On Bone-Forming Cells and Blood Vessels in Bone Development

Cell Metabolism

Previews intracellular calcium levels in cells from a patient with IPAH obtained at the time of lung transplantation. Taken together, these findings provide a link between an intrinsic metabolic abnormality of pulmonary vascular cellular mitochondria, affecting acute vasoconstriction and resistance to apoptosis, and vascular remodeling. While the studies by Sutendra et al. provide important connections between a variety of independent observations and lead to a plausible mechanism of upstream pathogenesis of PAH, there are a number of unanswered questions: 1. The degree of vasculopathy seen in chronic hypoxia is quite modest compared to that observed in PAH, and the former is generally reversible upon restoration of normoxic conditions, while the latter is, at least for now, irreversible. Accordingly, the extrapolation from hypoxic pulmonary hypertension to PAH is tenuous. 2. The translation of findings in animal models of PAH, particularly monocrotaline-induced PAH, to the clinical arena has been disappointing, with a number of drugs or other interventions demonstrating dramatic results in animals and either no effect or, at best, a modest effect in patients. 3. The role of the endothelial cell in the pathogenesis of PAH remains

unclear, and these experiments did not include studies of these cells. Thus, it remains unclear whether endothelial dysfunction is an early or late event in the pathogenesis of PAH or whether an endothelial-smooth muscle interaction early in the disease process is important. 4. The impact on proliferation of human cells exposed to the metabolic inhibitors was not investigated, an observation that would have provided a much stronger link between altering intracellular calcium and remodeling. 5. The authors examined cells from a single patient with ‘‘IPAH.’’ Whether their observations will be reinforced with experiments from cells obtained from other patients with this or other forms of PAH is unclear. Despite these limitations, Sutendra et al. have advanced our understanding of the pathogenesis of pulmonary vascular disease and have pointed us in the direction of a new and selective target for therapy. While more work needs to be done before a formal clinical trial of metabolic inhibitors should be entertained, the prospect of reversing the vasculopathy of PAH is exciting and brings a new set of colleagues—experts in metabolism—into the battle.

REFERENCES Chin, K.M., and Rubin, L.J. (2008). J. Am. Coll. Cardiol. 51, 1527–1538. Krick, S., Platoshyn, O., McDaniel, S.S., Rubin, L.J., and Yuan, J.X. (2001). Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L887–L894. Morrell, N.W. (2010). Adv. Exp. Med. Biol. 661, 251–264. Rai, P.R., Cool, C.D., King, J.A., Stevens, T., Burns, N., Winn, R.A., Kasper, M., and Voelkel, N.F. (2008). Am. J. Respir. Crit. Care Med. 178, 558– 564. Remillard, C.V., Tigno, D.D., Platoshyn, O., Burg, E.D., Brevnova, E.E., Conger, D., Nicholson, A., Rana, B.K., Channick, R.N., Rubin, L.J., et al. (2007). Am. J. Physiol. Cell Physiol. 292, C1837– C1853. Sutendra, G., Bonnet, S., Rochefort, G., Haromy, A., Folmes, K.D., Lopaschuk, G.D., Dyck, J.R., and Michelakis, E.D. (2010). Sci. Transl. Med. 2, 44ra58. Yu, Y., Fantozzi, I., Remillard, C.V., Landsberg, J.W., Kunichika, N., Platoshyn, O., Tigno, D.D., Thistlethwaite, P.A., Rubin, L.J., and Yuan, J.X.-J. (2004). Proc. Natl. Acad. Sci. USA 101, 13861– 13866. Yuan, J.X.J., and Rubin, L.J. (2005). Circulation 111, 534–538. Yuan, X.J., Goldman, W.F., Tod, M.L., Rubin, L.J., and Blaustein, M.P. (1993). Am. J. Physiol. 264, L116–L123. Yuan, J.X.-J., Aldinger, A.M., Juhaszova, M., Wang, J., Conte, J.V., Jr., Gaine, S.P., Orens, J.B., and Rubin, L.J. (1998). Circulation 98, 1400– 1406.

