RASA1 mosaic mutations in patients with capillary malformation-arteriovenous malformation.

BACKGROUND: Capillary malformation-arteriovenous malformation is an autosomal dominant disorder, characterised by capillary malformations and increased risk of fast-flow vascular malformations, caused by loss-of-function mutations in the RASA1 or EPHB4 genes. Around 25% of the patients do not seem to carry a germline mutation in either one of these two genes. Even if other genes could be involved, some individuals may have mutations in the known genes that escaped detection by less sensitive techniques. We tested the hypothesis that mosaic mutations could explain some of previously negative cases. METHODS: DNA was extracted from peripheral blood lymphocytes, saliva or vascular malformation tissues from four patients. RASA1 and EPHB4 coding regions and exon/intron boundaries were analysed by targeted custom gene panel sequencing. A second panel and/or Sanger sequencing were used to confirm the identified mutations. RESULTS: Four distinct mosaic RASA1 mutations, with an allele frequency ranging from 3% to 25%, were identified in four index patients with classical capillary malformation-arteriovenous malformation phenotype. Three mutations were known, one was novel. In one patient, a somatic second hit was also identified. One index case had three affected children, illustrating that the mosaicism was also present in the germline. CONCLUSION: This study shows that RASA1 mosaic mutations can cause capillary malformation-arteriovenous malformation. Thus, highly sensitive sequencing techniques should be considered as diagnostic tools, especially for patients with no family history. Even low-level mosaicism can cause the classical phenotype and increased risk for offspring. In addition, our study further supports the second-hit pathophysiological mechanism to explain the multifocality of vascular lesions in this disorder.

Membrane trafficking and exocytosis are upregulated in port wine stain blood vessels.

INTRODUCTION: Port wine stain (PWS) is characterized as a progressive dilatation of immature venule-like vasculatures which result from differentiation-impaired endothelial cells. In this study, we aimed to identify the major biological pathways accounting for the pathogenesis of PWS. METHODS: Sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to identify differentially expressed proteins in PWS lesions, followed by confirmative studies with immunohistochemistry, immunoblot and transmission electron microscopy (TEM). RESULTS: 107 out of 299 identified proteins showed differential expressions in PWS lesions as compared to normal skin, mainly involving the functions of biosynthesis, membrane trafficking, cytoskeleton and cell adhesion/migration. The confirmative studies showed that expressions of membrane trafficking/exocytosis related proteins such as VAT1, IQGAP1, HSC70, clathrin, perlecan, spectrin α1 and GDIR1 were significantly increased in PWS blood vessels as compared to normal ones; while collagen subtypes 6A1 and 6A3 were decreased in PWS skin. Furthermore, TEM studies showed there is a significant upregulation of extracellular vesicle exocytosis from PWS blood vessels as compared to control. CONCLUSIONS: The biological process of membrane trafficking and exocytosis is enhanced in PWS blood vessels. Our results imply that the extracellular vesicles released by lesional endothelial cells may act as potential intercellular signaling mediators to contribute to the pathogenesis of PWS.

A dose-finding study for hemoporfin in photodynamic therapy for port-wine stain: A multicenter randomized double-blind phase IIb trial.

BACKGROUND/PURPOSE: Previous studies have shown that hemoporfin-mediated photodynamic therapy (PDT) was a treatment for port-wine stain (PWS). Our current study aimed to identify optimal hemoporfin dose. METHODS: A prospective, multicenter, double-blind, randomized clinical trial was conducted. Patients were assigned into low- or high-dose hemoporfin (2.5 mg/kg and 5 mg/kg intravenously, respectively), or control (placebo) group, at a rate of 2:2:1. Treatment efficacy was evaluated at week 8. Then, patients in control group were randomly assigned into either high- or low-dose hemoporfin group. Treatment reactions and adverse events were analyzed at week 16. RESULTS: A total of 100 patients (40, 40, 20 in low-, high-dose hemoporfin, and control group, respectively) were enrolled. Compared to low dose (40%) and control group (15%), a higher proportion of patients in high-dose group (75%) had achieved skin lesion improvements. Treatment satisfactions were graded highest in high-dose group. Compared to low-dose group (14.3%), high-dose group (46.0%) had more frequent skin hyperpigmentation, which disappeared 3-6 months after treatment. Other treatment reactions and adverse events were comparable between two groups. CONCLUSIONS: Photodynamic therapy with 5 mg/kg hemoporfin could be an effective and safe treatment for PWS.

Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

BACKGROUND: Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. OBJECTIVES: To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. METHODS: Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. RESULTS: Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. CONCLUSIONS: PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133+ /CD166+ /EphB1+ /EfnB2+ , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures.

Determination of Optical and Microvascular Parameters of Port Wine Stains Using Diffuse Reflectance Spectroscopy.

Characterizing port wine stains (PWS) with its optical parameters [i.e. absorption coefficient (µ a) and reduced scattering coefficient (µ s')] and microvascular parameters [i.e. blood volume fraction (BVF), mean vessel diameter (MVD), and oxygen saturation (StO2)] is extremely important for elucidating the mechanisms for its light-based treatments, such as pulsed dye laser and photodynamic therapy. In this study, a customized diffuse reflectance spectroscopy (DRS) probe with an appropriate source-detector distance was used to measure the diffuse reflectance spectra of PWS lesions in clinical practice. The results demonstrate that optical parameters of different types of PWS lesions can be accurately extracted by fitting the DRS with diffusion equation. Since the sampling depth of the probe coincides with the depth distribution of abnormal vasculature in PWS, the obtained microvascular parameters of PWS lesions that changed from pink to purple are in agreement with the corresponding physiological conditions. This study suggests that DRS can be utilized to quantitatively determine the optical and microvascular parameters of PWS lesions, which have the potential for planning the protocol and predicting the efficiency for light-based PWS treatments.

Angiolymphoid hyperplasia with eosinophilia developing within a port wine stain.

A 19-year-old male with a port wine stain on the base of his neck presented with a 5-month history of gradual thickening of the involved skin which interfered with clothing and caused repeated bleeding. The lesion was excised and histopathologic examination revealed angiolymphoid hyperplasia with eosinophilia (ALHE) arising from the pre-existing port wine stain - a rare finding with only one previously reported case. Additionally the lesion was associated with elevated serum renin levels which virtually normalized following excision of the lesion. We further demonstrated the expression of angiotensin converting enzyme and angiotensin II receptors 1 and 2 by the lesion and discuss the possible role of the renin-angiotensin system in this condition.

A common factor suppresses thickening in young women with malar area port wine stains and delays low density lipoprotein elevation: is it estrogen?

Port wine stains in the malar area of the face can develop thickening in early adult life. We began a study with a hypothesis that this thickening can be associated with elevation of low density lipoprotein. In a retrospective review, we divided 53 subjects with malar port wine stains into 4 groups, adults 25-39 years of age with thickening, that age group without thickening, adults 40+ years of age with thickening, and that age group without thickening. Low density lipoprotein levels in the subjects were compared to age and sex matched controls randomly selected from the general Dermatology clinic. The younger subjects with thickening demonstrated significantly higher low density lipoprotein levels than their controls (p .0082) and without thickening lower low density lipoprotein levels than their controls with great significance (p .00058). The subjects without thickening also consisted mainly of women. The low density lipoprotein levels in the older age groups, whether thickened or not, demonstrated no significant difference in low density lipoprotein levels between subjects and controls. This led to a new hypothesis that there is a factor in a subgroup of young adult women with malar port wine stains that suppresses thickening and delays the elevation of low density lipoprotein and that this factor might be estrogen. The implications of this hypothesis are that it could define a marker for a subset of the population that might be protected from the diseases associated with early elevation of low density lipoprotein and provide a source of cutaneous tissue for studying the basic science of this protection (although limited by cosmetic considerations). Future laboratory research to test the new hypothesis might include testing blood of women with malar port wine stains with or without thickening for estrogen and other sex hormones. It might also include skin biopsies to study receptors for estrogen, other sex hormones, and angiogenic factors in malar port wine stains with or without thickening. Future clinical research might include a long term prospective project to study the development of low density lipoprotein related diseases in women with malar port wine stains with or without thickening over years.

Simulating light transport through skin for color prediction of port wine stain lesions: a review.

