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Port Wine Birthmarks (PWBs) are a congenital vascular malformation on the skin, occurring in 1–3 per 1000 live births. We have recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, and so we aim to explore them in this study. Metabolites were separated by ultra-performance liquid chromatography and screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant, multivariate, and univariate analyses were used to identify differential metabolites (DMs). KEGG analysis was used to determine the enrichment of metabolic pathways. A total of 339 metabolites was identified. There were 22 DMs, among which nine were downregulated—including sphingosine—and 13 were upregulated, including glutathione in PWB iPSCs, as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and the downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key molecules associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1α (HIF1α), glutathione S-transferase Pi 1 (GSTP1), γ-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skin. Other significantly affected metabolic pathways in PWB iPSCs included pentose and glucuronate interconversions; amino sugar and nucleotide sugars; alanine, aspartate, and glutamate; arginine, purine, D-glutamine, and D-glutamate; arachidonic acid, glyoxylate, and dicarboxylate; nitrogen, aminoacyl-tRNA biosynthesis, pyrimidine, galactose, ascorbate, and aldarate; and starch and sucrose. Our data demonstrated that there were perturbations in sphingolipid and cellular redox homeostasis in PWB vasculatures, which could facilitate cell survival and pathological progression. Our data implied that the upregulation of glutathione could contribute to laser-resistant phenotypes in some PWB vasculatures.

Lesional iPSCs-derived ECs can resemble pathological vascular phenotypes of PWB. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated. This data suggests new therapeutics to be developed for targeting such dysregulated pathways for treatment of PWB.

BACKGROUND: Port wine birthmark (PWB) is a congenital vascular malformation resulting from developmentally defective endothelial cells (ECs). Developing clinically relevant disease models for PWB studies is currently an unmet need. OBJECTIVE: Our study aims to generate PWB-derived induced pluripotent stem cells (iPSCs) and iPSC-derived ECs that preserve disease-related phenotypes. METHODS: PWB iPSCs were generated by reprogramming lesional dermal fibroblasts and differentiated into ECs. RNA-seq was performed to identify differentially expressed genes (DEGs) and enriched pathways. The functional phenotypes of iPSC-derived ECs were characterized by capillary-like structure (CLS) formation in vitro and Geltrex plug-in assay in vivo . RESULTS: Human PWB and control iPSC lines were generated through reprogramming of dermal fibroblasts by introducing the "Yamanaka factors" (Oct3/4, Sox2, Klf4, c-Myc) into them; the iPSCs were successfully differentiated into ECs. These iPSCs and their derived ECs were validated by expression of a series of stem cell and EC biomarkers, respectively. PWB iPSC-derived ECs showed impaired CLS in vitro with larger perimeters and thicker branches as compared to control iPSC-derived ECs. In the plug-in assay, perfused human vasculature formed by PWB iPSC- derived ECs showed bigger perimeters and greater densities than those formed by control iPSC- derived ECs in severe combined immune deficient (SCID) mice. The transcriptome analysis showed that dysregulated pathways of stem cell differentiation, Hippo, Wnt, and focal adhesion persisted through differentiation of PWB iPSCs to ECs. Functional enrichment analysis showed that Hippo and Wnt pathway-related PWB DEGs are enriched for vasculature development, tube morphology, endothelium development, and EC differentiation. Further, members of the zinc finger (ZNF) gene family were overrepresented among the DEGs in PWB iPSCs. ZNF DEGs confer significant functions in transcriptional regulation, chromatin remodeling, protein ubiquitination, and retinoic acid receptor signaling. Furthermore, NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were dysregulated in PWB ECs as readouts of impaired differentiation. CONCLUSIONS: PWB iPSC-derived ECs render a novel and clinically-relevant disease model by retaining pathological phenotypes. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated, which may contribute to the development of differentiation-defective ECs in PWB. BULLETED STATEMENTS: What is already known about this topic?: Port Wine Birthmark (PWB) is a congenital vascular malformation with an incidence rate of 0.1 - 0.3 % per live births.PWB results from developmental defects in the dermal vasculature; PWB endothelial cells (ECs) have differentiational impairments.Pulse dye laser (PDL) is currently the preferred treatment for PWB; unfortunately, the efficacy of PDL treatment of PWB has not improved over the past three decades.What does this study add?: Induced pluripotent stem cells (iPSCs) were generated from PWB skin fibroblasts and differentiated into ECs.PWB ECs recapitulated their pathological phenotypes such as forming enlarged blood vessels in vitro and in vivo.Hippo and Wnt pathways were dysregulated in PWB iPSCs and ECs.Zinc-finger family genes were overrepresented among the differentially expressed genes in PWB iPSCs.Dysregulated NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were enriched in PWB ECs.What is the translational message?: Targeting Hippo and Wnt pathways and Zinc-finger family genes could restore the physiological differentiation of ECs.Targeting NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways could mitigate the pathological progression of PWB.These mechanisms may lead to the development of paradigm-shifting therapeutic interventions for PWB.

