Publications by authors named "Michael Ginsberg"

29 Publications

  • Page 1 of 1

Reversal of emphysema by restoration of pulmonary endothelial cells.

J Exp Med 2021 Aug 21;218(8). Epub 2021 Jul 21.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Weill Cornell Medicine, New York, NY.

Chronic obstructive pulmonary disease (COPD) is marked by airway inflammation and airspace enlargement (emphysema) leading to airflow obstruction and eventual respiratory failure. Microvasculature dysfunction is associated with COPD/emphysema. However, it is not known if abnormal endothelium drives COPD/emphysema pathology and/or if correcting endothelial dysfunction has therapeutic potential. Here, we show the centrality of endothelial cells to the pathogenesis of COPD/emphysema in human tissue and using an elastase-induced murine model of emphysema. Airspace disease showed significant endothelial cell loss, and transcriptional profiling suggested an apoptotic, angiogenic, and inflammatory state. This alveolar destruction was rescued by intravenous delivery of healthy lung endothelial cells. Leucine-rich α-2-glycoprotein-1 (LRG1) was a driver of emphysema, and deletion of Lrg1 from endothelial cells rescued vascular rarefaction and alveolar regression. Hence, targeting endothelial cell biology through regenerative methods and/or inhibition of the LRG1 pathway may represent strategies of immense potential for the treatment of COPD/emphysema.
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http://dx.doi.org/10.1084/jem.20200938DOI Listing
August 2021

Comparative engraftment and clonality of macaque HSPCs expanded on human umbilical vein endothelial cells versus non-expanded cells.

Mol Ther Methods Clin Dev 2021 Mar 15;20:703-715. Epub 2021 Feb 15.

Center for Cellular Engineering, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.

hematopoietic stem and progenitor cell (HSPC) expansion platforms are under active development, designed to increase HSPC numbers and thus engraftment ability of allogeneic cord blood grafts or autologous HSPCs for gene therapies. Murine and models have not correlated well with clinical outcomes of HSPC expansion, emphasizing the need for relevant pre-clinical models. Our rhesus macaque HSPC competitive autologous transplantation model utilizing genetically barcoded HSPC allows direct analysis of the relative short and long-term engraftment ability of lentivirally transduced HSPCs, along with additional critical characteristics such as HSPC clonal diversity and lineage bias. We investigated the impact of expansion of macaque HSPCs on the engineered endothelial cell line (E-HUVECs) platform regarding safety, engraftment of transduced and E-HUVEC-expanded HSPC over time compared to non-expanded HSPC for up to 51 months post-transplantation, and both clonal diversity and lineage distribution of output from each engrafted cell source. Short and long-term engraftment were comparable for E-HUVEC expanded and the non-expanded HSPCs in both animals, despite extensive proliferation of CD34 cells during 8 days of culture for the E-HUVEC HSPCs, and optimization of harvesting and infusion of HSPCs co-cultured on E-HUVEC in the second animal. Long-term hematopoietic output from both E-HUVEC expanded and unexpanded HSPCs was highly polyclonal and multilineage. Overall, the comparable HSPC kinetics of macaques to humans, the ability to study post-transplant clonal patterns, and simultaneous multi-arm comparisons of grafts without the complication of interpreting allogeneic effects makes our model ideal to test HSPC expansion platforms, particularly for gene therapy applications.
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http://dx.doi.org/10.1016/j.omtm.2021.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937567PMC
March 2021

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

J Exp Med 2020 06;217(6)

Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY.

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.
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http://dx.doi.org/10.1084/jem.20190730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971135PMC
June 2020

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery.

J Vis Exp 2018 05 16(135). Epub 2018 May 16.

Center for Reproductive Medicine and Infertility, Weill Cornell Medical College; Tri-Institutional Stem Cell Derivation Laboratory, Weill Cornell Medical College;

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.
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http://dx.doi.org/10.3791/57472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101226PMC
May 2018

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

Microcirculation 2018 07 30;25(5):e12455. Epub 2018 May 30.

Bioengineering Program, Fred DeMatteis School of Engineering and Applied Science, Hofstra University, Hempstead, NY, USA.

Objective: Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response.

Methods: Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness.

Results: Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness.

Conclusions: Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease.
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http://dx.doi.org/10.1111/micc.12455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407863PMC
July 2018

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration.

Sci Immunol 2018 01;3(19)

Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of , a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as , a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.
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http://dx.doi.org/10.1126/sciimmunol.aal2736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795617PMC
January 2018

Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue.

