Bromelain Protects Critically Perfused Musculocutaneous Flap Tissue from Necrosis.

Bromelain has previously been shown to prevent ischemia-induced necrosis in different types of tissues. In the present study, we, therefore, evaluated for the first time, the tissue-protective effects of bromelain in musculocutaneous flaps in mice. Adult C57BL/6N mice were randomly assigned to a bromelain treatment group and a control group. The animals were treated daily with intraperitoneal injections of 20 mg/kg bromelain or saline (control), starting 1 h before the flap elevation throughout a 10-day observation period. The random-pattern musculocutaneous flaps were raised on the backs of the animals and mounted into a dorsal skinfold chamber. Angiogenesis, nutritive blood perfusion and flap necrosis were quantitatively analyzed by means of repeated intravital fluorescence microscopy over 10 days after surgery. After the last microscopy, the flaps were harvested for additional histological and immunohistochemical analyses. Bromelain reduced necrosis of the critically perfused flap tissue by ~25%. The bromelain-treated flaps also exhibited a significantly higher functional microvessel density and an elevated formation of newly developed microvessels in the transition zone between the vital and necrotic tissues when compared to the controls. Immunohistochemical analyses demonstrated a markedly lower invasion of the myeloperoxidase-positive neutrophilic granulocytes and a significantly reduced number of cleaved caspase 3-positive apoptotic cells in the transition zone of bromelain-treated musculocutaneous flaps. These findings indicate that bromelain prevents flap necrosis by maintaining nutritive tissue perfusion and by suppressing ischemia-induced inflammation and apoptosis. Hence, bromelain may represent a promising compound to prevent ischemia-induced flap necrosis in clinical practice.

Tissue-Protective Mechanisms of Bioactive Phytochemicals in Flap Surgery.

Despite careful preoperative planning, surgical flaps are prone to ischemic tissue damage and ischemia-reperfusion injury. The resulting wound breakdown and flap necrosis increase both treatment costs and patient morbidity. Hence, there is a need for strategies to promote flap survival and prevent ischemia-induced tissue damage. Phytochemicals, defined as non-essential, bioactive, and plant-derived molecules, are attractive candidates for perioperative treatment as they have little to no side effects and are well tolerated by most patients. Furthermore, they have been shown to exert beneficial combinations of pro-angiogenic, anti-inflammatory, anti-oxidant, and anti-apoptotic effects. This review provides an overview of bioactive phytochemicals that have been used to increase flap survival in preclinical animal models and discusses the underlying molecular and cellular mechanisms.

Linalool inhibits the angiogenic activity of endothelial cells by downregulating intracellular ATP levels and activating TRPM8.

Angiogenesis crucially contributes to various diseases, such as cancer and diabetic retinopathy. Hence, anti-angiogenic therapy is considered as a powerful strategy against these diseases. Previous studies reported that the acyclic monoterpene linalool exhibits anticancer, anti-inflammatory and anti-oxidative activity. However, the effects of linalool on angiogenesis still remain elusive. Therefore, we investigated the action of (3R)-(-)-linalool, a main enantiomer of linalool, on the angiogenic activity of human dermal microvascular endothelial cells (HDMECs) by a panel of angiogenesis assays. Non-cytotoxic doses of linalool significantly inhibited HDMEC proliferation, migration, tube formation and spheroid sprouting. Linalool also suppressed the vascular sprouting from rat aortic rings. In addition, Matrigel plugs containing linalool exhibited a significantly reduced microvessel density 7 days after implantation into BALB/c mice. Mechanistic analyses revealed that linalool promotes the phosphorylation of extracellular signal-regulated kinase (ERK), downregulates the intracellular level of adenosine triphosphate (ATP) and activates the transient receptor potential cation channel subfamily M (melastatin) member (TRPM)8 in HDMECs. Inhibition of ERK signaling, supplementation of ATP and blockade of TRPM8 significantly counteracted linalool-suppressed HDMEC spheroid sprouting. Moreover, ATP supplementation completely reversed linalool-induced ERK phosphorylation. In addition, linalool-induced ERK phosphorylation inhibited the expression of bone morphogenetic protein (BMP)-2 and linalool-induced TRPM8 activation caused the inhibition of ß1 integrin/focal adhesion kinase (FAK) signaling. These findings indicate an anti-angiogenic effect of linalool, which is mediated by downregulating intracellular ATP levels and activating TRPM8.

