Functional Redundancy of SWI/SNF Catalytic Subunits in Maintaining Vascular Endothelial Cells in the Adult Heart

Rationale: Mating type switching/sucrose non-fermenting (SWI/SNF) chromatin-remodeling complexes utilize either BRG1 or BRM as a catalytic subunit to alter nucleosome position and regulate gene expression. BRG1 is required for vascular endothelial cell (VEC) development and embryonic survival, whereas BRM is dispensable. Objective: To circumvent embryonic lethality and study Brg1 function in adult tissues, we used conditional gene targeting. To evaluate possible Brg1-Brm redundancy, we analyzed Brg1 mutant mice on wild-type and Brm-deficient backgrounds. Methods and Results: The inducible Mx1-Cre driver was used to mutate Brg1 in adult mice. These conditional-null mutants exhibited a tissue-specific phenotype and unanticipated functional compensation between Brg1 and Brm. Brg1 single mutants were healthy and had a normal lifespan, whereas Brg1/Brm double mutants exhibited cardiovascular defects and died within 1 month. BRG1 and BRM were required for the viability of VECs but not other cell types where both genes were also knocked out. The VEC phenotype was most evident in the heart, particularly in the microvasculature of the outer myocardium, and was recapitulated in primary cells ex vivo. VEC death resulted in vascular leakage, cardiac hemorrhage, secondary death of cardiomyocytes due to ischemia, and ventricular dissections. Conclusions: BRG1-catalyzed SWI/SNF complexes are particularly important in cardiovascular tissues. However, in contrast to embryonic development, in which Brm does not compensate, Brg1 is required in adult VECs only when Brm is also mutated. These results demonstrate for the first time that Brm functionally compensates for Brg1 in vivo and that there are significant changes in the relative importance of BRG1- and BRM-catalyzed SWI/SNF complexes during the development of an essential cell lineage.     

We Offer Renal Replacement Therapy to Patients Who Are Not Benefitted by It

Nephrologists offer renal replacement therapy (RRT) to patients who are unlikely to benefit in part because of our discomfort discussing goals of care in the setting of an uncertain prognosis for a given individual. Permanent neurological impairment, terminal illness (life expectancy <6 months), medical conditions precluding the safe delivery of dialysis, elderly patients with poor prognosis, and those who begin “early” RRT are categories of patients for whom dialysis may not be beneficial. Successful use of time‐limited trials of dialysis may reduce the number of patients who are started on RRT without significant benefit. However, clear achievable milestones and goals need to be incorporated into plans for time‐limited trials to ensure that continuing RRT beyond the trial period is appropriate. The lack of information on outcomes and symptom management using a “palliative approach” to dialysis suggests this should not be a clinical option until additional study is done and efficacy data available. Clinical practice guidelines are available to assist nephrologists in the appropriate withholding of RRT.     

Migration of Mouse 3T3 Fibroblasts in Response to a Serum Factor

A serum factor that promotes migration of (mouse) 3T3 fibroblast cells is shown to be distinct from the growth-promoting and cell-survival factors of serum. The factor promotes migration of cells from the edge of a wound in confluent 3T3 cells, but cell migration under these conditions does not lead to the initiation of DNA synthesis. Subsequent addition of serum initiates DNA synthesis in the migrating cells. The results establish that breaking contacts between quiescent 3T3 cells is not sufficient to initiate DNA synthesis. The DNA synthesis observed in migrating 3T3 cells in the typical "woundhealing" experiment presumably results because the migrating cells have an increased ability to use serum factors. Serum-factor requirements for "wound healing" in cultured 3T3 cells are discussed.     

