Angiogenesis and proliferation of endothelial cells in hypertrophic and nodular port-wine stain

Wenxin Yu ,Jiafang Zhu ,Lizhen Wang ,Gang Ma ,Xiaoxi Lin

a Department of Laser and Aesthetic Medicine,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China

b Shanghai Key Laboratory of Stomatology,Department of Oral Pathology,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China

c Department of Oral Pathology,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China

Keywords:Port-wine stain Capillary malformation Hypertrophy Nodule Angiogenesis Proliferation Pathology

ABSTRACT Background: Port-wine stain (PWS)has been classified not as the hyperplasia of cells,but rather,as an expansion of malformed vessels.However,previous studies have reported upregulated expression of proangiogenic factors in PWS.Several studies have indicated that the pathology exhibits proliferation of numerous endothelial cells in hypertrophic/nodular PWS.This study aimed to determine the expression of vascular epithelial growth factor(VEGF),matrix metalloproteinase-9(MMP-9),angiopoietin-2(ANG-2),and basic fibroblast growth factor(bFGF)in hypertrophic PWS.Methods: Immunohistochemistry was used to analyze skin samples from 33 patients with hypertrophic PWS.Expression levels of VEGF,MMP-9,ANG-2,and bFGF in hypertrophic PWS were determined by multiplying the intensity by the percentage of immunoreactive cells.Immunoreactivity scores were classified as follows:negative(0),low (1),moderate (2,3,and 4),or high (6).Results: Based on pathological characteristics,hypertrophic PWS was divided into vascular malformation and pyogenic granuloma(PG)types.VEGF,MMP-9,ANG-2,and bFGF were significantly activated in the blood vessels of PG-type PWS samples compared with their counterparts in blood vessels of vascular malformation-type PWS samples and controls.PG-type hypertrophic PWS,which exhibited proliferation of endothelial cells,showed the strongest activation.Conclusion: The exuberant proliferation of endothelial cells in PG-type hypertrophic PWS may be associated with the regulation of proangiogenic factors during development.These proangiogenic factors that function in the angiogenesis and proliferation of endothelial cells may play an important role in the pathogenesis and progression of PWS.Furthermore,these factors may be dynamic and behave differently in various types of hypertrophic PWS.

1.Introduction

Port-wine stain (PWS) is a congenital capillary malformation that occurs in 0.3%-0.5%of newborns.1PWS lesions typically appear flat and pink initially and then become darker,nodular,and hypertrophic with age.The majority of PWS cases (70%) are located in the face and neck areas,which can lead to physical and psychosocial problems.2,3

PWS is characterized by ectatic changes in vascular morphology rather than by the abnormal proliferation of endothelial cells.4However,previous studies have indicated that vascular epithelial growth factor(VEGF)is upregulated in PWS.5-8In a study by Emre Vural et al.,5results of the immunohistochemical analysis of PWS and control samples showed statistically significant overexpression of both VEGF and VEGF-R2 molecules in the PWS samples relative to those in the control samples.Several pathological studies have revealed the proliferation of a large number of endothelial cells in nodular PWS.9-13Meanwhile,VEGF,matrix metalloproteinase-9(MMP-9),angiopoietin-2(ANG-2),and basic fibroblast growth factor (bFGF) are considered important angiogenesis regulation factors.However,studies on the role of VEGF,MMP-9,ANG-2,and bFGF in the pathogenesis and progression of hypertrophic PWS lesions have rarely been conducted.An intensive study on the influence of angiogenesis and proliferation of endothelial cells on hyperplasia and reconstruction of PWS can elucidate the mechanisms of hypertrophic PWS and provide a theoretical foundation for the prevention and treatment of PWS.

In this study,we evaluated the expression of proangiogenic mediators,VEGF,MMP-9,ANG-2,and bFGF in hypertrophic PWS to investigate the pathogenesis of PWS progression using histological and immunohistochemical approaches.

2.Methods

2.1.Patient data

This study was approved by the Institutional Review Board of the Shanghai Ninth People’s Hospital.Skin samples were taken from 33 patients with untreated hypertrophic PWS lesions and 11 patients with normal-appearing skin as controls(Fig.1).Patient ages ranged from 14 to 73 years.A total of 33 hypertrophic PWS lesions were located in the face and neck areas.Hypertrophic PWS was divided according to morphology into vascular malformation and pyogenic granuloma(PG)type.13Clinical information for each PWS sample is presented in Table 1.Immunohistochemistry for VEGF,MMP-9,ANG-2,and bFGF was performed using routine procedures.For VEGF,MMP-9,ANG-2,and bFGF,for each case and antibody,the immunoreactivity score was determined by multiplying the intensity by the percentage of immunoreactive cells.Immunoreactivity scores are classified as follows:negative (0),low (1),moderate(2,3,and 4),or high(6).14,15

2.2.Data analysis

All data were analyzed using Stata 14.0 and GraphPad Prism 5(GraphPad Software,Inc.,Carlsbad,CA,USA).Comparison of VEGF,MMP-9,ANG-2,and bFGF activation among different groups was performed using Fisher’s exact χ2test.AP-value of ≤0.05 was considered to indicate statistical significance in all comparisons.

