell factor). Increase in bio-available sKitL enhances cycling and morility of CXCR4+VEGFR1+ cells, setting up the stage for mobilization to the circulation. We have preliminary evidence demonstrating that in mice, left lung pneumonectomy results in mobilization and incorporation of hemangiogenic progenitor cells into regenerating lung. Based on these studies, we hypothesize that the regenerating lung through plasma elevation of SDF-1 and VEGFs support the recruitment of marrow derived pro-angiogenic CXCR4+VEGFR1+ hematopoietic cells thereby facilitating incorporation of VEGFR2+ endothelial cells into functional regenerating lung vasculature. This hypothesis will be tested through studying the following specific aims: 1) Determine the temporal, and spatial recruitment patterns and functional contribution of CXCR4+VEGFR1+ progenitors during lung regeneration and compare their incorporation pattern into mice with diminished hemangiogenic potential including, Idl^TdS"'", P1GF"'", VEGF^/", and VEGFigg"'" mice. 2) Assess the physiological significance of SDF-1 and VEGFs in accelerating lung revascularization through recruitment of CXCR4+VEGFR1+ cells. 3) Evaluate the contribution of BM-derived VEGFR2+, and VEGFR1+CXCR4+ cells to revascularization during lung regeneration. The proposed studies will take advantage of the expertise of Dr. RG Crystal (PI) in lung regeneration and emphysema models and the applicant's experience in hematopoiesis and endothelial cell biology to define the mechanism and identify molecular mediators that support recruitment of CXCR4+VEGFR1+ progenitors thereby supporting regeneration of pulmonary vasculature. These studies will lay the foundation for designing clinical protocols to deliver BM derived cells, including CXCR4-t-VEGFRl+ and VEGFR2+ cells, or to introduce SDF-1 and angiogenic factors, to enhance lung revascularization in patients suffering from emphysema and COPD.

DESCRIPTION (provided by applicant): Accumulating evidence from our group and others suggest that bone marrow (BM) sinusoidal endothelial cells (SECs) represent a dynamic vascular niche, which may provide the cellular platform for the reconstitution of hematopoiesis after myelosuppression. Using technical advances in bone marrow (BM) preparation, we have recently established a comprehensive phenotypic and functional signature of BM SECs at steady state and during hemangiogenic regeneration. We have recently shown that, after moderate to severe myelosuppression rapid regeneration of the regressed SECs is essential for engraftment and replenishment of the transplanted long-term hematopoietic stem cells (LT-HSCs) and reconstitution of hematopoiesis. Most likely, transplanted HSC and their lineage committed hematopoietic progenitor cells (HPCs) by releasing of neo- angiogenic factors contribute to the regeneration of SEC. However, the precise mechanism by which angiogenic factors released by the pro-angiogenic hematopoietic cells, such as CXCR4+VEGFR1+ myeloid and megakaryocytic progenitors cells support reconstruction of the SECs is not known. The broad long-term objective of this proposal is to identify the molecular pathways and to define the mechanism whereby angiogenic factors, specifically the VEGF-A isoforms, PlGF and SDF1 elaborated by specific subsets of the hematopoietic cells support assembly and remodeling of BM's Vascular Niche, thereby supporting reconstitution of HSCs and hematopoiesis after myelosuppression. Therefore, we hypothesize that within BM, the VE- cadherin+VEGFR2+VEGFR3+Sca1- SECs establish a vascular niche, which is a dynamic cellular microenvironment essential for the reconstitution of HSC, and hematopoiesis after myelosuppression. Regenerating pro-angiogenic CXCR4+VEGFR1+ hematopoietic cells through release of VEGF-A SDF-1, and as yet unrecognized angiogenic factors accelerate regeneration of the SECs thereby accelerating the reconstitution of the LT- HSCs and hematopoiesis. This hypothesis will be tested through: 1) determining the mechanism by which pro-angiogenic hematopoietic cells by elaborating VEGF-A and SDF-1 support the regeneration of regressed SECs thereby reconstituting LT-HSCs and hematopoiesis: 2) assessing the relative contribution of preexisting CXCR4+ endothelial cells versus transplanted CXCR4+VEGFR1+ hematopoietic cells to the revascularization of the ischemic limbs and 3) evaluating the physiological significance of enforced expression of angiogenic factors in accelerating the regeneration of BM SECs and reconstitution of hematopoiesis. We anticipate that understanding the mechanism by which angiogenic factors regulate hematopoiesis and HSC self-renewal will offer new strategies to treat BM failure states, including aplastic anemia, myelodysplastic syndromes and accelerate BM reconstitution after chemotherapy, irradiation and transplantation. PUBLIC HEALTH RELEVANCE: We hypothesize that bone marrow's vascular niche is a dynamic cellular microenvironment that is essential for the maintenance and reconstitution of hematopoiesis after myelosuppression. Regenerating CXCR4+VEGFR1+ hematopoietic cells through release of angiogenic factors, including VEGF-A, PlGF, SDF-1 and FGF-2 support regeneration of the sinusoidal endothelial cells into functional vascular niche thereby accelerating the restoration of hematopoietic stem cells and reconstitution of hematopoiesis. We anticipate that understanding the mechanism by which angiogenic factors regulate the reconstruction of the vascular niche in the bone marrow will offer new strategies to treat hematopoietic failure states, including aplastic anemia, myelodysplastic syndromes and accelerate BM reconstitution after chemotherapy, irradiation and bone marrow transplantation.

