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Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(β)—two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection—but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09823-4.

The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.

Islet transplantation has shown promise as a curative therapy for type 1 diabetes (T1D). However, the side effects of systemic immunosuppression and limited long‐term viability of engrafted islets, together with the scarcity of donor organs, highlight an urgent need for the development of new, improved, and safer cell‐replacement strategies. Induction of local immunotolerance to prevent allo‐rejection against islets and stem cell derived β cells has the potential to improve graft function and broaden the applicability of cellular therapy while minimizing adverse effects of systemic immunosuppression. In this mini review, recent developments in non‐encapsulation, local immunomodulatory approaches for T1D cell replacement therapies, including islet/β cell modification, immunomodulatory biomaterial platforms, and co‐transplantation of immunomodulatory cells are discussed. Key advantages and remaining challenges in translating such technologies to clinical settings are identified. Although many of the studies discussed are preliminary, the growing interest in the field has led to the exploration of new combinatorial strategies involving cellular engineering, immunotherapy, and novel biomaterials. Such interdisciplinary research will undoubtedly accelerate the development of therapies that can benefit the whole T1D population., An overview of different local immunomodulatory approaches in β cell replacement therapies is provided. Strategies in islet/β cell modification with immunomodulatory proteins immobilized on the cell surface or released to the local microenvironment, biomaterial platforms presenting or delivering immunomodulatory signals, and co‐transplantation of immunomodulatory cells are discussed.

Pharmacotherapy of vascular anomalies has limited efficacy and potentially limiting toxicity. Targeted nanoparticle (NP) drug delivery systems have the potential to accumulate within tissues where the vasculature is impaired, potentially leading to high drug levels (increased efficacy) in the diseased tissue and less in off-target sites (less toxicity). Here, we investigate whether NPs can be used to enhance drug delivery to bioengineered human vascular networks (hVNs) that are a model of human vascular anomalies. We demonstrate that intravenously injected phototargeted NPs enhanced accumulation of NPs and the drug within hVNs. With phototargeting we demonstrate 17 times more NP accumulation within hVNs than was detected in hVNs without phototargeting. With phototargeting there was 10-fold more NP accumulation within hVNs than in any other organ. Phototargeting resulted in a 6-fold increase in drug accumulation (doxorubicin) within hVNs in comparison to animals injected with the free drug. Nanoparticulate approaches have the potential to markedly improve drug delivery to vascular anomalies.

Hemophilia A (HA) is a bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). Current treatments are based on regular infusions of FVIII concentrates throughout a patient’s life. Alternatively, viral gene therapies that directly deliver F8 in vivo have shown preliminary successes. However, hurdles remain, including lack of infection specificity and the inability to deliver the full-length version of F8 due to restricted viral cargo sizes. Here, we developed an alternative nonviral ex vivo gene-therapy approach that enables the overexpression of full-length F8 in patients’ endothelial cells (ECs). We first generated HA patient–specific induced pluripotent stem cells (HA-iPSCs) from urine epithelial cells and genetically modified them using a piggyBac DNA transposon system to insert multiple copies of full-length F8. We subsequently differentiated the modified HA-iPSCs into competent ECs with high efficiency, and demonstrated that the cells (termed HA-FLF8-iECs) were capable of producing high levels of FVIII. Importantly, following subcutaneous implantation into immunodeficient hemophilic (SCID-f8ko) mice, we demonstrated that HA-FLF8-iECs were able to self-assemble into vascular networks, and that the newly formed microvessels had the capacity to deliver functional FVIII directly into the bloodstream of the mice, effectively correcting the clotting deficiency. Moreover, our implant maintains cellular confinement, which reduces potential safety concerns and allows effective monitoring and reversibility. We envision that this proof-of-concept study could become the basis for a novel autologous ex vivo gene-therapy approach to treat HA.

BACKGROUND Endothelial-to-mesenchymal-transition (EndMT) plays a major role in cardiac fibrosis, including endocardial fibroelastosis but the stimuli are still unknown. We developed an endothelial cell (EC) culture and a whole heart model to test whether mechanical strain triggers TGF-β-mediated EndMT. METHODS Isolated ECs were exposed to 10% uniaxial static stretch for 8 h (stretch) and TGF-β-mediated EndMT was determined using the TGF-β-inhibitor SB431542 (stretch + TGF-β-inhibitor), BMP-7 (stretch + BMP-7) or losartan (stretch + losartan), and isolated mature and immature rats were exposed to stretch through a weight on the apex of the left ventricle. Immunohistochemical staining for double-staining with endothelial markers (VE-cadherin, PECAM1) and mesenchymal markers (αSMA) or transcription factors (SLUG/SNAIL) positive nuclei was indicative of EndMT. RESULTS Stretch-induced EndMT in ECs expressed as double-stained ECs/total ECs (cells: 46 ± 13%; heart: 15.9 ± 2%) compared to controls (cells: 7 ± 2%; heart: 3.1 ± 0.1; p

Transplantation of stem cell derived β (SC-β) cells represents a promising new therapy for type 1 diabetes (T1D). However, the delivery of these cells, ideally without immunosuppression and in a retrievable device that is both functional and safe, remains a challenge. Indeed, prioritizing safety in cell delivery devices often compromises cell viability and function. Herein, we report the design of a safe and functional device composed of a highly porous, durable nanofibrous skin and an immunoprotective hydrogel core. The device made by electrospinning a medical grade thermoplastic silicone-polycarbonate-urethane is soft but tough (~15 MPa at a rupture strain of > 2). Tuning the nanofiber size to < ~500 nm prevented cell penetration while maintaining maximum mass transfer and interestingly decreased the cellular overgrowth on blank devices to as low as a single cell layer (~3 μm thick) in peritoneal cavity of mice. Using bioluminescence imaging (BLI), we confirmed the safety, indicated as continuous containment of even proliferative cells, within the device for 5 months. With either syngeneic, allogeneic or xenogeneic rodent islets, the device corrected chemically induced diabetes in mice and remained functional for up to 200 days. Most importantly, the device supported the function of human SC-β cells and reversed diabetes almost immediately (within a week) in both immunodeficient mice (for up to 120 days) and immunocompetent mice (for up to 60 days). Lastly, we demonstrated the scalability and retrievability of the device in dogs where we also observed viable human SC-β cells despite xenogeneic immune responses. The nanofibrous device design may therefore provide a translatable solution to the balance between safety and functionality in developing stem cell-based therapies for T1D.

