Bleomycin inhibits CLEC14A to attenuate the progression of extracranial arteriovenous malformations

Congzhen Qiao, Yajing Qiu, Yun Zou, Chen Hua, Yuanbo Li, Yunbo Jin, Xiaoxi Lin

Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

Keywords:Bleomycin Extracranial arteriovenous malformation CLEC14A RNA-Sequencing

ABSTRACT Background: We previously reported that interstitial injection of bleomycin (BLM) reduces the size of early-stage extracranial arteriovenous malformation (AVM).Here,we sought to investigate the potential mechanism of BLM in treating extracranial AVM.Methods: Samples of human extracranial AVM (n=3) with no pharmacological treatment were harvested.AVM endothelial cells were isolated and cultured in primary cell culture.The transcriptome was examined using RNAsequencing, and differentially expressed C-type lectin domain family 14 member A (CLEC14A) was validated at the transcriptomic and protein levels.Immunocytochemical staining of CLEC14A was performed in samples of human extracranial AVM, with and without BLM treatment.Results: Through second-generation sequencing, we found that the expression of 5 689 genes were differentially increased or decreased following 24-h BLM stimulation.We found that CLEC14A may play an important role in the progression of AVM and can be inhibited by BLM treatment.Conclusion: BLM inhibited CLEC14A expression to attenuate the progression of AVM.

1.Introduction

Extracranial arteriovenous malformation (AVM) is a congenital disease caused by the lack of a normal capillary bed between the arteries and veins.Although the incidence is low, about 0.07–0.25/1 000,1it is the most dangerous and refractory type in the field.2Regarded as a congenital structural change of vasculature,AVM shows continuous and irreversible progression.Clinically,we find that simple artery ligation or embolization fails to relieve symptoms; rather, it accelerats the progression of extracranial AVM, causing damage to the appearance.If not controlled, the skin lesions become ulcerated in some cases.Once ulceration occurs,it is unlikely to heal and often results in deadly arterial bleeding.This remodeling of AVM suggests that it is actively progressing rather than passively expanding.3The current predicament is that not only the development of AVM and the mechanisms of progression are poorly understood,but little is known about treatment options and efficacy as well.

We reported that intra-interstitial injection of bleomycin (BLM) in extracranial AVMs had an effective rate of 84.4%,showing good efficacy in attenuating AVM development in a pilot clinical study of 32 cases of early extracranial AVM.3Although BLM is used to treat low-flow vascular diseases such as venous malformation and lymphatic malformation,4the mechanism is not well understood.Here, we investigated the potential mechanism of BLM in the attenuation of AVM progression.

2.Materials and methods

2.1.Sample collection and primary cell isolation

All clinical samples were collected under the approval of the Scientific Research Ethics Committee of our hospital.Primary endothelial cells were isolated from the samples of surgically resected extracranial AVMs not exposed to any previous medication or interventional therapy.For comparison purposes,lesions that had been treated only with BLM(HY-17565, MedChemExpress, NJ, USA) were also collected.Three tissue samples were collected from the core of the lesions immediately after surgical removal.All samples were rinsed using phosphate-buffered saline (PBS).One sample was fixed with cold 4% paraformaldehyde(158127, Sigma, MO, USA) for subsequent sectioning and immunofluorescence staining.Another sample was transferred to a 1.5 mL centrifuge tube and stored in liquid nitrogen for future protein collection.The final sample was placed in Hank’s solution(14025092,Gibco,MI,USA)before being minced on a sanitized bench and digested with collagenase to isolate vascular endothelial-type calcium-dependent adhesion molecule(CD31)and human endothelial cells(130-091-935,Miltenyi Biotec,CA,USA).

2.2.Immunohistochemistry

After fixation with paraformaldehyde for 20 min, the samples were rinsed with PBS three times and completely dehydrated with a concentration gradient of sucrose solution.OCT (62550, Sakura Finetek, NJ,USA)was added to the tissue samples for sectioning.The pericytes were marked with FSP-1 (072274, EMD Millipore, MA, USA), and the endothelial cells were marked with VE-cadherin (AF938,R&D Systems, MN,USA).The sections were immunostained for the comparison of the effects of BLM on arteriovenous malformations by co-staining the pericyte marker with FSP-1 and the endothelial cell marker with VE-cadherin.

2.3.Observation of the morphology of the primary AVM endothelial cells

Cell morphology was observedin vitro.After 24 h of cell culture of primary AVM endothelial cells,unattached cells and free magnetic beads were removed by washing with PBS.Two mL of EGM2 medium(Lonza,Basel, Switzerland) with 15% fetal bovine serum (FBS; Cellsera, NSW,Australia)was added to each dish to continue the culture,and the solution was changed every 3 days.When the cells reached 90%confluency,they were passaged.

2.4.Differential expression analysis of the primary AVM endothelial cells by BLM stimulation

Primary endothelial cells of AVMs were obtained and stimulated with 10 μM BLM (HY-17565, MedChemExpress, NJ, USA) after secondary passage.The differential expression products in the cell transcription pool were harvested using the RNeasy Mini kit(QIAGEN,CA,USA)in a time-dependent manner (4 h, 16 h, 24 h, and 48 h) and detected by second-generation sequencing to explore the evidence of BLM-regulated blood flow-sensitive angiogenic factors.

