Spontaneous gene repair, also called revertant mosaicism, has been documented in several genetic disorders involving organs that undergo self-regeneration, including the skin. Genetic reversion may occur through different mechanisms, and in a single individual, the mutation can be repaired in various ways. Here we describe a disseminated pattern of revertant mosaicism observed in 6 patients with Kindler syndrome (KS), a genodermatosis caused by loss of kindlin-1 (encoded by FERMT1) and clinically characterized by patchy skin pigmentation and atrophy. All patients presented duplication mutations (c.456dupA and c.676dupC) in FERMT1, and slipped mispairing in direct nucleotide repeats was identified as the reversion mechanism in all investigated revertant skin spots. The sequence around the mutations demonstrated high propensity to mutations, favoring both microinsertions and microdeletions. Additionally, in some revertant patches, mitotic recombination generated areas with homozygous normal keratinocytes. Restoration of kindlin-1 expression led to clinically and structurally normal skin. Since loss of kindlin-1 severely impairs keratinocyte proliferation, we predict that revertant cells have a selective advantage that allows their clonal expansion and, consequently, the improvement of the skin condition.
Dimitra Kiritsi, Yinghong He, Anna M.G. Pasmooij, Meltem Onder, Rudolf Happle, Marcel Jonkman, Leena Bruckner-Tuderman, Cristina Has
BM-derived endothelial progenitor cells (EPCs) are critical and essential for neovascularization in tissue repair and tumorigenesis. EPCs migrate from BM to tissues via the bloodstream, but specific chemotactic cues have not been identified. Here we show in mice that the absence of CCR5 reduced vascular EPC accumulation and neovascularization, but not macrophage recruitment, and eventually delayed healing in wounded skin. When transferred into Ccr5–/– mice, Ccr5+/+ BM cells, but not Ccr5–/– cells, accumulated in the wound site, were incorporated into the vasculature, and restored normal neovascularization. Consistent with these observations, CCL5 induced in vitro EPC migration in a CCR5-dependent manner. Moreover, expression of VEGF and TGF-β was substantially diminished at wound sites in Ccr5–/– mice, which suggests that EPCs are important not only as the progenitors of endothelial cells, but also as the source of growth factors during tissue repair. Taken together, these data identify the CCL5/CCR5 interaction as what we believe to be a novel molecular target for modulation of neovascularization and eventual tissue repair.
Yuko Ishida, Akihiko Kimura, Yumi Kuninaka, Masanori Inui, Kouji Matsushima, Naofumi Mukaida, Toshikazu Kondo
Zinc deficiency can be an inherited disorder, in which case it is known as acrodermatitis enteropathica (AE), or an acquired disorder caused by low dietary intake of zinc. Even though zinc deficiency diminishes cellular and humoral immunity, patients develop immunostimulating skin inflammation. Here, we have demonstrated that despite diminished allergic contact dermatitis in mice fed a zinc-deficient (ZD) diet, irritant contact dermatitis (ICD) in these mice was more severe and prolonged than that in controls. Further, histological examination of ICD lesions in ZD mice revealed subcorneal vacuolization and epidermal pallor, histological features of AE. Consistent with the fact that ATP release from chemically injured keratinocytes serves as a causative mediator of ICD, we found that the severe ICD response in ZD mice was attenuated by local injection of soluble nucleoside triphosphate diphosphohydrolase. In addition, skin tissue from ZD mice with ICD showed increased levels of ATP, as did cultured wild-type keratinocytes treated with chemical irritants and the zinc-chelating reagent TPEN. Interestingly, numbers of epidermal Langerhans cells (LCs), which play a protective role against ATP-mediated inflammatory signals, were decreased in ZD mice as well as samples from ZD patients. These findings suggest that upon exposure to irritants, aberrant ATP release from keratinocytes and impaired LC-dependent hydrolysis of nucleotides may be important in the pathogenesis of AE.