Introduction: Endocardial Fibroelastosis (EFE) is a condition defined by the formation of fibrotic tissue on the endocardium, stunting cardiac development. The presence of EFE restricts diastolic compliance, leading to systolic and diastolic heart failure, and contributing to higher morbidity and mortality, particularly in Hypoplastic Left Heart Syndrome. While research suggests that EFE forms through aberrant endothelial-to-mesenchymal transition (EndoMT), the trigger of its formation is still speculative. Anecdotally, however, we have observed EFE development in patients with abnormal left ventricular flow. Objective: Our objective is to study whether the absence of normal flow induces EndoMT in neonatal endocardium and promotes EFE development, and whether losartan treatment, which can decrease TGFβ production to impair EndoMT, can abrogate EFE formation. Methods and Results: Using a rodent model of EFE, we establish three flow conditions in neonatal hearts: Normal Flow (NF), Static/no flow (S), and Regurgitant Flow (RF). Histologic analysis demonstrates EFE development in S and RF conditions, but not in NF conditions. Using qPCR, Western Blot Assay, and immunohistochemistry, we demonstrate that S and RF significantly increase mRNA and protein expression of EndoMT markers (αSMA, SNAIL), and EndoMT-related signaling pathways (TGFβ, NOTCH1) whereas NF conditions do not. With losartan treatment, we observe a significant decrease in EFE in neonatal hearts documented by histology and decreased mRNA and protein expression of EndoMT markers. To determine whether our findings are translatable to humans, human endocardial endothelial cells were isolated from left ventricular tissue and subjected to two flow conditions: static and laminar shear stress (LSS). By RNAseq analysis, we demonstrate that laminar shear stress suppresses the expression of genes critical for mesenchymal differentiation and Notch signaling, which are both associated with EndoMT progression. Conclusions: Our data demonstrates that the absence of normal flow provides an environment that promotes EFE development through activated EndoMT. Further, we identify losartan as a potential therapy for EFE, as it is able to abrogate EFE development in our animal model.

The capability of forming functional blood vessel networks is critical for the characterization of endothelial cells. In this chapter, we will review a modified in vivo vascular network forming assay by replacing traditional mouse tumor-derived Matrigel with a well-defined collagen-fibrin hydrogel. The assay is reliable and does not require special equipment, surgical procedure, or a skilled person to perform. Moreover, investigators can modify this method on-demand for testing different cell sources, perturbation of gene functions, growth factors, and pharmaceutical molecules, and for the development and investigation of strategies to enhance neovascularization of engineered human tissues and organs.

The 2020 Congress of the International Society of Thrombosis and Haemostasis (ISTH) was held virtually July 12-15, 2019, due to the coronavirus disease 2019 pandemic. The congress convenes annually to discuss clinical and basic topics in hemostasis and thrombosis. Each year, the program includes State of Art (SOA) lectures given by prominent scientists. Presenters are asked to create Illustrated Capsules of their talks, which are concise illustrations with minimal explanatory text. Capsules cover major themes of the presentation, and these undergo formal peer review for inclusion in this article. Owing to the shift to a virtual congress this year, organizers reduced the program size. There were 39 SOA lectures virtually presented, and 29 capsules (9 from talks omitted from the virtual congress) were both submitted and successful in peer review, and are included in this article. Topics include the roles of the hemostatic system in inflammation, infection, immunity, and cancer, platelet function and signaling, platelet function disorders, megakaryocyte biology, hemophilia including gene therapy, phenotype tests in hemostasis, von Willebrand factor, anticoagulant factor V, computational driven discovery, endothelium, clinical and basic aspects of thrombotic microangiopathies, fibrinolysis and thrombolysis, antithrombotics in pediatrics, direct oral anticoagulant management, and thrombosis and hemostasis in pregnancy. Capsule authors invite virtual congress attendees to refer to these capsules during the live presentations and participate on Twitter in discussion. Research and Practice in Haemostasis and Thrombosis will release 2 tweets from @RPTHJournal during each presentation, using #IllustratedReview, #CoagCapsule and #ISTH2020. Readers are also welcome to utilize capsules for teaching and ongoing education., peer-reviewed