DESCRIPTION (provided by applicant): Self-renewal is essential for the homeostasis and lifetime maintenance of many organ systems. The process of self-renewal is carried out by rare populations of adult stem cells whose key features are multilineage potential and repopulating capacity. Blood cell development is driven by the successive restriction of cell fate as multipotent hematopoietic stem cells (HSCs) give rise to all mature red and white blood cell types. Current models suggest that the accumulation of cell-type specific patterns of DNA methylation correlates with cell fate specification, with the pattern of marks both creating a record of a cell's lineage and locking that cell into a particular fate due to the innate heritability of such marks. We propose to produce genome-wide reference maps of DNA methylation throughout the human and mouse genomes during HSC differentiation. Our studies will compare the state of the murine and human methylomes in purified populations of long-term repopulating HSCs. We will assess lineage-specific patterns of marks in the earliest progenitor cells for which fate is phenotypically specified, and follow the subsequent remodeling of the epigenome through blood cell maturation. This study will provide the first comprehensive examination of the state of the epigenome through a well characterized developmental cascade. Though comparative studies in two distinct models, we hope to separate the key events that drive fate specification and restriction decisions from those that might simply correlate with cell differentiation. PUBLIC HEALTH RELEVANCE: The differentiation of long-term hematopoietic stem cells into mature red and white blood cells is one of the best studied developmental processes in animals. As more primitive cells turn into more differentiated and specialized cell types, accompanying changes in the epigenetic state of the genome are thought to reinforce these decisions. We propose to examine the state of the epigenome in both stem cells and mature cells within the hematopoietic system in humans and mice. In this way, we will reveal general principles of cell fate determination and understand how cells become increasingly restricted in their potential. These studies may also reveal strategies to create specific types of hematopoietic cells for therapeutic purposes.

DESCRIPTION (provided by applicant): Lineage-specific cytokines, including thrombopoietin (TPO), fail to reconstitute early phases of myelosuppression induced by irradiation, leading to prolonged life-threatening thrombocytopenia and bleeding complications. The long-term objective of this proposal is to demonstrate that pro- angiogenic factors with chemotactic activity, including Stromal Derived Factor-1 (SDF-1), SDF-1 analogue (CTCE-0214, Chemokine Therapeutics), FGF-4 and FGF-20 (CG53135, CuraGen), Kit-ligand (Amgen), VEGF-A and angiopoietin-1 by rapid regeneration of the bone marrow's (BM) vascular niche and recruitment of stem and progenitor cells could restore thrombopoiesis and facilitate engraftment of transplanted BM, after high dose radiation exposure. Within the BM, hematopoietic stem and progenitor cells reside in defined "niches", where they receive molecular and cellular instructions for proliferation, and differentiation. We have shown that recruitment of the megakaryocytic progenitors to the BM's vascular niche, supports their differentiation into functional platelets. Irradiation exposure to the BM not only induces rapid apoptosis of hematopoietic stem cells, but also damages the BM's vascular microenvironment. This leads to long-lasting life-threatening thrombocytopenia and engraftment failure after BM transplantation. Based on these studies, we hypothesize that angiogenic factors with stem and progenitor chemokine activity, such as SDF-1, FGF-4 and their analogues by rapid reconstitution of the vascular niche accelerates thrombopoiesis thereby protecting against life-threatening radiation-induced thrombocytopenia. In addition, angiogenic factors, including VEGF-A, Kit-ligand and angiopoietin-1 by enhancing the regeneration and stabilization of the vascular niche not only contribute to rapid restoration of thrombopoiesis, but also support engraftment of transplanted stem cells thereby augmenting restoration of thrombopoiesis after irradiation. These goals will be achieved through executing the following pre-clinical mechanistic studies: 1) Assess the potential of pro-angiogenic chemokines in accelerating thrombopoiesis after radiation-induced myelosuppression: 2) Determine the role of the angiogenic factors, VEGF-A, soluble Kit-ligand and angiopoietin-1, in regeneration of the BM's vascular niche to restore platelet recovery after irradiation 3) Evaluate the effectiveness of pre-conditioning of the BM