Expression of the Components of the Renin–Angiotensin System in Venous Malformation

Background Venous malformation (VM) is the most common form of vascular malformation, consisting of a network of thin-walled ectatic venous channels with deficient or absent media. This study investigated the expression of the components of the renin–angiotensin system (RAS), namely, (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (AIITR2) in subcutaneous (SC) and intramuscular (IM) VM. Materials and methods SC (n = 7) and IM (n = 7) VM were analyzed for the expression of PRR, ACE, ATIIR1, and ATIIR2 using 3,3-diaminobenzidine and immunofluorescent (IF) immunohistochemical (IHC) staining and NanoString gene expression analysis. Results IHC staining showed expression of PRR, ACE, and ATIIR1, and faint expression of ATIIR2 in the endothelium of SC and IM VM. Furthermore, ATIIR2 was expressed by cells away from the endothelium in both SC and IM VM lesions examined. NanoString analysis demonstrated the presence of PRR, ACE, and ATIIR1 but not ATIIR2. Conclusion The presence of PRR, ACE, ATIIR1, and potentially ATIIR2, in both SC and IM VM, suggests a role for the RAS in the biology of VM. This novel finding may lead to a mechanism-based therapy for VM.

inTrODUcTiOn Vascular anomalies are classified by the International Society for the Study of Vascular Anomalies classification system into vascular tumors and vascular malformations (1). Infantile hemangioma (IH) is the most common type of vascular tumor (1). Vascular malformation affects arteries, veins, lymphatics, and capillaries singly or in combinations, with venous malformation (VM) being the most common (1).
Venous malformation affects 1.5% of the population (2) and is characterized by thin-walled ectatic venous channels lined by flat endothelial cells (EC) with deficient or absent smooth muscle cells (SMC) (3,4). VM may involve any body site and tissue (4), commonly in subcutaneous (SC) and less commonly in intramuscular (IM) locations (5). Although VM is present at birth, it may not become apparent until later in life (3)(4)(5). Its clinical presentation depends on the location and size of the lesion. SC lesions typically present as compressible masses with a bluish hue, whereas IM lesions often present with a swelling and/or pain (4). VM may cause cosmetic concerns and/or functional deficits, such as obstructive sleep apnea as in the case of oropharyngeal lesions (6). VM grows proportionately with the growth of the child and may suddenly expand in response to hormonal changes or trauma, including incomplete surgical excision (3)(4)(5).
About 1-2% of VM cases are familial caused by a TIE2 mutation (7). Half of the sporadic cases also consists a TIE2 somatic mutation (7). These mutations have been shown to result in ligand-independent hyper-phosphorylation of the TIE2 receptor (7). Vikkula et al. (8) suggest that the TIE2 mutation in EC in VM may reduce SMC ligand expression causing a local uncoupling between normal SMC recruitment and the proliferation of EC.
Furthermore, more recent demonstration of hormone receptor proteins, such as follicle-stimulating hormone receptor (9) and the growth hormone receptor (10) within VM, highlights other potential pathways that may play a critical role in the biology of VM.
Previous work has identified the presence of primitive mesodermal cells with a neural crest phenotype in IH (12), regulated by the RAS (13). These discoveries of the stem cell nature of IH, regulated by the RAS, underscore the natural involution and accelerated involution of this tumor, induced by β-blockers and ACE inhibitors (14)(15)(16).
This study aimed to identify the expression of the components of the RAS, namely, PRR, ACE, ATIIR1, and ATIIR2, in both SC and IM VM. All DAB IHC-stained slides were mounted in Surgipath Micromount (cat#3801732, Leica). All IF IHC-stained slides were mounted in Vectashield HardSet antifade mounting medium with DAPI (cat#H-1500, Vector Laboratories).

Tissues
Positive control samples were selected based on the previously reported expression of the relevant proteins: placenta for PRR (18), kidney for ACE (19) and ATIIR2 (20), and liver for ATIIR1 (20). To determine the specificity of the primary antibodies, staining of VM sections was performed by omitting the primary antibodies.

image analysis
All DAB and IF IHC-stained slides were viewed and imaged using the Olympus BX53 microscope fitted with an Olympus DP21 digital camera (Olympus, Tokyo, Japan) and an Olympus FV1200 biological confocal laser-scanning microscope (Olympus, Tokyo, Japan), respectively. IF IHC images were processed with cellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).

