Calcimycin Suppresses S100A4 Expression and Inhibits the Stimulatory Effect of Transforming Growth Factor ß1 on Keloid Fibroblasts.

Recent researches have indicated that S100A4 participates in tissue fibrosis, whereas calcimycin inhibits this process as a novel S100A4 transcription inhibitor. However, the relationship and mechanisms between calcimycin and S100A4 in keloid fibroblasts (KFs) remain unknown. The present research was aimed to evaluate the effect of calcimycin on S100A4 expression and pathogenesis in KFs. Keloid fibroblasts were cultured and exposed to different concentrations of calcimycin in the absence or presence of transforming growth factor ß1 (TGF-ß1). The results showed that the expression of S100A4 was significantly increased in keloid derived fibroblasts compared with normal skin fibroblasts. Calcimycin depressed S100A4 in a concentration- and time-dependent manner. Moreover, calcimycin suppressed TGF-ß1-induced collagen type I, fibronectin, and α-smooth muscle actin expression and cell viability in cultured KFs. Furthermore, calcimycin modulated expression of TGF-ß/Smad target genes Smad7 and phosphorylation of TGF-ß1-induced Smad2/3. This research for the first time confirmed the presence of S100A4 in KFs. Calcimycin inhibits the expression of S100A4, as well as KF proliferation and migration and extracellular matrix (ECM) synthesis. Taken together, these results indicate that calcimycin might be a therapeutic candidate to keloid or other related fibrotic disorders.

Role of Homeodomain-Interacting Protein Kinase 2 in the Pathogenesis of Tissue Fibrosis in Keloid-Derived Keratinocytes.

Epithelial-mesenchymal transition (EMT) plays a critical role in fibrotic keloid formation, which is characterized by excessive collagen and extracellular matrix synthesis and deposition. Growing evidence suggests that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) acts upstream of several major fibrosis signaling pathways; however, the role of HIPK2 in the keloid fibrogenesis remains unknown. In the current study, we investigated the roles of HIPK2 in the pathogenesis of keloids. Primary normal skin and keloid keratinocytes were cultured and pretreated with transforming growth factor (TGF)-ß1. Next, keratinocytes were transfected with scrambled small interfering RNA (siRNA) and anti-HIPK2 siRNA. The TGF-ß1-associated HIPK2 alterations were investigated by quantitative real-time polymerase chain reaction. Protein levels were analyzed by western blotting. The HIPK2 was markedly increased in the keloid-derived keratinocytes compared with normal skin keratinocytes. In addition, HIPK2 induced the expression of EMT markers in normal skin keratinocytes by TGF-ß1-SMAD family member 3 (SMAD3). The effect of TGF-ß1-related EMT markers and SMAD3 phosphorylation in response to added TGF-ß1 was significantly abrogated when the cells were transfected with HIPK2 siRNA. We conclude that HIPK2 is a crucial factor in the pathogenesis of keloids, suggesting that HIPK2 might be a novel potential drug target for antikeloid therapy.

NADPH oxidase-2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis.

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor-beta1 (TGF-ß1) exerts pro-oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF-ß1-induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real-time Q-PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock-down analysis. Hydrogen peroxide production and 2',7'-dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non-phagocytic NADPH oxidase mRNA. TGF-ß1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF-ß1 phosphorylated MAPKs and increased activator protein-1 (AP-1) in a redox-sensitive manner, and NOX2 suppression inhibited baseline and TGF-ß1-mediated stimulation of Smad2 phosphorylation. Moreover, TGF-ß1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI-1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.

Effects of elevated carbon dioxide (CO2)and ozone (O3)concentrations on ectoenzyme activities in rice rhizospheric soil.