microenvironment with FGFs, VEGF-A and Kit-ligand to accelerate regeneration of the vascular niche to enhance engraftment of stem cells after BM transplantation into the irradiated BM. We anticipate that clinical availability of the SDF-1 analogue, CTCE-0214 (Chemokine Therapeutics), FGF-20 (CuraGen, CG53135), Kit-ligand and upcoming clinical grade pro-angiogenic factors that are currently being evaluated in phase I clinical trials, would provide the platform for assessing the role of these agents in amelioration and rapid restoration of thrombocytopenia induced by irradiation as it might occur as a consequence of a nuclear fall out. Exposure to irradiation results in major damage to the bone marrow resulting in failure in blood production. This leads to development of major life-threatening bleeding and infection. Currently, the factors that could promote blood production, specifically platelets, are not effective in restoring bone marrow's function. We have discovered specific "angiogenic" factors that by activating the formation of the blood vessels in the bone marrow will also induce rapid generation of platelets thereby ameliorating fatal bleeding complications associated with high doses of radiation exposure. Furthermore, these angiogenic factors by facilitating rapid assembly of the blood vessels provide for a fertile ground for the engraftment of transplanted stem cells into the bone marrow's microenvironment. This will result in enhanced restoration of blood production and platelet recovery. Collectively, we predict that our studies will determine the optimal means to deliver these angiogenic factors to restore blood production, platelet recovery and augment stem cell engraftment thereby diminishing complications associated with a nuclear fall out.

DESCRIPTION (provided by applicant): Liver transplantation is the mainstay of treatment for patients with end-stage liver disease. However, the paucity in genetically matched donors and technical hurdles associated with expanding hepatocytes has limited the number patients that could have otherwise been treated effectively with liver or hepatocyte transplantation. Therefore, identification of the molecular and cellular pathways that allow expansion and engraftment of hepatocytes and augment liver regeneration will have significant therapeutic impact. We have found that after 70% partial hepatectomy (PH), activation of liver sinusoidal endothelial cells (LSECs) by production of paracrine factors, defined as angiocrine factors, induce liver regeneration. We have defined phenotypic and operational definition of LSECs and have shown that LSECs compose of a specialized vascular network that are in direct cellular contact with hepatocytes, supporting liver regeneration (Ding et al, Nature 2010). After PH, activation of LSECs initiates and sustains the regeneration of remaining lobes of the liver. The activation of the VEGF-A tyrosine kinase receptor (VEGFR2) and Id1 pathway in LSECs upregulated the angiocrine expression of Wnt2 and hepatocyte growth factor (HGF) stimulating hepatic proliferation. However, the mechanism by which PH induces LSECs to produce hepatocyte-active angiocrine factors is unknown. We show that after PH subsets of the hematopoietic cells, (i.e. platelets), are recruited to liver sinusoids and activate LSECs by depositing VEGF-A and SDF-1. In addition to VEGFR2, chemokine receptor for SDF-1, CXCR7, is upregulated specifically on LSECs. Based on these data, we hypothesize that after PH, hematopoietic cells, specifically activated platelets, are recruited to liver LSECs and by deploying VEGF-A and SDF-1 stimulate VEGFR2+CXCR7+ LSECs to produce hepatocyte-active angiocrine factors that initiate and maintain liver regeneration. We will employ liver regeneration and angiogenic models developed in our laboratory to examine these hypotheses by executing the following experiments: Aim 1. Determine the mechanism by which activation of CXCR7 in VEGFR2+ LSECs support angiocrine-mediated hepatocyte regeneration. Aim 2. Define the role of recruited hematopoietic cells, specifically platelets in mediating LSEC activation driving liver regeneration. Aim 3. Determine the role of reciprocal interaction between platelets and Akt-activated LSECs in angiocrine factor induction and accelerating hepatocyte proliferation. Our approach to improve expansion of hepatocytes by unraveling the mechanism by which LSECs support liver regeneration will pave the way for identification of angiocrine factors that support long-term proliferation of functional hepatocytes and engraftment into the liver. In addition, our proposed experiments will allow for development of strategies in which by proper activation of LSECs, will set the stage for accelerating liver regeneration in the clinical setting.

