Song BW et al. (2022), Regulation of Inflammation-Mediated Endothelial...

ECB-ART-51105

Mar Drugs 2022 Nov 30;2012:. doi: 10.3390/md20120756.

Show Gene links Show Anatomy links

Regulation of Inflammation-Mediated Endothelial to Mesenchymal Transition with Echinochrome a for Improving Myocardial Dysfunction.

Song BW , Kim S , Kim R , Jeong S , Moon H , Kim H , Vasileva EA , Mishchenko NP , Fedoreyev SA , Stonik VA , Lee MY , Kim J , Kim HK , Han J , Chang W .

???displayArticle.abstract???
Endothelial-mesenchymal transition (EndMT) is a process by which endothelial cells (ECs) transition into mesenchymal cells (e.g., myofibroblasts and smooth muscle cells) and induce fibrosis of cells/tissues, due to ischemic conditions in the heart. Previously, we reported that echinochrome A (EchA) derived from sea urchin shells can modulate cardiovascular disease by promoting anti-inflammatory and antioxidant activity; however, the mechanism underlying these effects was unclear. We investigated the role of EchA in the EndMT process by treating human umbilical vein ECs (HUVECs) with TGF-β2 and IL-1β, and confirmed the regulation of cell migration, inflammatory, oxidative responses and mitochondrial dysfunction. Moreover, we developed an EndMT-induced myocardial infarction (MI) model to investigate the effect of EchA in vivo. After EchA was administered once a day for a total of 3 days, the histological and functional improvement of the myocardium was investigated to confirm the control of the EndMT. We concluded that EchA negatively regulates early or inflammation-related EndMT and reduces the myofibroblast proportion and fibrosis area, meaning that it may be a potential therapy for cardiac regeneration or cardioprotection from scar formation and cardiac fibrosis due to tissue granulation. Our findings encourage the study of marine bioactive compounds for the discovery of new therapeutics for recovering ischemic cardiac injuries.

???displayArticle.pubmedLink??? 36547903
???displayArticle.pmcLink??? PMC9781361
???displayArticle.link??? Mar Drugs
???displayArticle.grants??? [+]

???attribute.lit??? ???displayArticles.show???