nanostring gene expression analysis
Total RNA was extracted from ~10 mg of snap-frozen IM (n = 3) and SC (n = 3) VM tissues from the same cohorts of patients included in DAB IHC staining, using RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantitated with the NanoDrop 2000 Spectrophotometer (Thermo Scientific). The samples with A260/ A230 ≥1.8 and A260/A280 ≥1.9 were used for further analysis. The integrity of the RNA was assessed by Agilent 2100 BioAnalyser (Agilent Technologies, Santa Clara, CA, USA). The isolated RNA was then subjected to NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA) and completed by New Zealand Genomics, Ltd.

statistical analyses
Raw NanoString data were analyzed using SPSS (v22, IBM), corroborated with nSolver™ software (NanoString Technologies) using standard settings, and normalized against the housekeeping gene. To determine the level of confidence between the IM and SC VM samples, two-tailed Student's t-test was performed. Charts were made with Excel (Microsoft Office 2013). PRR was expressed on the endothelium of both IM (Figure 2A, brown) and SC (Figure 2B, brown) VM. ACE, which converts ATI to ATII, was expressed on the endothelium of IM (Figure 2C, brown) and SC (Figure 2D, brown) VM. To demonstrate a potential receptor available for downstream signaling of ATII, staining for ATIIR1 and ATIIR2 was performed. This demonstrated the expression of ATIIR1 on the endothelium of IM (Figure 2E, brown) and SC (Figure 2F, brown) VM. There was faint staining of ATIIR2 of the endothelium and cells away from the endothelium of both IM (Figure 2G, brown) and SC (Figure 2H, brown) VM lesions studied.
There was relatively more intense staining for ATIIR1, in both IM and SC VM, in the smaller lesional vessels (Figures 3A,B, brown, thin arrow) compared to the dilated vessels (Figures 3A,B, brown, thick arrow).
Positive controls for PRR ( Figure

immunoflourescent immunohistochemical staining
The endothelium of all IM (Figures 4A,C,E,G) and SC (Figures 4B,D,F,H) VM were identified by either CD34 (Figures 4A,B,G,H, green) or ERG (Figures 4C-F, red). IF IHC staining demonstrated the expression of PRR on the endothelium of IM (Figure 4A, red) and SC (Figure 4B, red) VM. The expression of ACE was demonstrated on the endothelium of IM (Figure 4C, green) and SC (Figure 4D, green) VM. ATIIR1 was expressed on the endothelium of IM (Figure 4E, green) and SC (Figure 4F, green) VM. ATIIR2 was also demonstrated on cells of the endothelium as well as cells away from the endothelium in both IM (Figure 4G, red) and SC (Figure 4H, red) VM. Cell nuclei were highlighted in blue (Figures 4A-H). Individual IF IHC staining for each of the aforementioned proteins is shown in Figure S3 in Supplementary Material.
Negative controls for IF IHC staining demonstrated appropriate specificity of the primary antibodies in anti-mouse ( Figure  S4A in Supplementary Material, green) and anti-rabbit ( Figure  S4B in Supplementary Material, red) combinations.

nanostring gene analysis
NanoString gene analysis showed that mRNA corresponding to PRR, ACE, and ATIIR1, but not ATIIR2, was detected in IM ( Figure 5A) and SC ( Figure 5B) VM tissue samples. The following average counts were calculated as expression relative to GAPDH, with their respective SEM for IM VM, PRR 0.00844 ± 0.0050, ACE 0.00064 ± 0.0002, and ATIIR1 0.00271 ± 0.0057; SC VM PRR 0.0809 ± 0.0238, ACE 0.0502 ± 0.0473, and ATIIR1 0.0191 ± 0.0087. Expression of PRR was higher in SC than IM samples, and this was statistically significant (p < 0.05).

