Publications by authors named "Yevgeny Brudno"

21 Publications

  • Page 1 of 1

Regenerating Antithrombotic Surfaces through Nucleic Acid Displacement.

ACS Biomater Sci Eng 2020 Apr 3;6(4):2159-2166. Epub 2020 Mar 3.

John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, Massachusetts 02138, United States.

Blood-contacting devices are commonly coated with antithrombotic agents to prevent clot formation and to extend the lifespan of the device. However, in vivo degradation of these bioactive surface agents ultimately limits device efficacy and longevity. Here, a regenerative antithrombotic catheter surface treatment is developed using oligodeoxynucleotide (ODN) toehold exchange. ODN strands modified to carry antithrombotic payloads can inhibit the thrombin enzyme when bound to a surface and exchange with rapid kinetics over multiple cycles, even while carrying large payloads. The surface-bound ODNs inhibit thrombin activity to significantly reduce fibrinogen cleavage and fibrin formation, and this effect is sustained after ODN exchange of the surface-bound strands with a fresh antithrombotic payload. This study presents a unique strategy for achieving a continuous antithrombotic state for blood-contacting devices using an ODN-based regeneration method.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00038DOI Listing
April 2020

Targeting Using Arylboronate/Nopoldiol Click Conjugation.

Bioconjug Chem 2020 10 30;31(10):2288-2292. Epub 2020 Sep 30.

Joint Department of Biomedical Engineering, University of North Carolina - Chapel Hill and North Carolina State University Raleigh, 1840 Entrepreneur Drive, Raleigh, North Carolina 27695, United States.

Bioorthogonal click reactions yielding stable and irreversible adducts are in high demand for applications, including in biomolecular labeling, diagnostic imaging, and drug delivery. Previously, we reported a novel bioorthogonal "click" reaction based on the coupling of ortho-acetyl arylboronates and thiosemicarbazide-functionalized nopoldiol. We now report that a detailed structural analysis of the arylboronate/nopoldiol adduct by X-ray crystallography and B NMR reveals that the bioorthogonal reactants form, unexpectedly, a tetracyclic adduct through the cyclization of the distal nitrogen into the semithiocarbazone leading to a strong B-N dative bond and two new 5-membered rings. The cyclization adduct, which protects the boronate unit against hydrolytic breakdown, sheds light on the irreversible nature of this polycondensation. The potential of this reaction to work in a live animal setting was studied through capture of fluorescently labeled molecules . Arylboronates were introduced into tissues through intradermal injection of their activated NHS esters, which react with amines in the extracellular matrix. Fluorescently labeled nopoldiol molecules were administered systemically and were efficiently captured by the arylboronic acids in a location-specific manner. Taken together, these proof-of-concept studies establish arylboronate/nopoldiol bioorthogonal chemistry as a candidate for wide array of applications in chemical biology and drug delivery.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00453DOI Listing
October 2020

Scaffold-Mediated Static Transduction of T Cells for CAR-T Cell Therapy.

Adv Healthc Mater 2020 07 11;9(14):e2000275. Epub 2020 Jun 11.

Joint Department of Biomedical Engineering, University of North Carolina - Chapel Hill and North Carolina State University - Raleigh, 1840 Enterpreneur Way, Raleigh, NC, 27695, USA.

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive clinical responses in patients with B-cell malignancies. Critical to the success of CAR-T cell therapies is the achievement of robust gene transfer into T cells mediated by viral vectors such as gamma-retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost-effective CAR-T cell therapies. Herein, a low-cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition is described. CAR-T cells produced by macroporous scaffold-mediated viral transduction exhibit >60% CAR expression, retain effector phenotype, expand to clinically relevant cell numbers, and eradicate CD19 lymphoma in vivo. Efficient transduction is dependent on scaffold macroporosity. Taken together, the data show that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.
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http://dx.doi.org/10.1002/adhm.202000275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518635PMC
July 2020

Click cross-linking improves retention and targeting of refillable alginate depots.

Acta Biomater 2020 08 1;112:112-121. Epub 2020 Jun 1.

