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 thismay lead to late complications. Defect wound healing in diabetic patients is poorlyunderstood, but impaired granulation is observed clinically. We have therefore decidedto study an in vltro system using cultured fibroblasts. These were derived from biopsiesfrom human diabetic and non-diabetic wounds and uninjured skin, Goto-Kakazaki ratsand Wistar rats. In addition Swiss 3T3 mouse fibroblasts were studied.

Fibroblasts from chronic non-diabetic and insulin dependent / non-insulin dependentdiabetic chronic wounds show decreased adhesion and proliferation compared to thosefrom uninjured skin. However, chronic diabetic wound fibroblasts have even loweradhesion and proliferation than chronic non-diabetic wound fibroblasts. Fibroblastsfrom human uninjured skin, non-diabetic or diabetic, have similar rates of adhesionand proliferation. Fibroblasts from GK rats have decreased adhesion compared to thosefrom Wistar rats. These results suggest that the chronic wound fibroblasts displayaltered characteristics, but also that the diabetic condition influences dermal fibroblastgrowth characteristics.

High glucose inhibits human and Swiss 3T3 fibroblast proliferation, and causesa growth factor and insulin resistance in human fibroblasts. Protein kinase C inhibitors,antioxidants and protein tyrosine phosphatase inhibitors independently reversed thehigh glucose induced growth factor resistance.

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

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

Non-diabetic uninjured skin (NU) fibroblast proliferation is inhibited when addingmedia from chronic non-insulin dependent diabetic wound (DW) fibroblasts. The DWmedia and media from GK rat fibroblasts show elevated L-lactate levels. When addingL-lactate to the NU fibroblasts, proliferation is decreased. This indicates thatthe elevated levels of L-lactate in the media derived from DW fibroblasts, are atleast partly responsible for the inhibition of NU fibroblasts, and for the decreasedbasal proliferation of DW fibroblasts. During high glucose conditions, L-lactatelevels are increased in NU, GK and Wistar rat fibroblasts, indicating increased utilisationof anaerobic metabolic pathways. Additionally, ß-hydroxybutyrate, similarlyto L-lactate, diminishes NU fibroblast proliferation, indicating an increase in cellularNADH levels.

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

KEY WORDS: diabetes, fibroblasts, wound healing, proliferation, protein kinaseC, lactate, growth factors, resistance.