Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis.

The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment.

Hyaluronan in the healthy and malignant hematopoietic microenvironment.

The fate of both endogenous and transplanted stem cells is dependent on the functional status of the regulatory local microenvironment, which is compromised by disease and therapeutic intervention. The glycosaminoglycan hyaluronan (HA) is a critical component of the hematopoietic microenvironment. We summarize recent advances in our understanding of the role of HA in regulating mesenchymal stem cells, osteoblasts, fibroblasts, macrophages, and endothelium in bone marrow (BM) and their crosstalk within the hematopoietic microenvironment. HA not only determines the volume, hydration, and microfluidics of the BM interstitial space, but also, via interactions with specific receptors, regulates multiple cell functions including differentiation, migration, and production of regulatory factors. The effects of HA are dependent on the polymer size and are influenced by the formation of complexes with other molecules. In healthy BM, HA synthases and hyaluronidases form a molecular network that maintains extracellular HA levels within a discrete physiological window, but HA homeostasis is often perturbed in pathological conditions, including hematological malignancies. Recent studies have suggested that HA synthases may have functions beyond HA production and contribute to the intracellular regulatory machinery. We discuss a possible role for HA synthases, intracellular and extracellular HA in the malignant BM microenvironment, and resistance to therapy.

Homing of neural stem cells from the venous compartment into a brain infarct does not involve conventional interactions with vascular endothelium.

Human neural stem cells (hNSCs) hold great potential for treatment of a wide variety of neurodegenerative and neurotraumatic conditions. Heretofore, administration has been through intracranial injection or implantation of cells. Because neural stem cells are capable of migrating to the injured brain from the intravascular space, it seemed feasible to administer them intravenously if their ability to circumvent the blood-brain barrier was enhanced. In the present studies, we found that interactions of hNSCs in vitro on the luminal surface of human umbilical vein endothelial cells was enhanced following enforced expression of cutaneous lymphocyte antigen on cell surface moieties by incubation of hNSCs with fucosyltransferase VI and GDP-fucose (fhNSCs). Interestingly, ex vivo fucosylation of hNSCs not only did not improve the cells homing into the brain injured by stroke following intravenous administration but also increased mortality of rats compared with the nonfucosylated hNSC group. Efforts to explain these unexpected findings using a three-dimensional flow chamber device revealed that transmigration of fhNSCs (under conditions of physiological shear stress) mediated by stromal cell-derived factor 1α was significantly decreased compared with controls. Further analysis revealed that hNSCs poorly withstand physiological shear stress, and their ability is further decreased following fucosylation. In addition, fhNSCs demonstrated a higher frequency of cellular aggregate formation as well as a tendency for removal of fucose from the cell surface. In summary, our findings suggest that the behavior of hNSCs in circulation is different from that observed with other cell types and that, at least for stroke, intravenous administration is a suboptimal route, even when the in vitro rolling ability of hNSCs is optimized by enforced fucosylation.

A novel three-dimensional flow chamber device to study chemokine-directed extravasation of cells circulating under physiological flow conditions.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

Hyaluronan inhibits postchemotherapy tumor regrowth in a colon carcinoma xenograft model.

Bone marrow hypoplasia and pancytopenia are among the most undesirable sequelae of chemotherapy for the treatment of cancer. We recently showed that hyaluronan (HA) facilitates hematopoietic recovery in tumor-free animals receiving chemotherapeutic agents. However, following a chemotherapeutic regimen in tumor-bearing animals, it is possible that residual tumor cells might respond to systemic injections of HA. Thus, in this study, we investigated the effect of HA on the regrowth of residual tumor cells following chemotherapy. As a model, we used the HCT-8 human colon carcinoma cell line, which expresses the HA receptor CD44, binds exogenous HA, and is susceptible to a chemotherapy protocol containing irinotecan and 5-fluorouracil in a human/mouse xenograft model. HCT-8 cells were implanted in severe combined immunodeficient mice, followed by irinotecan/5-fluorouracil treatment. After three rounds of chemotherapy, residual tumors were allowed to regrow in the presence or absence of HA. The dynamics of tumor regrowth in the group treated with HA was slower compared with the control group. By week 5 after tumor implantation, the difference in the size of regrown tumors was statistically significant and correlated with lower proliferation and higher apoptosis in HA-treated tumors as compared with controls. This finding provides evidence that HA treatment does not stimulate but delays the growth of residual cancer cells, which is an important parameter in establishing whether the use of HA can enhance current chemotherapeutic strategies.