Magnetically powered microwheel thrombolysis of occlusive thrombi in zebrafish.

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here, we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-functionalized µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-functionalized µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Genetic duplication of tissue factor reveals subfunctionalization in venous and arterial hemostasis.

Tissue factor (TF) is an evolutionarily conserved protein necessary for initiation of hemostasis. Zebrafish have two copies of the tissue factor gene (f3a and f3b) as the result of an ancestral teleost fish duplication event (so called ohnologs). In vivo physiologic studies of TF function have been difficult given early lethality of TF knockout in the mouse. We used genome editing to produce knockouts of both f3a and f3b in zebrafish. Since ohnologs arose through sub- or neofunctionalization, they can unmask unknown functions of non-teleost genes and could reveal whether mammalian TF has developmental functions distinct from coagulation. Here we show that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF results in lethal hemorrhage by 2-4 months despite normal embryonic and vascular development. Larval vascular endothelial injury reveals predominant roles for TFa in venous circulation and TFb in arterial circulation. Finally, we demonstrate that loss of TF predisposes to a stress-induced cardiac tamponade independent of its role in fibrin formation. Overall, our data suggest partial subfunctionalization of TFa and TFb. This multigenic zebrafish model has the potential to facilitate study of the role of TF in different vascular beds.

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation.

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of ß-globinopathies (sickle cell disease and ß-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor-like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

A monoallelic-to-biallelic T-cell transcriptional switch regulates GATA3 abundance.

Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene.

Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development.

Notch1 signaling must elevate to high levels in order to drive the proliferation of CD4-CD8- double-negative (DN) thymocytes and progression to the CD4+CD8+ double-positive (DP) stage through ß-selection. During this critical phase of pre-T-cell development, which is also known as the DN-DP transition, it is unclear whether the Notch1 transcriptional complex strengthens its signal output as a discrete unit or through cofactors. We previously showed that the protein inhibitor of activated STAT-like coactivator Zmiz1 is a context-dependent cofactor of Notch1 in T-cell leukemia. We also showed that withdrawal of Zmiz1 generated an early T-lineage progenitor (ETP) defect. Here, we show that this early defect seems inconsistent with loss-of-Notch1 function. In contrast, at the later pre-T-cell stage, withdrawal of Zmiz1 impaired the DN-DP transition by inhibiting proliferation, like withdrawal of Notch. In pre-T cells, but not ETPs, Zmiz1 cooperatively regulated Notch1 target genes Hes1, Lef1, and Myc. Enforced expression of either activated Notch1 or Myc partially rescued the Zmiz1-deficient DN-DP defect. We identified residues in the tetratricopeptide repeat (TPR) domain of Zmiz1 that bind Notch1. Mutating only a single residue impaired the Zmiz1-Notch1 interaction, Myc induction, the DN-DP transition, and leukemic proliferation. Similar effects were seen using a dominant-negative TPR protein. Our studies identify stage-specific roles of Zmiz1. Zmiz1 is a context-specific cofactor for Notch1 during Notch/Myc-dependent thymocyte proliferation, whether normal or malignant. Finally, we highlight a vulnerability in leukemic cells that originated from a developmentally important Zmiz1-Notch1 interaction that is hijacked during transformation from normal pre-T cells.

High-Throughput Single-Cell Sequencing of both TCR-ß Alleles.

Allelic exclusion is a vital mechanism for the generation of monospecificity to foreign Ags in B and T lymphocytes. In this study, we developed a high-throughput barcoded method to simultaneously analyze the VDJ recombination status of both mouse TCR-ß alleles in hundreds of single cells using next-generation sequencing.

GATA-3 regulates hematopoietic stem cell maintenance and cell-cycle entry.

