Ultraviolet A irradiation induces ultraweak photon emission with characteristic spectral patterns from biomolecules present in human skin.

Oxidative stress is associated with photoaging of the skin as well as with skin cancer, and is therefore, critical to monitor. Ultraweak photon emission (UPE) is extremely weak light generated during the oxidative process in the living body and has been used as a non-invasive and label-free marker for the evaluation of oxidative stress. However, the mechanism of UPE generation is not clear. Therefore, we aimed to elucidate the molecular mechanism underlying UPE generation by analyzing the spectra of UPE generated from biomolecules in the skin during ultraviolet A (UVA) exposure. The spectra of UVA-induced UPE generated from linoleic acid, linolenic acid, elastin, phospholipids, and 5,6-dihydroxyindole-2-carboxylic acid were measured, and the spectrum of human skin tissue was also obtained. The spectral patterns varied for the different biomolecules and the peaks were distinct from those of the skin tissue. These results suggested that the UPE generated from skin tissue is a collection of light emitted by biomolecules. Moreover, we proposed that UPE is generated through a photosensitization reaction and energy transfer. The identified characteristic spectral patterns of UPE can be useful to elucidate UVA-induced oxidative stress in the skin, with implications for prevention and treatment of photoaging and skin diseases.

Histologic and Clinical Changes in Vulvovaginal Tissue After Treatment With a Transcutaneous Temperature-Controlled Radiofrequency Device.

BACKGROUND: Although transcutaneous temperature-controlled radiofrequency (TTCRF) may effectively treat vulvovaginal laxity (VVL), atrophic vaginitis (AV), orgasmic dysfunction (OD), and stress urinary incontinence (SUI), there is a lack of histopathologic evidence to validate its use. OBJECTIVE: Evaluate clinical and histological changes induced by vulvovaginal TTCRF. MATERIALS AND METHODS: This was a prospective, nonrandomized trial. Ten female subjects with mild-to-moderate VVL, with or without AV, OD, and/or SUI underwent 3 TTCRFs at 4-week intervals. Five subjects underwent pre- and post-treatment biopsies of the labia majora and vaginal canal for histology. Assessments were performed at baseline and Days 10, 30, 60, and 120. RESULTS: Investigator-rated VVL improved significantly from baseline to Day 10, with improvement maintained through Day 120 (p = .001 and .001, respectively). Sexual satisfaction improved significantly by Day 60 (p = .001). Improvement in AV reached significance at Day 120 (p = .048). Although OD and SUI improved steadily, the difference in improvement did not reach statistical significance. Histology revealed that post-treatment increases in collagen, elastin, vascularity, and small nerve fibers. CONCLUSION: Transcutaneous temperature-controlled RF resulted in significant improvements in AV, VVL, and sexual satisfaction with milder improvements in OD and SUI. Post-treatment histology demonstrated neocollagenesis, neoelastogenesis, neoangiogenesis, and the first reported finding of TTCRF-related neurogenesis.

Treatment of pseudoxanthoma elasticum-like papillary dermal elastolysis with nonablative fractional resurfacing laser resulting in clinical and histologic improvement in elastin and collagen.

OBJECTIVE: Pseudoxanthoma elasticum-like papillary dermal elastolysis (PXE-PDE) is a rare skin condition seen predominantly in elderly females. These asymptomatic lesions are brought to the dermatologist's attention due to patient's displeasure in their appearance. We report a case of a 28-year-old female with PXE-PDE on the right neck treated with nonablative fractional resurfacing (NAFR). CASE: The patient on examination had yellow-to-skin-colored papules that coalesced into a cobblestone-appearing plaque on her lateral neck. The area was treated with NAFR to improve texture and cosmesis. After three treatments, the patient noted at least a 50% improvement of appearance and texture in the affected skin. Adverse effects were mild without development of serious side effects, such as scarring and/or permanent dyspigmenation. CONCLUSION: Established therapies for elastin disorders like PXE-PDE have yielded unsatisfactory results by patient and clinician standards. Positive results with NAFR, illustrated in our PXE-PDE case, further provides a role for NAFR in elastin disorders.