	Figure 1. EchA inhibits EndMT in HUVECs. (A) EndMT is induced by TGF-β2 and IL-1β in HUVECs. Untreated HUVECs or TGF-β2 and IL-1β-treated HUVECs were examined for morphological changes using a light microscope (100 × magnification). (B) The chemical structure of EchA. (C) TGF-β2 and IL-1β mRNA expression levels were quantified in the sham control and MI groups. Compared with the sham control group, TGF-β2 and IL-1β mRNA expression levels in the MI group were increased. (D) The cells were pretreated with different concentrations of EchA (0, 1, 3, 5, and 10 μM) for 24 h. Cell viability was determined by the CCK-8 assay (n = 5). (E,F) The relative density of CD31, VE-cadherin and α-SMA, in addition to fibronectin protein bands, were analyzed by Western blot with β-actin as the control. (G) Fluorescence microscopic images of FL-LDL uptake in HUVECs incubated with LDL (40× magnification). (H,I) Permeability tests were performed after HUVECs were incubated with TGF-β2 and IL-1β and with different concentrations of EchA (0, 1, 3, 5, and 10 μM) for 24 h. EchA inhibits TGF-β2- and IL-1β-induced monolayer permeability of HUVECs. ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control. N.S., not significant.
	Figure 2. EchA suppresses migration of TGF-β2- and IL-1β-stimulated HUVECs. (A) Representative images of HUVECs after treatment with different EchA concentrations (0, 1, 3, 5, and 10 μM) for 24 h. Serum-starved HUVECs were first scratched using a 200 μL tip, and subsequently treated with TGF-β2 and IL-1β for 24 h. The images of the wounds were obtained at 0 and 24 h using a light microscope (40× magnification). (B) The area between the wound edges was measured and compared among the groups. (C) Representative images of migrated TGF-β2- and IL-1β-stimulated HUVECs in transwell chamber assay after treatment with different concentrations (0, 1, 3, 5, and 10 μM) of EchA for 24 h using a light microscope (40× magnification). (D) The number of migrated cells was measured and compared among the groups. (E,F) Relative density analysis of the RhoA protein bands by Western blot with β-actin as the control. Relative density analysis of phosphorylated Cdc42 and cofilin protein bands by Western blot with Cdc42 and cofilin as the controls. # p < 0.05, ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control.
	Figure 3. EchA induces anti-inflammatory and anti-oxidative responses in HUVECs. (A) ROS production in HUVECs was determined by DCFH-DA assay. The HUVECs were pretreated with different concentrations of EchA for 24 h prior to treatment with TGF-β2 and IL-1β. Images were taken using a fluorescence microscope (40× magnification). (B) The bar graph shows the statistical results of fluorescence intensity. The mean intensity of DCFH-DA was quantified using ImageJ software. (C,D) Relative density analysis of the NF-κB p-p65, p-IκBα, and p-smad2/3 protein bands by Western blot with NF-κB p65, IκBα, and smad2/3 as the control. Relative density analysis of the TGFβR1 protein bands by Western blot with β-actin as the control. ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control.
	Figure 4. EchA improves mitochondrial dysfunction of EndMT in HUVECs. (A) Mitochondrial membrane potential in HUVECs was determined by TMRE assay. The HUVECs were pretreated with different concentrations of EchA for 24 h prior to treatment with TGF-β2 and IL-1β. Images were captured using a fluorescence microscope (200× magnification). (B) The bar graph shows the statistical results of fluorescence intensity. The mean intensity of TMRM was quantified using ImageJ software. (C,D) The mtDNA/nDNA ratio was analyzed to quantify mitochondrial DNA. (E) Effect of EchA on the mRNA expression levels of COXⅡ, TUFM, SSBP1, TFB2M, POLG, and TFAM, as measured by RT-qPCR and normalized to β-actin. ## p < 0.01, ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control.
	Figure 5. EchA regulates EndMT in the infarcted heart. (A) The timeline of the animal study is presented. After MI modeling, mice were administered EchA for 3 days, and histological and cardiac function analyses for EndMT factor were performed 1 and 4 weeks after surgery, respectively. (B) RT-qPCR was performed to analyze the expression of EMT- or EndMT-associated genes Col1a, Fsp1, Snail, Tgfb, and Vimentin. (C) Immunofluorescence image of EndMT in the infarcted area (green: VE-cadherin (endothelial origin), red: α-SMA (fibrosis), blue: DAPI, dotted white line: ischemic area, white arrow: VE-cadherin and α-SMA double positive cells) (10× and 100× magnification). (D,E) Relative density analysis of the collagen I, collagen III, MMP-2, and MMP-9 protein bands by Western blot with GAPDH as the control. * p < 0.05, ** p < 0.001 vs. normal group. Data are expressed as X-fold induction compared to normal control. All values are mean ± standard deviation. Statistical significance was assessed by one-way ANOVA.
	Figure 6. EchA exerts cardioprotective effects after myocardial infarction. (A) Representative photo and column scatter plot of infarct size determined through TTC staining. Infarct size (%) was calculated as the ratio of the infarcted area (pale) to the risk area (deep red). Distance between white columns = 1 cm. (B) TUNEL assay (TUNEL-positive cells, pink; DAPI, blue) was performed 1 week following MI. (C) Masson’s trichrome staining of cardiac sections 1 week after establishing MI (collagen fibers, light blue; muscle fibers, red). (D) Immunofluorescence staining for CD31 1 week after MI (CD31-positive microvessels, red; DAPI, blue). (E) Ejection fraction. (F) Representative pressure–volume loop. (G) End-systolic pressure–volume relationship (ESPVR). (H) A schematic summary of the regulation of EndMT by treating EchA in ischemic hearts. * p < 0.05 vs. MI group. Data are expressed as X-fold induction compared to normal control. All values are mean ± standard deviation. Statistical significance was assessed by one-way ANOVA.

References [+] :

Aisagbonhi, Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition. 2011, Pubmed