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference. Fil: Nowak Sliwinska, Patrycja. Université of Lausanne; Suiza. Univeristé of Geneve; Suiza Fil: Alitalo, Kari. Katholikie Universiteit Leuven; Bélgica Fil: Allen, Elizabeth. Katholikie Universiteit Leuven; Bélgica Fil: Anisimov, Andrey. Katholikie Universiteit Leuven; Bélgica Fil: Aplin, Alfred C.. University of Washington; Estados Unidos Fil: Auerbach, Robert. University of Wisconsin; Estados Unidos Fil: Augustin, Hellmut G.. Heidelberg University; Alemania. German Cancer Consortium; Alemania Fil: Bates, David O.. University of Nottingham; Reino Unido Fil: Beijnum, Judy R. van. Cancer Center Amsterdam; Países Bajos Fil: Bender, R. Hugh F.. University of California; Estados Unidos Fil: Bergers, Gabriele. Katholikie Universiteit Leuven; Bélgica Fil: Bikfalvi, Andreas. Universite de Bordeaux; Francia Fil: Bischoff, Joyce. Harvard Medical School; Estados Unidos Fil: Böck, Barbara C.. Heidelberg University; Alemania. German Cancer Consortium; Alemania Fil: Brooks, Peter C.. Maine Medical Center Research Institute; Estados Unidos Fil: Bussolino, Federico. Università di Torino; Italia. Candiolo Cancer Institute; Italia Fil: Cakir, Bertan. Harvard Medical School; Estados Unidos Fil: Carmeliet, Peter. Katholikie Universiteit Leuven; Bélgica Fil: Castranova, Daniel. Harvard Medical School; Estados Unidos Fil: Cimpean, Anca M.. Victor Babes University of Medicine and Pharmacy; Rumania Fil: Cleaver, Ondine. University Of Texas At Brownsville; Estados Unidos Fil: Coukos, George. Universida de Lausanne; Suiza Fil: Davis, George E.. University of Missouri; Estados Unidos Fil: De Palma, Michele. Swiss Federal Institute of Technology; Suiza Fil: Dimberg, Anna. Uppsala University; Suiza Fil: Dings, Ruud P. M.. University of Arkansas for Medical Sciences; Estados Unidos Fil: Djonov, Valentin. University of Bern; Suiza Fil: Dudley, Andrew C.. University of Virginia; Estados Unidos Fil: Dufton, Neil P.. Imperial College London; Reino Unido Fil: Fendt, Sarah-Maria. VIB Center for Cancer Biology; Bélgica Fil: Ferrara, Napoleone. University of California at San Diego; Estados Unidos Fil: Fruttiger, Marcus. University College London; Estados Unidos Fil: Fukumura, Dai. Harvard Medical School; Estados Unidos Fil: Ghesquière, Bart. Harvard Medical School; Estados Unidos Fil: Gong, Yan. Harvard Medical School; Estados Unidos Fil: Griffin, Robert J.. VIB Center for Cancer Biology; Bélgica Fil: Harris, Adrian L.. University of Oxford; Reino Unido Fil: Hughes, Christopher C. W.. University of California at Irvine; Estados Unidos Fil: Hultgren, Nan W.. University of California at Irvine; Estados Unidos Fil: Iruela-Arispe, M. Luisa. University of California at Los Angeles; Estados Unidos Fil: Irving, Melita. Universida de Lausanne; Suiza Fil: Maidana, Agostina Jainen. Harvard Medical School; Estados Unidos Fil: Kalluri, Raghu. Texas A&M University; Estados Unidos Fil: Kalucka, Joanna. Katholikie Universiteit Leuven; Bélgica Fil: Kerbel, Robert S.. University of Toronto; Canadá Fil: Kitajewski, Jan. University of Illinois; Estados Unidos Fil: Klaassen, Ingeborg. University of Amsterdam; Países Bajos Fil: Kleinmann, Hynda K.. The George Washington University; Estados Unidos Fil: Koolwijk, Pieter. Fondation Asile des Aveugles; Suiza. Universida de Lausanne; Suiza Fil: Kuczynski, Elisabeth. University of Toronto; Canadá Fil: Kwak, Brenda R.. University of Geneva; Suiza Fil: Koen, Marien. HistoGeneX; Bélgica Fil: Melero Martin, Juan M.. University of Liège; Bélgica Fil: Munn, Lance L.. Harvard Medical School; Estados Unidos Fil: Nicosia, Roberto F.. VA Puget Sound Health Care System; Estados Unidos Fil: Noel, Agnes. University of Liège; Bélgica Fil: Nurro, Jussi. University of Eastern Finland; Finlandia Fil: Olsson, Anna-Karin. Uppsala University; Suiza Fil: Petrova, Tatiana V.. Ludwig Institute for Cancer Research Lausanne; Suiza Fil: Pietras, Kristian. Division of Translational Cancer Research; Suecia Fil: Pili, Roberto. Indiana University Simon Cancer Center; Estados Unidos Fil: Pollard, Jeffrey W.. University of Edinburgh; Reino Unido Fil: Post, Mark J.. Maastricht University; Países Bajos Fil: Quax, Paul H. A.. Einthoven Laboratory for Experimental Vascular Medicine; Países Bajos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Raica, Marius. Victor Babes University of Medicine and Pharmacy; Rumania Fil: Randi, Anna M.. Imperial College London; Reino Unido Fil: Ribatti, Domenico. Università degli Studi di Bari; Italia Fil: Ruegg, Curzio. University of Fribourg; Suiza Fil: Schlingemann, Reinier O.. University of Amsterdam; Países Bajos Fil: Schulte Merker, Stefan. Institute of Cardiovascular Organogenesis and Regeneration; Alemania Fil: Smith, Lois E. H.. Harvard Medical School; Estados Unidos Fil: Song, Jonathan W.. Ohio State University; Estados Unidos Fil: Stacker, Steven A.. University of Melbourne; Australia Fil: Stalin, Jimmy. Institute of Cardiovascular Organogenesis and Regeneration; Alemania Fil: Stratman, Amber N.. National Institutes of Health; Estados Unidos Fil: Van de Velde, Maureen. University of Liège; Bélgica Fil: van Hinsbergh, Victor W. M.. Universida de Lausanne; Suiza Fil: Vermeulen, Peter B.. HistoGeneX; Bélgica. University of Antwerp; Bélgica Fil: Waltenberger, Johannes. University of Münster; Alemania Fil: Weinstein, Brant M.. National Institutes of Health; Estados Unidos Fil: Xin, Hong. University of California at San Diego; Estados Unidos Fil: Yetkin Arik, Bahar. University of Amsterdam; Países Bajos Fil: Yla Herttuala, Seppo. University of Eastern Finland; Finlandia Fil: Yoder, Mervin C.. Indiana University; Estados Unidos Fil: Griffioen, Arjan W.. VU University Medical Center; Países Bajos

In article number 2100070, Juan M. Melero-Martin, Ruei-Zeng Lin, and co-workers present a biphasic scaffold that allows self-sustained endochondral ossification. Following seeding with human vascular and mesenchymal stem cells, this biomimetic material enables the rapid generation of osteovascular niches and ossification centers, leading to the formation of vascularized bone tissue in vivo.

Regeneration of large bones remains a challenge in surgery. Recent developmental engineering efforts aim to recapitulate endochondral ossification (EO), a critical step in bone formation. However, this process entails the condensation of mesenchymal stem cells (MSCs) into cartilaginous templates, which requires long-term cultures and is challenging to scale up. Here, we develop a biomimetic scaffold that allows rapid and self-sustained EO without initial hypertrophic chondrogenesis. The design comprises a porous chondroitin sulfate (CS) cryogel decorated with whitlockite calcium phosphate nanoparticles, and a soft hydrogel occupying the porous space. This composite scaffold enables human endothelial colony-forming cells (ECFCs) and MSCs to rapidly assemble into osteovascular niches in immunodeficient mice. These niches contain ECFC-lined blood vessels and perivascular MSCs that differentiate into RUNX2(+)OSX(+) pre-osteoblasts after one week in vivo. Subsequently, multiple ossification centers are formed, leading to de novo bone tissue formation by eight weeks, including mature human OCN(+)OPN(+) osteoblasts, collagen-rich mineralized extracellular matrix, hydroxyapatite, osteoclast activity, and gradual mechanical competence. The early establishment of blood vessels are essential, and grafts that do not contain ECFCs fail to produce osteovascular niches and ossification centers. Our findings suggest a novel bioengineering approach to recapitulate EO in the context of human bone regeneration.

Timely tissue vascularization and integration of engineered tissues into a patient plays an important role in the successful translation of engineered tissues into clinically relevant therapies. To decrease the time needed to vascularize an engineered adipose tissue, suitable local microenvironments provided by hydrogels to support cell-based functional vascular network formation have been investigated. Using the same biomolecule in solution two types of hydrogels can be obtained: a “physical hydrogel” which is thermal-induced self-assemble fibril initiation and growth, due to amino and carboxyl telopeptides on collagen chains, and a “chemical hydrogel” which results from the covalently cross-linking of the side chains induced by one step enzyme mediation in aqueous solution. In this paper, we compare the capability of engineering vascular network and large-sized vascularized adipose tissue in vivo in different types of collagen hydrogels, physical and chemical crosslinking. The relationships between vascular network formation and hydrogel properties for the two types of hydrogels are discussed. Finally, we successfully engineered a vascularized adipose tissue construct (~877.6 adipocytes/mm(2); 94% area of a construct) in the absence of exogenous cytokines in chemical covalently crosslinking cell-laden hydrogel. These results show manipulating the polymerized methods of a hydrogel could not only modulate vascular network formation, but also regenerate adipose tissue in vivo.

