In vitro studies of the diabetic condition using cultured fibroblasts with focus on wound healing

Abstract

This thesis focuses on the diabetic condition at the cellular level, and how this may lead to late complications. Defect wound healing in diabetic patients is poorly understood, but impaired granulation is observed clinically. We have therefore decided to study an in vitro system using cultured fibroblasts. These were derived from biopsies from human diabetic and non-diabetic wounds and uninjured skin, Goto-Kakazaki rats and Wistar rats. In addition Swiss 3T3 mouse fibroblasts were studied.

Fibroblasts from chronic non-diabetic and insulin dependent / non-insulin dependent diabetic chronic wounds show decreased adhesion and proliferation compared to those from uninjured skin. However, chronic diabetic wound fibroblasts have even lower adhesion and proliferation than chronic non-diabetic wound fibroblasts. Fibroblasts from human uninjured skin, non-diabetic or diabetic, have similar rates of adhesion and proliferation. Fibroblasts from GK rats have decreased adhesion compared to those from Wistar rats. These results suggest that the chronic wound fibroblasts display altered characteristics, but also that the diabetic condition influences dermal fibroblast growth characteristics. High glucose inhibits human and Swiss 3T3 fibroblast proliferation, and causes a growth factor and insulin resistance in human fibroblasts. Protein kinase C inhibitors, antioxidants and protein tyrosine phosphatase inhibitors independently reversed the high glucose induced growth factor resistance.

The studies on post receptor mechanisms for glucocorticoid-induced resistance to insulin-like peptides in Swiss 3T3 fibroblasts, indicate that the activation of MAP kinase may be dissociated from IGF-I-induced anabolic pathways and tyrosine phosphorylation of IRS-I. Additionally the dexamethasone-induced reduction of IRS-I expression maybe important for the impaired activation of MAP kinase by insulin-like peptides in steroid treated cells.

Heparin and low-molecular derivatives of heparin but not de-N-sulphated heparin stimulate human and Swiss 3T3 fibroblast proliferation. This effect is dose-dependent, and does not require the presence of serum. Antibodies to basic FGF totally abolishes this stimulatory effect in Swiss 3T3 mouse fibroblasts, indicating that one of the mechanisms for heparin in these cells is to facilitate binding of bFGF to its high-affinity receptors. However, heparin may also affect other growth factors by prolonging or increasing their activity. Hyaluronic acid is also effective in promoting growth, especially in GK and Wistar rat fibroblasts. The effect of hyaluronic acid could be through increased adhesion, but also through a direct metabolic effect.

Non-diabetic uninjured skin (NU) fibroblast proliferation is inhibited when adding media from chronic non-insulin dependent diabetic wound (DW) fibroblasts. The DW media and media from GK rat fibroblasts show elevated L-lactate levels. When adding L-lactate to the NU fibroblasts, proliferation is decreased. This indicates that the elevated levels of L-lactate in the media derived from DW fibroblasts, are at least partly responsible for the inhibition of NU fibroblasts, and for the decreased basal proliferation of DW fibroblasts. During high glucose conditions, L-lactate levels are increased in NU, GK and Wistar rat fibroblasts, indicating increased utilisation of anaerobic metabolic pathways. Additionally, ß-hydroxybutyrate, similarly to L-lactate, diminishes NU fibroblast proliferation, indicating an increase in cellular NADH levels.

In summary, fibroblasts from chronic wounds in NIDDM and IDDM patients and from GK rats show decreased adhesion and proliferation. Our results suggest that this may depend on increased glucose utilisation through non-oxidative anaerobic glycolysis as well as on increased NADH in the cytosol and the mitochondria. Additionally, we speculate that resistance to growth promoting factors or low amounts of these maybe responsible for the observed effects. We believe that these in vitro systems may be of value for the understanding of the mechanisms behind impaired wound healing in diabetes.