Rapid method for isolation of normal human peripheral blood eosinophils on discontinuous Percoll gradients and comparison with neutrophils

Previous studies on human eosinophils often have used cells from patients with hypereosinophilia syndrome or parasitosis owing to the difficulty in isolating pure populations of eosinophils from normal individuals. In the present study, human eosinophils were isolated with a purity of 97%, with 70% recovery from normal individuals with blood eosinophil counts of less than 3%. Human eosinophils are denser than neutrophils, but the range of densities of the two cell types overlap, making purification of eosinophils by density-gradient centrifugation difficult. However, if neutrophils were exposed to the chemotactic peptide (f-Met-Leu-Phe), which did not stimulate eosinophils, the neutrophils' density decreased, shifting them away from the density of eosinophils. Whole normal blood anticoagulated with EDTA was incubated at 37 degrees C for 15 minutes with 10(-6) mol/L f-Met-Leu-Phe and then layered over a discontinuous Percoll gradient (65% and 75% in diluted phosphate-buffered saline) and centrifuged at 400 g for 25 minutes at 22 degrees C. The cell layer between the 65% and 75% Percoll was collected and washed, and hypotonic lysis was used to remove erythrocytes. This cell layer contained 97.3 +/- 0.7% eosinophils (N = 8) with a yield of 4.9 X 10(4) eosinophils per milliliter of whole blood, or 70% of the total eosinophil count. The isolated eosinophils were in a quiescent state but responded to Escherichia coli endotoxin- activated serum with shape change and chemotaxis, membrane depolarization, and reduced nitroblue tetrazolium (96.0 +/- 1.0%), when stimulated with phorbol myristate acetate. In phagocytic assays, 89.3 +/- 1.3% of the eosinophils ingested Candida albicans v 96.0% +/- 1.0% of neutrophils. In contrast, the eosinophils did not respond chemotactically, alter membrane potential, or reduce nitroblue tetrazolium when treated with f-Met-Leu-Phe, and studies with f-Met-Leu- [3H]Phe showed that normal eosinophils lacked expression of receptors for f-Met-Leu-Phe. In control studies, normal eosinophils that were not exposed to f-Met-Leu-Phe during purification also failed to respond to f-Met-Leu-Phe, indicating intrinsic differences between normal eosinophils and neutrophils. Thus, exposure of whole blood to f-Met-Leu- Phe, followed by separation on Percoll is a simple method for rapid isolation of normal human eosinophils.     

Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.     

B220- bone marrow progenitor cells from New Zealand black autoimmune mice exhibit an age-associated decline in Pre-B and B-cell generation

New Zealand Black (NZB) autoimmune mice exhibit progressive, age- dependent reduction in bone marrow pre-B cells. To ascertain the capacity of NZB bone marrow B220- cells to generate pre-B cells in a supportive environment, B-lineage (B220+) cell-depleted and T-cell- depleted bone marrow cells from NZB mice at 1 to 3, 6, and 10 to 11 months of age were adoptively transferred into irradiated (200R) C.B17 severe combined immunodeficient (SCID) mice. Bone marrow pre-B cells (sIgM- CD43[S7]- B220+) were assessed 3 and 10 weeks posttransfer. Pre- B cells and B cells were reconstituted in SCID recipients of older NZB progenitor cells by 10 weeks posttransplant, in contrast to the very low numbers of pre-B cells present in the donor bone marrow. However, B220- bone marrow progenitor cells from greater than 10-month-old NZB donors were deficient in the reconstitution of both pre-B and B cells in SCID recipients at 3 weeks post-transfer. This reflected a slower kinetics of repopulation, because older NZB-->SCID recipients had numbers of both pre-B and B cells similar to recipients of young NZB progenitor cells by 10 weeks posttransplant. Adoptive transfer of equal mixtures of BALB/c and older NZB bone marrow B220- progenitor cells into irradiated C.B17 SCID recipients failed to demonstrate active suppression. These results suggest that, with age, NZB bone marrow has reduced numbers and/or function of early B220- B-lineage progenitors. Consistent with this hypothesis, B220- bone marrow cells from older NZB mice were deficient in progenitors capable of yielding interleukin-7 (IL-7) responsive pre-B cells in vitro on stimulation with the pre-B- cell potentiating factor, insulin-like growth factor 1 (IGF-1).