Nobuko Kawaguchi

Autografting of fat pads has a long history in plastic and reconstructive surgery for augmentation of lost soft tissue. However, the results are disappointing because of absorption of the grafts with time. The fate of transplanted fat is linked to adipose precursor cells distributed widely in connective tissues. Adipocyte precursor cells can proliferate and mature into adipocytes even in the adult body depending on microenvironment. When reconstituted basement membrane, Matrigel, supplemented with more than 1 ng/ml bFGF was injected s.c. into 6-week-old mice, the neovascularization induced within 1 week was followed by migration of endogenous adipose precursor cells, and a clearly visible fat pad was formed. The pad grew until 3 weeks after the injection and persisted for at least 10 weeks. Such de novo adipogenesis was induced reproducibly by s.c. injection of Matrigel and bFGF over the chest, lateral abdomen, or head. Adipogenesis could be induced even in ear cartilage or in muscle. Thus, our results demonstrated that an abundant population of adipose precursor cells is distributed widely in connective tissues of the adult body and that they migrate into the neovascularized plug of Matrigel for proliferation and maturation. These results suggest a technique of augmenting lost soft tissue in plastic and reconstructive surgery.

Changes in cell shape are a morphological hallmark of differentiation. In this study we report that the expression of ADAM12, a disintegrin and metalloprotease, dramatically affects cell morphology in preadipocytes, changing them from a flattened, fibroblastic appearance to a more rounded shape. We showed that the highest levels of ADAM12 mRNA were detected in preadipocytes at the critical stage when preadipocytes become permissive for adipogenic differentiation. Furthermore, as assessed by immunostaining, ADAM12 was transiently expressed at the cell surface concomitant with the reduced activity of beta1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/beta1 integrin complexes in these preadipocytes. Overexpression of ADAM12 at the cell surface of 3T3-L1 preadipocytes achieved by transient transfection or retroviral transduction led to the disappearance of the extensive network of actin stress fibers that are characteristic of these cells, and its reorganization into a cortical network located beneath the cell membrane. The cells became more rounded, exhibited fewer vinculin-positive focal adhesions, and adhered less efficiently to fibronectin in attachment assays. Moreover, ADAM12-expressing cells were more prone to apoptosis, which could be prevented by treating the cells with beta1-activating antibodies. A reduced and re-organized fibronectin-rich extracellular matrix accompanied these changes. In addition, beta1 integrin was more readily extracted with Triton X-100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary for early adipocyte differentiation.

Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis.

Subcutaneous injection of reconstituted basement membrane (Matrigel) in combination with basic fibroblast growth factor induces de novo adipogenesis in which endogenous precursor cells invade the artificially formed Matrigel space, proliferate and differentiate to form adipose tissue. Since this adipogenesis offers us a novel approach for soft-tissue reconstruction without transplanting preadipocytes, the early process was examined by optical and electron microscopy. Formation of multiple layers of fibroblast-like cells at the surface of Matrigel implant was the first response of connective tissue. The cells within four to five layers proximal to Matrigel implant acquired a thick cytoplasm and an enlarged nucleus, and they invaded Matrigel space together with endothelial cells which caused neovascularization. Phagocytotic incorporation and digestion of Matrigel components by well-developed lysosomes appeared to be a stimulus of fibroblast-like cells to mature depending on proximity to Matrigel. The fibroblast-like cells often contacted to the outer surface of capillary over a large area and rapidly accumulated lipid droplets. Electron microscopy of the developing adipocytes showed a well-organized smooth endoplasmic reticulum and mitochondria. This investigation thus revealed the characteristics of adipocyte precursor cells, which can be recruited for regenerative engineering of soft tissues.