A survey of the literature is presented regarding the simulation of port wine stain (PWS) skin color. Knowledge of PWS features, such as the depths and diameters of affected vessels, is essential for informing laser treatment. These may be determined through the inverse application of a skin model. The techniques which have been applied to achieve this are analyzed in detail. Radiative transfer (RT) is found to be the preferred method of simulation. By far the most common approximations to RT are the diffusion approximations, which have been applied successfully in the past and Monte Carlo techniques, which are now the methods of choice. As the requirements for improvement of laser treatment on an individual basis continues, the needs for further work towards accurate estimations of individual optical coefficients and robust, flexible simulation techniques are identified.

Capillary malformation of port-wine stain: differentiation from early arteriovenous malformation by histopathological clues.

BACKGROUND: Cutaneous arteriovenous malformations (AVMs) of stage I may mimic port-wine stains (PWSs) clinicopathologically; therefore, it may be misdiagnosed and mistreated as being PWS. OBJECTIVE: To suggest the clinicopathological differential clues between early AVMs and PWSs. METHODS: A set of 10 radiologically proven AVMs of stage I was selected in conjunction with a set of 10 age-/sex-matched PWSs as a control. Their clinical features, hematoxylin and eosin, CD31, and smooth muscle actin immunohistochemistry were then compared. RESULTS: Four pathological clues for differential diagnosis with statistical significance (P < 0.05) were found: the vessel density (CD31), presence of vascular luminal red blood cells, elongation and haphazard branching of vessels, and thickened vessel walls highlighted by smooth muscle actin. CONCLUSION: Therefore, 4 differential clues with respect to stage I AVM and PWS in their earlier developmental stages have been proposed.

Stem cell marker upregulation in normal cutaneous vessels following pulsed-dye laser exposure and its abrogation by concurrent rapamycin administration: implications for treatment of port-wine stain birthmarks.

Port-wine stains (PWS) represent a group of vascular malformations that are usually accompanied by psychological distress for affected patients, often reflected in high treatment demand. Although the pulsed-dye laser (PDL) was established as standard therapy for PWS more than a decade ago, therapeutic outcome may be unsatisfactory. One of the main drawbacks to successful PDL therapy is PWS revascularization shortly after laser exposure. Therefore, inhibition of revascularization should improve therapeutic outcome of PDL therapy. In this study, we first evaluated the effects of various light energies on normal cutaneous vessels over a period of 14 days, particularly the proliferation and stem cell marker expression of dermal endothelial cells, which were found to be highest 8 days following laser exposure. We found that PDL exposure induced dose-dependent damage of dermal vessels up to energy densities of 6 J/cm(2), above which no increase in PDL-induced effects were observed with the energies employed in this study. In dermal endothelial cells of PDL-exposed skin, we found strong expression of the proliferation marker Ki-67 as well as the stem cell marker nestin but not other stem cell markers such as CD133 and CD166. The influence of rapamycin (RPM), used as an adjuvant to PDL exposure, was also investigated. RPM administration reduced Ki-67 and nestin expression in dermal endothelial cells and increased PDL-induced destruction of dermal vessels, indicating that the use of RPM after PDL exposure may be an interesting new approach for prolonging and improving PWS laser therapeutic outcome.

Fluorescence monitoring of a photosensitizer and prediction of the therapeutic effect of photodynamic therapy for port wine stains.

In this study, a fluorescence method was established to obtain the local concentration of a photosensitizer (PS) based on the realtime fluorescence measurement of skin with port wine stain (PWS) during photodynamic therapy (PDT). This algorithm corrected for the distortions of PS fluorescence spectra imposed by the absorption of melanin and hemoglobin in skin and other factors, which yields a semi-quantitative measurement of PS concentration. Based on this information, a therapeutic effect correlation index (TECI) was proposed as the area under the PS concentration-time curve during PDT. The correlation between TECI and PDT treatment outcome was analyzed from 31 PWS patients. The measured PS fluorescence spectra showed that under the same PS dose, there were clear variations in the concentrations of the PS during PDT. Statistical analysis showed that TECI has a positive correlation with PDT outcome. Patients with a higher TECI value had a better treatment outcome. These results suggest that fluorescence spectroscopy can be used in situ to monitor skin PS concentration during PDT and to provide a valuable diagnostic tool to predict PDT outcome.

Treatment of resistant port-wine stains with a pulsed dual wavelength 595 and 1064 nm laser: a histochemical evaluation of the vessel wall destruction and selectivity.

