Extracellular vesicle-encapsulated CC16 as novel nanotherapeutics for treatment of acute lung injury.

Acute lung injury (ALI) is still associated with high mortality. Growing evidence suggests that Club Cell Protein 16 (CC16) plays a protective role against ALI. However, the doses of recombinant CC16 (rCC16) used in preclinical studies are supraphysiological for clinical applications. Extracellular vesicles (EVs) are nanovesicles endogenously generated by mammalian cells. Our study demonstrated that CC16 is released via small EVs and EV-encapsulated CC16 (sEV-CC16) and has anti-inflammatory activities, which protect mice from lipopolysaccharide (LPS) or bacteria-induced ALI. Additionally, sEV-CC16 can activate the DNA damage repair signaling pathways. Consistent with this activity, we observed more severe DNA damage in lungs from Cc16 knockout (KO) than wild-type (WT) mice. Mechanistically, we elucidated that CC16 suppresses nuclear factor κB (NF-κB) signaling activation by binding to heat shock protein 60 (HSP60). We concluded that sEV-CC16 could be a potential therapeutic agent for ALI by inhibiting the inflammatory and DNA damage responses by reducing NF-κB signaling.

AKT Isoforms in the Immune Response in Cancer.

AKT is a protein kinase that exists in three isoforms: AKT1, AKT2, and AKT3. Though similar in structure, these isoforms display different effects. AKT is activated downstream of PI3K, and together, this signaling pathway helps regulate cellular processes including cell growth, proliferation, metabolism, survival, and apoptosis. Disruption in these pathways has been associated with disorders including cardiovascular diseases, developmental disorders, inflammatory responses, autoimmune diseases, neurologic disorders, type 2 diabetes, and several cancers. In cancer, deregulation in the PI3K/AKT pathway can be manifested as tumorigenesis, pathological angiogenesis, and metastasis. Increased activity has been correlated with tumor progression and resistance to cancer treatments. Recent studies have suggested that inhibition of the PI3K/AKT pathway plays a significant role in the development, expansion, and proliferation of cells of the immune system. Additionally, AKT has been found to play an important role in differentiating regulatory T cells, activating B cells, and augmenting tumor immunosurveillance. This emphasizes AKT as a potential target for inhibition in cancer therapy. This chapter reviews AKT structure and regulation, its different isoforms, its role in immune cells, and its modulation in oncotherapy.

Plasma matrix metalloproteinase-3 predicts mortality in acute respiratory distress syndrome: a biomarker analysis of a randomized controlled trial.

BACKGROUND: Matrix metalloproteinase-3 (MMP-3) is a proteolytic enzyme involved in acute respiratory distress syndrome (ARDS) pathophysiology that may serve as a lung-specific biomarker in ARDS. METHODS: This study was a secondary biomarker analysis of a subset of Albuterol for the Treatment of Acute Lung Injury (ALTA) trial patients to determine the prognostic value of MMP-3. Plasma sample MMP-3 was measured by enzyme-linked immunosorbent assay. The primary outcome was the area under the receiver operating characteristic (AUROC) of MMP-3 at day 3 for the prediction of 90-day mortality. RESULTS: A total of 100 unique patient samples were evaluated and the AUROC analysis of day three MMP-3 showed an AUROC of 0.77 for the prediction of 90-day mortality (95% confidence interval: 0.67-0.87), corresponding to a sensitivity of 92% and specificity of 63% and an optimal cutoff value of 18.4 ng/mL. Patients in the high MMP-3 group (≥ 18.4 ng/mL) showed higher mortality compared to the non-elevated MMP-3 group (< 18.4 ng/mL) (47% vs. 4%, p < 0.001). A positive difference in day zero and day three MMP-3 concentration was predictive of mortality with an AUROC of 0.74 correlating to 73% sensitivity, 81% specificity, and an optimal cutoff value of + 9.5 ng/mL. CONCLUSIONS: Day three MMP-3 concentration and difference in day zero and three MMP-3 concentrations demonstrated acceptable AUROCs for predicting 90-day mortality with a cut-point of 18.4 ng/mL and + 9.5 ng/mL, respectively. These results suggest a prognostic role of MMP-3 in ARDS.

