- Email: [email protected]
The expression of vascular endothelial growth factor and its receptors in port-wine stains
Otolaryngology–Head and Neck Surgery (2008) 139, 560-564
ORIGINAL RESEARCH—PEDIATRIC OTOLARYNGOLOGY
The expression of vascular endothelial growth factor and its receptors in port-wine stains Emre Vural, MD, Jeevan Ramakrishnan, MD, Neslihan Cetin, MD, Lisa Buckmiller, MD, James Y. Suen, MD, and Chun-Yang Fan, MD, PhD, Little Rock, AR OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in portwine stains (PWSs). DESIGN: An immunohistochemistry (IHC) study on formalinfixed, paraffin-embedded specimens. METHODS: Representative sections from surgical resection specimens of 12 PWS patients and 12 control specimens stained with routine IHC by using polyclonal anti-VEGF and anti– VEGF-R2 antibodies. Slides were evaluated semiquantitatively for the intensity of staining for VEGF and VEGF-R2 by using a scoring system varying from 0 to 3⫹. RESULTS: PWS specimens showed statistically significant overexpression of both VEGF and VEGF-R2 molecules when compared with control specimens (P ⬍ 0.005). CONCLUSIONS: VEGF and its receptor may play an important role in the pathogenesis of PWS. It is possible that PWS may progress by hyperplasia in addition to hypertrophy. VEGF-R blockade may have a potential role as a targeted approach in the treatment of this disfiguring condition in the future. © 2008 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
P
ort-wine stain (PWS) is a vascular malformation that affects approximately 0.3 percent of newborns and causes significant negative psychological and social impacts on the patients and their families because of worsening of the signs and symptoms by age.1 Although repetitive flashlamp pulsed-dye laser (FLPD) treatments have been successfully used for the management of this disfiguring condition in certain patients, overall complete clearance rate of PWS is still less than 20 percent with FLPD.2 Because the head and neck are the most commonly involved regions with this pathology; PWS unresponsive to laser treatments usually progresses into significantly disfiguring and destructive tumors necessitating complex and highly morbid surgical resections, which may be devastating to the patient in terms of cosmesis and function.3 Apparently, there is still a need for a more effective treatment for this invasive and progressive disease. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and permeability factor that has
been shown to stimulate angiogenesis, vascular remodeling, and new tissue growth.4,5 The effects of VEGF in revascularization of avascular tissue, improving ischemic skin flaps, and tumor angiogenesis have been widely investigated.6-8 It has also been shown that VEGF could have a potential role in the development of other vascular malformations such as hemangiomas and arteriovenous malformations.9,10 However, the potential role of VEGF in the pathogenesis of PWS is unknown. The purpose of this study was to investigate the expression of VEGF and its most potent receptor (VEGFR2) in PWS as a potential mechanism of progression, which may be a subject of targeted treatment in the future.
METHODS After obtaining an exempt status letter from the Institutional Review Board at the University of Arkansas for Medical Sciences for this immunohistochemistry (IHC) study, the departmental vascular anomalies database was searched for PWS patients who underwent resection of their lesions. Twelve patients with PWS were identified with adequate data. The database of the pathology department was also searched for control specimens, and 12 discarded skin specimens without known pathologic skin conditions were identified. The pathologist of the vascular anomalies team (CYF) examined the hematoxylin-eosin slides for each PWS and control case to identify the representative paraffin blocks of formalin-fixed specimens. Paraffin-embedded skin and PWS tissues were sectioned at 4 m, deparaffinized, and rehydrated.
IHC and Interpretation of the Results Routine IHC stainings were performed on each specimen at the Histology Core Laboratory at the University of Arkansas for Medical Sciences. Briefly in order, slides were treated with target retrieval solution (pH ⫽ 6.0) and peroxidase-blocking reagent (Dako North America Inc, Carpentaria, CA) followed by 10 percent normal rabbit serum (Vector
Received May 14, 2008; revised July 2, 2008; accepted July 10, 2008.
