Perlecan Diversely Regulates the Migration and Proliferation of Distinct Cell Types in vitro.

Perlecan is a multifunctional component of the extracellular matrix. It shows different effects on distinct cell types, and therefore it is thought to show potential for therapies targeting multiple cell types. However, the full range of multifunctionality of perlecan remains to be elucidated. We cultured various cell types, which were derived from epithelial/endothelial, connective and muscle tissues, in the presence of either antiserum against perlecan or exogenous perlecan, and examined the effects of perlecan on cell migration and proliferation. Cell migration was determined using a scratch assay. Blocking of perlecan by anti-perlecan antiserum inhibited the migration of vascular endothelial cells (VECs) and bone marrow-derived mesenchymal stem cells, and exogenous perlecan added to the culture medium promoted the migration of these cell types. The migration of other cell types was inhibited or was not promoted by exogenous perlecan. Cell proliferation was measured using a water-soluble tetrazolium dye. When cells were cultured at low densities, perlecan blocking inhibited the proliferation of VECs, and exogenous perlecan promoted the proliferation of keratinocytes. In contrast, the proliferation of fibroblasts, pre-adipocytes and vascular smooth muscle cells cultured at low densities was inhibited by exogenous perlecan. When cells were cultured at high densities, perlecan blocking promoted the proliferation of most cell types, with the exception of skeletal system-derived cells (chondrocytes and osteoblasts), which were inhibited by exogenous perlecan. Our results provide an overview of the multiple functions of perlecan in various cell types, and implicate a potential role of perlecan to inhibit undesirable activities, such as fibrosis, obesity and intimal hyperplasia.

Disruption of endogenous perlecan function improves differentiation of rat articular chondrocytes in vitro.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) are necessary for normal cartilage development and chondrocyte differentiation. However, recent studies demonstrated that HSPG accelerate dedifferentiation and catabolism in chondrocytes from degenerative cartilage. In this study, we investigated the inhibitory effect of HSPG on chondrocyte differentiation in vitro. Rat articular chondrocytes were cultured at low (0.3 × 10(4) cells/cm(2) ) and high (1.5 × 10(5) cells/cm(2) ) density in the presence or absence of heparitinase I, an HS degrading enzyme. Cells cultured at low density dedifferentiated and exhibited an elongated morphology, and treatment with heparitinase I precluded cell elongation. Conversely, populations of chondrocytes cultured at high density exhibited either a dedifferentiated or differentiated phenotype. Glycosaminoglycan accumulation increased in heparitinase I-treated cells. To determine the function of perlecan, an important HSPG for cartilage development, in chondrocyte differentiation, rat chondrocyte cultures were exposed to an anti-perlecan antiserum to inhibit perlecan function. Western blotting analysis indicated that preventing perlecan activity increased type II collagen synthesis. Our results suggest that HSPG are negative regulators of chondrocyte differentiation in vitro and that perlecan contributes to chondrocyte dedifferentiation in vitro.

Diverse functions of perlecan in central nervous system cells in vitro.

Therapeutic treatment targeting one cell type is considered ineffective in remedying any injury to the central nervous system (CNS). Perlecan, a multi-functional, heparan sulfate proteoglycan, shows diverse effects on distinct cell types, suggesting that it is one of the candidates that can augment the regenerative mechanisms in the injured CNS. Therefore, we examined the functions of perlecan in CNS cells in vitro by using perlecan purified from bovine kidney. Perlecan-coated cell culture plates, unlike their type I/III collagen-coated counterparts, did not inhibit the adhesion of neural stem/progenitor cells (NS/PCs) and neurons. The coated perlecan and the perlecan added to the culture medium suppressed astrocyte proliferation; however, perlecan added to the medium promoted NS/PC proliferation. Neurons were promoted to extend their neurites on the perlecan-coated substrate, and perlecan added to the medium also showed a similar effect. NS/PC proliferation and neurite extension is a major regenerative reaction in CNS injury, whereas excess proliferation of astrocytes cause hypertrophy of glial scars, which repels neurons. Our in vitro study suggests that perlecan is an attractive candidate to promote regenerative mechanisms and to suppress reactions that hamper regenerative processes in cases of CNS injury.

Changes in the composition of the extracellular matrix accumulated by mesenchymal stem cells during in vitro expansion.

One of the approaches to preserve the properties of mesenchymal stem cells (MSCs) during in vitro expansion is to use cell culture substrates. MSCs are known to generate the extracellular matrix (ECM) proper to preserve their proliferative capacity in vitro, but extensive expansion is considered to deprive MSCs of the capacity to prepare such ECM. In order to examine the features of ECM proper that is required to preserve the proliferative capacity of MSCs, we analyzed the changes in the composition of ECM accumulated by MSCs during in vitro expansion. Biochemical and immunological analysis showed that collagen and laminin content decreased during expansion. Immunofluorescence and ultrastructural analyses showed that the ECM structure changed from a dynamic fibrous, porous and steric structure to a static, crammed, and planar one. The results of Western blotting analysis suggested loose intermolecular association in ECM molecules accumulated by extensively proliferated MSCs. The ECM prepared by extensively proliferated MSCs was less effective to recover their proliferative capacity than that prepared by less proliferated cells. Our results suggest that a cell culture substrate to expand MSCs requires abundance in collagen and basement membrane components, and steric, porous and fibrous structure in which ECM molecules are tightly associated.

Contrasting effect of perlecan on adipogenic and osteogenic differentiation of mesenchymal stem cells in vitro.

Perlecan, a basement membrane component, shows diverse functions in different organs and tissues. However, the role of perlecan in differentiation of mesenchymal stem cells (MSCs) has been barely investigated. In this study, we examined the effect of perlecan on adipogenic and osteogenic differentiation of MSCs in vitro by adding extrinsic perlecan to culture media or blocking the function of intrinsic perlecan expressed into culture media by differentiating MSCs. Extrinsic perlecan suppressed adipogenic differentiation; however, it promoted osteogenic differentiation. These functions were further confirmed by a study of blocking intrinsic perlecan. Perlecan treated with heparitinase-I also showed the suppressive effect on adipogenic differentiation. In contrast, the promotive effect on osteogenic differentiation was found to be heparan sulfate-dependent. Intrinsic perlecan was suggested to be effective at the late stage of adipogenic differentiation by a study of perlecan-blocking performed at distinct periods, but was suggested to be effective at the early stage of osteogenic differentiation. Our results showed perlecan has contrasting effect on adipogenic and osteogenic differentiation of MSCs due to its diverse actions. Based on these outcomes, we recognized that employing extrinsic perlecan or blocking intrinsic perlecan is effective for regulating adipogenic and osteogenic differentiation of MSCs by restricting its direction.