Search

Tumor-infiltrating lymphocytes (TIL) that can recognize and kill tumor cells have curative potential in subsets of patients treated with adoptive cell transfer (ACT). However, lack of TIL therapeutic efficacy in many patients may be due in large part to a paucity of tumor-reactive T cells in TIL and the exhausted and terminally differentiated status of those tumor-reactive T cells. We sought to reprogram exhausted TIL that possess T-cell receptors (TCR) specific for tumor antigens into induced pluripotent stem cells (iPSC) to rejuvenate them for more potent ACT. We first attempted to reprogram tumor neoantigen-specific TIL by αCD3 Ab prestimulation which resulted in failure of establishing tumor-reactive TIL-iPSCs, instead, T cell–derived iPSCs from bystander T cells were established. To selectively activate and enrich tumor-reactive T cells from the heterogenous TIL population, CD8+ PD-1+ 4-1BB+ TIL population were isolated after coculture with autologous tumor cells, followed by direct reprogramming into iPSCs. TCR sequencing analysis of the resulting iPSC clones revealed that reprogrammed TIL-iPSCs encoded TCRs that were identical to the pre-identified tumor-reactive TCRs found in minimally cultured TIL. Moreover, reprogrammed TIL-iPSCs contained rare tumor antigen-specific TCRs, which were not detectable by TCR sequencing of the starting cell population. Thus, reprogramming of PD-1+ 4-1BB+ TIL after coculture with autologous tumor cells selectively generates tumor antigen-specific TIL-iPSCs, and is a distinctive method to enrich and identify tumor antigen-specific TCRs of low frequency from TIL. Significance: Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.

The list of antibodies used in the study

TIL-tumor cell coculture activated tumor-reactive T cells and generated tumor-reactive TIL-iPSCs from TIL of patient 3784. A, FACS panels showing the gating strategy to sort PD1+ 4-1BB+ CD8+ T cells after TIL-tumor cell coculture. Top panel shows the phenotype of TIL without coculture. Dump gate is a mixture of different T lineage markers (CD25, CD56, and TCRγδ) to exclude regulatory T cells (CD25+), natural killer cells (CD56+), and gamma-delta T cells (TCRγδ+). B, A summary table of the TCRβ sequence of eight preidentified tumor-reactive TCRs indicating their CDR3β amino acid sequence, Vβ family, frequency in bulk (before coculture) and in sorted PD1+ 4-1BB+ (DP) population, enrichment (DP/bulk) and presence in established iPSC clones. C, A bar graph showing the relative frequency of TCR clones, which were present in bulk population (starting cells) and reprogrammed to iPSCs, in four sorted populations [PD1− 4-1BB− (DN), PD1+ 4-1BB− (PD1+ SP), PD1− 4-1BB+ (4-1BB+ SP), PD1+ 4-1BB+ (DP)] after TIL-tumor cell coculture relative to bulk frequency of each TCR clone before coculture. Please see Supplementary Table S1 for the details of the TCR clones (TCR 2, 3, 6, 9, 11, 12, 15, and 17).

TCR stimulation is necessary for T cells to be reprogrammed to T-iPSCs. A, Schematic representation of PBMC derived T-cell reprogramming into iPSC. Whole PB T cells or the sorted populations were stimulated by αCD3/28 beads, and transduced with Sendai virus containing four Yamanaka factors and SV40 large T antigen. Approximately after 20–25 days’ culture in iPSC culture condition, the number of established iPSC colonies were counted. B, A bar graph indicates the numbers of AP-positive ES cell-like iPSC colonies derived from 1E+5 T cells with or without TCR stimulation. N = 3 for each donor. SeV containing four Yamanaka factors (OSKM) or OSKM and SV40 were used. C, Gating strategy to sort four subsets of CD8+ T cells (Naïve, CM, EM, and EMRA). PBMC were stained by antibodies against CD3, CD8, CD62L, CD45RO, CCR7, and CD45RA. CD8+ T cells were first gated on the basis of CD45RO and CD62L expression, followed by the second gate based on CD45RA and CCR7 expression. Four subsets of CD8+ T cells (Naïve, CM, EM, and EMRA) were sorted according to the gates indicated in the flow cytometry panels. D, A summary table showing which subset (Naïve, CM, EM, and EMRA) were reprogrammed to iPSCs by different stimulation methods. Sorted CD8+ T-cell subsets (Naïve, CM, EM, and EMRA) were stimulated by different methods (OKT-3 or αCD3/28 beads) before SeV infection. E, Representative AP, phase contrast, and bright field images were shown to describe the methods of iPSC colonies enumeration from different subsets (Naïve, CM, EM, and EMRA) of CD8 T cells reprogrammed into iPSC in each experiments following transduction of Sendai virus containing four Yamanaka factors and SV40 large T antigen. Each well was seeded with 1E+5 T cells. F, A bar graph indicates the numbers of AP-positive ES cell-like colonies derived from 1E+5 sorted CD8+ naïve/CM/EM/EMRA T cells activated by αCD3/28 beads. N = 3 for each donor.