0194-5998/$34.00 © 2008 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2008.07.015
Vural et al
The expression of vascular endothelial growth . . .
Figure 1 A bar chart showing immunostaining scores for VEGF in PWS and control groups.
Laboratories, Burlingame, CA). Polyclonal antihuman VEGF R2 (KDR) and antihuman VEGF antibodies were used at 1:25 dilution as primary antibodies (R&D Systems Inc, Minneapolis, MN; catalog numbers AF357 and AB-293-NA, respectively). Slides were then treated with biotinylated rabbit antigoat at 1:400 dilution as the secondary antibody followed by the ABC kit (Vector Laboratories) and DAB⫹ (Dako North America Inc). Counterstaining was performed by using hematoxylin. Human placenta was used for positive staining control. The VEGF and VEGF-R2 IHC staining results were interpreted by the pathologist of the vascular anomalies team (CYF) semiquantitatively by scoring the staining intensity of VEGF-R2– and VEGF-positive endothelial cells (strong staining: 3, moderate staining: 2, weak staining: 1, and no staining: 0) by using low-power (⫻200) and high power (⫻400) magnification. A Wilcoxon two-sample test was performed to compare the immunostaining scores for VEGF and VEGF-R2 between controls and PWS specimens.
561
This was probably because of poor quality of the specimen embedded in the paraffin block or faulty embedding process. Therefore, this specimen was excluded from the analysis. In this study, both antihuman VEGF-R2 and antihuman VEGF antibodies specifically stained the endothelial cells. Smooth muscle cells of blood vessels and fibroblasts in the stroma of control skin and PWS were not reactive with either antibody. Because of scant cytoplasm of the endothelial cells, it was impossible to obtain a good membranous staining pattern for the antihuman VEGF-R2 antibody. Thus, the staining pattern appeared similar for both antihuman VEGF R2 and antihuman VEGF antibodies. Scores for the intensity of immunostaining for VEGF and VEGF-R2 are summarized in Figures 1 and 2, respectively. The average immunostaining scores for VEGF in controls and PWS were 0.83 (minimum ⫽ 0, maximum ⫽ 2, ⫾0.8 SD) and 2.63 (minimum ⫽ 2, maximum ⫽ 3, ⫾0.5 SD), respectively. The average immunostaining scores for VEGF-R2 in controls and PWS specimens were 0.92 (minimum ⫽ 0, maximum ⫽ 2, ⫾0.7 SD) and 2.72 (minimum ⫽ 2, maximum ⫽ 3, ⫾0.5 SD), respectively. PWS showed significantly higher staining scores than the controls for both VEGF and VEGF-R2 (P ⬍ 0.005) (Fig 3).
DISCUSSION
RESULTS
VEGFs are a group of glycoproteins composed of seven variants, specifically VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor, and VEGF-F.11 VEGF-A is the most potent member of this family that induces proliferation, sprouting, and tube formation of endothelial cells.12 Additionally, VEGF-A stimulates vasodilatation by inducing endothelial nitric oxide synthase.11 VEGF shows these effects by binding to a variety of transmembrane tyrosine kinase receptors (VEGFR1, VEGF-R2, and VEGF-R3), and VEGF-R2 seems to be the main receptor responsible for proangiogenic effects of VEGF.12 Because hemangiomas and vascular malformations are angiogenesis dependent, the role of VEGF and its recep-
PWS specimens came from a patient group composed of eight males and four females, with an average age of 21.8 (minimum 3, maximum 39). Four of 12 patients received at least one FLPD laser treatment before their surgeries. The indication for surgery was cosmetic reasons in all cases. Five of 12 patients underwent surgery for simultaneous functional deficits in addition to cosmetic deformity in the form of drooling (1 patient), poor oral intake (2 patients), and bleeding (2 patients). None of the patients had additional comorbidities, except one patient who had SturgeWeber syndrome. The antibodies for both VEGF and VEGF-R2 worked properly in all representative slides except one PWS specimen in which immunostaining was unsuccessful for either antibody despite repetitive attempts.