Tatsuyoshi Kawamura, Youichi Ogawa, Yuumi Nakamura, Satoshi Nakamizo, Yoshihiro Ohta, Hajime Nakano, Kenji Kabashima, Ichiro Katayama, Schuichi Koizumi, Tatsuhiko Kodama, Atsuhito Nakao, Shinji Shimada
Wounds that fail to heal in a timely manner, for example, diabetic foot ulcers, pose a health, economic, and social problem worldwide. For decades, conventional wisdom has pointed to growth factors as the main driving force of wound healing; thus, growth factors have become the center of therapeutic developments. To date, becaplermin (recombinant human PDGF-BB) is the only US FDA-approved growth factor therapy, and it shows modest efficacy, is costly, and has the potential to cause cancer in patients. Other molecules that drive wound healing have therefore been sought. In this context, it has been noticed that wounds do not heal without the participation of secreted Hsp90α. Here, we report that a 115-aa fragment of secreted Hsp90α (F-5) acts as an unconventional wound healing agent in mice. Topical application of F-5 peptide promoted acute and diabetic wound closure in mice far more effectively than did PDGF-BB. The stronger effect of F-5 was due to 3 properties not held by conventional growth factors: its ability to recruit both epidermal and dermal cells; the fact that its ability to promote dermal cell migration was not inhibited by TGF-β; and its ability to override the inhibitory effects of hyperglycemia on cell migration in diabetes. The discovery of F-5 challenges the long-standing paradigm of wound healing factors and reveals a potentially more effective and safer agent for healing acute and diabetic wounds.
Chieh-Fang Cheng, Divya Sahu, Fred Tsen, Zhengwei Zhao, Jianhua Fan, Rosie Kim, Xinyi Wang, Kathryn O’Brien, Yong Li, Yuting Kuang, Mei Chen, David T. Woodley, Wei Li
Pemphigus vulgaris (PV) is a severe autoimmune disease involving blistering of the skin and mucous membranes. It is caused by autoantibodies against desmoglein 3 (Dsg3), an adhesion molecule critical for maintaining epithelial integrity in the skin, oral mucosa, and esophagus. Knowing the antigen targeted by the autoantibodies renders PV a valuable model of autoimmunity. Recently, a role for Dsg3-specific CD4+ T helper cells in autoantibody production was demonstrated in a mouse model of PV, but whether these cells exert cytotoxicity in the tissues is unclear. Here, we analyzed 3 Dsg3-specific TCRs using transgenic mice and retrovirus induction. Dsg3-specific transgenic (Dsg3H1) T cells underwent deletion in the presence of Dsg3 in vivo. Dsg3H1 T cells that developed in the absence of Dsg3 elicited a severe pemphigus-like phenotype when cotransferred into immunodeficient mice with B cells from Dsg3–/– mice. Strikingly, in addition to humoral responses, T cell infiltration of Dsg3-expressing tissues led to interface dermatitis, a distinct form of T cell–mediated autoimmunity that causes keratinocyte apoptosis and is seen in various inflammatory/autoimmune skin diseases, including paraneoplastic pemphigus. The use of retrovirally generated Dsg3-specific T cells revealed that interface dermatitis occurred in an IFN-γ– and TCR avidity–dependent manner. This model of autoimmunity demonstrates that T cells specific for a physiological skin-associated autoantigen are capable of inducing interface dermatitis and should provide a valuable tool for further exploring the immunopathophysiology of T cell–mediated skin diseases.
Hayato Takahashi, Michiyoshi Kouno, Keisuke Nagao, Naoko Wada, Tsuyoshi Hata, Shuhei Nishimoto, Yoichiro Iwakura, Akihiko Yoshimura, Taketo Yamada, Masataka Kuwana, Hideki Fujii, Shigeo Koyasu, Masayuki Amagai
Androgenetic alopecia (AGA), also known as common baldness, is characterized by a marked decrease in hair follicle size, which could be related to the loss of hair follicle stem or progenitor cells. To test this hypothesis, we analyzed bald and non-bald scalp from AGA individuals for the presence of hair follicle stem and progenitor cells. Cells expressing cytokeratin15 (KRT15), CD200, CD34, and integrin, α6 (ITGA6) were quantitated via flow cytometry. High levels of KRT15 expression correlated with stem cell properties of small cell size and quiescence. These KRT15hi stem cells were maintained in bald scalp samples. However, CD200hiITGA6hi and CD34hi cell populations — which both possessed a progenitor phenotype, in that they localized closely to the stem cell–rich bulge area but were larger and more proliferative than the KRT15hi stem cells — were markedly diminished. In functional assays, analogous CD200hiItga6hi cells from murine hair follicles were multipotent and generated new hair follicles in skin reconstitution assays. These findings support the notion that a defect in conversion of hair follicle stem cells to progenitor cells plays a role in the pathogenesis of AGA.
