Introduction
Lymphatic malformation (LM), a low-flow vascular malformation,1,2 affects 1 of 5000 births,3 mostly the head and neck.3 Fifty percent of LM are diagnosed perinatally, with the reminder presenting as slow-growing swellings.2 LM may cause cosmetic concerns, exudates, pruritis, and pain and are prone to infection2 Organ involvement3 may cause airway obstruction,1 visual impingement, and lymphedema.4
LM is categorized as macrocystic, microcystic, or mixed.2,4 Histologically, LM consists of thin-walled, irregular lymphatic vessels with disorganized endothelial cells,1 surrounded by sparse stroma.3
Microcystic LM (mLM) consists of cysts separated by septa and tends to infiltrate tissues,2,4 posing treatment challenges.1 Treatment options include sclerotherapy with alcohol, OK-432, or bleomycin,2 sirolimus,5 and/or surgical excision,2,6 with recurrence rates of 22%–59%.4
The pathogenesis of LM is poorly understood and may result from absent drainage due to dysregulated embryonic lymphangiogenesis.4,6 Vascular endothelial growth factor-C (VEGF-C) and VEGF receptor-3 (VEGFR-3) are critical in supporting lymphatic endothelial cells (LEC) through mitogen-activated protein kinase and PIK3CA7–9 considered the main driver of lymphangiogenesis,10 demonstrated in rat6 and mouse11 models.
In the classical renin-angiotensin system (RAS), renin catalyzes proteolysis of angiotensinogen to generate angiotensin I (ATI), which is further cleaved to form angiotensin II (ATII) by angiotensin-converting enzyme (ACE).12 Prorenin receptor (PRR), binds both prorenin and renin,13 increasing its catalytic activity 5-fold.14 ACE2 balances these actions by cleaving ATII to form the peptide Ang (1–7).15 The RAS regulates blood pressure and fluid volume homeostasis,16 and is involved in other physiological processes including angiogenesis, tissue remodeling, and inflammation.16–18
The RAS also acts in paracrine fashion,14 being biologically active in local tissues.19,20 This functions either independently or synergistically with the endocrine RAS,13,14,17 possibly contributing to local tissue dysfunction and disease.17 In vascular anomalies, components of the RAS are expressed by embryonic stem cell (ESC)–like cells in infantile hemangioma,21 pyogenic granuloma,22 and venous malformation (VM).23 The presence of the RAS in LM is unknown.
We have recently demonstrated the presence of cells that express stemness-associated markers OCT4, SOX2, NANOG, KLF4, and c-MYC on the endothelium and stroma of lesional vessels in mLM.24 We hypothesized that the RAS was present in mLM and was present on this primitive population.
Materials and methods
Tissue samples
mLM tissue samples from 18 patients including those from our previous study24 (Supplemental Digital Content, Table 1, https://links.lww.com/JV9/A22) were sourced from the Gillies McIndoe Research Institute Tissue Bank for this study, which was approved by the Central Health and Disability Ethics Committee (Ref. 13/CEN/130), with written informed consent from all participants.
Histology and immunohistochemical staining
Hematoxylin and eosin (H&E) staining was performed on 4-μm-thick formalin-fixed paraffin-embedded sections of mLM tissue samples from 18 patients. The presence of mLM was confirmed on H&E-stained slides and by the expression of D2-40 (1:100, cat#M3619, Dako, Glostrup, Denmark) by an anatomical pathologist. Immunohistochemical staining was then performed with primary antibodies for angiotensinogen (1:50, cat#79299, Cell Signaling, Danvers, MA), renin (1:500, cat#14291-1-AP, Proteintech, Rosemont, IL), PRR (1:500; cat#ab40790, Abcam, Cambridge, United Kingdom), ACE (1:100; cat#MCA2056, Bio-Rad, Hercules, CA), ACE2 (1:200; cat#MAB933 R&D Systems, Minneapolis, MN), and angiotensin II receptor 2 (AT2R; 1:2000; cat#NBP1-77368, Novus Biologicals, Littleton, CO). 3,3’-diaminobenzidine was used as the chromogen, using the Leica BOND RX auto-stainer (Leica, Nussloch, Germany). Slides were mounted in Dako Mounting Medium (cat#CS703, Dako). There are no suitable commercially available antibodies for AT1R.25–28
Normal human tissues used for positive controls were kidney (angiotensinogen, renin, ACE, ACE2, and AT2R) and placenta (PRR). Our internal antibody validation process involves staining on tissue negative controls using salivary gland (angiotensinogen, renin, and AT2R), colon (PRR), and skin (ACE and ACE2).