Fluorescence spectroscopy in the visible range for the assessment of UVB radiation effects in hairless mice skin.

Ultraviolet (UV) radiation may induce skin alterations as observed in photoaging. Some recognized modifications are epidermal hyperplasia, amorphous deposition of degraded elastic fibers and reduction in the number of collagen fibers. They alter the tissue biochemical properties that can be interrogated by steady state fluorescence spectroscopy (SSFS). In this study, we monitored the changes in endogenous fluorescence emission from hairless mice skin during a protocol of photoaging using UVB irradiation. To perform the fluorescence spectroscopy, it was used a violet laser (408nm) to induce the native fluorescence that is emitted in the visible range. Under 408nm excitation, the emission spectrum showed bands with peaks centered around 510, 633 and 668nm for irradiated and control groups. A relative increase of the fluorescence at 633nm emission on the flank was observed with time when compared to the ventral skin at the same animal and the non-irradiated control group. We correlated the emission at 633nm with protoporphyrin IX (PpIX), and our hypothesis is that the PpIX metabolism in the photoaged and aged skin are different. PpIX fluorescence intensity in the photoaged skin is higher and more heterogeneous than in the aged skin. Notwithstanding, more spectroscopic and biochemistry studies investigating the 510 and 633nm emission are needed to confirm this hypothesis.

Molecular Mechanisms of Stress-Responsive Changes in Collagen and Elastin Networks in Skin.

Collagen and elastin networks make up the majority of the extracellular matrix in many organs, such as the skin. The mechanisms which are involved in the maintenance of homeostatic equilibrium of these networks are numerous, involving the regulation of genetic expression, growth factor secretion, signalling pathways, secondary messaging systems, and ion channel activity. However, many factors are capable of disrupting these pathways, which leads to an imbalance of homeostatic equilibrium. Ultimately, this leads to changes in the physical nature of skin, both functionally and cosmetically. Although various factors have been identified, including carcinogenesis, ultraviolet exposure, and mechanical stretching of skin, it was discovered that many of them affect similar components of regulatory pathways, such as fibroblasts, lysyl oxidase, and fibronectin. Additionally, it was discovered that the various regulatory pathways intersect with each other at various stages instead of working independently of each other. This review paper proposes a model which elucidates how these molecular pathways intersect with one another, and how various internal and external factors can disrupt these pathways, ultimately leading to a disruption in collagen and elastin networks.

Flash photolysis and pulse radiolysis studies on elastin hydrolysates.

The formation of reactive species and free radicals in water soluble elastin hydrolysates have been investigated by pulse radiolysis and flash photolysis. Elastin hydrolysates were obtained by hydrolysis of elastin extracted from aorta. An investigation of the photochemical properties of elastin hydrolysates in water was carried out using nanosecond laser irradiation. The transient spectra of elastin hydrolysates solution excited at 266 nm showed two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The reactions of hydrated electrons and ˙OH radicals with elastin have been studied by pulse radiolysis. In the absorption spectra of products resulting from the reaction of elastin with e(aq)(-) small maximum absorption in UV and visible light was observed. In the absorption spectra of products resulting from the reaction of the hydroxyl radicals with elastin two bands were observed. The first one at 320 nm and the second one at 410 nm. Reaction of OH radicals with elastin hydrolysates lead to formation of Tyr phenoxyl radicals with absorption at 410 nm. The influence of the addition of sodium azide NaN3 on the formation of the transients was evaluated.

Long-term evaluation of collagen and elastin following infrared (1100 to 1800 nm) irradiation.

BACKGROUND: Infrared irradiation stimulates collagen production, but histological differences in its long-term effects on type I versus type III collagen and elastin in human tissue are unclear. OBJECTIVE: To investigate the effects of infrared irradiation. METHODS AND MATERIALS: In vivo human tissues in sun-protected and sun-exposed areas were irradiated with infrared. Histological samples were analyzed, and visual changes were assessed up to 90 days post-treatment. RESULTS: Infrared irradiation provided long-term increases in collagen and elastin levels on post-irradiation days 30, 60 and 90 compared to controls. Significant increases in type I collagen persisted until 30 and 60 days, and in sun-protected and exposed skin biopsies, respectively. Significant increases in type III collagen and elastin persisted until 90 days in both sun-protected and sun-exposed skin biopsies. CONCLUSION: Infrared irradiation provides safe and effective long-term stimulation of collagen I and III and elastin, which is beneficial for improving skin laxity and wrinkles.