Notwithstanding remarkable progress in vascular network engineering, implanted bioengineered microvessels largely fail to form anastomoses with the host vasculature. Here, we demonstrate that implants containing assembled human vascular networks (A-Grafts) fail to engraft due to their inability to engage non-inflammatory host neutrophils upon implantation into mice. In contrast, unassembled vascular cells (U-Grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-Grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-Grafts reengaged neutrophils and enhanced revascularization in A-Grafts, a process that was recapitulated by blocking Notch signaling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues.

Here we investigated whether endothelial colony forming cells (ECFC) and mesenchymal progenitor cells (MPC) form vascular networks and restore blood flow in ischemic skeletal muscle, and whether host myeloid cells play a role. ECFC + MPC, ECFC alone, MPC alone, or vehicle alone were injected into the hind limb ischemic muscle one day after ligation of femoral artery and vein. At day 5, hind limbs injected with ECFC + MPC showed greater blood flow recovery compared with ECFC, MPC, or vehicle. Tail vein injection of human endothelial specific Ulex europaeus agglutinin-I demonstrated an increased number of perfused human vessels in ECFC + MPC compared with ECFC. In vivo bioluminescence imaging showed ECFC persisted for 14 days in ECFC + MPC-injected hind limbs. Flow cytometric analysis of ischemic muscles at day 2 revealed increased myeloid lineage cells in ECFC + MPC-injected muscles compared to vehicle-injected muscles. Neutrophils declined by day 7, while the number of myeloid cells, macrophages, and monocytes did not. Systemic myeloid cell depletion with anti-Gr-1 antibody blocked the improved blood flow observed with ECFC + MPC and reduced ECFC and MPC retention. Our data suggest that ECFC + MPC delivery could be used to reestablish blood flow in ischemic tissues, and this may be enhanced by coordinated recruitment of host myeloid cells.

Many common diseases involve the injury, loss, or death of organ tissues. For these patients, organ transplantation is often the only viable solution. Nonetheless, organ transplantation is seriously limited by the relative scarcity of living and non-living donors, a situation that is worsening with aging of the world population. Tissue Engineering (TE) is a research discipline in regenerative medicine that aims to generate tissues in the laboratory that can replace diseased and damaged tissues in patients. Crucially, engineered tissues must have a vascular network that guarantees adequate nutrient supply, gas exchange, and elimination of waste products. Therefore, the search for clinically relevant sources of vasculogenic cells and the subsequent development of methods to achieve rapid vascularization is of utmost importance. We and others have previously shown that human blood-derived endothelial colony-forming cells (ECFCs) have the required vasculogenic capacity to form functional vascular networks in vivo. These studies demonstrated that, in the presence of an appropriate source of perivascular cells, ECFCs can self-assemble into microvascular networks and connect to the host vasculature, a process that takes approximately 7 days in vivo. The prospect is to incorporate these vascular networks into future engineered tissues. However, engineered tissues must have a functional vasculature immediately after implantation in order to preserve viability and function. Thus, it is critical to further develop strategies for rapid formation of perfused vascular network in vivo. Here, we describe a methodology to deliver ECFCs and bone marrow-derived mesenchymal stem cells (MSCs) subcutaneously into immunodeficient mice in the presence of fibroblast growth factor-2 (FGF-2). This approach significantly reduces the time needed to achieve functional anastomoses between bioengineered human blood vessels and the host vasculature. This methodology includes (1) isolation, characterization and culture of ECFCs, (2) isolation, characterization and culture of MSCs, and (3) implantation of ECFCs and MSCs, in the presence of FGF-2, into immunodeficient mice to generate perfused vascular networks.

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality world-wide. Whether endogenous repair and regenerative ability could be augmented by drug administration is an important issue for generation of novel therapeutic approach. Recently it was reported that in mice pretreated with thymosin beta 4 (TB4) and subsequently subjected to experimental MI, a subset of epicardial cells differentiated into cardiomyocytes. In clinical settings, epicardial priming with TB4 prior to MI is impractical. Here we tested if TB4 treatment after MI could reprogram epicardium into cardiomyocytes and augment the epicardium's injury response. Using epicardium genetic lineage trace line Wt1CreERT2/+ and double reporter line Rosa26mTmG/+, we found post-MI TB4 treatment significantly increased the thickness of epicardium and coronary capillary density. However, epicardium-derived cells did not adopt cardiomyocyte fate, nor did they migrate into myocardium to become coronary endothelial cells. Our result thus indicates that TB4 treatment after MI does not alter epicardial cell fate to include the cardiomyocyte lineage, providing both cautions and insights for the full exploration of the potential benefits of TB4 in the clinical settings. This article is part of a Special Issue entitled ‘Possible Editorial’.

The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.

The field of tissue engineering seeks to create metabolically demanding, functional tissues, which will require blood vessel networks capable of forming rapidly in a variety of extracellular matrix (ECM) environments. We tested whether human endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) could form microvascular networks in type I collagen, fibrin and an engineered peptide hydrogel, PuraMatrix, in 7 days in vivo in immune-deficient mice. These results are compared to those previously published, based on the Matrigel ECM. Perfused blood vessels formed in all three types of ECM within 7 days. Collagen at 5 and 6 mg/ml and 10 mg/ml fibrin supported vessel formation at 30-60 vessels/mm(2), and PuraMatrix enabled vessel formation to 160 vessels/mm(2), significantly greater than collagen or fibrin. Vessels were composed of EPCs with perivascular cells on their abluminal surfaces. EPCs injected alone formed a low density of blood vessels in collagen and PuraMatrix, while MPCs injected alone resulted in sparse vessel networks in all ECMs tested. A rheometer was used to determine whether the ECMs which supported vascularization had bulk physical properties similar to or distinct from Matrigel. Collagen and fibrin were the stiffest matrices to support extensive vascularization, with storage moduli in the range 385-510 Pa, while Matrigel, at 80 Pa, and PuraMatrix, at 5 Pa, were far more compliant. Thus, EPCs and MPCs were capable of vasculogenesis in environments having disparate physical properties, although vascular density was greater in more compliant ECMs. We propose that EPC/MPC-mediated vascularization is a versatile technology which may enable the development of engineered organs.