2.5.Materials and related reagents

Primary antibodies against CD144(AF938)and C-type lectin domain family 14 member A (CLEC14A; AF4968) for immunocytochemistry were purchased from R&D Systems (Minneapolis, MN, USA).The primary antibody(072274)for S100A4 was purchased from EMD Millipore(Burlington, MA, USA).Secondary donkey anti-mouse IgG and donkey anti-goat IgG were purchased from Jackson ImmunoResearch (West Grove,PA,USA).

3.Results

We collected samples from patients with extracranial AVM.First,we observed the microscopic differences before and after local BLM treatment from the same patient (Fig.1), followed by immunofluorescence staining to compare the difference between the BLM-treated group and the untreated group(Fig.2).As expected,it was found that in the BLMtreated group, the intravascular lesions were significantly occluded compared to the untreated group,and the number of fibroblasts around the malformed vessels was significantly increased in the treated group.

Next, we isolated primary endothelial cells from surgically resected AVM specimens.Endothelial cells at the secondary passage were stimulated with 10 μM BLM and analyzed using time as a variable.Differential expression products in the endothelial cell transcriptional pool were detected using second-generation sequencing.All BLM-treated and control groups that were sequenced by the second generation were subjected to principal component analysis (PCA), and it was found that the BLMtreated group was significantly different from the control group(Fig.3).PCA is a characterized analysis that reduces the dimensionality of a dataset while maintaining the largest contribution to the variance within the dataset.The heatmap analysis also showed the similarities between different samples in each group(Fig.4).

We performed BLM stimulation of primary AVM-ECs and compared them with those of the control group.Through second-generation sequencing and differential gene expression analysis, we found that CLEC14A was significantly reduced in the BLM-treated group in a time-dependent manner (Fig.5).Interestingly, CLEC14A was significantly expressed under oscillatory shear stress (Fig.6), and CLEC14A has been reported to exert a strong angiogenic effect.5,6CLEC14A binds to MMRN2 and plays a role in inducing angiogenesis during tumor growth.6Lee et al.revealed that CLEC14A plays a role in vascular homeostasis by refining vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 signaling in endothelial cells.It has been shown to be related to the pathogenesis of human diseases associated with angiogenesis.7

Fig.1.Immunohistochemistry.CD31(vascular endothelial-type calcium-dependent adhesion molecule)is an endothelial cell-specific expression marker;Vimentin is a pericyte marker (100× magnification).BLM, bleomycin.

Fig.2.Immunofluorescent staining of non-treated(NT)and BLM-treated extracranial AVM samples under laser confocal microscopy.After BLM treatment,multiple malformed vessels in the AVM lesions were occluded,and multiple fibroblasts were present around the occluded deformed blood vessels.DAPI(blue)shows the nucleus;FSP-1(fibrillar fine protein,red)is a fibroblast-specific expression marker;VE-Cadherin(vascular endothelial-type calcium-dependent adhesion molecule,yellow) is endothelial cell-specific expression Marker; Elastin (green); Merged, fused image (purple).Scale: 100 μm.BLM, bleomycin; AVM, arteriovenous malformation.

Fig.3.Principal component analysis of gene expression profiles for each sample from different groups. PC1: principal component 1 (52%); PC2: principal component 2(12%).B:bleomycin;C:control.4 h,16 h,24 h,and 48 h refer to samples at different time points;1,2,and 3 are the same number of repeated samples at the same time point.

Next, we studied the difference between the expression of CLEC14A in the untreated AVM lesions and the BLM-treated group.As expected,the fluorescence intensity of CLEC14A expression in the BLM group was significantly lower than that in the untreated group (Fig.7), indicating that BLM can inhibit its expression and suggesting that heightened expression of CLEC14A may be one of the causes of AVM progression.

We compared the protein expression of CLEC14A in the BLM-treated and untreated groups (Fig.8).In the BLM-treated group,the expression of CLEC14A was significantly decreased,revealing that BLM can regulate the expression of CLEC14A.The inhibition of angiogenesis under oscillatory shear stress is of great significance for the treatment of AVM.

4.Discussion

Blood flow is an important factor in the progression of AVM.The discovery by Baeyens et al.8in the zebrafish arteriovenous model demonstrated that blood flow affects the transforming growth factor beta(TGF-β) pathway in hereditary hemorrhagic telangiectasia (HHT), and that blood flow enhances BMP9-activated activin receptor-like kinase 1(ALK1) signaling by enhancing the association of ALK1 with ENG.This discovery has shed light on the study of AVM,convincing us to seek the pathogenesis of AVM progression affected by blood flow.Tu et al.9found that Notch signaling activation contributes to the angiogenesis of AVM by increasing the velocity of blood flow in rats.This finding,by means of the Notch signaling pathway, reveals the mechanism of potential AVM formation, suggesting that blood flow should be able to activate different signaling pathways leading to the formation of AVM.Galie et al.10used a microfluidic controller to regulate local fluid mechanical forces,revealing that proper blood flow shear stress can trigger angiogenesis.We believe that angiogenesis caused by changes in blood flow is one of the main causes of accelerated progression after palliative treatment for AVM.Therefore, we searched for genes related to angiogenesis and cross-matched these differentially expressed genes stimulated by shear stress to investigate the potential mechanisms of rapid progression in residual AVM.

