Jonni S. Moore

Hyperbaric oxygen stimulates vasculogenic stem cell growth and differentiation in vivo

We hypothesized that oxidative stress from hyperbaric oxygen (HBO(2), 2.8 ATA for 90 min daily) exerts a trophic effect on vasculogenic stem cells. In a mouse model, circulating stem/progenitor cell (SPC) recruitment and differentiation in subcutaneous Matrigel were stimulated by HBO(2) and by a physiological oxidative stressor, lactate. In combination, HBO(2) and lactate had additive effects. Vascular channels lined by CD34(+) SPCs were identified. HBO(2) and lactate accelerated channel development, cell differentiation based on surface marker expression, and cell cycle entry. CD34(+) SPCs exhibited increases in thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3, phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and stromal cell-derived factor-1. Cell recruitment to Matrigel and protein synthesis responses were abrogated by N-acetyl cysteine, dithioerythritol, oxamate, apocynin, U-0126, neutralizing anti-vascular endothelial growth factor, or anti-stromal cell-derived factor-1 antibodies, and small inhibitory RNA to Trx reductase, lactate dehydrogenase, gp91(phox), HIF-1 or -2, and in mice conditionally null for HIF-1 in myeloid cells. By causing an oxidative stress, HBO(2) activates a physiological redox-active autocrine loop in SPCs that stimulates vasculogenesis. Thioredoxin system activation leads to elevations in HIF-1 and -2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on SPCs.

Lactate Stimulates Vasculogenic Stem Cells via the Thioredoxin System and Engages an Autocrine Activation Loop Involving Hypoxia-Inducible Factor 1

ABSTRACT The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34+ SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34+ cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34+ SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45+ leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors.

Flow cytometric measurement of circulating endothelial cells: The effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations

Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition, numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD), we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs), EPCs, and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset.

Peripheral blood MDS score: A new flow cytometric tool for the diagnosis of myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders diagnosed using morphologic and clinical findings supported by cytogenetics. Because abnormalities may be subtle, diagnosis using these approaches can be challenging. Flow cytometric (FCM) approaches have been described; however the value of bone marrow immunophenotyping in MDS remains unclear due to the variability in detected abnormalities. We sought to refine the FCM approach by using peripheral blood (PB) to create a clinically useful tool for the diagnosis of MDS.

CD34 + , kit + , rhodamine123 low phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells

We hypothesized that human hematopoietic cells displaying a CD34+, kit+, rhodamine123low phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123low cells comprised from 0.05–0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33−, CD38−, CD20−, and glycophorin A−. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 × 103 candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine–human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.

Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis

Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum‐free cell‐culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum‐free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL‐3 was TPO able to stimulate a small increase (∼25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34+ cells were cultured for 7 d in serum‐free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre‐cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit‐erythroid (BFU‐E) colonies as cells which were pre‐cultured in the absence of TPO. Further, when primitive CD34+, Kit+ MNC were cultured for 3–7 d under serum‐free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.

Replacement of graft-resident donor-type antigen presenting cells alters the tempo and pathogenesis of murine cardiac allograft rejection.

BACKGROUND Graft-resident antigen presenting cells (APCs) are potent stimulators of the alloresponse. To test whether replacement of graft-resident donor-type APCs with those of recipient-type alters allorecognition and the pathogenesis of both acute and chronic rejection, we created chimeric hearts for transplantation into naive recipients. METHODS To replace donor-type APCs with those of recipient-type, chimeric animals were created by bone marrow transplantation (BMT) in fully allogeneic mouse and rat strain combinations. The degree of APC replacement in chimeric organs was assessed phenotypically and functionally. Chimeric hearts were transplanted heterotopically into untreated recipients. RESULTS Flow cytometric and immunohistochemical analysis did not detect residual bone marrow recipient-type APCs in mouse BMT chimeras. Although semi-quantitative reverse transcription polymerase chain reaction detected 0.001-0.01% residual cells, APCs isolated from chimeric organs were functionally unable to stimulate donor-type cells. When transplanted into naive recipients, chimeric mouse hearts had significantly prolonged survival but were nevertheless rejected acutely. Similar results were obtained in the ACI --> LEW rat strain combination. However, in the PVG --> DA rat model, the majority of chimeric hearts survived >100 days and all long-surviving hearts developed cardiac allograft vasculopathy. CONCLUSIONS BMT leads to near complete replacement of organ-resident APCs. The virtual absence of donor-type APCs in chimeric hearts delays or prevents acute rejection in a strain-dependent manner. In contrast, this type of graft modification does not prevent cardiac allograft vasculopathy. This suggests that, although the CD4+ direct pathway may play a role in acute rejection, it is not essential for the development of chronic rejection in rodent cardiac allografts.