Bipolar fractional radiofrequency treatment induces neoelastogenesis and neocollagenesis.

BACKGROUND: We recently introduced Renesis, a novel minimally invasive radiofrequency (RF) device, for the treatment of human skin. The wound healing response post-fractional RF (FRF) treatment was examined in human subjects. STUDY DESIGN: The FRF system delivered RF energy directly within the dermis via 5 micro-needle electrode pairs. Tissue temperature was held at 72 degrees C for 4 seconds using an intelligent feedback system. The wound healing response was evaluated histologically and by RT-PCR up to 10 weeks post-RF treatment. Neoelastogenesis and the role of heat shock proteins (HSPs) were assessed by immunohistochemistry. RESULTS: FRF treatment generated a RF thermal zone (RFTZ) pattern in the reticular dermis that consisted of zones of denatured collagen separated by zones of spared dermis. RFTZs were observed through day 28 post-treatment but were replaced by new dermal tissue by 10 weeks. HSP72 expression rapidly diminished after day 2 while HSP47 expression increased progressively through 10 weeks. Reticular dermal volume, cellularity, hyaluronic acid, and elastin content increased. RT-PCR studies revealed an immediate increase in IL-1beta, TNF-alpha, and MMP-13 while MMP-1, HSP72, HSP47, and TGF-beta levels increased by 2 days. We also observed a marked induction of tropoelastin, fibrillin, as well as procollagens 1 and 3 by 28 days post-treatment. CONCLUSION: Our study revealed a vigorous wound healing response is initiated post-treatment, with progressive increase in inflammatory cell infiltration from day 2 through 10 weeks. An active dermal remodeling process driven by the collagen chaperone HSP47 led to complete replacement of RFTZs with new collagen by 10 weeks post-treatment. Furthermore, using both immunohistochemical and PCR studies, we successfully demonstrated for the first time evidence of profound neoelastogenesis following RF treatment of human skin. The combination of neoelastogenesis and neocollagenesis induced by treatment with the FRF system may provide a reliable treatment option for skin laxity and/or rhytids.

Mechanistic effects of long-term ultraviolet B irradiation induce epidermal and dermal changes in human skin xenografts.

UVB irradiation has been reported to induce photoaging and suppress systemic immune function that could lead to photocarcinogenesis. However, because of the paucity of an UVB-induced photodamaged skin model, precise and temporal mechanism(s) underlying the deleterious effects of long-term UVB exposure on human skin have yet to be delineated. In this study, we established a model using human skin xenografted onto severe combined immunodeficient mice, which were subsequently challenged by repeated UVB irradiation for 6 weeks. Three-dimensional optical image analysis of skin replicas and noninvasive biophysical measurements illustrated a significant increase in skin surface roughness, similar to premature photoaging, and a significant loss of skin elasticity after long-term UVB exposure. Resembling authentically aged skin, UVB-exposed samples exhibited significant increases in epithelial keratins (K6, K16, K17), elastins, and matrix metalloproteinases (MMP-1, MMP-9, MMP-12) as well as degradation of collagens (I, IV, VII). The UVB-induced deterioration of fibrous keratin intermediate filaments was also observed in the stratum corneum. Additionally, similarities in gene expression patterns between our model and chronologically aged skin substantiated the plausible relationship between photodamage and chronological age. Furthermore, severe skin photodamage was observed when neutralizing antibodies against TIMP-1, an endogenous inhibitor of MMPs, were administered during the UVB exposure regimen. Taken together, these findings suggest that our skin xenograft model recapitulates premature photoaged skin and provides a comprehensive tool with which to assess the deleterious effects of UVB irradiation.

Histological and clinical studies on the effects of low to medium level infrared light therapy on human and mouse skin.

