Mouse model for protoporphyria. II. Cellular and subcellular events in the photosensitivity flare of the skin.

Acute phototoxic reactions were induced by long-wave ultraviolet light (UV-A) in mice with griseofulvin-induced protoporphyria. The clinical response was characterized by erythema, pronounced edema, and purpura. Tracer experiments and electron microscopy revealed pronounced vascular damage and leakage of vascular contents, whereas the epidermis and all other dermal components were intact. There was selective destruction of endothelial cells and damage of the basal lamina of the vessels. This striking vascular injury was absent from nonprotoporphyric UV-A-irradiated mice and from protoporphyric and nonprotoporphyric mice exposed to short-wave ultraviolet light (UV-B). Patients with erythropoietic protoporphyria (EPP) exhibit an identical, selective damage of blood vessels when irradiated with UV-A or sunlight but not with UV-B alone. It is hypothesized that in both murine protoporphyria and EPP, endothelial cells are photosensitized by protoporphyrin circulating in the serum and that photosensitized endothelia represent the primary cellular target of the photochemical reaction induced by UV-A.     

Clinically uninvolved skin in AIDS: evidence of atypical dermal vessels similar to early lesions observed in Kaposi''s sarcoma

The clinically uninvolved skin of 4 patients with well-developed AIDS was investigated by electron microscopy. All biopsy specimens had vascular abnormalities: protruding endothelial cells, vascular channels reduced to slits, gaps within the vascular walls, and extravasated erythrocytes. These features are similar to those described in early lesions of Kaposi's sarcoma. These findings suggest that blood vessels of the clinically uninvolved skin of AIDS patients are potential sites of Kaposi's sarcoma lesions.     

Vascular changes in erythropoietic protoporphyria: histopathologic and immunohistochemical study

BACKGROUND: Erythropoietic protoporphyria (EPP) is an inherited disease caused by deficient activity of ferrochelatase in the heme biosynthetic pathway. Accumulation of protoporphyrins and light exposure results in acute phototoxic skin reactions. The histopathologic findings of the light-exposed skin are thickening of the superficial dermal vessel walls and amorphous deposits around the vessels, but the origin and detailed composition of the perivascular material have been unclear. OBJECTIVE: The vascular morphology and composition of the perivascular material were studied in the skin samples of patients with EPP. METHODS: Skin biopsy specimens of 8 patients with EPP representing 7 Finnish EPP families with different genotypes were studied by means of light and electron microscopy and immunohistochemical methods. RESULTS: The characteristic finding was thickened, periodic acid-Schiff-positive vessel walls caused by concentric reduplication of basal lamina and excess of fine granular material at the basal membrane zone in the superficial dermis. The perivascular deposits in the vicinity of vessel walls had a homogeneous or fine granular appearance without filaments. Direct immunofluorescence showed constant IgG deposits together with IgA, IgM, and C3 in the vessel walls. In immunohistochemistry, collagen IV and laminin could be demonstrated at the vascular basal membrane together with serum amyloid P protein, kappa and lambda light chains, and a 90-kd glycoprotein. CONCLUSION: The vascular involvement indicates that the blood vessel walls in the papillary dermis are the primary tissues affected during an acute photoreaction. The repeated acute damage and repair processes in the basement membrane zone result in thickening of the vessel walls. Perivascular deposits are a secondary and irreversible phenomenon resulting from the leakage and accumulation of different serum components. These changes were not found in the nonexposed skin, indicating that an increased level of erythrocyte protoporphyrin per se is not responsible for the cutaneous manifestations, but the interaction of solar radiation is mandatory. Amorphous deposits distinguish EPP from variegate porphyria and porphyria cutanea tarda; a histopathologic examination may be a helpful tool in differentiating porphyric and nonporphyric photosensitivity.     