Figure 2 A bar chart showing immunostaining scores for VEGF-R2 in PWS and control groups.
Role of the Funding Source Financial support for purchasing supplies used in the study was obtained through the Medical Research Endowment Award at the University Arkansas for Medical Sciences.
562
Otolaryngology–Head and Neck Surgery, Vol 139, No 4, October 2008
Figure 3 The expression of VEGF and VEGF-R2 in skin controls and PWS specimens is shown. Note the strong staining for VEGF (B) and VEGF-R2 (D) in PWS when compared with controls (A and C) (magnification ⫻400).
tors was widely investigated as potential contributors to the pathogenesis of these anomalies. It has been shown that VEGF promoted endothelial cell proliferation in hemangiomas even stronger than estrogen.13 Another study showed that VEGF messenger RNA was upregulated in proliferating hemangiomas.14 Significantly high circulating levels of VEGF were also found in patients with proliferating hemangiomas.9 The overexpression of VEGF and VEGF-R was also shown in intracranial arteriovenous malformations.10,15,16 All these studies provided evidence regarding the possible involvement of VEGF and its receptors in the development and/or progression of hemangiomas and vascular malformations and the possibility of suppressing of these lesions by antiangiogenic modulation as shown in many solid tumors.17 Surprisingly, the possible role of VEGF and its receptors has never been investigated in the pathogenesis of another vascular malformation, PWS, probably because of the common belief that PWS progresses by ectasia of the normal number of vessels forming this pathology rather than proliferation of the vessels.2 The reason of the widespread ectasia was attributed to an abnormal neural supply to these capillaries because of the decreased number of nerve fibers shown in PWS compared with normal skin in histopathological studies.18 The current study showed overexpression of VEGF and VEGF-R2 compared with controls, suggesting that angiogenic stimulus may contribute to the pathogenesis of PWS. This may be in the form of hypertrophy,
hyperplasia, or both because VEGF induces both proliferation and vasodilatation.11,12 Despite the advances in laser technology, the gold standard treatment of PWS by using FLPD laser often fails to eradicate this disease.2,19 The main disadvantage of FLPD laser treatment is the fact that only less than 20% of PWS respond completely to this treatment, and the vast majority of nonresponsive or incompletely responsive lesions progress into hypertrophied and disfiguring lesions. It is known that certain areas such as the central face were even less responsive to FLPD laser treatment.20 There are multiple reasons for the ineffective clearance of PWS by FLPD, and these are mainly attributed to the inadequate penetration of the FLPD laser into the dermis because of wavelength limitations, inadequate damage to the vessel wall because of limited heat conduction from the central portion of the vessel to the peripheral walls causing subsequent regeneration of the vessels, inadequate amount of target chromophore in smaller vessels causing insufficient absorption of the light, and inadequate fluence.2 It is obvious that more effective treatment modalities are necessary for eradication of this disfiguring disease when FLPD fails, especially for the lesions in the head and neck region because of their visibility and interference with important physiologic functions such as breathing, vision, and oral intake. Antiangiogenic treatment using VEGF receptor blocking agents recently gained popularity with promising results.21
Vural et al
The expression of vascular endothelial growth . . .
Although this approach is mainly being investigated for the treatment of a wide variety of malignant tumors, it has been suggested that antiangiogenic treatments may have future applications in the treatment of vascular anomalies.17 This may be especially useful in AVMs in which a dominant feeding vessel could be identified angiographically that may act as a route for targeted delivery of the antiangiogenic agents. As we have shown in this IHC study, overexpression of VEGF and VEGF-R2 in PWS may be an important factor in the development and/or progression of this pathology, and patients with PWS may be candidates for targeted antiangiogenic treatments. Although how to deliver VEGF-R blockers specifically to the target vascular malformations such as PWS without affecting angiogenesis in healthy tissues remains a question, especially in the pediatric age groups; advances in nanotechnology and molecular biology may solve this problem in the future. There is definitely a need to develop an animal model for PWS because such a model currently does not exist.