Joint Department of Biomedical Engineering, University of North Carolina - Chapel Hill and North Carolina State University - Raleigh, 1840 Entrepreneur Drive, Raleigh, NC 27695, USA. Electronic address:

Injectable alginate hydrogels have demonstrated utility in tissue engineering and drug delivery applications due in part to their mild gelation conditions, low host responses and chemical versatility. Recently, the potential of these gels has expanded with the introduction of refillable hydrogel depots - alginate gels chemically decorated with click chemistry groups to efficiently capture prodrug refills from the blood. Unfortunately, high degrees of click group substitution on alginate lead to poor viscoelastic properties and loss of ionic cross-linking. In this work, we introduce tetrabicyclononyne (tBCN) agents that covalently cross-link azide-modified alginate hydrogels for tissue engineering and drug delivery application in vivo. Adjusting cross-linker concentration allowed tuning the hydrogel mechanical properties for tissue-specific mechanical strength. The bioorthogonal and specific click reaction creates stable hydrogels with improved in vivo properties, including improved retention at injected sites. Azide-alginate hydrogels cross-linked with tBCN elicited minimal inflammation and maintained structural integrity over several months and efficiently captured therapeutics drug surrogates from the circulation. Taken together, azide-alginate hydrogels cross-linked with tBCN convey the benefits of alginate hydrogels for use in tissue engineering and drug delivery applications of refillable drug delivery depots. STATEMENT OF SIGNIFICANCE: Ionically cross-linked, injectable alginate biomaterials hold promise in many different clinical settings. However, adding new chemical functionality to alginate can disrupt their ionic cross-linking, limiting their utility. We have developed a "click" cross-linking strategy to improve the mechanical properties and tissue function of modified alginate biomaterials and enable them to capture small molecule drugs from the blood. We show that click cross-linked materials remain in place better than ionically cross-linked materials and efficiently capture payloads from the blood. Development of click cross-linking for refillable depots represents a crucial step toward clinical application of this promising drug delivery platform.
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http://dx.doi.org/10.1016/j.actbio.2020.05.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365769PMC
August 2020

Extracellular-Matrix-Anchored Click Motifs for Specific Tissue Targeting.

Mol Pharm 2020 02 27;17(2):392-403. Epub 2019 Dec 27.

Joint Department of Biomedical Engineering , University of North Carolina, Chapel Hill and North Carolina State University , Raleigh. 911 Oval Drive , Raleigh , North Carolina 27695 , United States.

Local presentation of cancer drugs by injectable drug-eluting depots reduces systemic side effects and improves efficacy. However, local depots deplete their drug stores and are difficult to introduce into stiff tissues, or organs, such as the brain, that cannot accommodate increased pressure. We present a method for introducing targetable depots through injection of activated ester molecules into target tissues that react with and anchor themselves to the local extracellular matrix (ECM) and subsequently capture systemically administered small molecules through bioorthogonal click chemistry. A computational model of tissue-anchoring depot formation and distribution was verified by histological analysis and confocal imaging of cleared tissues. ECM-anchored click groups do not elicit any noticeable local or systemic toxicity or immune response and specifically capture systemically circulating molecules at intradermal, intratumoral, and intracranial sites for multiple months. Taken together, ECM anchoring of click chemistry motifs is a promising approach to specific targeting of both small and large therapeutics, enabling repeated local presentation for cancer therapy and other diseases.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00589DOI Listing
February 2020

Clickable, acid labile immunosuppressive prodrugs for in vivo targeting.

Biomater Sci 2020 Jan 6;8(1):266-277. Epub 2019 Nov 6.

Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA. and Wyss Institute for Biologically Inspired Engineering, Cambridge, Massachusetts 02138, USA.

Allotransplantation offers the potential to restore the anatomy and function of injured tissues and organs, but typically requires life-long, systemic administration of immunosuppressive drugs to prevent rejection, which can result in serious complications. Targeting the immunosuppressive drug to the graft favors local tissue concentration versus systemic drug exposure and end-organ toxicity. This could reduce the overall dose and dosing frequency of immunosuppressive drugs, and improve the safety and efficacy of treatment. Here, we developed dibenzocyclooctyne (DBCO)-modified prodrugs of the immunosuppressive drugs tacrolimus, rapamycin and mycophenolic acid, and demonstrated their targeted conjugation both in vitro and in vivo to azido-modified hydrogels via Click chemistry. Such azido-modified hydrogels placed in transplanted tissues enable sustained local release of drugs, and could be repeatedly refilled with systemically administered acid-labile prodrugs after drug exhaustion. Thus, clickable prodrugs with degradable linkers provide new possibilities for graft targeted immunosuppression in the context of allotransplantation.
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http://dx.doi.org/10.1039/c9bm01487jDOI Listing
January 2020

Platelet-Inspired Nanocells for Targeted Heart Repair After Ischemia/Reperfusion Injury.