Plants as source of new therapies for endometriosis: a review of preclinical and clinical studies.

BACKGROUND: Given the disadvantages and limitations of current endometriosis therapy, there is a progressive increase in studies focusing on plant-derived agents as a natural treatment option with the intention of achieving high efficiency, avoiding adverse effects and preserving the chance for successful pregnancy. The heterogeneity of these studies in terms of evaluated agents, applied approaches and outcomes illustrates the need for an up-to-date summary and critical view on this rapidly growing field in endometriosis research. OBJECTIVE AND RATIONALE: This review provides a comprehensive overview of plant-derived agents and natural treatment strategies that are under preclinical or clinical investigation and critically evaluates their potential for future endometriosis therapy. SEARCH METHODS: An English language PubMed literature search was performed using variations of the terms 'endometriosis', 'natural therapy', 'herb/herbal', 'plant', 'flavonoid', 'polyphenol', 'phytochemical', 'bioactive', 'Kampo' and 'Chinese medicine'. It included both animal and human studies. Moreover, the Clinicaltrials.gov database was searched with the term 'endometriosis' for clinical trials on plant-derived agents. No restriction was set for the publication date. OUTCOMES: Natural therapies can be assigned to three categories: (i) herbal extracts, (ii) specific plant-derived bioactive compounds and (iii) Chinese herbal medicine (CHM). Agents of the first category have been shown to exert anti-proliferative, anti-inflammatory, anti-angiogenic and anti-oxidant effects on endometrial cells and endometriotic lesions. However, the existing evidence supporting their use in endometriosis therapy is quite limited. The most studied specific plant-derived bioactive compounds are resveratrol, epigallocatechin-3-gallate, curcumin, puerarin, ginsenosides, xanthohumol, 4-hydroxybenzyl alcohol, quercetin, apigenin, carnosic acid, rosmarinic acid, wogonin, baicalein, parthenolide, andrographolide and cannabinoids, with solid evidence about their inhibitory activity in experimental endometriosis models. Their mechanisms of action include pleiotropic effects on known signalling effectors: oestrogen receptor-α, cyclooxygenase-2, interleukin-1 and -6, tumour necrosis factor-α, intercellular adhesion molecule-1, vascular endothelial growth factor, nuclear factor-kappa B, matrix metalloproteinases as well as reactive oxygen species (ROS) and apoptosis-related proteins. Numerous studies suggest that treatment with CHM is a good choice for endometriosis management. Even under clinical conditions, this approach has already been shown to decrease the size of endometriotic lesions, alleviate chronic pelvic pain and reduce postoperative recurrence rates. WIDER IMPLICATIONS: The necessity to manage endometriosis as a chronic disease highlights the importance of identifying novel and affordable long-term safety therapeutics. For this purpose, natural plant-derived agents represent promising candidates. Many of these agents exhibit a pleiotropic action profile, which simultaneously inhibits fundamental processes in the pathogenesis of endometriosis, such as proliferation, inflammation, ROS formation and angiogenesis. Hence, their inclusion into multimodal treatment concepts may essentially contribute to increase the therapeutic efficiency and reduce the side effects of future endometriosis therapy.

Calligonum comosum (Escanbil) extract exerts anti-angiogenic, anti-proliferative and anti-inflammatory effects on endometriotic lesions.

