Platelets Regulate the Migration of Keratinocytes via Podoplanin/CLEC-2 Signaling during Cutaneous Wound Healing in Mice.

Podoplanin is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), which is expressed on platelets. Recent evidence indicates that this specific marker of lymphatic endothelial cells is also expressed by keratinocytes at the edge of wounds. However, whether podoplanin or platelets play a role in keratinocyte activity during wound healing remains unknown. We evaluated the effect of podoplanin expression levels on keratinocyte motility using cultured primary normal human epidermal keratinocytes (NHEKs). Down-regulation of podoplanin in NHEKs via transfection with podoplanin siRNA inhibited their migration, indicating that podoplanin plays a mandatory role in this process. In addition, down-regulation of podoplanin was correlated with up-regulation of E-cadherin, suggesting that podoplanin-mediated stimulation of keratinocyte migration is associated with a loss of E-cadherin. Both the addition of platelets and treatment with CLEC-2 inhibited the migration of NHEKs. The down-regulation of RhoA activity and the up-regulation of E-cadherin in keratinocytes were also induced by CLEC-2. In conclusion, these results suggest that podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Altering the regulation of keratinocyte migration by podoplanin might be a novel therapeutic approach to improve wound healing.

Malignant melanoma showing a rapid response to nivolumab.

Malignant melanoma is a highly aggressive skin tumour, with a recent rise in incidence. Nivolumab is a recently developed anti-programmed cell death-1 immune checkpoint inhibitor and its usage has resulted in a significant improvement in the overall survival of patients with metastatic melanomas. We report a case of advanced melanoma that showed a significant and rapid response to nivolumab treatment. The patient displayed multiple melanoma-associated vitiligo prior to treatment; this symptom was theorised to indicate potentially immunoreactive melanoma and the need for nivolumab. In addition, interferon-ß was injected prior to nivolumab treatment. The significant rapid response to nivolumab suggested the induction of a marked immune response against melanoma by interferon-ß. Therefore, interferon-ß could be a useful and effective adjuvant for nivolumab therapy.

Podoplanin expression in cancer-associated fibroblasts predicts aggressive behavior in melanoma.

BACKGROUND: Recent studies have showed podoplanin expression in several tumors, which has been associated with lymph node metastasis and poor prognosis. Podoplanin expression in cancer-associated fibroblasts also correlates with tumor progression. However, the association of podoplanin expression with melanomas remains unclear. METHODS: To clarify the prognostic significance of podoplanin in melanoma, podoplanin expression in tumor cells and cancer-associated fibroblasts was examined by immunohistochemistry in tissue samples collected from 55 melanoma patients. RESULTS: Podoplanin expression in tumor cells was identified in 38 patients (69.1%) but did not show correlation with characteristics of tumor progression such as tumor thickness (p = 0.52) and sentinel lymph node (SLN) metastasis (p = 0.79). Podoplanin expression in cancer-associated fibroblasts was observed in 25 patients (45.5%), 11 of whom (44.0%) had SLN metastasis. In contrast, only 4 of 30 patients (13.3%) with podoplanin-negative cancer-associated fibroblasts exhibited SLN metastasis. Podoplanin-positive cancer-associated fibroblasts were associated with increased tumor thickness and SLN metastasis. Furthermore, patients with podoplanin-positive cancer-associated fibroblasts had poorer survival than those with podoplanin-negative cancer-associated fibroblasts (p = 0.0148). CONCLUSION: The presence of podoplanin expression in cancer-associated fibroblasts correlates with aggressive behavior in melanoma and might therefore serve as a useful prognostic factor for patients with melanoma.

Topical simvastatin accelerates wound healing in diabetes by enhancing angiogenesis and lymphangiogenesis.