Pharmacological Inhibition of Spermine Oxidase Suppresses Excitotoxicity Induced Neuroinflammation in Mouse Retina.

Polyamine oxidation plays a major role in neurodegenerative diseases. Previous studies from our laboratory demonstrated that spermine oxidase (SMOX, a member of the polyamine oxidase family) inhibition using MDL 72527 reduced neurodegeneration in models of retinal excitotoxicity and diabetic retinopathy. However, the mechanisms behind the neuroprotection offered by SMOX inhibition are not completely studied. Utilizing the experimental model of retinal excitotoxicity, the present study determined the impact of SMOX blockade in retinal neuroinflammation. Our results demonstrated upregulation in the number of cells positive for Iba-1 (ionized calcium-binding adaptor molecule 1), CD (Cluster Differentiation) 68, and CD16/32 in excitotoxicity-induced retinas, while MDL 72527 treatment reduced these changes, along with increases in the number of cells positive for Arginase1 and CD206. When retinal excitotoxicity upregulated several pro-inflammatory genes, MDL 72527 treatment reduced many of them and increased anti-inflammatory genes. Furthermore, SMOX inhibition upregulated antioxidant signaling (indicated by elevated Nrf2 and HO-1 levels) and reduced protein-conjugated acrolein in excitotoxic retinas. In vitro studies using C8-B4 cells showed changes in cellular morphology and increased reactive oxygen species formation in response to acrolein (a product of SMOX activity) treatment. Overall, our findings indicate that the inhibition SMOX pathway reduced neuroinflammation and upregulated antioxidant signaling in the retina.

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.

Akt-independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS-CoV2 infection.

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.

Delayed Akt suppression in the lipopolysaccharide-induced acute lung injury promotes resolution that is associated with enhanced effector regulatory T cells.

The adaptive immune response could play a major role in the resolution of lung injury. Although regulatory T cells (Tregs) have been implicated in promoting the resolution of lung injury, therapeutic strategies to enhance Treg quantity and activity at the site of injury need further exploration. In the current study, Akt inhibition using triciribine (TCBN), given 48 h after lipopolysaccharide (LPS) administration, increased Tregs-promoted resolution of acute lung injury (ALI). TCBN treatment enhanced the resolution of LPS-induced ALI on day 7 by reducing pulmonary edema and neutrophil activity associated with an increased number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs, specifically in the injured lungs and not in the spleen. Treatment of EL-4 T-lymphocytes with two Akt inhibitors (TCBN and MK-2206) for 72 h resulted in increased FoxP3 expression in vitro. On the other end, Treg-specific PTEN knockout (PTENTreg KO) mice that have a higher Akt activity in its Tregs exhibited a significant impairment in ALI resolution, increased edema, and neutrophil activity associated with a reduced number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs as compared with the control group. In conclusion, our study identifies a potential target for the treatment of late-stage ALI by promoting resolution through effector Treg-mediated suppression of inflammation.

Patients with acute respiratory distress syndrome exhibit increased stromelysin1 activity in the blood samples.

Enzyme activity analyses in the blood are expected to be reliable, non-invasive diagnostic as well as prognostic markers to reflect disease progression in acute lung injury (ALI). The objective of the current study was to evaluate the enzymatic activity of stromelysin1 (matrix metalloprotease-3) in the plasma/serum samples collected from ALI patients compared to the samples collected from healthy controls. Gene expression omnibus (GEO) database analysis indicated a correlation between increased stromelysin1 gene expression and the incidence of ALI in various animal models. Our analysis of patient plasma/serum samples from healthy controls and ALI patients revealed a significant, 3-fold increase in stromelysin1 activity in ALI plasma/serum compared to healthy subjects with no difference in stromelysin1 activity between the serum and plasma samples. Interestingly, no significant difference in stromelysin1 activity between non-smoking and smoking subjects was observed. These findings provide fundamental information on the potential reliability of stromelysin1 activity analysis, combined with other biomarkers in development, in blood samples for the early detection of ALI.

Regulation of blood-retinal barrier cell-junctions in diabetic retinopathy.

