Hyaluronan Expressed by Bone Marrow Mesenchymal Cells Regulates Functions of the Hematopoietic Microenvironment

Abstract 1243

While the quantity and quality of transplanted hematopoietic stem cells (HSC) are important for the recovery of hematopoiesis, the functional status of the regulatory hematopoietic microenvironment is a critical parameter that determines the regenerative function of HSCs. The quality of the microenvironment, i.e. its ability to support hematopoiesis, may be compromised under pathological circumstances such as during disease development or as a result of therapeutic interventions. Thus, the hematopoietic microenvironment should be allowed to recover prior to HSC transplantation. To effectively prepare the marrow microenvironment for HSC transplantation it is important to understand which of the molecular pathways regulating the function of the microenvironment are disrupted under the specific pathological condition.

The involvement of hyaluronan (HA) in regulation of hematopoiesis has been previously suggested. However, whether HA contributes to the regulatory network of the hematopoietic microenvironment is not well understood. Since HA is highly susceptible to irradiation, which induces HA degradation and depolymerization leading to HA chain fragmentation and affecting its three-dimensional structure, sublethally irradiated mice (6Gy) were used to test the effect of exogenous HA on hematopoietic recovery. We found that administration of HA shortened the period of cytopenia compared to control mice which received vehicle only. To investigate whether the depletion of HA from the microenvironment has negative effects of hematopoietic homeostasis, knockout mice of three hyaluronan synthase genes (Has1, Has2, Has3) were generated as a mouse model of targeted HA deficiency in the hematopoietic microenvironment. Specifically, we generated double Has knockout (KO) mice (dHAS1/3 KO, Has1^–/–;Has3^–/–) and triple Has KO mice (tHAS1/2/3 KO, Prx1-Cre;Has2^flox/flox;Has1^–/–;Has3^–/–). In the following study, wild type (WT), dHAS1/3 and tHAS1/2/3 KO mice were sublethally irradiated (6Gy) and the dynamics of hematopoietic recovery were tested. We found that the recovery of leukocytes in tHAS1/2/3 KO mice was significantly delayed as compared to WT and dHAS1/3 KO mice. This finding suggests that the HA-deficient microenvironment cannot support hematopoietic recovery following irradiation. Additional tests demonstrated that the number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of tHAS1/2/3 KO mice as compared to WT and dHAS1/3/KO mice. In line with this observation, decreased hematopoietic activity was observed in long-term bone marrow cultures (LTBMC) from tHAS1/2/3 KO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dHAS1/3/KO cultures was the same. 4-methylumbelliferone (4-MU) 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 HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of HSC in response to SDF-1. In addition, pharmacological inhibition of HA synthesis decreased homing of transplanted HSC into the marrow and interactions of HSC with endothelial cells under conditional physiological shear stress.

Our findings demonstrate 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. Collectively, our results strongly suggest that HA is a biologically active component of the hematopoietic microenvironment and is involved in regulating hematopoietic homeostasis. Since some treatments or compounds reduce HA concentrations in tissues and some conditions are associated with increased levels of HA, it may prove clinically useful to monitor the dynamics of endogenous HA recovery to aid in identifying the optimal time for stem cell transplantation. Our data also suggest that biologically active exogenous HA polymers of the correct size, source, and conformation as well as HA synthesis inhibitors may have potential use in clinical hematology to correct misbalanced HA levels.