ETHNOPHARMACOLOGICAL RELEVANCE: Calligonum comosum is a desert plant that is applied in traditional folkloric medicine for the treatment of abnormally heavy or prolonged menstruation and menstrual cramps. Moreover, it has been suggested for the treatment of infertility-causing conditions. Its bioactive chemical constituents inhibit multiple processes, such as angiogenesis, inflammation and invasive tissue growth, which may be beneficial in the therapy of endometriosis. AIM OF THE STUDY: We investigated the effects of Calligonum comosum on the development of endometriotic lesions. MATERIALS AND METHODS: We evaluated the anti-angiogenic activity of Calligonum comosum ethyl acetate fraction (CCEAF) in different in vitro angiogenesis assays. Moreover, we surgically induced endometriotic lesions in BALB/c mice, which received 50 mg/kg Calligonum comosum total extract (CCTE) or vehicle (control) over 4 weeks. The growth, cyst formation, vascularization and immune cell infiltration of the lesions were assessed with high-resolution ultrasound imaging, caliper measurements, histology and immunohistochemistry. RESULTS: CCEAF doses of up to 10 µg/mL did not impair the viability of human dermal microvascular endothelial cells (HDMEC), but dose-dependently suppressed their migration, tube formation and sprouting, indicating a substantial anti-angiogenic effect of CCEAF. Furthermore, CCTE significantly inhibited the growth and cyst formation of developing murine endometriotic lesions when compared to vehicle-treated controls. This was associated with a reduced vascularization, cell proliferation and immune cell infiltration. CONCLUSIONS: Our findings show that Calligonum comosum targets multiple, fundamental processes in the pathogenesis of endometriosis, which may be beneficial for the treatment of this common gynecological disorder.

Soy Intake Since the Prepubertal Age May Contribute to the Pathogenesis of Endometriosis in Adulthood.

High prevalence of endometriosis was reported in Asian women as a result of their traditionally high intake of soy foods during infancy. Soy is widely used in infant feeding after weaning from breast milk or cow milk. This study thus aimed to determine to what extent soy intake before puberty may contribute to the development of endometriosis. For this purpose, immature (6-week old) female rats were fed with various soy formulas (0%, 10%, 20%, 30%, 40%, 50%, and 60%). Normal control animals were fed with a soy-free diet. At 13 weeks of age, animals (except the normal control) underwent a transplantation surgery to establish endometriosis. Estradiol valerate and oxytocin were used to induce pelvic pain. Endometrial implant levels of glutathione (GSH) and malondialdehyde (MDA) allowed estimating tissue oxidative status. Physiological ovarian function was assessed by histological analysis of ovaries. Results showed that soy-fed animals grew faster than animals receiving a soy-free diet (P < .001). In animals supplemented with more than 10% of soy, the intensity of pelvic pain increased (P < .001) as well as the volume of ectopic foci. In addition, tissue levels of MDA and GSH increased (P < .001). The ovarian function was altered and the number of luteinized unruptured follicles increased. In conclusion, although animals supplemented with soy at the prepubertal stage displayed a good growth performance, regular soy consumption may promote the development and progress of endometriosis in adulthood, especially when soy content in food is more than 10%.

VEGF-loaded mineral-coated microparticles improve bone repair and are associated with increased expression of epo and RUNX-2 in murine non-unions.

A poor vascular supply of the fracture gap is a key factor for the development of atrophic non-unions. Mineral-coated microparticles (MCM) represent a sophisticated carrier system for the delivery of vascular endothelial growth factor (VEGF). Hence, we investigated whether VEGF-loaded MCM improve bone repair in non-unions. For this purpose, we analyzed binding and release kinetics of MCM for VEGF in vitro. Moreover, we applied VEGF-loaded or -unloaded MCM in a murine non-union model in vivo and studied the process of bone healing by means of biomechanical, radiological, histomorphometric, and Western blot techniques. MCM-free non-unions served as controls. The binding efficiency of MCM for VEGF was 46 ± 3% and the release profile revealed an initial minor burst release followed by a sustained release over a 50-day study period, thus, mimicking the physiological expression profile of VEGF during bone healing. In vivo, bone defects treated with VEGF-loaded MCM exhibited a higher bending stiffness, a higher fraction of bone volume/tissue volume and a larger callus area on days 14 and 70 when compared to the other groups. Western blot analyses on day 14 revealed a higher expression of VEGF, erythropoietin (EPO), and runt-related transcription factor 2, but not of EPO-receptor in bone defects treated with VEGF-loaded MCM. These findings demonstrate that the use of MCM for VEGF delivery shows great potential due to the ability to maintain protein stability and functionality in vivo. Moreover, the application of VEGF-loaded MCM represent a promising strategy for the treatment of non-unions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Indole-3-carbinol is a potent inhibitor of ischemia-reperfusion-induced inflammation.