To confirm coexpression of proteins, immunofluorescence staining was performed on 2 representative mLM tissue samples from the 18 patients included in immunohistochemical staining, using the same primary antibodies at the same concentrations, and costaining with OCT4 (1:30; cat#309M-16, Cell Marque, Rocklin, CA) as a surrogate marker of the ESC-like population previously identified in mLM.24 Secondary antibodies and amplification kits used were Alexa Fluor anti-mouse 488 (1:500; cat#A-21202, Life Technologies, Carlsbad, CA), Alexa Fluor anti-rabbit 594 (1:500; cat#A-21207, Life Technologies), VectaFluor Excel anti-mouse 488 (ready-to-use; cat#DK-2488, Vector Laboratories, Burlingame, CA), and VectaFluor Excel anti-rabbit 594 (ready-to-use; cat#DK-1594, Vector Laboratories). All antibodies were diluted with BOND primary antibody diluents (cat#AR9352, Leica). Immunofluorescence-stained slides were mounted using Vectashield hardset medium with 4’,6-diamidino-2-phenylindole (cat#H-1500, Vector Laboratories).
Negative controls for immunohistochemical and immunofluorescence staining were mLM sections stained with primary isotype mouse (ready-to-use; cat#IR750, Dako) and rabbit (ready-to-use; cat#IR600, Dako) antibodies. We were unable to validate a suitable antibody for AT1R and have therefore excluded this marker from protein-level analysis.25–28 Positive and negative control tissues were selected based on manufacturer recommendations, and the Human Protein Atlas.29
Image capture and analysis
Immunohistochemical-stained slides were viewed and imaged using an Olympus BX53 light microscope with an Olympus SC100 digital camera (Olympus, Tokyo, Japan), and processed with cellSens 2.0 software (Olympus). Immunofluorescence-stained slides were viewed with an Olympus FV1200 biological confocal laser-scanning microscope (Olympus) and processed with cellSens Dimension 1.11 software (Olympus).
Western blotting
Total protein was extracted from 6 available snap-frozen mLM tissue samples of the 18 patients by pestle homogenization (cat#80-6483-37, GE Healthcare, Piscataway, NJ) in ice-cold radioimmunoprecipitation assay buffer (cat#R0278, Sigma-Aldrich, St. Louis, MO) supplemented with 1× HALT Protease and Phosphatase Inhibitor Cocktail (cat#78440, Pierce Biotechnology, Rockford, IL) and 10 mM dithiothreitol (cat#R0861, Thermo Fisher Scientific, Waltham, MA). Protein was quantified using a bicinchoninic acid assay (cat#23227, Pierce Biotechnology), and diluted in an equal volume of 2× Bolt lithium dodecyl sulfate sample buffer (cat#B0007, Life Technologies). Equal amounts of protein (~20 μg total protein per sample) were denatured at 70°C for 10 minutes in sample buffer and resolved by 4%–12% 1D-PAGE (cat#NW04120BOX, Invitrogen, Carlsbad, CA) and transferred to a polyvinylidene fluoride membrane (cat#IB24001, Invitrogen) using an iBlot 2 device (cat#IB21001, Thermo Fisher Scientific). Blotted membranes were blocked for 5 minutes at room temperature in 1× iBind Flex FD solution per manufacturer’s instructions (cat#SLF2019, Thermo Fisher Scientific) and probed for angiotensinogen (1:1000, cat#79299, Cell Signaling), PRR (1:500; cat#HPA003156, Sigma Aldrich), ACE (1:200; cat#sc-12184, Santa Cruz, Dallas, TX), ACE2 (1:500, cat#MAB933, R&D Systems), AT2R (1:500; cat#ab92445, Abcam), or α-tubulin (1:1000; cat#62204, Invitrogen), using the iBind Flex Western device (cat#SLF2000, Invitrogen). Secondary antibodies used were goat anti-rabbit