CONCLUSIONS VEGF and VEGF-R2 expression is significantly increased in PWS compared with controls. These results may suggest that VEGF and VEGF-R could contribute to the pathogenesis of PWS by inducing vessel proliferation, vasodilatation, or both. Antiangiogenic treatments using VEGF blocking agents may be useful in the treatment of FLPD laser-resistant cases.
ACKNOWLEDGEMENTS The authors would like to thank Ms Jennifer James of Arkansas Cancer Research Center’s Core Facility Lab for her invaluable help in performing immunohistochemistry stainings.
AUTHOR INFORMATION From the Departments of Otolaryngology–Head and Neck Surgery (Drs Vural, Ramakrishnan, Buckmiller, and Suen) and Pathology (Drs Cetin and Fan), University of Arkansas for Medical Sciences, Little Rock, Arkansas; and Department of Pathology, John McClellan VA Hospital, Little Rock, AR. Corresponding author: Dr Emre Vural, Department of OtolaryngologyHead and Neck Surgery, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 543, Little Rock, AR 72205. E-mail address: [email protected]. Accepted for presentation in the American Academy of Otolaryngology– Head Neck Surgery Annual Meeting, September 21-24, 2008, Chicago, IL.
AUTHOR CONTRIBUTIONS Emre Vural, study design, obtaining funding source, data collection, manuscript preparation; Jeevan Ramakrishnan, data collection, manu-
563
script preparation; Neslihan Cetin, data collection, manuscript preparation; Lisa Buckmiller, data collection; James Y. Suen, data collection, critical review; Chun-Yang Fan, immunohistochemical analysis.
FINANCIAL DISCLOSURE Supported by the Medical Research Endowment Award at the University of Arkansas for Medical Sciences.
REFERENCES 1. Miller AC, Pit-Ten Cate IM, Watson HS, et al. Stress and family satisfaction in parents of children with facial port-wine stains. Pediatr Dermatol 1999;16:190 –7. 2. Jasim ZF, Handley JM. Treatment of pulsed dye laser-resistant port wine stain birthmarks. J Am Acad Dermatol 2007;57:677– 82. 3. Waner M, Suen JY. Hemangiomas and vascular malformations of the head and neck. 1st ed. New York: Wiley-Liss; 1999. 4. Taub PJ, Silver L, Weinberg H. Plastic surgical perspectives on vascular endothelial growth factor as gene therapy for angiogenesis. Plast Reconstr Surg 2000;105:1034 – 42. 5. Nissen NN, Polverini PJ, Koch AE, et al. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. Am J Pathol 1998;152:1445–52. 6. Richter GT , Fan CY, Ozgursoy O, et al. Effect of vascular endothelial growth factor on skin graft survival in Sprague Dawley rats. Arch Otolaryngol Head Neck Surg 2006;132:637– 41. 7. Taub PJ, Marmur JD, Zhang WX, et al. Locally administered vascular endothelial growth factor cDNA increases survival of ischemic experimental skin flaps. Plast Reconstr Surg 1998;102: 2033–9. 8. Kyzas PA, Stefanou D, Batistatou A, et al. Prognostic significance of VEGF immunohistochemical expression and tumor angiogenesis in head and neck squamous cell carcinoma. J Cancer Res Clin Oncol 2005;131:624 –30. 9. Zhang L, Lin X, Wang W, et al. Circulating level of vascular endothelial growth factor in differentiating hemangioma from vascular malformation patients. Plast Reconstr Surg 2005;116:200 – 4. 10. Koizumi T, Shiraishi T, Hagihara N, et al. Expression of vascular endothelial growth factors and their receptors in and around intracranial arteriovenous malformations. Neurosurgery 2002;50: 117–24. 11. Otrock ZK, Makarem JA, Shamseddine AI. Vascular endothelial growth factor family of ligands and receptors: review. Blood Cells Mol Dis 2007;38:258 – 68. 12. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669 –76. 13. Xiao X, Liu J, Sheng M. Synergistic effect of estrogen and VEGF on the proliferation of hemangioma vascular endothelial cells. J Pediatr Surg 2004;39:1107–10. 14. Chang J, Most D, Bresnick S, et al. Proliferative hemangiomas: analysis of cytokine gene expression and angiogenesis. Plast Reconstr Surg 1999;103:1–9. 15. Kilic T, Pamir MN, Kullu S, et al. Expression of structural proteins and angiogenic factors in cerebrovascular anomalies. Neurosurgery 1999; 46:1179 –91. 16. Sonstein WJ, Kader A, Michelsen WJ, et al. Expression vascular endothelial growth factor in pediatric and adult cerebral arteriovenous malformations: an immunocytochemical study. J Neurosurg 1996;85: 838 – 45. 17. Marler JJ, Fishman SJ, Kilroy SM, et al. Increased urinary matrix metalloproteinases parallels the extent and activity of vascular anomalies. Pediatrics 2005;116:38 – 45.