Table S1 indicating TIL-iPSCs were established from T cells of low frequency along with major tumor neoantigen specific T cells from patient 3784

TIL-tumor cell coculture resulted in establishment of TIL-iPSCs from tumor-reactive T-cell clones. A, Schematic representation of TIL-tumor cell coculture to isolate tumor-reactive T cells and subsequent reprogramming into iPSCs. Autologous tumor cell line and minimally cultured TIL were cocultured for 16 hours, sorted for PD1+ 4-1BB+ CD8+ T cells, and transduced with Sendai virus containing four Yamanaka factors and SV40 large T antigen. On days 20–21 when cells formed domed shaped ES cell-like colonies, they were individually collected as clones and expanded. B, A summary table of nine different TCRs identified from 13 different master tubes containing 221 TIL-iPSC clones by using immunosequencing analysis. CDR3β amino acid sequence, Vβ family, frequency in bulk (before coculture in starting material) and in sorted PD1+ 4-1BB+ (DP) population, enrichment (DP/Bulk), the least number of iPSC clones established and the type of TCR were noted. PIR, preidentified reactive TCR; NF, newly found TCR (unknown reactivity). C, A summary table of the TCRβ sequence analysis of six preidentified tumor-reactive TCRs (PIR-TCR I–VI) indicating the CDR3β amino acid sequence, Vβ family, frequency in bulk (before coculture) and in the sorted PD1+ 4-1BB+ (DP) population after TIL-tumor cell coculture, enrichment (DP/Bulk) and presence in established iPSC clones. * means the TCR was not found in established T-iPSC clones but detected in the remaining clones in the dish after picking up iPS cell colonies (mother dish). D, A graph indicates the frequency of PIR-TCRs in starting cells (Bulk) and in sorted PD1+ 4-1BB+ (DP) cells. E, A bar graph indicates the enrichment analysis (the ratio of DP/Bulk) of those six PIR-TCRs.

Nonspecific TCR stimulation by αCD3 Ab is not suitable for selective reprogramming of tumor-reactive T cells. A, A summary table of top 10 frequent TCRβ sequences in the starting cells, that is, expanded TIL of patient 4069, indicating the CDR3β amino acid sequence, Vβ family and frequency. The most frequent TCR was the patient's neoantigen-specific TCR (indicated in red). B, A summary table of TCRβ sequences of established TIL-iPSCs by αCD3 Ab stimulation, indicating the CDR3β amino acid sequence, Vβ family and the number of TIL-iPSC clones established. C, A summary table of six candidate TCR pairs including five newly found TCRs (NF TCR 1–5) and one PIR-TCR which were sequenced individually from master tube 6 containing 14 individual iPSC clones. The table indicates TCR pairs, CDR3β sequence, Vβ family, CDR3α sequence, and Vα family. NF, newly found TCR (unknown reactivity); PIR, preidentified reactive TCR. D and E, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 2C). T cells were cocultured with autologous DC pulsed with wild-type (WT) peptide (16 mer or 9 mer) or mutant peptide (9 mer). D shows the representative FACS plots and E shows the percentage of 4-1BB+ cells in each condition. Representative data of three independent experiments from 3 different healthy donor T cells. Mock indicates empty vector transduced T cells used as negative control. F and G, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 2C). T cells were cocultured with autologous, or HLA-matched allogeneic tumor cells derived from the PDXs model. F shows the representative FACS plots and G shows the percentage of 4-1BB+ cells in each condition. Representative data of three independent experiments from 3 different healthy donor T cells. Mock indicates empty vector transduced T cells used as negative control. H, A graph showing the real-time cell growth monitoring (cell count/image) of RFP-overexpressed PDX-derived tumor cell line cultured with T cells transduced with PIR-TCR analyzed by the Incucyte live imaging and analysis system (effector-to-target ratio of 2:1). Empty vector transduced T cells (Mock) and tumor cell alone (TC) were used as negative controls. The results are shown as mean ± SD (N = 4).

