Phosphodiesterase 4 is overexpressed in keloid epidermal scars and its inhibition reduces keratinocyte fibrotic alterations.

BACKGROUND: Epidermal remodeling and hypertrophy are hallmarks of skin fibrotic disorders, and keratinocyte to mesenchymal (EMT)-like transformations drive epidermis alteration in skin fibrosis such as keloids and hypertrophic scars (HTS). While phosphodiesterase 4 (PDE4) inhibitors have shown effectiveness in various fibrotic disorders, their role in skin fibrosis is not fully understood. This study aimed to explore the specific role of PDE4B in epidermal remodeling and hypertrophy seen in skin fibrosis. METHODS: In vitro experiments examined the effects of inhibiting PDE4A-D (with Roflumilast) or PDE4B (with siRNA) on TGFß1-induced EMT differentiation and dedifferentiation in human 3D epidermis. In vivo studies investigated the impact of PDE4 inhibition on HOCl-induced skin fibrosis and epidermal hypertrophy in mice, employing both preventive and therapeutic approaches. RESULTS: The study found increased levels of PDE4B (mRNA, protein) in keloids > HTS compared to healthy epidermis, as well as in TGFß-stimulated 3D epidermis. Keloids and HTS epidermis exhibited elevated levels of collagen Iα1, fibronectin, αSMA, N-cadherin, and NOX4 mRNA, along with decreased levels of E-cadherin and ZO-1, confirming an EMT process. Inhibition of both PDE4A-D and PDE4B prevented TGFß1-induced Smad3 and ERK1/2 phosphorylation and mesenchymal differentiation in vitro. PDE4A-D inhibition also promoted mesenchymal dedifferentiation and reduced TGFß1-induced ROS and keratinocyte senescence by rescuing PPM1A, a Smad3 phosphatase. In vivo, PDE4 inhibition mitigated HOCl-induced epidermal hypertrophy in mice in both preventive and therapeutic settings. CONCLUSIONS: Overall, the study supports the potential of PDE4 inhibitors, particularly PDE4B, in treating skin fibrosis, including keloids and HTS, shedding light on their functional role in this condition.

Increased Expression of Galectin-3 in Skin Fibrosis: Evidence from In Vitro and In Vivo Studies.

Skin fibrosis is a hallmark of a wide array of dermatological diseases which can greatly impact the patients' quality of life. Galectin-3 (GAL-3) has emerged as a central regulator of tissue fibrosis, playing an important pro-fibrotic role in numerous organs. Various studies are highlighting its importance as a skin fibrotic diseases biomarker; however, there is a need for further studies that clarify its role. This paper aims to ascertain whether the expression of GAL-3 is increased in relevant in vitro and in vivo models of skin fibrosis. We studied the role of GAL-3 in vitro using normal human dermal fibroblasts (NHDF) and fibrocytes. In addition, we used a skin fibrosis murine model (BALB/c mice) and human biopsies of healthy or keloid tissue. GAL-3 expression was analyzed using real time PCR, Western blot and immunostaining techniques. We report a significantly increased expression of GAL-3 in NHDF and fibrocytes cell cultures following stimulation with transforming growth factor ß1 (TGFß1). In vivo, GAL-3 expression was increased in a murine model of systemic sclerosis and in human keloid biopsies. In sum, this study underlines the involvement of GAL-3 in skin fibrosis using several models of the disease and highlights its role as a relevant target.

Phosphodiesterase 4 is overexpressed in human keloids and its inhibition reduces fibroblast activation and skin fibrosis.

There is a pressing medical need for improved treatments in skin fibrosis including keloids and hypertrophic scars (HTS). This study aimed to characterize the role of phosphodiesterase 4 (PDE4), specifically PDE4B in fibrotic skin remodeling in vitro and in vivo. In vitro, effects of PDE4A-D (Roflumilast) or PDE4B (siRNA) inhibition on TGFß1-induced myofibroblast differentiation and dedifferentiation were studied in normal (NHDF) and keloid (KF) human dermal fibroblasts. In vivo, the role of PDE4 on HOCl-induced skin fibrosis in mice was addressed in preventive and therapeutic protocols. PDE4B (mRNA, protein) was increased in Keloid > HTS compared to healthy skin and in TGFß-stimulated NHDF and KF. In Keloid > HTS, collagen Iα1, αSMA, TGFß1 and NOX4 mRNA were all elevated compared to healthy skin confirming skin fibrosis. In vitro, inhibition of PDE4A-D and PDE4B similarly prevented TGFß1-induced Smad3 and ERK1/2 phosphorylation and myofibroblast differentiation, elevated NOX4 protein and proliferation in NHDF. PDE4A-D inhibition enabled myofibroblast dedifferentiation and curbed TGFß1-induced reactive oxygen species and fibroblast senescence. In KF PDE4A-D inhibition restrained TGFß1-induced Smad3 and ERK1/2 phosphorylation, myofibroblast differentiation and senescence. Mechanistically, PDE4A-D inhibition rescued from TGFß1-induced loss in PPM1A, a Smad3 phosphatase. In vivo, PDE4 inhibition mitigated HOCl-induced skin fibrosis in mice in preventive and therapeutic protocols. The current study provides novel evidence evolving rationale for PDE4 inhibitors in skin fibrosis (including keloids and HTS) and delivered evidence for a functional role of PDE4B in this fibrotic condition.