Loss of the blood-retinal barrier (BRB) integrity and subsequent damage to the neurovascular unit in the retina are the underlying reasons for diabetic retinopathy (DR). Damage to BRB eventually leads to severe visual impairment in the absence of prompt intervention. Diabetic macular edema and proliferative DR are the advanced stages of the disease where BRB integrity is altered. Primary mechanisms contributing to BRB dysfunction include loss of cell-cell barrier junctions, vascular endothelial growth factor, advanced glycation end products-induced damage, and oxidative stress. Although much is known about the involvement of adherens and tight-junction proteins in the regulation of vascular permeability in various diseases, there is a significant gap in our knowledge on the junctional proteins expressed in the BRB and how BRB function is modulated in the diabetic retina. In this review article, we present our current understanding of the molecular composition of BRB, the changes in the BRB junctional protein turnover in DR, and how BRB functional modulation affects vascular permeability and macular edema in the diabetic retina.

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.

Liposomes Targeting P21 Activated Kinase-1 (PAK-1) and Selective for Secretory Phospholipase A2 (sPLA2) Decrease Cell Viability and Induce Apoptosis in Metastatic Triple-Negative Breast Cancer Cells.

P21 activated kinases (or group I PAKs) are serine/threonine kinases whose expression is altered in prostate and breast cancers. PAK-1 activity is inhibited by the small molecule "Inhibitor targeting PAK-1 activation-3" (IPA-3), which has selectivity for PAK-1 but is metabolically unstable. Secretory Group IIA phospholipase A2 (sPLA2) expression correlates to increased metastasis and decreased survival in many cancers. We previously designed novel liposomal formulations targeting both PAK-1 and sPLA2, called Secretory Phospholipase Responsive liposomes or SPRL-IPA-3, and demonstrated their ability to alter prostate cancer growth. The efficacy of SPRL against other types of cancers is not well understood. We addressed this limitation by determining the ability of SPRL to induce cell death in a diverse panel of cells representing different stages of breast cancer, including the invasive but non-metastatic MCF-7 cells, and metastatic triple-negative breast cancer (TNBC) cells such as MDA-MB-231, MDA-MB-468, and MDA-MB-435. We investigated the role of sPLA2 in the disposition of these liposomes by comparing the efficacy of SPRL-IPA-3 to IPA-3 encapsulated in sterically stabilized liposomes (SSL-IPA-3), a formulation shown to be less sensitive to sPLA2. Both SSL-IPA-3 and SPRL-IPA-3 induced time- and dose-dependent decreases in MTT staining in all cell lines tested, but SPRL-IPA-3-induced effects in metastatic TNBC cell lines were superior over SSL-IPA-3. The reduction in MTT staining induced by SPRL-IPA-3 correlated to the expression of Group IIA sPLA2. sPLA2 expression also correlated to increased induction of apoptosis in TNBC cell lines by SPRL-IPA-3. These data suggest that SPRL-IPA-3 is selective for metastatic TNBC cells and that the efficacy of SPRL-IPA-3 is mediated, in part, by the expression of Group IIA sPLA2.

The Roles of CCN1/CYR61 in Pulmonary Diseases.

CCN1 (cysteine-rich 61, connective tissue growth factor, and nephroblastoma-1), previously named CYR61 (cysteine-rich angiogenic inducer 61) belongs to the CCN family of matricellular proteins. CCN1 plays critical roles in the regulation of proliferation, differentiation, apoptosis, angiogenesis, and fibrosis. Recent studies have extensively characterized the important physiological and pathological roles of CCN1 in various tissues and organs. In this review, we summarize both basic and clinical aspects of CCN1 in pulmonary diseases, including acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), lung fibrosis, pulmonary arterial hypertension (PAH), lung infection, and lung cancer. We also emphasize the important challenges for future investigations to better understand the CCN1 and its role in physiology and pathology, as well as the questions that need to be addressed for the therapeutic development of CCN1 antagonists in various lung diseases.

The unconventional role of Akt1 in the advanced cancers and in diabetes-promoted carcinogenesis.