ObjectiveWe retrospectively studied the clinical presentations and outcomes of endovascular management in a mixed pediatric and adult Klippel-Trenaunay syndrome (KTS) population at a single academic medical center.MethodsWe performed a retrospective study of patients with KTS who had been referred for endovascular intervention after evaluation and diagnosis by a multidisciplinary team at a single academic medical center during a 10-year period. The patient demographics, areas affected, presenting symptoms, previous treatments, imaging modalities, endovascular treatment types, number of treatments, and complications were assessed. The technical and clinical success rates were calculated.ResultsTwenty-six patients with suspected KTS were evaluated. Of these 26 patients, 20, aged 2 to 75 years, had been diagnosed with KTS using the International Society for the Study of Vascular Anomalies criteria and referred for endovascular management. The left lower extremity was affected most often. The presenting symptoms were pain (80%), edema (70%), bleeding (10%), numbness (25%), and claudication (25%). Of the 20 patients, 16 (80%) had undergone treatment of KTS before presenting to our institution. Magnetic resonance imaging and ultrasound (US) were the most common imaging modalities. Fifteen patients underwent 46 endovascular treatments during the study period. The treatments included 5 endovenous ablations only, 4 US-guided sclerotherapies with endovenous ablation, 5 US-guided sclerotherapies only, and 32 catheter-directed venograms with additional interventions. Localized intravascular coagulopathy was the only procedure-related complication and occurred in one patient after three treatments. The technical success rate was 97.8%, and the clinical success rate was 100%.ConclusionsEndovascular intervention is safe and effective for KTS patients for whom conservative management has failed. Pain and edema were the most common presenting symptoms. Presenting symptoms may be related to pathology of anomalous veins, orthotopic superficial veins or deep veins. Venous claudication can be present in those with KTS despite patency of the deep venous system. Magnetic resonance imaging and duplex US are frequently used modalities for venous assessment. The complications of endovascular treatment are rare but include localized intravascular coagulopathy.

ImportanceSturge-Weber syndrome (SWS) is a neurocutaneous syndrome involving the skin, brain, and eyes. Consensus recommendations for management are lacking.ObjectiveTo consolidate the current literature with expert opinion to make recommendations that will guide treatment and referral for patients with port-wine birthmarks (PWBs).Evidence reviewIn this consensus statement, 12 nationally peer-recognized experts in dermatology with experience treating patients with SWS were assembled. Key topics and questions were formulated for each group and included risk stratification, optimum treatment strategies, and recommendations regarding light-based therapies. A systematic PubMed search was performed of English-language articles published between December 1, 2008, and December 1, 2018, as well as other pertinent studies identified by the expert panel. Clinical practice guidelines were recommended.FindingsTreatment of PWBs is indicated to minimize the psychosocial impact and diminish nodularity and potentially tissue hypertrophy. Better outcomes may be attained if treatments are started at an earlier age. In the US, pulsed dye laser is the standard for all PWBs regardless of the lesion size, location, or color. When performed by experienced physicians, laser treatment can be safe for patients of all ages. The choice of using general anesthesia in young patients is a complex decision that must be considered on a case-by-case basis.Conclusions and relevanceThese recommendations are intended to help guide clinical practice and decision-making for patients with SWS and those with isolated PWBs and may improve patient outcomes.

Lymphatic malformations (LMs) are rare congenital anomalies. LMs are often refractory to standard treatments, including surgical resection, debulking, and sclerotherapy. Use of sildenafil, a phosphodiesterase-5 inhibitor, for treatment of pediatric LMs has been reported with demonstrated benefit to some patients. This case series reports treatment of three patients (aged 14-37 years) suffering from complicated or refractory LMs with a low-dose oral phosphodiesterase-5 inhibitor, resulting in significant clinical improvement.

Erythrocyte-based carriers can serve as theranostic platforms for delivery of imaging and therapeutic payloads. Engineering these carriers at micro- or nanoscales makes them potentially useful for broad clinical applications ranging from vascular diseases to tumor theranostics. Longevity of these carriers in circulation is important in delivering a sufficient amount of their payloads to the target. We have investigated the circulation dynamics of micro (∼4.95 μm diameter) and nano (∼91 nm diameter) erythrocyte-derived carriers in real time using near-infrared fluorescence imaging, and evaluated the effectiveness of such carrier systems in mediating photothermolysis of cutaneous vasculature in mice. Fluorescence emission half-lives of micro- and nanosized carriers in response to a single intravenous injection were ∼49 and ∼15 min, respectively. A single injection of microsized carriers resulted in a 3-fold increase in signal-to-noise ratio that remained nearly persistent over 1 h of imaging time. Our results also suggest that a second injection of the carriers 7 days later can induce a transient inflammatory response, as manifested by the apparent leakage of the carriers into the perivascular tissue. The administration of the carriers into the mice vasculature reduced the threshold laser fluence to induce photothermolysis of blood vessels from >65 to 20 J/cm2. We discuss the importance of membrane physicochemical and mechanical characteristics in engineering erythrocyte-derived carriers and considerations for their clinical translation.