BACKGROUND: Ischemia-reperfusion (I/R) induces tissue inflammation, which is characterized by an increased leukocyte-endothelial cell interaction and leukocyte transmigration. These processes are mediated by the activation of the nuclear factor (NF)κB signaling pathway, resulting in an elevated expression of specific adhesion molecules. The phytochemical indole-3-carbinol (I3C) has been shown to exert anti-inflammatory effects by interfering with NFκB signal transduction. The aim of the present study was to investigate whether I3C is capable of counteracting the pathogenesis of I/R injury. MATERIALS AND METHODS: We investigated the inhibitory effect of I3C on endothelial surface protein expression during hypoxia and reoxygenation by flow cytometry. Moreover, the subcellular localization of NFκB was analyzed by immunofluorescence and Western blot. Adhesion protein levels on leukocytes after tumor necrosis factor-α stimulation were determined using flow cytometry. Finally, leukocyte-endothelial cell interaction and leukocyte transmigration during I/R was investigated in dorsal skinfold chambers of BALB/c mice by means of repetitive intravital fluorescence microscopy and immunohistochemistry. RESULTS: I3C suppressed the expression of E-selectin and intercellular adhesion molecule-1 on human dermal microvascular endothelial cells by reducing the transcriptional activity of NFκB. Furthermore, surface protein levels of macrophage-1 antigen as well as activated lymphocyte function-associated antigen-1 were markedly reduced on I3C-treated leukocytes. In vivo, I3C treatment decreased the numbers of adherent and transmigrated leukocytes. This was associated with a reduced macromolecular leakage when compared with vehicle-treated controls. CONCLUSIONS: These novel results indicate that I3C reduces the expression of endothelial and leukocytic adhesion proteins, resulting in attenuated leukocyte-endothelial cell interactions during I/R. Accordingly, dietary supplements containing I3C may be beneficial for the treatment of I/R-induced inflammation.

Lead Optimization Generates CYP11B1 Inhibitors of Pyridylmethyl Isoxazole Type with Improved Pharmacological Profile for the Treatment of Cushing's Disease.

Cushing's disease, characterized by elevated plasma cortisol levels, can be controlled by inhibition of 11ß-hydroxylase (CYP11B1). The previously identified selective and potent CYP11B1 inhibitor 5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine Ref 7 (IC50= 2 nM) exhibited promutagenic potential as well as very low oral bioavailability in rats (F = 2%) and was therefore modified to overcome these drawbacks. Successful lead optimization resulted in similarly potent and selective 5-((5-methoxypyridin-3-yl)methyl)-3-phenylisoxazole 25 (IC50 = 2 nM, 14-fold selectivity over CYP11B2), exhibiting a superior pharmacological profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1 (IC50 = 2 µM) and showed a high oral bioavailability (F = 50%) and sufficient plasma concentrations in rats, providing an excellent starting point for a proof-of-principle study.

Geraniol Suppresses Angiogenesis by Downregulating Vascular Endothelial Growth Factor (VEGF)/VEGFR-2 Signaling.

Geraniol exerts several direct pharmacological effects on tumor cells and, thus, has been suggested as a promising anti-cancer compound. Because vascularization is a major precondition for tumor growth, we analyzed in this study the anti-angiogenic action of geraniol. In vitro, geraniol reduced the migratory activity of endothelial-like eEND2 cells. Western blot analyses further revealed that geraniol downregulates proliferating cell nuclear antigen (PCNA) and upregulates cleaved caspase-3 (Casp-3) expression in eEND2 cells. Moreover, geraniol blocked vascular endothelial growth factor (VEGF)/VEGFR-2 signal transduction, resulting in a suppression of downstream AKT and ERK signaling pathways. In addition, geraniol significantly reduced vascular sprout formation in a rat aortic ring assay. In vivo, geraniol inhibited the vascularization of CT26 tumors in dorsal skinfold chambers of BALB/c mice, which was associated with a smaller tumor size when compared to vehicle-treated controls. Immunohistochemical analyses confirmed a decreased number of Ki67-positive cells and CD31-positive microvessels with reduced VEGFR-2 expression within geraniol-treated tumors. Taken together, these findings indicate that geraniol targets multiple angiogenic mechanisms and, therefore, is an attractive candidate for the anti-angiogenic treatment of tumors.