Tumor-infiltrating lymphocytes (TIL) that can recognize and kill tumor cells have curative potential in subsets of patients treated with adoptive cell transfer (ACT). However, lack of TIL therapeutic efficacy in many patients may be due in large part to a paucity of tumor-reactive T cells in TIL and the exhausted and terminally differentiated status of those tumor-reactive T cells. We sought to reprogram exhausted TIL that possess T-cell receptors (TCR) specific for tumor antigens into induced pluripotent stem cells (iPSC) to rejuvenate them for more potent ACT. We first attempted to reprogram tumor neoantigen-specific TIL by αCD3 Ab prestimulation which resulted in failure of establishing tumor-reactive TIL-iPSCs, instead, T cell–derived iPSCs from bystander T cells were established. To selectively activate and enrich tumor-reactive T cells from the heterogenous TIL population, CD8+ PD-1+ 4-1BB+ TIL population were isolated after coculture with autologous tumor cells, followed by direct reprogramming into iPSCs. TCR sequencing analysis of the resulting iPSC clones revealed that reprogrammed TIL-iPSCs encoded TCRs that were identical to the pre-identified tumor-reactive TCRs found in minimally cultured TIL. Moreover, reprogrammed TIL-iPSCs contained rare tumor antigen-specific TCRs, which were not detectable by TCR sequencing of the starting cell population. Thus, reprogramming of PD-1+ 4-1BB+ TIL after coculture with autologous tumor cells selectively generates tumor antigen-specific TIL-iPSCs, and is a distinctive method to enrich and identify tumor antigen-specific TCRs of low frequency from TIL.Significance:Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.

TIL stimulated with autologous tumor cell line generated antigen specific iPSC whereas αCD3/28 beads mediated stimulation did not. A, Summary of three candidate TCR pairs identified in TIL-iPSC lines established by different stimulation methods. Bead Stim TCR: The most dominant clone in the TIL-iPSCs established by αCD3/28 beads stimulation, Tumor Stim TCR: The TCR identified in the TIL-iPSC clones established by coculture with autologous tumor cells (TCR 4 in Fig. 3B), and PIR-TCR III: preidentified reactive TCR clone detected in the TIL-iPSC clones established by coculture with autologous tumor cells. The table includes TCR type, CDR3β amino acid sequences, Vβ family, CDR3α amino acid sequences, Vα family are described. B and C, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 4A). T cells were cocultured with autologous or allogeneic tumor cell lines. B shows the representative FACS plots and C shows the percentage of 4-1BB+ cells in each condition. N = 3 for each condition. Representative data of four independent experiments. Mock: empty vector transduced PBL as a negative control, PIR-TCR III as a positive control. D, A bar graph showing the results of ELISA IFNγ production assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 4A). T cells were cocultured with autologous or allogeneic tumor cell lines for 16 hours and culture soup were analyzed for IFNγ by ELISA. N = 3 for each condition. Representative data of four independent experiments. E, A graph showing the real-time cell growth monitoring (cell count/image) of RFP-overexpressed tumor cells from pt. 1913 cocultured with different TCRs (listed in Fig. 4A), analyzed by the Incucyte live imaging and analysis system at effector-to-target ratio of 2:1. Mock indicates empty vector transduced T cells used as a negative control. The results are shown as the mean ± SD of N = 4 each condition. A representative data of four independent experiments.

Figure S1: A summary table of the patient samples used and the schema of the study

Summary: Induced pluripotent stem cell (iPSC)-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs). iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies. : A barrier for clinical application of iPSC-derived CD8 T cells using OP9/DLL1 is their abnormal biology. Vizcardo et al. show that a 3D thymic culture system enables the generation of a homogeneous antigen-specific T cell subset, named iTEs, which closely mimics naive T cells and exhibits potent anti-tumor activity. Keywords: thymopoiesis, T cell differentiation, iPSC differentiation, adoptive cell transfer, naïve T cell, recent rhymic emigrants, fetal thymus organ culture, immunotherapy, 3D culture, tumor antigen specific T cell

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from naïve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8αβ(+) antigen-specific T cells with a naïve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

Despite the long-standing observation that herpes simplex virus (HSV) latency-associated transcript (LAT) promoter deletion viruses show impaired recurrence phenotypes in relevant animal models, the mechanism by which these sequences exert this phenotypic effect is unknown. We constructed and evaluated four mutant HSV-2 isolates with targeted mutations in the LAT promoter and LAT-associated microRNAs (miRNAs) affecting (i) the LAT TATA box; (ii) the LAT ICP4-binding site; (iii) miRNA I (miR-I) and miR-II (miR-I/II), which both target ICP34.5; and (iv) miR-III, which targets ICP0. While the LAT TATA box mutant caused milder acute infections than wild-type (WT) virus, there was no difference in the recurrence phenotype between these viruses. LAT and miRNA expression during latency was not impaired by this mutation, suggesting that other promoter elements may be more important for latent HSV-2 LAT expression. Mutation of the LAT ICP4-binding site also did not cause an in vivo phenotypic difference between mutant and WT viruses. Acute infection and reactivation from latency of the miR-I/II mutant were similar to those of its rescuant, although the acute infection was slightly reduced in severity relative to that caused by the wild-type virus. The miR-III mutant also exhibited WT phenotypes in acute and recurrent phases of infection. While they do not rule out an effect of these elements in human latency or reactivation, these findings do not identify a specific role for LAT or LAT-associated miRNAs in the HSV-2 LAT promoter deletion phenotype in guinea pigs. Thus, other sequences in this region may play a more important role in the long-studied LAT-associated phenotype in animals. IMPORTANCE While it has been known for several decades that specific HSV-1 and HSV-2 sequences near the LAT promoter are required for efficient viral reactivation in animal models, the mechanism is still not known. We constructed four mutant viruses with the goal of identifying critical sequence elements and of specifically testing the hypothesis that microRNAs that are expressed during latency play a role. Determination that specific LAT promoter sequences and miRNA sequences do not influence viral reactivation of HSV-2 helps to narrow down the search for the mechanism by which the virus controls its latency and recurrence phenotype.