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18. Smoller BR, Rosen S. Port-wine stains: a disease of altered neural modulation of blood vessels? Arch Dermatol 1986;122: 177–9. 19. Lanigan SW, Taibjee SM. Recent advances in laser treatment of port-wine stains. Br J Dermatol 2004;151:527–33.
20. Sevila A, Nagore E, Botella-Estrada R, et al. Videomicroscopy of venular malformations (port-wine stain type): prediction of response to pulsed dye laser. Pediatr Dermatol 2004;21:589 –96. 21. John AR, Bramhall SR, Eggo MC. Antiangiogenic therapy and surgical practice. Br J Surg 2008;95:281–93.
ORIGINAL RESEARCH—PEDIATRIC OTOLARYNGOLOGY
The expression of vascular endothelial growth factor and its receptors in port-wine stains Emre Vural, MD, Jeevan Ramakrishnan, MD, Neslihan Cetin, MD, Lisa Buckmiller, MD, James Y. Suen, MD, and Chun-Yang Fan, MD, PhD, Little Rock, AR OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in portwine stains (PWSs). DESIGN: An immunohistochemistry (IHC) study on formalinfixed, paraffin-embedded specimens. METHODS: Representative sections from surgical resection specimens of 12 PWS patients and 12 control specimens stained with routine IHC by using polyclonal anti-VEGF and anti– VEGF-R2 antibodies. Slides were evaluated semiquantitatively for the intensity of staining for VEGF and VEGF-R2 by using a scoring system varying from 0 to 3⫹. RESULTS: PWS specimens showed statistically significant overexpression of both VEGF and VEGF-R2 molecules when compared with control specimens (P ⬍ 0.005). CONCLUSIONS: VEGF and its receptor may play an important role in the pathogenesis of PWS. It is possible that PWS may progress by hyperplasia in addition to hypertrophy. VEGF-R blockade may have a potential role as a targeted approach in the treatment of this disfiguring condition in the future. © 2008 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
P
ort-wine stain (PWS) is a vascular malformation that affects approximately 0.3 percent of newborns and causes significant negative psychological and social impacts on the patients and their families because of worsening of the signs and symptoms by age.1 Although repetitive flashlamp pulsed-dye laser (FLPD) treatments have been successfully used for the management of this disfiguring condition in certain patients, overall complete clearance rate of PWS is still less than 20 percent with FLPD.2 Because the head and neck are the most commonly involved regions with this pathology; PWS unresponsive to laser treatments usually progresses into significantly disfiguring and destructive tumors necessitating complex and highly morbid surgical resections, which may be devastating to the patient in terms of cosmesis and function.3 Apparently, there is still a need for a more effective treatment for this invasive and progressive disease. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and permeability factor that has
been shown to stimulate angiogenesis, vascular remodeling, and new tissue growth.4,5 The effects of VEGF in revascularization of avascular tissue, improving ischemic skin flaps, and tumor angiogenesis have been widely investigated.6-8 It has also been shown that VEGF could have a potential role in the development of other vascular malformations such as hemangiomas and arteriovenous malformations.9,10 However, the potential role of VEGF in the pathogenesis of PWS is unknown. The purpose of this study was to investigate the expression of VEGF and its most potent receptor (VEGFR2) in PWS as a potential mechanism of progression, which may be a subject of targeted treatment in the future.