Adv Funct Mater 2019 Jan 13;29(4). Epub 2018 Nov 13.

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North, Carolina State University, Raleigh, NC 27695, USA.

Cardiovascular disease is the leading cause of mortality worldwide. While reperfusion therapy is vital for patient survival post-heart attack, it also causes further tissue injury, known as myocardial ischemia/reperfusion (I/R) injury in clinical practice. Exploring ways to attenuate I/R injury is of clinical interest for improving post-ischemic recovery. A platelet-inspired nanocell (PINC) that incorporates both prostaglandin E2 (PGE)-modified platelet membrane and cardiac stromal cell-secreted factors to target the heart after I/R injury is introduced. By taking advantage of the natural infarct-homing ability of platelet membrane and the overexpression of PGE receptors (EPs) in the pathological cardiac microenvironment after I/R injury, the PINCs can achieve targeted delivery of therapeutic payload to the injured heart. Furthermore, a synergistic treatment efficacy can be achieved by PINC, which combines the paracrine mechanism of cell therapy with the PGE/EP receptor signaling that is involved in the repair and regeneration of multiple tissues. In a mouse model of myocardial I/R injury, intravenous injection of PINCs results in augmented cardiac function and mitigated heart remodeling, which is accompanied by the increase in cycling cardiomyocytes, activation of endogenous stem/progenitor cells, and promotion of angiogenesis. This approach represents a promising therapeutic delivery platform for treating I/R injury.
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http://dx.doi.org/10.1002/adfm.201803567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111457PMC
January 2019

Replenishable drug depot to combat post-resection cancer recurrence.

Biomaterials 2018 09 6;178:373-382. Epub 2018 May 6.

Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Cir., Boston, MA 02115, USA; School of Engineering and Applied Sciences, Harvard University, 29 Oxford St., Cambridge, MA 02138, USA. Electronic address:

Local drug presentation made possible by drug-eluting depots has demonstrated benefits in a vast array of diseases, including in cancer, microbial infection and in wound healing. However, locally-eluting depots are single-use systems that cannot be refilled or reused after implantation at inaccessible sites, limiting their clinical utility. New strategies to noninvasively refill drug-eluting depots could dramatically enhance their clinical use. In this report we present a refillable hydrogel depot system based on bioorthogonal click chemistry. The click-modified hydrogel depots capture prodrug refills from the blood and subsequently release active drugs locally in a sustained manner. Capture of the systemically-administered refills serves as an efficient and non-toxic method to repeatedly refill depots. Refillable depots in combination with prodrug refills achieve sustained release at precancerous tumor sites to improve cancer therapy while eliminating systemic side effects. The ability to target tissues without enhanced permeability could allow the use of refillable depots in cancer and many other medical applications.
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http://dx.doi.org/10.1016/j.biomaterials.2018.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075722PMC
September 2018

On-demand drug delivery from local depots.

J Control Release 2015 Dec 14;219:8-17. Epub 2015 Sep 14.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138. Electronic address:

Stimuli-responsive polymeric depots capable of on-demand release of therapeutics promise a substantial improvement in the treatment of many local diseases. These systems have the advantage of controlling local dosing so that payload is released at a time and with a dose chosen by a physician or patient, and the dose can be varied as disease progresses or healing occurs. Macroscale drug depot can be induced to release therapeutics through the action of physical stimuli such as ultrasound, electric and magnetic fields and light as well as through the addition of pharmacological stimuli such as nucleic acids and small molecules. In this review, we highlight recent advances in the development of polymeric systems engineered for releasing therapeutic molecules through physical and pharmacological stimulation.
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http://dx.doi.org/10.1016/j.jconrel.2015.09.011DOI Listing
December 2015

In vivo targeting through click chemistry.

ChemMedChem 2015 Apr 20;10(4):617-20. Epub 2015 Feb 20.

Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Cir., Boston, MA 02115 (USA); School of Engineering and Applied Sciences, Harvard University, 29 Oxford St., Cambridge, MA 02138 (USA).

Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug-eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.
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http://dx.doi.org/10.1002/cmdc.201402527DOI Listing
April 2015

Three-dimensional human tissue models that incorporate diabetic foot ulcer-derived fibroblasts mimic in vivo features of chronic wounds.

Tissue Eng Part C Methods 2015 May 31;21(5):499-508. Epub 2015 Mar 31.

1 Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University , Boston, Massachusetts.

Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound-healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix deposition. In addition to reflecting clinical attributes of DFUs, the wound-healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically relevant platform for elucidating the cell-cell and cell-matrix-related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications.
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http://dx.doi.org/10.1089/ten.TEC.2014.0414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410281PMC
May 2015

Refilling drug delivery depots through the blood.

Proc Natl Acad Sci U S A 2014 Sep 19;111(35):12722-7. Epub 2014 Aug 19.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138;

Local drug delivery depots have significant clinical utility, but there is currently no noninvasive technique to refill these systems once their payload is exhausted. Inspired by the ability of nanotherapeutics to target specific tissues, we hypothesized that blood-borne drug payloads could be modified to home to and refill hydrogel drug delivery systems. To address this possibility, hydrogels were modified with oligodeoxynucleotides (ODNs) that provide a target for drug payloads in the form of free alginate strands carrying complementary ODNs. Coupling ODNs to alginate strands led to specific binding to complementary-ODN-carrying alginate gels in vitro and to injected gels in vivo. When coupled to a drug payload, sequence-targeted refilling of a delivery depot consisting of intratumor hydrogels completely abrogated tumor growth. These results suggest a new paradigm for nanotherapeutic drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.
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http://dx.doi.org/10.1073/pnas.1413027111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156738PMC
September 2014

Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart.

Biomaterials 2014 Aug 23;35(25):6850-6858. Epub 2014 May 23.

School of Engineering and Applied Sciences, Harvard University, 29 Oxford street, Cambridge, MA 02138, USA.

Cell delivery to the infarcted heart has emerged as a promising therapy, but is limited by very low acute retention and engraftment of cells. The objective of this study was to compare a panel of biomaterials to evaluate if acute retention can be improved with a biomaterial carrier. Cells were quantified post-implantation in a rat myocardial infarct model in five groups (n = 7-8); saline injection (current clinical standard), two injectable hydrogels (alginate, chitosan/β-glycerophosphate (chitosan/ß-GP)) and two epicardial patches (alginate, collagen). Human mesenchymal stem cells (hMSCs) were delivered to the infarct border zone with each biomaterial. At 24 h, retained cells were quantified by fluorescence. All biomaterials produced superior fluorescence to saline control, with approximately 8- and 14-fold increases with alginate and chitosan/β-GP injectables, and 47 and 59-fold increases achieved with collagen and alginate patches, respectively. Immunohistochemical analysis qualitatively confirmed these findings. All four biomaterials retained 50-60% of cells that were present immediately following transplantation, compared to 10% for the saline control. In conclusion, all four biomaterials were demonstrated to more efficiently deliver and retain cells when compared to a saline control. Biomaterial-based delivery approaches show promise for future development of efficient in vivo delivery techniques.
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http://dx.doi.org/10.1016/j.biomaterials.2014.04.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051834PMC
August 2014

Sustained delivery of VEGF maintains innervation and promotes reperfusion in ischemic skeletal muscles via NGF/GDNF signaling.

Mol Ther 2014 Jul 28;22(7):1243-1253. Epub 2014 Apr 28.

School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA; Wyss Institute for Biologically Inspired Engineering, Cambridge, Massachusetts, USA. Electronic address:

Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.
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http://dx.doi.org/10.1038/mt.2014.76DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089004PMC
July 2014

Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

PLoS One 2013 30;8(12):e83755. Epub 2013 Dec 30.

Program in Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875480PMC
September 2014

Enhancing microvascular formation and vessel maturation through temporal control over multiple pro-angiogenic and pro-maturation factors.

Biomaterials 2013 Dec 22;34(36):9201-9. Epub 2013 Aug 22.