Rising atmospheric carbon dioxide (CO2) and ozone (O3) concentrations are the main global change drivers. Soil ectoenzymes play an important role in maintaining soil ecosystem services. Exploring the responses of soil ectoenzymes to elevated CO2 and O3 concentrations is important for combating global climate change. In this study, we simulated elevated CO2 concentrations (+200 µmol·mol-1, eCO2), elevated O3 concentrations (0.04 µmol·mol-1, eO3), and their combination (eCO2+eO3) in open-top chambers (OTCs), and investigated the responses of rhizospheric soil ectoenzyme activities. The results showed that eCO2 significantly increased the ß-D-Glucosidase (ßG) activity by 73.0%, and decreased that of polyphenol oxidase (PHO), peroxidase (PEO), and acid phosphatase (AP) by 48.9%, 46.6% and 72.9% respectively, but did not affect that of cellulose hydrolase (CBH) and ß-N-Acetylglucosaminidase (NAG). eO3 significantly reduced the activities of CBH and AP by 34.2% and 30.4%, respectively. The activities of PHO and AP were reduced by 87.3% and 32.3% under the eCO2+eO3 compared with the control, respectively. Results of the principal coordinate analysis, permutation multivariate analysis of variance and redundancy analysis showed that both elevated CO2 and O3 significantly affected soil ectoenzyme activities, with stronger effects of elevated CO2 than elevated O3. Root nitrogen content, root carbon to nitrogen ratio, soil microbial biomass carbon and nitrate nitrogen were the main drivers of soil ectoenzyme activities under elevated CO2 and O3. Elevated O3 could partially neutralize the effects of elevated CO2 on soil ectoenzyme activities. In conclusion, elevated CO2 and O3 restrained the activities of most soil ectoenzyme, suggesting that climate change would threat soil ecosystem services and functions in the agroecosystem.

Pre-expanded muscle-sparing Latissimus Dorsi free flap: An ideal option for the reconstruction of extensive anterior knee contractures.

BACKGROUND: Extensive and complex contractures in the anterior knee area can pose a significant challenge for reconstruction due to insufficient skin and soft tissue coverage and poor cosmetic and functional outcomes using traditional methods. We presented our experience with pre-expanded muscle-sparing latissimus dorsi (LD) free flap as an alternative option for large-scale anterior knee reconstruction. METHODS: From January 2016 to December 2020, we applied this surgical technique in six patients with large postburn or post-traumatic contractures of the anterior knee. After tissue expansion of several months, the expanded muscle-sparing LD free flap was harvested and transferred to resurface the lesions. Operative procedures, postoperative complications, and long-term outcomes were evaluated. RESULTS: A total of six patients aged 7 to 32 years (mean: 20 years) were reconstructed successfully without any major complication. The flap ranged from 20 × 8 cm to 40 × 16 cm. All donor sites were primarily closed. Follow-up (range: 12 to 24 months) evaluation showed satisfactory results in both cosmetic and functional aspects. CONCLUSIONS: Pre-expanded muscle-sparing LD free flap is a reliable and effective choice for extensive anterior knee contracture reconstruction with satisfactory esthetic and functional outcome. It can provide substantial amount of soft tissue coverage with minimal complications and donor-site morbidity. Furthermore, it offers a good basis for next-step orthopedic surgery, such as total knee arthroplasty (TKA).

Effects of NF-kappaB inhibitor on titanium particulate-induced inflammation in a murine model.

BACKGROUND: Activation of nuclear factor kappa B (NF-kappaB) signaling in response to implant particulates may be critical in the pathogenesis of implant loosening after joint arthroplasty. The purpose of this study was to investigate the inhibitory effects of pyrrolidine dithiocarbamate (PDTC) in a murine model of inflammation induced by titanium (Ti) particulates. MATERIALS AND METHODS: Ti particulates were introduced into established air pouches on C57BL/6J mice. Mice were injected intraperitoneally with either high-dose PDTC (100 mg/kg) or low-dose PDTC (50 mg/kg). Mice without drug treatment, as well as mice injected with saline alone were included. Each group consisted of sixteen mice. The membranes and lavage fluid were harvested 2 d or 7 d after injection of particulate suspension for histological and molecular analysis. RESULTS: Histologic analysis showed that PDTC reduced inflammatory responses in air pouches, that is, thinner membrane and decreased cellular infiltration. In addition, PDTC reduced the release of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the lavage fluid or supernatant of homogenates as evaluated by ELISA. CONCLUSION: These results suggest that PDTC inhibits Ti particulate-induced inflammatory responses in the murine model; thus it represents a promising therapeutic candidate for the prevention and treatment of implant loosening.

Angiotensin II activates connective tissue growth factor and induces extracellular matrix changes involving Smad/activation and p38 mitogen-activated protein kinase signalling pathways in human dermal fibroblasts.