3.Results

The 23 patients with vascular malformation-type hypertrophic PWS were characterized by numerous ectatic dermal blood vessels with thick vessel walls,while the remaining 10 patients with the PG-type hyperplastic PWS showed many proliferating endothelial cells with“mass-like”distribution.

There was negative/low activation of VEGF,MMP-9,ANG-2,and bFGF in the control samples and vascular malformation type of hyperplastic PWS,while significant activation of VEGF,MMP-9,ANG-2,and bFGF was found in the PG type of hyperplastic PWS.VEGF,MMP-9,ANG-2,and bFGF in the PG-type hypertrophic PWS exhibited the strongest activation among all sample types(P＜0.001)(Figs.2 and 3 and Tables 1 and 2).

Table 1 Patient description of hypertrophic PWS samples.

4.Discussion

Based on clinical and cellular studies,vascular birthmarks are divided into two major categories:1) hemangiomas or lesions that demonstrate endothelial hyperplasia and malformations,and 2) lesions that exhibit normal endothelial turnover.4In modern usage,the suffix“-oma”is used to define a lesion arising from cellular overgrowth.Therefore,hemangiomas are defined as vascular lesions that exhibit hyperplasia.Endothelial cells normally show numerous mitotic features and short doubling times.Meanwhile,vascular malformations are generally restricted to abnormal differentiation and morphogenesis of the vascular and lymphatic channels.The normal rate of endothelial turnover is the determinant feature of an unperturbed“vascular malformation”,literally“badly formed vessels”.16

Although vascular malformations occur as structural anomalies,they are not mutable.The PWS changes slowly with time.In the head and neck region,the dull-pink color characteristic of infancy gradually darkens to bright red in young adulthood and then to deep crimson or purple in middle age.By the third or fourth decade of life,the surface of the stain typically becomes raised(“cobblestone-like”),and fibrovascular nodules often appear.17,18

Fig.1.Patient enrollment and analysis.

Vascular malformation vessels in hypertrophic PWS

In this type of PWS,the lesion is composed of ectatic vessels exhibiting a venular morphology,located mostly in the papillary and upper reticular dermis.Extension into the deep dermis,subcutaneous fat,and skeletal muscle is occasionally observed.The anomalous vessels tend to be round,with flat,elongated,mitotically inactive endothelium and thin walls with minimal collagen and pericytes.With increasing age,these channels become progressively ectatic and occupy a greater portion of the dermal area.Mural thickness and extension of vessels deeper into the dermis increase only minimally with age.18,19In general,thickening of the vascular wall is caused by multilamination of the basement membrane and an increase in collagen and pericytes.20The mural thickness may vary within a sample.

Fig.3.No/Low activation of VEGF,MMP-9,ANG-2,and bFGF in normal skin (A-D,400×),vascular malformation-type hypertrophic PWS (E-H,200×),and significant activation of PG-type hypertrophic PWS (I-K,200×;L,100×).

Age-related changes in PWS are attributed to progressive dilatation of ectatic dermal vessels and epidermal and adnexal hyperplasia with increased fibrous tissue.These alterations have been characterized as epithelial,neural,and mesenchymal“hamartomatous”changes,suggesting a field defect in multiple germ layers.18,21

In 23 vascular malformation-type hypertrophic PWS samples,the histopathologic features associated with cobblestoning and nodularity include extension of abnormal vessels into the deep dermis,subcutis,and skeletal muscle,nodular aggregates of vessels,exaggerated vascular ectasia,thick-walled vessels,dermal fibrosis,appendageal hyperplasia,and follicular plugging and perifollicular microabscesses.19,21

Proliferation of endothelial cells in hypertrophic PWS

Histopathologically,vascular malformations are lined with a flat endothelium lying on a thin basal lamina.Examination of older lesions shows progressive ectasia and mural thickening in structurally abnormal slow-flow vessels and increased tortuosity of malformed fast-flow vessels.Vascular malformations may exhibit endothelial and/or smooth muscular hyperplasia (angiogenesis) when triggered by hormonal stimulation,trauma,thrombosis,surgical or endovascular intervention,or unknown stimuli.

