Endothelial cell activation and neovascularization are prominent in dermatomyositis

Abstract

Background: While vascular and immune abnormalities are common in juvenile and adult dermatomyositis (DM), the molecular changes that contribute to these abnormalities are not clear. Therefore, we investigated pathways that facilitate new blood vessel formation and dendritic cell migration in dermatomyositis.

Methods: Muscle biopsies from subjects with DM (9 children and 6 adults) and non-myositis controls (6 children and 7 adults) were investigated by immunohistochemistry using antibodies that recognize existing (anti-CD146) and newly formed blood vessels (anti-alphaVbeta3) and mature dendritic cells (anti-DC-LAMP). Blood vessel quantification was performed by digitalized image analysis. Additional muscle biopsies from subjects with adult DM and non-myositis controls were used for global gene expression profiling experiments.

Results: A significant increase in neovascularization was found in muscle biopsies of DM patients; neovascularization (alphaVbeta3 positive capillaries and vessels per muscle fiber) was much higher in juvenile than in adult DM patients (control vs juvenile DM: Mean +/- SE: 0.06 +/- 0.01 vs 0.6 +/- 0.05; p < 0.0001 and control vs adult DM: Mean +/- SE: 0.60 +/- 0.1 vs 0.75 +/- 0.1; p = 0.051). Gene expression analysis demonstrated that genes that participate not only in angiogenesis but also in leukocyte trafficking and the complement cascade were highly up regulated in DM muscle in comparison to age matched controls. DC-LAMP positive dendritic cells were highly enriched at perivascular inflammatory sites in juvenile and adult DM patients along with molecules that facilitate dendritic cell transmigration and reverse transmigration (CD142 and CD31).

Conclusion: These results suggest active neovascularization and endothelial cell activation in both juvenile and adult DM. It is likely that close association of monocytes with endothelial cells initiate rapid dendritic cell maturation and an autoimmune response in DM.

Figure 1

Expression pattern of CD146 in juvenile dermatomyositis (DM), adult DM, and control muscle biopsies. Frozen muscle sections from myositis and control samples were stained with antibodies that recognize pan endothelial marker CD146. Representative staining patterns for CD146 in normal human muscle control (panels a&c) and DM (panel b) and juvenile DM (panel d). Note strong endothelial staining in the capillaries of both DM and juvenile DM patients.

Figure 2

Expression pattern of αVβ3 in juvenile DM, adult DM, and control muscle biopsies. Frozen muscle sections from myositis and normal human muscle control samples were stained with antibodies that recognize αVβ3. Representative staining patterns for αVβ3 in control (panels a&c) and DM (panel b) and juvenile DM (panel d).

Figure 3

Members of the angiogenic, anti-angiogenic, lymphocyte trafficking and complement activation pathways are highly up-regulated in DM patients compared to normal human muscle (NHM) controls. Gene-expression profiles generated from disease and control groups were analyzed using high-density oligonucleotide arrays. Gene expression values were obtained following Affymetrix MAS5 normalization. Manually grouped genes were further clustered with GeneSpring to visualize clusters of genes with similar expression pattern. Gene tree was colored by the expression of average over the whole group. Gene function was based on information available in public databases or in the literature.

Figure 4

DC-LAMP positive dendritic cells are enriched in perivascular area of inflammation in juvenile DM patients. Juvenile DM and control biopsies were stained with antibodies that recognize dendritic cell marker DC-LAMP along with an isotype matched control (upper panels). Consecutive sections stained with DC-LAMP and CD146 showing close proximity of DC-LAMP positive cells to blood vessels. Normal biopsies showed no staining with DC-LAMP antibodies (data not shown).

Figure 5

Expression pattern of CD142, CD31 and DC-LAMP in juvenile DM patients. Serial sections of juvenile DM biopsies stained with anti-tissue factor (CD142) and PECAM1 (CD31) antibodies (upper panel). Serial sections stained with CD31 and DC-LAMP showing close proximity of DC-LAMP positive cells to CD31 positive blood vessels (arrows).