ErbB Signalling is a Potential Therapeutic Target for Vascular Lesions with Fibrous Component
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The authors address an important clinical entity and an important area of unmet clinical need. The authors use a combination of in vitro and in vivo experiments to learn how stromal cells surrounding vessels in venous malformations (VM) and angiomatosis of soft tissue (AST) contribute to the angiogenic activities driving the vascular lesions. They discovered that secretion of transforming growth factor-alpha (TGFa) from both endothelial cells and stromal cells, shows evidence for EGF-receptor phosphorylation. In addition, they show that afatinib, a pan-ErbB TKI inhibitor may have therapeutic benefits.
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Abstract
Background: Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to high recurrence of the lesions more effective therapies are needed. Methods: As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. Results: We report, for the first time, expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of VEGF-A paracrinally, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts. Conclusions: Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. Funding: Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, and Department of Musculosceletal and Plastic Surgery, Helsinki University Hospital.
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Author Response
Reviewer #1 (Public Review):
The authors present a strong set of experiments to uncover what type of role non-mutant stromal cells might be playing in the development of VM and AST, two vascular lesions that share some similarities.
Questions about experimental design.
- For quantification of gene expression in VM and AST specimens in Figure 2, the methods say qPCR data were normalized to housekeeping genes, but it would be helpful to normalize to endothelial content. It might be that increased TGFa is due to increased endothelium.
We thank the Reviewer for this excellent suggestion. We have now added this new data as suggested with normalization of TGFA mRNA to the endothelial marker PECAM-1/CD31 mRNA. A trend towards an increased expression of TGFA mRNA was detected in VM/AST specimens in comparison to the …
Author Response
Reviewer #1 (Public Review):
The authors present a strong set of experiments to uncover what type of role non-mutant stromal cells might be playing in the development of VM and AST, two vascular lesions that share some similarities.
Questions about experimental design.
- For quantification of gene expression in VM and AST specimens in Figure 2, the methods say qPCR data were normalized to housekeeping genes, but it would be helpful to normalize to endothelial content. It might be that increased TGFa is due to increased endothelium.
We thank the Reviewer for this excellent suggestion. We have now added this new data as suggested with normalization of TGFA mRNA to the endothelial marker PECAM-1/CD31 mRNA. A trend towards an increased expression of TGFA mRNA was detected in VM/AST specimens in comparison to the control group. We also show in the manuscript that besides CD31-positive vascular structures, TGFA is expressed in intervascular areas, i.e. between the vessels, in the patients’ lesions (Fig.2) and in lesion-derived CD31negative intervascular stromal cells. These data altogether demonstrate that i) TGFA is expressed also in other cell types than endothelial cells and ii) indicates that the increased expression of TGFA in lesion samples is not only due to increased vasculature/endothelium in the patient samples.
The new RT-qPCR data has now been added to the manuscript as a new Fig. 2 - figure supplement 1.
- The mutant allelic frequency for the HUVEC-PIK3CA WT versus HUVEC-PIK3CA H1047R should be provided. This is critically needed for the interpretation of the results.
Thank you for this valuable comment. To confirm that PIK3CA H1047R is still present in transduced HUVECs at the end-point of the mouse xenograft experiment, we performed a new ddPCR analysis detecting fractional abundance of PIK3CA p.H1047R from the matrigel plug-in samples. In this new data, mean fractional abundance of PIK3CA p.H1047R in fibroblast containing PIK3CA H1047R EC plugs was shown to be 27.1 % (variation 26.5-27.8 %; n=2 mice in duplicates). This corresponds to ~54 % of PIK3CA p.H1047R mutation positive cells in the plug, assuming a single copy of the mutation in each cell. As a control group, no positivity was detected in samples with fibroblast and in PIK3CAwt EC, as all the cells express the wildtype form of the PIK3CA gene. Please see Author Response Image 1 representative 2D amplification plots of the mutation analysis. Fractional abundances of PIK3CA mutations in the patient tissue samples and patient-derived CD31+ cells can also be seen in Table 1 and were in a range of 5-12 % (whole tissue) and 44-51 % (EC fraction).
- From Figure 5, it appears that the human primary fibroblasts are not required for the mutant ECs to form perfused vessels (panel H).
We thank the Reviewer for the comment and agree that based on our H&E staining and erythrocyte analysis, perfused vessels are evident in PIK3CA mutant plugs containing ECs with fibroblasts but also in plugs containing ECs alone. This was expected as PIK3CA mutation in ECs alone has shown to be a driver of venous malformation. However, prior to our study the role of fibroblasts in PIK3CA-driven lesions had not been studied. To better understand the role of fibroblasts in lesion formation, we have now added new data to the manuscript containing example images of the PIK3CA H1047R plugs with or without fibroblasts, and added a new quantitation of their erythrocyte amount. Please see Author Response Image 2. Our data demonstrates that there are significantly: i) more CD31-positive vascular structures (Fig. 5E-G), ii) larger lumens (Fig. 5D-F) and iii) more erythrocyte-containing regions, indicative for perfused vessels (new Fig. 5H) in lesions with fibroblasts in comparison to plugs containing ECs alone. This implies that fibroblasts further induce PIK3CA-driven EC lesion formation.
Author Response Image 2. Vascular structures formed with PIK3CA H1047R ECs alone and PIK3CA H1047R ECs + FBs in mouse xenograft plugs. In the figure panel, H&E staining on each individual plug in these groups is presented. Equal size close-up images were taken from the middle of each plug covering > 50% of plug area (scale bar 250µm). More erythrocytes (red) are seen in the plugs with fibroblasts in comparison to ECs alone. Scanned images of the H&E stained whole tissue sections can be seen in the Fig. 5 – source file.
