Endothelial‐to‐mesenchymal transition (EndMT) was first reported in the embryogenesis. Recent studies show that EndMT also occurs in the disease progression of atherosclerosis, cardiac and pulmonary ...fibrosis, pulmonary hypertension, diabetic nephropathy, and cancer. Although transforming growth factor β (TGFβ) is crucial for EndMT, it is not clear which isoform elicits a predominant effect. The current study aims to directly compare the dose‐dependent effects of TGFβ1, TGFβ2, and TGFβ3 on EndMT and characterize the underlying mechanisms. In our results, all three TGFβ isoforms induced EndMT in human microvascular endothelial cells after 72 hr, as evidenced by the increased expression of mesenchymal markers N‐cadherin and α‐smooth muscle actin as well as the decreased expression of endothelial nitric oxide synthase. Interestingly, the effect of TGFβ2 was the most pronounced. At 1 ng/ml, only TGFβ2 treatment resulted in significantly increased phosphorylation (activation) of Smad2/3 and p38‐MAPK and increased expression of mesenchymal transcription factors Snail and FoxC2. Intriguingly, we observed that treatment with 1 ng/ml TGFβ1 and TGFβ3, but not TGFβ2, resulted in an increased expression of TGFβ2, thus indicating that EndMT with TGFβ1 and TGFβ3 treatments was due to the secondary effects through TGFβ2 secretion. Furthermore, silencing TGFβ2 using small interfering RNA blunted the expression of EndMT markers in TGFβ1‐ and TGFβ3‐treated cells. Together, our results indicate that TGFβ2 is the most potent inducer of EndMT and that TGFβ1‐ and TGFβ3‐induced EndMT necessitates a paracrine loop involving TGFβ2.
Our study demonstrates that transforming growth factor β2 (TGFβ2) is the most potent inducer of endothelial‐to‐mesenchymal transition (EndMT) and that TGFβ1‐ and TGFβ3‐induced EndMT necessitates a paracrine loop involving TGFβ2. Silencing TGFβ2 using small interfering RNA blunted the expression of EndMT markers in TGFβ1‐ and TGFβ3‐treated cells.
The Src-family kinases (SFKs), an intracellularly located group of non-receptor tyrosine kinases are involved in oncogenesis. The importance of SFKs has been implicated in the promotion of tumor cell ...motility, proliferation, inhibition of apoptosis, invasion and metastasis. Recent evidences indicate that specific effects of SFKs on epithelial-to-mesenchymal transition (EMT) as well as on endothelial and stromal cells in the tumor microenvironment can have profound effects on tumor microinvasion and metastasis. Although, having been studied extensively, these novel features of SFKs may contribute to greater understanding of benefits from Src inhibition in various types of cancers. Here we review the novel role of SFKs, particularly c-Src in mediating EMT, modulation of tumor endothelial-barrier, transendothelial migration (microinvasion) and metastasis of cancer cells, and discuss the utility of Src inhibitors in vascular normalization and cancer therapy.
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Endothelial‐to‐mesenchymal transition (EndMT) and fibroblast‐to‐myofibroblast (FibroMF) differentiation are frequently reported in organ fibrosis. Stromelysin1, a matrix metalloprotease‐3 (MMP3) has ...been indicated in vascular pathologies and organ injuries that often lead to fibrosis. In the current study, we investigated the role of stromelysin1 in EndMT and FibroMF differentiation, which is currently unknown. In our results, whereas TGFβ2 treatment of endothelial cells (ECs) induced EndMT associated with increased expression of stromelysin1 and mesenchymal markers such as α‐smooth muscle actin (αSMA), N‐cadherin, and activin linked kinase‐5 (ALK5), inhibition of stromelysin1 blunted TGFβ2‐induced EndMT. In contrast, treatment of NIH‐3T3 fibroblasts with TGFβ1 promoted FibroMF differentiation accompanied by increased expression of αSMA, N‐cadherin, and ALK5. Intriguingly, stromelysin1 inhibition in TGFβ1‐stimulated myofibroblasts further exacerbated fibroproliferation with increased FibroMF marker expression. Gene Expression Omnibus (GEO) data analysis indicated increased stromelysin1 expression associated with EndMT and decreased stromelysin1 expression in human pulmonary fibrosis fibroblasts. In conclusion, our study has identified that EndMT and FibroMF differentiation are reciprocally regulated by stromelysin1.