Decades of research have elucidated the critical role of Akt isoforms in cancer as pro-tumorigenic and metastatic regulators through their specific effects on the cancer cells, tumor endothelial cells and the stromal cells. The pro-cancerous role of Akt isoforms through enhanced cell proliferation and suppression of apoptosis in cancer cells and the cells in the tumor microenvironment is considered a dogma. Intriguingly, studies also indicate that the Akt pathway is essential to protect the endothelial-barrier and prevent aberrant vascular permeability, which is also integral to tumor perfusion and metastasis. To complicate this further, a flurry of recent reports strongly indicates the metastasis suppressive role of Akt, Akt1 in particular in various cancer types. These reports emanated from different laboratories have elegantly demonstrated the paradoxical effect of Akt1 on cancer cell epithelial-to-mesenchymal transition, invasion, tumor endothelial-barrier disruption, and cancer metastasis. Here, we emphasize on the specific role of Akt1 in mediating tumor cell-vasculature reciprocity during the advanced stages of cancers and discuss how Akt1 differentially regulates cancer metastasis through mechanisms distinct from its pro-tumorigenic effects. Since Akt is integral for insulin signaling, endothelial function, and metabolic regulation, we also attempt to shed some light on the specific effects of diabetes in modulating Akt pathway in the promotion of tumor growth and metastasis.

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.

Endothelial Akt1 loss promotes prostate cancer metastasis via ß-catenin-regulated tight-junction protein turnover.

BACKGROUND: Cancer research, in general, is focused on targeting tumour cells to limit tumour growth. These studies, however, do not account for the specific effects of chemotherapy on tumour endothelium, in turn, affecting metastasis. METHODS: We determined how endothelial deletion of Akt1 promotes prostate cancer cell invasion in vitro and metastasis to the lungs in vivo in endothelial-specific Akt1 knockdown mice. RESULTS: Here we show that metastatic human PC3 and DU145 prostate cancer cells invade through Akt1-deficient human lung endothelial cell (HLEC) monolayer with higher efficiency compared to control HLEC. Although the endothelial Akt1 loss in mice had no significant effect on RM1 tumour xenograft growth in vivo, it promoted metastasis to the lungs compared to the wild-type mice. Mechanistically, Akt1-deficient endothelial cells exhibited increased phosphorylation and nuclear translocation of phosphorylated ß-catenin, and reduced expression of tight-junction proteins claudin-5, ZO-1 and ZO-2. Pharmacological inhibition of ß-catenin nuclear translocation using compounds ICG001 and IWR-1 restored HLEC tight-junction integrity and inhibited prostate cancer cell transendothelial migration in vitro and lung metastasis in vivo. CONCLUSIONS: Here we show for the first time that endothelial-specific loss of Akt1 promotes cancer metastasis in vivo involving ß-catenin pathway.

cGMP Signaling Increases Antioxidant Gene Expression by Activating Forkhead Box O3A in the Colon Epithelium.

Signaling through cGMP has therapeutic potential in the colon, where it has been implicated in the suppression of colitis and colon cancer. In this study, we tested the ability of cGMP and type 2 cGMP-dependent protein kinase (PKG2) to activate forkhead box O (FoxO) in colon cancer cells and in the colon epithelium of mice. We show that activation of PKG2 in colon cancer cells inhibited cell proliferation, inhibited AKT, and activated FoxO. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and reduced epithelial reduction-oxidation (redox) stress. FoxO3a was the most prominent isoform in the distal colon epithelium, with prominent luminal staining. FoxO3a levels were reduced in Prkg2-/- animals, and FoxO target genes were unaffected by 8Br-cGMP challenge in vitro. Treatment of mice with the phosphodiesterase-5 inhibitor vardenafil (Levitra) mobilized FoxO3a to the nucleus of luminal epithelial cells, which corresponded to increased FoxO target gene expression, reduced redox stress, and increased epithelial barrier integrity. Treatment of human colonic biopsy specimens with 8Br-cGMP also activated catalase and manganese superoxide dismutase expression, indicating that this pathway is conserved in humans. Taken together, these results identify a novel signaling pathway in the colon epithelium, where FoxO tumor suppressors could provide protection from redox stress. Moreover, this pathway is regulated by endogenous cGMP/PKG2 signaling, and can be targeted using phosphodiesterase-5 inhibitors.

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.

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.

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.