Impaired wound healing is a major complication of diabetes. Recent studies have reported reduced lymphangiogenesis and angiogenesis during diabetic wound healing, which are thought to be new therapeutic targets. Statins have effects beyond cholesterol reduction and can stimulate angiogenesis when used systemically. However, the effects of topically applied statins on wound healing have not been well investigated. The present study tested the hypothesis that topical application of simvastatin would promote lymphangiogenesis and angiogenesis during wound healing in genetically diabetic mice. A full-thickness skin wound was generated on the back of the diabetic mice and treated with simvastatin or vehicle topically. Simvastatin administration resulted in significant acceleration of wound recovery, which was notable for increases in both angiogenesis and lymphangiogenesis. Furthermore, simvastatin promoted infiltration of macrophages, which produced vascular endothelial growth factor C in granulation tissues. In vitro, simvastatin directly promoted capillary morphogenesis and exerted an antiapoptotic effect on lymphatic endothelial cells. These results suggest that the favorable effects of simvastatin on lymphangiogenesis are due to both a direct influence on lymphatics and indirect effects via macrophages homing to the wound. In conclusion, a simple strategy of topically applied simvastatin may have significant therapeutic potential for enhanced wound healing in patients with impaired microcirculation such as that in diabetes.

Intractable diffuse alopecia caused by multifactorial side-effects in treatment of acute lymphocytic leukemia: connection to iatrogenic failure of estrogen secretion.

Treatment of infantile acute lymphocytic leukemia (ALL) may cause failure to thrive and hypogonadism due to hypopituitarism induced by chemotherapy and whole-brain radiotherapy. We report the case of a 22-year-old girl with a genetic predisposition to pattern hair loss who developed inveterate diffuse alopecia. The patient had onset of ALL at 8 years old and underwent bone marrow transplantation (BMT). Diffuse alopecia gradually advanced over her whole body. Her vellus scalp hair gradually came out, and hair loss progressed again at 8 years, after BMT. She later developed iatrogenic failure of secretion of estrogen and was treated with estrogen substitution therapy for 14 months from the age of 20. There was a small increase in the volume of hair during therapy, but alopecia returned to the former level after the therapy was suspended. Histopathologic examinations of the scalp performed during estrogen substitution therapy and 2 years after suspension of the therapy showed a 60% decrease in the number of hair follicles and prominent development of vellus hair. We conclude that estrogen influenced hair growth in the context of a genetic predisposition for pattern hair loss in this case.

Granuloma annulare-like reaction to the bacillus Calmette-Guerin vaccination.

We report the case of a 6-month-old girl with a granuloma annulare (GA)-like reaction to the bacillus Calmette-Guerin (BCG) vaccination. The eruption developed at the vaccination site 1 month after vaccination and the lesion gradually disseminated over the body within 2 months. A biopsy specimen of the skin lesion showed degenerated collagen bundles surrounded by imperfect palisading histiocytes, lymphocytes and epithelioid cells in the dermis, which led to a diagnosis of GA-like reaction as a secondary reaction to BCG inoculation. The eruption at the vaccination site and the scattered GA reaction resolved after 1 month of treatment with prednisolone valerate acetate ointment, leaving only pigmentation.

Topical application of ex vivo expanded endothelial progenitor cells promotes vascularisation and wound healing in diabetic mice.

Impaired wound healing leading to skin ulceration is a serious complication of diabetes and may be caused by defective angiogenesis. Endothelial progenitor cells (EPCs) can augment neovascularisation in the ischaemic tissue. Experiments were performed to test the hypothesis that locally administered EPCs can promote wound healing in diabetes. Full-thickness skin wounds were created on the dorsum of diabetic mice. EPCs were obtained from bone marrow mononuclear cells (BMMNCs) and applied topically to the wound immediately after surgery. Vehicle and non-selective BMMNCs were used as controls. Wound size was measured on days 5, 10 and 14 after treatment, followed by resection, histological analysis and quantification of vascularity. Topical application of EPCs significantly promoted wound healing, as assessed by closure rate and wound vascularity. Immunostaining revealed that transplanted EPCs induced increased expression of vascular endothelial growth factor and basic fibroblast growth factor. Few EPCs were observed in the neovasculature based on in vivo staining of the functional vasculature. Ex vivo expanded EPCs promote wound healing in diabetic mice via mechanisms involving increased local cytokine expression and enhanced neovascularisation of the wound. This strategy exploiting the therapeutic capacity of autologously derived EPCs may be a novel approach to skin repair in diabetes.

Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling.

Sonic hedgehog (Shh) is a crucial regulator of organ development during embryogenesis. We investigated whether intramyocardial gene transfer of naked DNA encoding human Shh (phShh) could promote a favorable effect on recovery from acute and chronic myocardial ischemia in adult animals, not only by promoting neovascularization, but by broader effects, consistent with the role of this morphogen in embryogenesis. After Shh gene transfer, the hedgehog pathway was upregulated in mammalian fibroblasts and cardiomyocytes. This resulted in preservation of left ventricular function in both acute and chronic myocardial ischemia by enhanced neovascularization, and reduced fibrosis and cardiac apoptosis. Shh gene transfer also enhanced the contribution of bone marrow-derived endothelial progenitor cells to myocardial neovascularization. These data suggest that Shh gene therapy may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by triggering expression of multiple trophic factors and engendering tissue repair in the adult heart.

Immunohistological analysis of peptide-induced delayed-type hypersensitivity in advanced melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination.

BACKGROUND: In melanoma patients vaccinated with monocyte-derived melanoma peptide-pulsed dendritic cells (DC), the delayed-type hypersensitivity (DTH) reactions have been examined as a surrogate marker to determine if acquired immunity is induced by DC vaccination. To date, however, only limited information has been reported as for histopathological analyses of DTH. OBJECTIVE: To evaluate tumor-specific immunomonitoring histopathologically after DC vaccination in melanoma patients. METHODS: Seven patients previously vaccinated with monocyte-derived melanoma peptide-pulsed DCs were challenged with recall antigenic peptide injection in the skin of the forearm. Using immunohistochemical techniques, the presence of immune cells and the expression of CD4, CD8, interleukin (IL)-2, IL-4, IL-10, Foxp3, CD1a, CD1d, and interferon (IFN)-gamma was investigated at the site of injection where a DTH reaction developed. RESULTS: Strong DTH reactions from infiltrated erythema to bullae formation were detected in all 7 cases. Biopsies taken from the DTH site revealed heavy infiltration of mononuclear cells and eosinophils in the dermis and subcutaneous tissue. Cells staining positively for CD4, CD8, IL-2, IL-4, Foxp3, CD1d, and IFN-gamma were increased at the site 48h after antigen injection in all cases. Cells positive for IL-10 were never found in any patient. Regulatory T cells appeared 6h after injection and reached their maximum at day 7. CONCLUSIONS: The significant induction of CD8(+)T cells as well as both Th1 and Th2-type cells at the site of DTH suggests that effective antigen presentation leading to anti-tumor immune responses has taken place. Inhibitory mechanisms may also develop as the disappearance of the DTH response could be related to an increase in Foxp3+ cells.

Symmetrical drug-related intertriginous and flexural exanthema caused by valacyclovir.

Drug-related eruptions that appear only on intertriginous or flexural folds and in gluteal areas have recently been termed symmetrical drug-related intertriginous and flexural exanthema (SDRIFE). We report a case of a 56-year-old woman with acute erythematous rash in the intertriginous areas after treatment with the L-valine ester of acyclovir, valacyclovir. Oral-challenge tests resulted in erythematous pruritic rash in the intertriginous area by valacyclovir. The patient was diagnosed as having SDRIFE due to valacyclovir.

Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.