Angiotensin II (Ang II) stimulation has been shown to regulate proliferation of skin fibroblasts and the production of extracellular matrix, which are very important processes in skin wound healing and fibrosis; however, there is little knowledge about the mechanisms involved in this process. We investigated the molecular aspects of this system with regards to Ang II in human dermal fibroblasts (HDF) and its potential role in fibrosis. Fibroblasts derived from human skin were subjected to examine differential relative gene and protein expression after transfection with specific reporter expression vectors and Ang II in vitro. In growth-arrested HDFs, Ang II treatment for 20 min caused acute activation of Smad2 phosphorylation, Smad overexpression and Smad-dependent gene transcription. The angiotensin type 1 (AT1) antagonist losartan diminished Ang II-induced Smad activation. The blockade of endogenous transforming growth factor-beta1 did modify the activation of Smad caused by Ang II. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 diminished Ang II-induced Smad2 phosphorylation. Transient transfection with Smad7, which interferes with receptor-mediated activation of Smad2, diminished Ang II-induced connective tissue growth factor promoter activation, gene and protein expression and fibronectin, type I procollagen and type III procollagen overexpression, showing that Smad activation is involved in Ang II-induced dermal fibrosis. Our results show that Ang II activation of Smad2 occurs via the AT1 receptor, but not the AT2 receptor. Activation of Smad2 required p38 MAPK but not p42/p44 MAPK or the epidermal growth factor receptor.

Effect of camptothecin on collagen synthesis in fibroblasts from patients with keloid.

Keloids are distinguished by substantial deposition of collagen in the dermis, resulting in an imbalanced production and aggregation of extra cellular matrix. This study was undertaken to evaluate the effects of the topoisomerase I inhibitor camptothecin (CPT) on collagen synthesis in the activated dermal fibroblasts from healthy donors and patients with keloid. The fibroblasts were cultured in the presence or absence of CPT. Cellular toxicity assay was determined by MTT analysis. The expression of type I collagen and type III collagen was studied both at the transcriptional and post-transcriptional levels, using conventional quantitative real-time reverse transcription PCR and Western blotting. Results showed that there was predominantly a clear and dose-dependent decrease in the synthesis of collagen 1, not collagen 3, in keloid fibroblasts without significantly cellular toxicity. The CPT had an activity on the regulation of the ratio of type I/III collagen in the metabolism of keloid fibroblasts by inhibiting the secretion of type I collagen. The data suggest that the inhibitory effect of CPT, a topoisomerase I inhibitor, on collagen synthesis may be an effective treatment for limiting fibrosis in keloid patients.

[Skeletal biomechanical effectiveness of retinoic acid on induction of osteoporotic rats treated by alendronate and qianggu capsules].

OBJECTIVE: To study the effectiveness of retinoic acid on induction of osteoporotic rats treated by either alendronate or qianggu capsules and co-administration. METHODS: Sixty-five female SD rats were treated with retinoic acid 80 mg x kg(-1) x d(-1) by gastric lavage for 15 days. Then 5 rats were confirmed cases of osteoporosis and the remaining 60 were randomly divided into 4 groups 15 each: (1) control group with NS 8 ml x kg(-1) x w(-1); (2) alendronate group with alendronate 40 mg x kg(-1) x w(-1); (3) qianggu group with qianggu capsules 90 mg x kg(-1) x d(-1); (4) co-medicated group with alendronate 40 mg x kg(-1) x w(-1) and qianggu capsules 90 mg x kg(-1) x d(-1). Five rats in each group were sacrificed at week 2, 4 and 6 respectively to carry out the biomechanic tests, histopathologic examination and bony callus volume calculation. RESULTS: Biomechanical properties of femur changed significantly after the treatment by alendronate or qianggu capsules and co-medication as compared with that of NS after 4 weeks (P < 0.05); the bony callus were larger when treated by alendronate (P < 0.05) and smaller by qianggu capsules (P > 0.05); the bone trabecula formed and rebuilding were slower by alendronate and quicker by qianggu capsules. CONCLUSION: Alendronate or qianggu capsules and co-medication can improve biomechanical properties of femur by retinoic acid on induction of osteoporotic rats. Qianggu capsules can improve bone union.

Thyroxine (T4) may promote re-epithelialisation and angiogenesis in wounded human skin ex vivo.