Background Bacterial sepsis is still a complication in patients transfused with stored platelets (PLTs). We have recently demonstrated that selected antimicrobial peptides (AMPs) have bactericidal activity in bacteria-spiked PLTs. In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice. Here we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail), and evaluated their effect on the in vitro properties of PLTs. Study Design and Methods Leukoreduced ABO- and D-identical whole blood–derived PLT concentrates were pooled and divided into two 60-mL aliquots in CLX storage bags. On Day 0, one bag received a peptide cocktail of RW3 plus RW4 at 0.01 mmol/L final concentration (test) and the other bag received only phosphate-buffered saline (PBS), the AMP solvent (control). The treated PLTs were stored for 7 days at 20 to 24°C. Samples were collected on Days 1, 5, and 7 to evaluate the in vitro properties of PLTs with standard assays. Results In vitro properties of the RW3 plus RW4 cocktail–treated PLTs were similar to those incubated with PBS only. There were no significant differences between the control and test PLTs during the 7-day storage. Conclusion Leukoreduced whole blood–derived PLTs treated with a mixture of RW3 and RW4 peptides maintain their in vitro properties during 7 days of storage.

T cells are potentially curative for patients with metastatic cancer, but many patients with cancer have T cells that are 'terminally differentiated', a condition associated with treatment failure. We have observed that less differentiated T cells have a greater capacity to proliferate, persist and destroy large cancer deposits. Advances in regenerative medicine might allow the generation of rejuvenated T cells from induced pluripotent stem cells (iPSC). We have previously reported that T cells can be generated from iPSC in vitro by co-culturing them OP9 stromal cells expressing Notch-1 ligand, Delta-like-1 (OP9/DLL1). These cells have limited tumor-specificity but also exhibit unconventional and NK cell-like properties demonstrating lineage diversion into alternative lymphoid development pathways, with unknown consequences for their safety and efficacy. To generate iPSC-derived T cells with more naturalistic tumor-specific T cell programs, we sought to restore physiologic signals for selection, maturation and survival. We employed a novel 3D thymic culture system using fetal thymic tissue and generated a novel type of T cell, 'iPSC-derived thymic emigrants' (iTE). Antigen-specific CD8αβ+ iTE exhibited functional properties in vitro that were almost indistinguishable from natural naïve CD8αβ+ T cells, including vigorous expansion and robust anti-tumor activity. iPSC-derived immature T cells generated using OP9/DLL1 and 'educated' in fetal thymic organoids in a 3D culture system resembled naturally-occurring 'young' T cells, as analyzed using whole genome RNA-seq techniques. iTE recapitulated many of the transcriptional programs of naïve T cells in vivo and revealed a striking capacity for engraftment, memory formation and efficient tumor destruction. Although many milestones remain, our data show that 'Next-Gen' autologous tumor-specific T cells can realistically be generated from iPSC using 3D thymic organ tissue. Our next goal is now to employ these cells to treat patients with metastatic cancer because iPSC-derived T cells have a potentially unlimited capacity for proliferation, engraftment and anti-tumor activity. Disclosures Vizcardo: NCI: Patents & Royalties: International Patent Application PCT/US2017/65986. Klemen:NIH/NCI: Patents & Royalties: International Patent Application PCT/US2017/65986. Restifo:NIH/NCI: Patents & Royalties: International Patent Application PCT/US2017/65986.

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1α-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1α deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1α deficiency reduced TNF-α, IL-1β, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1β mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights.

The motor neuron disease spinal muscular atrophy (SMA) causes profound muscle weakness that most often leads to early death. At autopsy, SMA is characterized by loss of motor neurons and muscle atrophy, but the initial cellular events that precipitate motor unit dysfunction and loss remain poorly characterized. Here, we examined the function and corresponding structure of neuromuscular junction (NMJ) synapses in a mouse model of severe SMA (hSMN2/delta7SMN/mSmn−/−). Surprisingly, most SMA NMJs remained innervated even late in the disease course; however they showed abnormal synaptic transmission. There was a two-fold reduction in the amplitudes of the evoked endplate currents (EPCs), but normal spontaneous miniature EPC (MEPC) amplitudes. These features in combination indicate reduced quantal content. SMA NMJs also demonstrated increased facilitation suggesting a reduced probability of vesicle release. By electron microscopy, we found a decreased density of synaptic vesicles that is likely to contribute to the reduced release probability. In addition to presynaptic defects, there were postsynaptic abnormalities. EPC and MEPC decay time constants were prolonged because of a slowed switch from the fetal acetylcholine receptor (AChR) γ-subunit to the adult ε-subunit. There was also reduced size of AChR clusters and small myofibers, which expressed an immature pattern of myosin heavy chains. Together these results indicate that impaired synaptic vesicle release at NMJs in severe SMA is likely to contribute to failed postnatal maturation of motor units and muscle weakness.