In the inflamed cornea, there is a parallel outgrowth of blood and lymphatic vessels into the normally avascular cornea. We tested whether adaptive and/or innate immune cells were actively involved in the genesis of new lymphatic vessels. Our results indicate that innate immune cells (CD11b+ macrophages, but not CD11c+ dendritic cells) physically contributed to lymphangiogenesis under pathological conditions and that bone marrow-derived CD11b+ macrophages expressed lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions in the corneal stromata of mice. Furthermore, blood vascular endothelial cells that expressed the Tie2 promoter did not contribute to newly formed lymphatic vessels under inflamed conditions. Our in vitro experiments demonstrated that CD11b+ macrophages alone were capable of forming tube-like structures that expressed markers of lymphatic endothelium such as LYVE-1 and podoplanin. The novel finding that CD11b+ macrophages are critical for the development of inflammation-dependent lymphangiogenesis in the eye suggests a new mechanism of lymphangiogenesis.

Endothelial progenitor thrombospondin-1 mediates diabetes-induced delay in reendothelialization following arterial injury.

Delayed reendothelialization contributes to restenosis after angioplasty and stenting in diabetes. Prior data have shown that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to endothelial recovery after arterial injury. We investigated the hypothesis that the EPC contribution to reendothelialization may be impaired in diabetes, resulting in delayed reendothelialization. Reendothelialization was significantly reduced in diabetic mice compared with nondiabetic mice in a wire-induced carotid denudation model. The EPC contribution to neoendothelium was significantly reduced in Tie2/LacZ BM-transplanted diabetic versus nondiabetic mice. BM from diabetic and nondiabetic mice was transplanted into nondiabetic mice, revealing that reendothelialization was impaired in the recipients of diabetic BM. To examine the relative roles of denuded artery versus EPCs in diabetes, we injected diabetic and nondiabetic EPCs intravenously after arterial injury in diabetic and nondiabetic mice. Diabetic EPCs recruitment to the neoendothelium was significantly reduced, regardless of the diabetic status of the recipient mice. In vitro, diabetic EPCs exhibited decreased migration and adhesion activities. Vascular endothelial growth factor and endothelial NO synthase expressions were also significantly reduced in diabetic EPCs. Notably, thrombospondin-1 mRNA expression was significantly upregulated in diabetic EPCs, associating with the decreased EPC adhesion activity in vitro and in vivo. Reendothelialization is impaired by malfunctioning EPCs in diabetes. Diabetic EPCs have phenotypic differences involving thrombospondin-1 expression compared with nondiabetic EPCs, revealing potential novel mechanistic insights and therapeutic targets to improve reendothelialization and reduce restenosis in diabetes.

Ecthyma gangrenosum without pseudomonas septicemia in a kidney transplant recipient.

We report a case of ecthyma gangrenosum (EG) without septicemia in a renal transplant recipient who presented with a 1-month history of painful ulcers, vesicles and bullae on the face and extremities. Histopathological findings revealed subepidermal bullae covered by a necrotic epidermis containing an infiltrate of a moderate number of lymphocytes, neutrophils and necrotic collagen. Many dilated and congested capillaries were also present due to thrombi beneath the bullae, with alteration of collagen fibers through the superficial to middle dermis with some infiltrate. A culture from the ulcers revealed the presence of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus, whereas the results of repeated blood cultures were negative. The ulcers were completely cured by early appropriate i.v. antibiotic therapy with granulocyte colony-stimulating factor, without progression to EG with septicemia. An immunocompromised state due to immunosuppressive drugs, in addition to diabetes mellitus, hypogammaglobulinemia and hypoproteinemia, may have caused the EG and herpes zoster may have exacerbated the condition.

Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial progenitor cell-mediated microvascular remodeling.