The hypothesis that bone marrow–derived, circulating endothelial cells incorporate into tumor blood vessels is unresolved. We have measured the numbers of bone marrow–derived versus resident endothelial cells in spontaneous prostate cancers during different stages of tumor progression and in age-matched normal prostates. Bone marrow–derived endothelial cells were rare in dysplasia and in well differentiated cancers representing between 0 and 0.04% of the total tumor mass. Instead, approximately 99% of all tumor-associated bone marrow–derived cells were CD45+ hematopoietic cells, including GR-1+, F4-80+, and CD11b+ myeloid cells. Similar to peripheral blood mononuclear cells, these tumor-associated myeloid cells expressed matrix metalloproteinases (MMPs), consistent with their proposed catalytic role during tumor angiogenesis. Furthermore, freshly isolated CD11b+ cells stimulated tumor endothelial cell cord formation by 10-fold in an in vitro angiogenesis assay. The bone marrow is, therefore, a reservoir for cells that augment tumor angiogenesis, but the tumor endothelium is derived primarily from the local environment.

Objectives: Creating functional small-diameter tissue-engineered blood vessels has not been successful to date. Moreover, the processes underlying the in vivo remodeling of these grafts and the fate of cells seeded onto scaffolds remain unclear. Here we addressed these unmet scientific needs by using intravital molecular imaging to monitor the development of tissue-engineered vascular grafts (TEVG) implanted in mouse carotid artery. Methods and Results: Green fluorescent protein–labeled human bone marrow–derived mesenchymal stem cells and cord blood–derived endothelial progenitor cells were seeded on polyglycolic acid–poly-L-lactic acid scaffolds to construct small-caliber TEVG that were subsequently implanted in the carotid artery position of nude mice (n = 9). Mice were injected with near-infrared agents and imaged using intravital fluorescence microscope at 0, 7, and 35 days to validate in vivo the TEVG remodeling capability (Prosense680; VisEn, Woburn, MA) and patency (Angiosense750; VisEn). Imaging coregistered strong proteolytic activity and blood flow through anastomoses at both 7 and 35 days postimplantation. In addition, image analyses showed green fluorescent protein signal produced from mesenchymal stem cell up to 35 days postimplantation. Comprehensive correlative histopathological analyses corroborated intravital imaging findings. Conclusions: Multispectral imaging offers simultaneous characterization of in vivo remodeling enzyme activity, functionality, and cell fate of viable small-caliber TEVG.

The recruitment of myeloid cells has been consistently associated with the formation of new blood vessels during pathological angiogenesis. However, the participation of myeloid cells in bioengineered vascular networks remains unclear. Therefore, we tested whether host myeloid cells play a role in the formation of bioengineered vascular networks that occurs in vivo upon coimplantation of blood-derived endothelial progenitor cells and bone-marrow-derived mesenchymal progenitor cells, suspended as single cells in Matrigel, into immune-deficient mice. We observed an influx of spatially organized host CD11b(+) myeloid cells into the Matrigel implant 1 to 3 days after implantation, which was shown to be cell mediated rather than a nonspecific response. Myeloid cells were significantly reduced once the implants were fully vascularized at days 6 and 7, suggesting an active role during steps that precede formation of functional anastomoses and perfused vessels. Importantly, depletion of circulating myeloid cells resulted in a significant reduction in microvessel density in the implants. In summary, the recruitment of myeloid cells occurs rapidly after coimplantation of endothelial and mesenchymal progenitor cells and is necessary for full vascularization in this model. This is the first demonstration of a role for recruited myeloid cells in the formation of bioengineered vascular networks.

Vascularization of tissues is a major challenge of tissue engineering (TE). We hypothesize that blood-derived endothelial progenitor cells (EPCs) have the required proliferative and vasculogenic activity to create vascular networks in vivo. To test this, EPCs isolated from human umbilical cord blood or from adult peripheral blood, and human saphenous vein smooth muscle cells (HSVSMCs) as a source of perivascular cells, were combined in Matrigel and implanted subcutaneously into immunodeficient mice. Evaluation of implants at one week revealed an extensive network of human-specific lumenal structures containing erythrocytes, indicating formation of functional anastomoses with the host vasculature. Quantitative analyses showed the microvessel density was significantly superior to that generated by human dermal microvascular endothelial cells (HDMECs) but similar to that generated by human umbilical vein endothelial cells (HUVECs). We also found that as EPCs were expanded in culture, their morphology, growth kinetics, and proliferative responses toward angiogenic factors progressively resembled those of HDMECs, indicating a process of in vitro maturation. This maturation correlated with a decrease in the degree of vascularization in vivo, which could be compensated for by increasing the number of EPCs seeded into the implants. Our findings strongly support the use of human EPCs to form vascular networks in engineered organs and tissues.

In situ analysis of fetal semilunar valve leaflets has revealed cells coexpressing endothelial and mesenchymal markers along the endothelium, with diminished frequency seen in adult valves. To determine whether such cells are progenitor cells, we isolated clonal populations from human pulmonary valves. The clones expressed endothelial markers but showed potential to further differentiate into endothelium in response to vascular endothelial growth factor (VEGF)-A. When exposed to transforming growth factor (TGF)-β 2 , individual clones adopted a mesenchymal phenotype to varying degrees and expressed markers of endothelial to mesenchymal transformation (EMT). Both VEGF- and TGFβ 2 –induced phenotypic changes were partially reversible, indicating the plasticity of these cells. When challenged with VEGF or TGFβ 2 , a hierarchy of endothelial/mesenchymal potential could be seen among the clonal populations: cells initially closer to an endothelial phenotype showed a strong response to TGFβ 2 that could be inhibited by VEGF, whereas cells closer to a mesenchymal phenotype responded to TGFβ 2 but were resistant to endothelial-inducing effects of VEGF. These findings suggest the presence of bipotential valve progenitor cells with ability to differentiate into either endothelial or interstitial cells of the valve leaflet. Understanding the differentiation potential and function of these cells may be important for understanding heart valve disease and may also be applied to current paradigms for creating tissue-engineered heart valves.

Infantile hemangiomas are composed of endothelial cells (ECs), endothelial progenitor cells (EPCs), as well as perivascular and hematopoietic cells. Our hypothesis is that hemangioma-derived EPCs (HemEPCs) differentiate into the mature ECs that comprise the major compartment of the tumor. To test this, we isolated EPCs (CD133+/Ulex europeus– I+) and mature ECs (CD133–/Ulex europeus–I+) from proliferating hemangiomas and used a previously described property of hemangioma-derived ECs (HemECs), enhanced migratory activity in response to the angiogenesis inhibitor endostatin, to determine if HemEPCs share this abnormal behavior. Umbilical cord blood–derived EPCs (cbEPCs) were analyzed in parallel as a normal control. Our results show that HemEPCs, HemECs, and cbEPCs exhibit increased adhesion, migration, and proliferation in response to endostatin. This angiogenic response to endostatin was consistently expressed by HemEPCs over several weeks in culture, whereas HemECs and cbEPCs shifted toward the mature endothelial response to endostatin. Similar mRNA-expression patterns among HemEPCs, HemECs, and cbEPCs, revealed by microarray analyses, provided further indication of an EPC phenotype. This is the first demonstration that human EPCs, isolated from blood or from a proliferating hemangioma, are stimulated by an angiogenesis inhibitor. These findings suggest that EPCs respond differently from mature ECs when exposed to angiogenic or antiangiogenic signals.