Autophagy is a process by which cytoplasmic organelles can be catabolized either to remove defective structures or as a means of providing macromolecules for energy generation under conditions of nutrient starvation. In this study we demonstrate that mitochondrial autophagy is induced by hypoxia, that this process requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, and that in cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death.

Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1α gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.

Aims We investigated whether hypoxia-inducible factor 1α (HIF-1α) plays a role in the acute phase of ischaemic preconditioning (IPC). Methods and results Hearts from wild-type (WT) mice and mice heterozygous for a null allele at the locus encoding HIF-1α (HET) were subjected to IPC (10-min ischaemia/5 min reperfusion, or two cycles of 5 min ischaemia/5 min reperfusion), followed by 30 min ischaemia and reperfusion. Left ventricular-developed pressure, heart rate, and coronary flow rate were measured continuously. Apoptosis and infarct size were assessed by TUNEL assay, cleaved caspase 3 immunohistochemistry, and triphenyltetrazolium chloride staining. Production of reactive oxygen species (ROS) in isolated cardiac mitochondria was measured by a chemiluminescence assay. The phosphatase and tensin homologue (PTEN) and AKT (protein kinase B) were analysed by immunoblot assay. IPC improved functional recovery and limited infarct size and apoptosis after prolonged ischaemia–reperfusion in WT hearts, but not in HET hearts. Mitochondrial ROS production, PTEN oxidation, and AKT phosphorylation were impaired in HET hearts. WT and HET hearts were protected by adenosine, which acts via an ROS-independent mechanism. Conclusion HIF-1α is required for IPC-induced mitochondrial ROS production and myocardial protection against ischaemia–reperfusion injury.

Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH-evoked cardio-respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia-inducible factor-1 (HIF-1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF-1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF-1 and if so whether it contributes to cardio-respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild-type (WT) and heterozygous (HET) mice partially deficient in HIF-1α, the O2 regulated subunit of the HIF-1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h−1, 8 h day−1) or to 10 days of 21% O2 (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long-term facilitation (sLTF) of the chemoreceptor activity in CIH-exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH-exposed HET mice. Analysis of cardio-respiratory responses in CIH-exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH-exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH-induced increases in ROS but also CIH-evoked HIF-1α up-regulation in WT mice. These results indicate that: (a) HIF-1 activation is critical for eliciting CIH-induced carotid body-mediated cardio-respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF-1 and ROS.

Bone marrow–derived stromal cells have engendered interest because of their therapeutic potential for promoting tissue vascularization and repair. When mononuclear cells isolated from mouse bone marrow were cultured in DMEM supplemented with 10% fetal bovine serum, cell populations arose that showed rapid proliferation and loss of contact inhibition. These cells formed invasive soft tissue sarcomas after i.m. injection into nude or scid mice. I.v. injection resulted in the formation of tumor foci in the lungs. The tumors were transplantable into syngeneic immunocompetent mice. Direct injection of cultured cells into immunocompetent mice also resulted in tumor formation. Karyotype analysis showed that increased chromosome number and multiple Robertsonian translocations occurred at passage 3 coincident with the loss of contact inhibition. The remarkably rapid malignant transformation of cultured mouse bone marrow cells may have important implications for ongoing clinical trials of cell therapy and for models of oncogenesis. (Cancer Res 2006; 66(22): 10849-54)

We have previously shown that exposure of C57BL/6J mice to intermittent hypoxia (IH) leads to 1) hypertriglyceridemia due to upregulation of pathways of lipid biosynthesis, including sterol regulatory element binding protein (SREBP)-1 and stearoyl CoA desaturase (SCD)-1; and 2) hypercholesterolemia due to impaired cholesterol uptake. The goal of the present study was to examine whether hypoxia-inducible factor (HIF)-1 is implicated in changes in lipid metabolism induced by IH. Lean HIF-1α ( Hif1a)+/−mice, which are heterozygous for a null allele at the locus encoding the HIF-1α subunit, and their wild-type (WT) Hif1a+/+littermates were exposed to IH or control conditions for 5 days. IH increased fasting blood glucose, serum total cholesterol, and high-density lipoprotein-cholesterol, phospholipids, triglycerides (TG), and leptin in mice of both genotypes, whereas serum insulin and interleukin-6 were elevated only in WT mice. The impact of IH on serum TG levels in WT mice was significantly greater than that in Hif1a+/−mice (95 ± 9 vs. 66 ± 6 mg/dl, P < 0.05), whereas cholesterol and glucose levels were affected independently of genotype. Under hypoxic conditions, mRNA and protein levels of SREBP cleavage-activating protein (SCAP) and SCD-1 and protein levels of nuclear isoform of SREBP-1 in the liver were induced to significantly higher levels in WT mice than in Hif1a+/−mice. We conclude that 1) the effect of IH on serum TG levels is mediated through HIF-1, 2) HIF-1 may impact on posttranscriptional regulation of SREBP-1, and 3) the effect of IH on serum cholesterol levels was not altered by partial HIF-1α deficiency.