ß2-adrenoreceptors control human skin microvascular reactivity.

Topical α1- and α2-adrenoreceptor (ADRA1 and 2) agonists are effective in alleviating permanent vasodilation and facial erythema associated with rosacea by inducing skin vasoconstriction. Although ß-adrenoreceptor (ADRB) antagonists are used off-label for rosacea, pharmacological and pharmacodynamic data pertaining to these receptors in skin micro-vessels are lacking. Objectives: To analyse the expression of different adrenergic receptors and their contribution to vasoreactivity in skin micro-vessels. Small arteries (500-800 µm) and arterioles (<200 µm) were studied in human foreskin tissue. Specifically, ADR-A1, -A2, -B1 and -B2 expression was assayed by immunofluorescence, polymerase chain reaction (PCR), and western blotting. Small skin artery reactivity was evaluated using ex vivo myography (500-800 µm) or a visible microscope perfusion system with precision-cut skin slices (<200 µm). ADRB2 was the most highly expressed receptor in small skin arteries and arterioles, followed by ADRA2. ADRA2 activation via brimonidine-induced vasoconstriction was greater in skin arterioles than in small skin arteries, and more potent than that with norepinephrine (NE). The use of prazosin (ADRA1 inhibitor) partially attenuated brimonidine-induced vasoconstriction, indicating some activation of ADRA1 by brimonidine, at least at 10-µM concentrations. Small skin arteries and arterioles, pre-treated with prazosin and stimulated with NE, exhibited ADRB2-mediated vasodilation, which was inhibited by the beta blockers, propranolol or timolol. This study shows that ADRB2 is predominantly expressed in small skin arteries and arterioles, and that ADRBs plays a functional role in vasodilation. The data presented here indicate that ADRBs can be a therapeutic target for the treatment of rosacea.

IFATS collection: Identification of hemangioblasts in the adult human adipose tissue.

The stromal-vascular fraction (SVF) of human adipose tissue contains, among other cell types, mesenchymal stem cells and precursors of adipocyte and endothelial cells. Here we show that, in addition, the nonhematopoietic fraction of the SVF has hematopoietic activity, since all types of hematopoietic colony-forming units (CFUs) developed when cultured in methylcellulose-based medium. This hematopoietic activity was restricted to the CD45(-)CD105(+) cell subset, well correlated with KDR(+) cell content, and increased after culture with a combination of early-acting hematopoietic cytokines. Most of the CD45(-)KDR(+)CD105(+) cells were nonadherent and did not express CD31, and this subset included both CD34(-) and CD34(+) cells. Moreover, these nonadherent cells migrated in response to KDR gradient, and when they were cultured in the presence of both hematopoietic and endothelial growth factors, a wave of CFUs was followed by a wave of mixed colonies comprising adherent elongated and nonadherent round hematopoietic cells. These mixed hematopoietic-endothelial (Hem-End) colonies were able to generate secondary Hem-End colonies and exhibited both hematopoietic and endothelial activity, as demonstrated by in vitro functional assays. These findings demonstrate for the first time the existence of primitive mesodermal progenitors within the SVF of human adipose tissue that exhibit in vitro hematopoietic and hemangioblastic activities, susceptible to being used in cell therapy and basic cell research. Disclosure of potential conflicts of interest is found at the end of this article.

Fibroblast-Negative CD34-Negative Cells from Human Adipose Tissue Contain Mesodermal Precursors for Endothelial and Mesenchymal Cells.

There is considerable evidence that stem/progenitor cells reside in the vasculature during the prenatal and postnatal stages. The stromal vascular fraction (SVF) of human adipose tissue is markedly rich in blood vessels, and it is a source of mesenchymal/stromal cells (MSCs). Therefore, we hypothesized that, in addition to MSCs, the SVF may contain other mesodermal precursors. However, the SVF has a high content of CD34(+) cells with high proliferative capacity, which can prevent the growth of the most quiescent cells. By using an antifibroblast (FIB) antibody coupled to microbeads, we show that ∼ 90%-95% of the nonhematopoietic CD34(+) cells were retained in the CD45(-)FIB(+) fraction. Reverse transcription-polymerase chain reaction analysis revealed that the CD45(-)FIB(-)CD34(-) cell fraction expressed higher mRNA levels of KDR and GATA2 than its complementary CD45(-)FIB(-)CD34(+) cell fraction, which contained the SVF endothelial cells. Surprisingly, when CD45(-)FIB(-)CD34(-) cells were cultured in endothelial growth medium, they gave rise to endothelial colonies and mesenchymal colonies. Moreover, when CD45(-)FIB(-)CD34(-) cells were cultured in embryonic stem cell expansion medium, they gave rise to cells exhibiting the full range of phenotypes observed in the freshly isolated SVF, including CD34(+) and CD31(+) cells. Together, these results suggest that the CD45(-)FIB(-)CD34(-) cells within the SVF of human adipose tissue function as mesodermal precursors of mesenchymal and endothelial cells.