Sci Rep 2017 08 15;7(1):8203. Epub 2017 Aug 15.

Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, New York, NY, 10065, United States.

Despite major advances in tissue cryopreservation and auto-transplantation, reperfusion ischemia and hypoxia have been reported as major obstacles to successful recovery of the follicular pool within grafted ovarian tissue. We demonstrate a benefit to follicular survival and function in human ovarian tissue that is co-transplanted with exogenous endothelial cells (ExEC). ExECs were capable of forming functionally perfused vessels at the host/graft interface and increased both viability and follicular volume in ExEC-assisted grafts with resumption of antral follicle development in long-term grafts. ExECs that were engineered to constitutively express anti-mullerian hormone (AMH) induced a greater proportion of quiescent primordial follicles than control ExECs, indicating suppression of premature mobilization that has been noted in the context of ovarian tissue transplantation. These findings present a cell-based strategy that combines accelerated perfusion with direct paracrine delivery of a bioactive payload to transplanted ovarian tissue.
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http://dx.doi.org/10.1038/s41598-017-08491-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557862PMC
August 2017

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

Nat Commun 2017 05 19;8:15374. Epub 2017 May 19.

Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medicine, 1300 York Avenue, New York, New York 10065, USA.

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.
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http://dx.doi.org/10.1038/ncomms15374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454459PMC
May 2017

Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

Nature 2017 05 17;545(7655):439-445. Epub 2017 May 17.

Ansary Stem Cell Institute, Division of Regenerative Medicine, Department of Medicine, Weill Cornell Medicine, New York, New York 10065, USA.

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1 FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.
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http://dx.doi.org/10.1038/nature22326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794215PMC
May 2017

Catheter-directed Intraportal Delivery of Endothelial Cell Therapy for Liver Regeneration: A Feasibility Study in a Large-Animal Model of Cirrhosis.

Radiology 2017 10 11;285(1):114-123. Epub 2017 May 11.

From the Department of Radiology (K.S.L., D.L., A.R., K.S., D.C.M.), Laboratory of Comparative Pathology (S.F.S.), and Department of Genetic Medicine (B.S.D., S.R.), Weill Cornell Medicine, 525 E 68th St, Payson Pavilion 5, New York, NY 10065; and Angiocrine Bioscience, San Diego, Calif (M.D.G.).

Purpose To demonstrate the feasibility of imaging-guided catheter-directed delivery of endothelial cell therapy in a porcine model of cirrhosis for liver regeneration. Materials and Methods After approval from the institutional animal care and use committee, autologous liver endothelial cells were grown from core hepatic specimens from swine. Cirrhosis was induced in swine by means of transcatheter infusion of ethanol and iodized oil into the hepatic artery. Three weeks after induction of cirrhosis, the swine were randomly assigned to receive autologous cell therapy (endothelial cells, n = 4) or control treatment (phosphate-buffered saline, n = 4) by means of imaging-guided transhepatic intraportal catheterization. Fluorescence-activated cell sorting analysis was performed on biopsy samples 1 hour after therapy. Three weeks after intraportal delivery of endothelial cells, the swine were euthanized and the explanted liver underwent quantitative pathologic examination. Statistical analysis was performed with an unpaired t test by using unequal variance. Results Liver endothelial cells were successfully isolated, cultured, and expanded from eight 20-mm, 18-gauge hepatic core samples to 50 × 10 autologous cells per pig. Intraportal delivery of endothelial cell therapy or saline was technically successful in all eight swine, with no complications. Endothelial cells were present in the liver for a minimum of 1 hour after intraportal infusion. Swine treated with endothelial cell therapy showed mean levels of surrogate markers of hepatobiliary injury that were consistent with decreases in hepatic fibrosis and biliary ductal damage relative to the control animals, although statistical significance was not met in this pilot study: The mean percentage of positive pixels at Masson trichrome staining was 7.28% vs 5.57%, respectively (P = .20), the mean proliferation index with cytokeratin wide-spectrum was 2.55 vs 1.13 (P = .06), and the mean proliferation index with Ki67 was 7.08 vs 4.96 (P = .14). Conclusion The results confirm the feasibility of imaging-guided catheter-directed endothelial cell therapy with an intraportal technique for the treatment of cirrhosis in a porcine model. A trend toward decreased liver fibrosis with endothelial cell therapy was observed. Larger animal studies and human studies are necessary to confirm significance. RSNA, 2017.
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http://dx.doi.org/10.1148/radiol.2017162617DOI Listing
October 2017

Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

Stem Cells Transl Med 2017 03 14;6(3):864-876. Epub 2016 Oct 14.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34 cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34 C38 HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34 cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34 cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876.
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http://dx.doi.org/10.5966/sctm.2016-0240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442761PMC
March 2017

Sox17 drives functional engraftment of endothelium converted from non-vascular cells.