Endothelial‐to‐mesenchymal transition (EndMT) and fibroblast‐to‐myofibroblast (FibroMF) differentiation contribute to organ fibrosis. Stromelysin1, a matrix metalloprotease‐3 (MMP3) has been indicated in organ injuries and fibrosis. Our study reports that EndMT and FibroMF differentiation are reciprocally regulated by stromelysin1.
Abstract Akt1 is essential for the oncogenic transformation and tumor growth in various cancers. However, the precise role of Akt1 in advanced cancers is conflicting. Using a neuroendocrine ...TRansgenic Adenocarcinoma of the Mouse Prostate ( TRAMP ) model, we first show that the genetic ablation or pharmacological inhibition of Akt1 in mice blunts oncogenic transformation and prostate cancer (PCa) growth. Intriguingly, triciribine (TCBN)-mediated Akt inhibition in 25-week old, tumor-bearing TRAMP mice and Akt1 gene silencing in aggressive PCa cells enhanced epithelial to mesenchymal transition (EMT) and promoted metastasis to the lungs. Mechanistically, Akt1 suppression leads to increased expression of EMT markers such as Snail1 and N-cadherin and decreased expression of epithelial marker E-cadherin in TRAMP prostate, and in PC3 and DU145 cells. Next, we identified that Akt1 knockdown in PCa cells results in increased production of TGFβ1 and its receptor TGFβ RII, associated with a decreased expression of β-catenin. Furthermore, treatment of PCa cells with ICG001 that blocks nuclear translocation of β-catenin promoted EMT and N-cadherin expression. Together, our study demonstrates a novel role of the Akt1-β-catenin-TGFβ1 pathway in advanced PCa.
The suppression of Akt1 activity by LPS in pulmonary endothelial cells results in increased FoxO1/3a activation, in turn, leading to increased stromelysin1 expression/activity and reduced expression ...of tight junction proteins, particularly claudin5. Pharmacologically targeting FoxO or stromelysin activity inhibits LPS-induced pulmonary vascular injury and inflammation.
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Enhanced vascular permeability is associated with inflammation and edema in alveoli during the exudative phase of acute respiratory distress syndrome (ARDS). Mechanisms leading to the endothelial contribution on the early exudative stage of ARDS are not precise. We hypothesized that modulation of endothelial stromelysin1 expression and activity by Akt1-forkhead box-O transcription factors 1/3a (FoxO1/3a) pathway could play a significant role in regulating pulmonary edema during the initial stages of acute lung injury (ALI). We utilized lipopolysaccharide (LPS)-induced mouse ALI model in vivo and endothelial barrier resistance measurements in vitro to determine the specific role of the endothelial Akt1-FoxO1/3a-stromelysin1 pathway in ALI. LPS treatment of human pulmonary endothelial cells resulted in increased stromelysin1 and reduced tight junction claudin5 involving FoxO1/3a, associated with decreased trans-endothelial barrier resistance as determined by electric cell-substrate impedance sensing technology. In vivo, LPS-induced lung edema was significantly higher in endothelial Akt1 knockdown (EC-Akt1–/–) compared to wild-type mice, which was reversed upon treatment with FoxO inhibitor (AS1842856), stromelysin1 inhibitor (UK356618) or with shRNA-mediated FoxO1/3a depletion in the mouse lungs. Overall, our study provides the hope that targeting FoxO and styromelysin1 could be beneficial in the treatment of ALI.
