Hematopoietic disorders such as bone marrow failure, immune dysfunction, and certain cancers, may involve a link between the endochondral skeleton and marrow. The objective of this renewal is to provide a mechanistic basis for the skeleto-hematopoietic link, by addressing how a hematopoietic marrow environment may form by replacement of hypertrophic cartilage by bone and marrow during endochondral ossification (EO). This proposal stems from our transgenic (Tg) and null mice for collagen X, a matrix protein expressed in hypertrophic cartilage prior to EO. Murine skeleto-hematopoietic defects include growth plate compressions, osteopenia, marrow aplasia, and altered blood cell differentiation. We propose that collagen X provides a structural network that is stabilized by proteoglycans (PGs)/glycosaminoglycans (GAGs), and sequesters cytokines. Network disruption causes redistribution of growth plate components and a cytokine imbalance; the latter may cause the hemato-poietic defects. To test this hypothesis we will: 1) Identify the defective cellular component in the marrow environment of collagen X mice by co-cultures of hematopoietic stern cells (HSCs) with stromal cells, hypertrophic chondrocytes, and osteoblasts, and test if marrow transplantation can rescue the hematopoietic defects; 2) Identify which PGs/GAGs are altered in growth plate/marrow junctions by immunohistochemistry and electron microscopy, test if they bind collagen X by affinity coelectrophoreisis, and assess the role of these interactions in hematopoiesis by co-cultures of HSCs and hypertrophic chondrocytes GAG biosynthesis modulators; 3) Examine the relationship between the severity of the skeleto-hematopoietic phenotype and dysregulated cytokine expression by macrophage cytokine production, identify the defect in immune response initiation/regulation by endotoxin challenge, ascertain dysregulated cytokines by blot arrays, identify the tissue source of aberrant cytokine production, and induce/block the disease phenotype by injection of cytokines or neutralizing antibodies; and 4) Test if other murine models with altered hypertrophic cartilage (Grg5 null & Snell dwarfs) have similar hematopoietic defects caused by cytokine dysregulation. ? ?