DiscUssiOn
This report provides novel findings of the presence of PRR, ACE, ATIIR1, and potentially ATIIR2 within both IM and SC VM.
The demonstration of PRR on the endothelium of VM highlights the putative role of this receptor in the biology of VM, including binding pro-renin and renin and downstream signaling    through MAP kinases ERK1 and ERK2 (18). Pro-renin bound to PRR undergoes a conformational change, exposing the active sites and rendering it enzymatically active (11). Renin bound to PRR leads to a fourfold increase in catalytic activity (18). The presence of PRR on the endothelium of VM indicates a potential role for facilitating increased local conversion of angiotensinogen to ATI, indicating a role for this peptide in VM biology.
ACE has been reported as a marker for identifying primitive hemangioblasts derived from human pluripotent stem cells (21). It is exciting to speculate that the endothelium of VM, which we have demonstrated to express this marker, may reflect a relative primitive phenotype for this endothelium; however, this is the topic of our current investigation.
It is intriguing that both DAB and IF IHC analysis revealed positive staining for ATIIR2 on the endothelium as well as cells away from the endothelium, but that its expression was not demonstrated by NanoString gene expression analysis. The reasons for these include possible non-specific binding of the primary antibody used for IHC staining, despite appropriate controls being used, or that the probe used for the NanoString analysis failed to cover all the possible mRNA splice variants for this protein. Furthermore, it is interesting to note that the DAB IHC analysis of the SC and IM VM samples revealed relatively increased staining for ATIIR1 on the endothelium of smaller vessels compared to the dilated vessels, the reasons for which extend beyond this study.
The presence of PRR, ACE, ATIIR1, and potentially ATIIR2 in VM indicates a potential role for the RAS in this condition. Nguyen Dinh Cat et al. (22) have demonstrated the components of the RAS including PRR, ACE, ATIIR1, and ATIIR2 in normal vascular tissues. ATIIR1 and ATIIR2 have been shown to serve distinct functions, but share ATII as a common ligand (20). The demonstration of ATIIR1, and potentially ATIIR2, in VM presented here does not delve into the functional roles of these proteins in VM, as this is the topic of further study. However, given ATIIR1 is responsible for the proangiogenic effects of ATII (23), this may, in part, explain the increased density of abnormal venous channels within VM. The presence of ATIIR2 may indicate cellular differentiation determination, as previously proposed by Zambidis et al. (21).
A recent case report observing reduction of rectal bleeding and anemia from a VM affecting the rectosigmoid junction following administration of propranolol and celecoxib (24) was attributed by the authors to the vasoconstrictive effect of propranolol, a β-blocker, and the inhibitory effect of celecoxib, a COX-2 inhibitor, on vascular endothelial growth factor (VEGF) (24). The serendipitous observed clinical response in this case could potentially be explained by the inhibitory effects of the β-blocker and COX-2 inhibitor on the RAS. Our study demonstrates the expression of PRR on the endothelium of VM, and we hypothesize that the observed effect of these medications is via the reduction in plasma renin levels by a β-blocker (25) and mitigation of ATII-induced expression of PRR by a COX-2 inhibitor (26).
The putative primitive endothelial phenotype in VM is partially supported by the demonstration of positive cytoplasmic staining for stem cell growth factor receptor, c-kit, in small lesional vessels, but not in dilated vessels, of VM (27). We have reported a similar pattern of stronger staining for ATIIR1 on the endothelium of smaller lesional vessels, and it is exciting to speculate that the absence of c-kit in dilated lesional vessels may indicate that the latter are more mature and can no longer maintain a putative stem cell population, with the smaller vessels as potential precursors. However, this is a topic of our ongoing research. This observation would therefore suggest that the smaller lesional vessels are the precursors of the more mature and ectatic vessels, but this is beyond the scope of this study. TIE2 gene mutations have been implicated in VM (7), although the exact mechanism leading to the development of VM has yet to be fully elucidated. Vikkula et al. (8) propose a role in coupling of EC proliferation and SMC recruitment.
A previous study shows that ATII, acting through ATIIR1, stimulates the production of angiopoietin-2 (Ang-2), a ligand for TIE2 (28). Given that Ang-2 modulates the effect of angiopoietin-1 (Ang-1) on TIE2 (29) as well as increases the effects of VEGF. Based on our findings, it is exciting to speculate that increased signaling via ATIIR1 results in increased production of Ang-2, ultimately leading to the development of VM, via activation of TIE2 (8,29). Figure 6 shows our proposed model for the pathogenesis of VM incorporating a role for the RAS.
Current treatments of VM aim to relieve symptoms caused by VM, but they often neither halt nor reverse the underlying process that leads to the development and expansion of these lesions, limiting their efficacy (30), and depending on the location of the lesion, may not be feasible.
To the best of our knowledge, this is the first report demonstrating the presence of PRR, ACE, ATIIR1, and potentially ATIIR2 in IM and SC VM. This novel finding suggests the RAS as a potential therapeutic target for VM using RAS modulators.

DeclaraTiOns
The content of this article has not been submitted or published elsewhere. There was no source of funding for the article. The authors declare that there is no source of financial or other support, or any financial or professional relationships, which may pose a competing interest. All authors contributed to the preparation of this manuscript. The manuscript has been seen and approved by all authors.