BACKGROUND: Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial-mesenchymal interaction during embryonic development. Recent observations indicate that exogenous administration of Shh can induce angiogenesis and may accelerate repair of ischemic myocardium and skeletal muscle. Because angiogenesis plays a pivotal role in wound repair, we hypothesized that activation of the hedgehog pathway may promote a favorable effect on microvascular remodeling during cutaneous wound healing and thereby accelerate wound closure. Because diabetes is associated with impaired wound healing, we tested this hypothesis in a diabetic model of cutaneous wound repair. METHODS AND RESULTS: In Ptc1-LacZ mice, cutaneous injury resulted in LacZ expression, indicating that expression of the Shh receptor Patched was induced and therefore that the Shh signaling pathway was intact postnatally and upregulated in the process of wound repair. In diabetic mice, topical gene therapy with the use of naked DNA encoding for Shh resulted in significant local gene expression and acceleration of wound recovery. The acceleration in wound healing was notable for increased wound vascularity. In bone marrow transplantation models, the enhanced vascularity of the wound was shown to be mediated, at least in part, by enhanced recruitment of bone marrow-derived endothelial progenitor cells. In vitro, Shh promoted production of angiogenic cytokines from fibroblasts as well as proliferation of dermal fibroblasts. Furthermore, Shh directly promoted endothelial progenitor cell proliferation, migration, adhesion, and tube formation. CONCLUSIONS: These findings suggest that a simple strategy of topically applied Shh gene therapy may have significant therapeutic potential for enhanced wound healing in patients with impaired microcirculation such as occurs in diabetes.

Primary cutaneous anaplastic large cell lymphoma associated with vascular endothelial growth factor arising from a burn scar.

We present a case of primary cutaneous anaplastic large cell lymphoma manifesting with widespread erythema around an ulcer. Skin biopsy from the ulcer showed CD30-positive atypical lymphocytes throughout the dermis, and immunostaining demonstrated that these cells produced vascular endothelial growth factor, a potent stimulus for angiogenesis. Skin biopsy from the erythema revealed rich neovascularization, which was probably associated with vascular endothelial growth factor.

Podoplanin expression in peritumoral keratinocytes predicts aggressive behavior in extramammary Paget's disease.

BACKGROUND: Recent studies have demonstrated podoplanin expression in several tumors, which has been associated with lymph node metastasis and poor prognosis. Podoplanin expression in peritumoral cells such as cancer-associated fibroblasts also correlates with tumor progression in several cancers. However, podoplanin expression and its association with extramammary Paget's disease (EMPD) remain unclear. OBJECTIVE: In this study, we examined whether the presence of podoplanin expression in tumor cells or peritumoral basal keratinocytes correlated with aggressive behavior in patients with EMPD and investigated the mechanisms of podoplanin-mediated tumor invasion in this disorder. METHODS: Skin samples of 37 patients with EMPD were investigated by immunohistochemical analysis. The functions of podoplanin in keratinocytes were examined in vitro by RT-PCR and with invadopodia gelatin-degradation assays using HaCaT cells. RESULTS: Podoplanin was not identified in tumor cells in all cases. Podoplanin expression in peritumoral basal keratinocytes was observed in 25 patients (67.6%). In in situ EMPD, 50% of cases (9 in 18) exhibited podoplanin-positive keratinocytes, whereas 84.2% (16 in 19) demonstrated positive staining in invasive EMPD (P<0.05). Podoplanin expression in peritumoral keratinocytes was also associated with tumor thickness (P<0.005). By immunohistochemical analysis, podoplanin-positive peritumoral keratinocytes were found to be negative for E-cadherin, one of the major adhesion molecules of keratinocytes, which might contribute to tumor invasion into the dermis through a crack in the basal cell layer induced by down-regulation of cell adhesion therein. We further found that podoplanin-positive keratinocytes exhibited invadopodia, which are thought to function in the migration of cancer cells through tissue barriers, indicating that podoplanin-positive peritumoral basal keratinocytes might assist tumor invasion by degrading the extracellular matrix. CONCLUSION: The presence of podoplanin expression in peritumoral keratinocytes correlates with aggressive behavior in EMPD and might therefore serve as a useful prognostic marker for patients with EMPD.

Dibutyryl cAMP influences endothelial progenitor cell recruitment during wound neovascularization.