Fig.4.Gene expression profiling similarity heat map for each sample from different groups.B:bleomycin;C:control.4 h,16 h,24 h,and 48 h refer to samples at different time points; 1, 2, and 3 are the same number of repeated samples at the same time point.

Fig.5.Second generation sequencing to detect the expression level of CLEC14A under bleomycin stimulation. The expression of CLEC14A in primary endothelial cells of AVM exhibited a time-dependent effect under 10 μM BLM stimulation. P＜0.0001 (n=3) data are average values ± standard deviation.＊＊P＜0.01,＊＊＊＊P＜0.0001(n=3).CLEC14A,C-type lectin domain family 14 member A; AVM, arteriovenous malformations; BLM, bleomycin.FPKM, the number of fragments read per million bases per million bases of transcription.

Fig.6.Second generation sequencing to detect the expression of CLEC14A under oscillatory shear stress.Under oscillatory shear stress,the expression of CLEC14A in the primary endothelial cells of coronary artery increased.All data are average values ± standard deviation.＊＊＊＊P＜0.0001 (n=4).CLEC14A, Ctype lectin domain family 14 member A; FPKM, the number of fragments read per million bases per million bases of transcription; OS, oscillatory shear stress;LS, laminar shear stress.

Fig.7.Immunofluorescence staining of BLM-treated and non-treated extracranial AVM samples under fluorescence microscopy.After BLM treatment,many malformed vessels in the AVM lesions were occluded, and the expression of CLEC14A on the occluded vascular endothelial cells decreased.DAPI (blue) shows the nucleus; CD31 (vascular endothelial-type calcium-dependent adhesion molecule, green) is an endothelial cell-specific expression marker; CLEC14A (C-type lectin domain 14A, red); Merged, Merged image in purple.Scale: 50 μm.BLM, bleomycin; AVM, arteriovenous malformations; CLEC14A, C-type lectin domain family 14 member A.

Fig.8.Protein expression levels of CLEC14A in EC groups with different treatment. The internal control is glyceraldehyde 3-phosphate dehydrogenase.(n=4).CLEC14A, C-type lectin domain family 14 member A; BLM, bleomycin.

Through second-generation sequencing,we found that the expression of 5 689 genes were differentially increased or decreased following 24-h BLM stimulation.BLM significantly inhibited the expression of CLEC14A in a time-dependent manner, indicating that BLM directly regulates CLEC14A expression.We were fortunate to find the CLEC14A gene in this transcriptome, not only for its involvement in cancer angiogenesis, but also for its important role in the progression of AVM.Bocci et al.revealed a genetic network associated with activin A receptor like type 1(ACVRL1) (a gene encoding ALK1) in human cancer tissues.11They found eight genes co-expressed with ACVRL1 in different tumor types and found that CLEC14A is a potential downstream target of ACVRL1.ALK1 is a signaling molecule of the TGF-β family.Mutations can cause hereditary HHT,which is an important genetic model for studying AVMs.Sara et al.12also found the tumor proangiogenic marker CLEC14A from the proteomics of the formation stage of human primary endothelial cells.Given that BLM can effectively attenuate the progression of residual AVM clinically,we postulate that stimulation of oscillatory shear stress in the deformed lumen is responsible for the progression of AVM, and inhibition of CLEC14A by BLM attenuates AVM progression.

5.Conclusion

This study is the first to report that CLEC14A may be responsible for AVM progression.In future studies, we will look for the signaling transduction responsible for triggering high expression of CLEC14A under oscillatory shear stress and use BLM as a tool to identify key factors in AVM.We hope to provide a new target for AVM treatment in the future.

Ethics declarations

Ethics approval and consent to participate

This study received ethical approval from the Ethics Committee of Shanghai Ninth People’s Hospital (approval no.SH9H-2020-TK214-1).All participants provided written informed consent prior to study enrollment.

Consent for publication

All the patients gave written informed consent to publish the data contained within this study.

Competing interests

The authors declare that they have no competing interests.

Authors’contributions

Qiao C:Conceptualization,Writing-Original draft,Methodology.Qiu Y: Data curation, Formal analysis, Validation.Zou Y: Visualization,Investigation.Hua C: Methodology, Resources.Li Y: Software, Investigation.Jin Y: Conceptualization, Supervision, Writing-Reviewing and Editing.Lin X: Project administration, Writing-Reviewing and Editing,Funding acquisition.

Acknowledgments

This research was supported, in whole or in part, by the Project of Biobank (grant no.YBKA201902) from Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; Multi-center Clinical Research Programs (grant no.DLY201613), Clinical Research Center, Shanghai Jiao Tong University School of Medicine; and Rare Disease Registration Platform of Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine (grant no.JYHJB02).