The high prevalence of Herpesviruses in the population and the maintenance of lifelong latent reservoirs are challenges to the control of herpetic diseases, despite the availability of antiviral pharmaceuticals that target viral DNA replication. In addition to oral and genital lesions, herpes simplex virus infections and recurrent reactivations from the latent pool can result in severe pathology including neonatal infection and mortality, blindness due to ocular keratitis, and viral-induced complications in immunosuppressed individuals. Herpesviruses, like their cellular hosts, are subject to the regulatory impacts of chromatin and chromatin modulation machinery that promotes or suppresses gene expression. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 and JMJD2s. Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and a block to infection and reactivation in vitro. Here, the concept of epigenetic suppression of viral infection is demonstrated in three animal models of herpes simplex virus infection and disease. Inhibition of LSD1 via treatment of animals with the monoamine oxidase inhibitor tranylcypromine results in suppression of viral lytic infection, subclinical shedding, and reactivation from latency in vivo. Phenotypic suppression is correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Given the expanding development of epipharmaceuticals, this approach has substantial potential for anti-herpetic treatments with distinct advantages over the present pharmaceutical options.

In order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans -activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation. IMPORTANCE The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5′ ends and evaluation of ICP4 response. These findings provide further insight into the virus' strategy to tightly control expression of lytic cycle genes (especially the neurovirulence factor, ICP34.5) and suggest a mechanism (via ICP4) for the transition from latency to reactivated productive infection.

Hyperhomocyst(e)inemia (HH) is an established independent risk factor for coronary, cerebral and peripheral vascular diseases. Recent studies have indicated that certain cardiovascular risk factors, including diabetes and hypercholesterolemia, impair expression of vascular endothelial growth factor (VEGF) and endogenous angiogenesis. In this study, we investigate the impact of moderate HH on angiogen- esis and VEGF pathway in a mouse model of hindlimb ischemia. Upon induction of unilateral hindlimb ischemia, endogenous angiogenesis, expression of VEGF, and phosphorylation of the VEGF receptor Flk-1 were evaluated in mice heterozygous for a deletion of the cystathionine �-synthase gene (CBS) and compared with those observed in CBS�/� mice. CBS�/� mice exhibit moderate HH, as demonstrated by measuring plasma total homocyst(e)ine (tHcy) levels, which were significantly higher in these animals compared with CBS�/� mice (4.77 � 0.82 vs 2.10 � 0.28, p � 0.01). Twenty-eight days after induction of ischemia, hindlimb blood flow was significantly reduced in CBS�/� mice compared with CBS�/� animals (0.49 � 0.03, n � 12 vs 0.71 � 0.09, n � 10; p � 0.05). In addition, there was a significant negative correlation between plasma homocyst(e)ine levels and the laser Doppler perfusion ratio in CBS�/� mice (p � 0.0087, r �� 0.7171). While VEGF expression and Flk-1 phosphorylation were not impaired in the ischemic muscles of CBS�/� mice, phos- phorylation of the endothelial cell survival factor Akt was significantly inhibited by homocyst(e)ine in a dose-dependent manner in human umbilical vein endothelial cell (HUVECs) in vitro. In conclusion, our findings demonstrate that endogenous angiogenesis is inversely related to plasma levels of homocyst(e)ine in genetically engineered, heterozygous mice with moderate HH. This impairment, however, is not dependent on reduced expression of VEGF or impaired phosphorylation of its recep- tor Flk-1. In contrast, our data suggest that impaired Akt phosphorylation mediates the impairment of angiogenesis associated with HH.

The physiologic ability of peripheral nerves to regenerate after injury is impaired with aging. However, the mechanisms responsible for this phenomenon are still incompletely characterized. In this study, we investigated whether aging influences the intraneural angiogenic response that occurs after injury and during regeneration of peripheral nerves. We performed a crush injury of the sciatic nerve in old and senescence accelerated mice and found that the peripheral nerves of these animals are unable to locally upregulate vascular endothelial growth factor (VEGF), a prototypical angiogenic cytokine, after injury and have substantial deficits in mounting an appropriate intraneural angiogenic response during nerve regeneration. Our findings provide new evidence of possible interdependent relationships between aging, VEGF, angiogenesis, and nerve regeneration and suggest that vascular abnormalities might play a role in aging-associated neurological dysfunction, with potentially important fundamental and clinical implications.