Although numerous studies have implicated Akt and Src kinases in vascular endothelial growth factor (VEGF) and Angiopoietin‐1 (Ang‐1)‐induced endothelial‐barrier regulation, a link between these two ...pathways has never been demonstrated. We determined the long‐term effects of Akt inhibition on Src activity and vice versa, and in turn, on the human microvascular endothelial cell (HMEC) barrier integrity at the basal level, and in response to growth factors. Our data showed that Akt1 gene knockdown increases gap formation in HMEC monolayer at the basal level. Pharmacological inhibition of Akt, but not Src resulted in exacerbated VEGF‐induced vascular leakage and impaired Ang‐1‐induced HMEC‐barrier protection in vitro at 24 hr. Whereas inhibition of Akt had no effect on VEGF‐induced HMEC gap formation in the short term, inhibition of Src blunted this process. In contrast, inhibition of Akt disrupted the VEGF and Ang‐1 stabilized barrier integrity in the long‐term while inhibition of Src did not. Interestingly, both long‐term Akt inhibition and Akt1 gene knockdown in HMECs resulted in increased Tyr416 phosphorylation of Src. Treatment of HMECs with transforming growth factor‐β1 (TGFβ1) that inhibited Akt Ser473 phosphorylation in the long‐term, activated Src through increased Tyr416 phosphorylation and decreased HMEC‐barrier resistance. The effect of TGFβ1 on endothelial‐barrier breakdown was blunted in Akt1 deficient HMEC monolayers, where endothelial‐barrier resistance was already impaired compared to the control. To our knowledge, this is the first report demonstrating a direct cross‐talk between Akt and Src in endothelial‐barrier regulation.
The current study identifies the long‐term effects of VEGF and Ang‐1 on HMEC‐barrier protection via increased Akt and normalized Src activities, and that sustained treatment with TGFβ1 promotes endothelial‐barrier breakdown associated with inhibition of Akt and activation of Src. Direct inhibition of Akt activity using triciribine or gene silencing lead to Src activation in the long‐term indicating that Akt and Src are reciprocally regulated in the long‐term endothelial‐barrier function.
Fibroblast-to-myofibroblast (FibroMF) differentiation is crucial for embryogenesis and organ fibrosis. Although transforming growth factor-β (TGFβ) is the primary mediator of FibroMF differentiation, ...the type-I receptor (TGFβRI) responsible for this has not yet been confirmed. In the current study, we investigated the ALK1 and ALK5 expressions in TGFβ1-stimulated NIH 3T3 fibroblasts to compare with the data from the Gene Expression Omnibus (GEO) repository. In our results, whereas TGFβ1 treatment promoted FibroMF differentiation accompanied by increased ALK5 expression and reduced ALK1 expression, TGFβ1-induced FibroMF differentiation and increased α-smooth muscle actin (αSMA) and ALK5 expression were inhibited by co-treatment with ALK5 inhibitor SB431542. GEO database analysis indicated increased ALK5 expression and reduced ALK1 expression in fibrotic compared to normal mouse or human tissues correlating with organ fibrosis progression. Finally, the inhibitors of Akt, mTOR, and β-catenin suppressed TGFβ1-induced ALK5 expression, indicating that the Akt pathway promotes FibroMF differentiation via ALK5 expression and fibrosis.
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Dostopno za:
DOBA, FSPLJ, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Vascular permeability regulated by the vascular endothelial growth factor (VEGF) through endothelial-barrier junctions is essential for inflammation. Mechanisms regulating vascular permeability ...remain elusive. Although ‘Akt’ and ‘Src’ have been implicated in the endothelial-barrier regulation, it is puzzling how both agents that protect and disrupt the endothelial-barrier activate these kinases to reciprocally regulate vascular permeability. To delineate the role of Akt1 in endothelial-barrier regulation, we created endothelial-specific, tamoxifen-inducible Akt1 knockout mice and stable ShRNA-mediated Akt1 knockdown in human microvascular endothelial cells. Akt1 loss leads to decreased basal and angiopoietin1-induced endothelial-barrier resistance, and enhanced VEGF-induced endothelial-barrier breakdown. Endothelial Akt1 deficiency resulted in enhanced VEGF-induced vascular leakage in mice ears, which was rescued upon re-expression with Adeno-myrAkt1. Furthermore, co-treatment with angiopoietin1 reversed VEGF-induced vascular leakage in an Akt1-dependent manner. Mechanistically, our study revealed that while VEGF-induced short-term vascular permeability is independent of Akt1, its recovery is reliant on Akt1 and FoxO-mediated claudin expression. Pharmacological inhibition of FoxO transcription factors rescued the defective endothelial barrier due to Akt1 deficiency. Here we provide novel insights on the endothelial-barrier protective role of VEGF in the long term and the importance of Akt1-FoxO signaling on tight-junction stabilization and prevention of vascular leakage through claudin expression.