The role of VEGF and thrombospondins in skin angiogenesis

The vasculature in adult skin remains normally quiescent, due to the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. However, skin retains the capacity for brisk initiation of angiogenesis, the growth of new blood vessels from preexisting vessels, during tissue repair and in numerous diseases, including inflammatory skin diseases such as psoriasis and skin cancers such as cutaneous squamous cell carcinomas. Moreover, cyclic vascular expansion occurs during the growth phase of the hair follicle. Recent evidence suggests vascular endothelial growth factor as the major skin angiogenesis factor. During skin angiogenesis, expression of vascular endothelial growth factor is induced in epidermal keratinocytes by several stimuli including transforming growth factor-alpha and hypoxia, leading to increased vascularization of the dermis. In contrast, vascular endothelial growth factor-C induces skin lymphangiogenesis. Thrombospondin-1 and thrombospondin-2 are endogenous inhibitors of angiogenesis that are expressed in normal skin, maintaining the quiescence of cutaneous vessels. Both inhibitors potently inhibit skin cancer growth via inhibition of tumor angiogenesis. Targeting cutaneous blood vessels represents a promising new therapeutic approach for the treatment of a variety of skin diseases.     

Adult cutaneous hemangiomas are composed of nonreplicating endothelial cells

Thirty-four human "cherry" dermal hemangiomas were studied by electron microscopy, immunohistochemistry, and cell culture to assess the neoplastic nature of these lesions. Electron microscopy of nine hemangiomas revealed a pronounced thickening of the basement membrane (0.6 to 14 micron) in 93% of the total 158 vascular structures examined within the lesions. This increase was caused mainly by multiple layers of basal lamina, which were irregular in outline and frequently associated with pericytes. Basement membrane changes were present both in the periphery of the hemangiomas, as well as in the center of the lesions. Immature vessels could not be identified and mitoses were absent in all endothelial cells. Using an immunohistochemical marker (Ki67) specific for proliferating cells in G2 and S phases, positive staining was not found in the endothelial cells lining the hemangiomatous vessels, whereas basal epidermal keratinocytes in the same preparations and cultured microvascular endothelial cells expressed the antigen. Endothelial cells of nine hemangiomas did not stain with an activation-related antibody (E12) specific for endothelial cells. When endothelial cells from 14 hemangiomas were isolated and cultured under conditions that support the growth of normal human skin microvascular endothelial cells, the cells of hemangiomatous origin failed to grow. We conclude that the adult hemangiomas may not be true neoplasms, but a tissue overgrowth composed of mature vessels resembling dermal venules, lined by endothelial cells with virtually no turnover.     

Leukocytoclastic vasculitis masking chronic vascular changes in previously undiagnosed erythropoietic protoporphyria

A 31‐year old man presented with swelling and purpura of the dorsum of the hands following sun exposure. There was a preceding lifelong history of photosensitivity, but this episode, which occurred after the recent commencement of oral iron therapy, and after recent alcohol ingestion, was much more severe than any preceding episode. Skin biopsy performed 48 h after the onset of symptoms showed features consistent with the early stages of leukocytoclastic vasculitis. Direct immunofluorescence showed homogeneous thick staining of the vessel walls with IgG, IgM and IgA, together with abundant perivascular fibrinogen. A subsequent periodic acid‐Schiff (PAS) stain on the skin biopsy revealed thickening of the walls of dermal vessels, which was not discernible in routinely stained (hematoxylin/eosin) sections. The diagnosis of erythropoietic protoporphyria (EPP) was confirmed by significantly elevated erythrocyte protoporphyrin levels and positive plasma fluorimetry. The diagnosis of porphyria may have been missed by routine skin microscopy if not for the additional information provided by clinical history, direct immunofluorescence and PAS stain. The pathogenesis and histopathology of acute and chronic vascular changes in EPP are reviewed.     