Maintaining hematopoietic stem cell (HSC) quiescence is a critical property for the life-long generation of blood cells. Approximately 75% of cells in a highly enriched long-term repopulating HSC (LT-HSC) pool (Lin(-)Sca1(+)c-Kit(hi)CD150(+)CD48(-)) are quiescent, with only a small percentage of the LT-HSCs in cycle. Transcription factor GATA-3 is known to be vital for the development of T cells at multiple stages in the thymus and for Th2 differentiation in the peripheral organs. Although it is well documented that GATA-3 is expressed in HSCs, a role for GATA-3 in any prethymic progenitor cell has not been established. In the present study, we show that Gata3-null mutant mice generate fewer LT-HSCs and that fewer Gata3-null LT-HSCs are in cycle. Furthermore, Gata3 mutant hematopoietic progenitor cells fail to be recruited into an increased cycling state after 5-fluorouracil-induced myelosuppression. Therefore, GATA-3 is required for the maintenance of a normal number of LT-HSCs and for their entry into the cell cycle.

Magnetically Powered Microwheel Thrombolysis of Occlusive Thrombi in Zebrafish.

Tissue plasminogen activator (tPA) is the only FDA approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Providing a living microfluidic analog, transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Interactions between multiple cell types in parallel microfluidic channels: monitoring platelet adhesion to an endothelium in the presence of an anti-adhesion drug.

A simple method for immobilizing endothelial cells in the channels of a microfluidic device fabricated with soft lithography is presented that requires no surface oxidation of the substrate material used in conjunction with the microfluidic device and is operable even with a reversible seal. Specifically, optimal conditions for culturing bovine pulmonary artery endothelial cells (bPAECs) to the surface of a Petri dish were investigated. The parameters investigated included fibronectin concentration, temperature, seeding density, and immobilization time. To enhance the utility of the device, all optimization studies, and studies involving platelet adhesion to the immobilized endothelium, were performed in parallel channels, thereby enabling improved throughput over a single channel device. The optimal conditions for cell immobilization included coating the Petri dish with 100 microg/mL fibronectin, a seeding cell density of 1.00 x 10(5) cells mL(-1), and an immobilization time of 90 min at 37 degrees C. The device was then employed to monitor the physical interaction (adhesion) of platelets to the immobilized endothelium in the presence of a known platelet activator (ADP) and a drug inhibitor of platelet activation. The number of platelets adhering to the endothelial cells in the channels increased from 17.0 +/- 2.3 in the absence of ADP to 63.2 +/- 2.4 in the presence of 5.00 microM ADP. Moreover, the data presented here also shows that inhibition of endothelium nitric oxide (NO) production, a recognized inhibitor of platelet adhesion to the endothelium, increased the number of platelets adhering to the surface to 35.4 +/- 1.0. In the presence of NO inhibition and 5.00 microM ADP, the affect on platelet adhesion was further increased to 127 +/- 5.2. Finally, this device was employed to investigate the effect of a drug known to inhibit platelet adhesion (clopidogrel) and, in the presence of the drug, the platelet adhesion due to activation by 5.00 microM ADP decreased to 24.0 +/- 3.8. This work is the first representation of multiple cell types physically interacting in the channels of a microfluidic device and further demonstrates the potential of these devices in the drug discovery process and drug efficacy studies.

GATA3 Abundance Is a Critical Determinant of T Cell Receptor ß Allelic Exclusion.

Allelic exclusion describes the essential immunological process by which feedback repression of sequential DNA rearrangements ensures that only one autosome expresses a functional T or B cell receptor. In wild-type mammals, approximately 60% of cells have recombined the DNA of one T cell receptor ß (TCRß) V-to-DJ-joined allele in a functional configuration, while the second allele has recombined only the DJ sequences; the other 40% of cells have recombined the V to the DJ segments on both alleles, with only one of the two alleles predicting a functional TCRß protein. Here we report that the transgenic overexpression of GATA3 leads predominantly to biallelic TCRß gene (Tcrb) recombination. We also found that wild-type immature thymocytes can be separated into distinct populations based on intracellular GATA3 expression and that GATA3LO cells had almost exclusively recombined only one Tcrb locus (that predicted a functional receptor sequence), while GATA3HI cells had uniformly recombined both Tcrb alleles (one predicting a functional and the other predicting a nonfunctional rearrangement). These data show that GATA3 abundance regulates the recombination propensity at the Tcrb locus and provide new mechanistic insight into the historic immunological conundrum for how Tcrb allelic exclusion is mediated.

Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs.

The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

Conditional Gata2 inactivation results in HSC loss and lymphatic mispatterning.

The transcription factor GATA-2 plays vital roles in quite diverse developmental programs, including hematopoietic stem cell (HSC) survival and proliferation. We previously identified a vascular endothelial (VE) enhancer that regulates GATA-2 activity in pan-endothelial cells. To more thoroughly define the in vivo regulatory properties of this enhancer, we generated a tamoxifen-inducible Cre transgenic mouse line using the Gata2 VE enhancer (Gata2 VECre) and utilized it to temporally direct tissue-specific conditional loss of Gata2. Here, we report that Gata2 VECre-mediated loss of GATA-2 led to anemia, hemorrhage, and eventual death in edematous embryos. We further determined that the etiology of anemia in conditional Gata2 mutant embryos involved HSC loss in the fetal liver, as demonstrated by in vitro colony-forming and immunophenotypic as well as in vivo long-term competitive repopulation experiments. We further documented that the edema and hemorrhage in conditional Gata2 mutant embryos were due to defective lymphatic development. Thus, we unexpectedly discovered that in addition to its contribution to endothelial cell development, the VE enhancer also regulates GATA-2 expression in definitive fetal liver and adult BM HSCs, and that GATA-2 function is required for proper lymphatic vascular development during embryogenesis.

The dual nature of extracellular ATP as a concentration-dependent platelet P2X1 agonist and antagonist.

Patient groups subject to higher occurrence of stroke (e.g., people with diabetes, cystic fibrosis, pulmonary hypertension) have reduced release of ATP from their erythrocytes (ERYs) when subjected to flow-induced deformation or pharmacological stimuli. These same groups also have platelets that are more adhesive in comparison to controls. Here we show platelet aggregation, and inhibition of that aggregation, is affected by free Ca(2+) entering the platelet through the ATP-gated P2X1 receptor. The addition of ATP (10 microM) increased the platelet NO by 26.7 +/- 7.7%. This value was decreased significantly to below basal levels in the presence of NF 449 (p < 0.001), an inhibitor of the P2X1 receptor on the platelet. Aggregation profiles measured in the presence of ATP revealed that when the P2X1 receptor was blocked, or when the measurements were performed in Ca(2+) free buffer, platelet aggregation was nearly eliminated. Our findings employing standard aggregation measurements suggest that ATP behaves as a platelet inhibitor below 1.6 x 10(-19) moles ATP per platelet; however, above this value, ATP behaves as a platelet activator. These findings suggesting a dual nature of ATP with regard to platelet behavior were confirmed by passing platelets over endothelial cells that were coated in the channels of a microfluidic device. Importantly, it was determined that ERY-derived ATP release was a major determinant of platelet adhesion to the endothelium. These findings may have implications in anti-platelet drug design as most current therapies focus on the inhibition of P2Y-type receptors. Moreover, through the use of microfluidic technologies, we have provided in vitro evidence for a possible relationship between ERY properties and platelet behavior in vivo.

A Molecular Level Understanding of Zinc Activation of C-peptide and its Effects on Cellular Communication in the Bloodstream.

Inspired by previous reports, our group has recently demonstrated that C-peptide exerts beneficial effects upon interactions with red blood cells (RBCs). These effects can be measured in RBCs obtained from animal models of both type 1 diabetes and type 2 diabetes, though to different extents. To date, the key metrics that have been measured involving C-peptide and RBCs include an increase in glucose uptake by these cells and a subsequent increase in adenosine triphosphate (ATP) release. Importantly, to date, our group has only been able to elicit these beneficial effects when the C-peptide is prepared in the presence of Zn2+. The C-peptide-induced release of ATP is of interest when considering that ATP is a purinergic signaling molecule known to stimulate the production of nitric oxide (NO) in the endothelium and in platelets. This NO production has been shown to participate in smooth muscle relaxation and subsequent vessel dilation. Furthermore, NO is a well-established platelet inhibitor. The objective of this review is to provide information pertaining to C-peptide activity on RBCs. Special attention is paid to the necessity of Zn2+ activation, and the origin of that activation in vivo. Finally, a mechanism is proposed that explains how C-peptide is exerting its effects on other cells in the bloodstream, particularly on endothelial cells and platelets, via its ability to stimulate the release of ATP from RBCs.