A new quantitative analysis of erythrocyte positive area in relation to whole plug area using SproutAngio quantification tool was additionally performed (). Analysis was done on a blinded manner and showed significantly increased erythrocyte amount in the plugs containing PIK3CA H1047R ECs and fibroblasts (in comparison to EC alone). Describtion of the analysis has now been added in the manuscript (p. 42, rows 839-843) Figures 5G and 5H in the manuscript were updated to show statistics and automated intensitybased quantification of the erythrocyte positive area/ plug instead of erythrocyte scoring (scale 0-3).
Is it possible that TGFa from the ECs is sufficient to drive vascular malformation?
Mutations in genes such as PIK3CA, TEK and KRAS have been shown to drive formation of vascular anomalies. Thus it is unlikely that a single growth factor, such as increased expression of TGFA, would drive this process alone. That being said, our data shows that TGFA is able to regulate proliferation of PIK3CA mutated ECs via secondary mechanism (Fig. 4F), and we show that inhibition of EGFR pathway is able to reduce PIK3CA-driven lesion growth in mice (Fig. 7). As our bulk RNA-sequencing data from patient-derived cells, showed expression of also other growth factors in lesion ECs (Table 3), it is likely that multiple angiogenic growth factors are involved in lesion formation similarly as in tumors and their expression is primarily driven by mutated cells and secondary by cell-cell crosstalk with other lesion cell types. Thus, targeting of multiple signalling pathways could be a beneficial treatment strategy in the future.
Reviewer #2 (Public Review):
In this manuscript, Ilmonen H. et al explored potential crosstalk between endothelial cells and fibroblasts in a context of sporadic vascular malformation (venous malformation and angiomatoses of soft tissue). With a high level of evidence, they found that mutated endothelial cells secrete TGFA that will activate surrounding fibroblasts, leading in turn to VEGFA secretion that will stimulate endothelial cell sprouting and vascular malformation development. Experiments are well-designed and support their hypothesis. Some controls are missing, particularly in Fig. 2. Indeed, it is mandatory to provide data from healthy skin biopsies (that are available in many laboratories): TGFa, CD31, P-EGFR staining.
We thank the Reviewer for the comments. Although it is common that VM presents in skin, in this work we solely focused on intramuscular and subcutaneous AST and VM patient samples and excluded the samples containing skin from this study. We did TGFA immunostainings from healthy skeletal muscle that can be seen Figure 2 – figure supplement 2B. CD31 staining of vessels in healthy skeletal muscle near the resection margin can be seen in Figure 1B. Please see below also tissue locations of all VM and AST samples in this study:
• Intramuscular, 42.1 % of lesions (n=16)
• Intramuscular and subcutaneous, 21.1 % of lesions (n=8)
• Intramuscular, subcutaneous and synovial membrane, 5.3 % of lesions (n=2)
• Intramuscular and synovial membrane, 2.6 % of lesions (n=1)
• Subcutaneous and synovial membrane, 2.6 % of lesions (n=1)
• Subcutaneous only, 26.3 % of lesions (n=10)
• Skin, none of the lesions
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eLife assessment
The authors address an important clinical entity and an important area of unmet clinical need. The authors use a combination of in vitro and in vivo experiments to learn how stromal cells surrounding vessels in venous malformations (VM) and angiomatosis of soft tissue (AST) contribute to the angiogenic activities driving the vascular lesions. They discovered that secretion of transforming growth factor-alpha (TGFa) from both endothelial cells and stromal cells, shows evidence for EGF-receptor phosphorylation. In addition, they show that afatinib, a pan-ErbB TKI inhibitor may have therapeutic benefits.
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Reviewer #1 (Public Review):
The authors present a strong set of experiments to uncover what type of role non-mutant stromal cells might be playing in the development of VM and AST, two vascular lesions that share some similarities.
Questions about experimental design.
- For quantification of gene expression in VM and AST specimens in Figure 2, the methods say qPCR data were normalized to housekeeping genes, but it would be helpful to normalize to endothelial content. It might be that increased TGFa is due to increased endothelium.
- The mutant allelic frequency for the HUVEC-PIK3CA WT versus HUVEC-PIK3CA H1047R should be provided. This is critically needed for the interpretation of the results.
- From Figure 5, it appears that the human primary fibroblasts are not required for the mutant ECs to form perfused vessels (panel H). Is it possible …
Reviewer #1 (Public Review):
The authors present a strong set of experiments to uncover what type of role non-mutant stromal cells might be playing in the development of VM and AST, two vascular lesions that share some similarities.
Questions about experimental design.
- For quantification of gene expression in VM and AST specimens in Figure 2, the methods say qPCR data were normalized to housekeeping genes, but it would be helpful to normalize to endothelial content. It might be that increased TGFa is due to increased endothelium.
- The mutant allelic frequency for the HUVEC-PIK3CA WT versus HUVEC-PIK3CA H1047R should be provided. This is critically needed for the interpretation of the results.
- From Figure 5, it appears that the human primary fibroblasts are not required for the mutant ECs to form perfused vessels (panel H). Is it possible that TGFa from the ECs is sufficient to drive vascular malformation?
-
Reviewer #2 (Public Review):
In this manuscript, Ilmonen H. et al explored potential crosstalk between endothelial cells and fibroblasts in a context of sporadic vascular malformation (venous malformation and angiomatoses of soft tissue). With a high level of evidence, they found that mutated endothelial cells secrete TGFA that will activate surrounding fibroblasts, leading in turn to VEGFA secretion that will stimulate endothelial cell sprouting and vascular malformation development.
Experiments are well-designed and support their hypothesis.
Some controls are missing, particularly in Fig. 2. Indeed, it is mandatory to provide data from healthy skin biopsies (that are available in many laboratories): TGFa, CD31, P-EGFR staining.
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