DESCRIPTION (provided by applicant): Allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation has the potential to cure hematologic disease. However, many patients do not have an HLA matched donor, and graft-versus-host disease is a significant problem. Autologous patient HSPCs can be genetically corrected to cure the disease, but low yields of autologous HSPCs and ex vivo manipulation cause a loss of stemness leading to reduced engraftment. Thus, HSPC production from patient-specific induced pluripotent stem cells (iPSCs) would solve these problems and represent an unlimited cell source. To advance clinical translation of iPSC therapeutics, we propose a novel strategy to expand iPSC-derived HSPCs for hematopoietic transplantation. Specifically, we propose to engineer endothelial cells (ECs) for generation, expansion, and engraftment of putative HSPCs from pigtail macaque (Mn)iPSCs in the clinically relevant nonhuman primate model. In a promising collaboration with Dr. Shahin Rafii, we developed an effective, novel platform to expand macaque CD34+ LT- HSCs up to 25-fold by co-culture with Akt-activated human endothelial cells (E4+ECs). We recently found that iPSC-HSPCs expanded on E4+ECs have high levels of engraftment in NSG mice (up to 50% CD45+ cells). This evidence substantiates our novel approach to alter iPSC-HSPC biology through direct contact culture with angiocrine/hematopoietic signals unique to E4+ECs. The proposed studies will translate these findings to nonhuman primates and thus provide a major step toward producing iPSC-HSPCs with the capacity for hematopoietic reconstitution and correction of genetic diseases.

DESCRIPTION (provided by applicant): Current therapeutic approaches for the repair of the injured lung tissue have had no major long-term benefit in restoring pulmonary function. We have set forth the concept that pulmonary capillary endothelial cells (PCECs) are not just passive conduits that deliver oxygen and nutrients but rather by establishing a supportive niche play a key role in lung regeneration and repair. The overarching goal of this project is to define the mechanism by which after lung injury, activated PCECs through production of growth factors, defined as angiocrine factors, support alveolar regeneration without provoking aberrant fibrosis. We have established the phenotypic definition of PCECs and have shown that after left lung pneumonectomy (PNX), activation of the VEGF-A receptor-2 (VEGFR2) and FGFR1 expressed on the PCECs leads to upregulation of the metalloproteinase MMP14. MMP14 via unmasking cryptic EGF-receptor ligand domains stimulate alveolar regeneration. Notably, transplantation and engraftment of wild-type PCECs expressing MMP14 into the lung of VEGFR2/FGFR1 deficient mice restores lung alveolarization without stimulating fibrosis. However, the mechanism by which PNX activates PCECs to produce angiocrine factors is unknown. Our preliminary data indicate that after PNX, hyperoxia and bleomycin-induced lung injury, myeloid cells and platelets are recruited to the injured lung tissue and by deposition of VEGF-A and stromal derived factor-1 (SDF- 1, CXCl12) activate their cognate receptors VEGFR2 and CXCR4 expressed on PCECs to produce angiocrine factors initiating lung repair. Based on these data, we hypothesize that after lung injury, hematopoietic cells are recruited to pulmonary capillary vessels and by deploying VEGF-A and SDF-1 stimulate VEGFR2+CXCR4+ PCECs to produce alveolar-active angiocrine factors that support lung repair, while preventing aberrant fibrosis. We plan to leverage lung injury models and technologies developed in our lab, including a new approach to reprogram amniotic cells into vascular endothelial cells (rAC-VECs) to execute the following experiments: Aim 1. Dissect the mechanism by which activation of CXCR4 in VEGFR2+ PCECs elicits and maintains angiocrine-mediated lung repair. Aim 2. Examine the role of recruited hematopoietic cells to damaged lung vessels in mediating PCEC activation, angiocrine factor production and lung repair while preventing aberrant fibrosis. Aim 3. Determine the role of reciprocal crosstalk between hematopoietic cells and PCECs in inducing angiocrine signaling and accelerating alveolar regeneration and repair. Our proposed experiments will set the stage for development of pre-clinical strategies in which by proper activation of PCECs or transplantation of lung-specific engineered PCECs will allow for stimulating lung repair, thus improving respiratory functions and minimizing maladaptive remodeling into fibrotic tissues.