Nat Commun 2017 01 16;8:13963. Epub 2017 Jan 16.

Division of Regenerative Medicine, Department of Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, 1300 York Avenue, Room A-863, New York, New York 10065, USA.

Transplanting vascular endothelial cells (ECs) to support metabolism and express regenerative paracrine factors is a strategy to treat vasculopathies and to promote tissue regeneration. However, transplantation strategies have been challenging to develop, because ECs are difficult to culture and little is known about how to direct them to stably integrate into vasculature. Here we show that only amniotic cells could convert to cells that maintain EC gene expression. Even so, these converted cells perform sub-optimally in transplantation studies. Constitutive Akt signalling increases expression of EC morphogenesis genes, including Sox17, shifts the genomic targeting of Fli1 to favour nearby Sox consensus sites and enhances the vascular function of converted cells. Enforced expression of Sox17 increases expression of morphogenesis genes and promotes integration of transplanted converted cells into injured vessels. Thus, Ets transcription factors specify non-vascular, amniotic cells to EC-like cells, whereas Sox17 expression is required to confer EC function.
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http://dx.doi.org/10.1038/ncomms13963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5260855PMC
January 2017

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis.

Nat Commun 2016 12 21;7:13829. Epub 2016 Dec 21.

Department of Genetic Medicine, Ansary Stem Cell Institute, Weill Cornell Medical College, New York, New York 10021, USA.

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.
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http://dx.doi.org/10.1038/ncomms13829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187502PMC
December 2016

Transplantation of Endothelial Cells to Mitigate Acute and Chronic Radiation Injury to Vital Organs.

Radiat Res 2016 Aug 26;186(2):196-202. Epub 2016 Jul 26.

Weill Cornell Medical College Ansary Stem Cell Institute, Department of Medicine, Division of Regenerative Medicine, New York, New York.

Current therapeutic approaches for treatment of exposure to radiation involve the use of antioxidants, chelating agents, recombinant growth factors and transplantation of stem cells (e.g., hematopoietic stem cell transplantation). However, exposure to high-dose radiation is associated with severe damage to the vasculature of vital organs, often leading to impaired healing, tissue necrosis, thrombosis and defective regeneration caused by aberrant fibrosis. It is very unlikely that infusion of protective chemicals will reverse severe damage to the vascular endothelial cells (ECs). The role of irradiated vasculature in mediating acute and chronic radiation syndromes has not been fully appreciated or well studied. New approaches are necessary to replace and reconstitute ECs in organs that are irreversibly damaged by radiation. We have set forth the novel concept that ECs provide paracrine signals, also known as angiocrine signals, which not only promote healing of irradiated tissue but also direct organ regeneration without provoking fibrosis. We have developed innovative technologies that enable manufacturing and banking of human GMP-grade ECs. These ECs can be transplanted intravenously to home to and engraft to injured tissues where they augment organ repair, while preventing maladaptive fibrosis. In the past, therapeutic transplantation of ECs was not possible due to a shortage of availability of suitable donor cell sources and preclinical models, a lack of understanding of the immune privilege of ECs, and inadequate methodologies for expansion and banking of engraftable ECs. Recent advances made by our group as well as other laboratories have breached the most significant of these obstacles with the development of technologies to manufacture clinical-scale quantities of GMP-grade and human ECs in culture, including genetically diverse reprogrammed human amniotic cells into vascular ECs (rAC-VECs) or human pluripotent stem cells into vascular ECs (iVECs). This approach provides a path to therapeutic EC transplantation that can be infused concomitantly or sequentially with hematopoietic stem cell transplantation more than 24 h after irradiation to support multi-organ regeneration, thereby improving immediate and long-term survival, while limiting long-term morbidity resulting from nonregenerative damage repair pathways.
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http://dx.doi.org/10.1667/RR14461.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028179PMC
August 2016

Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis.

Nat Med 2016 Feb 18;22(2):154-62. Epub 2016 Jan 18.

Ansary Stem Cell Institute, Weill Cornell Medicine, New York, New York, USA.