Background and objectivesPort wine stain (PWS) is a congenital vascular malformation of the human skin. Laser is the treatment of choice for PWS. Laser-resistant PWS is one crucial factor accounting for inadequate treatment outcome, which needs to be fully characterized. This study aims to quantitatively characterize the morphology of laser-resistant PWS blood vessels in the upper papillary dermis using in vivo reflectance confocal microscopy (RCM).Study design/materials and methodsA total of 42 PWS subjects receiving laser treatment from August 2016 through July 2018 were enrolled into this study. Thirty-three subjects had facial PWS; nine had extremity PWS. All subject's PWS received multiplex 585/1,064 nm laser treatment. RCM images were taken before and after treatment. The density, diameter, blood flow, and depth of PWS blood vessels were analyzed.ResultsWe found 44.4% PWS on the extremities (four out of nine subjects) were laser-resistant, which was significantly higher (P < 0.001) when compared with those PWS on the face (15.2%, 5 out of 33 subjects). The laser-resistant facial PWS blood vessels had significantly higher blood flow (1.35 ± 0.26 U vs. 0.89 ± 0.22 U, P < 0.001), larger blood vessel diameters (109.60 ± 18.24 µm vs. 84.36 ± 24.04 µm, P = 0.033) and were located deeper in the skin (106.01 ± 13.87 µm vs. 87.82 ± 12.57 µm, P < 0.001) in the skin when compared with laser-responsive PWS on the face. The average PWS blood vessel density (17.01 ± 4.63/mm2 vs. 16.61 ± 4.44/mm2 , P = 0.857) was not correlated to the laser resistance.ConclusionsLaser-resistant PWS blood vessels had significantly higher blood flow, larger diameters, and were located deeper in the skin. RCM can be a valuable tool for a prognostic evaluation on laser-resistant lesions before treatment, thereby providing guidance for tailored laser treatment protocols, which may improve the therapeutic outcome. The limitations for this study include relative small sample size and acquisitions of different blood vessels before and after 2 months of treatment. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Port wine stain (PWS) is a congenital vascular malformation involving human skin. Approximately 15-20% of children a facial PWS involving the ophthalmic (V1) trigeminal dermatome are at risk for Sturge Weber syndrome (SWS), a neurocutaneous disorder with vascular malformations in the cerebral cortex on the same side of the facial PWS lesions. Recently, evidence has surfaced that advanced our understanding of the pathogenesis of PWS/SWS, including discoveries of somatic genetic mutations (GNAQ, PI3K), MAPK and PI3K aberrant activations, and molecular phenotypes of PWS endothelial cells. In this review, we summarize current knowledge on the etiology and pathology of PWS/SWS based on evidence that the activation of MAPK and/or PI3K contributes to the malformations, as well as potential futuristic treatment approaches targeting these aberrantly dysregulated signaling pathways. Current data support that: (1) PWS is a multifactorial malformation involving the entire physiological structure of human skin; (2) PWS should be pathoanatomically re-defined as "a malformation resulting from differentiation-impaired endothelial cells with a progressive dilatation of immature venule-like vasculatures"; (3) dysregulation of vascular MAPK and/or PI3K signaling during human embryonic development plays a part in the pathogenesis and progression of PWS/SWS; and (4) sporadic low frequency somatic mutations, such as GNAQ, PI3K, work as team players but not as a lone wolf, contributing to the development of vascular phenotypes. We also address many crucial questions yet to be answered in the future research investigations.

IntroductionPort 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.MethodsSequential 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).Results107 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.ConclusionsThe 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.

Pulsed dye laser irradiation in the wavelength range of 585 to 600 nm is currently the gold standard for treatment of port-wine stains (PWSs). However, this treatment method is often ineffective for deeply seated blood vessels and in individuals with moderate to heavy pigmentation. Use of optical particles doped with the FDA-approved near-infrared (NIR) absorber, indocyanine green (ICG), can potentially provide an effective method to overcome these limitations. Herein, we theoretically investigate the effectiveness of particles derived from erythrocytes, which contain ICG, in mediating photothermal destruction of PWS blood vessels. We refer to these particles as NIR erythrocyte-derived transducers (NETs). Our theoretical model consists of a Monte Carlo algorithm to estimate the volumetric energy deposition, a finite elements approach to solve the heat diffusion equation, and a damage integral based on an Arrhenius relationship to quantify tissue damage. The model geometries include simulated PWS blood vessels as well as actual human PWS blood vessels plexus obtained by the optical coherence tomography. Our simulation results indicate that blood vessels containing micron- or nano-sized NETs and irradiated at 755 nm have higher levels of photothermal damage as compared to blood vessels without NETs irradiated at 585 nm. Blood vessels containing micron-sized NETs also showed higher photothermal damage than blood vessels containing nano-sized NETs. The theoretical model presented can be used in guiding the fabrication of NETs with patient-specific optical properties to allow for personalized treatment based on the depth and size of blood vessels as well as the pigmentation of the individual's skin.

Laser treatment for posttraumatic injury offers the clinician the unique opportunity for early intervention in mediating early scar formation, or for reducing the appearance of scars after maturation. In this review, the authors focus on the mechanisms by which lasers exert their therapeutic effects, highlighting several popular lasers and dosimetry used, and underscoring the power of combined surgical scar revision in managing posttraumatic facial scars.

Various methods are available for refining scars of the external nose and optimal scar revision frequently requires the utilization of multiple techniques. Differing anatomy of nasal subunits and their underlying structural framework limit surgical options in nasal scar revision compared with other areas of the face. An understanding of a variety of laser technologies and their specific applications can vastly aid in fine, controlled scar revision. Achieving optimal scar reduction regularly requires multiple stages of intervention, close follow-up, and repeat procedures.

Here, we review our current knowledge on the etiology and treatment of port-wine stain (PWS) birthmarks. Current treatment options have significant limitations in terms of efficacy. With the combination of 1) a suitable preclinical microvascular model, 2) laser speckle imaging (LSI) to evaluate blood-flow dynamics, and 3) a longitudinal experimental design, rapid preclinical assessment of new phototherapies can be translated from the lab to the clinic. The combination of photodynamic therapy (PDT) and pulsed-dye laser (PDL) irradiation achieves a synergistic effect that reduces the required radiant exposures of the individual phototherapies to achieve persistent vascular shutdown. PDL combined with anti-angiogenic agents is a promising strategy to achieve persistent vascular shutdown by preventing reformation and reperfusion of photocoagulated blood vessels. Integration of LSI into the clinical workflow may lead to surgical image guidance that maximizes acute photocoagulation, is expected to improve PWS therapeutic outcome. Continued integration of noninvasive optical imaging technologies and biochemical analysis collectively are expected to lead to more robust treatment strategies.