Aisagbonhi, Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition. 2011, Pubmed
Apostolatos, Inhibition of atypical protein kinase C‑ι effectively reduces the malignancy of prostate cancer cells by downregulating the NF-κB signaling cascade. 2018, Pubmed
Bansal, Integrin alpha 11 in the regulation of the myofibroblast phenotype: implications for fibrotic diseases. 2017, Pubmed
Chen, Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells. 2016, Pubmed
Chen, Endothelial-to-mesenchymal transition drives atherosclerosis progression. 2015, Pubmed
Cooley, TGF-β signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling. 2014, Pubmed
Crawford, Origin of developmental precursors dictates the pathophysiologic role of cardiac fibroblasts. 2012, Pubmed
Czubryt, Common threads in cardiac fibrosis, infarct scar formation, and wound healing. 2012, Pubmed
Doerr, Differential effect of hypoxia on early endothelial-mesenchymal transition response to transforming growth beta isoforms 1 and 2. 2016, Pubmed
Du, Knockdown on aPKC-ι inhibits epithelial-mesenchymal transition, migration and invasion of colorectal cancer cells through Rac1-JNK pathway. 2019, Pubmed
Edgley, Targeting fibrosis for the treatment of heart failure: a role for transforming growth factor-β. 2012, Pubmed
Elliott, Foramen ovale closure is a process of endothelial-to-mesenchymal transition leading to fibrosis. 2014, Pubmed
Evrard, Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability. 2016, Pubmed
Fedoreyev, Antiviral and Antioxidant Properties of Echinochrome A. 2018, Pubmed , Echinobase
Forteza, Conditional knockout of polarity complex (atypical) PKCι reveals an anti-inflammatory function mediated by NF-κB. 2016, Pubmed
Frantz, Transforming growth factor beta inhibition increases mortality and left ventricular dilatation after myocardial infarction. 2008, Pubmed
Guan, CXCR7 attenuates the TGF-β-induced endothelial-to-mesenchymal transition and pulmonary fibrosis. 2017, Pubmed
He, Role of the endothelial-to-mesenchymal transition in renal fibrosis of chronic kidney disease. 2013, Pubmed
Jeong, Echinochrome A protects mitochondrial function in cardiomyocytes against cardiotoxic drugs. 2014, Pubmed , Echinobase
Jeong, Echinochrome a increases mitochondrial mass and function by modulating mitochondrial biogenesis regulatory genes. 2014, Pubmed , Echinobase
Jimenez, Endothelial to mesenchymal transition (EndoMT) in the pathogenesis of Systemic Sclerosis-associated pulmonary fibrosis and pulmonary arterial hypertension. Myth or reality? 2016, Pubmed
Kim, Regulation of alternative macrophage activation by MSCs derived hypoxic conditioned medium, via the TGF-β1/Smad3 pathway. 2020, Pubmed
Kim, Multifaceted Clinical Effects of Echinochrome. 2021, Pubmed , Echinobase
Kim, Exosomes derived from microRNA-584 transfected mesenchymal stem cells: novel alternative therapeutic vehicles for cancer therapy. 2018, Pubmed
Kim, Epigenetic modulation as a therapeutic approach for pulmonary arterial hypertension. 2015, Pubmed
Kim, A Novel Atypical PKC-Iota Inhibitor, Echinochrome A, Enhances Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells. 2018, Pubmed , Echinobase
Kim, Echinochrome A regulates phosphorylation of phospholamban Ser16 and Thr17 suppressing cardiac SERCA2A Ca²⁺ reuptake. 2015, Pubmed , Echinobase
Kovacic, Endothelial to Mesenchymal Transition in Cardiovascular Disease: JACC State-of-the-Art Review. 2019, Pubmed
Lebedev, Antioxidant properties, autooxidation, and mutagenic activity of echinochrome a compared with its etherified derivative. 2001, Pubmed