Although the lung can undergo self-repair after injury, fibrosis in chronically injured or diseased lungs can occur at the expense of regeneration. Here we study how a hematopoietic-vascular niche regulates alveolar repair and lung fibrosis. Using intratracheal injection of bleomycin or hydrochloric acid in mice, we show that repetitive lung injury activates pulmonary capillary endothelial cells (PCECs) and perivascular macrophages, impeding alveolar repair and promoting fibrosis. Whereas the chemokine receptor CXCR7, expressed on PCECs, acts to prevent epithelial damage and ameliorate fibrosis after a single round of treatment with bleomycin or hydrochloric acid, repeated injury leads to suppression of CXCR7 expression and recruitment of vascular endothelial growth factor receptor 1 (VEGFR1)-expressing perivascular macrophages. This recruitment stimulates Wnt/β-catenin-dependent persistent upregulation of the Notch ligand Jagged1 (encoded by Jag1) in PCECs, which in turn stimulates exuberant Notch signaling in perivascular fibroblasts and enhances fibrosis. Administration of a CXCR7 agonist or PCEC-targeted Jag1 shRNA after lung injury promotes alveolar repair and reduces fibrosis. Thus, targeting of a maladapted hematopoietic-vascular niche, in which macrophages, PCECs and perivascular fibroblasts interact, may help to develop therapy to spur lung regeneration and alleviate fibrosis.
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http://dx.doi.org/10.1038/nm.4035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872630PMC
February 2016

Direct conversion of human amniotic cells into endothelial cells without transitioning through a pluripotent state.

Nat Protoc 2015 Dec 5;10(12):1975-85. Epub 2015 Nov 5.

Division of Regenerative Medicine, Department of Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, New York, New York, USA.

Endothelial cells (ECs) have essential roles in organ development and regeneration, and therefore they could be used for regenerative therapies. However, generation of abundant functional endothelium from pluripotent stem cells has been difficult because ECs generated by many existing strategies have limited proliferative potential and display vascular instability. The latter difficulty is of particular importance because cells that lose their identity over time could be unsuitable for therapeutic use. Here, we describe a 3-week platform for directly converting human mid-gestation lineage-committed amniotic fluid-derived cells (ACs) into a stable and expandable population of vascular ECs (rAC-VECs) without using pluripotency factors. By transient expression of the ETS transcription factor ETV2 for 2 weeks and constitutive expression the ETS transcription factors FLI1 and ERG1, concomitant with TGF-β inhibition for 3 weeks, epithelial and mesenchymal ACs are converted, with high efficiency, into functional rAC-VECs. These rAC-VECs maintain their vascular repertoire and morphology over numerous passages in vitro, and they form functional vessels when implanted in vivo. rAC-VECs can be detected in recipient mice months after implantation. Thus, rAC-VECs can be used to establish a cellular platform to uncover the molecular determinants of vascular development and heterogeneity and potentially represent ideal ECs for the treatment of regenerative disorders.
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http://dx.doi.org/10.1038/nprot.2015.126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881298PMC
December 2015

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells.

J Clin Invest 2015 Mar 9;125(3):1243-54. Epub 2015 Feb 9.

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC-derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina-induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain-null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood-derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence.
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http://dx.doi.org/10.1172/JCI79328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362238PMC
March 2015

Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells.

Nat Biotechnol 2014 Nov 12;32(11):1151-1157. Epub 2014 Oct 12.

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA.

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.
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http://dx.doi.org/10.1038/nbt.3048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318247PMC
November 2014

Activation of the vascular niche supports leukemic progression and resistance to chemotherapy.

Exp Hematol 2014 Nov 29;42(11):976-986.e3. Epub 2014 Aug 29.

Department of Genetic Medicine, Weill Cornell Medical College, New York, NY, 10065, USA.

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents.
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http://dx.doi.org/10.1016/j.exphem.2014.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254082PMC
November 2014

Akt suppression of TGFβ signaling contributes to the maintenance of vascular identity in embryonic stem cell-derived endothelial cells.

Stem Cells 2014 Jan;32(1):177-90

Department of Genetic Medicine, Ansary Stem Cell Institute, Howard Hughes Medical Institute, Weill Cornell Medical College, New York, New York, USA.

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (ECs) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates.
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http://dx.doi.org/10.1002/stem.1521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886558PMC
January 2014

Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.