BACKGROUND AND OBJECTIVES: Pulsed dye laser (PDL) is the current treatment of choice for port-wine stains (PWS), but 25-50% of treated lesions do not demonstrate a significant improvement. Hybrid lasers may improve treatment efficacy, especially those using the synergies between PDL and Nd:YAG 1064 nm laser. The objectives of this study were to assess vessel wall damage and epidermal sparing after a dual wavelength treatment with the two lasers, using different laser parameters. MATERIAL AND METHODS: Post-treatment biopsies, after using a laser platform that allows sequential pulses of PDL and Nd:YAG 1064 nm lasers, were performed in five patients with PWS resistant to PDL. The biopsies were stained with nitroblue-tetrazolium chloride (NBTC), using enzymatic activity that stops immediately after cell death and allows a better identification of viable cells. RESULTS: Five patients with PWS and a median age of 33 years were enrolled in this study. Selectivity and efficacy was observed with this dual wavelength approach, with the best results observed with PDL pulses shorter than 10 ms, use of the 10 mm spot, and a second pass with PDL only. CONCLUSIONS: Histochemical studies with NBTC stain can help the laser surgeon establish the best treatment parameters and understand some of the unwanted side effects. The dual wavelength used in this study showed efficacy, but better assessment of treatment parameters, such as the delay between the two lasers, is needed to avoid side effects.

The expression of vascular endothelial growth factor and its receptors in port-wine stains.

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in port-wine stains (PWSs). DESIGN: An immunohistochemistry (IHC) study on formalin-fixed, 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.

Autism with facial port-wine stain: a new syndrome?

The hallmark of Sturge-Weber syndrome is leptomeningeal angiomatosis. Over 15 years, four children were identified (2 boys, age 2.9-6 years) with unilateral facial port-wine stain, referred for presumable Sturge-Weber syndrome but who were also autistic. Computed tomography and magnetic resonance imaging scans failed to show evidence of leptomeningeal angioma in all four children. Three of the children had a history of seizures. Detailed neuropsychologic testing of three children revealed a similar presentation, characterized by developmental disturbance, particularly involving delayed onset of language, and early-emerging social atypicality. Positron emission tomography scanning of cerebral glucose metabolism revealed hypometabolism in the bilateral medial temporal regions, anterior cingulate gyrus, frontal cortex, right temporal cortex, and cerebellum. The pattern of glucose hypometabolism differed from that of 12 children with infantile autism (age 2.7-7.9 years) who had mild left medial temporal but more severe right temporal cortical hypometabolism and showed a reversal of normal frontotemporal asymmetry of glucose metabolism. Unilateral facial port-wine stain and autism with no intracranial angioma on conventional imaging may represent a rare clinical entity distinct from both infantile autism and previously described variants of Sturge-Weber syndrome.

Methemoglobin formation during laser induced photothermolysis of vascular skin lesions.

BACKGROUND AND OBJECTIVE: Monitoring dynamic changes during laser induced photothermolysis of vascular skin lesions is essential for obtaining an optimal therapeutic result. Rapid photoinduced thermal damage occurs at a threshold temperature of about 70 degrees C. It is therefore, relevant to identify markers to indicate if this threshold temperature has been reached. Methemoglobin, which is formed by a photo-induced oxidation of hemoglobin, indicates that the temperature has reached this threshold value. This study presents a proof of concept of a method for monitoring the in vivo presence of methemoglobin immediately after laser exposure. STUDY DESIGN/MATERIALS AND METHODS: The present study was designed to investigate the in vivo temperature dependence of hemoglobin absorption in the 450-800 nm spectrum range. In vivo diffuse reflectance measurements of port-wine stain (PWS) and telangiectasia were performed prior to, and immediately after, laser treatment with a pulsed dye laser (PDL) at 585 nm wavelength. RESULTS: In vivo measurements following laser treatment of vascular skin lesions showed an immediate increase in the optical absorption of blood. This effect, caused by thermal stress, is a result of an increased dermal blood volume fraction and methemoglobin formation. The effect is light dose dependent, and reflectance spectra revealed methemoglobin formation in patients treated with fluences above 5 J/cm2 at 585 nm wavelength. CONCLUSIONS: It was proved that methemoglobin can be measured in vivo by reflectance spectroscopy. Measurements of the average methemoglobin concentrations immediately after laser exposure may be a valuable diagnostic tool to verify that the blood temperature has been sufficiently high to induce thermal damage to the vessel wall.