Lebedev, Iron chelators and free radical scavengers in naturally occurring polyhydroxylated 1,4-naphthoquinones. 2008, Pubmed
Lee, Therapeutic implications of microRNAs in pulmonary arterial hypertension. 2014, Pubmed
Lennikov, Amelioration of endotoxin-induced uveitis treated with the sea urchin pigment echinochrome in rats. 2014, Pubmed , Echinobase
Li, Endothelial-to-Mesenchymal Transition: A Potential Mechanism for Atherosclerosis Plaque Progression and Destabilization. 2017, Pubmed
Li, Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases. 2018, Pubmed
Li, Mitochondrial dysfunction in fibrotic diseases. 2020, Pubmed
Ma, The aPKCι blocking agent ATM negatively regulates EMT and invasion of hepatocellular carcinoma. 2014, Pubmed
Ma, TGF-β-Induced Endothelial to Mesenchymal Transition in Disease and Tissue Engineering. 2020, Pubmed
Ma, Cardiac Fibroblast Activation Post-Myocardial Infarction: Current Knowledge Gaps. 2017, Pubmed
Maddaluno, EndMT contributes to the onset and progression of cerebral cavernous malformations. 2013, Pubmed
Mahler, Inflammatory cytokines promote mesenchymal transformation in embryonic and adult valve endothelial cells. 2013, Pubmed
Maleszewska, IL-1β and TGFβ2 synergistically induce endothelial to mesenchymal transition in an NFκB-dependent manner. 2013, Pubmed
Manavski, Clonal Expansion of Endothelial Cells Contributes to Ischemia-Induced Neovascularization. 2018, Pubmed
Mihira, TGF-β-induced mesenchymal transition of MS-1 endothelial cells requires Smad-dependent cooperative activation of Rho signals and MRTF-A. 2012, Pubmed
Mischenko, Echinamines A and B, first aminated hydroxynaphthazarins from the sea urchin Scaphechinus mirabilis. 2005, Pubmed , Echinobase
Ratnayake, Two novel atypical PKC inhibitors; ACPD and DNDA effectively mitigate cell proliferation and epithelial to mesenchymal transition of metastatic melanoma while inducing apoptosis. 2017, Pubmed
Salvatore, Effects of Metformin in Heart Failure: From Pathophysiological Rationale to Clinical Evidence. 2021, Pubmed
Shu, Suppression of PGC-1α Drives Metabolic Dysfunction in TGFβ2-Induced EMT of Retinal Pigment Epithelial Cells. 2021, Pubmed
Streef, Epicardial Contribution to the Developing and Injured Heart: Exploring the Cellular Composition of the Epicardium. 2021, Pubmed
Sun, TGF-β1 attenuates mitochondrial bioenergetics in pulmonary arterial endothelial cells via the disruption of carnitine homeostasis. 2020, Pubmed
Tian, Role of vasodilator-stimulated phosphoprotein in human cytomegalovirus-induced hyperpermeability of human endothelial cells. 2018, Pubmed
Tombor, Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction. 2021, Pubmed
Xiao, Fine-tuning vascular fate during endothelial-mesenchymal transition. 2017, Pubmed
Yang, Macrophages-aPKCɩ-CCL5 Feedback Loop Modulates the Progression and Chemoresistance in Cholangiocarcinoma. 2022, Pubmed
Yi, Mitochondrial dysfunction induces EMT through the TGF-β/Smad/Snail signaling pathway in Hep3B hepatocellular carcinoma cells. 2015, Pubmed
Yoon, Spinochrome D Attenuates Doxorubicin-Induced Cardiomyocyte Death via Improving Glutathione Metabolism and Attenuating Oxidative Stress. 2018, Pubmed , Echinobase
Yoshimatsu, Emerging roles of inflammation-mediated endothelial-mesenchymal transition in health and disease. 2022, Pubmed
Zeisberg, Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. 2007, Pubmed
Zeisberg, Biomarkers for epithelial-mesenchymal transitions. 2009, Pubmed
Zhang, Epithelial-mesenchymal transition of rat peritoneal mesothelial cells via Rhoa/Rock pathway. 2011, Pubmed