The objective of this work was to investigate the effect of devitalized human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) seeded on mineralized nanofiber microsheets on protein release, osteogenesis, vasculogenesis, and macrophage polarization. Calcium phosphate nanocrystals were grown on the surface of aligned, functionalized nanofiber microsheets. The microsheets were seeded with hMSCs, ECFCs, or a mixture of hMSCs+ECFCs, cultured for cell attachment, differentiated to the osteogenic or vasculogenic lineage, and devitalized by lyophilization. The release kinetic of total protein, bone morphogenetic protein-2 (BMP2), and vascular endothelial growth factor (VEGF) from the devitalized microsheets was measured. Next, hMSCs and/or ECFCs were seeded on the devitalized cell microsheets and cultured in the absence of osteo-/vasculo-inductive factors to determine the effect of devitalized cell microsheets on hMSC/ECFC differentiation. Human macrophages were seeded on the microsheets to determine the effect of devitalized cells on macrophage polarization. Based on the results, devitalized undifferentiated hMSC and vasculogenic-differentiated ECFC microsheets had highest sustained release of BMP2 and VEGF, respectively. The devitalized hMSC microsheets did not affect M2 macrophage polarization while vascular-differentiated, devitalized ECFC microsheets did not affect M1 polarization. Both groups stimulated higher M2 macrophage polarization compared to M1.

Introduction: Endothelial colony-forming cells (ECFCs) are endothelial progenitors that circulate in peripheral blood and are currently the subject of intensive investigation due to their therapeutic potential. However, in adults, ECFCs comprise a very small subset among circulating cells, which makes their isolation a challenge. Materials and Methods: Currently, the standard method for ECFC isolation relies on the separation of mononuclear cells and erythrocyte lysis, steps that are time consuming and known to increase cell loss. Alternatively, we previously developed a novel disposable microfluidic platform containing antibody-functionalized degradable hydrogel coatings that is ideally suited for capturing low-abundance circulating cells from unprocessed blood. In this study, we reasoned that this microfluidic approach could effectively isolate rare ECFCs by virtue of their CD34 expression. Results: We conducted preclinical experiments with peripheral blood from four adult volunteers and demonstrated that the actual microfluidic capture of circulating CD34+ cells from unprocessed blood was compatible with the subsequent differentiation of these cells into ECFCs. Moreover, the ECFC yield obtained with the microfluidic system was comparable to that of the standard method. Importantly, we unequivocally validated the phenotypical and functional properties of the captured ECFCs, including the ability to form microvascular networks following transplantation into immunodeficient mice. Discussion: We showed that the simplicity and versatility of our microfluidic system could be very instrumental for ECFC isolation while preserving their therapeutic potential. We anticipate our results will facilitate additional development of clinically suitable microfluidic devices by the vascular therapeutic and diagnostic industry.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1’s larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (i) pre-culturing human ECFC on laminin, fibronectin, and collagen coated plates; and (ii) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.

Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony–forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation, and migration toward vascular endothelial growth factor-A and fibroblast growth factor-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies ( P =0.04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events.

Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that are particularly abundant in umbilical cord blood. We sought to determine whether ECFC abundance in cord blood is associated with maternal body-mass index (BMI) in nonpathologic pregnancies.We measured the level of ECFCs in the cord blood of neonates (n = 27) born from non-obese healthy mothers with nonpathologic pregnancies and examined whether ECFC abundance correlated with maternal BMI. We also examined the effect of maternal BMI on ECFC phenotype and function using angiogenic and vasculogenic assays.We observed variation in ECFC abundance among subjects and found a positive correlation between prepregnancy maternal BMI and ECFC content (r = 0.51, P = .007), which was independent of other obstetric factors. Despite this variation, ECFC phenotype and functionality were deemed normal and highly similar between subjects with maternal BMI25 kg/m(2) and BMI between 25-30 kg/m(2), including the ability to form vascular networks in vivo.This study underlines the need to consider maternal BMI as a potential confounding factor for cord blood levels of ECFCs in future comparative studies between healthy and pathologic pregnancies.

There are numerous diseases associated with a malfunction of the endothelium, including ischemic injuries that follow thrombotic events, visual loss due to a defective cornea endothelium, and endothelial cell (EC) dysfunction in patients with diabetes. Some of these diseases are susceptible of cell therapies that aim to replace the defective endothelium. Thus, there is a need for a robust, clinically suitable source of autologous ECs. In principle, ECs can be obtained from a variety of autologous tissues, including small diameter veins and the microvasculature of tissues such as skin and adipose. However, the clinical use of mature ECs is limited by site morbidity and low cell proliferation potential. These limitations have motivated the search for other sources of ECs with more proliferative and vasculogenic activities such as those derived from embryonic stem cell (ESCs) and induced pluripotent stem cells (iPSCs). However, ethical considerations along with a poor understanding of the mechanisms controlling the differentiation of embryonic stem cells are hurdles that need to be overcome before these cells can be used in a clinical setting. Alternatively, the existence of postnatal endothelial progenitor cells (EPCs) in circulation represents a promising opportunity to non-invasively obtain large quantities of autologous ECs. However, the process for obtaining EPC-derived ECs has not been straightforward in part due to a lack of consensus regarding EPC definition, origin, and function of these cells. As a result, the term EPCs has been used to define two distinct subpopulations of cells, one with hematopoietic (early EPCs) and the other with endothelial (late EPCs) characteristics. Despite the often-confusing terminology, the functional distinction between these two very different types of EPCs is increasingly better understood. In particular, the ability of late EPCs to generate large amount of endothelial colony-forming cells (ECFCs) is now well recognized. Blood-derived ECFCs represent a robust population of cells with endothelial phenotype and ability to function as bona fide ECs both in vitro and in vivo, and thus, they constitute a promising source of cells for future vascular cell therapies.

