Regulation of the spatial organization of mesenchymal connective tissue: effects of cell-associated versus released isoforms of platelet-derived growth factor

Abstract

Platelet-derived growth factor (PDGF), a mitogen and chemoattractant for mesenchymal cells, occurs as cell-associated or released isoforms. To investigate their in vivo role, human keratinocytes, which normally synthesize both types of PDGF, were genetically modified to overexpress either wild-type PDGF-B (cell-associated) or the truncation mutant PDGF-B211 (released). Cells expressing the mutant isoform released 20 times more PDGF (145 ng/hour/10(7) cells) than cells expressing the wild-type isoform (6 ng/ hour/10(7) cells). When grafted as epithelial sheets onto athymic mice, modified cells formed a stratified epithelium and induced a connective tissue response that differed depending on the PDGF isoform expressed. Expression of PDGF-B211 induced a thick connective tissue with increased numbers of fibroblasts, mononuclear cells, and blood vessels evenly distributed throughout the connective tissue layer, whereas expression of PDGF-B induced a zone of fibroblasts and mononuclear cells localized to the interface of the epidermis and connective tissue, which often disrupted the continuity of the basement membrane. Immunostaining revealed that wild-type PDGF protein was deposited in the basement membrane region. These data suggest that the different binding properties of PDGF isoforms control the spatial organization of cellular events in regenerating mesenchymal tissue in vivo.

Figure 1.

Modified keratinocytes synthesize PDGF-B proteins. A: Time course of PDGF synthesis and release was determined by removing portions of the culture medium of a confluent culture of modified keratinocytes as indicated over a 4-day period, and PDGF-BB protein was measured by ELISA. B: Cell-associated PDGF was determined by treatment of cells with 1 mol/L NaCl in DMEM and measuring released PDGF by ELISA.

Figure 2.

Wild-type and mutant PDGF produced by keratinocytes are bioactive. Culture medium conditioned for 30 hours by unmodified and modified keratinocytes was harvested in DMEM 5% PPP, and PDGF protein levels were measured by ELISA. Various doses of keratinocyte-produced PDGF were assayed for the ability to stimulate [³H]thymidine incorporation by quiescent Swiss 3T3 cells. Triplicate samples and ±SEM are presented.

Figure 3.

Wild-type and mutant PDGF induce different connective tissue responses in vivo. Cultures of confluent epithelia generated from unmodified (A, D, G, and J), MFG-PDGF-B (B, E, H, and K), or MFG-PDGF-B211 (C, F, I, and L) transduced keratinocytes, were grafted to athymic mice. Seven days later, grafts were fixed and sections (5 μm) stained with H&E (A to F) and visualized under low (A to C) and high (D to F) magnification. Other grafts were frozen, and cryosections were stained for laminin (D to F) or hPDGF-BB (G to I). Note the lack of continuity of the basement membrane in grafts overexpressing wild-type PDGF-B (H) and the deposition of PDGF-BB in nondisrupted regions of the basement membrane (K) when visualized under high magnification.

Figure 4.

Thickness of connective tissue subjacent to grafts expressing PDGF-B211 is enhanced. The thickness of the tissue subjacent to the 7-day-old grafts of three strains of keratinocytes were measured by image analysis of H&E sections. For each field of view, the area of the mouse connective tissue layer subjacent to the human epithelium was determined and the thickness computed. Measurements were taken for 10 fields of view per graft, and each point represents one graft. From each group the mean is presented. *P < 0.05; strain D, n = 8; strain C, n = 8; strain A, n = 7.

Figure 5.

Wild-type PDGF-BB is deposited in the basement membrane zone. Cryosections of 7-day grafts of cells expressing wild-type PDGF-B were simultaneously stained with a rabbit antibody to hPDGF-BB (B and D) and mouse antibodies to either collagen IV (A) or laminin (C). Staining was separately visualized with a fluorescein-conjugated affinity-purified goat-anti-rabbit IgG antibody or lissamine-rhodamine-conjugated affinity-purified goat anti-mouse IgG antibody.