Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture.

OBJECTIVE: The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. METHODS: Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). RESULTS: Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. CONCLUSIONS: Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

The fibroproliferative response of arterial smooth muscle cells to balloon catheter injury is associated with increased hyaluronidase production and hyaluronan degradation.

Hyaluronan (HA) is a glycosaminoglycan found in greatest amounts in the extra-cellular matrix of loose connective tissue. HA has been shown to be closely involved in arterial smooth muscle cell (ASMC) proliferation and migration. No studies have examined the degradation of HA in the vessel wall during proliferation of ASMC. The aim of our study was to determine whether HA degradation was modulated in the injured rat aorta with a catheter balloon. To evaluate HA degradation we quantified the activity of the enzyme which degrades HA (hyaluronidase) and determined HA molecular mass in the aorta. Aorta was analyzed in sham operated aorta (D0) and 14 (D14) days after injury. Intima-media wet weight and DNA content, a parameters reflecting ASMC response to injury, were significantly increased at D14 (+35.5 and +40.8%). HA increased at D14 (+87%) and was mainly expressed in the neointima. Hyaluronidase activity also increased in the aorta at D14 (+25.5%). In the normal aorta, HA was mainly present in a high molecular mass form (2000 kDa). Two low molecular mass HA were also detected (29 and <20 kDa). At D14, the form of 2000 kDa was dramatically increased in comparison to that in normal aorta. In addition, the injured aorta contained a large number of low molecular mass form of HA. To know whether hyaluronidase production in the injured aorta was associated with appearance of new isoforms, we determined the molecular mass of this enzyme. Only one form of hyaluronidase (78 kDa) was present in both groups (D0 and D14). In conclusion, the proliferative response of ASMC to injury in the rat was found to be associated with increased HA degradation.

Increased hyaluronan and hyaluronidase production and hyaluronan degradation in injured aorta of insulin-resistant rats.

Diabetic patients have a greater incidence of restenosis, which has been shown to be related to exaggerated intimal hyperplasia. Hyaluronan (HA) has been shown to be closely involved in arterial smooth muscle cell proliferation and migration, which provoke intimal hyperplasia after balloon catheter injury. Our aim was to determine the effect of fructose feeding, which produces certain characteristics of non-insulin-dependent diabetes (ie, insulin resistance, hyperinsulinemia, and hypertriglyceridemia), on production of HA and hyaluronidase and degradation of HA in rat aorta. Treated rats received fructose (25% in tap water) 12 weeks before balloon catheter injury and 14 days afterward. Fructose-fed rats had hyperinsulinemia and hypertriglyceridemia. Injury increased intima-media wet weight (7.5%) and DNA content (20%) in control rats. This increase was significantly greater in fructose-fed rats (22% for wet weight and 34% for DNA content) and was associated with greater HA and hyaluronidase production (123% and 41%, respectively) than in control rats (49% and 7%, respectively). Determination of HA molecular mass showed that balloon catheter injury increased the number of HA fragments in the aorta of control rats. Normal aorta of fructose-fed rats contained more HA fragments than that of control rats. Injury to the aorta of fructose-fed rats increased HA fragments and induced the appearance of a very-high-molecular-mass (>2000 kDa) HA. In conclusion, fructose treatment, which induced hyperinsulinemia and hypertriglyceridemia, increased HA and hyaluronidase production and HA degradation in injured aorta. This finding suggests that HA, which has been shown to play a crucial role in proliferation and migration of arterial smooth muscle cells, may be involved in the promotional effect of long-term fructose feeding on arterial wall reaction to injury.

Effects of diabetes and insulin treatment of diabetic rats on hyaluronan and hyaluronectin production in injured aorta.

The present study was conducted to determine the effect of diabetes with and without insulin treatment on the production of hyaluronen (HA) and distribution of hyaluronectin (HN) in the rat aorta 14 days after injury with a catheter balloon. Injury increased intima-media wet weight (+11%) and DNA content (+37.5%). This increase was slightly enhanced in untreated diabetic rats (+14.7% for wet weight and +48.9% for DNA content) and was significantly greater in diabetic rats treated with insulin (+28.9% for wet weight and +54% for DNA content). HA content increase in the injured aorta of nondiabetic rats (+43.6%) was similar in untreated diabetic (+44.7%) and more pronounced in diabetic rats treated with insulin (+91.3%). HA was markedly expressed in the neointima of nondiabetic rats, particularly near the lumen of the aorta. In untreated diabetic rats, HA was present throughout the neointima and not mainly close to the lumen. HA staining in the neointima of diabetic rats treated with insulin was similar to that in nondiabetic rats. HN was strongly expressed throughout the neointima of all groups. Injury enhanced the production of a high molecular mass HN (>400 kDa); this was not observed either in untreated or in insulin-treated diabetic rats. In conclusion, insulin treatment promoted the proliferative response of aorta to injury and this was associated mainly with increased HA production. This finding suggests that HA, which has been shown to play a crucial role in smooth muscle cell proliferation and migration, may be involved in the promoting effect of insulin treatment on arterial wall reaction to injury.