Femoral microarchitecture and endosteal niche alterations in protein malnutrition: possible effects on hematopoiesis?

Abstract

Hematopoiesis is a dynamic process that leads to blood cells production. At the centre of this process sits the hematopoietic stem cells (HSC), which preferentially reside along the trabecular metaphysis of long bones. Evidences suggest that specific microenvironments could support HSC self-renewal and differentiation. The endosteal niche hosts HSC in quiescence or self-renewing state. Protein malnutrition (PM) is associated with pancytopenia, bone marrow (BM) hypoplasia and changes in bone microenvironment. In this way, we hypothesized that PM may change the endosteal niche with a consequent impairment in the number and function of long-term HSC (LT-HSC). To test this hypothesis, C57Bl/6J mice were randomly assigned in control and malnourished groups which received normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5 weeks period. We characterized femoral bone tissue changes in vivo by measuring bone mineral density (BMD) and ex vivo, using computed microtomography. Compared to controls, malnourished animals (MA) presented a decrease in the BMD of different regions of the femur and a significant decrease in the trabecular bone volume (BV/TV), and trabecular number (Tb.N) of the distal metaphysis of the femur, followed by an increase in the trabecular separation (Tb.Sp) and a prevalence of rod-like trabeculae. Furthermore, osteoblasts and LT-HSC were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry. We found a smaller amount of LT-HSC in the BM of MA. Osteoblasts from MA showed decreased mRNA expression of type I collagen and osteocalcin, however no difference was found on CXCL-12, angiopoetin, Jagged-2 or N-cadherin mRNA expression. Therefore, we may conclude that PM compromises trabecular bone structure and reduces the number of LT-HSC cells in the BM. Hematopoiesis is a dynamic process that leads to blood cells production. At the centre of this process sits the hematopoietic stem cells (HSC), which preferentially reside along the trabecular metaphysis of long bones. Evidences suggest that specific microenvironments could support HSC self-renewal and differentiation. The endosteal niche hosts HSC in quiescence or self-renewing state. Protein malnutrition (PM) is associated with pancytopenia, bone marrow (BM) hypoplasia and changes in bone microenvironment. In this way, we hypothesized that PM may change the endosteal niche with a consequent impairment in the number and function of long-term HSC (LT-HSC). To test this hypothesis, C57Bl/6J mice were randomly assigned in control and malnourished groups which received normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5 weeks period. We characterized femoral bone tissue changes in vivo by measuring bone mineral density (BMD) and ex vivo, using computed microtomography. Compared to controls, malnourished animals (MA) presented a decrease in the BMD of different regions of the femur and a significant decrease in the trabecular bone volume (BV/TV), and trabecular number (Tb.N) of the distal metaphysis of the femur, followed by an increase in the trabecular separation (Tb.Sp) and a prevalence of rod-like trabeculae. Furthermore, osteoblasts and LT-HSC were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry. We found a smaller amount of LT-HSC in the BM of MA. Osteoblasts from MA showed decreased mRNA expression of type I collagen and osteocalcin, however no difference was found on CXCL-12, angiopoetin, Jagged-2 or N-cadherin mRNA expression. Therefore, we may conclude that PM compromises trabecular bone structure and reduces the number of LT-HSC cells in the BM.