There is a pressing need for improved preclinical model systems in which to study human skin wound healing. Here, we report the development and application of a serum-free full thickness human skin wound healing model. Not only can re-epithelialization (epidermal repair) and angiogenesis be studied in this simple and instructive model, but the model can also be used to identify clinically relevant wound-healing promoting agents, and to dissect underlying candidate mechanisms of action in the target tissue. We present preliminary ex vivo data to suggest that Thyroxine (T4), which reportedly promotes skin wound healing in rodents in vivo, may promote key features of human skin wound healing. Namely, T4 stimulates re-epithelialisation and angiogenesis, and modulates both wound healing-associated epidermal keratin expression and energy metabolism in experimentally wound human skin. Functionally, the wound healing-promoting effects of T4 are at least partially mediated via fibroblast growth factor/fibroblast growth factor receptor-mediated signalling, since they could be significantly antagonized by bFGF-neutralizing antibody. Thus, this pragmatic, easy-to-use full-thickness human skin wound healing model provides a useful preclinical research tool in the search for clinically relevant candidate wound healing-promoting agents. These ex vivo data encourage further pre-clinical testing of topical T4 as a cost-efficient, novel agent in the management of chronic human skin wounds.

Inhibition of vascular endothelial growth factor expression in keloid fibroblasts by vector-mediated vascular endothelial growth factor shRNA: a therapeutic potential strategy for keloid.

Vascular endothelial growth factor (VEGF) plays important roles in the regulation of angiogenesis and inflammation in both pathological and physiological wound repair. Several strategies have been developed for keloid therapy; however, a universally effective treatment has not been explored to date. In this study, three potential short interfering RNA (siRNA) sequences for the VEGF gene were cloned into expression plasmids and transfected into keloid fibroblasts. PGC-VEGF shRNA 2 (short hairpin RNA 2) plasmid significantly inhibited VEGF expression determined by real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and fibroblasts growth was also significantly by (methyl thiazolyl tetrazolium) MTT assay and apoptosis detection, whereas the control transfection showed no obviously effects. Plasminogen activator inhibitor-1 (PAI-1) expression in pGC-VEGF shRNA2 group was also obviously downregulated when compared to the pGC-VEGF shRNA negative control and mock group. These results suggest that modulation of VEGF production by vector-based RNAi in keloid fibroblasts may be a therapeutic potential strategy for keloid.

Proliferation hemangiomas formation through dual mechanism of vascular endothelial growth factor mediated endothelial progenitor cells proliferation and mobilization through matrix metalloproteinases 9.

Infantile hemangioma is the most common tumor of infancy and the mechanism leading to proliferation hemangiomas formation is poorly understood and currently no successful treatment modality exists. We hypothesize that EPCs formed during proliferation hemangiomas, as the result of vascular endothelial growth factor (VEGF) stimulation through MMP9, play the major role in the control of cell proliferation and capillary-like vessels production. Accepting the hypothesis to be correct, a therapy that inhibits EPC mobilization and proliferation can be used to prevent the proliferation hemangiomas formation. Current therapies are only partially effective and safe because they could not eliminate all the relative factors of proliferation hemangiomas formation at all, such as: EPCs in the peripheral blood, and at the same time inducing death (apoptosis and necrosis) of other normal cells. A more efficient prevention of proliferation hemangiomas could be achieved using specific drugs or biologic methods that inhibit EPC mobilization and proliferation. Therapy based on gene therapy, capable to specifically inhibit VEGF and MMP9 expression in gene level, can be possibly effective.

Possibilities and potential roles of estrogen in the pathogenesis of proliferation hemangiomas formation.

Hemangiomas, often categorized as angiogenic diseases, are the most common tumors of infancy, the life span of which is generally divided into proliferating phase, involuting phase, and involuted phase. Despite their high prevalence, the mechanism leading to proliferation hemangiomas formation is poorly understood and the best approach to their management remains controversial. None of the current therapeutic modalities is ideal, partly because the pathogenesis of hemangioma and the mechanism of its proliferation are far from clear. Many clues reveal that estrogen has an important role in developing the vascular system, experimental and clinical evidences accumulated in recent years also suggest the potential for estrogen to influence neovascularization. Based on those, we hypothesize that estrogen play a potential role in the development of hemangiomas, mainly by regulating some key angiogenic factors, including MMP-9, EPCs, VEGF, NO, etc. Accepting the hypothesis to be correct, a therapy that identify estrogen as a potential target for the design of new, more specific treatments can be used to prevent the proliferation hemangiomas formation. The hypothesis may lead a new direction in the study of mechanisms for proliferation hemangiomas formation, and further study of the precise mechanisms for estrogen-induced hemangiomas will produce effective antiestrogens and estrogen receptor antagonists as new medication for the very difficult problem.