Delayed wound healing is one of the major complications of diabetes, and is caused by delayed cellular infiltration, reduced angiogenesis, and decreased formation and organization of collagen fibers. Recently, endothelial progenitor cells (EPC) isolated from peripheral blood were shown to accumulate at sites of neovascularization during wound healing. The present study tested the hypothesis that sodium N-6,2'-O-dibutyryl adenosine-3',5'-cyclic phosphate (DBcAMP), which has been shown to accelerate wound healing, promotes recruitment of EPC into wounds and contributes to the stimulation of neovascularization in genetically diabetic mice. Topical application of DBcAMP resulted in significant acceleration of wound healing and wound vascularization partly via enhanced recruitment of EPC. EPC in DBcAMP-treated wounds were mainly localized to cell clusters at the border of the granulation tissue, a site where blood supply is most insufficient. DBcAMP treatment increased the mRNA expression of angiogenic cytokines vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1alpha (SDF-1alpha) in vivo in wound tissue and in cultured fibroblasts and macrophages, in vitro. Culture supernatants of DBcAMP-treated cells enhanced EPC migration. Taken together, these results indicate that DBcAMP promotes neovascularization in wound healing, at least partly by increasing the accumulation of EPC at wound sites.

Cutaneous tissue angiotensin-converting enzyme may participate in pathologic scar formation in human skin.

BACKGROUND: Many studies have shown that up-regulation of angiotensin-converting enzyme (ACE) participates in adverse fibrous remodeling. Although this has become an accepted fact in the cardiovascular field, the relationship between ACE and cutaneous fibrous remodeling, such as keloid or hypertrophic scars, remains unknown. OBJECTIVE: We sought to investigate ACE in normal skin, wounded skin, and pathologic scars. METHODS: Ten samples undergoing a normal wound-healing process, 14 samples of pathologic scar tissue, and 15 samples of normal skin were used in this study. Cutaneous tissue ACE activities were measured with high-pressure liquid chromatography. Localization of ACE was assessed by immunohistochemistry. RESULTS: The ACE activity in pathologic scar tissue was significantly higher than in normal and wounded skin. Immunohistochemical studies demonstrated that myofibroblasts were stained with anti-ACE antibody. LIMITATIONS: The study is small. CONCLUSIONS: These results suggest that up-regulated ACE may participate in cutaneous pathologic scar formation the same as the cardiovascular system.

Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate-pulsed mature, monocyte-derived dendritic cells.

We performed a clinical trial to assess the feasibility and efficacy of immunotherapy with peptides, tumor lysate or both peptides and tumor lysate-pulsed mature, monocyte-derived dendritic cells (DC) for advanced malignant melanoma patients that are resistant to conventional therapies. Sixteen patients were enrolled in this trial. All patients received DC vaccines i.d. in the proximal thigh, close to the inguinal lymph nodes, one treatment per week or 2 weeks. Several factors such as clinical findings, computed tomography (CT) images, delayed type hypersensitivity (DTH) response, enzyme-linked immunosorbent spot (ELISPOT) assay, and immunohistochemistry in primary, metastatic lesions and the DTH site were evaluated. Clinical results through DC vaccination were as follows: in 11 evaluable cases, three stable disease, six progression of disease and two disease-free from the time of study entry to the completion of one vaccination course. One patient showed reduction of the tumors in the metastases on chest CT during the first and second course of DC vaccination. Ten out of 14 evaluable cases showed positive DTH responses to more than one treatment with melanoma peptides or tumor lysate. Eight out of 13 evaluable cases showed positive immunological responses to more than one treatment with melanoma peptides or tumor lysate in an ELISPOT assay. As for the experiences with toxicity and adverse reactions, autosensitization dermatitis-like eruptions appeared in five cases during DC vaccination. No severe adverse effects were seen in any of the patients. In our study, the clinical efficacy in prolongation of the patients' survival was confirmed. At the same time, cancer immunoediting of the tumor was also found. It will be necessary to improve the tumor-specificity of this therapeutic approach and to analyze the mechanism(s) of tumor escape from immunosurveillance in melanoma.