Red blood cell stimulation of platelet nitric oxide production indicated by quantitative monitoring of the communication between cells in the bloodstream.

ATP is a recognized stimulus of nitric oxide synthase and is released from red blood cells (RBCs) upon deformation. The objective of this work is to demonstrate that RBCs stimulate nitric oxide production in platelets by employing a continuous flow analysis system in which the stream contains both RBCs and platelets. Here, two drugs known to improve blood flow in vivo (pentoxyfilline and iloprost) are shown to increase both the release of RBC-derived ATP and the production of platelet-derived NO. A flow-based chemiluminescence assay (in vitro) was employed to quantitatively determine the amount of ATP released from erythrocytes subjected to flow-induced deformation. Prior to being subjected to flow, erythrocytes were incubated in the absence or presence of 4.8 microM pentoxyfilline or 80 nM iloprost. Erythrocytes obtained from rabbits (n=22) that were subjected to flow released 239 +/- 29 nM ATP. When treated with pentoxyfilline, the ATP released from the flowing RBCs increased to 450 +/- 94 nM ATP. An increase in RBC-derived ATP was also measured for iloprost-incubated RBCs in flow (362 +/- 45 nM ATP). Importantly, platelets that were loaded with diaminofluorofluorescein diacetate, an intracellular fluorescence probe for NO, exhibited increases in fluorescence intensity by 16% in the presence of RBCs treated with pentoxyfilline and a 10% increase when treated with iloprost. When the ATP release from the RBCs was inhibited with glybenclamide, the platelet fluorescence intensity decreased by 25 and 51% for RBCs incubated with pentoxyfilline and iloprost, respectively. In an experiment not involving the RBC, inhibition of the P2x receptor on the platelets (an ATP receptor) resulted in no increase in platelet NO production, suggesting that the NO production in the activated platelet is due to ATP.

Fluorescence determination of nitric oxide production in stimulated and activated platelets.

Nitric oxide (NO) is quantitatively determined in platelets prior to, and after, stimulation with adenosine triphosphate (ATP) or activation with adenosine diphosphate (ADP). Platelets obtained from the whole blood of rabbits were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent NO production was measured as a fluorescent benzotriazole. Experiments were performed to determine the effect of probe concentration and probe incubation time in the platelets prior to measurement of the fluorescence. This information, combined with the method of multiple standard additions, was then employed to determine the moles of intracellular NO in the platelets (2.7 +/- 0.3) x 10(-16) mol of NO/platelet and the basal level of extracellular NO in the platelet sample (9.9 +/- 2.2) x 10(-18) mol of NO/platelet. Moreover, this method was used to quantitatively determine the amount of NO released from platelets whose NO production was stimulated with ATP (a nitric oxide synthase stimulus) or ADP, a substance known to result in NO production through platelet aggregation. When stimulated with ATP, the NO released from the platelets was determined to be (2.0 +/- 0.1) x 10(-17) mol of NO/platelet. When activated with ADP, the platelets released (2.8 +/- 0.3) x 10(-17) mol of NO/platelet. The difference between the extracellular basal levels of NO and that after stimulation with either ATP or ADP is in agreement with current estimates of NO release from platelets. Therefore, we conclude that a fluorescence determination of NO using the DAF family of probes, in combination with the method of multiple standard additions, can be employed to quantitatively determine the basal levels of NO in platelets, as well as the amount of NO released from stimulated and/or activated platelets.