Apolipoprotein E-knockout (apoE KO) mice have peripheral sensory nerve defects, reduced and delayed response to noxious thermal stimuli, abnormal morphology of unmyelinated fibers, and impaired blood-nerve and blood-brain barriers. In this study, we show that, compared to wild-type mice, peripheral nerves of apoE KO mice have impaired ability to respond to ischemia, as demonstrated by measurement of motor and sensory conduction velocity. In addition, mice lacking apoE exhibit a deficit of reinnervation of ischemic epidermis, evaluated by immunofluorescent staining for the pan-neuronal marker PGP 9.5. Also regional nerve blood flow, measured by laser Doppler, and intraneural angiogenesis after ischemia are significantly compromised in apoE-deficient mice. Finally, upregulation of the angiogenic cytokine vascular endothelial growth factor (VEGF), which physiologically occurs after ischemia in the peripheral nerve of wild-type mice, is severely impaired in apoE KO mice. Among the several neural defects that have already been described in mice lacking apoE, this is the first demonstration that functional recovery to ischemia is impaired in the peripheral nerves of these animals. This deficit is mirrored by the inability of upregulating VEGF and mounting an appropriate intraneural angiogenic response following injury. These findings provide new evidence of possible interdependent relationships between VEGF, angiogenesis, and nerve function and regeneration and may provide new important information on the role of apoE in the nervous system.

Background— Hedgehog (Hh) proteins are morphogens regulating epithelial–mesenchymal signaling during several crucial processes of embryonic development, including muscle patterning. Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog constitute the repertoire of Hh genes in humans. The activities of all 3 are transduced via the Patched (Ptc1) receptor. Recent observations indicate that exogenous administration of Shh induces angiogenesis. Here, we studied whether the endogenous Hh pathway, in addition to its functions during embryogenesis, plays a physiological role in muscle regeneration after ischemia in adults. Methods and Results— We found that skeletal muscle ischemia induces strong local upregulation of Shh mRNA and protein. In addition, the Ptc1 receptor is activated in interstitial mesenchymal cells within the ischemic area, indicating that these cells respond to Shh and that the Shh pathway is functional. We also found that Shh-responding cells produce vascular endothelial growth factor under ischemic conditions and that systemic treatment with a Shh-blocking antibody inhibits the local angiogenic response and the upregulation of vascular endothelial growth factor. Conclusions— Our study shows that the Hh signaling may be recapitulated postnatally in adult and fully differentiated muscular tissues and has a regulatory role on angiogenesis during muscle regeneration after ischemia. These findings demonstrate a novel biological activity for the Hh pathway with both fundamental and potential therapeutic implications.

Nitric oxide (NO) is a signaling molecule that can trigger adaptive (physiological) or maladaptive (pathological) responses to stress stimuli in a context-dependent manner. We have previously reported that NO may signal axonal injury to neighboring glial cells. In this study, we show that mice deficient in neuronal nitric oxide synthase (nNOS −/− ) are more vulnerable than WT mice to toxin-induced peripheral neuropathy. The administration of NO donors to primary dorsal root ganglion cultures prevents axonal degeneration induced by acrylamide in a dose-dependent manner. We demonstrate that NO-induced axonal protection is dependent on hypoxia-inducible factor (HIF)-1–mediated transcription of erythropoietin (EPO) within glial (Schwann) cells present in the cultures. Transduction of Schwann cells with adenovirus AdCA5 encoding a constitutively active form of HIF-1α results in amelioration of acrylamide-induced axonal degeneration in an EPO-dependent manner. Mice that are partially deficient in HIF-1α (HIF-1α +/− ) are also more susceptible than WT littermates to toxic neuropathy. Our results indicate that NO→HIF-1→EPO signaling represents an adaptive mechanism that protects against axonal degeneration.

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.

Diabetes is a major risk factor for ischemic disease. Treatment options for diabetic patients with peripheral arterial disease when revascularization is not possible are limited, resulting in a high incidence of limb amputation. We evaluated the therapeutic potential of AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, in a diabetic model of critical limb ischemia. Diabetic db / db and nondiabetic db /+ mice were subjected to unilateral femoral artery ligation. Limb perfusion, tissue viability, and motor function were more severely impaired in db / db mice. Intramuscular injection of AdCA5 into the ischemic limb of db / db mice increased the recovery of limb perfusion and function, reduced tissue necrosis, rescued the diabetes-associated impairment of circulating angiogenic cells, enhanced endothelial nitric oxide synthase activation, and increased vessel density and luminal area in the ischemic limb.

Early treatment with the histone deacetylase inhibitor, trichostatin A, plus nutritional support extended median survival of spinal muscular atrophy mice by 170%. Treated mice continued to gain weight, maintained stable motor function, and retained intact neuromuscular junctions long after trichostatin A was discontinued. In many cases, ultimate decline of mice appeared to result from vascular necrosis, raising the possibility that vascular dysfunction is part of the clinical spectrum of severe spinal muscular atrophy. Early spinal muscular atrophy disease detection and treatment initiation combined with aggressive ancillary care may be integral to the optimization of histone deacetylase inhibitor treatment in human patients.