Background and objectivePort-wine stain (PWS) birthmarks affect ∼22 million people worldwide. After several treatment sessions, complete disappearance of the PWS occurs in only ∼10% of treated patients. There is a need to develop a new strategy to improve the efficacy of each treatment session and the overall treatment outcome. The study objective was to determine how intraoperative measurements of blood flow correlate with treatment response assessed several weeks post treatment.Study design/materials and methodsWe employed Laser Speckle Imaging (LSI) to measure intraoperative blood-flow dynamics. We collected data from 24 subjects undergoing laser therapy for facial PWS birthmarks. Photographs were taken before treatment and at a follow-up visit, and analyzed by two expert observers.ResultsIntraoperative LSI enables real-time monitoring of blood-flow dynamics in response to laser treatment and can inform clinicians on the need for focused re-treatment. The degree of PWS blanching achieved is positively correlated with the log-transformed acute blood-flow reduction (P = 0.022).ConclusionLSI is a simple, intraoperative monitoring tool during laser therapy of PWS birthmarks. LSI provides a single value for blood flow that correlates well with the degree of blanching achieved with laser therapy.

Background/objectivesSimilar to conventional cryogen spray cooling, carbon dioxide (CO2) spray may be used in combination with laser cartilage reshaping (LCR) to produce cartilage shape change while minimizing cutaneous thermal injury. Recent ex vivo evaluation of LCR with CO2 cooling in a rabbit model has identified a promising initial parameter space for in vivo safety and efficacy evaluation. This pilot study aimed to evaluate shape change and cutaneous injury following LCR with CO2 cooling in 5 live rabbits.Study design/materials and methodsThe midportion of live rabbit ears were irradiated with a 1.45 µm wavelength diode laser (12 J/cm(2)) with simultaneous CO2 spray cooling (85 millisecond duration, 4 alternating heating/cooling cycles per site, 5 to 6 irradiation sites per row for 3 rows per ear). Experimental and control ears (no LCR) were splinted in the flexed position for 30 days following exposure. A total of 5 ears each were allocated to the experimental and control groups.ResultsShape change was observed in all irradiated ears (mean 70 ± 3°), which was statistically different from control (mean 37 ± 11°, P = 0.009). No significant thermal cutaneous injury was observed, with preservation of the full thickness of skin, microvasculature, and adnexal structures. Confocal microscopy and histology demonstrated an intact and viable chondrocyte population surrounding irradiated sites.ConclusionsLCR with CO2 spray cooling can produce clinically significant shape change in the rabbit auricle while minimizing thermal cutaneous and cartilaginous injury and frostbite. This pilot study lends support for the potential use of CO2 spray as an adjunct to existing thermal-based cartilage reshaping modalities. An in vivo systematic evaluation of optimal laser dosimetry and cooling parameters is required.

We present an uncommon case of nasal alar and facial necrosis following calcium hydroxylapatite filler injection performed elsewhere without direct physician supervision. The patient developed severe full-thickness necrosis of cheek and nasal alar skin 24 h after injections into the melolabial folds. Management prior to referral included oral antibiotics, prednisone taper, and referral to a dermatologist (day 3) who prescribed valacyclovir for a presumptive herpes zoster reactivation induced by the injection. Referral to our institution was made on day 11, and after herpetic outbreak was ruled out by a negative Tzanck smear, debridement with aggressive local wound care was initiated. After re-epithelialization and the fashioning of a custom intranasal stent to prevent vestibular stenosis, pulsed dye laser therapy was performed for wound modification. The patient healed with an acceptable cosmetic outcome. This report underscores the importance of facial vasculature anatomy, injection techniques, and identification of adverse events when using fillers. A current treatment paradigm for such events is also presented.

Background and objectivesPort-wine stain (PWS) is a congenital, progressive vascular malformation of the dermis. The use of optical coherence tomography (OCT) for the characterization of blood vessels in PWS skin has been demonstrated by several groups. In the past few years, advances in OCT technology have greatly increased imaging speed. Sophisticated numerical algorithms have improved the sensitivity of Doppler OCT dramatically. These improvements have enabled the noninvasive, high-resolution, three-dimensional functional imaging of PWS skin. Here, we demonstrate high-resolution, three-dimensional, microvasculature imaging of PWS and normal skin using Doppler OCT technique.Study design/materials and methodsThe OCT system uses a swept source laser which has a central wavelength of 1,310 nm, an A-line rate of 50 kHz and a total average power of 16 mW. The system uses a handheld imaging probe and has an axial resolution of 9.3 µm in air and a lateral resolution of approximately 15 µm. Images were acquired from PWS subjects at the Beckman Laser Institute and Medical Clinic. Microvasculature of the PWS skin and normal skin were obtained from the PWS subject.ResultsHigh-resolution, three-dimensional microvasculature of PWS and normal skin were obtained. Many enlarged PWS vessels are detected in the dermis down to 1.0 mm below the PWS skin surface. In one subject, the blood vessel diameters range from 40 to 90 µm at the epidermal-dermal junction and increase up to 300-500 µm at deeper regions 700-1,000 µm below skin surface. The blood vessels close to the epidermal-dermal junction are more uniform, in terms of diameter. The more tortuous and dilated PWS blood vessels are located at deeper regions 600-1,000 µm below the skin surface. In another subject example, the PWS skin blood vessels are dilated at very superficial layers at a depth less than 500 µm below the skin surface. The PWS skin vessel diameters range from 60 to 650 µm, with most vessels having a diameter of around 200 µm.ConclusionsOCT can be used to quantitatively image in vivo skin micro-vasculature. Analysis of the PWS and normal skin blood vessels were performed and the results can provide quantitative information to optimize laser treatment on an individual patient basis.