	Figure 1. EchA inhibits EndMT in HUVECs. (A) EndMT is induced by TGF-β2 and IL-1β in HUVECs. Untreated HUVECs or TGF-β2 and IL-1β-treated HUVECs were examined for morphological changes using a light microscope (100 × magnification). (B) The chemical structure of EchA. (C) TGF-β2 and IL-1β mRNA expression levels were quantified in the sham control and MI groups. Compared with the sham control group, TGF-β2 and IL-1β mRNA expression levels in the MI group were increased. (D) The cells were pretreated with different concentrations of EchA (0, 1, 3, 5, and 10 μM) for 24 h. Cell viability was determined by the CCK-8 assay (n = 5). (E,F) The relative density of CD31, VE-cadherin and α-SMA, in addition to fibronectin protein bands, were analyzed by Western blot with β-actin as the control. (G) Fluorescence microscopic images of FL-LDL uptake in HUVECs incubated with LDL (40× magnification). (H,I) Permeability tests were performed after HUVECs were incubated with TGF-β2 and IL-1β and with different concentrations of EchA (0, 1, 3, 5, and 10 μM) for 24 h. EchA inhibits TGF-β2- and IL-1β-induced monolayer permeability of HUVECs. ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control. N.S., not significant.
	Figure 2. EchA suppresses migration of TGF-β2- and IL-1β-stimulated HUVECs. (A) Representative images of HUVECs after treatment with different EchA concentrations (0, 1, 3, 5, and 10 μM) for 24 h. Serum-starved HUVECs were first scratched using a 200 μL tip, and subsequently treated with TGF-β2 and IL-1β for 24 h. The images of the wounds were obtained at 0 and 24 h using a light microscope (40× magnification). (B) The area between the wound edges was measured and compared among the groups. (C) Representative images of migrated TGF-β2- and IL-1β-stimulated HUVECs in transwell chamber assay after treatment with different concentrations (0, 1, 3, 5, and 10 μM) of EchA for 24 h using a light microscope (40× magnification). (D) The number of migrated cells was measured and compared among the groups. (E,F) Relative density analysis of the RhoA protein bands by Western blot with β-actin as the control. Relative density analysis of phosphorylated Cdc42 and cofilin protein bands by Western blot with Cdc42 and cofilin as the controls. # p < 0.05, ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control.
	Figure 3. EchA induces anti-inflammatory and anti-oxidative responses in HUVECs. (A) ROS production in HUVECs was determined by DCFH-DA assay. The HUVECs were pretreated with different concentrations of EchA for 24 h prior to treatment with TGF-β2 and IL-1β. Images were taken using a fluorescence microscope (40× magnification). (B) The bar graph shows the statistical results of fluorescence intensity. The mean intensity of DCFH-DA was quantified using ImageJ software. (C,D) Relative density analysis of the NF-κB p-p65, p-IκBα, and p-smad2/3 protein bands by Western blot with NF-κB p65, IκBα, and smad2/3 as the control. Relative density analysis of the TGFβR1 protein bands by Western blot with β-actin as the control. ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control.
	Figure 4. EchA improves mitochondrial dysfunction of EndMT in HUVECs. (A) Mitochondrial membrane potential in HUVECs was determined by TMRE assay. The HUVECs were pretreated with different concentrations of EchA for 24 h prior to treatment with TGF-β2 and IL-1β. Images were captured using a fluorescence microscope (200× magnification). (B) The bar graph shows the statistical results of fluorescence intensity. The mean intensity of TMRM was quantified using ImageJ software. (C,D) The mtDNA/nDNA ratio was analyzed to quantify mitochondrial DNA. (E) Effect of EchA on the mRNA expression levels of COXⅡ, TUFM, SSBP1, TFB2M, POLG, and TFAM, as measured by RT-qPCR and normalized to β-actin. ## p < 0.01, ### p < 0.001 vs. HUVECs without EchA and TGF-β2 and IL-1β treatment; * p < 0.05, p < 0.01, * p < 0.001 vs. TGF-β2- and IL-1β-induced HUVECs without EchA treatment. Data are expressed as X-fold induction compared to normal control.
	Figure 5. EchA regulates EndMT in the infarcted heart. (A) The timeline of the animal study is presented. After MI modeling, mice were administered EchA for 3 days, and histological and cardiac function analyses for EndMT factor were performed 1 and 4 weeks after surgery, respectively. (B) RT-qPCR was performed to analyze the expression of EMT- or EndMT-associated genes Col1a, Fsp1, Snail, Tgfb, and Vimentin. (C) Immunofluorescence image of EndMT in the infarcted area (green: VE-cadherin (endothelial origin), red: α-SMA (fibrosis), blue: DAPI, dotted white line: ischemic area, white arrow: VE-cadherin and α-SMA double positive cells) (10× and 100× magnification). (D,E) Relative density analysis of the collagen I, collagen III, MMP-2, and MMP-9 protein bands by Western blot with GAPDH as the control. * p < 0.05, ** p < 0.001 vs. normal group. Data are expressed as X-fold induction compared to normal control. All values are mean ± standard deviation. Statistical significance was assessed by one-way ANOVA.
	Figure 6. EchA exerts cardioprotective effects after myocardial infarction. (A) Representative photo and column scatter plot of infarct size determined through TTC staining. Infarct size (%) was calculated as the ratio of the infarcted area (pale) to the risk area (deep red). Distance between white columns = 1 cm. (B) TUNEL assay (TUNEL-positive cells, pink; DAPI, blue) was performed 1 week following MI. (C) Masson’s trichrome staining of cardiac sections 1 week after establishing MI (collagen fibers, light blue; muscle fibers, red). (D) Immunofluorescence staining for CD31 1 week after MI (CD31-positive microvessels, red; DAPI, blue). (E) Ejection fraction. (F) Representative pressure–volume loop. (G) End-systolic pressure–volume relationship (ESPVR). (H) A schematic summary of the regulation of EndMT by treating EchA in ischemic hearts. * p < 0.05 vs. MI group. Data are expressed as X-fold induction compared to normal control. All values are mean ± standard deviation. Statistical significance was assessed by one-way ANOVA.