The role of endothelial cell apoptosis in the effect of etanercept in psoriasis

Background Psoriasis is a chronic inflammatory skin disease associated with abnormal vascular expansion in the papillary dermis. Tumour necrosis factor (TNF)‐α is a proinflammatory cytokine that can induce antiapoptotic proteins and endothelial cell activation factors in psoriasis. Objectives The present study investigated the effect of the anti‐TNF‐α agent etanercept on the expression of endothelial nuclear factor‐κB (NF‐κB), angiogenic vascular endothelial growth factor (VEGF), endothelial cell marker CD31, antiangiogenic factor thrombospondin‐1 (TSP‐1), and antiapoptotic factors Bcl‐2 and Bcl‐xL in psoriasis. Methods Sixteen patients with moderate‐to‐severe psoriasis were included in the study and treated with etanercept 50 mg twice weekly subcutaneously for 12 weeks. Biopsies of lesional skin (baseline, weeks 3, 6 and 10) were obtained and immunohistochemically stained with antibodies for CD31, VEGF, TSP‐1, NF‐κB, Bcl‐2 and Bcl‐xL. Double immunofluorescence staining for VEGF and CD31 was evaluated with confocal laser microscopy. The terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labelling (TUNEL) assay was applied for apoptosis detection. Results Etanercept caused a statistically significant time‐dependent reduction in the number of dermal blood vessels, the number of CD31+ cells and VEGF in psoriatic lesions, with induction of endothelial cell apoptosis and statistically significant upregulation of TSP‐1 in psoriatic vessels. Immunohistochemical analysis showed significant reduction of NF‐κB, Bcl‐2 and Bcl‐xL expression in endothelial cells during treatment. These changes were accompanied by a marked clinical response. Conclusions The present findings suggest that treatment with etanercept induces apoptosis, reduces apoptosis‐inhibiting factors in psoriatic endothelial cells, and decreases angiogenesis in psoriatic skin.     

Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor

Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial growth factor, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified or control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial growth factor mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial growth factor-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial growth factor protein throughout the entire culture period. The bioactivity of vascular endothelial growth factor protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial growth factor-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of keratinocytes in cultured skin substitutes can lead to increased vascular endothelial growth factor expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications.     

Erythropoietic protoporphyria: synopsis of 20 patients

The authors present 20 patients (9 men, 11 women) with erythropoietic protoporphyria (EPP). The diagnosis was made on the basis of photosensitivity and porphyrin analysis. The disease first became apparent in the first years of life. The following acute symptoms were induced after exposure to sunlight: pruritus with or without skin changes, burning, pain and erythema, sometimes with petechiae, vesiculation and, in two cases, systemic symptoms. Chronic skin changes included hyalinosis cutis-like skin lesions, scarring, and also petechiae. Phototesting provoked only subjective symptoms, and none of the skin lesions characteristic of EPP could be induced. Postnatal diagnosis was attempted in three newborns, each of whom had one parent with proven EPP, by measuring the porphyrins in erythrocytes of cord blood. In all three normal porphyrin values were determined, and during an observation period of 3 years none has developed EPP. Therapy with carotenoids has yielded good to very good results in two-thirds of the patients. So far, a diagnosis of EPP has been established in 30 patients in Düsseldorf: one has died of liver cirrhosis and another has liver damage.     

ICAM-1 and LFA-1 Expressions in the Lesional Skin of Annular Erythema Associated with Sjögren Syndrome

ICAM-1 and LFA-1 expression was studied in the lesional skin of ten cases of annular erythema associated with Sjögren syndrome. Most of the infiltrating mononuclear cells around blood vessels expressed LFA-1 in addition to its strong expression on vascular endothelial cells and focal expression on the epidermal basal cell layer in 3 cases. ICAM-1 expression on vascular endothelial cells was similar to LFA-1, although relatively focal and weak expression was observed on mononuclear cells. ICAM-1 expression on keratinocytes was focal and limited to the basal cell layer in annular erythema. These findings suggest that strong expression of ICAM-1 on endothelial cells but not keratinocytes and LFA-1 on mononuclear cells might play some role in the induction of skin lesions in annular erythema associated with Sjögren syndrome.     