BackgroundPresently the heart rate is monitored in the Neonatal Intensive Care Unit with contact sensors: electrocardiogram or pulse oximetry. These techniques can cause injuries and infections, particularly in very premature infants with fragile skin. Camera based plethysmography was recently demonstrated in adults as a contactless method to determine heart rate.AimTo investigate the feasibility of this technique for NICU patients and identify challenging conditions.Study design and participantsVideo recordings using only ambient light were made of 19 infants at two NICUs in California and The Netherlands. Heart rate can be derived from these recordings because each cardiovascular pulse wave induces minute pulsatile skin color changes, invisible to the eye but measurable with a camera.ResultsIn all infants the heart beat induced photoplethysmographic signal was strong enough to be measured. Low ambient light level and infant motion prevented successful measurement from time to time.ConclusionsContactless heart rate monitoring by means of a camera using ambient light was demonstrated for the first time in the NICU population and appears feasible. Better hardware and improved algorithms are required to increase robustness.

Laser cartilage reshaping (LCR) with cryogen spray cooling is a promising modality for producing cartilage shape change while reducing cutaneous thermal injury. However, LCR in thicker tissues, such as auricular cartilage, requires higher laser power, thus increasing cooling requirements. To eliminate the risks of freeze injury characteristic of high cryogen spray pulse rates, a carbon dioxide (CO2) spray, which evaporates rapidly from the skin, has been proposed as the cooling medium. This study aims to identify parameter sets which produce clinically significant reshaping while producing minimal skin thermal injury in LCR with CO2 spray cooling in ex vivo rabbit auricular cartilage. Excised whole rabbit ears were mechanically deformed around a cylindrical jig and irradiated with a 1.45-μm wavelength diode laser (fluence 12-14 J/cm(2) per pulse, four to six pulse cycles per irradiation site, five to six irradiation sites per row for four rows on each sample) with concomitant application of CO2 spray (pulse duration 33-85 ms) to the skin surface. Bend angle measurements were performed before and after irradiation, and the change quantified. Surface temperature distributions were measured during irradiation/cooling. Maximum skin surface temperature ranged between 49.0 to 97.6 °C following four heating/cooling cycles. Significant reshaping was achieved with all laser dosimetry values with a 50-70 °C difference noted between controls (no cooling) and irradiated ears. Increasing cooling pulse duration yielded progressively improved gross skin protection during irradiation. CO2 spray cooling may potentially serve as an alternative to traditional cryogen spray cooling in LCR and may be the preferred cooling medium for thicker tissues. Future studies evaluating preclinical efficacy in an in vivo rabbit model are in progress.

Although low-level laser therapy (LLLT) has been a valuable therapeutic technology in the clinic, its efficacy may be reduced in deep tissue layers due to strong light scattering which limits the photon density. In order to enhance the photon density in deep tissue layers, this study developed an optical tissue clearing (OTC) laser probe (OTCLP) system which can utilize four different OTC methods: 1) tissue temperature control from 40 to 10°C; 2) laser pulse frequency from 5 to 30 Hz; 3) glycerol injection at a local region; and 4) a combination of the aforementioned three methods. The efficacy of the OTC methods was evaluated and compared by investigating laser beam profiles in ex-vivo porcine skin samples. Results demonstrated that total (peak) intensity at full width at half maximum of laser beam profile when compared to control data was increased: 1) 1.21(1.39)-fold at 10°C; 2) 1.22 (1.49)-fold at a laser pulse frequency of 5 Hz; 3) 1.64 (2.41)-fold with 95% glycerol injection; 4) 1.86 (3.4)-fold with the combination method. In conclusion, the OTCLP system successfully improved the laser photon density in deep tissue layers and may be utilized as a useful tool in LLLT by increasing laser photon density.

Background and objectivesPort-wine stains (PWSs) are capillary vascular malformations that are commonly resistant to treatment. Currently, the pulsed dye laser (PDL) is the treatment of choice. Multiple treatments are required and complete blanching after laser irradiation is rarely achieved. We review current therapeutic modalities for PWSs and recent developments for enhanced clearance.Study design/materials and methodsRelevant literature was reviewed including PDL modifications for improved efficacy, alternative laser devices for treatment-resistant PWSs, and the addition of agents to modulate the wound-healing response after laser irradiation.ResultsAlthough PDL is the treatment of choice for PWSs, increased understanding of interactions between PWSs and PDL has led to improvements in therapeutic outcome in terms of lesion blanching.ConclusionsPreliminary evidence of combination therapy using antiangiogenic agents after laser irradiation appears promising and could lead to the development of a new standard of care for PWSs.