Dev Cell 2013 Jul 18;26(2):204-19. Epub 2013 Jul 18.

Department of Genetic Medicine, Howard Hughes Medical Institute, Weill Cornell Medical College, New York, NY 10065, USA.

Microvascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration.
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http://dx.doi.org/10.1016/j.devcel.2013.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873200PMC
July 2013

Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.

Blood 2013 Jan 20;121(5):770-80. Epub 2012 Nov 20.

Department of Genetic Medicine, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, USA.

Unlabelled: Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation.

Key Points: Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.
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http://dx.doi.org/10.1182/blood-2012-07-444208DOI Listing
January 2013

Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFβ suppression.

Cell 2012 Oct 18;151(3):559-75. Epub 2012 Oct 18.

Howard Hughes Medical Institute, Ansary Stem Cell Institute, Department of Genetic Medicine, Weill Cornell Medical College, New York, NY, 10065, USA.

ETS transcription factors ETV2, FLI1, and ERG1 specify pluripotent stem cells into induced vascular endothelial cells (iVECs). However, iVECs are unstable and drift toward nonvascular cells. We show that human midgestation c-Kit(-) lineage-committed amniotic cells (ACs) can be reprogrammed into vascular endothelial cells (rAC-VECs) without transitioning through a pluripotent state. Transient ETV2 expression in ACs generates immature rAC-VECs, whereas coexpression with FLI1/ERG1 endows rAC-VECs with a vascular repertoire and morphology matching mature endothelial cells (ECs). Brief TGFβ-inhibition functionalizes VEGFR2 signaling, augmenting specification of ACs into rAC-VECs. Genome-wide transcriptional analyses showed that rAC-VECs are similar to adult ECs in which vascular-specific genes are expressed and nonvascular genes are silenced. Functionally, rAC-VECs form stable vasculature in Matrigel plugs and regenerating livers. Therefore, short-term ETV2 expression and TGFβ inhibition with constitutive ERG1/FLI1 coexpression reprogram mature ACs into durable rAC-VECs with clinical-scale expansion potential. Banking of HLA-typed rAC-VECs establishes a vascular inventory for treatment of diverse disorders.
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http://dx.doi.org/10.1016/j.cell.2012.09.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507451PMC
October 2012

Attention-deficit/hyperactivity disorder: a personal perspective.

Adolesc Med State Art Rev 2008 Aug;19(2):339-51, xi-xii

Department of Pediatrics, Jacobi Medical Center, 1400 Pelham Parkway S, Room 817-A, Bronx, NY 10461, USA.

Children with ADHD can achieve academic and personal success in adulthood. In this article the author shares personal perspectives on his own struggles with ADHD as a child and shares insights, tips, and anecdotes from his own life. These insights, when viewed in light of the scientific literature regarding ADHD, offer new directions for research into the management of the disorder in children and adolescents.
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August 2008

Amino acid residues required for physical and cooperative transcriptional interaction of STAT3 and AP-1 proteins c-Jun and c-Fos.

Mol Cell Biol 2007 Sep 16;27(18):6300-8. Epub 2007 Jul 16.

Laboratory of Molecular Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

Cooperation between STAT3 and c-Jun in driving transcription during transfection of reporter constructs is well established, and both proteins are present on some interleukin-6 (IL-6) STAT3-dependent promoters on chromosomal loci. We report that small interfering RNA knockdown of c-Jun or c-Fos diminishes IL-6 induction of some but not all STAT3-dependent mRNAs. Specific contact sites in STAT3 responsible for interaction of a domain of STAT3 with c-Jun were known. Here we show that the B-zip domain of c-Jun interacts with STAT3 and that c-Jun mutation R261A or R261D near but not in the DNA binding domain blocks in vitro STAT3-c-Jun interaction and decreases costimulation of transcription in transfection assays. Cooperative binding to DNA of tyrosine-phosphorylated STAT3 and both wild-type and R261A mutant c-Jun was observed. Even c-Jun mutant R261D, which on its own did not bind DNA, bound DNA weakly in the presence of STAT3. We conclude that a functional interaction between STAT3 and c-Jun while bound to chromosomal DNA elements exists and is necessary for driving transcription on at least some STAT3 target genes. Identifying such required interactive protein interfaces should be a stimulus to search for compounds that could ultimately inhibit the activity of STAT3 in tumors dependent on persistently active STAT3.
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http://dx.doi.org/10.1128/MCB.00613-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2099614PMC
September 2007

PKA-dependent binding of mRNA to the mitochondrial AKAP121 protein.