Luis A. Garza, Chao-Chun Yang, Tailun Zhao, Hanz B. Blatt, Michelle Lee, Helen He, David C. Stanton, Lee Carrasco, Jeffrey H. Spiegel, John W. Tobias, George Cotsarelis
Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice with epidermal expression of human TGF-β1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein–coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue. High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders.
John R. Zibert, Katrin Wallbrecht, Margarete Schön, Lluis M. Mir, Grete K. Jacobsen, Veronique Trochon-Joseph, Céline Bouquet, Louise S. Villadsen, Ruggero Cadossi, Lone Skov, Michael P. Schön
Amputation as a result of impaired wound healing is a serious complication of diabetes. Inadequate angiogenesis contributes to poor wound healing in diabetic patients. Endothelial progenitor cells (EPCs) normally augment angiogenesis and wound repair but are functionally impaired in diabetics. Here we report that decreased expression of manganese superoxide dismutase (MnSOD) in EPCs contributes to impaired would healing in a mouse model of type 2 diabetes. A decreased frequency of circulating EPCs was detected in type 2 diabetic (db/db) mice, and when isolated, these cells exhibited decreased expression and activity of MnSOD. Wound healing and angiogenesis were markedly delayed in diabetic mice compared with normal controls. For cell therapy, topical transplantation of EPCs onto excisional wounds in diabetic mice demonstrated that diabetic EPCs were less effective than normal EPCs at accelerating wound closure. Transplantation of diabetic EPCs after MnSOD gene therapy restored their ability to mediate angiogenesis and wound repair. Conversely, siRNA-mediated knockdown of MnSOD in normal EPCs reduced their activity in diabetic wound healing assays. Increasing the number of transplanted diabetic EPCs also improved the rate of wound closure. Our findings demonstrate that cell therapy using diabetic EPCs after ex vivo MnSOD gene transfer accelerates their ability to heal wounds in a mouse model of type 2 diabetes.
Eric J. Marrotte, Dan-Dan Chen, Jeffrey S. Hakim, Alex F. Chen
Pemphigus is a life-threatening autoimmune disease in which antibodies specific for desmogleins (Dsgs) cause loss of keratinocyte cell adhesion and blisters. In order to understand how antibodies cause pathogenicity and whether there are commonalities among antibodies in different patients that could ultimately be used to target specific therapy against these antibodies, we characterized Dsg-specific mAbs cloned by phage display from 3 patients with pemphigus vulgaris and 2 with pemphigus foliaceus. Variable heavy chain gene usage was restricted, but similar genes were used for both pathogenic and nonpathogenic mAbs. However, the heavy chain complementarity-determining region 3 (H-CDR3) of most pathogenic, but not nonpathogenic, mAbs shared an amino acid consensus sequence. Randomization of the H-CDR3 and site-directed mutagenesis indicated that changes in this sequence could block pathogenicity but not necessarily binding. In addition, for 2 antibodies with longer H-CDR3s, a tryptophan was critical for pathogenicity but not binding, a result that is consistent with blocking the tryptophan acceptor site that is thought to be necessary for Dsg-mediated adhesion. These studies indicate that H-CDR3 is critical for pathogenicity of a human autoantibody, that a small region (even 1 amino acid) can mediate pathogenicity, and that pathogenicity can be uncoupled from binding in these antibodies.
Jun Yamagami, Aimee S. Payne, Stephen Kacir, Ken Ishii, Don L. Siegel, John R. Stanley
Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications.
Sandra Chapman, Xuefeng Liu, Craig Meyers, Richard Schlegel, Alison A. McBride