In vitro studies of the diabetic condition using cultured fibroblasts with focus on wound healing
Author: Hehenberger, Karin M.
Date: 1997-12-19
Location: Karolinska sjukhusets aula
Time: 9.00
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.
Issue date: 1997-11-28
Publication year: 1997
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