Effects of a synthetic retinoid on skin structure, matrix metalloproteinases, and procollagen in healthy and high-risk subjects with diabetes

Abstract

Background: In diabetes, foot ulceration may result from increased skin fragility. Retinoids can reverse some diabetes-induced deficits of skin structure and function, but their clinical utility is limited by skin irritation. The effects of diabetes and MDI 301, a nonirritating synthetic retinoid, and retinoic acid have been evaluated on matrix metalloproteinases (MMPs), procollagen expression, and skin structure in skin biopsies from nondiabetic volunteers and diabetic subjects at risk of foot ulceration using organ culture techniques.

Methods: Zymography and enzyme-linked immunosorbent assay were utilized for analysis of MMP-1, -2, and -9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) and immunohistochemistry for type I procollagen protein abundance. Collagen structure parameters were assessed in formalin-fixed, paraffin-embedded tissue sections.

Results: The % of active MMP-1 and -9 was higher and TIMP-1 abundance was lower in subjects with diabetes. Type 1 procollagen abundance was reduced and skin structural deficits were increased in diabetes. Three μM MDI 301 reduced active MMP-1 and -9 abundance by 29% (P < .05) and 40% (P < .05), respectively, and increased TIMP-1 by 45% (P = .07). MDI 301 increased type 1 procollagen abundance by 40% (P < .01) and completely corrected structural deficit scores. Two μM retinoic acid reduced MMP-1 but did not significantly affect skin structure.

Conclusions: These data indicate that diabetic patients at risk of foot ulceration have deficits of skin structure and function. MDI 301 offers potential for repairing this skin damage complicating diabetes.

Figure 1. Effect of MDI 301 and retinoic acid on MMP-1 activity in organ culture

Organ culture fluid from untreated, 1 μM and 3 μM MDI 301-treated or 2 μM RA-treated skin was collected on day 4 and assayed for MMP-1 by β–casein zymography. Zymographic images were scanned and digitized, and negative images quantified. Upper Panel: Representative β-casein zymogram demonstrating MMP-1 in organ culture fluid from untreated diabetic skin and skin treated with 1 μM and 3 μM MDI-301 and 2 μM retinoic acid (RA). A higher percentage of MMP-1 in the active form can be seen in culture fluid from untreated skin as compared to MDI 301 and RA treated skin. Lower Panel. Active enzyme expressed as a percentage of total enzyme (densitometry values from active forms divided by values from active + latent forms). Values shown are means and standard errors based on organ cultures from 12 normal and 27 diabetic volunteers. Statistical significance of the differences among the four groups was determined by analysis of variance followed by paired-group comparisons. *p<0.05 vs untreated samples

Figure 2. Effect of MDI 301 and retinoic acid on MMP-9 activity in organ culture

Organ culture fluid from untreated, 1 μM and 3 μM MDI 301-treated or 2 μM RA-treated skin was collected on day 4 and assayed for MMP-9 by gelatin zymography. Zymographic images were scanned and digitized, and negative images quantified. Upper Panel: Representative gelatin zymogram demonstrating MMP-9 (and MMP-2) in organ culture fluid from untreated diabetic skin and skin treated with 1 μM and 3 μM MDI-301 and 2 μM retinoic acid (RA). A higher percentage of MMP-9 in the active form can be seen in culture fluid from untreated skin as compared to MDI 301 and RA-treated skin. MMP-2 latent and active forms can also be seen and there is a reduction of active MMP-2 in MDI-301 and RA-treated samples. Lower Panel. Active enzyme expressed as a percentage of total enzyme (densitometry values from active forms divided by values from active + latent forms). Values shown are means and standard errors based on organ cultures from 12 normal and 27 diabetic volunteers. Statistical significance of the differences among the four groups was determined by analysis of variance followed by paired-group comparisons. *p<0.05 vs untreated samples

Figure 3. Effect of MDI 301 and retinoic acid on type I procollagen abundance

Skin tissue from non-diabetic and diabetic subjects was maintained in organ culture for 8 days under serum-free, growth factor-free conditions in the absence or presence of 1 μM and 3 μM MDI 301 or 2 μM RA. At the end of the incubation period, sections were dewaxed blocked with 10% horse serum and incubated with antibodies to type 1 pro-collagen and the immunoreactivity revealed using a Vectastain universal elite ABC kit (Vector Laboratories, Orton Southgate, Peterborough, Cambridgeshire, UK) and diaminobenzidine (DAKO, Ely, Cambridgeshire, UK). The area and density of staining within an area of 1000X1000 pixels was determined by image analysis after subtraction of background staining and the product calculated and presented as the overall abundance (in intensity units). Values shown are means and standard errors based on sections from 12 normal and 27 diabetic volunteers. Statistical significance of the differences among the four groups was determined by analysis of variance followed by paired-group comparisons. RA= Retinoic Acid. Results expressed as mean ± SEM. *p<0.01 vs untreated samples

Figure 4. Immunohistochemcial staining for type 1 procollagen in untreated and MDI 301-treated non-diabetic and diabetic skin in organ culture

Skin tissue from non-diabetic and diabetic subjects was maintained in organ culture for 8 days under serum-free, growth factor-free conditions in the absence or presence of 3 μM MDI 301. At the end of the incubation period, sections are dewaxed blocked with 10% horse serum and incubated with antibodies to type 1 pro-collagen and the immunoreactivity revealed using a Vectastain universal elite ABC kit (Vector Laboratories, Orton Southgate, Peterborough, Cambridgeshire, UK) and diaminobenzidine (DAKO, Ely, Cambridgeshire, UK). A: Normal control. B: Normal 3 μM MDI 301-treated. C: Diabetic control. D: Diabetic 3 μM MDI-301-treated. Reduced abundance of type 1 procollagen can be seen in the untreated diabetic skin which is increased by 3 μM MDI 301 treatment (x200)

Figure 5. Histological features of untreated and MDI 301-treated non-diabetic and diabetic skin in organ culture

Skin tissue from non-diabetic and diabetic subjects was maintained in organ culture for 8 days under serum-free, growth factor-free conditions in the absence or presence of 3 μM MDI 301. At the end of the incubation period, the tissue was fixed in 10% buffered formalin and examined at the light microscopy level after sectioning and staining with hematoxylin and eosin. A: Normal control. B: Normal 3 μM MDI 301-treated. C: Diabetic control. D: Diabetic 3 μM MDI-301-treated. Epidermal hyperplasia can be seen in the 3 μM MDI 301 treated diabetic skin (x200).

Figure 5. Histological features of untreated and MDI 301-treated non-diabetic and diabetic skin in organ culture

Skin tissue from non-diabetic and diabetic subjects was maintained in organ culture for 8 days under serum-free, growth factor-free conditions in the absence or presence of 3 μM MDI 301. At the end of the incubation period, the tissue was fixed in 10% buffered formalin and examined at the light microscopy level after sectioning and staining with hematoxylin and eosin. A: Normal control. B: Normal 3 μM MDI 301-treated. C: Diabetic control. D: Diabetic 3 μM MDI-301-treated. Epidermal hyperplasia can be seen in the 3 μM MDI 301 treated diabetic skin (x200).