Background and objectiveObjective methods to assess port wine stain (PWS) response to laser treatment have been the subject of various research efforts for several years. Herein, we present a pilot study using a newly developed, light emitting diode (LED) based spatial frequency domain imaging (SFDI) device to record quantitatively biochemical compositional changes in PWS after laser therapy.Study design/patients and methodsA SFDI system was used to image before, and after, five PWS treatment sessions [n = 4 subjects (one subject was imaged before and after two consecutive laser treatments)]. SFDI derived wide-field optical properties (absorption and scattering) and tissue chromophore concentrations including oxy-hemoglobin (ctO(2) Hb), deoxy-hemoglobin (ctHHb), total hemoglobin (ctTHb), and tissue oxygen saturation (stO(2) ) are presented for skin imaged prior to and immediately after laser treatment. The SFDI derived images were analyzed by comparing the above measurements in PWS to those of normal skin and tracking changes immediately after laser exposure.ResultsElevated oxy-hemoglobin (>20%) and tissue oxygen saturation (>5%) were measured in all PWS lesions and compared to values for normal skin prior to treatment. Laser treatment resulted in an increase in deoxy-hemoglobin (>100%), decrease in tissue oxygen saturation (>10%), and reduced scattering (>15%) in all PWS lesions. One subject was followed before and after two consecutive laser treatments and the overall improvement in PWS lesion blanching was quantitatively assessed by measuring a 45% decrease in dermal blood volume.ConclusionSFDI is a rapid non-contact wide-field optical technique that shows potential as an imaging device that can be used to quantify biochemical compositional changes in PWS after laser therapy. Future work will investigate the potential of SFDI to provide intra-operative guidance for laser therapy of PWS lesions on an individual patient basis.

During the last three decades, several laser systems, ancillary technologies, and treatment modalities have been developed for the treatment of port wine stains (PWSs). However, approximately half of the PWS patient population responds suboptimally to laser treatment. Consequently, novel treatment modalities and therapeutic techniques/strategies are required to improve PWS treatment efficacy. This overview therefore focuses on three distinct experimental approaches for the optimization of PWS laser treatment. The approaches are addressed from the perspective of mechanical engineering (the use of local hypobaric pressure to induce vasodilation in the laser-irradiated dermal microcirculation), optical engineering (laser-speckle imaging of post-treatment flow in laser-treated PWS skin), and biochemical engineering (light- and heat-activatable liposomal drug delivery systems to enhance the extent of post-irradiation vascular occlusion).

Background/purposeConventional treatment methods for acne vulgaris have various side effects such as the development of bacterial resistance, phototoxicity, vertigo, gastro-intestinal problems, and drug eruptions. To minimize such side effects, light and thermal methods have been alternately suggested. This study characterized a new acne vulgaris treatment device (AVTD) that combines both light and thermal methods and evaluated its clinical efficacy.MethodsWe characterized the thermal and light properties of the AVTD itself and evaluated its thermal characteristics in ex vivo porcine skin samples. The Arrhenius equation was used to calculate the skin thermal injury coefficient to confirm the skin safety of the AVTD. Finally, the clinical efficacy of the AVDT was evaluated by analyzing cross-polarization and erythema index images, which were obtained from 13 volunteers undergoing treatment with the AVTD.ResultsThe temperature of the AVTD itself was maintained at 49.1 °C on the tip and 39.7 °C in the porcine skin samples. The peak intensity of the light-emitting diode (LED) light was observed at 468 nm. The skin safety of the AVTD was confirmed and 84.2% of the volunteers presented positive treatment results.ConclusionThe treatment of acne using the AVTD resulted in a high treatment rate in a clinical study, minimizing side effects. On the basis of these results, we can be sure that the AVTD may be effectively used for the treatment of acne vulgaris.

Background/objectivesCurrent laser therapy of port wine stain (PWS) birthmarks with a single laser pulse (SLP) does not produce complete lesion removal in the majority of patients. To improve PWS therapeutic efficacy, we evaluated the performance of an approach based on multiple laser pulses (MLP) to enhance blood vessel photocoagulation.Study designThe hamster dorsal window chamber model was used. Radiant exposure (RE), pulse repetition rate (f(r)), total number of pulses (n(p)), and length of vessel irradiated were varied. Blood vessels in the window were irradiated with either SLP with RE of 4-7 J/cm(2) or MLP with RE per pulse of 1.4-5.0 J/cm(2), f(r) of 0.5-26.0 Hz, and n(p) of 2-5. The laser wavelength was 532 nm and pulse duration was 1 ms. Either a 2 mm vessel segment or entire vessel branch was irradiated. Digital photographs and laser speckle images of the window were recorded before and at specific time points after laser irradiation to monitor laser-induced blood vessel structural and functional changes, respectively.ResultsWe found that: (1) for a SLP approach, the RE required to induce blood vessel photocoagulation was 7 J/cm(2) as compared to only 2 J/cm(2) per pulse for the MLP approach; (2) for MLP, two pulses at a repetition rate of 5 Hz and a RE of 3 J/cm(2) can induce photocoagulation of more than 80% of irradiated blood vessel; and (3) irradiation of a longer segment of blood vessel resulted in lower reperfusion rate.ConclusionsThe MLP approach can induce blood vessel photocoagulation at much lower RE per pulse as compared to SLP. The 5 Hz f(r) and the need for two pulses are achievable with modern laser technology, which makes the MLP approach practical in the clinical management of PWS birthmarks.