METHODS After obtaining an exempt status letter from the Institutional Review Board at the University of Arkansas for Medical Sciences for this immunohistochemistry (IHC) study, the departmental vascular anomalies database was searched for PWS patients who underwent resection of their lesions. Twelve patients with PWS were identified with adequate data. The database of the pathology department was also searched for control specimens, and 12 discarded skin specimens without known pathologic skin conditions were identified. The pathologist of the vascular anomalies team (CYF) examined the hematoxylin-eosin slides for each PWS and control case to identify the representative paraffin blocks of formalin-fixed specimens. Paraffin-embedded skin and PWS tissues were sectioned at 4 m, deparaffinized, and rehydrated.
IHC and Interpretation of the Results Routine IHC stainings were performed on each specimen at the Histology Core Laboratory at the University of Arkansas for Medical Sciences. Briefly in order, slides were treated with target retrieval solution (pH ⫽ 6.0) and peroxidase-blocking reagent (Dako North America Inc, Carpentaria, CA) followed by 10 percent normal rabbit serum (Vector
Received May 14, 2008; revised July 2, 2008; accepted July 10, 2008.
0194-5998/$34.00 © 2008 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2008.07.015
Vural et al
The expression of vascular endothelial growth . . .
Figure 1 A bar chart showing immunostaining scores for VEGF in PWS and control groups.
Laboratories, Burlingame, CA). Polyclonal antihuman VEGF R2 (KDR) and antihuman VEGF antibodies were used at 1:25 dilution as primary antibodies (R&D Systems Inc, Minneapolis, MN; catalog numbers AF357 and AB-293-NA, respectively). Slides were then treated with biotinylated rabbit antigoat at 1:400 dilution as the secondary antibody followed by the ABC kit (Vector Laboratories) and DAB⫹ (Dako North America Inc). Counterstaining was performed by using hematoxylin. Human placenta was used for positive staining control. The VEGF and VEGF-R2 IHC staining results were interpreted by the pathologist of the vascular anomalies team (CYF) semiquantitatively by scoring the staining intensity of VEGF-R2– and VEGF-positive endothelial cells (strong staining: 3, moderate staining: 2, weak staining: 1, and no staining: 0) by using low-power (⫻200) and high power (⫻400) magnification. A Wilcoxon two-sample test was performed to compare the immunostaining scores for VEGF and VEGF-R2 between controls and PWS specimens.
561
This was probably because of poor quality of the specimen embedded in the paraffin block or faulty embedding process. Therefore, this specimen was excluded from the analysis. In this study, both antihuman VEGF-R2 and antihuman VEGF antibodies specifically stained the endothelial cells. Smooth muscle cells of blood vessels and fibroblasts in the stroma of control skin and PWS were not reactive with either antibody. Because of scant cytoplasm of the endothelial cells, it was impossible to obtain a good membranous staining pattern for the antihuman VEGF-R2 antibody. Thus, the staining pattern appeared similar for both antihuman VEGF R2 and antihuman VEGF antibodies. Scores for the intensity of immunostaining for VEGF and VEGF-R2 are summarized in Figures 1 and 2, respectively. The average immunostaining scores for VEGF in controls and PWS were 0.83 (minimum ⫽ 0, maximum ⫽ 2, ⫾0.8 SD) and 2.63 (minimum ⫽ 2, maximum ⫽ 3, ⫾0.5 SD), respectively. The average immunostaining scores for VEGF-R2 in controls and PWS specimens were 0.92 (minimum ⫽ 0, maximum ⫽ 2, ⫾0.7 SD) and 2.72 (minimum ⫽ 2, maximum ⫽ 3, ⫾0.5 SD), respectively. PWS showed significantly higher staining scores than the controls for both VEGF and VEGF-R2 (P ⬍ 0.005) (Fig 3).