BACKGROUND AND OBJECTIVE: Deep heating or denaturation of collagen has been reported to be necessary for nonablative skin rejuvenation. The purpose of this study was to examine whether thermally damaged collagen is an indispensable factor to increase the amount of collagen in vivo. Epidermal and dermal responses to infrared light therapy using a Titan source were examined with the aim of correlating histological and clinical responses in human and amelanotic mouse skin. STUDY DESIGN/MATERIALS AND METHODS: Ten, 20, or 30 J/cm2 infrared light were irradiated on the human subject's skin (thigh), while 5, 10, 20, or 30 J/cm2 were used on amelanotic mouse skin. Biopsies were taken and analyzed using hematoxylin and eosin (H&E) and Elastica von Gieson stain. RESULTS: Ten or 20 J/cm2 infrared light increased the amount of both collagen and elastin in all layers of the dermis without denaturing the collagen in human skin. A higher dose of 30 J/cm2 also increased the amount of collagen and elastin, but denatured the collagen in human skin. (In addition to the thigh, 2 treatments of 10 J/cm2 infrared light improved skin toning and texture on the subject's face). In mouse skin, 5 or 10 J/cm2 remarkably increased the amount of both collagen and elastin, and of epidermal cells. Twenty or 30 J/cm increased the amount of collagen and elastin and the number of keratinocytes, but caused some vacuolated degeneration of keratinocytes. The presence of denatured collagen was not evident due to the high density of collagen. CONCLUSIONS: This study shows that the denaturation of collagen is not required to increase the amounts of collagen or elastin in vivo in human skin. The activation of the mitochondria as well as the denaturation of collagen may play important roles in infrared phototherapy.

Spectroscopic studies into the influence of UV radiation on elastin in the presence of collagen.

An investigation into the influence of UV irradiation on elastin hydrolysates in the presence of collagen was carried out using UV-Vis spectroscopy and spectrofluorometry. It was found that the absorbance of elastin hydrolysates in solution increased during irradiation more than the absorbance of the elastin/collagen blend. The fluorescence of elastin hydrolysates was observed at 305nm and at 380nm after excitation at 270nm. For the elastin/collagen mixture in solution, fluorescence spectrum shows only one maximum at 305nm. UV irradiation caused fluorescence fading at 305nm. For irradiated elastin the fluorescence at 305nm decreased faster than for the irradiated elastin/collagen mixture. The maximum of the fluorescence peak was shifted for elastin by 4nm, whereas for the elastin/collagen blends the shift was only 1-2nm. All the obtained results point out the ability of mixing elastin and collagen, and suggest that the elastin/collagen mixture in solution is less sensitive to UV irradiation than elastin hydrolysates alone.

Nitroxide conjugate of a thermally responsive elastin-like polypeptide for noninvasive thermometry.

Hyperthermia, as an adjuvant with radiation and chemotherapy, has shown promise in the treatment of cancer. The relevant biological effects of a hyperthermia treatment are both time and temperature-dependent, creating a need for accurate thermometry. We present a novel noninvasive thermometry modality that combines a temperature responsive biopolymer, the elastin-like polypeptide (ELP), and nitroxide to produce an ELP-nitroxide conjugate. When examined with electron paramagnetic resonance (EPR) spectroscopy, the ELP-nitroxide conjugate has temperature-dependent spectral line widths whose predictive accuracy is approximately 0.3 degrees C (80 microM). We believe that the temperature-dependent changes observed in the EPR spectrum are due to the combined effect of temperature, viscosity and effective radius on the rotational correlation time of the ELP-nitroxide conjugate.

The effect of ultraviolet-B exposure scheduling on the photodamage of hairless mouse skin.

In a mouse model, we investigated whether different exposure protocol of ultraviolet-B with the same total doses could induce a different degree of photodamage in mouse skin. Two different exposure frequencies, three times or six times a week, were applied under the condition of weekly same cumulative irradiation dose equally for 10 weeks. Then the photodamage parameters such as skin wrinkling, histochemical dermal change and epidermal and dermal thickness were evaluated. Wrinkle grade, histological assessment by score, and dermal thickness did not reveal any difference between the two groups. However, at irradiation week 10 epidermal thickness of the three times a week irradiation group was significantly thicker than that of the six times a week irradiation group. The same cumulative dose resulted in different epidermal thickness. Our results suggested that exposure frequency or scheduling could influence the epidermal damage by ultraviolet radiation even though the cumulative dose is equal.