horse radish peroxidase (HRP) conjugate (1:1000, cat#111-035-045, Jackson ImmunoResearch Laboratories, West Grove, PA) for angiotensinogen, PRR, ACE, and AT2R, goat anti-mouse HRP (1:1000, cat#ab6789, Abcam) for ACE2, or donkey anti-mouse Alexa Fluor 488 (1:2000; cat#A-21202, Life Technologies) for α-tubulin. Clarity Western ECL (cat#170-5061, Bio-Rad) was used as the substrate for visualizing HRP-detected protein bands. The ChemiDoc MP Imaging system and Image Lab 6.0 software (Bio-Rad) were used for both HRP and fluorescent band detection and analysis. Positive controls used were human plasma (angiotensinogen), human tonsil (PRR), mouse lung (ACE), human kidney (ACE2), and mouse heart (AT2R). Blotting for renin was abandoned as no antibody was found to produce a single specific band. There are no suitable antibodies commercially available for AT1R.25–28
Reverse transcription quantitative polymerase chain reaction
Total RNA was isolated from 5 snap-frozen mLM tissue samples of the 18 patients. Approximately 20 mg of each tissue sample was homogenized using the Omni Tissue Homogenizer (Omni International, Kennesaw, GA), and RNA prepared using the RNeasy Mini Kit following manufacturer’s instructions (cat#74104, Qiagen, Hilden, Germany). An on-column DNase digest step was included (cat#79254, Qiagen). RNA was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Transcript expression was analyzed in triplicate using the Rotor-Gene Multiplex RT-PCR Kit (cat#204974, Qiagen) and TaqMan Gene Expression Assay primer probes on a Rotor Gene Q (Qiagen). The primer probes used were angiotensinogen (Hs01586213_m1), renin (Hs00982555_m1), PRR (Hs00997145_m1), ACE (Hs00174179_m1), ACE2 (Hs01085333_m1), AT1R (Hs00258938_m1), AT2R (Hs00169126_m1), and the reference genes GAPDH (Hs99999905_m1) and PUM1 (Hs00206469_m1) (cat#4331182, Thermo Fisher Scientific). Universal human reference RNA (UHR; cat#CLT636690, Takara, Shiga, Japan), total RNA from a range of healthy adult human tissues, was used as the calibrator for the 2ΔΔCt analysis. Nuclease-free water was added for the no template control. Positive controls used were HepG2 cells (angiotensinogen and ACE2), uterine fibroid tissue (PRR, ACE, AT1R, and AT2R), and PC3 cells (renin). Endpoint amplification products were confirmed using 2% agarose gel electrophoresis (cat#G402002, Thermo Fisher Scientific) and imaged using the ChemiDoc MP (Bio-Rad). Graphs were generated using GraphPad Prism (v8.0.2, San Diego, CA) with results expressed as fold change relative to UHR. A biologically relevant fold-change cut-off was set at 2.0 for upregulated, and 0.5 for downregulated genes.
Results
Histology and immunohistochemical staining
H&E staining showed all 18 mLM samples consisted of irregularly outlined, dilated, thin-walled lymphatic vessels lined by flat endothelium (Figure 1A), positive for D2-40 (Figure 1B). Except positive staining of PRR in the pericyte layer of the lesional lymphatic vessels in 1 case, angiotensinogen (Figure 1C), renin (Figure 1D), PRR (Figure 1E), ACE (Figure 1F), and ACE2 (Figure 1G) were not present in any of the mLM samples. PRR (Supplemental Digital Content, Figure 1A, https://links.lww.com/JV9/A17) and ACE (Supplemental Digital Content, Figure 1B, https://links.lww.com/JV9/A17) were present only on the endothelium of normal vessels. AT2R (Figure 1H) was present on the endothelium and the pericyte layer of the lesional lymphatic vessels and cells within the stroma.