On Bone-Forming Cells and Blood Vessels in Bone Development Claire Clarkin1 and Bjorn R. Olsen1,* 1Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2010.09.009

Replacement of nonvascular cartilage by bone and bone marrow is a critical step in bone development. In a recent issue of Developmental Cell, Maes et al. (2010) report that a distinct population of immature precursors of bone-forming cells migrate into the cartilage in intimate association with invading blood vessels. The development of most bones, such as bones in the limbs and spine, proceeds via a two-stage process known as endochondral ossification. The architectural

modeling of a bone takes place in the location of the future bone via assembly of a template. The template consists of hyaline cartilage, a nonvascular tissue

314 Cell Metabolism 12, October 6, 2010 ª2010 Elsevier Inc.

composed of chondrocytes dispersed within a complex extracellular matrix. As the template grows and takes on the shape of the future bone, chondrocytes

Cell Metabolism

Previews in the central region of the they could have additional template stop proliferating; fates and functions. express a characteristic set of Remarkably, as the two transcription factors, cytocell populations were folkines, and matrix molecules; lowed during endochondral increase in size (become ossification, 70% of Osx/ hypertrophic); and activate lacZ+ cells were found apoptotic cell-death mechain the spongy/trabecular nisms. These changes set in bone region inside the motion a process that results developing bone, whereas in the conversion of the Col1/lacZ+ cells remained nonvascular cartilage template predominantly on the to an organ with compact outside, in the cortical rebone tissue on the surface gion. Thus, a major subset (cortical bone) and spongy of Osx/lacZ+ cells serve (trabecular) bone tissue and as precursors of the osteohighly vascularized bone blasts that form the primary marrow inside. During the past spongiosa. Maes et al. Figure 1. Schematic Drawing of the Middle Portion of a Developing Endochondral Bone 25 years, a number of signifi(2010) also examined As described in the text, mature osteoblasts (Col1/lacZ+ cells) form cortical bone cant studies have provided whether hypertrophic chonon the outside, while immature precursors (Osx/lacZ+ cells) invade the cartilage insights into the molecular drocytes make a contribuand reach the primary ossification center in association with blood vessels. mechanisms regulating differtion to trabecular bone entiation of chondrocytes and formation during endoosteoblasts from mesenchymal precursor and start synthesizing extracellular com- chondral ossification, a possibility that cells and signals that control penetration ponents such as collagen type I. Taking has long been considered, by following of blood vessels into hypertrophic carti- advantage of the difference in timing of the fate of hypertrophic chondrocytes. lage during endochondral ossification. Osterix and collagen I expression, Maes They found that although some of the However, the origin of the osteoblastic et al. (2010) generated two transgenic chondrocyte-like cells at the cartilage/ cells that produce the spongy/trabecular mouse lines in which Cre recombinase perichondrial junction may contribute to bone (also referred to as the primary (controlled either by the Osterix or bone formation, the majority of the hyperspongiosa) in which hematopoietic bone a collagen I promoter) could be activated trophic chondrocytes do not become marrow niches are established has re- in a tamoxifen-dependent manner to osteoblasts in the central region of the mained uncertain. Many cellular sources induce expression of b-galactosidase/ primary spongiosa, at least in mice (and, have been considered, including peri- lacZ in osteoblastic lineage cells. By as pointed out by Maes et al., in the chondrial cells on the cartilage surface, controlling the timing of exposure of ‘‘time span of our studies’’). hypertrophic chondrocytes, pericytes developing transgenic embryos in the An important question raised by these associated with blood vessels pene- two lines to a pulse of tamoxifen relative data is the mechanism by which only trating into the cartilage, and progenitor to the timing of endochondral ossification immature osteoblastic cells are recruited cells circulating in the blood. Experi- in the limbs, the investigators labeled from the perichondrium into the primary mental support for the notion that osteo- early- (Osx/lacZ+) and late-stage (Col1/ ossification center together with vascular blasts may be generated from all these lacZ+) osteoblastic lineage cells as they endothelial cells and osteoclasts. Based sources under various conditions exists, differentiated in the perichondrial regions on previous studies (Zelzer et al., 2001, but no study providing definitive identifi- of developing bones. Electron micros- 2004) demonstrating that hypertrophic cation of the major origin of the cells that copy suggested that about 60% of the chondrocytes express high levels of form the primary spongiosa during bone Osx/lacZ+ cells were immature cells VEGFA, a potent stimulator of both endoformation has been published, until now. (large nuclei and sparse endoplasmic thelial and osteoclastic migration, it In a recent study, Maes et al. (2010) reticulum), and about a third of these cells seems likely that the mechanism includes advance the field by presenting compel- were found in pericyte-like fashion along VEGF-associated activities. One possiling data in support of the conclusion endothelial cells in blood vessels. The bility suggested by the present study is that osteoblasts in the primary spongiosa remaining Osx/lacZ+ cells were classified piggybacking of immature osteoblast are derived from immature precursors as early osteoblasts. In contrast, Col1/ precursors on vascular endothelial cells that invade into the cartilage in intimate lacZ+ cells appeared to be fully differenti- as the vessels sprout and extend from association with blood vessels (Figure 1). ated osteoblasts, engaged in the process the perichondrial region toward the hyperDifferentiation of osteoblasts from of forming cortical bone. Not surprisingly, trophic cartilage inside the forming bone. mesenchymal cells requires the transcrip- further analyses indicated that some of Interestingly, Maes et al. (2010) provide tion factor Osterix (Osx) (Nakashima et al. the immature Osx/lacZ+ cells serve as preliminary data suggesting that Osx2002). Osterix is expressed at an early precursors of the mature osteoblasts. positive cells express high levels of stage in the osteoblastic lineage, before However, the intimate association with VEGF and of Angiopoietin-1 and PDGRb, cells reach the fully differentiated stage blood vessels raised the possibility that molecules that play a role in interactions Cell Metabolism 12, October 6, 2010 ª2010 Elsevier Inc. 315

