Endochondral ossification is the process where cartilage forms prior to ossification and in which new bone forms during both fracture healing and ectopic bone formation. Transitioning to ossification is a highly coordinated process between hypertrophic chondrocytes, vascular endothelial cells, osteoblasts and osteoclasts. A critical biological process that is central to the interactions of these various cell types is angiogenesis. Although it is well established that angiogenesis is crucial for fracture repair, less is known pertaining to the role of angiogenesis in ectopic bone formation. Furthermore, fracture repair models are complicated by extensive trauma, subsequent inflammatory responses and concurrent repair processes in multiple tissues. In order to more definitively characterize the relationship between angiogenesis and postnatal endochondral ossification, a model of ectopic bone formation was used. Human demineralized bone matrix (DBM) was implanted in immune-deficient mice (rag null (B6.129S7-Rag1tm1/MOM/J)) to induce ectopic bone. Inhibition of angiogenesis with either a small molecule (TNP-470) or a targeted biological (Vascular Endothelial Growth Factor Receptor type 2 [VEGFR2] blocking antibody) prevented ectopic bone formation by 83% and 77%, respectively. Most striking was that the progression of chrondrogenesis was halted during very early phases of chondrocyte differentiation between condensation and prehypertrophy (TNP-470) or the proliferative phase (VEGFR2 blockade) prior to hypertrophy, while osteoclast recruitment and resorption were almost completely inhibited. Our results demonstrate angiogenesis plays a developmental role in endochondral bone formation at a much earlier phase of chondrogenesis than suggested by prior findings.

Caveolae are cell membrane invaginations that are highly abundant in adipose tissue, endothelial cells and the lung. The formation of caveolae is dependent on the expression of various structural proteins that serve as scaffolding for these membrane invaginations. Cavin1 is a newly identified structural protein whose deficiency in mice leads to loss of caveolae formation and to development of a lipodystrophic phenotype. In this study, we sought to investigate the functional role of Cavin1 in the lung. Cavin1 deficient mice possessed dramatically altered distal lung morphology and exhibited significant physiological alterations, notably, increased lung elastance. The changes in distal lung architecture were associated with hypercellularity and the accumulation of lung macrophages. The increases in lung macrophages occurred without changes to circulating numbers of mononuclear cells and without evidence for increased proliferation. However, the increases in lung macrophages were associated with higher levels of macrophage chemotactic factors CXCL2 and CCL2 in BAL fluid from Cavin1−/− mice suggesting a possible mechanism by which these cells accumulate. In addition, lung macrophages from Cavin1−/− mice were larger and displayed measurable differences in gene expression when compared to macrophages from wild-type mice. Interestingly, macrophages were also increased in adipose tissue but not in liver, kidney or skeletal muscle from Cavin1−/− mice, and similar tissue specificity for macrophage accumulation was observed in lungs and adipose tissue from Caveolin1−/− mice. In conclusion, this study demonstrates an important role for Cavin1 in lung homeostasis and suggests that caveolae structural proteins are necessary for regulating macrophage number and phenotype in the lung.

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