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Figure 1.: A representative hematoxylin and eosin–stained section (A) and immunohistochemical-stained sections (B–H) of microcystic lymphatic malformation showing characteristic ectatic lymphatic channels lined by flat endothelial cells (A) which stained positively for D2–40 (B, brown). Angiotensinogen (C, brown), renin (D, brown), PRR (E, brown), ACE (F, brown), and ACE2 (G, brown) were not detected on the lesional lymphatic vessels. AT2R (H, brown) was present on the endothelium of the lesional lymphatic vessels. Nuclei were counter-stained with hematoxylin (A–H, blue). Original magnification: ×200, inset ×400. ACE indicates angiotensin-converting enzyme; AT2R, angiotensin II receptor 2; PRR, prorenin receptor.
Human tissues used for positive controls for immunohistochemical staining showed the expected staining patterns for angiotensinogen (Supplemental Digital Content, Figure 2A, https://links.lww.com/JV9/A18), renin (Supplemental Digital Content, Figure 2B, https://links.lww.com/JV9/A18), ACE (Supplemental Digital Content, Figure 2D, https://links.lww.com/JV9/A18), ACE2 (Supplemental Digital Content, Figure 2E, https://links.lww.com/JV9/A18), and AT2R (Supplemental Digital Content, Figure 2F, https://links.lww.com/JV9/A18) in kidney and PRR (Supplemental Digital Content, Figure 2C, https://links.lww.com/JV9/A18) in placenta. Isotype controls (Supplemental Digital Content, Figure 2G, https://links.lww.com/JV9/A18) showed no staining, confirming the specificity of the secondary antibodies. Tissue negative controls (Supplemental Digital Content, Figure 3, https://links.lww.com/JV9/A19) showed no significant nonspecific staining of the primary antibodies.
Immunofluorescence staining
Immunofluorescence staining demonstrated expression of AT2R (Figure 2, red) on the OCT4+ (Figure 2, green) cells on the endothelium and media of the lesional lymphatic vessels and cells within the stroma. Split images of the stains presented in Figure 2 are shown in Supplemental Digital Content, Figure 4A and B, https://links.lww.com/JV9/A20. Minimal staining on the negative control (Supplemental Digital Content, Figure 4C, https://links.lww.com/JV9/A20) confirmed specificity of the secondary antibodies.
Figure 2.: Representative immunofluorescence-stained section of microcystic lymphatic malformation demonstrating cytoplasmic expression of AT2R (red) on the OCT4+ (green) cells on the endothelium (long arrow) and media (short arrow) of the lesional lymphatic vessels and the stroma (arrowhead). Cell nuclei were counter-stained with 4’,6-diamidino-2-phenylindole (blue). Original magnification: ×400. AT2R indicates angiotensin II receptor 2.
Western blotting
Western blot (WB) performed on 6 snap-frozen mLM tissue samples from the original cohort of 18 patients demonstrated expression of angiotensinogen (Figure 3A) in all 6 samples at 60 kDa, close to the expected weight of 55 kDa. PRR (Figure 3B) was detected in all samples at 35 kDa, with 4 samples also showing bands at the size for its soluble form at 27 kDa. ACE was detected at 195 kDa in 5 of the 6 samples (Figure 3C, red). ACE2 (Figure 3D, red) and AT2R (Figure 3E, red) were detected in the positive controls, but none of the samples. Equivalent total protein loading for all 6 mLM tissue samples was confirmed by α-tubulin (Figure 3F, red).
Figure 3.: Full-length western blot images of total protein extracted from microcystic lymphatic malformation tissue samples from 6 patients demonstrating the presence of angiotensinogen (A, red) in all 6 samples at the expected size of ~60 kDa. PRR (B, red) was detected in all samples at the appropriate size of ~35 kDa, with bands representing the soluble form of PRR at ~27 kDa in 4 samples. ACE (C, red) was detected in 5 of the 6 tissue samples at ~195 kDa. ACE2 (D, red) was detected in the positive control, but none of the samples. AT2R (E, red) was similarly confirmed in the positive control but was not detected in the tissue samples. α-tubulin loading control (F, red) confirmed approximate equal loading. ACE indicates angiotensin-converting enzyme; AT2R, angiotensin II receptor 2; PRR, prorenin receptor.