This article is a clinically practical review structured around the specific applications of laser technologies used in acute management of soft tissue injuries in surgical incisions and trauma. Surgical and traumatic incisions and injuries provide the clinician with the unique opportunity to follow the progression and maturation of the wound healing response from a very early stage. There has been a recent interest in early cosmetic optimization of surgical and traumatic wounds on the face using optical technologies. Early clinical results for acute laser intervention starting immediately after suture removal or the first several weeks after repair have been very promising.

Background/purposeDigital color image analysis is currently considered as a routine procedure in dermatology. In our previous study, a multimodal facial color imaging modality (MFCIM), which provides a conventional, parallel- and cross-polarization, and a fluorescent color image, was introduced for objective evaluation of various facial skin lesions. This study introduces a commercial version of MFCIM, DermaVision-PRO, for routine clinical use in dermatology and demonstrates its dermatological feasibility for cross-evaluation of skin lesions.Methods/resultsSample images of subjects with actinic keratosis or non-melanoma skin cancers were obtained at four different imaging modes. Various image analysis methods were applied to cross-evaluate the skin lesion and, finally, to extract valuable diagnostic information. DermaVision-PRO is potentially a useful tool as an objective macroscopic imaging modality for quick prescreening and cross-evaluation of facial skin lesions.ConclusionDermaVision-PRO may be utilized as a useful tool for the cross-evaluation of widely distributed facial skin lesions and as an efficient database management of patient information.

Background and objectiveDespite application of cryogen spray (CS) precooling, customary treatment of port wine stain (PWS) birthmarks with a single laser pulse does not result in complete lesion blanching for a majority of patients. One obvious reason is nonselective absorption by epidermal melanin, which limits the maximal safe radiant exposure. Another possible reason for treatment failure is screening of laser light within large PWS vessels, which prevents uniform heating of the entire vessel lumen. Our aim is to identify the parameters of sequential CS cooling and laser irradiation that will allow optimal photocoagulation of various PWS blood vessels with minimal risk of epidermal thermal damage.Study design and methodsLight and heat transport in laser treatment of PWS are simulated using a custom 3D Monte Carlo model and 2D finite element method, respectively. Protein denaturation in blood and skin are calculated using the Arrhenius kinetic model with tissue-specific coefficients. Simulated PWS vessels with diameters of 30-150 µm are located at depths of 200-600 µm, and shading by nearby vessels is accounted for according to PWS histology data from the literature. For moderately pigmented and dark skin phototypes, PWS blood vessel coagulation and epidermal thermal damage are assessed for various parameters of sequential CS cooling and 532-nm laser irradiation, i.e. the number of pulses in a sequence (1-5), repetition rate (7-30 Hz), and radiant exposure.ResultsSimulations of PWS treatment in darker skin phototypes indicate specific cooling/irradiation sequences that provide significantly higher efficacy and safety as compared to the customary single-pulse approach across a wide range of PWS blood vessel diameters and depths. The optimal sequences involve three to five laser pulses at repetition rates of 10-15 Hz.ConclusionsApplication of the identified cooling/irradiation sequences may offer improved therapeutic outcome for patients with resistant PWS, especially in darker skin phototypes.

Various physico-chemical tissue optical clearing (TOC) methods have been suggested to maximize photon density in tissue. In order to enhance photon density, a compression-controlled low-level laser probe (CCLLP) system was developed by utilizing the principle of mechanical tissue compression. Negative compression (NC) was applied to the laser probes built in various diameters and simultaneously the laser was irradiated into ex-vivo porcine skin samples. Laser photon density (LPD) was evaluated as a function of NC and probe diameter by analyzing 2D diffusion images of the laser exposures. The CCLLP system resulted in a concentrated laser beam profile, which means enhancement of the LPD. As indicators of LPD, the laser peak intensity increased and the full width at half maximum (FWHM) decreased as a function of NC. The peak intensity at –30 kPa increased 2.74, 3.22, and 3.64 fold at laser probe diameters of 20, 30, and 40 mm, respectively. In addition, sample temperature was measured with a thermal camera and increased 0.4 K at –30 kPa after 60 s of laser irradiation as a result of enhanced LPD. The CCLLP system effectively demonstrated enhancement of the LPD in tissue and potentially its clinical feasibility.

Background and objectivesVarious physical methods, such as microneedling, laser ablation, sonophoresis, and sandpaper, have been widely studied to enhance the transdermal delivery of tissue optical clearing (TOC) agents. A previous study demonstrated that the microneedling method could effectively enhance the permeability of a TOC agent through the skin barrier.Study design/materials and methodsIn this study, we introduce a new physical combination method which utilizes both microneedling and sonophoresis to further enhance the transdermal delivery of a TOC agent, glycerol. Porcine skin samples were divided into a control group treated only with the microneedle roller and a test group treated with both the microneedle roller and sonophoresis. Glycerol was applied topically after microneedling. The optimal concentration and transdermal delivery efficacy of glycerol were quantitatively evaluated.ResultsA 70% glycerol solution was determined to be the optimal concentration for the combination method. The combination method resulted in approximately a 2.3-fold higher transdermal diffusion rate of glycerol when compared to the microneedling method alone.ConclusionThe combination method and optimal glycerol concentration effectively enhanced transdermal delivery of glycerol by accelerating the diffusion rate through the skin barrier.