Disruption of the structure of the Golgi apparatus and the function of the secretory pathway by mutants G93A and G85R of Cu, Zn superoxide dismutase (SOD1) of familial amyotrophic lateral sclerosis

The Golgi apparatus of motor neurons (GA) is fragmented in sporadic amyotrophic lateral sclerosis (ALS), in familial ALS with SOD1 mutations, and in mice that express SOD1G93A of familial ALS, in which it was detected months before paralysis. In paralyzed transgenic mice expressing SOD1G93A or SOD1G85R, mutant proteins aggregated not only in the cytoplasm of motor neurons, but also in astrocytes and oligodendrocytes. Furthermore, aggregation of the G85R protein damaged astrocytes and was associated with rapidly progressing disease. In order to gain insight into the functional state of the fragmented GA, we examined the effects of S0D1 mutants G93A and G85R in Chinese Hamster Ovary Cells (CHO). In contrast to cells expressing the wt and G93A, the G85R expressers had no SOD1 activity. However, cells expressing both mutants, and to a lesser degree the wt, showed decreased survival, fragmentation of the GA, and dysfunction of the secretory pathway, which was assessed by measuring the amount of cell surface co-expressed CD4, a glycoprotein processed through the GA. The G93A and wt proteins were partially recovered in detergent insoluble fractions; while the recovery of G85R was minimal. Both mutants showed equal reductions of cell survival and function of the secretory pathway, in comparison to the wt and cells expressing mutant alsin, a protein found in rare cases of fALS. These results are consistent with the conclusion that the two SOD1 mutants, by an unknown mechanism, promote the dispersion of the GA and the dysfunction of the secretory pathway. This and other in vitro models of mutant SOD1 toxicity may prove useful in the elucidation of pathogenetic mechanisms.

A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes

Murine lymphocytes readily undergo spontaneous and glucocortocoid-induced apoptosis in vitro. It has been previously demonstrated that during apoptosis, many cell types including lymphocytes, enzymatically cleave their DNA, thus demonstrating a sub-G0 DNA peak when stained with propidium iodide and analyzed by flow cytometry. In a mixed population, it is often desirable to phenotypically identify distinct populations or subsets undergoing apoptosis, thus requiring multiparameter analysis of surface phenotype and DNA content. Paraformaldehyde fixation procedures, although common for surface evaluation, have not been extensively used in methods quantifying apoptosis. To measure apoptosis in a mixed lymphocyte population, we evaluated a gentle detergent permeabilization and paraformaldehyde fixation procedure combined with propidium iodide (PI) DNA staining, adapted from existing methods for cell cycle studies. With this method and rigorous gating techniques which we defined, we detected both apoptotic and debris fractions within the sub-G0 cell cycle region of a glucocortocoid-treated murine lymphocyte cell line. Using this cell line, WEHI 231.7, as a lymphocyte model, we developed a logical gating strategy to exclude debris from analysis. We further demonstrated that apoptosis in freshly isolated murine lymphocytes detected with paraformaldehyde fixation and PI staining was quantitatively comparable to PI staining with ethanol fixation, or nick translation labeling of DNA strand breaks (TUNEL). Finally, using fresh murine spleen cells, we demonstrated that paraformaldehyde fixation preserves surface protein staining, allowing multiparameter analysis of immunophenotype and apoptotic or cell cycle status in a mixed lymphocyte population. Thus, this method offers an inexpensive and technically simple alternative for assessing apoptosis and surface phenotype.

Endothelial microparticles: Sophisticated vesicles modulating vascular function

Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation.