143 Galunisertib Exerts Targeted Anti-Fibrotic Effects in In Vitro Models of Burn Wound Healing

2021 ◽  
Vol 42 (Supplement_1) ◽  
pp. S95-S95
Author(s):  
Joshua M Peterson ◽  
Anesh Prasai ◽  
Jayson W Jay ◽  
Steven E Wolf ◽  
Amina El Ayadi
Keyword(s):  
Wound Healing ◽  
Protein Expression ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Burn Injury ◽  
Dependent Manner ◽  
Burn Wound ◽  
Concentration Dependent Manner ◽  
Burn Scar

Abstract Introduction Inhibition of TGF-β has shown promising in vitro and in vivo results for reduction of hypertrophic scarring after burn injury. However, TGF-β regulates diverse cellular pathways apart from fibrosis, including physiologic wound healing, cell cycle control, and homeostasis. Galunisertib, a novel small molecular tyrosine kinase inhibitor of TGF-beta receptor type 1, specifically targets downstream pro-fibrotic pathways of TGF-β signaling, has an excellent adverse effect profile, and minimal off-target effects. We hypothesized that galunisertib diminishes fibrotic phenotypes in a targeted fashion, making it a promising candidate drug for prevention of hypertrophic burn scar and contracture. Methods Commercially available fibroblasts were treated with TGF-β at concentrations typical of burn wounds to induce fibrotic phenotype fibroblasts (FPF). FPF cells were treated with galunisertib for 0, 1, 2, 3, and 7 days at concentrations ranging from 0.01 μM to 100 μM. FPF viability and proliferation were assessed with MTT assay. Modulation of FPF cell fibrotic gene and was analyzed using quantitative real-time PCR (qRT-PCR) of COL1A1, COL3A1, FN1, αSMA, CTGF, DCN, MMP1, and MMP13. Analysis of phenotype modulation was performed by western blotting of P-Smad2/3, collagen-1, fibronectin, and αSMA. Statistical analysis performed with students t-test and ANOVA. Results TGF-β treated FPF cells had significantly increased proliferation relative to commercially available fibroblasts, which was attenuated by treatment with 2.5–10 μM galunisertib at 2 or 3 days after treatment in a concentration dependent manner (p < 0.05) while also not causing a relative decrease in proliferation. Expression of FPF downstream pro-fibrotic genes underwent significant fold changes (FN1, αSMA, CTGF: p < 0.05). Protein expression showed decrease in both SMAD2/3 phosphorylation and fibrotic protein expression. Scratch assay analysis is ongoing. Conclusions Galunisertib exerts targeted dose-dependent inhibition of fibrotic gene expression and phenotypes in vitro without hindering cellular proliferation.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mohammad Reza Shiran ◽  
Elham Mahmoudian ◽  
Abolghasem Ajami ◽  
Seyed Mostafa Hosseini ◽  
Ayjamal Khojasteh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Animal Model ◽  
Protein Expression ◽  
Western Blot ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

scholarly journals The Efficacy of Sunitinib Treatment of Renal Cancer Cells Is Associated with the Protein PHAX In Vitro

Biology ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 74 ◽  
Author(s):  
Rafia S. Al-Lamki ◽  
Nicholas J. Hudson ◽  
John R. Bradley ◽  
Anne Y. Warren ◽  
Tim Eisen ◽  
...  
Keyword(s):  
Organ Culture ◽  
Protein Expression ◽  
Cancer Progression ◽  
Kinase Inhibitor ◽  
Kidney Tissue ◽  
Computational Prediction ◽  
Dependent Manner ◽  
Normal Kidney ◽  
Cell Renal Cell Carcinoma

Anti-angiogenic agents, such as the multi-tyrosine kinase inhibitor sunitinib, are key first line therapies for metastatic clear cell renal cell carcinoma (ccRCC), but their mechanism of action is not fully understood. Here, we take steps towards validating a computational prediction based on differential transcriptome network analysis that phosphorylated adapter RNA export protein (PHAX) is associated with sunitinib drug treatment. The regulatory impact factor differential network algorithm run on patient tissue samples suggests PHAX is likely an important regulator through changes in genome-wide network connectivity. Immunofluorescence staining of patient tumours showed strong localisation of PHAX to the microvasculature consistent with the anti-angiogenic effect of sunitinib. In normal kidney tissue, PHAX protein abundance was low but increased with tumour grade (G1 vs. G3/4; p < 0.01), consistent with a possible role in cancer progression. In organ culture, ccRCC cells had higher levels of PHAX protein expression than normal kidney cells, and sunitinib increased PHAX protein expression in a dose dependent manner (untreated vs. 100 µM; p < 0.05). PHAX knockdown in a ccRCC organ culture model impacted the ability of sunitinib to cause cancer cell death (p < 0.0001 untreated vs. treated), suggesting a role for PHAX in mediating the efficacy of sunitinib.

scholarly journals Reversal of Cancer Multidrug Resistance (MDR) Mediated by ATP-Binding Cassette Transporter G2 (ABCG2) by AZ-628, a RAF Kinase Inhibitor

2020 ◽  
Vol 8 ◽  
Author(s):  
Jing-Quan Wang ◽  
Qiu-Xu Teng ◽  
Zi-Ning Lei ◽  
Ning Ji ◽  
Qingbin Cui ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kinase Inhibitor ◽  
Efflux Pump ◽  
Simulation Analysis ◽  
Chemotherapeutic Agents ◽  
Dynamics Simulation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Raf Kinase

Overexpression of ABCG2 remains a major impediment to successful cancer treatment, because ABCG2 functions as an efflux pump of chemotherapeutic agents and causes clinical multidrug resistance (MDR). Therefore, it is important to uncover effective modulators to circumvent ABCG2-mediated MDR in cancers. In this study, we reported that AZ-628, a RAF kinase inhibitor, effectively antagonizes ABCG2-mediated MDR in vitro. Our results showed that AZ-628 completely reversed ABCG2-mediated MDR at a non-toxic concentration (3 μM) without affecting ABCB1-, ABCC1-, or ABCC10 mediated MDR. Further studies revealed that the reversal mechanism was by attenuating ABCG2-mediated efflux and increasing intracellular accumulation of ABCG2 substrate drugs. Moreover, AZ-628 stimulated ABCG2-associated ATPase activity in a concentration-dependent manner. Docking and molecular dynamics simulation analysis showed that AZ-628 binds to the same site as ABCG2 substrate drugs with higher score. Taken together, our studies indicate that AZ-628 could be used in combination chemotherapy against ABCG2-mediated MDR in cancers.

Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix

1997 ◽  
Vol 110 (7) ◽  
pp. 861-870 ◽  
Author(s):  
D. Greiling ◽  
R.A. Clark
Keyword(s):  
Wound Healing ◽  
In Vitro Model ◽  
Collagen Gel ◽  
Fibrin Clot ◽  
Dependent Manner ◽  
Fibrin Gel ◽  
Concentration Dependent Manner ◽  
Vitro Model ◽  
Early Wound

After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway

2000 ◽  
Vol 278 (1) ◽  
pp. C33-C39 ◽  
Author(s):  
Lina Paolucci ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt
Keyword(s):  
Protein Kinase ◽  
Pertussis Toxin ◽  
Lysophosphatidic Acid ◽  
Kinase Inhibitor ◽  
Structural Features ◽  
Protein Kinase D ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Swiss 3T3

Protein kinase D (PKD) is a serine-threonine protein kinase with distinct structural features and enzymological properties. Herein we demonstrate that lysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss 3T3 and Rat-1 cells. LPA induced PKD activation in a concentration-dependent fashion with maximal stimulation (7.6-fold) achieved at 5 μM. Treatment of Swiss 3T3 cells with the protein kinase C (PKC) inhibitors GF-I, Ro-31–8220, and Gö-7874 completely abrogated PKD activation induced by LPA at concentrations that did not inhibit PKD activity when added directly to the in vitro kinase assays. PKD activation induced by LPA was attenuated markedly and selectively by prior exposure of either Swiss 3T3 or Rat-1 cells to pertussis toxin (PTx) in a concentration-dependent manner. In contrast, treatment with the protein tyrosine kinase inhibitor genistein, the MEK inhibitor PD-098059, or the phosphoinositide 3-kinase inhibitor wortmannin did not affect PKD activation in response to LPA. These results provide the first example of PTx-sensitive and PKC-dependent PKD activation and identify a novel Gi-dependent event in the action of LPA.

scholarly journals Role of H2S Supplementation on Burn Wound Healing and Molecular Chaperones

