Diabetic pigmented spots on the extrapretibial region: immunohistochemical study of type IV collagen in dermal vessel wall

ARTICLE

It is now generally accepted that microangiopathy characteristics of diabetes mellitus occur in the dermal vasculature and may result in various cutaneous lesions, although the pathogenic relationship between blood vessel changes and cutaneous lesions remains to be established [1, 2]. Diabetic dermopathy, also called shin spots or pigmented pretibial patches because of their usual location, is the most common finding in diabetes mellitus. Shin spots are usually atrophic, hyperpigmented and oval. Histologically, they show thick-walled capillaries with PAS-positive material in the papillary dermis and extravasated erythrocytes [3-7].

The morphological hallmark of diabetic microvascular disease is a generalized basement membrane thickening with a progressive increase in permeability. A possible explanation for this phenomenon is the non-enzymatic glycosylation of the proteins of the basement membrane including type IV collagen, laminin and heparan sulfate proteoglycans [8, 9].

Type IV collagen is a major component of basement membrane and is composed of six genetically distinct chains (alpha₁-alpha₆) [10]. Recently, chain-specific monoclonal antibodies for type IV collagen have been established, and type IV collagen in the dermal vascular basement membrane has been reported to consist of alpha₁ (IV) and alpha₂ (IV) chains [11].

We report a relatively rare case of diabetic dermopathy occurring in a location other than pretibial, and have investigated whether type IV collagen in the vessel wall consists of alpha₁ and alpha₂ chains like normal dermal vascular basement membrane, or contains specific alpha chains other than alpha₁ and alpha₂ chains.

Case report

A 53-year-old Japanese female visited our department complaining of skin lesions on the trunk. Multiple pigmented macules with a diameter of 2-6 mm were observed on the thighs and lower abdominal area (Fig. 1). She said the pigmented macules began as red spots but could not give a clear account of their onset. They were completely asymptomatic. She denied any trauma or insect bite prior to the onset of the spots. There was no family history of diabetes mellitus. Fasting blood sugar level was high (569 mg/dl). HbA1c and C peptide were 13.4% (normal range; 4.3-5.8) and 3.2 ng/ml (normal range; 0.6-2.8), respectively. Laboratory studies disclosed normal values for the following: blood cell count, sedimentation rate, immunoglobulins (Ig), liver function tests, electrolytes, cholesterol, urea and renal function tests. Anti-insulin antibody was negative.

Histology and immunohistochemistry

Histological examination was performed on the pigmented macule and normal-appearing skin on the thigh. Histopathology of the pigmented spot with H&E staining showed an increase in the number of dilated vessels in the papillary dermis. There was a perivascular infiltrate consisting of lymphocytes, histiocytes and some extravasated red cells. A small amount of hemosiderin was seen. There was no leukocytoclasis suggestive of vasculitis. No significant basal melanosis in the epidermis was observed. Specific vascular changes were revealed by a PAS stain. Vessel walls in the papillary dermis were thickened and showed abundant PAS-positive material distributed in a fibrillar fashion (Fig. 2a). Histopathology of non-lesional skin revealed essentially similar findings to that of a pigmented spot except for the absence of extravasated red cells and hemosiderin (Fig. 2b).

Monoclonal antibodies for alpha₁-alpha₆ chains of type IV collagen were produced as previously described [11]. Lesional and normal-appearing skin of the patient, as well as normal control skin was frozen in liquid nitrogen and cut into 5 µm sections, then fixed in 4% paraformaldehyde. The slides were covered with the monoclonal antibodies (1:1 dilution) for 1 hr at room temperature, then incubated with anti-rat IgG (Dako) at a 1:100 dilution for 30 min at room temperature [11]. Antigenic sites were demonstrated by the avidin-biotin complex method. The antibodies for alpha₁ and alpha₂ chains stained the basement membranes of both the dermo-epidermal junction (DEJ) and dermal blood vessels in the lesional and non-lesional skin (Fig. 3a). In normal control skin, antibodies for alpha₁ and alpha₂ chains stained DEJ and blood vessels similarly but to a lesser extent than those of patient skin (Fig. 3b). The antibodies for alpha₃ and alpha₄ chains exhibited no positive staining in lesional, non-lesional or control skin (not shown). The antibodies for alpha₅ and alpha₆ chains stained only the basement membrane of DEJ in the lesional and non-lesional skin (Fig. 3c). Antibodies for alpha₅ and alpha₆ chains stained DEJ alone in control skin as was observed in patient skin, but the extent was lesser than in the patient skin.

Discussion

Although the pigmented spots in this patient were smaller in size and unusual in their location, they were similar to so-called shin spots on the basis of histological findings. Similar cases have been reported as unusual diabetic pigmented pretibial patches by Bauer et al. [4]. Histopathology of pigmented spot and non-lesional skin showed similar vascular changes. The histological and immunohistological differences between pigmented spot and normal-appearing skin in the patient were the presence of extravasated red cells and hemosiderin in the pigmented spots. Pigmented spots, therefore, may be caused by extravasated red cells and hemosiderin deposits.

Disproportionate expression of alpha₁-alpha₆ (IV) chains has been reported in several immature tissues and diseased states. The alpha₅ (IV) chain was consistently detected in fetal epithelium but not in adult epithelium of the human small intestine [12]. Desjardins et al. [13] found that the labelling intensity of alpha₁ (IV), alpha₂ (IV) and alpha₃ (IV) but not alpha₄ (IV) were markedly increased along the tubular basement membrane of diabetic kidney, while alpha₁-alpha₄ (IV) were detected equally in the normal condition. These previous data suggest that the diabetic state may lead to disproportionate increases in individual type IV collagen chains. This tempted us to characterize the type IV collagen chains in the PAS-positive component deposited in the dermal vessels by immunohistochemical procedures. The results demonstrated that the PAS-positive component in vessel walls in the pigmented and non-lesional skin consisted of alpha₁ and alpha₂ chains of type IV collagen which are normal constituents of dermal capillary basement membrane. These results indicate that proportionate increases of alpha₁ and alpha₂ chains of type IV collagen in the dermal vascular basement membrane may occur in diabetic dermopathy.

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