Gene expression profile of AIDS-related Kaposi's sarcoma

Abstract

Background: Kaposi's Sarcoma (KS) is a proliferation of aberrant vascular structures lined by spindle cells, and is caused by a gammaherpes virus (HHV8/KSHV). Its course is aggravated by co-infection with HIV-1, where the timing of infection with HIV-1 and HHV8 is important for the clinical outcome.

Methods: In order to better understand the pathogenesis of KS, we have analysed tissue from two AIDS-KS lesions, and from normal skin by serial analysis of gene expression (SAGE). Semi-quantitative RT-PCR was then used to validate the results.

Results: The expression profile of AIDS-related KS (AIDS-KS) reflects an active process in the skin. Transcripts of HHV8 were found to be very low, and HIV-1 mRNA was not detected by SAGE, although it could be found using RT-PCR. Comparing the expression profile of AIDS-KS tissue with publicly available SAGE libraries suggested that AIDS-KS mRNA levels are most similar to those in an artificially mixed library of endothelial cells and leukocytes, in line with the description of KS lesions as containing spindle cells with endothelial characteristics, and an inflammatory infiltrate. At least 64 transcripts were found to be significantly elevated, and 28 were statistically downregulated in AIDS-KS compared to normal skin. Five of the upregulated mRNAs, including Tie 1 and sialoadhesin/CD169, were confirmed by semi-quantitative PCR to be elevated in additional AIDS-KS biopsies. Antibodies to sialoadhesin/CD169, a known marker of activated macrophages, were shown to specifically label tumour macrophages.

Conclusion: The expression profile of AIDS-KS showed 64 genes to be significantly upregulated, and 28 genes downregulated, compared with normal skin. One of the genes with increased expression was sialoadhesin (CD169). Antibodies to sialoadhesin/CD169 specifically labelled tumour-associated macrophages, suggesting that macrophages present in AIDS-KS lesions belong to a subset of human CD169+ macrophages.

Figure 1

Histological staining of AIDS-KS and normal tissues Hematoxylin-eosin staining of normal skin tissue (panel A), and the two advanced-stage, nodular AIDS-KS tissues (panels D/G). Endothelial cells were stained with anti-CD31 (Dako) (panel B = normal skin, panels E/H = AIDS-KS tissue) and lymphocyte subpopulations were stained with mAbs to CD3 (panel C = normal skin, panels F/I = AIDS-KS tissue).

Figure 2

Scatterplot analysis of SAGE libraries Scatterplots of KSa versus KSb (A), KSa versus normal skin NS (B) and KSb versus NS (C) were generated using the SAS version 6.12 software. The tag frequency for each (per 50,000) is plotted on a logarithmic scale for KSa and KSb. The Pearson correlation coefficient of each comparison was calculated.

Figure 3

Semi-quantitative RT-PCR analysis of differentially expressed genes PCR-analysis was performed for six genes on cDNA generated from normal skin tissues or different AIDS-KS tissues. Expression level indicated the 10-fold dilution step starting with 500 ng total RNA/DNase treated. Error bars are shown. * P < 0.05.

Figure 4

Photomicrographs of anti-CD68 and anti-CD169 stained sections of selected tissues The two frozen skin biopsies show a similar histopathological picture of nodular stage Kaposi's sarcoma. Left panels (A/D): HE staining; middle panels (B/E): αCD68 staining; Right panels (C/F): αCD169 staining. Throughout the dermis, vascular proliferations are found surrounded by irregular, rather dense areas of atypical spindle cell proliferations. Both lesions contain sparse inflammatory cell infiltrates, mainly consisting of CD68+ macrophages.