Epub ahead of print.-- The final publication is available at link.springer.com, Blood-derived endothelial colony-forming cells (ECFCs) have robust vasculogenic potential that can be exploited to bioengineer long-lasting human vascular networks in vivo. However, circulating ECFCs are exceedingly rare in adult peripheral blood. Because the mechanism by which ECFCs are mobilized into circulation is currently unknown, the reliability of peripheral blood as a clinical source of ECFCs remains a concern. Thus, there is a need to find alternative sources of autologous ECFCs. Here we aimed to determine whether ECFCs reside in the vasculature of human white adipose tissue (WAT) and to evaluate if WAT-derived ECFCs (watECFCs) have equal clinical potential to blood-derived ECFCs. We isolated the complete endothelial cell (EC) population from intact biopsies of normal human subcutaneous WAT by enzymatic digestion and selection of CD31+ cells. Subsequently, we extensively compared WAT-derived EC phenotype and functionality to bonafide ECFCs derived from both umbilical cord blood and adult peripheral blood. We demonstrated that human WAT is indeed a dependable source of ECFCs with indistinguishable properties to adult peripheral blood ECFCs, including hierarchical clonogenic ability, large expansion potential, stable endothelial phenotype, and robust in vivo blood vessel-forming capacity. Considering the unreliability and low rate of occurrence of ECFCs in adult blood and that biopsies of WAT can be obtained with minimal intervention in an ambulatory setting, our results indicate WAT as a more practical alternative to obtain large amounts of readily available autologous ECFCs for future vascular cell therapies., This work was supported by a National Institutes of Health Grant (R00EB009096, J. M.-M).

The final publication is available at link.springer.com, Mesenchymal stem cells (MSCs) can generate multiple end-stage mesenchymal cell types and constitute a promising population of cells for regenerative therapies. Additionally, there is increasing evidence supporting other trophic activities of MSCs, including the ability to enable formation of vasculature in vivo. Although MSCs were originally isolated from the bone marrow, the presence of these cells in the stromal vascular fraction of multiple adult tissues has been recently recognized. However, it is unknown whether the capacity to modulate vasculogenesis is ubiquitous to all MSCs regardless of their tissue of origin. Here, we demonstrated that tissue-resident MSCs isolated from four distinct tissues have equal capacity to modulate endothelial cell function, including formation of vascular networks in vivo. MSCs were isolated from four murine tissues, including bone marrow, white adipose tissue, skeletal muscle, and myocardium. In culture, all four MSC populations secreted a plethora of pro-angiogenic factors that unequivocally induced proliferation, migration, and tube formation of endothelial colony-forming cells (ECFCs). In vivo, co-implantation of MSCs with ECFCs into mice generated an extensive network of blood vessels with ECFCs specifically lining the lumens and MSCs occupying perivascular positions. Importantly, there were no differences among all four MSCs evaluated. Our studies suggest that the capacity to modulate the formation of vasculature is a ubiquitous property of all MSCs, irrespective of their original anatomical location. These results validate multiple tissues as potential sources of MSCs for future cell-based vascular therapies. © 2012 Springer Science+Business Media B.V., This work was supported by a grant from the National Institutes of Health (R00EB009096) to J.M.-M, and by grants from the Instituto de Salud Carlos III and Junta de Andalucía, Consejería de Salud to R.M.-L.

Tumor-associated stroma is typified by a persistent, non-resolving inflammatory response that enhances tumor angiogenesis, growth and metastasis. Inflammation in tumors is instigated by heterotypic interactions between malignant tumor cells, vascular endothelium, fibroblasts, immune and inflammatory cells. We found that tumor-associated adipocytes also contribute to inflammation. We have analyzed peritumoral adipose tissue in a syngeneic mouse melanoma model. Compared to control adipose tissue, adipose tissue juxtaposed to implanted tumors exhibited reduced adipocyte size, extensive fibrosis, increased angiogenesis and a dense macrophage infiltrate. A mouse cytokine protein array revealed up-regulation of inflammatory mediators including IL-6, CXCL1, MCP-1, MIP-2 and TIMP-1 in peritumoral versus counterpart adipose tissues. CD11b(+) macrophages contributed strongly to the inflammatory activity. These macrophages were isolated from peritumoral adipose tissue and found to over-express ARG1, NOS2, CD301, CD163, MCP-1 and VEGF, which are indicative of both M1 and M2 polarization. Tumors implanted at a site distant from subcutaneous, anterior adipose tissue were strongly growth-delayed, had fewer blood vessels and were less populated by CD11b(+) macrophages. In contrast to normal adipose tissue, micro-dissected peritumoral adipose tissue explants launched numerous vascular sprouts when cultured in an ex vivo model. Thus, inflamed tumor-associated adipose tissue fuels the growth of malignant cells by acting as a proximate source for vascular endothelium and activated pro-inflammatory cells, in particular macrophages.

As in normal tissues, solid tumors require vascular structures to form conduits for blood, oxygen, and nutrients. Tumors induce sprouting of new blood vessels (angiogenesis) or co-opt preexisting ones. Tumor blood vessels are dysfunctional and characterized by excessive branching and sprouting, leakiness, and inflammation. These abnormalities can contribute to tumor progression. For example, in a process resembling healing wounds, tumor cells and tumor blood vessels secrete factors that mobilize pro-inflammatory, hematopoietic progenitor cells from the bone marrow. These hematopoietic cells are, generally, proangiogenic, and their activity may be partially responsible for the dysfunctional features of tumor blood vessels. Thus, identifying key pathways controlling communication between tumors and bone marrow-derived pro-inflammatory cells will improve our understanding of vascular dysfunction in tumors and contribute to the identification of new therapeutic targets for anti-angiogenesis strategies.

For decades, autologous ex vivo gene therapy has been postulated as a potential alternative to parenteral administration of recombinant proteins. However, achieving effective cellular engraftment of previously retrieved patient cells is challenging. Recently, our ability to engineer vasculature in vivo has allowed for the introduction of instructions into tissues by genetically modifying the vascular cells that build these blood vessels. In the present study, we genetically engineered human blood–derived endothelial colony-forming cells (ECFCs) to express erythropoietin (EPO) under the control of a tetracycline-regulated system, and generated subcutaneous vascular networks capable of systemic EPO release in immunodeficient mice. These ECFC-lined vascular networks formed functional anastomoses with the mouse vasculature, allowing direct delivery of recombinant human EPO into the bloodstream. After activation of EPO expression, erythropoiesis was induced in both normal and anemic mice, a process that was completely reversible. This approach could relieve patients from frequent EPO injections, reducing the medical costs associated with the management of anemia. We propose this ECFC-based gene-delivery strategy as a viable alternative technology when routine administration of recombinant proteins is needed.

Umbilical cord blood (UCB) is recognized as an enriched source of endothelial progenitor cells (EPCs) with potential therapeutic value. Because cryopreservation is the only reliable method for long-term storage of UCB cells, the clinical application of EPCs depends on our ability to acquire them from cryopreserved samples; however, the feasibility of doing so remains unclear. In this study we demonstrate that EPCs can be isolated from cryopreserved UCB-derived mononuclear cells (MNCs). The number of outgrowth EPC colonies that emerged in culture from cryopreserved samples was similar to that obtained from fresh UCB. Furthermore, EPCs obtained from cryopreserved MNCs were phenotypically and functionally indistinguishable from freshly isolated ones, including the ability to form blood vessels in vivo. Our results eliminate the necessity of performing cell isolation procedures ahead of future clinical needs and suggest that EPCs derived from cryopreserved UCB may be suitable for EPC-related therapies.