Local reactions to tick bites

A retrospective histological and immunohistochemical study has been carried out in 25 cases of tick bites recorded in our Departments. The samples that included an attached tick showed a cement cone anchoring the mouthparts to the skin and a blood-soaked, spongiform appearance of the superficial dermis, with a mild neutrophilic and eosinophilic infiltration. The vessels displayed a loose multilayered endothelial proliferation, with plump endothelia, permeated with erythrocytes. A few of them were severed, allowing copious blood extravasation. The established lesions included the following: erythema chronicum migrans-like cases, foreign body granulomas-sometimes containing remnants of the mouthparts-cutaneous lymphoid hyperplasia, either of the T-cell or the B-cell type, and tick-bite alopecia. In both the T-cell and B-cell pseudolymphomas, several vessels showed concentric endothelial and perithelial proliferation similar to that seen in the acute lesions. In the tick-bite alopecia, a lymphocytic infiltrate attacked the permanent portion of the hair follicles, whose reaction was a noticeable hyperplasia of the fibrous sheaths, although only a minority of the hairs was destroyed. The observed alterations are specific in the acute lesions and in the alopecia, where they directly arise as a result of the interactions between the host's tissues and the antihemostatic, anti-inflammatory, and immunomodulatory chemicals contained in the tick saliva. In the other lesions, the changes seem less characteristic, although the fragments of mouthparts and the special vascular changes provide a clue to their etiology.     

Toll样受体2和4在银屑病皮损中的表达

目的研究Toll样受体（TLR）2和4在银屑病皮损中的表达，探讨其与银屑病发病的关系。方法选用16例滴状银屑病、13例斑块状银屑病患者及10例正常人皮肤的石蜡切片．用免疫组化的方法研究TLR2和TLR4的表达。结果10例正常人皮肤的基底层均有较弱的TLR2表达而无TLR4表达．真皮血管内皮细胞未见TLR2及TLR4表达。所有16例滴状银屑病、13例斑块状银屑病皮损的基底细胞层均可见明显的TLR2表达，棘层也有弱表达；TLR4则呈现表皮全层的弥漫性强表达。银屑病真皮浅层血管内皮细胞可见明显的TLR2及TLR4表达。结论TLR2、TLR4在银屑病皮损均有表达，TLR4的表达更高：提示感染相关免疫与银屑病发病关系密切。     

Immunohistological evaluation of endothelial markers and basement membrane components in port-wine stains

As shown by an immunohistological study, the endothelial cells in the lesions of port-wine stains (PWSs), investigated for factor VIII-related antigen and Ulex europaeus agglutinin I lectin, were found to be swollen or flattened corresponding to mild or pronounced dilatation of vessels, respectively. Furthermore, dilated vessels in the lesional skin exhibited a broader staining for type IV collagen, laminin and fibronectin, which, however, was not considered to be of primary pathogenetic importance for vessel dilatation in PWSs. As concluded from the number of vessels, no vascular proliferation occurred, which indicates that PWS is not a true hemangioma but a special clinical type of telangiectasia.     

Vascular endothelial cell distribution and adhesion molecule expression in xanthoma

BACKGROUND: The migration of circulating monocytes into the dermis is considered to be essential for both the initiation and the progression of xanthoma. The contribution of vascular endothelial cells to the migration process is unclear. METHODS: Twenty cases of xanthelasma and six cases of tuberous xanthoma lesions were analyzed using immunohistochemical staining. RESULTS: Xanthoma lesions contained up to 25-fold more von Willebrand factor-stained endothelial cells than normal skin. The prevalence of E-selectin-positive endothelial cells increased by up to threefold more in xanthoma lesions than in normal skin. In contrast, the prevalence of intercellular cell adhesion molecule-1 (ICAM-1) decreased up to 3.5-fold more in xanthoma lesions than in normal skin. In xanthoma lesions, almost all ICAM-1-positive endothelial cells co-expressed with E-selectin but many endothelial cells, which only expressed E-selectin, were also found in the lesions and the ratio of macrophages to endothelial cells was higher (10:1) than that in normal skin (5:1). CONCLUSIONS: Endothelial cells proliferate and express E-selectin rather than ICAM-1 under a microenvironment in which macrophages predominate rather than endothelial cells, thereby promoting macrophage migration into xanthoma lesions.     