Curcumin intake affects miRNA signature in murine melanoma with mmu-miR-205-5p most significantly altered.

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in 'o-glycan biosynthesis', 'endoplasmatic reticulum protein processing' and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.

Inhibition of angiogenesis: a novel antitumor mechanism of the herbal compound arctigenin.

Arctigenin, a functional ingredient of several traditional Chinese herbs, has been reported to have potential antitumor activity. However, its mechanisms of action are still not well elucidated. Because the establishment and metastatic spread of tumors is crucially dependent on angiogenesis, here we investigated whether arctigenin inhibits tumor growth by disturbing blood vessel formation. For this purpose, human dermal microvascular endothelial cells were exposed to different arctigenin doses to study their viability, proliferation, protein expression, migration, and tube formation compared with vehicle-treated controls. In addition, arctigenin action on vascular sprouting was analyzed in an aortic ring assay. Furthermore, we studied direct arctigenin effects on CT26.WT colon carcinoma cells. Spheroids of these tumor cells were transplanted into the dorsal skinfold chamber of arctigenin-treated and vehicle-treated BALB/c mice for the in-vivo analysis of tumor vascularization and growth by intravital fluorescence microscopy, histology, and immunohistochemistry. We found that noncytotoxic doses of arctigenin dose dependently reduced the proliferation of human dermal microvascular endothelial cells without affecting their migratory and tube-forming capacity. Arctigenin treatment also resulted in a decreased cellular expression of phosphorylated serine/threonine protein kinase AKT, vascular endothelial growth factor receptor 2, and proliferating cell nuclear antigen and inhibited vascular sprouting from aortic rings. In addition, proliferation, but not secretion of vascular endothelial growth factor, was decreased in arctigenin-treated tumor cells. Finally, arctigenin suppressed the vascularization and growth of engrafting CT26.WT tumors in the dorsal skinfold chamber model. Taken together, these results show for the first time an antiangiogenic action of arctigenin, which may contribute considerably toward its antitumor activity.

Local preconditioning by thermal stress accelerates microvascular thrombus formation.

Heme oxygenase 1 (HO-1) has been shown to suppress microvascular thrombus formation. Because stress conditioning induces HO-1 and, in addition, the anticoagulant thrombomodulin and thrombospondin 1, we studied the effect of hyperthermic and hypothermic local stress conditioning on microvascular thrombus formation. For local stress conditioning, the hindlimb of Sprague-Dawley rats was subjected to local heating (42.5 degrees C) or cooling (4 degrees C) for 30 min at 24 h before induction of thrombosis. Sham-exposed hindlimbs served as controls. Thrombosis was induced photochemically in arterioles and venules of the preconditioned tissue (muscle, subcutis, and periosteum) by continuous light exposure after injection of a fluorescent dye. Immunohistochemistry revealed that stress conditioning distinctly induced HO-1, thrombomodulin, and thrombospondin 1 but also von Willebrand factor in endothelial cells. Of interest, intravital fluorescence microscopic analysis of the kinetics of thrombus formation could not confirm an antithrombotic effect of stress conditioning but showed, in contrast, a significant acceleration of thrombosis (P < 0.05) in both arterioles and venules of either of the tissues studied. Although hypothermic and hyperthermic stress conditioning induces antithrombotic HO-1, thrombomodulin, and thrombospondin 1, it enhances endogenous thrombogenicity, most probably due to upregulation of the prothrombotic von Willebrand factor. Thus, preconditioning with local stress cannot be considered as a strategy to prevent thrombus formation.