DISCUSSION
RESULTS
VEGFs are a group of glycoproteins composed of seven variants, specifically VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor, and VEGF-F.11 VEGF-A is the most potent member of this family that induces proliferation, sprouting, and tube formation of endothelial cells.12 Additionally, VEGF-A stimulates vasodilatation by inducing endothelial nitric oxide synthase.11 VEGF shows these effects by binding to a variety of transmembrane tyrosine kinase receptors (VEGFR1, VEGF-R2, and VEGF-R3), and VEGF-R2 seems to be the main receptor responsible for proangiogenic effects of VEGF.12 Because hemangiomas and vascular malformations are angiogenesis dependent, the role of VEGF and its recep-
PWS specimens came from a patient group composed of eight males and four females, with an average age of 21.8 (minimum 3, maximum 39). Four of 12 patients received at least one FLPD laser treatment before their surgeries. The indication for surgery was cosmetic reasons in all cases. Five of 12 patients underwent surgery for simultaneous functional deficits in addition to cosmetic deformity in the form of drooling (1 patient), poor oral intake (2 patients), and bleeding (2 patients). None of the patients had additional comorbidities, except one patient who had SturgeWeber syndrome. The antibodies for both VEGF and VEGF-R2 worked properly in all representative slides except one PWS specimen in which immunostaining was unsuccessful for either antibody despite repetitive attempts.
Figure 2 A bar chart showing immunostaining scores for VEGF-R2 in PWS and control groups.
Role of the Funding Source Financial support for purchasing supplies used in the study was obtained through the Medical Research Endowment Award at the University Arkansas for Medical Sciences.
562
Otolaryngology–Head and Neck Surgery, Vol 139, No 4, October 2008
Figure 3 The expression of VEGF and VEGF-R2 in skin controls and PWS specimens is shown. Note the strong staining for VEGF (B) and VEGF-R2 (D) in PWS when compared with controls (A and C) (magnification ⫻400).
tors was widely investigated as potential contributors to the pathogenesis of these anomalies. It has been shown that VEGF promoted endothelial cell proliferation in hemangiomas even stronger than estrogen.13 Another study showed that VEGF messenger RNA was upregulated in proliferating hemangiomas.14 Significantly high circulating levels of VEGF were also found in patients with proliferating hemangiomas.9 The overexpression of VEGF and VEGF-R was also shown in intracranial arteriovenous malformations.10,15,16 All these studies provided evidence regarding the possible involvement of VEGF and its receptors in the development and/or progression of hemangiomas and vascular malformations and the possibility of suppressing of these lesions by antiangiogenic modulation as shown in many solid tumors.17 Surprisingly, the possible role of VEGF and its receptors has never been investigated in the pathogenesis of another vascular malformation, PWS, probably because of the common belief that PWS progresses by ectasia of the normal number of vessels forming this pathology rather than proliferation of the vessels.2 The reason of the widespread ectasia was attributed to an abnormal neural supply to these capillaries because of the decreased number of nerve fibers shown in PWS compared with normal skin in histopathological studies.18 The current study showed overexpression of VEGF and VEGF-R2 compared with controls, suggesting that angiogenic stimulus may contribute to the pathogenesis of PWS. This may be in the form of hypertrophy,
