Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Targeted deletion of the murine corneodesmosin gene delineates its essential role in skin and hair physiology.

Controlled proteolytic degradation of specialized junctional structures, corneodesmosomes, by epidermal proteases is an essential process for physiological desquamation of the skin. Corneodesmosin (CDSN) is an extracellular component of corneodesmosomes and, although considerable debate still exists, genetic studies have suggested that the CDSN gene in the major psoriasis-susceptibility locus (PSORS1) may be responsible for susceptibility to psoriasis, a human skin disorder characterized by excessive growth and aberrant differentiation of keratinocytes. CDSN is also expressed in the inner root sheath of hair follicles, and a heterozygous nonsense mutation of the CDSN gene in humans is associated with scalp-specific hair loss of poorly defined etiology. Here, we have investigated the pathogenetic roles of CDSN loss of function in the development of skin diseases by generating a mouse strain with targeted deletion of the Cdsn gene. Cdsn-deficient mouse skin showed detachment of the stratum corneum from the underlying granular layer and/or detachment within the upper granular layers due to the disrupted integrity of the corneodesmosomes. When grafted onto immunodeficient mice, Cdsn-deficient skin showed rapid hair loss together with epidermal abnormalities resembling psoriasis. These results underscore the essential roles of CDSN in hair physiology and suggest functional relevance of CDSN gene polymorphisms to psoriasis susceptibility.

Ephrin-A3 not only increases the density of hair follicles but also accelerates anagen development in neonatal mice.

BACKGROUND: Ephrins are cell-membrane-bound ligands for Eph receptor tyrosine kinases (Eph). Although ephrins are known to regulate a variety of developmental processes, little is known of their role in hair development. Previously, we studied the gene expression of dermal papilla cells from androgenetic alopecia and found that ephrin-A3 was significantly down-regulated. OBJECTIVE: To characterize the expression of ephrin-A3 in the hair cycle and evaluate the effect of ephrin-A3 on hair growth. METHODS: We investigated gene expression and protein expression of each ephrin-As and EphAs in the skin of neonatal mice through the first and second hair cycle using quantitative PCR and immunohistochemical analysis, respectively. We also injected ephrin-A3 protein into the skin of neonatal mice and demonstrated the effect of ephrin-A3 on hair follicle development. RESULTS: Expression of ephrin-A3 revealed a rapid increase at the beginning of the anagen phase, a peak during the mid-anagen, and a rapid fading during the telogen phase. In addition, we found ephrin-A3 protein was expressed in the developing hair follicles with a characteristic spatiotemporal localization. Furthermore, injection of ephrin-A3 into the skin of neonatal mice markedly accelerated the differentiation process of hair follicles. In addition, injection of ephrin-A3 unexpectedly increased the number of hair follicles. CONCLUSION: These findings demonstrated that ephrin-A3 not only accelerates anagen development but also increases the density of hair follicles, and also suggested that an ephrin-A-EphA signal pathway is closely involved in hair follicle development.

Adenosine increases anagen hair growth and thick hairs in Japanese women with female pattern hair loss: a pilot, double-blind, randomized, placebo-controlled trial.

Adenosine upregulates the expression of vascular endothelial growth factor and fibroblast growth factor-7 in cultured dermal papilla cells. It has been shown that, in Japanese men, adenosine improves androgenetic alopecia due to the thickening of thin hair due to hair follicle miniaturization. To investigate the efficacy and safety of adenosine treatment to improve hair loss in women, 30 Japanese women with female pattern hair loss were recruited for this double-blind, randomized, placebo-controlled study. Volunteers used either 0.75% adenosine lotion or a placebo lotion topically twice daily for 12 months. Efficacy was evaluated by dermatologists and by investigators and in phototrichograms. As a result, adenosine was significantly superior to the placebo according to assessments by dermatologists and investigators and by self-assessments. Adenosine significantly increased the anagen hair growth rate and the thick hair rate. No side-effects were encountered during the trial. Adenosine improved hair loss in Japanese women by stimulating hair growth and by thickening hair shafts. Adenosine is useful for treating female pattern hair loss in women as well as androgenetic alopecia in men.

Novel ALK5 inhibitor TP0427736 reduces TGF-ß　induced growth inhibition in human outer root sheath cells and elongates anagen phase in mouse hair follicles.

BACKGROUND: Androgenic alopecia (AGA) occurs as a result of the contraction of the anagen phase because of the action of androgens on hair follicles. TGF-ß production from dermal papillae is enhanced by androgens, and growth inhibition of hair-follicle cells is induced by TGF-ß, and the hair cycle progresses from the anagen phase to the catagen phase. We investigated both the in vitro and in vivo potency of the newly identified ALK5 inhibitor TP0427736 {6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole}. METHODS: For in vitro study, kinase inhibitory activity was evaluated with ELISA, and inhibitory activity against TGF-ß-induced Smad2/3 phosphorylation in A549 cells and TGF-ß-induced growth inhibition of human outer root sheath cells were assayed using ELISA. For in vivo study, we used a mouse model that had been synchronized through dorsal hair depilation. RESULTS: TP0427736 inhibited ALK5 kinase activity with an IC50 of 2.72nM; this effect was 300-fold higher than the inhibitory effect on ALK3. In cell-based assays, TP0427736 inhibited Smad2/3 phosphorylation in A549 cells and decreased the growth inhibition of human outer root sheath cells. The topical application of TP0427736 significantly decreased Smad2 phosphorylation in mouse skin, and its repeated application suppressed the shortening of average hair follicle length during the transition from the late anagen phase to the catagen phase. CONCLUSIONS: TP0427736, a potent ALK5 inhibitor with appropriate in vitro and in vivo profiles, may serve as a potential new therapy for AGA.　.

Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice.