2021 ◽  
Vol 6 (2) ◽  
pp. 171-176
Author(s):  
Saurabh Verma ◽  
Gaurav K. Keshri ◽  
Manish Sharma ◽  
Asheesh Gupta
Keyword(s):  
Wound Healing ◽  
Protein Expression ◽  
Molecular Chaperones ◽  
Wound Repair ◽  
Cellular Proliferation ◽  
Repair Process ◽  
Histopathological Analysis ◽  
Burn Wound ◽  
Burn Wound Healing

Treatment of non-healing burn injuries is a major challenge for the current scientific research. Hydrogen sulfide (H2S) is an endogenous gasotransmitter, which regulates redox homeostasis and cytoprotection during pathophysiological conditions. Similarly, heat shock proteins (HSPs) are molecular chaperones, which also confer cytoprotection during the wound repair process. Notably, the role of H2S as a regulator of HSPs during burn wound healing is still elusive. The present study investigated the effects of H2S supplementation on molecular chaperones during full-thickness, third-degree burn wound healing in the experimental rats. The animals were treated with sodium hydrosulphide (NaHS) as H2S donor (5 mg/kg body weight, intraperitoneal) daily for 10 days prior to burn-induction and continued till the fifth-day post-wounding. Histopathological analysis (Masson’s trichrome) revealed enhanced wound healing evident by increased collagen fiber deposition, cellular proliferation and re-epithelialisation in NaHS administered group as compared to the burn control. Furthermore, immunoblot analyses demonstrated significantly increased protein expression of molecular chaperons viz. HSP90, HSP70, HSP27, and GRP78 in H2S treated group as compared to control. Therefore, the present study signifies that H2S supplementation upregulates the protein expression levels of molecular chaperones, which could facilitate the cytoprotection during the tissue repair process and accelerates the burn wound healing.

scholarly journals Regulation of A Disintegrin And Metalloproteinase with ThromboSpondin Repeats-1 Expression in Human Endometrial Stromal Cells by Gonadal Steroids Involves Progestins, Androgens, and Estrogens

2006 ◽  
Vol 91 (12) ◽  
pp. 4825-4835 ◽  
Author(s):  
Jiadi Wen ◽  
Hua Zhu ◽  
Shuko Murakami ◽  
Peter C. K. Leung ◽  
Colin D. MacCalman
Keyword(s):  
Protein Expression ◽  
Stromal Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Endometrial Stromal Cells ◽  
Expression Levels ◽  
Mrna And Protein Expression ◽  
Regulatory Effects ◽  
A Disintegrin And Metalloproteinase

Abstract Context: Gonadal steroids are key regulators of the extracellular matrix remodeling events that occur in the human endometrium during each menstrual cycle. The spatiotemporal expression of A Disintegrin And Metalloproteinase with ThromboSpondin repeats (ADAMTS)-1 in human endometrial stroma in vivo suggests that this novel metalloproteinase may contribute to this tightly regulated developmental process. Objective: The objective of the study was to determine whether progesterone (P4), 17β-estradiol (E2), or the nonaromatizable androgen dihydrotestosterone (DHT), alone or in combination, is capable of regulating ADAMTS-1 mRNA and protein levels in human endometrial stromal cells in a concentration- and time-dependent manner. Design: A real-time quantitative PCR strategy and Western blotting were used to examine ADAMTS-1 mRNA and protein expression levels in primary cultures of human endometrial stromal cells. Results: P4 and DHT but not E2 increased the levels of the ADAMTS-1 mRNA transcript and protein species (110 kDa) present in endometrial stromal cells in vitro in a concentration- and time-dependent manner. A combination of P4 and DHT resulted in an additional increase in stromal ADAMTS-1 expression, whereas E2 attenuated the regulatory effects of P4 and DHT in a concentration-dependent manner. The antisteroidal compounds, mifepristone (RU486) and hydroxyflutamide, were also found to inhibit specifically the P4- and DHT-mediated increase in ADAMTS-1 mRNA and protein expression levels in these primary cell cultures in a concentration-dependent manner, respectively. Conclusions: These studies demonstrate that progestins, androgens, and estrogens, alone and in combination, have distinct regulatory effects on ADAMTS-1 mRNA and protein expression levels in human endometrial stromal cells in vitro.

scholarly journals Identification of Major Active Ingredients Responsible for Burn Wound Healing ofCentella asiaticaHerbs