hyperplasia, or both because VEGF induces both proliferation and vasodilatation.11,12 Despite the advances in laser technology, the gold standard treatment of PWS by using FLPD laser often fails to eradicate this disease.2,19 The main disadvantage of FLPD laser treatment is the fact that only less than 20% of PWS respond completely to this treatment, and the vast majority of nonresponsive or incompletely responsive lesions progress into hypertrophied and disfiguring lesions. It is known that certain areas such as the central face were even less responsive to FLPD laser treatment.20 There are multiple reasons for the ineffective clearance of PWS by FLPD, and these are mainly attributed to the inadequate penetration of the FLPD laser into the dermis because of wavelength limitations, inadequate damage to the vessel wall because of limited heat conduction from the central portion of the vessel to the peripheral walls causing subsequent regeneration of the vessels, inadequate amount of target chromophore in smaller vessels causing insufficient absorption of the light, and inadequate fluence.2 It is obvious that more effective treatment modalities are necessary for eradication of this disfiguring disease when FLPD fails, especially for the lesions in the head and neck region because of their visibility and interference with important physiologic functions such as breathing, vision, and oral intake. Antiangiogenic treatment using VEGF receptor blocking agents recently gained popularity with promising results.21
Vural et al
The expression of vascular endothelial growth . . .
Although this approach is mainly being investigated for the treatment of a wide variety of malignant tumors, it has been suggested that antiangiogenic treatments may have future applications in the treatment of vascular anomalies.17 This may be especially useful in AVMs in which a dominant feeding vessel could be identified angiographically that may act as a route for targeted delivery of the antiangiogenic agents. As we have shown in this IHC study, overexpression of VEGF and VEGF-R2 in PWS may be an important factor in the development and/or progression of this pathology, and patients with PWS may be candidates for targeted antiangiogenic treatments. Although how to deliver VEGF-R blockers specifically to the target vascular malformations such as PWS without affecting angiogenesis in healthy tissues remains a question, especially in the pediatric age groups; advances in nanotechnology and molecular biology may solve this problem in the future. There is definitely a need to develop an animal model for PWS because such a model currently does not exist.
CONCLUSIONS VEGF and VEGF-R2 expression is significantly increased in PWS compared with controls. These results may suggest that VEGF and VEGF-R could contribute to the pathogenesis of PWS by inducing vessel proliferation, vasodilatation, or both. Antiangiogenic treatments using VEGF blocking agents may be useful in the treatment of FLPD laser-resistant cases.
ACKNOWLEDGEMENTS The authors would like to thank Ms Jennifer James of Arkansas Cancer Research Center’s Core Facility Lab for her invaluable help in performing immunohistochemistry stainings.
AUTHOR INFORMATION From the Departments of Otolaryngology–Head and Neck Surgery (Drs Vural, Ramakrishnan, Buckmiller, and Suen) and Pathology (Drs Cetin and Fan), University of Arkansas for Medical Sciences, Little Rock, Arkansas; and Department of Pathology, John McClellan VA Hospital, Little Rock, AR. Corresponding author: Dr Emre Vural, Department of OtolaryngologyHead and Neck Surgery, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 543, Little Rock, AR 72205. E-mail address: [email protected]. Accepted for presentation in the American Academy of Otolaryngology– Head Neck Surgery Annual Meeting, September 21-24, 2008, Chicago, IL.
AUTHOR CONTRIBUTIONS Emre Vural, study design, obtaining funding source, data collection, manuscript preparation; Jeevan Ramakrishnan, data collection, manu-
563
script preparation; Neslihan Cetin, data collection, manuscript preparation; Lisa Buckmiller, data collection; James Y. Suen, data collection, critical review; Chun-Yang Fan, immunohistochemical analysis.
FINANCIAL DISCLOSURE Supported by the Medical Research Endowment Award at the University of Arkansas for Medical Sciences.
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