Distinct effects of pharmacological inhibition of stromelysin1 on endothelial-to-mesenchymal transition and myofibroblast differentiation.

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 TGFß2 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 TGFß2-induced EndMT. In contrast, treatment of NIH-3T3 fibroblasts with TGFß1 promoted FibroMF differentiation accompanied by increased expression of αSMA, N-cadherin, and ALK5. Intriguingly, stromelysin1 inhibition in TGFß1-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.

Differential regulation of TGFß type-I receptor expressions in TGFß1-induced myofibroblast differentiation.

Fibroblast-to-myofibroblast (FibroMF) differentiation is crucial for embryogenesis and organ fibrosis. Although transforming growth factor-ß (TGFß) is the primary mediator of FibroMF differentiation, the type-I receptor (TGFßRI) responsible for this has not yet been confirmed. In the current study, we investigated the ALK1 and ALK5 expressions in TGFß1-stimulated NIH 3T3 fibroblasts to compare with the data from the Gene Expression Omnibus (GEO) repository. In our results, whereas TGFß1 treatment promoted FibroMF differentiation accompanied by increased ALK5 expression and reduced ALK1 expression, TGFß1-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 ß-catenin suppressed TGFß1-induced ALK5 expression, indicating that the Akt pathway promotes FibroMF differentiation via ALK5 expression and fibrosis.

Pharmacological inhibition of ß-catenin prevents EndMT in vitro and vascular remodeling in vivo resulting from endothelial Akt1 suppression.

Endothelial to mesenchymal transition (EndMT), where endothelial cells acquire mesenchymal characteristics has been implicated in several cardiopulmonary, vascular and fibrotic diseases. The most commonly studied molecular mechanisms involved in EndMT include TGFß, Notch, interleukin, and interferon-γ signaling. As of today, the contributions of Akt1, an important mediator of TGFß signaling and a key regulator of endothelial barrier function to EndMT remains unclear. By using the ShRNA based gene silencing approach and endothelial-specific inducible Akt1 knockdown (ECKOAkt1) mice, we studied the role of Akt1 in EndMT in vitro and pathological vascular remodeling in vivo. Stable, Akt1 silenced (ShAkt1) human microvascular endothelial cells (HMECs) indicated increased expression of mesenchymal markers such as N-cadherin and α-SMA, phosphorylation of Smad2/3, cellular stress via activation of p38 MAP Kinase and the loss of endothelial nitric oxide synthase (eNOS) accompanied by a change in the morphology of HMECs in vitro and co-localization of endothelial and mesenchymal markers promoting EndMT in vivo. EndMT as a result of Akt1 loss was associated with increased expression of TGFß2, a potent inducer of EndMT and mesenchymal transcription factors Snail1, and FoxC2. We observed that hypoxia-induced lung vascular remodeling is exacerbated in ECKOAkt1 mice, which was reversed by pharmacological inhibition of ß-catenin. Thus, we provide novel insights into the role of Akt1-mediated ß-catenin signaling in EndMT and pathological vascular remodeling, and present ß-catenin as a potential target for therapy for various cardiopulmonary diseases involving vascular remodeling.

Endothelial stromelysin1 regulation by the forkhead box-O transcription factors is crucial in the exudative phase of acute lung injury.

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.

Isoform-specific effects of transforming growth factor ß on endothelial-to-mesenchymal transition.

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 ß (TGFß) 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 TGFß1, TGFß2, and TGFß3 on EndMT and characterize the underlying mechanisms. In our results, all three TGFß 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 TGFß2 was the most pronounced. At 1 ng/ml, only TGFß2 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 TGFß1 and TGFß3, but not TGFß2, resulted in an increased expression of TGFß2, thus indicating that EndMT with TGFß1 and TGFß3 treatments was due to the secondary effects through TGFß2 secretion. Furthermore, silencing TGFß2 using small interfering RNA blunted the expression of EndMT markers in TGFß1- and TGFß3-treated cells. Together, our results indicate that TGFß2 is the most potent inducer of EndMT and that TGFß1- and TGFß3-induced EndMT necessitates a paracrine loop involving TGFß2.

Suppression of Akt1-ß-catenin pathway in advanced prostate cancer promotes TGFß1-mediated epithelial to mesenchymal transition and metastasis.