The success of therapeutic vascularization and tissue engineering will rely on our ability to create vascular networks using human cells that can be obtained readily, can be expanded safely ex vivo, and can produce robust vasculogenic activity in vivo. Here we describe the formation of functional microvascular beds in immunodeficient mice by coimplantation of human endothelial and mesenchymal progenitor cells isolated from blood and bone marrow. Evaluation of implants after 1 week revealed an extensive network of human blood vessels containing erythrocytes, indicating the rapid formation of functional anastomoses within the host vasculature. The implanted endothelial progenitor cells were restricted to the luminal aspect of the vessels; mesenchymal progenitor cells were adjacent to lumens, confirming their role as perivascular cells. Importantly, the engineered vascular networks remained patent at 4 weeks in vivo. This rapid formation of long-lasting microvascular networks by postnatal progenitor cells obtained from noninvasive sources constitutes an important step forward in the development of clinical strategies for tissue vascularization.

Rapid and complete vascularization of ischemic tissues and thick engineered tissues is likely to require vasculogenesis. Therefore, the search for clinically relevant sources of vasculogenic cells and the subsequent development of experimental models of vasculogenesis is of utmost importance. Here, we describe a methodology adapted from the Matrigel plug assay to deliver human blood-derived endothelial progenitor cells (EPCs) and mature smooth muscle cells (SMCs) subcutaneously into immunodeficient mice. One week after implantation, an extensive microvascular network composed of the human EPCs and SMCs is formed within the Matrigel. The presence of human EPC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This is a very versatile assay that allows (1) dialing the final microvessel density by varying either the total number of cells in the original cell suspension or the ratio between EPCs and SMCs, (2) studying the effect of substituting another type of perivascular cell for mature SMCs or another type of endothelial cell, (3) tracking each of the implanted cell types by labeling (e.g., GFP tagging) prior to implantation, and (4) studying the effect of genetically modifying the cells prior to implantation. Additionally, this assay is relatively simple to perform and it does not require an incision or surgical procedure. This murine model of human vasculogenesis is ideally suited for studies on the cellular and molecular components of microvessel development, pathologic neovascular responses, and for the development and investigation of strategies to enhance neovascularization of engineered human tissues and organs.

Infantile hemangioma is a benign endothelial tumor composed of disorganized blood vessels. It exhibits a unique life cycle of rapid postnatal growth followed by slow regression to a fibrofatty residuum. Here, we have reported the isolation of multipotential stem cells from hemangioma tissue that give rise to hemangioma-like lesions in immunodeficient mice. Cells were isolated based on expression of the stem cell marker CD133 and expanded from single cells as clonal populations. The CD133-selected cells generated human blood vessels 7 days after implantation in immunodeficient mice. Cell retrieval experiments showed the cells could again form vessels when transplanted into secondary recipients. The human vessels expressed GLUT-1 and merosin, immunodiagnostic markers for infantile hemangioma. Two months after implantation, the number of blood vessels diminished and human adipocytes became evident. Lentiviral expression of GFP was used to confirm that the hemangioma-derived cells formed the blood vessels and adipocytes in the immunodeficient mice. Thus, when transplanted into immunodeficient mice, hemangioma-derived cells recapitulated the unique evolution of infantile hemangioma — the formation of blood vessels followed by involution to fatty tissue. In summary, this study identifies a stem cell as the cellular origin of infantile hemangioma and describes for what we believe is the first time an animal model for this common tumor of infancy.

disruption, and how these correlate with endogenous TGF‐β signaling. Methods: Rat aortic SMCs were cultured with IL1β+ or TNF‐α alone (0 and100 ng/ml) and together (50 ng/ml each) over 4–120 h and over 22 days. Based on an initial WNT PCR array, we further screened for Wnts 1, 3, 4, 5A, 7A, MMPs 2, 7, 9 and 24; TIMPS 1–4, TGF‐β; and matrix genes elastin, collagen, LOX, and fibrillin. TCF activation was studied through Topflash/Fopflash ratio analysis. Western blots were performed for select proteins and TEM used to gauge de novo elastic matrix deposition and quality at 22 days. Results: Bootstrapping analysis showed that IL1β+TNF‐α suppressed canonical Wnts (1,3A,4) and greatly enhanced non-canonical Wnts (5A,7A), gradually suppressing TGF-β. These temporally coincided with exponential increases in MMPs 2 and 9 mRNA and protein and elastin gene down-regulation, reflected in a sporadic, nonfibrillar elastic matrix. Conclusions: Our results suggest that elastin and cell homeostasis initially maintained by the canonical Wnts and TGF-β is perturbed by cytokine-activation of Wnts 5A and 7A resulting in enhanced disruption and suppressed regeneration of elastin matrix. The results suggest a potential mechanistic target for therapy to concurrently reverse both these effects to stabilize AAAs.

Objectives Clinical translation of tissue-engineered heart valves requires valve competency and lack of stenosis in the short and long term. Early studies of engineered valves showed promise, although lacked complete definition of valve function. Building on prior experiments, we sought to define the in vivo changes in structure and function of autologous engineered pulmonary valved conduits. Methods Mesenchymal stem cells were isolated from neonatal sheep bone marrow and seeded onto a bioresorbable scaffold. After 4 weeks of culture, valved conduits were implanted. Valve function, cusp, and conduit dimensions were evaluated at implantation (echocardiography), at the experimental midpoint (magnetic resonance imaging), and at explant, at 1 day, and 1, 6, 12, or 20 weeks postoperatively (direct measurement, echocardiography). Histologic evaluation was performed. Results Nineteen animals underwent autologous tissue-engineered valved conduit replacement. At implantation, valved conduit function was excellent; maximum transvalvular pressure gradient by Doppler echocardiography was 17 mm Hg; most valved conduits showed trivial pulmonary regurgitation. At 6 postoperative weeks, valve cusps appeared less mobile; pulmonary regurgitation was mild to moderate. At 12 weeks or more, valved conduit cusps were increasingly attenuated and regurgitant. Valved conduit diameter remained unchanged over 20 weeks. Dimensional measurements by magnetic resonance imaging correlated with direct measurement at explant. Conclusions We demonstrate autologous engineered tissue valved conduits that function well at implantation, with subsequent monitoring of dimensions and function in real time by magnetic resonance imaging. In vivo valves undergo structural and functional remodeling without stenosis, but with worsening pulmonary regurgitation after 6 weeks. Insights into mechanisms of in vivo remodeling are valuable for future iterations of engineered heart valves.