DESCRIPTION (provided by applicant): Organ regeneration promises unlimited access to replacement tissues. The current paradigm of organ regeneration is dependent on transplantation of adult tissue-restricted stem and progenitor cells to repair the damaged organ. However, healing injured organs often leads to fibrosis with little recovery of function. This proposal challenges the prevailing viewpoint and tests an alternative complimentary approach that regeneration could also be directed by tissue-specific vascular endothelial cells (ECs) functioning as an instructive niche to promote organ regeneration and repair without provoking maladaptive fibrosis. This notion is based on our finding that blood vessels are not just the passive plumbing for delivery of oxygen and nutrients, but are active participants in organ function. Indeed, our group has pioneered the transformative concept that tissue-specific ECs produce a defined set of non- fibrotic paracrine mediators, called Angiocrine Factors to directly induce organ regeneration without fibrosis. Yet, regenerative function and the repertoire of angiocrine factors elaborated by ECs depend upon the organ from which they originate. Indeed, the molecular determinants of angiocrine heterogeneity are unknown. Thus, we hypothesize that generic, unspecified, ECs acquire tissue specific function by a process of in vivo education wherein extra-vascular cues trigger transcriptional programs. Specified ECs are credentialed to deploy tissue-specific angiocrine growth factors that drive organ repair without aberrant pro-fibrotic remodeling. Our objective here is to identify transcription factors (TFs) regulating tissue-specification of EC angiocrine function so that generic ECs can be programmed to target particular vascular beds to promote regeneration. To test this transformative hypothesis and translate these concepts for clinical use we will address the following objectives: Aim 1) Identify molecular determinants of vascular heterogeneity and organotypic regenerative function; Aim 2) Determine and validate the molecular signals and angiocrine factors elaborated by tissue-specific ECs that promote organ repair without provoking fibrosis. We have developed technologies to propagate generic ECs derived from mouse and human pluripotent stem cells and those ECs transcriptionally reprogrammed from amniotic cells. The proposed work is expected to overturn the scientific conceptualization of a monofunctional, inert, microvasculature by revealing a dynamic, tissue-specified role for ECs in organ repair. Successful completion of the proposed studies will enable therapeutic use of educated, tissue-specified ECs that home to their native injured organs and supply tissue-specific angiocrine signals to orchestrate organ regeneration. Alternatively, once known the angiocrine factors could be delivered directly. This transformative approach opens new therapeutic avenues of research to stimulate organ repair without scarring.

PROJECT SUMMARY Hematopoietic stem cells (HSC) have well established clinical applications in the treatment of heritable and acquired blood disorders. However, their therapeutic potential could be significantly broadened by engineering novel methods to generate HSC de novo from pluripotent stem cells or from directly reprogrammed adult cells. Toward this goal, we have established endothelial cell (EC) niche based culture methods that provide the necessary conditions to support the specification and self-renewal of HSC from embryonic hemogenic precursors, and more recently, from adult ECs using transcription factor (TF)-mediated conversion that bypasses a pluripotent intermediate. We hypothesize that recreating the signals necessary and sufficient to develop a clinically meaningful system for HSC generation in vitro will necessitate a comprehensive, systems approach to deconstruct the niche provided signals required for HSC specification and self-renewal. Thus, the overall goal of this grant is to leverage unique expertise of the collaborating laboratories to elucidate the signaling interactions regulating HSC specification and self-renewal from embryonic hemogenic precursors or TF-reprogrammed adult EC in the context of the EC niche. Our approach consists of three overlapping aims. The first aim will identify EC niche-provided signals necessary for embryonic HSC specification and self-renewal. The second aim will identify the unique HSC programs induced by these signals that regulate the transition from embryonic hemogenic precursor to bone fide repopulating HSC. The third will identify comparable programs that regulate the transition from adult EC to HSC during TF-mediated reprogramming in the EC niche. Key to these studies will be innovative functional assays, transcriptional profiling methods, and computational approaches that will enable us to resolve cellular complexity of niche cells and their interactions with developing embryonic or reprogrammed HSC at the single cell level. The role of identified signal factors in stage-specific support of HSC specification will be validated and further refined in vitro by gain and loss of function studies in the context of niche EC. Furthermore, to extend these studies to stromal cell-free systems as a step toward clinical translation, we will also test the contribution of identified signal factors in HSC specification and self-renewal in the context of stage-specific modulation of Notch activation using engineered Notch agonists. To achieve the goals of this proposal, we have developed a multidisciplinary collaboration involving unique expertise in each of our laboratories, including basic HSC and EC niche cell biology, direct TF based cellular conversion, clinical HSC transplantation, genome wide assessment of rare stem cell populations at single cell resolution, and innovative computational approaches to deconstruct core signal pathways regulating developmental transitions. Altogether, we expect the proposed studies will ultimately guide the design of novel strategies for deriving and expanding HSC in vitro for therapeutic applications.

Summary/Abstract The 2017 Angiogenesis Gordon Conference will explore the very latest innovative research on the cellular principles and molecular regulation of vasculogenesis and angiogenesis in embryonic development, adulthood, aging and disease processes. Recent studies have uncovered the previously unrecognized pathways dictating endothelial cell heterogeneity, plasticity and adaptability of the tissue-specific endothelial cell population. This meeting will aim to integrate newly identified angiogenic pathways that enable endothelial cells to coordinate signalling and behaviour to establish functional, stable and durable organotypic capillary networks for therapeutic regeneration as well as tumor targeting. Presentations from fields of developmental, computational, biophysical and regenerative medicine will be employed to bring together the multipronged approaches to microfabricating long-lasting blood vessels. We will also seek to highlight new insights and approaches towards translation of angiogenic concepts into the clinic, including cancer therapy. This conference is organized to stimulate rich interactions between experts, senior and junior faculty, industry, clinicians and basic scientist and importantly provide educational opportunity for students and post-docs.

DESCRIPTION (provided by applicant): Organ regeneration promises unlimited access to replacement tissues. The current paradigm of organ regeneration is dependent on transplantation of adult tissue-restricted stem and progenitor cells to repair the damaged organ. However, healing injured organs often leads to fibrosis with little recovery of function. This proposal challenges the prevailing viewpoint and tests an alternative complimentary approach that regeneration could also be directed by tissue-specific vascular endothelial cells (ECs) functioning as an instructive niche to promote organ regeneration and repair without provoking maladaptive fibrosis. This notion is based on our finding that blood vessels are not just the passive plumbing for delivery of oxygen and nutrients, but are active participants in organ function. Indeed, our group has pioneered the transformative concept that tissue-specific ECs produce a defined set of non- fibrotic paracrine mediators, called Angiocrine Factors to directly induce organ regeneration without fibrosis. Yet, regenerative function and the repertoire of angiocrine factors elaborated by ECs depend upon the organ from which they originate. Indeed, the molecular determinants of angiocrine heterogeneity are unknown. Thus, we hypothesize that generic, unspecified, ECs acquire tissue specific function by a process of in vivo education wherein extra-vascular cues trigger transcriptional programs. Specified ECs are credentialed to deploy tissue-specific angiocrine growth factors that drive organ repair without aberrant pro-fibrotic remodeling. Our objective here is to identify transcription factors (TFs) regulating tissue-specification of EC angiocrine function so that generic ECs can be programmed to target particular vascular beds to promote regeneration. To test this transformative hypothesis and translate these concepts for clinical use we will address the following objectives: Aim 1) Identify molecular determinants of vascular heterogeneity and organotypic regenerative function; Aim 2) Determine and validate the molecular signals and angiocrine factors elaborated by tissue-specific ECs that promote organ repair without provoking fibrosis. We have developed technologies to propagate generic ECs derived from mouse and human pluripotent stem cells and those ECs transcriptionally reprogrammed from amniotic cells. The proposed work is expected to overturn the scientific conceptualization of a monofunctional, inert, microvasculature by revealing a dynamic, tissue-specified role for ECs in organ repair. Successful completion of the proposed studies will enable therapeutic use of educated, tissue-specified ECs that home to their native injured organs and supply tissue-specific angiocrine signals to orchestrate organ regeneration. Alternatively, once known the angiocrine factors could be delivered directly. This transformative approach opens new therapeutic avenues of research to stimulate organ repair without scarring.

Abstract Blood disorders could be cured by transplantation of hematopoietic stem and progenitor cells (HSPCs). However, because of lack of matched donors, many patients cannot undergo curative therapy. Thus, there is an unmet demand for engineering autologous HSPCs. The long-term goal of this project is to enable conversion of readily accessible autologous adult endothelial cells (ECs) into engraftable HSPCs. To enable this, we need to uncover the niche signals that drive the birth of human HSCs. To this end, we have devised a tractable in vitro method to reprogram readily accessible human and mouse adult ECs into hematopoietic cells. In this approach, we expressed four transcription factors (TFs), FOSB, GFI1, Runx1 and SPI1 (FGRS) in adult primary ECs along with coculture with vascular niche, to reprogram adult ECs into engraftable human HSPCs (rEC-HSPCs). While human rEC-HSPCs could engraft NSG mice, we could not assess whether these cells could give rise to functional T cells. Therefore, we isolated adult lung ECs from Runx1-IRES-GFP reporter mouse for conversion and to tract and modulate the emergence of HSPCs. We show that transient FGRS transduction of Runx1-IRES-GFP ECs along with vascular niche induction, over a 28 day period, enable transition through 3 steps of Induction-Specification-Expansion to generate repopulating mouse rEC- HSPCs. Notably, shutting off FGRS on day 28 before transplantation of CD45.2+ rEC-HSPCs was key to enable multi-lineage engraftment in 1o and 2o CD45.1+ lethally irradiated recipients. Engrafted T cells restore immune response in Rag1-/- mice eliciting adaptive immune response. Single cell clonal and limiting dilution transplantation of day 28 rEC-HSPCs show that these cells are capable of clonal expansion and contain 1/557 competitive repopulating units (CRU) thereby subsets of HSPCs represent long-term rEC-HSCs. Thus, we hypothesize that transient expression of FGRS in adult ECs along with induction by vascular and stromal niche signals will enable deciphering the hierarchy of signals that orchestrate accelerated transition of ECs to abundant engraftable long-term immunocompetent human rEC-HSPCs. These approaches set up the stage for generation of human rEC-HSCs. We will test this hypothesis by examining the following specific aims: 1) Employ non-integrative modified RNA (modRNA) delivery of FGRS TFs into adult human ECs to augment the efficiency, efficacy and safety of conversion of adult human ECs into rEC-HSPCs. 2) Identify tissue- specific specialized human vascular and stromal cell niches that orchestrate the induction, specification, and expansion of FGRS-transduced ECs into abundant immunocompetent long-term engraftable human rEC- HSPCs. 3) Decipher the role of human vascular and stromal niche derived signals, including Cxcl12:Cxcr4/Cxcr7 and BMP4/TGF-?1 in stepwise conversion of human adult ECs into rEC-HSPCs.! Collectively, our mechanistic studies will not only identify ontological pathways involved in differentiation of ECs to rEC-HSPCs, but also translate the potential of engineered autologous HSPCs to the clinical setting. !

