Expression of Cathepsins B, D, and G in Infantile Hemangioma

Abstract

Aims: The role of the renin-angiotensin system (RAS) in the biology of infantile hemangioma (IH) represents an emerging paradigm, particularly the involvement of renin, angiotensin converting enzyme, and angiotensin II. This study investigated the expression of cathepsins B, D, and G, enzymes that may modulate the RAS, in IH.

Materials and methods: The expression of cathepsins B, D, and G was examined using immunohistochemistry, enzyme activity assays, mass spectrometry, and NanoString gene expression assay in IH samples at different phases of development.

Results: Immunohistochemical staining showed the expression of cathepsins B, D, and G in proliferating and involuted IH samples. This was confirmed at the transcriptional level using NanoString gene expression assays. Mass spectrometry confirmed the identification of cathepsins D and G in all three phases of IH development, whereas cathepsin B was detected in 2/2 proliferating and 1/2 involuting lesions. Enzyme activity assays demonstrated the activity of cathepsins B and D, but not G, in all phases of IH development.

Conclusion: Our data demonstrated the presence of cathepsins B, D, and G in IH. Their role in modulating the RAS and the biology of IH offers potential novel targets for the management of this tumor.

Keywords: angiotensin converting enzyme; cathepsin; infantile hemangioma; paracrine; renin–angiotensin system.

Figure 1

The production of angiotensin II (ATII) involves the classical renin–angiotensin system (illustrated in bold letters) and paracrine bypass loops involving multiple enzymes (illustrated in unbold letters) in that angiotensinogen is converted to angiotensin I (ATI) either by cathepsin D or by renin, with renin converted from its precursor pro-renin form, by cathepsin B. ATI is then cleaved to form ATII by either angiotensin converting enzyme, chymase, or cathepsin G, which also can directly convert angiotensinogen to ATII.

Figure 2

Confocal immunofluorescent immunohistochemical images of proliferating (A, C, E) and involuted (B, D, F) IH samples. The expression of cathepsin B [(A,B), red] is seen on the endothelium [(A,B), thick arrows] as well as cells in the interstitium [(A,B), thin arrows] in proliferating and involuted IH. Cathepsin D was predominantly localized to cells of the interstitium [(C,D), green] and appeared distinct from the endothelium expressing GLUT-1 [(C,D), red]. Similarly, the expression of cathepsin G [(E,F), red] was also restricted to cells of the interstitium and is independent of the CD34⁺ endothelium [(E,F), green]. Cell nuclei [(A–F), blue] are stained with 4′,6′-diamidino-2-phenylindole dilactate.

Figure 3

Confocal immunofluorescent immunohistochemical images of proliferating IH showing distinct interstitial cellular populations expressing cathepsin B [red, (A)] and cathepsin D [green, (A)]. Cathepsin G [red, (B)] was expressed by the phenotypic mast cells that also expressed tryptase [(B), green)]. Cell nuclei [(A,B), blue] are stained with 4′,6′-diamidino-2-phenylindole dilactate.

Figure 4

Cathepsins B and D enzyme activity in infantile hemangioma tissues with enzyme activity assays performed in proliferating (n = 3), involuting (n = 3), and involuted (n = 3) samples. The data were subjected to statistical analysis using the Kruskal–Wallis one-way analysis of variance for independent samples. P < 0.05 was considered as statistically significant.

Figure 5

Relative protein abundance of cathepsins B, D, and G extracted from proliferating (n = 2), involuting (n = 2), and involuted (n = 2) infantile hemangioma (IH). Proteome Discoverer™ V1.4 and Scaffold 4.0 were used to establish relative protein abundance by spectral counting. Relative protein abundance of all three cathepsins was unchanged between all three IH samples.

Figure 6

Cathepsins B, D, and G mRNA levels in infantile hemangioma (IH) tissues. NanoString nCounter Gene Expression assay with specific probes for CTHB, CTHG, and CTHD genes was performed using proliferating (n = 6) and involuted (n = 6) IH tissue samples. The data are presented as mean ± SD, and statistical analyses showed no significance.