The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.

Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.

Chronic ultraviolet-B irradiation of the skin results in epidermal hyperplasia, degradation of extracellular matrix molecules, and formation of wrinkles. To characterize the biologic role of the vascular system in the mediation of ultraviolet-B-induced skin damage, we performed quantitative analyses of cutaneous blood vessels of mice after 10 wk of ultraviolet-B irradiation. Skin vascularization was greatly increased after chronic ultraviolet-B exposure with a significant increase of both the number and the size of dermal blood vessels, associated with upregulation of vascular endothelial growth factor expression in the hyperplastic epidermis. To directly study whether inhibition of angiogenesis may diminish ultraviolet-B-induced cutaneous damage, wild-type and transgenic mice with skin-specific overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 were subjected to the same ultraviolet-B irradiation regimen. Ultraviolet-B-irradiated thrombospondin-1 transgenic mice showed a significantly reduced skin vascularization, decreased endothelial cell proliferation, and increased endothelial cell apoptosis rates, compared with wild-type mice. Moreover, dermal photo-damage and wrinkle formation were greatly reduced in thrombospondin-1 transgenic mice. These results reveal an important role of the cutaneous vascular system in mediating ultraviolet-B-induced skin damage and suggest inhibition of angiogenesis as a potential new approach for the prevention of chronic cutaneous photo-damage.

An N-terminal 80 kDa recombinant fragment of human thrombospondin-2 inhibits vascular endothelial growth factor induced endothelial cell migration in vitro and tumor growth and angiogenesis in vivo.

We have previously shown that stable overexpression of the thrombospondin-2 (TSP-2) gene inhibited the tumor growth and angiogenesis of human squamous cell carcinoma xenotransplants. To investigate the potential antitumoral efficacy of systemic TSP-2 therapy, we expressed a recombinant 80 kDa fragment of human TSP-2 (TSP-2/NTF), encompassing the N-terminal globular region through the three type 1 repeats, in human kidney 293 EBNA cells, using a modified pCEP4 expression vector. Daily intraperitoneal injections of TSP-2/NTF resulted in a significant inhibition of the growth of human A431 squamous cell carcinomas in vivo and in reduced tumor vascularization. To further investigate possible mechanisms of the antiangiogenic activity of TSP-2/NTF, several in vitro angiogenesis assays were performed in human dermal microvascular endothelial cells. TSP-2/NTF inhibited vascular endothelial growth factor induced migration of human dermal microvascular endothelial cells and inhibited tube formation on Matrigel in vitro. TSP-2/NTF also inhibited vascular endothelial growth factor induced angiogenesis in an in vivo Matrigel assay. Moreover, TSP-2/NTF potently induced human dermal microvascular endothelial cell apoptosis in vitro but did not affect A431 tumor cell proliferation or apoptosis. These findings identify TSP-2/NTF as a potent systemic inhibitor of tumor growth and angiogenesis, acting by direct inhibition of several endothelial cell functions involved in neovascularization.

Keratinocytes can differentiate into eccrine sweat ducts in vitro: involvement of epidermal growth factor and fetal bovine serum.

BACKGROUND: in addition to formation of an epidermal sheet and dermal substitution, reconstruction of skin that possesses functionality is an important goal for dermatologists. OBJECTIVE: we attempted to regenerate eccrine sweat glands in vitro. METHODS: we constructed skin equivalent models with various combination of normal human keratinocytes and fibroblasts and also examined the effect of various growth factors. RESULTS: we found that keratinocytes invaded the collagen gels and formed eccrine duct-like structures, only when (i) the culture media contained at least 15 ng/ml of epidermal growth factor (EGF) and fetal bovine serum (FBS), (ii) the keratinocytes were derived from young donors, and (iii) fibroblasts were present in the gel. Interestingly, when cultured under the same conditions eccrine gland duct cells were unable to invade the gel. Immunohistochemical analyses revealed induction of carcinoembryonic antigen by EGF at the inner part of the eccrine duct-like structures. Proliferating cell nuclear antigen was expressed mainly in basal layers of the epithelia but was not observed in the deeply invaded part. Cytokeratin profiles of the reconstructed epithelia were consistent with those of the regenerating epidermis and partly with the eccrine sweat duct. CONCLUSIONS: although not perfect model, these results indicate that 'young' keratinocytes could differentiate into/toward eccrine sweat ducts in vitro in the presence of EGF and FBS in cooperation with dermal fibroblasts.

A critical role of placental growth factor in the induction of inflammation and edema formation.

Angiogenesis is a prominent feature of a number of inflammatory human diseases, including rheumatoid arthritis, psoriasis, and cutaneous delayed-type hypersensitivity (DTH) reactions. Up-regulation of placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, has been found in several conditions associated with pathologic angiogenesis; however, its distinct role in the control of angiogenesis has remained unclear. To directly investigate the biologic function of PlGF in cutaneous inflammation and angiogenesis, DTH reactions were investigated in the ear skin of wild-type mice, of PlGF-deficient mice, and of transgenic mice with targeted overexpression of human PlGF-2 in epidermal keratinocytes, driven by a keratin 14 promoter expression construct. Chronic transgenic delivery of PlGF-2 to murine epidermis resulted in a significantly increased inflammatory response, associated with more pronounced vascular enlargement, edema, and inflammatory cell infiltration than seen in wild-type mice. Conversely, PlGF deficiency resulted in a diminished and abbreviated inflammatory response, together with a reduction of inflammatory angiogenesis and edema formation. VEGF expression was up-regulated at a comparable level in the inflamed skin of all genotypes. These findings reveal that placental growth factor plays a critical role in the control of cutaneous inflammation, and they suggest inhibition of PlGF bioactivity as a potential new approach for anti-inflammatory therapy.