Recent advances in the understanding of the metastatic phenomenon in cancer have led to the description of a metastatic niche. This concept describes a site prepared for the tumor cells in areas frequently associated with metastasis for the individual tumor studied. This niche is a "soil" that allows for the tumor cell or "seed" to lodge and grow. Certain aspects of the biology of infantile hemangioma cells suggest a relationship to the placenta as a possible site of origin for the hemangioma precursor cells. In this article, a relationship between the placenta, with or without a chorangioma and the hemangioma sites of localization, is hypothesized. The placenta is suggested as the site of humoral factors that prepare a niche similar to the function of malignant tumor cells. If the hypothesis proves to be valid, clues for possible treatment are outlined.

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.

Background and objectivesComplete blanching of port wine stain (PWS) birthmarks after laser therapy is rarely achieved for most patients. We postulate that the low therapeutic efficacy or treatment failure is caused by regeneration and revascularization of photocoagulated blood vessels due to angiogenesis associated with the skin's normal wound healing response. Rapamycin (RPM), an antiangiogenic agent, has been demonstrated to inhibit growth of pathological blood vessels. Our objectives were to (1) investigate whether topical RPM can inhibit reperfusion of photocoagulated blood vessels in an animal model and (2) determine the effective RPM concentration required to achieve this objective.Study design/materials and methodsFor both laser-only and combined laser and RPM treated animals, blood vessels in the dorsal window chambers implanted on golden Syrian hamsters were photocoagulated with laser pulses. Structural and flow dynamics of blood vessels were documented with color digital photography and laser speckle imaging to evaluate photocoagulation and reperfusion. For the combined treatment group, topical RPM was applied to the epidermal side of the window daily for 14 days after laser exposure.ResultsIn the laser-only group, 23 out of 24 photocoagulated blood vessels reperfused within 5-14 days. In the combined treatment group with different RPM formulae and concentrations, the overall reperfusion rate of 36% was much lower as compared to the laser-only group. We also found that the reperfusion rate was not linearly proportional to the RPM concentration.ConclusionsWith topical RPM application, the frequency of vessel reperfusion was considerably reduced, which implies that combined light and topical antiangiogenic therapy might be a promising approach to improve the treatment efficacy of PWS birthmarks.

Background and objectiveDuring laser therapy of port wine stain (PWS) birthmarks, regions of perfusion may persist. We hypothesize that such regions are not readily observable even when laser surgery is performed by highly experienced clinicians. The objective of this study was to use objective feedback to assess the acute vascular response to laser therapy.Study design/materials and methodsA clinic-friendly laser speckle imaging (LSI) instrument was developed to provide the clinician with real-time images of blood flow during laser therapy. Images were acquired from patients undergoing laser therapy of PWS birthmarks at Scripps Clinic and the Beckman Laser Institute and Medical Clinic. Blood flow maps were extracted from the acquired imaging data. Histogram-based analysis was applied in grading the degree of heterogeneity present in the blood flow maps after laser therapy.ResultsCollectively, two types of patient responses were observed in response to laser exposure: (1) an immediate increase in perfusion within minutes after laser therapy; and (2) an overall decrease in blood perfusion approximately 1 hour after laser therapy, with distinct regions of persistent perfusion apparent in the majority of post-treatment blood-flow images. A comparison of blood flow in PWS and adjacent normal skin demonstrated that PWS blood flow can be greater than, or sometimes equivalent to, that of normal skin.ConclusionIn general, a decrease in skin perfusion is observed during pulsed laser therapy of PWS birthmarks. However, a heterogeneous perfusion map was frequently observed. These regions of persistent perfusion may be due to incomplete photocoagulation of the targeted vessels. We hypothesize that immediate retreatment of these regions identified with LSI, will result in enhanced removal of the PWS vasculature. Lasers Surg. Med. 41:563-571, 2009. (c) 2009 Wiley-Liss, Inc.

BackgroundAblative fractional carbon dioxide (CO(2)) laser treatments have gained popularity due to their efficacy, shortened downtime, and decreased potential for scarring in comparison to traditional ablative CO(2) resurfacing. To date, scarring with fractional CO(2) lasers has not been reported.ObjectiveFive patients treated with the same fractional CO(2) laser technology for photodamage of the neck were referred to our practices 1-3 months after treatment. Each patient developed scarring. Of the five cases, two are discussed in detail. The first was treated under general anesthesia on the face and anterior neck at a pulse energy of 30 mJ (859 microm depth) with 25% coverage. Eleven days after treatment, three non-healing areas along the horizontal skin folds of the anterior neck were noted. At 2 weeks after CO(2) ablative fractional resurfacing, these areas had become thickened. These raised areas were treated with a non-ablative fractionated 1,550 nm laser to modify the wound healing milieu. One week later, distinct firm pale papules in linear arrays with mild hypopigmentation had developed along involved neck skin folds. Skin biopsy was performed. For the second patient, the neck was treated at a pulse energy of 20 mJ (630 microm depth) with 30% coverage of the exposed skin, with a total treatment energy of 5.0 kJ. Minimal crusting was noted on the neck throughout the initial healing phase of 2 weeks. She then experienced tightness on her neck. Approximately 3 weeks after treatment, she developed multiple vertical and horizontal hypertrophic scars (HS).ResultsHistopathology for the first case confirmed the presence of a hypertrophic scar. The papules in this case completely resolved with mild residual hypopigmentation after treatment with topical corticosteroids. HS failed to resolve in the second case to date after 1 month.ConclusionAs with traditional ablative CO(2) laser resurfacing, HS is a potential complication of ablative fractional CO(2) laser resurfacing, particularly on the neck. With early diagnosis and appropriate treatment HS of neck skin may be reversible. We urge caution when treating the neck with this device and close attention to wound care in the post-operative period.