Cell Metabolism

Previews between endothelial cells and pericytes. Finally, it is possible that immature osteoblastic progenitors differ from mature osteoblasts in that they, like vascular endothelial cells and osteoclasts, are able to respond to migratory signals, such as VEGF. This study also provides a basis for addressing problems that go beyond the realm of bone formation. For example, with the osteoprogenitor population responsible for the formation of the trabecular bone inside endochondral bones defined and sorting techniques for the isolation of these cells from mixed populations available, the question of whether or how these cells and their extracellular matrix products contribute to the formation of hematopoietic stem cell niches may now be addressed using the subcap-

sular kidney assay that Chan et al. (2009) recently used to demonstrate that endochondral ossification is required for hematopoietic stem cell niche formation. Given that Nestin-positive mesenchymal stem cells in bone marrow were recently reported to constitute an essential component in the hematopoietic stem cell niche (Mendez-Ferrer et al., 2010), it will be important to examine whether they interact with or are related to these Osterixexpressing immature cells that give rise to the osteoblastic cells forming the primary spongiosa in endochondral bone.

Maes, C., Kobayashi, T., Selig, M.K., Torrekens, S., Roth, S.I., Mackem, S., Carmeliet, G., and Kronenberg, H.M. (2010). Dev. Cell 19, 329–344.

Mendez-Ferrer, S., Michurina, T.V., Ferraro, F., Mazloom, A.R., MacArthur, B.D., Lira, S.A., Scadden, D.T., Ma’ayan, A., Enikolopov, G.N., and Frenette, P.S. (2010). Nature 466, 829–834.

Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R., and deCrombrugghe, B. (2002). Cell 108, 17–29.

Zelzer, E., Glotzer, D.J., Hartmann, C., Thomas, D., Fukai, N., Soker, S., and Olsen, B.R. (2001). Mech. Dev. 106, 97–106.

REFERENCES Chan, C.K.F., Chen, C.C., Luppen, C.A., Kraft, D.L., Kim, J.B., DeBoer, A., Wei, K., and Weissman, I.L. (2009). Nature 457, 490–494.

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Zelzer, E., Mamluk, R., Ferrara, N., Johnson, R.S., Schipani, E., and Olsen, B.R. (2004). Development 131, 2161–2171.