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Fang Wu ◽  
Difei Bian ◽  
Yufeng Xia ◽  
Zhunan Gong ◽  
Qian Tan ◽  
...  
Keyword(s):  
Wound Healing ◽  
Burn Injury ◽  
Centella Asiatica ◽  
Asiatic Acid ◽  
Burn Wound ◽  
Active Constituent ◽  
Healthy Human ◽  
Burn Wound Healing

Centella asiaticaherbs have been prescribed as a traditional medicine for wound healing in China and Southeast Asia for a long time. They contain many kinds of triterpenoid compounds, mainly including glycosides (asiaticoside and madecassoside) and corresponding aglycones (asiatic acid and madecassic acid). To identify which is the major active constituent, a comprehensive and comparative study of these compounds was performed.In vitro, primary human skin fibroblasts, originating from healthy human foreskin samples, were treated with various concentrations of asiaticoside, madecassoside, asiatic acid, and madecassic acid, respectively. Cell proliferation, collagen synthesis, MMP-1/TIMP-1 balance, and TGF-β/Smad signaling pathway were investigated.In vivo, mice were orally administered with the four compounds mentioned above for two weeks after burn injury. The speed and quality of wound healing, as well as TGF-β1levels in skin tissues, were examined. Interestingly, in contrast to prevalent postulations, asiaticoside and madecassoside themselves, rather than their corresponding metabolites asiatic acid and madecassic acid, are recognized as the main active constituents ofC. asiaticaherbs responsible for burn wound healing. Furthermore, madecassoside is more effective than asiaticoside (P=0.0446for procollagen type III synthesisin vitro,P=0.0057for wound healing speed, andP=0.0491for wound healing patternin vivo, correspondingly).

scholarly journals Artemisinin and the Derivatives Play Novel Role in Treatment for Graves’ Orbitopathy as Conventional Antimalarials

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A483-A483
Author(s):  
Yan Guo ◽  
Yanbing Li
Keyword(s):  
Western Blot ◽  
Cellular Proliferation ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Novel Therapy ◽  
Concentration Dependent Manner ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts ◽  
Oil Red O Staining

Abstract Context: Graves’ orbitopathy (GO) an autoimmune disease in orbit, characterized with proptosis due to excessive proliferation, adipogenesis, fibrosis and hyaluronan secretion of orbital fibroblasts (OFs). OFs is potential to be a target for proptosis. But there are few effective therapies. Objectives: Our present purpose was to evaluate the effects of artemisinin (ARS) and the derivatives dihydroartemisinin (DHA), artesunate (ART) on OFs from GO patients in vitro. Design/Setting/Participants: OFs isolated from patients with GO (n = 10) were allowed to proliferate in the proliferation medium (PM); differentiate into adipocytes in the differentiation medium (DM) or differentiate into myofibroblast stimulated by TGF-β (10ng/ml); or produce hyaluronan stimulated by IL-1β (5ng/ml). Different dosages of ARS and the derivatives were administered in the above conditions. Main Outcome Measures: CCK-8 was used to assessed cell viability of OFs, EdU incorporation and flow cytometry were conducted to assess cellular proliferation. Adipogenesis was determined by Western blot and Oil Red O staining. Hyaluronan was quantified by ELISA. Fibrosis was assessed using Western blot. Results: ARS in concentrations lower than 100 μM, DHA &lt; 20 μM and ART &lt; 10 μM are nontoxic for OFs. Cellular proliferation of GO-OFs was halted by ARS and its derivatives at the maximum nontoxic dosage. ARS and its derivatives exerted an inhibitory action on adipogenesis of OFs in a concentration-dependent manner. Moreover, hyaluronan secretion was obviously decreased by ARS and its derivatives. Intriguingly, fibrosis markers were also decreased by the antimalarias in a dosage-dependent way. Conclusions: We elucidated the efficacies of ARS and its derivatives on proliferation, adipogenesis, fibrosis and hyaluronan production of OFs, supporting that ARS-based antimalarials play potential role as a novel therapy for GO from a perspective of in-vitro study.

An in vitro model for investigation of vitamin A effects on wound healing

2021 ◽  
pp. 1-6
Author(s):  
Hoda Keshmiri Neghab ◽  
Mohammad Hasan Soheilifar ◽  
Gholamreza Esmaeeli Djavid
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Vitamin A ◽  
In Vitro Model ◽  
Cellular Proliferation ◽  
Endothelial Cell Migration ◽  
Normal Fibroblast ◽  
Anti Inflammatory ◽  
Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inflammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inflammation responses.

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