Ischemic diseases such as stroke and proliferative retinopathy are characterized by hypoxia-driven release of angiogenic factors such as vascular endothelial growth factor (VEGF). However, ...revascularization of the ischemic areas is inadequate, resulting in impaired neuro-vascular function. We aim to examine the vascular protective effects of candesartan, an angiotensin receptor blocker, in an ischemic retinopathy mouse model. Vascular density, number of tip cells, and perfusions of capillaries were assessed. Activation of Muller glial cells and levels of peroxynitrite, VEGF, VEGFR2, inducible nitric oxide synthase, hemeoxygenase-1 (HO-1) were assessed. Proangiogenic effects of candesartan were examined in human endothelial cells (EC) that were cultured in normoxia or hypoxia and transduced with siRNA against HO-1. Candesartan (1 mg/kg) and (10 mg/kg) decreased hypoxia-induced neovascularization by 67 and 70 %, respectively. Candesartan (10 mg/kg) significantly stimulated the number of tip cells and physiological revascularization of the central retina (45 %) compared with untreated pups. The effects of candesartan coincided with reduction of hypoxia-induced Muller glial activation, iNOS expression and restoration of HO-1 expression with no significant change in VEGF levels. In vitro, silencing HO-1 expression blunted the ability of candesartan to induce VEGF expression under normoxia and VEGFR2 activation and angiogenic response under both normoxia and hypoxia. These findings suggest that candesartan improved reparative angiogenesis and hence prevented pathological angiogenesis by modulating HO-1 and iNOS levels in ischemic retinopathy. HO-1 is required for VEGFR2 activation and proangiogenic action of candesartan in EC. Candesartan, an FDA-approved drug, could be repurposed as a potential therapeutic agent for the treatment of ischemic diseases.
Doppler echocardiography has been demonstrated to be accurate in diagnosing valvular lesions in rheumatic heart disease (RHD) when compared to clinical evaluation alone.
To perform Doppler ...echocardiography in children clinically diagnosed by the Jones criteria to have acute rheumatic fever (ARF), and to then compare the effectiveness of echo in detecting single/multi-valvular lesions with that of the initial clinical evaluation.
We enrolled 93 children who were previously diagnosed with ARF by clinical examination. Presence of valvular lesions were enlisted, first by clinical auscultation, and then by performing Doppler echocardiography. We found that Doppler echocardiography was a sensitive technique, capable of detecting valvular lesions that were missed by clinical auscultation alone. Echocardiography of patients with carditis revealed mitral regurgitation to be the most common lesion present (53 patients, 56.98%), followed by aortic regurgitation in 21 patients (22.6%). The difference between clinical and echocardiographic diagnosis in ARF children with carditis was statistically significant for mitral regurgitation, aortic regurgitation and tricuspid regurgitation. Clinical auscultation alone revealed 4 cases of mitral stenosis, 39 mitral regurgitation, 14 aortic regurgitation, 9 tricuspid regurgitation; in contrast, echo revealed 5 cases of mitral stenosis, 53 mitral regurgitation, 21 aortic regurgitation, 18 tricuspid regurgitation.
Doppler echocardiography is a more sensitive technique for detecting valvular lesions. In the setting of ARF, echo enables a 46.9% higher detection level of carditis, as compared to the clinical examination alone. Echo was very significant in detecting regurgitation lesions, especially for cases of tricuspid regurgitation in the setting of multivalvular involvement. The results of our study are in accordance with previous clinical studies, all of which clearly demonstrate the advantages of Doppler echocardiography, paving the way for its probable inclusion as one of the Jones major criteria for diagnosing ARF.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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