Abstract Exposure to ionizing radiation is often fatal due to acute radiation syndromes (ARS) manifested as Gastrointestinal-ARS (GI-ARS) and Hematopoietic-ARS (H-ARS). Delayed effects of acute radiation exposure (DEARE) lead to multi-organ dysfunction syndrome (MODS). A common denominator of radiation induced multi-organ failure is due to damage to endothelial cells (ECs) and lymphatic ECs, resulting in leakiness, coagulopathy and inflammation, setting up stage for infection, sclerosis and tumorigenesis. The molecular basis of radiation-induced EC dysfunction is not well understood. Our goal is to capitalize on the regenerative function of ECs by intravenously transplanting readily-available, off-the- shelf, allogeneic human ECs to mitigate ARS, DEARE and MODS. Our central hypothesis is that radiation damaged blood vessel and lymphatic ECs become dysfunctional and fail to perform their vascular functions or supply the instructive signals required to promote tissue healing thereby leading to ARS and DEARE. We propose that transplantation of normal pro-regenerative ECs a day or days after radiation can rescue the multi-organ defects of radiation-injured ECs and promote scar-free healing. We have shown that tissue-specific ECs by producing angiocrine growth factors orchestrate the repair of injured organs without fibrosis. Intravenous transplantation of human ECs restores hematopoietic recovery in sublethally irradiated rodents and lethally irradiated pigtail macaque non-human primates (NHP) without fibrosis or tumorigenesis. The Rationale for the proposed experiments is that if we know how to efficiently generate abundant off-the-shelf GMP-grade human umbilical vein ECs (HUVECs) as a ?generic allogeneic vascular graft?, we will use NHP large animal model radiation models to determine the pharmacokinetics of HUVEC transplantation to use them as a definitive or intermediary radiation countermeasure to support organ repair post-radiation. We will test this hypothesis by addressing these Aims: 1) Manufacture of abundant functional clinical-grade master cell banks of monkey ECs (MUVECs) and human (HUVECs) for intravenous transplantation. 2) Identify the critical parameters for allogeneic/xenogeneic MUVEC and HUVEC transplantation into recipient mice to mitigate post-irradiation H-ARS and GI-ARS injury without provoking fibrosis..3) Employ pigtail macaque NHP radiation models to determine the scheduling, safety and efficacy of transplanting MUVECs and HUVECs to rejuvenate vascular niche for multi-organ repair without scarring. Completion of the proposed studies will enable therapeutic use of allogeneic off-the-shelf ?human ECs? that transiently home to the disrupted vascular beds of irradiated organs restoring angiogenesis and vascular niche functions promoting organ repair, scarring. The success of these studies will provide for a readily available medical counter measure (MCM) for the treatment of acute and chronic radiation syndromes preventing life threatening complications. 1

PROJECT ABSTRACT The overarching goal of our proposed research program is to develop a discovery pipeline that will enable identification of transcriptional codes for engineering tissue-specific endothelial cells (ECs) for therapeutic organ regeneration of heart, lung and blood. Therapies for organ regeneration promises unlimited access to the replacement tissues. However, despite breakthroughs in uncovering the molecular underpinnings of organ morphogenesis and organoid technology, translation of regenerative medicine to the clinic has confronted with hurdles. These bottlenecks are in part due to the lack of understanding as to how niche cells coordinate organ repair. Specifically, contribution of vascular niche cells that supply regenerative signals has not been realized. This R35 application builds upon the novel proposition that poor healing after organ damage is due to the dysfunction and loss of the tissue-specific ECs. This programmatic proposal examines the hypothesis that reconstitution of stem cells in injured organs is dependent on the pro-regenerative angiocrine signals supplied by tissue-specific vascular niche ECs. We have shown that organotypic ECs by deploying defined angiocrine factors support lung, cardiac, hepatic and hematopoietic regeneration. Thus, ECs perform actively as dynamic, tissue-specified niche cells critical for tissue homeostasis and repair. To test this and to set up the stage for therapies, we have engineered adaptable mouse, nonhuman primate and human ECs by transducing the transduction factor (TF) ETV2 into adult mature ECs (R-VECs) and differentiating human induced pluripotent stem cells (iPSCs) into generic fetal-like ECs (iVECs) that could inform on the pathways that induce organotypic TFs. These adaptive iVECs and R-VECs will be cocultured with heart, lung, and blood organoids in vitro or infused in vivo in mice undergoing organ repair to identify the induction of organotypic TFs in these cells. The educated iVECs and R-VECs will be recovered and subjected to RNA profiling and de novo motif discovery to identify induced tissue-specific TF(s). The identified TFs will be overexpressed or knocked down in ECs, to validate their function in sustaining organotypic and angiocrine profile for organ repair. We anticipate that transplantation of organotypic ECs will promote long-lasting tissue repair without provoking tumorigenesis or fibrosis. We have initiated FDA-approved human clinical trials to examine the safety and efficacy of allogeneic generic EC infusion for hematopoietic recovery. As a follow up, we intend to assess the contribution of R-VECs or iVECs-derived from nonhuman primates to regeneration in the pigtail macaque monkeys with the intention of translating the potential of organotypic ECs to clinic. The expected outcomes of the proposed research are identification of molecular signals and transcriptional determinants of tissue-specific vascular and angiocrine heterogeneity. Goals of this proposal fit with the mission of NHLBI R35 award to develop innovative regenerative discovery pipeline to promote safe and efficacious treatments for cardiac, pulmonary and blood maladies.