Spanish Multidisciplinary Melanoma Group (GEM) guidelines for the management of patients with advanced melanoma.

Advanced melanoma is a relatively uncommon condition whose therapeutic management has undergone major changes over the past four years. The present article aims to establish recommendations for the management of these patients based on the best available evidence reached by consensus of a group of professionals familiar in the treatment of these patients. These professionals, belonging to Spanish Multidisciplinary Melanoma Group, reviewed the diagnostic process and the incorporation of new techniques of molecular diagnosis of advanced disease; treatment and monitoring of stage III both as adjuvant locoregional treatments have been addressed, as well as new therapies for stage IV. We have reviewed the palliative treatment alternatives for disseminated disease, such as surgery, radiotherapy or non-cytotoxic systemic treatments. Finally, we have also reviewed the most relevant toxicities of new drugs and their management in clinical practice.

Human adipose tissue-resident monocytes exhibit an endothelial-like phenotype and display angiogenic properties.

INTRODUCTION: Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization. METHODS: The stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay. RESULTS: CD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells. Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs. Quantitative analysis of angiogenesis at 14 days after implantation demonstrated that neovascularization of the implants containing SVF CD14+ cells or PB monocytes previously co-cultured with MSCs was 3.5 or 2 times higher than that observed in the implants with SVF-derived MSCs. Moreover, immunofluorescence of Matrigel sections revealed that SVF CD14+ cells differentiated into endothelial cells and contributed to vascular endothelium. CONCLUSIONS: The results from this study suggest that adipose tissue-resident monocytes should contribute to tissue vascularization. Because SVF CD14+ cells were more efficient in inducing angiogenesis than SVF-derived MSCs, and differentiated into vascular endothelial cells, they may constitute a new cell source for cell-based therapeutic angiogenesis.

Human adipose tissue contains erythroid progenitors expressing fetal hemoglobin.

AIM: To investigate the origin of hematopoietic progenitors contained in the stromal vascular fraction (SVF) of human adipose tissue. METHODS: Tissue samples obtained from lipectomies were subjected to enzymatic digestion with collagenase to obtain a single-cell suspension. The centrifuged cell pellet, termed SVF, was separated immunomagnetically into CD45(+) and CD45(-) cells and cultured in serum-free medium containing hematopoietic cytokines. The freshly isolated and cultured cells were evaluated to determine their ability to form hematopoietic colony-forming units in clonogenic assays and for the expression of certain hematopoietic transcription factors by reverse transcription-polymerase chain reaction; the gene expression level was compared to that in CD34(+) hematopoietic progenitor cells from cord blood (CB) and adult peripheral blood (PB). To characterize erythroid progenitors, burst-forming units-erythroid (BFU-E) were developed in a semisolid medium under different culture conditions, and the hemoglobin composition and globin gene expression in the erythroid colonies were determined. RESULTS: The transcription factors SCL/TAL1, RUNX1, RUNX2 and GATA2 were expressed in both the CD45(+) and CD45(-) SVF populations; however, in contrast to our observations in the CD34(+) cells from CB and adult PB, GATA1 was not detected. Nevertheless, GATA1 could be detected in the SVF cells after seven days in culture, whereas its expression was upregulated in the CB CD34(+) cells. The analysis of BFU-E-derived colonies revealed that virtually all erythroid cells produced by SVF cells expressed fetal hemoglobin, and the γ-globin mRNA levels ranged between those obtained in the adult- and neonatal-derived erythroid cells. Moreover, the SVF-derived erythroid cells synthesized similar levels of α- and ß-globin mRNA, whereas the α-globin transcript levels were consistently higher those of ß-globin in the cells derived from CB or PB CD34(+) cells. Furthermore, although the cellular distribution of hemoglobin in the erythroid cells derived from the CD34(+) cells obtained from hematopoietic tissues was dependent on the presence or absence of serum in the culture medium, this did not affect the SVF-derived erythroid cells. CONCLUSION: Our results demonstrate that hematopoietic progenitors in SVF have molecular and functional features that differ from those exhibited by circulating progenitors, suggesting the possibility of a different origin.