Vascular structures in skin tumors: a dermoscopy study

OBJECTIVES: To describe the different vascular structures seen by dermoscopy and to evaluate their association with various melanocytic and nonmelanocytic skin tumors in a large series of cases. DESIGN: Digital dermoscopic images of the lesions were evaluated for the presence of various morphologic types of vessels. SETTING: Specialized university clinic. PATIENTS: From a larger database, 531 excised lesions (from 517 patients) dermoscopically showing any type of vascular structures were included. MAIN OUTCOME MEASURES: The frequency and positive predictive value of the different vascular structures seen in various tumors were calculated, and the differences were evaluated by the chi2 or Fisher exact test. RESULTS: Arborizing vessels were seen in 82.1% of basal cell carcinomas, with a 94.1% positive predictive value (P<.001). Dotted vessels were generally predictive for a melanocytic lesion (90.0%, P<.001), and were especially seen in Spitz nevi (77.8% of lesions). In melanoma, linear-irregular, dotted, and polymorphous/atypical vessels were the most frequent vascular structures, whereas milky-red globules/areas were the most predictive ones (77.8%, P = .003). The presence of erythema was most predictive for Clark nevus, whereas comma, glomerular, crown, and hairpin vessels were significantly associated with dermal/congenital nevi, Bowen disease, sebaceous hyperplasia, and seborrheic keratosis, respectively (P<.001 for all). CONCLUSIONS: Different morphologic types of vessels are associated with different melanocytic or nonmelanocytic skin tumors. Therefore, the recognition of distinctive vascular structures may be helpful for diagnostic purposes, especially when the classic pigmented dermoscopic structures are lacking.     

Vascularización en dermatoscopia

Under the right conditions, dermoscopy allows us to observe the vascular features of many different types of skin lesions. The visualization and identification of vessels with a characteristic morphology can be the key to diagnosis, especially in hypopigmented lesions in which the typical pigmented structures are not visible.Some of the more characteristic associations are the presence of crown vessels in sebaceous hyperplasia, arborizing telangiectasias in basal cell carcinoma, comma-shaped vessels in intradermal and compound nevi, dotted vessels in Spitz nevi and melanoma, and hairpin vessels in seborrheic keratoses.The recognition of distinctive vascular features can be of great help in the diagnosis of many types of skin lesions, and very often such patterns are the only key to the diagnosis of melanoma.     

The vascular characteristics of melasma

BACKGROUND: The pathogenesis of melasma is not yet fully understood. Previous studies indicate that dermal environment such as fibroblasts may have an important role in the development of melasma. Recently, it has been suggested that interactions between the cutaneous vasculature and melanocytes might have an influence on the development of pigmentation. OBJECTIVES: We investigated the vascular characteristics in melasma lesions. The expression of vascular endothelial growth factor (VEGF), a major angiogenic factor of the skin, was also investigated in melasma. METHODS: Erythema intensity was quantified by the increase of the a* parameter using a colorimeter. Skin samples were obtained from lesional and non-lesional facial skin of 50 Korean women with melasma. Immunohistochemistry was performed to determine the expression of factor VIIIa-related antigen and VEGF in melasma. RESULTS: The values of a* was significantly higher in the melasma lesion than that of perilesional normal skin. Computer-assisted image analyses of factor VIIIa-related antigen-stained sections revealed a significant increase of both the number and the size of dermal blood vessels in the lesional skin. There was significant relationship between the number of vessels and pigmentation in melasma. The expression of VEGF was significantly increased in melasma CONCLUSIONS: These data suggest that increased vascularity is one of the major findings in melasma. VEGF may be a major angiogenic factor for altered vessels in melasma.     