Reverse transcription quantitative polymerase chain reaction
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) demonstrated the presence of transcripts for angiotensinogen, PRR, ACE, and ACE2 in all 5 mLM tissue samples (Figure 4). Expression of AT1R was identified in 4, AT2R in 2, and renin in 1 of the 5 tissue samples (Figure 4). The mean level of transcript expression was downregulated relative to UHR for all components of the RAS. All markers except for PRR and ACE were significantly biologically downregulated, with the expression less than half that of UHR. There was 1 tissue sample each for PRR and ACE in which the expression level was approximately equivalent to UHR. Specific amplification was demonstrated by the expected sized amplicons on gel electrophoresis of qPCR products, with no products in the no template control reactions (Supplemental Digital Content, Figure 5, https://links.lww.com/JV9/A21).
Figure 4.: Fold-change (2ΔΔCT) in gene expression of components of the RAS: AGT, renin, PRR, ACE, ACE2, AT1R, and AT2R, determined by reverse transcription quantitative polymerase chain reaction on total RNA extracted from 5 microcystic lymphatic malformation tissue samples. Cycle threshold values were normalized to the reference genes GAPDH and PUM1. Expression was compared with UHR, with no change noted as the dashed horizontal line, and biologically significant up and downregulation as the dotted lines. Error bars represent 95% confidence intervals around the mean. ACE indicates angiotensin-converting enzyme; AT1R, angiotensin II receptor 2; AGT, angiotensinogen; PRR, prorenin receptor; RAS, renin-angiotensin system; UHR, universal human reference RNA.
Discussion
This study presents novel findings of the presence of AT2R in mLM. Immunohistochemical staining did not detect angiotensinogen or renin in any of the 18 mLM tissue samples. This was reflected by the RT-qPCR results, which showed transcript expression of renin in only 1 sample at a downregulated level, and downregulated transcript expression of angiotensinogen, relative to UHR. PRR was expressed on normal vasculature, with staining of the lesional lymphatic vessels in only 1 mLM sample. Transcript and protein expression as demonstrated by RT-qPCR and WB likely reflected this surrounding normal vasculature. Similarly, ACE expression was demonstrated only in surrounding normal vasculature by immunohistochemical staining, with WB confirming protein expression and RT-qPCR detection of transcripts likely reflecting its expression by this normal vasculature. ACE2 was not detected by immunohistochemical staining or WB, and transcripts were detected by RT-qPCR at significantly downregulated levels, relative to UHR. This could be due to translation below detectable levels using the protein techniques. AT2R was demonstrated on the lesional lymphatic vessels of mLM by immunohistochemical staining, and its transcript expression was confirmed by RT-qPCR, although its protein expression was below detectable levels on WB. This may be attributable to low protein levels within the mLM tissue samples and/or sampling bias. AT1R transcripts were detected by RT-qPCR in 4 of the 5 samples, although its protein expression in mLM could not be investigated due to a lack of suitable antibodies.25–28
The lack of expression of PRR, renin, and ACE in lesional lymphatic vessels does not necessarily preclude local conversion of angiotensinogen through to ATII. There are known bypass loops of the RAS, including chymase, aminopeptidases A, B, and N, aspartyl-aminopeptidase, and cathepsins.30–33 We have recently demonstrated the expression of cathepsins in mLM, suggesting the presence of such bypass loops.34
ACE2 has not been previously identified in mLM, and its direct effects on lymphangiogenesis have not been studied in detail. Its absence is significant, however, as it degrades ATII, leading to accumulation of ATII and a subsequent increase in signaling through its receptors. ATII has widespread effects throughout the body, including in the heart, brain, immune system, and vasculature.20 Both AT1R and AT2R bind ATII, with antagonistic actions.35–38 AT1R serves many physiological functions, including vasoconstriction, aldosterone secretion, and maintenance of sodium/potassium/water homeostasis.37 In contrast, AT2R mediates vasodilatation, nitric oxide release, and growth inhibition.35 AT2R is also more abundantly expressed in fetal tissue, playing an important role in fetal development.36 The presence of AT1R in mLM is significant due to its role in lymphangiogenesis through the stimulation of angiopoietin-2 (Ang-2) production via the protein kinase C and mitogen-activated protein kinase pathways.39
