Cultured fibroblasts from chronic diabetic wounds on the lower extremity (non-insulin-dependent diabetes mellitus) show disturbed proliferation

Abstract

Patients with diabetes mellitus experience impaired wound healing often resulting in chronic foot ulcers. Hospital discharge data indicate that 6-20% of all diabetic individuals hospitalized (mostly with type 2 diabetes) have a lower extremity ulcer. Maintaining glucose levels at acceptable levels (below 10 mmol/l) is considered to be an important part of the clinical treatment, but the exact mechanism by which diabetes delays wound repair is not yet known. We studied this phenomenon by determining the potential of fibroblasts isolated from the ulcer sites of four patients with non-insulin-dependent diabetes mellitus to proliferate in vitro. Controls were fibroblasts isolated from normal skin of the upper leg of five healthy age-matched volunteers and of six non-insulin-dependent diabetes patients. Proliferative capacity was analysed by evaluation of plates after trypsinization and [3H]thymidine incorporation. Fibroblast morphology was studied by light and transmission electron microscopy. Diabetic ulcer fibroblasts, measured by [3H]thymidine incorporation, proliferated significantly more slowly than the nonlesional control fibroblasts (P < 0.00047) and age-matched control fibroblasts (P < 0.00003). After culturing the fibroblasts for a prolonged period in high-glucose (27.5 mM) and low-glucose (5.5 mM, i.e. physiological) medium, this difference in proliferation rate between diabetic ulcer fibroblasts and nonlesional diabetic fibroblasts remained (P < 0.0001 for high-glucose and P < 0.0009 for low-glucose on day 7). Fibroblast proliferation in all three groups was slightly lower in high-glucose than in low-glucose medium, although not significantly at any time-point. Light microscopy showed diabetic ulcer fibroblasts to be large and widely spread. Transmission electron microscopy of cultured diabetic ulcer fibroblasts and nonlesional diabetic skin fibroblasts revealed a large dilated endoplasmic reticulum, a lack of microtubular structures and multiple lamellar and vesicular bodies. These results show a diminished proliferative capacity and abnormal morphology of fibroblasts derived from diabetic ulcers of non-insulin-dependent diabetes patients.

Fibroblasts from chronic wounds show altered TGF‐β‐signaling and decreased TGF‐β Type II Receptor expression

“…Evidence from studies of fibroblasts from diabetic ulcers have shown similar abnormalities (Loots et al, 1999;Loot et al, 2002). The microenvironment of the wound is likely to be another important determinant of cellular selection and phenotypic expression.…”

“…There is increasing evidence in support of this hypothesis, much of it developed in the context of diabetic and venous ulcers. Fibroblasts cultured from diabetic ulcers show decreased mitogenic potential and differential responses to certain cytokines, either along or in combination (Loots et al, 1999;Loot et al, 2002). It has been shown that dermal fibroblasts cultured from venous ulcers are senescent (Stanley and Osler, 2001), show decreased proliferative potential (Stanley et al, 1997), and are unresponsive to stimulation by such critical growth factors as transforming growth factor-b1 (TGF-b1) (Hasan et al, 1997) and platelet-derived growth factor BB (PDGF-BB) (Agren et al, 1999).…”

Chemokines in Wound Healing and as Potential Therapeutic Targets for Reducing Cutaneous Scarring

“…132 A consistent finding in pressure ulcers, the diabetic foot and venous stasis ulcers is an accumulation of senescent fibroblasts. [133][134][135] These fibroblasts demonstrate decreased proliferative rates and a dysfunctional chemokine secretory profile involving excess CCL2 release and reduced CXCL8 production compared with wounds healing normally. 136,137 Furthermore, high levels of functional CCL2, CCL5, CCL18, CCL20, CCL27, CXCL1, and CXCL12 have been reported in chronic wound debridement tissue.…”

Id3 is a novel regulator of p27kip1 mRNA in early G1 phase and is required for cell-cycle progression

“…Dermal fibroblast proliferation is a major feature of cutaneous wound healing (Martin, 1997), which is altered in several pathologies such as keloids (Calderon et al, 1996) and chronic wounds, which can evoluate toward cutaneous carcinoma (Loots et al, 1999;Chraibi et al, 2004). To date the mechanisms underlying cell proliferation in the cutaneous wound healing context remain poorly understood.…”