Immunofluorescence demonstration of type IV collagen and a noncollagenous glycoprotein in thickened vascular basal membranes in protoporphyria.

Specific rabbit antibodies to type IV collagen isolated from a basement membrane producing mouse tumor were used in indirect immunofluorescence tests to study the thickened vascular basement membrane in skin biopsies from patients with erythropoietic porphyria (EPP) and from protoporphyric mice. In addition, rabbit antibodies to a noncollagenous, basement membrane specific glycoprotein also derived from the mouse tumor were tested. It was shown that normal as well as altered vascular basement membranes in both the human and the murine skin specimens react with the anti-type IV collagen and the antiglycoprotein antibodies. A particular strong reaction in the diseases skin indicated that formation of new vascular basement membrane layers involved deposition of the major structural proteins which also constitute normal basement membrane matrices.     

Ultrastructural characterization of microvasculature in photoaging

The cutaneous microvasculature was examined by electron microscopy in order to compare its characteristics in photodamaged preauricular skin and in sun-protected postauricular sites of 15 Japanese women aged 58-81 years. The characteristic ultrastructural features of the microvasculature in photodamaged skin compared with those in sun-protected skin included dilated vessels embedded in elastin which depressed endothelial cells, vessels surrounded by a thick amorphous material composed of multiple laminations of a basement membrane-like material, and activated endothelial cells which had increased numbers of cytoplasmic organelles and pinocytotic vesicles. A novel finding of this study in photodamaged vessels was an increased formation of new vessels (angiogenesis) via two distinct pathways. In severe elastosis, activated endothelial cells with densely packed intracytoplasmic microfilaments extended large pseudopods into the elastotic material. In contrast, isolated mesenchymal cells, which possessed immature Weibel-Palade bodies, were scattered around pre-existing vessels within the Grenz zone. In some cases, many mesenchymal cells with electron-lucent cytoplasms aggregated and interconnected by cytoplasmic processes, which was followed by the formation of vascular structures. These results suggest that there are significant ultrastructural differences in vessels between photoaged and intrinsically aged facial skin and that the photodamaged microvascular system is characterized by the co-existence of regressive changes and angiogenesis.     

AKT1 overexpression in endothelial cells leads to the development of cutaneous vascular malformations in vivo

BACKGROUND: Vascular malformations are clinical disorders in which endothelial cells fail to remodel and/or undergo programmed cell death, leading to abnormal persistence of blood vessels. The abnormal persistence of vessels makes therapy difficult because these lesions are resistant to interventions that are effective against hemangiomas. Akt1 is a serine-threonine protein kinase, which is a key mediator of resistance to programmed cell death. Our objective was to determine whether sustained activation of Akt1 could lead to vascular malformation in mice. OBSERVATIONS: We examined the effect of constitutive activation of Akt1 in murine endothelial cells (MS1 cells). Overexpression of active AKT1 in MS1 cells led to the development of vascular malformations, characterized by wide endothelial lumens and minimal investment of smooth muscle surrounding the vessels. The histologic features of these vascular malformations is distinct from ras-transformed MS1 cells (angiosarcoma) and suggest that differing signal abnormalities give rise to human vascular malformations vs malignant vascular tumors. CONCLUSIONS: Inhibition of Akt signaling may be useful in the treatment of vascular malformations. Examination of problematic hemangiomas and vascular malformations for the presence of activated Akt or downstream targets of Akt, such as mammalian target of rapamycin (mTOR), may predict response to treatment with Akt inhibitors or rapamycin. This study provides a potential rationale for the systemic and topical use of these inhibitors for vascular malformations and hemangiomas.