ATII receptors have not been studied previously in mLM, with limited research into their role in lymphangiogenesis in other diseases and species. ACE inhibitors and AT1R blockers (ARBs) have been shown to reduce lymphangiogenesis via reduced VEGF-C levels in a murine gastric cancer model.40 In rats, this has also been shown in renal lymphangiogenesis of newborns,41 and a kidney transplant model.42 This is reflected by studies finding macrophage influx with cardiac lymphangiogenesis in response to ATII infusion in rats,43 and this same effect is inhibited by an ARB in mice.44,45 Investigation of the mechanisms of ATII-induced renal lymphangiogenesis in mice shows no expression of ATII receptors on LECs, or altered gene expression when cultured with ATII.46 However, splenocytes and CD11b+ cells cultured with ATII increases VEGF-C production, and subsequently altered gene expression of LECs in coculture.46 This is in contrast to findings from a more recent study showing that LECs express AT1R, and have increased proliferation and migration in response to ATII, which can be nullified by ARB treatment with 1385 differentially expressed genes on the LECs following ARB treatment, which can also be reversed ARB pretreatment.45 In breast cancer patients, AT1R is positively correlated with both VEGF-A and VEGF-D expression, however, the association with VEGF-C or lymphangiogenesis is not significant.47
Components to the RAS including PRR, ACE, AT1R, and AT2R have been demonstrated in VM,48 localized to the ESC-like population.23,49 VM and LM share similarities in etiological pathways.50-52 Ang-2 is a ligand for the TIE2 receptor, also implicated in VM pathogenesis,53 and is expressed on LECs.54 The Ang-TIE2 endothelial growth factor receptor signaling pathway plays an important role in both embryonic and pathologic lymphangiogenesis.53,55,56 Interestingly, Ang-2 can agonize or antagonize TIE2, depending on the context.53,54,56 Ang-2 knockout mice develop lymphatic defects, indicating a crucial role of Ang-2 in lymphatic vessel formation and stabilization.55 It is therefore likely that in the context of lymphangiogenesis, Ang-2 acts as an agonist to induce TIE2.54 Furthermore, upregulation of Ang-2 and the subsequent downregulation of Ang-1 in various conditions results in a loss of blood vessel stability.53 It is plausible that this could also occur in the context of lymphangiogenesis in mLM. As we have identified the presence of AT1R in mLM, we propose that ATII acts on AT1R, which may stimulate the production of VEGF-C, or Ang-2, thus inducing TIE2, resulting in pathological lymphangiogenesis and formation of mLM.
There is limited research on the involvement of the RAS in other vascular malformations, although there is a case report of Klippel-Trénaunay syndrome, responding to the treatment with propranolol.57
Some vascular tumors however have been found to demonstrate components of the RAS. As with mLM, infantile hemangioma also expresses AT2R, in addition to ACE,21 with cultured tissue sections demonstrating increased cellular proliferation in response to administration of ATII, an effect moderated by inhibitors of the RAS.58 Pyogenic granuloma also expresses AT2R alongside other components of the RAS.22 This suggests possible similarities in their disease processes.
We have recently demonstrated the presence of an ESC-like population on the endothelium and the stroma of mLM, which expresses the ESC markers OCT4, SOX2, NANOG, KLF4, and c-MYC.24 Greater expression of SOX2 and c-MYC, which are progenitor-associated markers,59 suggests a larger proportion of this population display a progenitor phenotype, with a smaller proportion of upstream ESC-like cells, which may give rise to the CD133+ progenitor population previously identified on LM cells.4
We here demonstrate the expression of AT2R on this ESC-like population, using OCT4 as a surrogate marker, suggesting that this primitive population may be driven by ATII receptors. Given the similarities between mLM and macrocystic LM, it is worth speculating in future research whether these findings also apply to macrocystic LM. In addition, functional experiments to investigate the effects of RAS inhibitors such as ACE inhibitors, ARBs, and β-blockers including propranolol on cell culture or other models of mLM may further reveal the role of the RAS in the pathology of mLM. This in turn may lead to novel therapeutic targeting of the primitive population within mLM by modulation of ATII receptors, by repurposing existing, affordable medications with well-established safety profiles.
Acknowledgments
We thank Ms Liz Jones of the Gillies McIndoe Research Institute for her assistance in H&E, immunohistochemical and immunofluorescence staining; and Kendra Boyes and Bede van Schaijik for performing the initial RT-qPCR experiments.
References
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