Fluorescence spectroscopic imaging to detect changes in collagen and elastin following laser tissue welding.

OBJECTIVE: A study was performed to evaluate the use of native fluorescence imaging to detect in situ molecular changes. SUMMARY BACKGROUND DATA: There is no ideal noninvasive method to monitor molecular changes in a local region at a laser weld joint without removing a section of tissue for histological examination. METHODS: Two sections of animal skin were welded together border to border using a Ti:sapphire laser beam (800 nm). Fluorescence imaging was performed on the cross section of the welded site at specific emission wavelengths (lambda c) for collagen at 380 nm and for elastin at 450 nm using excitation wavelengths (lambda e) of 340 nm, and 380 nm, respectively. RESULTS: A reduction of the collagen and elastin emission was observed in the fluorescence images of the welded region. These results were confirmed with histology using picrosirius red F3BA under polarized light and orcein stains. CONCLUSION: Optical spectroscopic imaging offers a new noninvasive detecting method for microscopic evaluation of laser tissue welding.

Photobleaching of arterial fluorescent compounds: characterization of elastin, collagen and cholesterol time-resolved spectra during prolonged ultraviolet irradiation.

To study the photobleaching of the main fluorescent compounds of the arterial wall, we repeatedly measured the time-resolved fluorescence of elastin, collagen and cholesterol during 560 s of excitation with nitrogen laser pulses. Three fluence rate levels were used: 0.72, 7.25 and 21.75 microW/mm2. The irradiation-related changes of the fluorescence intensity and of the time-resolved fluorescence decay constants were characterized for the emission at 390, 430 and 470 nm. The fluorescence intensity at 390 nm decreased by 25-35% when the fluence delivered was 4 mJ/mm2, a common value in fluorescence studies of the arterial wall. Cholesterol fluorescence photobleached the most, and elastin fluorescence photobleached the least. Photobleaching was most intense at 390 nm and least intense at 470 nm such that the emission spectra of the three compounds were markedly distorted by photobleaching. The time-resolved decay constants and the fluorescence lifetime were not altered by irradiation when the fluence was below 4 mJ/mm2. The spectral distortions associated with photobleaching complicate the interpretation of arterial wall fluorescence in terms of tissue content in elastin, collagen and cholesterol. Use of the time-dependent features of the emission that are not altered by photobleaching should increase the accuracy of arterial wall analysis by fluorescence spectroscopy.

Photo-enhanced modification of human skin elastin in actinic elastosis by N(epsilon)-(carboxymethyl)lysine, one of the glycoxidation products of the Maillard reaction.

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGEs), which are characterized by fluorescence, brown color, and cross-linking. Formation of AGEs in vitro requires oxygen and is dependent on transition metal-catalyzed oxidation of glucose or Amadori products. AGEs are thought to be involved in aging and age-enhanced diseases such as diabetic complications, atherosclerosis, dialysis-related amyloidosis, and Alzheimer's disease. Chronic exposure of the skin to sunlight induces hyperplasia of the elastic tissue in the upper dermis known as actinic elastosis. Herein we used a monoclonal anti-AGE antibody (6D12) whose epitope is N(epsilon)-(carboxymethyl)lysine (CML), one of the glycoxidation products of AGEs, and demonstrated that the lesions of actinic elastosis were modified by CML. Further immunohistochemical and immunoelectron microscopic examination with 6D12 demonstrated CML accumulates predominantly in elastic fibers especially in the amorphous electron-dense materials corresponding to photo-induced degenerated area rather than the electron-lucent region. Immunochemical analyses with enzyme-linked immunosorbent assay (ELISA) of elastase-soluble fractions demonstrated that the CML levels of the sun-exposed area were significantly higher than those of the sun-unexposed area. We conclude that ultraviolet-induced oxidation may accelerate CML formation in actinic elastosis of photoaged skin.