HIF-1α activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HIF-1α+/− fracture calluses are larger, stronger, and stiffer than HIF-1α+/+ calluses because of decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. Introduction: Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia after fracture activates hypoxia-inducible factor 1α (HIF-1α), leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1α is a key regulator of bone regeneration. Materials and Methods: Fixed femoral fractures were generated in mice with partial HIF-1α deficiency (HIF-1α+/−) and wildtype littermates (HIF-1α+/+). Fracture calluses and intact contralateral femurs from postfracture days (PFDs) 21 and 28 (N = 5–10) were subjected to μCT evaluation and four-point bending to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10, and 14 samples (N = 3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N = 2) to elucidate differential apoptosis. Results: Surprisingly, fracture calluses from HIF-1α+/− mice exhibited greater mineralization and were larger, stronger, and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, showing higher expression of proapoptotic protein phosphatase 2a (PP2A) and lower expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL2) in HIF-1α+/+ calluses. Subsequent TUNEL staining showed that HIF-1α+/+ calluses contained larger numbers of TUNEL+ chondrocytes and osteoblasts than HIF-1α+/− calluses. Conclusions: We conclude that partial HIF-1α deficiency results in decreased chondrocytic and osteoblastic apoptosis, thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration.

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.

Objective— The embryonic morphogen sonic hedgehog (SHh) has been shown to induce neovascularization of ischemic tissue but has not been shown to play a role in regulating vascular nerve supply. Accordingly, we investigated the hypothesis that systemic injection of SHh protein could improve nerve blood flow and function in diabetic neuropathy (DN). Methods and Results— Twelve weeks after induction of diabetes with streptozotocin, motor and sensory nerve conduction velocities (MCV and SCV) of the sciatic nerves were significantly reduced in diabetic rats. SHh-treated diabetic rats demonstrated marked improvement of both MCV and SCV ( P P Conclusions— These data indicate that Shh induces arteriogenesis and restores nerve function in DN.

Objective. – Until recently, prostacyclin (PGI 2 ) biological activities were thought to be exclusively mediated by cell surface receptors named IP. Recent studies have instead identified a novel pathway of PGI 2 signaling, occurring through activation of peroxisome proliferator-activated receptors (PPARs) located in the nucleus. The availability of stable PGI 2 analogs with different affinity for IP receptors and PPARs provides the possibility to test the importance and function of this dual pathway in vitro and in vivo. In this study, the in vivo angiogenic properties of different PGI 2 analogs and the potential relationship between PPAR-mediated pathways, vascular endothelial growth factor (VEGF), and angiogenesis were investigated. Methods and results. – By using the murine corneal model of angiogenesis, we found that PGI 2 analogs able to act on nuclear PPARs, such as iloprost and carbaprostacyclin (cPGI), induce angiogenesis in vivo. In contrast, cicaprost, a PGI 2 analog that only acts on IP receptors, has no in vivo angiogenic activity. Interestingly, angiogenesis induced by iloprost and cPGI does not differ in extent and morphology from that induced by VEGF and is associated with local increment of VEGF mRNA expression and protein levels. Finally, iloprost-induced angiogenesis is significantly decreased by systemic inhibition of VEGF activity, obtained by gene transfer of a soluble form of the VEGF receptor Flt-1. Conclusions. – These data demonstrate that stable PGI 2 analogs may have angiogenic properties in vivo, depending on their ability to act on PPARs. The resulting angiogenic process appears to be mediated by VEGF. These findings indicate that important physiological activities in the cardiovascular system, such as angiogenesis and VEGF induction, may be modulated by PGI 2 through specific activation of the PPAR signaling pathway in vivo, with potentially important fundamental and clinical implications.

Background— Stromal cell–derived factor-1 (SDF-1) is a chemokine considered to play an important role in the trafficking of hematopoietic stem cells. Given the close relationship between hematopoietic stem cells and endothelial progenitor cells (EPCs), we investigated the effect of SDF-1 on EPC-mediated vasculogenesis. Methods and Results— Flow cytometric analysis demonstrated expression of CXCR4, the receptor of SDF-1, by 66±3% of EPCs after 7 days in culture. In vitro modified Boyden chamber assay showed a dose-dependent EPC migration toward SDF-1 (control versus 10 ng/mL SDF-1 versus 100 ng/mL SDF-1, 24±2 versus 71±3 versus 140±6 cells/mm 2 ; P P 2 , P 2 ; P Conclusion— These findings indicate that locally delivered SDF-1 augments vasculogenesis and subsequently contributes to ischemic neovascularization in vivo by augmenting EPC recruitment in ischemic tissues.