Lobular capillary hemangioma (pyogenic granuloma),characterized by unusual progressive hypertrophy and fibrovascular overgrowth arising in capillary malformation,is widely known;however,whether this is attributed to an inherent capacity or rheologic influences remains unclear.9-13The study by Chen et al.,13in which 31 cases with nodular PWS were examined,indicated the mass distribution of PG endothelial cells in 61.2% of nodular PWS lesions.Such nodular types of lesions clearly exhibited the proliferation of endothelial cells,as well as angiogenesis of new vessels.In the present study,the proliferation of endothelial cells was also detected in 10 of the 33 cases of hypertrophic PWS,based on hematoxylin and eosin staining.

Angiogenesis/Neovasculogenesis in the progression of PWS

Enlargement of a vascular anomaly likely involves endothelial upregulation to form larger,and possibly new,channels.Vascular malformations are dynamic;they progress and often recur after resection or redarkening following laser therapy.21Pluripotent cells residing in vessel walls may play a primary role in progression and recurrence.

Huikeshoven21investigated 51 patients who received a median of seven additional treatment sessions since the last color measurement.When measured at follow-up with a median of 9.5 year,the stain became significantly darker than after the last five laser treatments.In recalcitrant PWS therapy,angiogenesis/neovasculogenesis likely occurs extensively during the short-term vascular remodeling phase and hampers the reduction in dermal blood content.20,21

VEGF,MMP-9,ANG-2,and bFGF,which are significant markers of cell proliferation and vascular formation,were used to confirm cell proliferation in the biopsies.

The activation of VEGF,its receptor,and ANG-2 has been detected in PWS.5,6Imiquimod and rapamycin,suppressors of cell proliferation,have been widely used to treat tumors.Enhanced efficacy was observed with the simultaneous use of imiquimod and rapamycin in pulsed dye laser treatment for PWS.22-25VEGF and ANG-2 were confirmed to be downregulated after treatment with this receptor antagonist.6

Nodularity of PWS also likely involves remodeling of the extracellular matrix and basement membranes that surround the vascular channels.MMPs degrade the components of the extracellular milieu to allow for cell proliferation and the formation of new blood vessels.Increased expression levels of MMPs and bFGF were associated with the occurrence and development of lesions in patients with vascular malformations,and Nguyen et al.found high expression of bFGF not only in patients with cancer,but also in the urine of patients with vascular malformations.6,7Marler8reported higher MMP and bFGF expression levels in the urine of patients with hemangiomas and vascular malformations than in normal patients.

Thus,the abnormal expression levels of these factors partially explain the abnormal angiogenesis and proliferation of endothelial cells in hypertrophic PWS.In the current study,VEGF,MMP-9,ANG-2,and bFGF showed significant levels of activation in the PG-type hyperplastic PWS,whereas no expression or low expression levels of these factors were detected in the control skin samples and the vascular malformation-type hyperplastic PWS samples.This study is the first to present VEGF,MMP-9,ANG-2,and bFGF activation profiles in different types of hypertrophic PWS.

Hypertrophy in PWS is typically regarded as the expansion of capillary vessels;increased deposition of basement membranous components such as type IV collagen,laminin,and fibronectin;and hyperplasia of skin appendages.20,26-28In the present study,activation levels of these factors were correlated with the proliferation of endothelial cells in hypertrophic PWS,which may contribute to both pathogenesis and progressive development of PG-type hypertrophic PWS.Meanwhile,they may exert several effects on vessel dilation,as detected in vascular malformation-type hypertrophic PWS.

This study has certain limitations,one of which is its small sample size.In addition,no studies have been conducted to observe redarkeningtype PWS after laser treatment.

5.Conclusion

The strong activation of VEGF,MMP-9,ANG-2,and bFGF in the PGtype PWS may be associated with the proliferation of endothelial cells and angiogenesis in hypertrophic PWS.Progressive dilatation of vessels and proliferation of endothelial cells are important for the progression of hypertrophic PWS.Thus,VEGF,MMP-9,ANG-2,and bFGF may be vital for the proliferation of endothelial cells in capillary malformation,but only slightly affect the progression of vascular dilation in ectatic PWS.

Ethics declarations

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Investigational Review Board of the Shanghai Ninth People’s Hospital (approval no.[2014]8).All participants provided written informed consent prior to enrollment in the study.

Consent for publication

All patients included in this study provided written informed consent to publish the data contained in this study.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Yu W:Conceptualization,Methodology,Software.Zhu J:Data curation,Writing-Original draft.Wang L:Visualization,Investigation.Ma G:Supervision.Lin X:Writing-Reviewing and Editing.

Acknowledgements

This study was supported by the National Natural Science Foundation of China(grant no.81602777).