School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA; Wyss Institute for Biological Inspired Engineering, Harvard University, Boston, MA 02115, USA.

Therapeutic stimulation of angiogenesis to re-establish blood flow in ischemic tissues offers great promise as a treatment for patients suffering from cardiovascular disease or trauma. Since angiogenesis is a complex, multi-step process, different signals may need to be delivered at appropriate times in order to promote a robust and mature vasculature. The effects of temporally regulated presentation of pro-angiogenic and pro-maturation factors were investigated in vitro and in vivo in this study. Pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) cooperatively promoted endothelial sprouting and pericyte detachment in a three-dimensional in vitro EC-pericyte co-culture model. Pro-maturation factors platelet-derived growth factor B (PDGF) and angiopoietin 1 (Ang1) inhibited the early stages of VEGF- and Ang2-mediated angiogenesis if present simultaneously with VEGF and Ang2, but promoted these behaviors if added subsequently to the pro-angiogenesis factors. VEGF and Ang2 were also found to additively enhance microvessel density in a subcutaneous model of blood vessel formation, while simultaneously administered PDGF/Ang1 inhibited microvessel formation. However, a temporally controlled scaffold that released PDGF and Ang1 at a delay relative to VEGF/Ang2 promoted both vessel maturation and vascular remodeling without inhibiting sprouting angiogenesis. Our results demonstrate the importance of temporal control over signaling in promoting vascular growth, vessel maturation and vascular remodeling. Delivering multiple growth factors in combination and sequence could aid in creating tissue engineered constructs and therapies aimed at promoting healing after acute wounds and in chronic conditions such as diabetic ulcers and peripheral artery disease.
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http://dx.doi.org/10.1016/j.biomaterials.2013.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811005PMC
December 2013

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.

Nature 2011 May 8;473(7347):394-7. Epub 2011 May 8.

Harvard Medical School, Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital Boston, Boston, Massachusetts 02115, USA.

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells.
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http://dx.doi.org/10.1038/nature10102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124347PMC
May 2011

An in vitro translation, selection and amplification system for peptide nucleic acids.

Nat Chem Biol 2010 Feb 27;6(2):148-55. Epub 2009 Dec 27.

Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template, (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures, (iii) selection of synthetic polymer libraries for desired binding or catalytic properties and (iv) amplification of template sequences that survive selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of 12 PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection and amplification on a library of 4.3 x 10(8) PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.
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http://dx.doi.org/10.1038/nchembio.280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808706PMC
February 2010

Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.

Science 2009 May 16;324(5929):930-5. Epub 2009 Apr 16.

Department of Pathology, Harvard Medical School and Immune Disease Institute, 200 Longwood Avenue, Boston, MA 02115, USA.

DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference-mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.
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http://dx.doi.org/10.1126/science.1170116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715015PMC
May 2009

Recent progress toward the templated synthesis and directed evolution of sequence-defined synthetic polymers.

Chem Biol 2009 Mar;16(3):265-76

Department of Chemistry and Chemical Biology and the Howard Hughes Medical Institute, 12 Oxford Street, Harvard University, Cambridge, MA 02138, USA.

Biological polymers such as nucleic acids and proteins are ubiquitous in living systems, but their ability to address problems beyond those found in nature is constrained by factors such as chemical or biological instability, limited building-block functionality, bioavailability, and immunogenicity. In principle, sequence-defined synthetic polymers based on nonbiological monomers and backbones might overcome these constraints; however, identifying the sequence of a synthetic polymer that possesses a specific desired functional property remains a major challenge. Molecular evolution can rapidly generate functional polymers but requires a means of translating amplifiable templates such as nucleic acids into the polymer being evolved. This review covers recent advances in the enzymatic and nonenzymatic templated polymerization of nonnatural polymers and their potential applications in the directed evolution of sequence-defined synthetic polymers.
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http://dx.doi.org/10.1016/j.chembiol.2009.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692969PMC
March 2009

DNA-templated polymerization of side-chain-functionalized peptide nucleic acid aldehydes.

J Am Chem Soc 2008 Apr 15;130(14):4646-59. Epub 2008 Mar 15.

Howard Hughes Medical Institute and the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step toward the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities.
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http://dx.doi.org/10.1021/ja0753997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2748799PMC
April 2008