Hideya Takenaka

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.

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. (Circulation. 2006;113:2413-2424.)

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.

Rituximab is effective for steroid-refractory sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) is the most common late complication following allogeneic stem cell transplantation, occurring in 25–80% of transplant recipients.1 It is becoming a more frequent problem due to the increasing recipient age at transplantation as well as the increasing use of alternative donors, peripheral blood stem cells, and donor lymphocyte infusions. The most widely employed first line therapy for chronic GVHD is a combination of cyclosporine (CSA) and prednisolone, but patients who failed to respond to the initial steroid-based therapy have a poor outcome.1 Therefore, various agents have been investigated as salvage therapy for chronic GVHD, but there is no standard approach that is uniformly accepted.

Spatial and temporal expression of basic fibroblast growth factor protein during wound healing of rat skin

Background Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key part in wound healing.

CXCR4 Antagonist AMD3100 Accelerates Impaired Wound Healing in Diabetic Mice

The antagonism of CXC-chemokine receptor 4 (CXCR4) with AMD3100 improves cardiac performance after myocardial infarction by augmenting the recruitment of endothelial progenitor cells (EPCs) from the bone marrow to the regenerating vasculature. We investigated whether AMD3100 may accelerate diabetes-impaired wound healing through a similar mechanism. Skin wounds were made on the backs of leptin-receptor–deficient mice and treated with AMD3100 or saline. Fourteen days after treatment, wound closure was significantly more complete in AMD3100-treated mice (AMD3100: 87.0±2.6%, Saline: 33.1±1.8%; P<0.0001) and was accompanied by greater collagen-fiber formation, capillary density, smooth-muscle-containing vessel density, and monocyte/macrophage infiltration. On day 7 after treatment, AMD3100 was associated with higher circulating EPC and macrophage counts and with significantly upregulated mRNA levels of stromal-cell–derived factor 1 and platelet-derived growth-factor B in the wound bed. AMD3100 also promoted macrophage proliferation and phagocytosis and the migration and proliferation of diabetic mouse primary dermal fibroblasts and 3T3 fibroblasts, which express very little CXCR4. In conclusion, a single topical application of AMD3100 promoted wound healing in diabetic mice by increasing cytokine production, mobilizing bone-marrow EPCs, and enhancing the activity of fibroblasts and monocytes/macrophages, thereby increasing both angiogenesis and vasculogenesis. Not all of the AMD3100-mediated effects evolved through CXCR4 antagonism.

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.

Merkel cell carcinoma with partial spontaneous regression: An immunohistochemical, ultrastructural, and TUNEL labeling study

We report a case of Merkel cell carcinoma that partially regressed after biopsy. A 76-year-old woman presented with an 1 month history of a rapidly enlarging nodule on her left cheek. After biopsy, the nodule reduced to almost half the size and was excised 1 month later. The excised specimen showed a dense cluster of lymphocytes and fibrosis around the tumor nests. In addition, lymphocytes showed apposition with tumor cells. An immunohistologically dense, even infiltration of CD4+ and CD8+ cells was found around the tumor nests, and more CD8+ cells than CD4+ cells were seen in the tumor nests. By electron microscopy (EM), apoptosis of tumor cells and lymphocytes was observed. Many apoptotic cells were also detected by in situ nick end-labeling (TUNEL) of DNA-breaks, especially in the marginal area of tumor nests surrounded by dense lymphocytic infiltrates. It seems likely that T-cell immunity, which induces apoptosis of tumor cells, may have been involved in tumor regression.

Keratinocyte Growth Inhibition through the Modification of Wnt Signaling by Androgen in Balding Dermal Papilla Cells

CONTEXT/OBJECTIVE Androgen induces androgenetic alopecia (AGA), which has a regressive effect on hair growth from the frontal region of the scalp. Conversely, Wnt proteins are known to positively affect mammalian hair growth. We hypothesized that androgen reduces hair growth via an interaction with the Wnt signaling system. The objective of this study was to investigate the effect of androgen on Wnt signaling in dermal papilla (DP) cells. DESIGN The effect of androgen and Wnt3a on keratinocyte proliferation was measured by use of a coculture system consisting of DP cells and keratinocytes. The molecular mechanisms of androgen and Wnt pathway interactions in DP cells were examined by analyzing the expression, intracellular localization, and activity of the androgen receptor (AR) and also downstream Wnt signaling molecules. RESULTS Wnt3a-dependent keratinocyte growth was suppressed by the addition of dihydrotestosterone in coculture with DP cells that were derived from AGA patients, but growth was not suppressed in coculture with DP cells from non-AGA males. Whereas DP cells from both scalp regions expressed AR protein, the expression levels of AR and cotranslocation with beta-catenin, a downstream Wnt signaling molecule, were higher in DP cells of AGA patients than in DP cells from non-AGA males. In addition, significant suppression of Wnt signal-mediated transcription in response to dihydrotestosterone treatment was observed only in DP cells from AGA patients. CONCLUSION These results suggest that Wnt signaling in DP cells is regulated by androgen and this regulation plays a pivotal role in androgens action on hair growth.

Glomeruloid hemangioma in POEMS syndrome shows two different immunophenotypic endothelial cells

The case of a Japanese woman with glomeruloid hemangioma, an initial marker for POEMS syndrome, is reported. Her cutaneous lesions were multiple and consisted of glomeruloid hemangiomas, cherry‐type capillary hemangiomas, and a mixture of both. The specimens of glomeruloid hemangiomas were studied by paraffin section immunohistochemistry with a large panel of antibodies and electron microscopy, respectively. The lesions, whose size ranged from minute foci to large nodules, were composed of anastomosing vascular channels resembling renal glomeruli and had irregular lumina, often featuring capillaries and sinusoid‐like spaces. The vascular channels were lined by a single layer of endothelial cells, which showed two types of cells. The capillary‐type endothelium possessed large vesicular nuclei with open chromatin and large amount of cytoplasm. The sinusoidal endothelium possessed small basal nuclei with dense chromatin as well as scant amount of cytoplasm. The former cells had a characteristic CD31+/CD34+/UEA I +/CD68− phenotype. Some of these cells ultrastructurally showed intracytoplasmic lumen formation. The latter cells had a characteristic CD31+/CD34−/UEA I−/CD68+ phenotype. The present study shows that glomeruloid hemangioma has unique morphologic and immunologic features that differ from the traditional hemangiomas as well as littoral cell angioma of the spleen.