All-trans-retinoic acid suppresses matrix metalloproteinase activity and increases collagen synthesis in diabetic human skin in organ culture

Abstract

Diabetes increases susceptibility to chronic skin ulceration. The etiology of chronic wound formation in diabetic individuals is multifactoral but may be accelerated by changes in the structure and function of the skin secondary to impaired fibroblast proliferation, decreased collagen synthesis, and increased matrix metalloproteinase (MMP) expression. This study explored the effects of all-trans-retinoic acid (RA) on cellular and biochemical features of diabetic human skin in organ culture. Two-mm skin biopsies from hip or ankle were obtained from diabetic subjects and incubated for 9 days in the absence or presence of 2 micro mol/L RA. Hip skin from non-diabetic individuals served as control. Following organ culture incubation, untreated and RA-treated tissue was examined histologically after staining with hematoxylin and eosin. In parallel, organ culture-conditioned medium collected on days 5 and 7 was assayed for levels of active and total MMP-1 (interstitial collagenase) and MMP-9 (gelatinase B). The same organ culture fluids were assayed for the presence of soluble collagen. In comparison with skin from non-diabetic individuals, diabetic skin demonstrated no major differences in overall epidermal thickness or collagen production (both were increased in RA-treated tissue as compared to non-RA-treated tissue). In contrast, levels of MMP-9 (active forms) were elevated in organ culture fluid from diabetic skin as compared to non-diabetic control skin. In the presence of RA, active forms of both MMP-1 and MMP-9 were reduced. Together, these data suggest that RA has the capacity to improve structure and function of diabetic skin, and that a major effect is on reduction of collagen-degrading MMPs.

Figure 1

Histological features of untreated and RA-treated non-diabetic and diabetic skin in organ culture. Tissue from normal and diabetic (hip) skin was maintained in organ culture for 9 days under serum-free, growth factor-free conditions in the absence or presence of 0.75 μg/ml RA. 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 RA-treated. C: Diabetic control. D: Diabetic RA-treated. Epidermal hyperplasia can be seen in the RA-treated skin from either normal or diabetic skin (×320).

Figure 2

MMP-9 elaboration in untreated and RA-treated non-diabetic and diabetic skin in organ culture. Organ culture fluid was collected on days 5 and 7 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 and RA-treated diabetic skin (day 5). A higher percentage of MMP-9 in the active form can be seen in culture fluid from untreated skin as compared to RA-treated skin. MMP-2 latent and active forms can also be seen but there is little difference between control 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 10 normal and 16 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 relative to non-diabetic skin. **P < 0.01 relative to non-RA-treated skin of same group.

Figure 3

MMP-1 elaboration in untreated and RA-treated non-diabetic and diabetic skin in organ culture. Organ culture fluid was collected on days 5 and 7 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 and RA-treated diabetic skin (day 5). A higher percentage of MMP-1 in the active form can be seen in culture fluid from untreated skin as compared to 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 10 normal and 10 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 relative to non-diabetic skin. **P < 0.01 relative to non-RA-treated skin of same group.

Figure 4

Comparison of RA and TIMP-1 for inhibition of MMP-9 activation. Organ culture fluid from control, RA-treated or TIMP-1 (1 μg/ml)-treated skin was collected on days 5 and 7 and assayed for MMP-9 by gelatin zymography. Zymographic images were scanned and digitized, and negative images were quantified. Upper panel: Total enzyme (densitometry values from combined latent and active forms). Lower panel: Active enzyme expressed as a percentage of total enzyme (values from active forms divided by values from active + latent forms). Values shown are means and standard errors based on organ cultures of hip skin from four diabetic volunteers. Statistical significance of the differences among the three groups was determined by analysis of variance followed by paired-group comparisons. *P < 0.05 relative to untreated control group.

Figure 5

TIMP-1 elaboration in organ cultures of untreated and RA-treated diabetic skin. Organ culture fluid was collected on days 5 and 7 from untreated and RA-treated diabetic skin and assayed for TIMP-1 by ELISA. Values shown are means and standard errors, based on organ cultures from four individuals. Statistical significance of the difference between untreated and RA-treated samples was determined using the Student’s t-test. *P < 0.01.