J Mol Biol 2003 Apr;327(4):885-97

Institute of Cancer Research, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032, USA.

Protein kinase A (PKA) anchoring proteins (AKAPs) tether PKA to various subcellular locations. AKAP121, which tethers PKAII to the outer mitochondrial membrane, includes a K homology (KH) RNA-binding motif. Purified AKAP121 KH domain binds the 3' untranslated regions (3'UTRs) of transcripts encoding the Fo-f subunit of mitochondrial ATP synthase and manganese superoxide dismutase (MnSOD). Binding requires a structural motif in the 3'UTR and is stimulated by PKA phosphorylation of the domain or a mutation that mimics this phosphorylation. AKAP121 expressed in HeLa cells promotes the translocation of MnSOD mRNA from cytosol to mitochondria and an increase in mitochondrial MnSOD. Both reactions are stimulated by cAMP. Thus, by focusing translation at the mitochondrial membrane, AKAP121 may facilitate import of mitochondrial proteins in response to cAMP stimulation.
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http://dx.doi.org/10.1016/s0022-2836(03)00173-6DOI Listing
April 2003

Specificity in DNA recognition by phage integrases.

Gene 2002 Oct;300(1-2):13-8

Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

The lambda-related (lambdoid) coliphages are related to one another by frequent natural recombination and maintain a high level of functional polymorphism for several activities of the phages. Arguments are presented that the polymorphism of the integration module results from selection (presumably frequency-dependent) for new (not improved) specificities of site recognition. Analysis of phages lambda and HK022 by Weisberg and collaborators previously showed that changes in five noncontiguous amino acids could switch site recognition specificity. Phage 21 and defective element e14, which integrate at the same site, differ in recognition specificity for both core and arm sites. In vitro assays of e14 and 21 insertion and excision confirm this conclusion. Inhibition by ds arm site oligonucleotides defines the sequence specificity more precisely.
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http://dx.doi.org/10.1016/s0378-1119(02)00846-6DOI Listing
October 2002

Essential role of A-kinase anchor protein 121 for cAMP signaling to mitochondria.

J Biol Chem 2003 Feb 8;278(6):4286-94. Epub 2002 Nov 8.

Dipartimento di Biologia e Patologia Molecolare e Cellulare, BioGeM Consortium, Istituto di Endocrinologia ed Oncologia Sperimentale CNR, Facoltà di Medicina, Universitá Federico II, via S. Pansini 5, 80131 Napoli, Italy.

A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.
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http://dx.doi.org/10.1074/jbc.M209941200DOI Listing
February 2003

A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs.

J Mol Biol 2002 Jul;320(3):663-75

Dipartimento di Biologia e Patologia Molecolare e Cellulare, BioGem Consortium, Instituto di Endocrinologia ed Oncologia Sperimentale CNR, Universitá Federico II, via S. Pansini 5, 80131 Naples, Italy.

A-kinase anchor proteins (AKAPs) assemble multi-enzyme signaling complexes in proximity to substrate/effector proteins, thus directing and amplifying membrane-generated signals. S-AKAP84 and AKAP121 are alternative splicing products with identical NH(2) termini. These AKAPs bind and target protein kinase A (PKA) to the outer mitochondrial membrane. Tubulin was identified as a binding partner of S-AKAP84 in a yeast two-hybrid screen. Immunoprecipitation and co-sedimentation experiments in rat testis extracts confirmed the interaction between microtubules and S-AKAP84. In situ immunostaining of testicular germ cells (GC2) shows that AKAP121 concentrates on mitochondria in interphase and on mitotic spindles during M phase. Purified tubulin binds directly to S-AKAP84 but not to a deletion mutant lacking the mitochondrial targeting domain (MT) at residues 1-30. The MT is predicted to form a highly hydrophobic alpha-helical wheel that might also mediate interaction with tubulin. Disruption of the wheel by site-directed mutagenesis abolished tubulin binding and reduced mitochondrial attachment of an MT-GFP fusion protein. Some MT mutants retain tubulin binding but do not localize to mitochondria. Thus, the tubulin-binding motif lies within the mitochondrial attachment motif. Our findings indicate that S-AKAP84/AKAP121 use overlapping targeting motifs to localize signaling enzymes to mitochondrial and cytoskeletal compartments.
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http://dx.doi.org/10.1016/s0022-2836(02)00479-5DOI Listing
July 2002
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