Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia

Abstract

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

Figure 1.

Migration of murine dermal fibroblasts after 20 hours of culture. Coverslips coated with colloidal gold salts and extracellular matrix molecules after fibroblast incubation viewed with ×40 magnification under dark-field optics. A: Wild-type murine dermal fibroblasts plated on 20 μg/ml of fibronectin. B: Diabetic db/db murine dermal fibroblasts plated on 20 μg/ml of fibronectin. The migration tracks (asterisk) are visible as black empty spaces against the background of bright gold salt particles. The cells are indicated by arrows. Note how the migration tracks of the wild-type cells are clearly larger than those of the diabetic cells.

Figure 2.

Diabetic fibroblasts show impaired migration on collagen and fibronectin compared to wild-type fibroblasts. Fibroblast migration assays were performed as described in Materials and Methods. In the gold-salt phagokinetic migration assay, fibroblasts were plated onto coverslips coated with varying concentrations of the matrix of interest and allowed to migrate for 20 hours. The figures depict mean migration track area ± SEM of fibroblasts from diabetic db/db and wild-type mice. Area is shown in pixels. A: Fibroblast migration on type I collagen. B: Fibroblast migration on fibronectin. *, P < 0.001 compared with wild-type control.

Figure 3.

Haptotaxis assays confirm impaired migration of diabetic fibroblasts on collagen and fibronectin. Modified Boyden chamber haptotaxis assays were prepared as described in Materials and Methods. A: Haptotaxis on collagen- and fibronectin-coated inserts. B: Up-regulation of haptotaxis by hypoxia (1% O₂) stimulation on collagen-coated dishes. *, P < 0.05 compared with wild-type control.

Figure 4.

Gelatin zymography shows that conditioned medium from cultured diabetic db/db fibroblasts contains elevated levels of pro-MMP-9 compared to wild-type fibroblasts. Gelatinase activity in conditioned medium from wild-type and diabetic db/db fibroblasts grown in culture on 10 μg/ml of type I collagen analyzed via gelatin zymography. Positions of molecular mass standards are indicated. A: Gelatin zymography showing enhanced 92-kd gelatinolytic activity of diabetic fibroblasts. Results show a typical 92/83-kd MMP-9 duplex and a 72/62-kd MMP-2 duplex. Lanes db1 and db2: Conditioned medium from diabetic fibroblast cultures. Lanes N1 and N2: Conditioned medium from wild-type control fibroblast cultures. No significant changes were detected in 72-kd (MMP-2) gelatinolytic activity. B: Densitometry analysis of 92-kd (latent MMP-9) gelatinolytic activity. Histograms represent the mean ± SEM of three separate samples. Wild-type control fibroblast MMP-9 densitometry is 100%. *, P < 0.001 compared with wild-type control.

Figure 5.

ELISA analysis of conditioned medium demonstrates total MMP-9 production is elevated in diabetic fibroblasts, while there is no difference in total MMP-2 production. MMP-2 and MMP-9 activity in conditioned medium from wild-type and diabetic db/db fibroblasts grown in culture. All experiments were repeated in triplicate. A: Histogram shows mean ± SEM total MMP-9 concentration in conditioned medium from three separate samples. *, P < 0.05 compared with wild-type control. B: Histogram shows mean ± SEM total MMP-2 concentration in conditioned medium from three separate samples.

Figure 6.

Diabetic fibroblasts production of VEGF is severely impaired compared to wild-type fibroblasts in normoxic and hypoxic conditions. Serum from wild-type and diabetic db/db fibroblasts examined for VEGF production after 20 hours in 21% O₂ (normoxia) or 1% O₂ (hypoxia). A: VEGF production by fibroblasts harvested from adult mice. Diabetic fibroblasts demonstrate severe impairments in VEGF production and VEGF up-regulation in response to hypoxia. Histogram represents the mean VEGF production ± SEM of three separate experiments. *, P < 0.001 compared with wild-type control.

Figure 7.

The diabetic phenotype is not apparent in neonatal mice and only begins to develop at 4 to 8 weeks of age. Weight and nonfasting blood glucose measurements were taken every 2 weeks until 16 weeks of age. Neonatal mice have none of the phenotypic changes observed in adult db/db mice. A: Weight in grams of diabetic and wild-type mice throughout time. B: Nonfasting blood glucose levels of diabetic and wild-type mice throughout time. Impaired wound healing begins between 4 to 8 weeks. Data points represent mean weight or blood glucose level ± SEM of four separate mice.

Figure 8.

Fibroblasts harvested from neonatal leptin receptor-deficient db/db mice show no impairment in production of VEGF or response to hypoxia compared to wild-type fibroblasts. Wild-type and diabetic db/db neonatal fibroblasts were grown in serum-free basal medium in either normoxic (21% O₂) or hypoxic (1% O₂) conditions. Conditioned medium was collected from the cells after 20 hours of incubation. There was no difference between VEGF production by neonatal db/db fibroblasts and wild-type neonatal fibroblasts in normoxic conditions. Furthermore, VEGF up-regulation by both cell types in hypoxia was equal and similar to that of adult fibroblasts. Histogram represents the mean VEGF production ± SEM of three separate experiments.