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 TGFß1 and its receptor TGFß RII, associated with a decreased expression of ß-catenin. Furthermore, treatment of PCa cells with ICG001 that blocks nuclear translocation of ß-catenin promoted EMT and N-cadherin expression. Together, our study demonstrates a novel role of the Akt1-ß-catenin-TGFß1 pathway in advanced PCa.

Modulation of long-term endothelial-barrier integrity is conditional to the cross-talk between Akt and Src signaling.

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-ß1 (TGFß1) that inhibited Akt Ser473 phosphorylation in the long-term, activated Src through increased Tyr416 phosphorylation and decreased HMEC-barrier resistance. The effect of TGFß1 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.

Novel roles of Src in cancer cell epithelial-to-mesenchymal transition, vascular permeability, microinvasion and metastasis.

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.

Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover.

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.

Genetic deletion and pharmacological inhibition of Akt1 isoform attenuates bladder cancer cell proliferation, motility and invasion.

Isoform specific expression, intracellular localization and function of Akt in bladder cancer are not known. In the current study, we identified Akt1, followed by Akt2 and Akt3 as the predominant Akt isoform in human T24 and UM-UC-3 metastatic bladder cancer cells. Whereas Akt1 is localized at the membrane, cytoplasm and nucleus, Akt2 is solely cytoplasmic and Akt3 is mostly localized in the nucleus in T24 cells. ShRNA-mediated Akt1 knockdown resulted in impaired T24 cell survival, proliferation, colony formation, migration and microinvasion. Whereas pharmacological inhibition of Akt1 resulted in impaired T24 and UM-UC-3 cell motility, viability and proliferation, effect of pharmacological inhibition by Akt2 inhibitor was limited to proliferation in T24, but not UM-UC-3 cells. Our data provide important clues on the therapeutic benefits of targeting Akt1 for bladder cancer therapy.

The Akt inhibitor, triciribine, ameliorates chronic hypoxia-induced vascular pruning and TGFß-induced pulmonary fibrosis.

BACKGROUND AND PURPOSE: Interstitial lung disease accounts for a group of chronic and progressive disorders associated with severe pulmonary vascular remodelling, peripheral vascular rarefaction and fibrosis, thus limiting lung function. We have previously shown that Akt is necessary for myofibroblast differentiation, a critical event in organ fibrosis. However, the contributory role of the Akt-mTOR pathway in interstitial lung disease and the therapeutic benefits of targeting Akt and mTOR remain unclear. EXPERIMENTAL APPROACH: We investigated the role of the Akt-mTOR pathway and its downstream molecular mechanisms in chronic hypoxia- and TGFß-induced pulmonary vascular pruning and fibrosis in mice. We also determined the therapeutic benefits of the Akt inhibitor triciribine and the mTOR inhibitor rapamycin for the treatment of pulmonary fibrosis in mice. KEY RESULTS: Akt1(-) (/) (-) mice were protected from chronic hypoxia-induced peripheral vascular pruning. In contrast, hyperactivation of Akt1 induced focal fibrosis similar to TGFß-induced fibrosis. Pharmacological inhibition of Akt, but not the Akt substrate mTOR, inhibited hypoxia- and TGFß-induced pulmonary vascular rarefaction and fibrosis. Mechanistically, we found that Akt1 modulates pulmonary remodelling via regulation of thrombospondin1 (TSP1) expression. Hypoxic Akt1(-) (/) (-) mice lungs expressed less TSP1. Moreover, TSP1(-) (/) (-) mice were resistant to adMyrAkt1-induced pulmonary fibrosis. CONCLUSIONS AND IMPLICATIONS: Our study identified Akt1 as a novel target for the treatment of interstitial lung disease and provides preclinical data on the potential benefits of the Akt inhibitor triciribine for the treatment of interstitial lung disease.

TNFα induces inflammatory stress response in microvascular endothelial cells via Akt- and P38 MAP kinase-mediated thrombospondin-1 expression.

Tumor necrosis factor-α (TNFα) and thrombospondin-1 (TSP-1) are well-known mediators of inflammation. However, a causal relationship between TNFα stimuli and TSP-1 expression in endothelial cell stress, and the underlying mechanisms has not yet been investigated. In our study, human microvascular endothelial cells (hMEC) were treated with TNFα and analyzed for endothelial dysfunction, TSP-1 expression, and associated mechanisms. TNFα treatment induced a dose-dependent increase in TSP-1 expression in hMEC associated with increased endothelial permeability, apoptosis, and reduced proliferation. Whereas TNFα activated Akt, ERK, and P38 mitogen-activated protein kinase (P38 MAPK) simultaneously in hMEC, inhibitors of Akt and P38 MAPK, but not ERK blunted TNFα-induced TSP-1 expression. Silencing of NFκB gene had no significant effect on TNFα-induced TSP-1 expression. Our study demonstrates the novel role of TNFα in inducing inflammatory stress response in hMEC through Akt- and P38 MAPK-mediated expression of TSP-1, independent of NFκB signaling.

p70 S6-kinase mediates the cooperation between Akt1 and Mek1 pathways in fibroblast-mediated extracellular matrix remodeling.

Previous studies have demonstrated both synergistic and opposing effects of Akt and Mek1/2 in various cell functions and disease states. Furthermore, Akt has been reported to inhibit and activate cRaf/Mek pathway, suggesting that their mutual interaction and cooperation may be cell type, stimuli and/or context specific. While PI3-kinase/Akt and cRaf/Mek pathways have been implicated in the regulation of extracellular matrix (ECM) remodeling, mutual interactions between these two pathways and their specific contributions to the events leading to ECM synthesis and assembly is not clear. We investigated the specific role of Akt1 and Mek1 in ECM synthesis and assembly by NIH 3T3 fibroblasts and how these effects were reconciled to mediate overall ECM remodeling. Our study identified that cooperation between Akt1 and Mek1 is necessary to mediate ECM synthesis. Whereas Akt1 activation resulted in Mek1 activation as evidenced by increased ERK1/2 phosphorylation, Mek1 inhibition using U0126 or DN-Mek1 resulted in enhanced Akt1 phosphorylation. Interestingly, both Akt1 and Mek1 activities were needed for the synthesis and assembly of ECM. The effect of Akt1 and Mek1 on ECM synthesis was reconciled through the activation of p70 S6-kinase via phosphorylation at T421/S424 and S411, respectively. Furthermore, Akt1 and Mek1 cooperated in mediating ECM assembly via activation of integrin ß1. Together, we show for the first time that Akt1 and Mek1 pathways cooperate in the regulation of ECM remodeling by the fibroblasts.

Candesartan stimulates reparative angiogenesis in ischemic retinopathy model: role of hemeoxygenase-1 (HO-1).

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.

Targeting Src-mediated Tyr216 phosphorylation and activation of GSK-3 in prostate cancer cells inhibit prostate cancer progression in vitro and in vivo.

Recent studies suggest a positive correlation between glycogen synthase kinase-3 (GSK-3) activation and tumor growth. Currently, it is unclear how both Akt that inhibits GSK-3 and active GSK-3 are maintained concurrently in tumor cells. We investigated the role of GSK-3 and the existence of an Akt-resistant pathway for GSK-3 activation in prostate cancer cells. Our data show that Src, a non-receptor tyrosine kinase is responsible for Y216GSK-3 phosphorylation leading to its activation even when Akt is active. Experiments involving mouse embryonic fibroblasts lacking cSrc, Yes and Fyn, as well as Src activity modulation in prostate cancer cells with constitutively active (CA-Src) and dominant negative Src (DN-Src) plasmids demonstrated the integral role of Src in Y216GSK-3 phosphorylation and activity modulation. Inhibition of GSK-3 with SB415286 in PC3 cells resulted in impaired motility, proliferation and colony formation. Treatment of PC3 cells with the Src inhibitor dasatinib reduced Y216GSK-3 phosphorylation and inhibited proliferation, invasion and micrometastasis in vitro. Dasatinib treatment of athymic nude mice resulted in impaired growth of PC3 cell tumor xenograft. Together, we provide novel insight into the Src-mediated Y216GSK-3 phosphorylation and activation in prostate cancer cells and reveal the potential benefits of targeting Src-GSK-3 axis using drugs such as dasatinib.

Doppler echocardiography imaging in detecting multi-valvular lesions: a clinical evaluation in children with acute rheumatic fever.

RATIONALE: Doppler echocardiography has been demonstrated to be accurate in diagnosing valvular lesions in rheumatic heart disease (RHD) when compared to clinical evaluation alone. OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.